piperidines and 4-diphenylacetoxy-1-1-dimethylpiperidinium

Although the molecular machinery and mechanism of cell secretion in acinar cells of the exocrine pancreas is well documented and clear, only recently has the pharmacophysiology of pancreatic exocrine secretion come to light. Therefore, we focus in this article on the current understanding of the pharmacophysiology of pancreatic exocrine secretion. The pancreatic secretory response to ingestion of a meal is mediated via a complex interplay of neural, humoral and paracrine mediators. A major role in the control of the intestinal phase of pancreatic secretion is attributed to vago-vagal enteropancreatic reflexes. In the scheme of this control mechanism, afferents originating in the duodenal mucosa, and efferents mediating central input on the pancreatic ganglia, activate intrapancreatic postganglionic neurons. Experiments utilizing specific receptor antagonists demonstrate the involvement of both muscarinic M1 and M3 receptors expressed in pancreatic acinar cells. Cholecystokinin (CCK), originally implicated in the humoral secretion of pancreatic enzymes, through a direct action on acinar CCK receptors, is also essential to the enteropancreatic reflex mechanism. CCK stimulation of the exocrine pancreatic secretion through excitation of sensory afferents of the enteropancreatic reflexes, is a paracrine mode of CCK action, and is probably the only one in humans and the predominant one in rats. In dogs, however, CCK acts on the pancreas via both the humoral and a paracrine route. More recent experiments suggest further possible sites of CCK action. Additionally, at the brain stem, vago-vagal enteropancreatic reflexes may be modulated by input from higher brain centres, particularly the hypothalamic-cholinergic system in the tonic stimulation of preganglionic neurons of the dorsal motor nucleus of the vagus projecting into the pancreas.

Topics: Animals; Cholecystokinin; Dogs; Guinea Pigs; Humans; Mice; Pancreas, Exocrine; Piperidines; Pirenzepine; Rats; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptors, Cholecystokinin; Reflex; Vagus Nerve

Topics: Animals; Delayed Rectifier Potassium Channels; Humans; Membrane Potentials; Myocardial Ischemia; Pilocarpine; Piperidines; Receptor, Muscarinic M3; Signal Transduction

Muscarinic agonists elicit contraction in the standard guinea pig ileum bioassay through activation of M3 muscarinic receptors that are also linked to phosphoinositide hydrolysis. Surprisingly, the most abundant muscarinic receptor in the ileum is the M2 which causes a specific inhibition of cyclic AMP accumulation elicited by the beta-adrenergic receptor. After most of the M3 receptors are inactivated, the ileum still retains high sensitivity to muscarinic agonists provided that the contractile responses are measured in the presence of histamine and forskolin, which together, have no effect on contraction. Under these conditions, the potencies of antagonists for blocking the contractile response are consistent with those expected for an M2 response. Moreover, the muscarinic contractile response measured in the presence of histamine and forskolin after inactivation of M3 receptors is pertussis toxin sensitive. In contrast, muscarinic contractions in the standard bioassay are pertussis toxin insensitive. These results demonstrate that the M2 muscarinic receptor can cause an indirect contraction of the guinea pig ileum by preventing the relaxing effect of agents that increase cAMP.

Topics: Animals; Colforsin; Cyclic AMP; Guinea Pigs; Heart; Histamine; Ileum; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Oxotremorine; Pertussis Toxin; Piperidines; Rats; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Signal Transduction; Submandibular Gland; Virulence Factors, Bordetella

The specific role played by different muscarinic receptor subtypes in sleep regulation is investigated and discussed. On the basis of the results obtained with intracerebroventricular injections of selective muscarinic antagonists into freely moving rats, it is suggested that each muscarinic receptor subtype induces different and specific changes in sleep phases and cortical desynchronization processes.

Topics: Animals; Diamines; Humans; Parasympatholytics; Piperidines; Receptors, Muscarinic; Sleep

Muscarinic M3 receptor antagonists have therapeutic potential for the treatment of disorders associated with altered smooth muscle contractility or tone. These include irritable bowel syndrome (IBS), chronic obstructive airways disease (COAD) and urinary incontinence. Zamifenacin is a potent muscarinic receptor antagonist on the guinea pig ileum (pA2 value 9.27) with selectivity over M2 receptors in the atria (135-fold) and M1/M4 receptors in the rabbit vas deferens (78-fold). In addition, zamifenacin had lower affinity for the M3 receptor in the salivary gland (pKi 7.97). In animals, zamifenacin potently inhibited gut motility in the absence of cardiovascular effects and with selectivity over inhibition of salivary secretion. In healthy volunteers, zamifenacin inhibited small and large bowel motility and increased the rate of gastric emptying over a dose range which was associated with minimal anticholinergic side effects. These data show that zamifenacin, a selective muscarinic M3 receptor antagonist, was well tolerated in man and was efficacious as an inhibitor of gut motility. Further studies in patients are required with muscarinic M3 receptor antagonists to confirm efficacy against symptoms in diseases associated with altered smooth muscle contractility.

Topics: Animals; Atrial Function; Atropine; Colon; Dicyclomine; Dioxoles; Dogs; Drug Evaluation, Preclinical; Gastrointestinal Motility; Guinea Pigs; Heart Atria; Humans; Ileum; Male; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Rabbits; Receptor, Muscarinic M3; Receptors, Muscarinic; Salivary Glands; Trachea; Vas Deferens

Topics: Animals; Animals, Newborn; Behavior, Animal; Carbachol; Chickens; Disease Models, Animal; Eating; Injections, Intraventricular; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M3

The rostromedial tegmental nucleus (RMTg) receives inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic nuclei. We tested the effects of RMTg M3 and M4 muscarinic cholinergic receptor antagonism in a conditioned place preference (CPP) paradigm in mice. RMTg infusions of the M3 muscarinic cholinergic receptor antagonist 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) do not result in the acquisition of CPP but increase locomotor activation. By contrast, RMTg infusions of the M4 muscarinic cholinergic receptor antagonist Tropicamide result in the acquisition of CPP but do not increase locomotor activation. The rewarding effects of RMTg Tropicamide infusions are dopamine-dependent as systemic pre-treatment with the broad-spectrum dopamine receptor antagonist flupenthixol prevents the acquisition of CPP induced by RMTg Tropicamide infusions. Under conditions of systemic dopamine receptor blockade, RMTg Tropicamide infusions significantly increase locomotor activation. These data provide further support for an important role of endogenous cholinergic input to the RMTg in reward function and suggest that the contributions of RMTg cholinergic input to rewarding and locomotor-activating effects involve differential contributions of RMTg M4 and M3 muscarinic receptors, respectively.

Topics: Animals; Behavior, Animal; Conditioning, Psychological; Dopamine Antagonists; Flupenthixol; Locomotion; Mice; Mice, Inbred C57BL; Muscarinic Antagonists; Piperidines; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Reward; Tropicamide; Ventral Tegmental Area

Pilocarpine is an M3 muscarinic agonist that is widely used for the treatment of xerostomia caused by various diseases and medical conditions. Pilocarpine induced the secretion of salivary fluid in perfused submandibular glands of rats. The secretion of salivary fluid observed after removal of pilocarpine was referred to as residual fluid secretion. The volume of fluid and time of the residual secretion depended on the dose of pilocarpine. Such a residual effect of pilocarpine was observed on fluid secretion via the paracellular pathway and oxygen consumption. When a muscarinic antagonist was added to the perfusate immediately after cessation of pilocarpine, residual secretion of salivary fluid did not occur. These observations indicate that the residual secretion of salivary fluid is a characteristics of the interaction of pilocarpine with muscarinic receptors.

Topics: Animals; Male; Muscarinic Antagonists; Oxygen Consumption; Perfusion; Pilocarpine; Piperidines; Rats, Wistar; Saliva; Submandibular Gland; Tetrodotoxin

Neuromodulation ensures that neural circuits produce output that is flexible whilst remaining within an optimal operational range. The neuromodulator acetylcholine is released during locomotion to regulate spinal motor circuits. However, the range of receptors and downstream mechanisms by which acetylcholine acts have yet to be fully elucidated. We therefore investigated metabotropic acetylcholine receptor-mediated modulation by using isolated spinal cord preparations from neonatal mice in which locomotor-related output can be induced pharmacologically. We report that M2 receptor blockade decreases the frequency and amplitude of locomotor-related activity, whilst reducing its variability. In contrast, M3 receptor blockade destabilizes locomotor-related bursting. Motoneuron recordings from spinal cord slices revealed that activation of M2 receptors induces an outward current, decreases rheobase, reduces the medium afterhyperpolarization, shortens spike duration and decreases synaptic inputs. In contrast, M3 receptor activation elicits an inward current, increases rheobase, extends action potential duration and increases synaptic inputs. Analysis of miniature postsynaptic currents support that M2 and M3 receptors modulate synaptic transmission via different mechanisms. In summary, we demonstrate that M2 and M3 receptors have opposing modulatory actions on locomotor circuit output, likely reflecting contrasting cellular mechanisms of action. Thus, intraspinal cholinergic systems mediate balanced, multimodal control of spinal motor output.

Topics: Acetylcholine; Action Potentials; Animals; Animals, Newborn; Diamines; In Vitro Techniques; Locomotion; Mice; Mice, Inbred C57BL; Motor Neurons; Muscarine; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Spinal Cord

Arecoline is the primary alkaloid in betel nuts, which are known as a risk factor for oral submucosal fibrosis and oral cancer. Lung cancer is a severe type of carcinoma with high cell motility that is difficult to treat. However, the detailed mechanisms of the correlation between Arecoline and lung cancer are not fully understood. Here, we investigated the effect of Arecoline on migration in lung cancer cell lines and its potential mechanism through the muscarinic acetylcholine receptor 3 (mAChR3)-triggered EGFR/Src/FAK pathway. Our results indicate that different concentrations of Arecoline treatment (10 µM, 20 µM, and 40 µM) significantly increased the cell migration ability in A549 and CL1-0 cells and promoted the formation of the filamentous actin (F-actin) cytoskeleton, which is a crucial element for cell migration. However, migration of H460, CL1-5, and H520 cell lines, which have a higher migration ability, was not affected by Arecoline treatment. The EGFR/c-Src/Fak pathway, which is responsible for cell migration, was activated by Arecoline treatment, and a decreased expression level of E-cadherin, which is an epithelial marker, was observed in Arecoline-treated cell lines. Blockade of the EGFR/c-Src/Fak pathway with the inhibitors of EGFR (Gefitinib) or c-Src (Dasatinib) significantly prevented Arecoline-promoted migration in A549 cells. Gefitinib or Dasatinib treatment significantly disrupted the Arecoline-induced localization of phospho-Y576-Fak during focal adhesion in A549 cells. Interestingly, Arecoline-promoted migration in A549 cells was blocked by a specific mAChR3 inhibitor (4-DAMP) or a neutralizing antibody of matrix metalloproteinase (MMP7 or Matrilysin). Taken together, our findings suggest that mAChR3 might play an essential role in Arecoline-promoted EGFR/c-Src/Fak activation and migration in an A549 lung cancer cell line.

Topics: Arecoline; Cell Line, Tumor; Cell Movement; ErbB Receptors; Focal Adhesion Kinase 1; Humans; Lung Neoplasms; Muscarinic Antagonists; Piperidines; Receptor, Muscarinic M3; Signal Transduction; src-Family Kinases

The search for new drugs remains an important focus for the safe and effective treatment of cardiovascular diseases. Previous evidence has shown that choline analogs can offer therapeutic benefit for cardiovascular complications. The current study investigates the effects of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) on cardiovascular function and cholinergic-nitric oxide signaling. Synthesized LQFM032 (0.3, 0.6, or 1.2 mg/kg) was administered by intravenous and intracerebroventricular routes to evaluate the potential alteration of mean arterial pressure, heart rate, and renal sympathetic nerve activity of normotensive and hypertensive rats. Vascular function was further evaluated in isolated vessels, while pharmacological antagonists and computational studies of nitric oxide synthase and muscarinic receptors were performed to assess possible mechanisms of LQFM032 activity. The intravenous and intracerebroventricular administration of LQFM032 elicited a temporal reduction in mean arterial pressure, heart rate, and renal sympathetic nerve activity of rats. The cumulative addition of LQFM032 to isolated endothelium-intact aortic rings reduced vascular tension and elicited a concentration-dependent relaxation. Intravenous pretreatment with L-NAME (nitric oxide synthase inhibitor), atropine (nonselective muscarinic receptor antagonist), pirenzepine, and 4-DAMP (muscarinic M1 and M3 subtype receptor antagonist, respectively) attenuated the cardiovascular effects of LQFM032. These changes may be due to a direct regulation of muscarinic signaling as docking data shows an interaction of choline analog with M1 and M3 but not nitric oxide synthase. Together, these findings demonstrate sympathoinhibitory, hypotensive, and antihypertensive effects of LQFM032 and suggest the involvement of muscarinic receptors.

Topics: Animals; Antihypertensive Agents; Aorta, Thoracic; Atropine; Blood Pressure; Heart Rate; Hypertension; Hypotension; Male; Muscarinic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Piperazines; Piperidines; Pirenzepine; Pyrazoles; Rats, Inbred SHR; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3

The ventral tegmental area (VTA) and the rostromedial tegmental nucleus (RMTg) each contribute to opiate reward and each receive inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic cell groups. We compared the contributions of VTA or RMTg muscarinic cholinergic receptors to locomotion induced by morphine infusions into the same sites. VTA co-infusion of atropine completely blocked VTA morphine-induced locomotion providing additional support for the important role of VTA muscarinic cholinergic receptors in the stimulant effects of opiates. By contrast, RMTg co-infusion of atropine increased RMTg morphine-induced locomotion. Furthermore, RMTg co-infusion of the M3-selective antagonist 4-DAMP, but not the M4-selective antagonist Tropicamide, strongly increased RMTg morphine-induced locomotion. RMTg infusions of 4-DAMP, but not of Tropicamide, by themselves strongly increased drug-free locomotion. Muscarinic cholinergic receptors in the RMTg thus also contribute to the stimulant effects of morphine, but in a way opposite to those in VTA. We suggest that the net effect of endogenous cholinergic input to the RMTg on drug-free and on RMTg morphine-induced locomotion is inhibitory.

Topics: Acetylcholine; Animals; Atropine; Locomotion; Mice; Mice, Inbred C57BL; Morphine; Muscarinic Antagonists; Piperidines; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Tropicamide; Ventral Tegmental Area

The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation.

Topics: Acetylcholine; Animals; Calcitonin Gene-Related Peptide; Dose-Response Relationship, Drug; Endothelium, Vascular; Hexamethonium; In Vitro Techniques; Male; Mesenteric Arteries; Piperidines; Pirenzepine; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Vasodilation

Studies have shown that muscarinic receptors 3 (M3R) plays key roles in regulating tumorigenesis and tumor growth. The aim of this study is to investigate the expression of M3R in human small cell lung cancer (SCLC) cell line SBC3 and to explore the effect of M3R antagonist on cell proliferation, apoptosis and adhesion.. SBC3 cells were cultured in vitro. RT-PCR and Western blot were used to detect the expression of M3R in SBC-3. MTT assay and flow cytometry were used to determine the effects of M3R antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) on the proliferation and apoptosis of SBC-3 cells. Flow cytometry was used to detect the integrin expression in SBC3 and alteration of integrin expression after treating the cells with cholinergic receptor agonist acetylcholine iodide (acetylcholine iodide, Ach) and 4-DAMP. Fibronectin (Fn) coated 96-well plates were used to detect the effect of Ach, 4-DAMP and anti-integrin antibody on cell adhesion.. M3R was expressed in SBC3 cells. 4-DAMP significantly inhibited SBC3 cells proliferation in a dose-dependent manner. 4-DAMP promoted cell apoptosis at a concentration of 10-4 M of 4-DAMP compared with control. αvβ1 and α5β1 integrin were expressed in SBC3 cells. 10-4 M Ach stimulated cell adhesion toward Fn significantly (P<0.01). This effect was completely abrogated by 10-5 M 4-DAMP, 5 μg/mL anti-β1 integrin antibody or anti-αv and anti-α5 integrin antibodies (P<0.01) and partially abrogated by 5 μg/mL anti-αv or anti-α5 integrin antibody (P<0.05). But Ach and 4-DAMP did not alter Fn receptor (αvβ1 or α5β1 integrin) expression.. M3R is expressed in SBC3 cell. The M3R antagonist inhibits SBC3 cell proliferation, adhesion and enhances cell apoptosis. M3R antagonist inhibiting SBC3 cell adhesion is presumably modulated by functional alteration of β1 containing integrins (αvβ1 and α5β1), but not by any variation in their expression.
.. 背景与目的 研究表明毒蕈碱胆碱受体3（muscarinic receptor 3, M3R）在多种肿瘤的发生、发展中发挥重要的作用。本研究旨在探讨M3R在人小细胞肺癌（small cell lung cancer, SCLC）细胞株SBC3的表达，M3R拮抗剂对细胞增殖、凋亡及粘附的影响。方法 体外培养SBC3细胞，RT-PCR和Western blot检测M3R的表达。MTT法及流式细胞法检测M3R拮抗剂（4-diphenylacetoxy-N-methylpiperidine methiodide, 4-DAMP）对细胞增殖和凋亡的影响。流式细胞法检测细胞整合素的表达及碘化乙酰胆碱（acetylcholine iodide, Ach）和4-DAMP对整合素表达的影响。纤维结合蛋白（Fn）包被的96孔板用以研究Ach、4-DAMP及整合素抗体对细胞粘附的作用。结果 SBC3细胞表达M3R，4-DAMP浓度依懒性抑制细胞增殖。与对照组比较，10-4 M 4-DAMP能够明显地增加SBC3细胞凋亡。SBC3细胞表达αvβ1和α5β1整合素，10-4 M Ach刺激细胞粘附（P<0.01）的作用几乎被10-5 M 4-DAMP、5 μg/mL抗-β1抗体或抗-αv和α5抗体完全阻断（P<0.01），但Ach及4-DAMP不影响αv、α5和β1的表达水平。结论 SBC3细胞表达M3R，M3R拮抗剂能抑制细胞的增殖并促进凋亡。其抑制粘附的作用是通过抑制细胞含β1的整合素（αvβ1 和 α5β1）的功能实现的。.

Topics: Apoptosis; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Piperidines; Receptor, Muscarinic M3; Small Cell Lung Carcinoma

Muscarinic and purinergic (P2X) receptors play critical roles in bladder urothelium under physiological and pathological conditions. Aim of present study was to characterize these receptors in rat bladder urothelium and detrusor muscle using selective radioligands of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) and αβ-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]αβ-MeATP). Similar binding parameters for each radioligand were observed in urothelium and detrusor muscle. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) mustard revealed co-existence of M2 and M3 receptors, with the number of M2 receptors being larger in the urothelium and detrusor muscle. Intravesical administration of imidafenacin and Dpr-P-4 (N → O) (active metabolite of propiverine) displayed significant binding of muscarinic receptors in the urothelium and detrusor muscle. The treatment with cyclophosphamide (CYP) or resiniferatoxin (RTX) resulted in a significant decrease in maximal number of binding sites (Bmax) for [(3)H]NMS and/or [(3)H]αβ-MeATP in the urothelium and detrusor muscle. These results demonstrated that 1) pharmacological characteristics of muscarinic and P2X receptors in rat bladder urothelium were similar to those in the detrusor muscle, 2) that densities of these receptors were significantly altered by pretreatments with CYP and RTX, and 3) that these receptors may be pharmacologically affected by imidafenacin and Dpr-P-4 (N → O) which are excreted in the urine.

Topics: Animals; Cyclophosphamide; Diterpenes; Imidazoles; Male; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Purinergic P2X Receptor Antagonists; Rats, Sprague-Dawley; Receptors, Muscarinic; Receptors, Purinergic P2X; Urinary Bladder; Urothelium

The activation of M3 cholinoceptors (M3 receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)-induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulation.. Rats were given choline (10 mg · kg(-1), i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), was injected (0.12 μg · kg(-1), i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca(2+)-cycling proteins were also assessed.. Choline treatment attenuated I/R-induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up-regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca(2+)-cycling proteins, including Na(+)Ca(2+) exchanger, inositol 1,4,5-trisphosphate receptor, sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4-DAMP.. Our data suggest that inhibition of the ROS-mediated CaMKII pathway and modulation of Ca(2+)-cycling proteins may be novel mechanisms underlying choline-induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M3 receptors in the vasculature, providing a new therapeutic strategy for I/R-induced vascular injury.

Topics: Administration, Intravenous; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Choline; Cholinergic Agonists; Dose-Response Relationship, Drug; Ischemia; Male; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Reperfusion Injury; Signal Transduction; Structure-Activity Relationship; Vascular System Injuries

The effects of selective activation of subtype 3 muscarinic (M3) receptors on electrical activity of isolated preparations of the atrial and ventricular myocardium of the newborn and 4-month-old rats were examined. Application of muscarinic receptor agonist pilocarpine (10(-5) M) in preparations with M2 cholinoreceptors blocked by selective antagonist methoctramine (10(-7) M) decreased the duration of action potentials in the atrial and ventricular myocardium. Selective blocker of M3 cholinoreceptors 4-DAMP (10(-8) M) prevented this effect. While stimulation of ventricular M3 cholinoreceptors with pilocarpine was significantly stronger in newborn pups than in mature rats, similar stimulation of atrial receptors revealed no significant difference in both groups.

Topics: Action Potentials; Age Factors; Animals; Animals, Newborn; Diamines; Heart Atria; Heart Ventricles; Male; Muscarinic Agonists; Muscarinic Antagonists; Pilocarpine; Piperidines; Rats; Receptor, Muscarinic M2; Receptor, Muscarinic M3

Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury.

Topics: Acetylcholine; AMP-Activated Protein Kinases; Apoptosis; Caspase 12; Cell Hypoxia; Cell Line; DNA Nucleotidylexotransferase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Heat-Shock Proteins; Human Umbilical Vein Endothelial Cells; Humans; Piperidines; Protein Serine-Threonine Kinases; Receptor, Muscarinic M3; Reperfusion Injury; RNA Interference; RNA, Small Interfering; Signal Transduction; Transcription Factor CHOP

Coupling reactions to make esters and amides are among the most widely used organic transformations. We report efficient procedures for amide bond formation and for the monoesterification of symmetrical diols in excellent yields without any requirement for high dilution or slow addition using resin-bound triarylphosphonium iodide. Easy purification, low moisture sensitivity, and good to excellent yields of the products are the major advantages of this protocol.

Topics: Amides; Esters; Indicators and Reagents; Organophosphorus Compounds; Piperidines

The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoo‑san (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The whole‑cell patch‑clamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+i) was analyzed in cultured ICCs using fura‑2‑acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4‑diphenylacetoxy‑N‑methyl‑piperidine methiodide muscarinic M3 receptor antagonist inhibited DS‑induced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'‑[β‑thio] diphosphate (GDP‑β‑S; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DS‑induced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DS‑induced pacemaker depolarizations. Pretreatment with Ca2+‑free solution and thapsigargin, a Ca2+‑ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DS‑induced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G protein‑dependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs.

Topics: Animals; Calcium-Transporting ATPases; Cells, Cultured; Diamines; Female; Gastrointestinal Motility; Guanosine Diphosphate; Interstitial Cells of Cajal; Intestine, Small; Male; Mice; Mice, Inbred BALB C; Pain; Phytotherapy; Piperidines; Plants, Medicinal; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Thapsigargin; Thionucleotides

Endocannabinoids (eCBs) are cannabis-like substances produced in the brain where their primary function is to regulate synaptic transmission by inhibiting neurotransmitter release in a retrograde fashion. We have recently demonstrated a novel mechanism regulating GABAergic transmission from neurons in the Substantia Nigra pars reticulata (SNr) to dopaminergic neurons in the Substantia Nigra pars compacta (SNc) mediated by eCBs. Production of eCBs was initiated by spillover of glutamate, yet the source of the glutamate was not determined (Freestone et al., 2014; Neuropharmacology 79 p467). The present study aimed at elucidating the potential role of glutamatergic terminals arising from neurons in the Subthalamic nucleus (STN) in driving the eCB-mediated modulation of this inhibitory transmission. GABAergic IPSCs or IPSPs evoked in SNc neurons by electrical stimuli delivered to the SNr region were transiently inhibited by electrical or pharmacological (U-tube application of muscarinic agonist carbachol [100 µM]) stimulation of the STN (to 74±5% and 69±4% respectively). In both stimulation protocols, the attenuation of GABAergic transmission was abolished by cannabinoid receptor 1 antagonist rimonabant (3 µM), and reduced by group 1 metabotropic glutamate receptor antagonist CPCCOEt (100 µM), consistent with a glutamate-initiated and eCB-mediated mechanism. The carbachol-induced attenuation of GABAergic transmission was abolished by M3 muscarinic receptor antagonist 4-DAMP (10 µM), confirming a specific activation of STN neurons. These results demonstrate that glutamatergic projection from the STN to dopaminergic SNc neurons underlies an eCB-mediated inhibition of GABAergic input to these neurons.

Topics: Animals; Carbachol; Chromones; Dopaminergic Neurons; Electric Stimulation; Endocannabinoids; Excitatory Postsynaptic Potentials; GABAergic Neurons; Inhibitory Postsynaptic Potentials; Pars Compacta; Pars Reticulata; Piperidines; Pyrazoles; Rats; Rimonabant; Subthalamic Nucleus; Synaptic Transmission

Having separated a sub-group of people with schizophrenia based on a marked loss of cortical [(3)H]pirenzepine binding (MRDS); we wished to determine if MRDS had lower levels of [(3)H]pirenzepine and other muscarinic receptor antagonist binding to the striatum and if this was due to loss of pre- or post-synaptic neurons or glia measured using surrogate markers (25 kilodalton synaptosomal-associated protein (SNAP 25), postsynaptic density protein 95 (PSD 95), glial fibrillary acidic protein (GFAP) 41/43) of cell number.. [(3)H]pirenzepine, [(3)H]AF-DX 384 and [(3)H]4-DAMP binding to the striatum from 37 subjects with schizophrenia (19 MRDS) and 20 controls as well as SNAP 25, PSD 95 and GFAP 41/43 in crude particulate membrane were measured.. [(3)H]pirenzepine and [(3)H]AF-DX 384 binding to the striatum were significantly lower in schizophrenia due to lower binding of both radioligands in the striatum from MRDS. Levels of PSD 95 were higher in schizophrenia, predominantly due to higher levels in MRDS.. Our data suggest muscarinic M1 ([(3)H]pirenzepine) and M2 and/or M4 receptors ([(3)H]AF-DX 384) are lower in the striatum from MRDS which could mediate inappropriate adaption to internal and external cues which, in turn, would affect motivation, cognition and motor control. Increased levels of PSD 95 could indicate increased post-synaptic boutons or changes in NMDA receptor-mediated signalling in MRDS.

Topics: Autoradiography; Blotting, Western; Corpus Striatum; Disks Large Homolog 4 Protein; Female; Glial Fibrillary Acidic Protein; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; Muscarinic Antagonists; Neuroglia; Neurons; Piperidines; Pirenzepine; Radioligand Assay; Radiopharmaceuticals; Receptors, Muscarinic; Schizophrenia; Synaptosomal-Associated Protein 25; Tritium

Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist.. Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses.. Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP.. Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs.

Topics: Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung; Piperidines; Receptors, Muscarinic; Respiratory Mucosa; Transforming Growth Factor beta1

To locate the muscarinic (M) M2 and M3 receptors in bladder interstitial cells of Cajal (ICCs) and to determine the effects of M2 and M3 agonists on bladder ICCs.. A total of 30 adult male Sprague-Dawley rats weighing 225-250 g were used in this study. Double-labeled fluorescence of muscarinic receptors and c-kit was performed for co-localization. To evaluate the effect of muscarinic agents on the excitation of bladder ICCs, we analyzed the inward current of bladder ICCs using the whole-cell patch clamp. The effect of muscarinic agents on the carbachol-induced inward currents was evaluated with the whole-cell patch clamp.. M2 and M3 receptors were confirmed in the stroma ICCs in rats' bladders with double-labeled immunofluorescence. Spontaneous action potential was observed in freshly isolated bladder ICCs. The carbachol-induced inward Ca2+ current in ICCs can be blocked by atropine. The M2 receptor antagonist methoctramine (1 μM) showed a weak inhibitory capability on the inward Ca2+ current [from 74.8 ± 9.6 to 63.3 ± 13.8 Pascal (pA), n = 12, P = .03]. While the M3 receptor antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) (1 μM) significantly inhibited the inward Ca2+ current (from 78.4 ± 11.2 to 17.3 ± 7.9 pA, n = 12, P < .001).. Bladder ICCs express M2 and M3 cholinergic receptors. Most muscarinic cholinergic receptor antagonists, especially the M3 antagonists, can effectively inhibit the carbamylcholine- induced inward current of bladder ICCs.

Topics: Action Potentials; Animals; Atropine; Calcium Channels; Carbachol; Cholinergic Agonists; Diamines; Interstitial Cells of Cajal; Male; Muscarinic Antagonists; Patch-Clamp Techniques; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Urinary Bladder

We previously identified a group of subjects with schizophrenia who, on average, have a 75% decrease in cholinergic receptor, muscarinic 1 (CHRM1) in Brodmann's area (BA) 9. To extend this finding, we determined i) if the decrease in CHRM1 was present in another functionally related CNS region (BA6), ii) whether the marked decrease in CHRM1 was accompanied by changes in levels of other CHRMs and iii) potential factors responsible for the decreased CHRM1 expression. We measured CHRM1 and CHRM3 using in situ radioligand binding with [(3)H]pirenzepine and [(3)H]4-DAMP respectively in BA6 from 20 subjects with schizophrenia who had low levels of CHRM1 in BA9 (SzLow[(3)H]PZP), 18 subjects with schizophrenia whose levels of CHRM1 were similar to controls (SzNormal[(3)H]PZP) and 20 control subjects. Levels of CHRM1, 3 and 4 mRNA were measured using qPCR and levels of the transcription factors, SP1 and SP3, were determined using Western blots. In BA6, the density of [(3)H]pirenzepine binding was decreased in subjects with SzLow[(3)H]PZP (p<0.001) compared to controls. The density of [(3)H]4-DAMP binding, levels of CHRM1, 3 and 4 mRNA and levels of SP1 and SP3 was not significantly different between the three groups. This study shows that the previously identified decrease in CHRM1 expression is not confined to the dorsolateral prefrontal cortex but is present in other cortical areas. The effect shows some specificity to CHRM1, with no change in levels of binding to CHRM3. Furthermore, this decrease in CHRM1 does not appear to be associated with low levels of CHRM1 mRNA or to simply be regulated by the transcription factors, SP1 and SP3, suggesting that other mechanisms are responsible for the decreased CHRM1 in these subjects.

Topics: Adult; Blotting, Western; Brain; Cohort Studies; Humans; Middle Aged; Piperidines; Pirenzepine; Polymerase Chain Reaction; Radioligand Assay; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Receptors, Muscarinic; RNA, Messenger; Schizophrenia; Sp1 Transcription Factor; Sp3 Transcription Factor; Tritium

Studies in humans and rodents support a role for muscarinic ACh receptor (mAChR) and nicotinic AChR in learning and memory, and both regulate hippocampal synaptic plasticity using complex and often times opposing mechanisms. Acetylcholinesterase (AChE) inhibitors are commonly prescribed to enhance cholinergic signaling in Alzheimer's disease in hopes of rescuing cognitive function, caused, in part, by degeneration of cholinergic innervation to the hippocampus and cortex. Unfortunately, therapeutic efficacy is moderate and inconsistent, perhaps due to unanticipated mechanisms. M1 mAChRs bidirectionally control synaptic strength at CA3-CA1 synapses; weak pharmacological activation using carbachol (CCh) facilitates potentiation, whereas strong agonism induces muscarinic long-term depression (mLTD) via an ERK-dependent mechanism. Here, we tested the prediction that accumulation of extracellular ACh via inhibition of AChE is sufficient to induce LTD at CA3-CA1 synapses in hippocampal slices from adult rats. Although AChE inhibition with eserine induces LTD, it unexpectedly does not share properties with mLTD induced by CCh, as reported previously. Eserine-LTD was prevented by the M3 mAChR-preferring antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), and pharmacological inhibition of MEK was completely ineffective. Additionally, pharmacological inhibition of p38 MAPK prevents mLTD but has no effect on eserine-LTD. Finally, long-term expression of eserine-LTD is partially dependent on a decrease in presynaptic release probability, likely caused by tonic activation of mAChRs by the sustained increase in extracellular ACh. Thus these findings extend current literature by showing that pharmacological AChE inhibition causes a prolonged decrease in presynaptic glutamate release at CA3-CA1 synapses, in addition to inducing a likely postsynaptic form of LTD.

Topics: Acetylcholine; Animals; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Cholinesterase Inhibitors; Enzyme Inhibitors; Extracellular Space; Long-Term Synaptic Depression; Male; MAP Kinase Kinase Kinases; Muscarinic Antagonists; p38 Mitogen-Activated Protein Kinases; Physostigmine; Piperidines; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Synapses; Tissue Culture Techniques

There is a remarkable paucity of studies analyzing the role of the endothelium-derived relaxing factors on the vascular effects of organophosphates. This study was carried out to evaluate the vascular effects of malathion and the role of nitric oxide (NO) and prostacyclin (PGI2).. Vascular reactivity measuring isometric forces in vitro ('organ chambers') and flow cytometry (cells loaded with DAF-FM DA) were used.. In rat thoracic aorta segments contracted with phenylephrine (Phe) (10(-7) mol/l), malathion (10(-10) to 10(-5) mol/l) induced concentration-dependent relaxation in arteries with intact endothelium (n = 7; p < 0.05). Malathion-mediated relaxation was blocked by N-nitro-L-arginine methyl ester (L-NAME; 10(-4) mol/l), a nonspecific NO synthase inhibitor, and/or indomethacin (10(-5) mol/l), a nonspecific cyclooxygenase inhibitor (n = 10, p < 0.05). In thoracic aorta rings, with and without endothelium, Phe (10(-10) to 10(-5) mol/l) evoked concentration-dependent contraction, which was reduced in the presence of malathion. In rings with or without endothelium, incubated with malathion, L-NAME and indomethacin, the Phe-induced contraction was restored. The role of NO was confirmed using flow cytometry. Malathion evokes endothelium-dependent relaxation through the M1 muscarinic receptor, since this relaxation was clearly blocked by atropine (M1 and M2 blocker) and pirenzepine (M1 blocker), but was less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker).. These findings suggest that the organophosphate compound effects on vascular reactivity depend of NO and PGI2.

Topics: Animals; Aorta, Thoracic; Atropine; Endothelium, Vascular; Epoprostenol; Gallamine Triethiodide; In Vitro Techniques; Indomethacin; Malathion; Male; Muscarinic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Pesticides; Phenylephrine; Piperidines; Pirenzepine; Rats, Wistar; Vasodilator Agents

We studied the effect of selective activation of muscarinic M3receptors on electrical activity in the isolated preparation of rat ventricular myocardium as well as contractile activity of the left ventricle of Langendorff-perfused isolated heart. Application of muscarinic agonist pilocarpine (10(-5)M) against the background of selective blockade of subtype 2 muscarinic receptors with methoctramine (10(-7)M) markedly shortened the duration of action potentials in the isolated ventricular myocardium and reduced the amplitude and maximum rates of left-ventricular pressure rise and decay in the isolated heart paced at a fixed rate. All these effects were significantly suppressed by selective M3receptor blocker 4-DAMP (10(-8)M), which attested to the involvement of M3muscarinic receptors.

Topics: Action Potentials; Animals; Diamines; Heart; Muscarinic Agonists; Muscarinic Antagonists; Myocardial Contraction; Myocardium; Parasympatholytics; Pilocarpine; Piperidines; Rats; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Ventricular Function

While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100μM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.

Topics: Aged; Atropine; Benzofurans; Carbachol; Cell Proliferation; Cells, Cultured; Cholinergic Agonists; Gene Expression; Humans; Male; Mitogen-Activated Protein Kinases; Muscarinic Antagonists; Myocytes, Smooth Muscle; Phosphatidylinositol 3-Kinases; Piperidines; Pirenzepine; Proto-Oncogene Proteins c-akt; Pyrrolidines; Receptors, Muscarinic; RNA, Messenger; Urinary Bladder

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca(2+) for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca(2+). In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM.

Topics: Amphotericin B; Animals; Carbachol; Cholinergic Agonists; Diamines; Evoked Potentials; Gene Expression Regulation; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Muscarinic Antagonists; Myocytes, Smooth Muscle; Patch-Clamp Techniques; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Urinary Bladder

Impairment of gastrointestinal tract (GI) function, including delayed gastric emptying and colonic dysmotility, are common features of primary Sjögren's syndrome (SS). However, the pathogenesis remains largely unknown. The aim of the current study was to investigate the role of functional autoantibodies to the muscarinic receptor in mediating GI dysfunction associated with primary SS. The effect of SS or normal immunoglobulin G (IgG) on smooth muscle (SM) motility was assessed by comparing the amplitude of carbachol (CCh) or electrical field stimulation (EFS) - induced muscle contraction before and after IgG application. Muscarinic receptor type 3 (M3R) played a dominant role in both colon and gastric SM contraction, while M2R was partly involved in gastric smooth muscle contraction. Preincubation for 1h of the colon and gastric SM strips with 1mg/ml purified IgG from the sera of four primary SS patients (SS IgG) significantly inhibited carbachol-induced smooth muscle contraction (CISC) over a range of CCh concentrations, whereas IgG from healthy controls had little effect. Incubation of the colon SM strips with SS IgG also inhibited EFS-induced colon muscle contraction, which was mimicked by the M3R-selective blocker, 4-DAMP. SR1403330, an NK1 antagonist, had little effect on EFS-mediated colonic SM contraction. The results suggest that autoantibodies isolated from primary SS patients' sera inhibit muscarinic receptor-mediated cholinergic neurotransmission in mouse colon and stomach, which may provide clues for explaining the GI dysfunction seen in patients with primary SS.

Topics: Animals; Autoantibodies; Carbachol; Cholinergic Agonists; Colon; Diamines; Dose-Response Relationship, Drug; Gastrointestinal Motility; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Sjogren's Syndrome; Stomach

[(3)H]4-DAMP is a radioligand that has been used to quantify levels of the muscarinic receptor CHRM3 protein in situ. However, in addition to high affinity binding to CHRM3, [(3)H]4-DAMP binds with low affinity to CHRM1 confounding the potential to discriminate between changes in these two muscarinic receptors. We have developed a [(3)H]4-DAMP binding assay, optimised for measuring CHRM3 protein levels in the cortex, with minimal selectivity towards CHRM1. The selectivity of our assay towards CHRM3 was confirmed using recombinant receptor-expressing, cell lysate preparations. [(3)H]4-DAMP binding levels were similar between wildtype and CHRM1 knockout mice, confirming that the amount of [(3)H]4-DAMP binding to CHRM1 was negligible. We used this assay to measure CHRM3 protein levels in the frontal pole, obtained post-mortem from subjects with bipolar disorder (n = 15), major depressive disorder (n = 15) and matched controls (n = 20) and showed that [(3)H]4-DAMP binding was not altered in either bipolar disorder or major depressive disorder. Western blotting confirmed that CHRM3 protein levels were unchanged in these subjects.

Topics: Adult; Aged; Animals; Cerebral Cortex; Dose-Response Relationship, Drug; Humans; Mice; Mice, Knockout; Middle Aged; Mood Disorders; Muscarinic Antagonists; Piperidines; Pirenzepine; Protein Binding; Radioligand Assay; Radionuclide Imaging; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptors, Muscarinic; Tritium

Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [(3)H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [(3)H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M3 selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M2-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP effect indicating that the M3AChR is the main receptor target of cGMP. Interestingly, these cGMP effects were potentiated by an activator (Sp-8-pCPT-cGMPS), and diminished by an inhibitor (Rp-8-pCPT-CGMPS), of cGMP-dependent protein kinase (PKG-II), which was detected by Western blotting using specific PKG II antibodies. Finally, plasma membrane M3AChRs were phosphorylated in a cGMP-dependent manner and this novel post-translational reversible modification at M3AChRs may act as a feedback mechanism to terminate the cGMP dependent muscarinic signal transduction cascades at the sarcolema of BTSM.

Topics: Animals; Cattle; Cell Membrane; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Feedback, Physiological; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Protein Processing, Post-Translational; Quinuclidinyl Benzilate; Receptors, Muscarinic; Signal Transduction; Trachea

The aim of the present study was to evaluate the role of a possible interaction between cannabinoid and muscarinic systems, both widely expressed in the ocular structure and involved in the control of bovine ciliary muscle contractility and intraocular pressure modulation. The ciliary muscle strips isolated by bovine eyes were exposed cumulatively to anandamide in the presence and in the absence of carbachol (5 nM), in a miograph system for isometric recording. The experiments were also conducted in the presence of AM251 (100 nM), 4-DAMP (100 nM), Pertussis toxin (500 ng/ml), U73122 (0.1 and 1 μM), chelerythrine (1 and 10 μM) and Y27632 (1 and 10 μM). Contractile responses were expressed as the percentage of 10 μM carbachol-induced contraction. The anandamide-induced contraction on bovine ciliary muscle strips was enhanced by the previous stimulation of Gq-protein-coupled muscarinic M3 receptors with carbachol. The contractile response to anandamide plus carbachol was affected by different inhibitors such as Pertussis toxin, phospholipase C, protein kinase C and Rho-kinase. The key results of the present study show that sequential activation of muscarinic M3 receptors and cannabinoid CB1 receptors produce synergistic contractile effects of the bovine ciliary muscle by involving the activation of Rho-kinase and protein kinase C.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Carbachol; Cattle; Cholinergic Agonists; Ciliary Body; Endocannabinoids; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Polyunsaturated Alkamides; Protein Kinase C; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Muscarinic M3; rho-Associated Kinases

Large airway bronchoconstriction acts mainly through cholinergic pathways via muscarinic M3 receptors with some contribution from M2 receptors. By contrast, the mechanisms of small airway contraction are largely unknown. This study used precision cut lung slices to examine the role of muscarinic M2 and M3 receptors in the contractile response of rat and human small airways. In rat small airways, the M3 preferential antagonist, 4-DAMP, inhibited carbachol-mediated contraction (1×10(-6) M) more than that of the M2 selective antagonist, AF-DX116 (pIC50 values: 8.85±0.18 and 6.31±0.19, n=6-8 respectively). Tiotropium, inhibited the contractile response to carbachol with (pIC50: 9.86±0.07, n=6), but could not distinguish between M2 and M3 mediated effects. Similar experiments using human small airways with tiotropium and AF-DX116, gave a pIC50 of 10.35±0.05 and a pKB of 6.37±0.13, n=5 respectively. Therefore, M3 receptors play a key role in muscarinic contraction of small airways in both rats and humans but the effect of M2 receptors cannot be excluded. To investigate the role of M2 receptors, carbachol-induced contraction of small airways was performed in the presence and absence of a β2-agonist in order to elevate intracellular cAMP levels prior to contraction. Isoproterenol-induced relaxation was significantly increased by AF-DX116 (P<0.001) in rat small airways and by AF-DX116 (P<0.01), gallamine (P<0.05) and pertussis toxin (P<0.05) in human small airways. Taken together, these data suggest that cholinergic antagonism of muscarinic receptors in human and rat small airways inhibits airway contraction via direct inhibition of contraction through M3 receptors, and by M2 receptor mediated inhibition of relaxation.

Topics: Animals; Carbachol; Cholinergic Agonists; Humans; In Vitro Techniques; Lung; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Scopolamine Derivatives; Tiotropium Bromide

Macrophages increase in number and are highly activated in chronic obstructive pulmonary disease (COPD). Muscarinic receptor antagonists inhibit acetylcholine-stimulated release of neutrophilic chemoattractants, suggesting that acetylcholine may regulate macrophage responses. Therefore, expression and function of components of the non-neuronal cholinergic system in monocyte-macrophage cells was investigated. RNA was isolated from monocytes, monocyte-derived macrophages (MDMs), lung and alveolar macrophages from nonsmokers, smokers and COPD patients, and expression of the high-affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter and muscarinic receptors (M(1)-M(5)) ascertained using real-time PCR. M(2) and M(3) receptor expression was confirmed using immunocytochemistry. Release of interleukin (IL)-8, IL-6 and leukotriene (LT)B(4) were measured by ELISA or EIA. All monocyte-macrophage cells expressed mRNA for components of the non-neuronal cholinergic system. Lung macrophages expressed significantly more M(1) mRNA compared with monocytes, and both lung macrophages and alveolar macrophages expressed the highest levels of M(3) mRNA. Expression of M(2) and M(3) protein was confirmed in MDMs and lung macrophages. Carbachol stimulated release of LTB(4) from lung macrophages (buffer 222.3 ± 75.1 versus carbachol 1,118 ± 622.4 pg · mL(-1); n = 15, p<0.05) but not IL-6 or IL-8. LTB(4) release was attenuated by the M(3) antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; half maximal effective concentration 5.2 ± 2.2 nM; n = 9). Stimulation of macrophage M(3) receptors promotes release of LTB(4), suggesting that anti-muscarinic agents may be anti-inflammatory.

Topics: Carbachol; Cells, Cultured; Choline O-Acetyltransferase; Humans; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Leukotriene B4; Macrophages; Membrane Transport Proteins; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pulmonary Disease, Chronic Obstructive; Receptors, Muscarinic; RNA; Smoking; Vesicular Acetylcholine Transport Proteins

When neostigmine is used to reverse muscle relaxants in patients with asthma without signs of airway inflammation, asthma attack is occasionally encountered. It is likely that abnormally increased electrical impulses traveling from the brain through cholinergic nerves to airway smooth muscles may be one of the pathogeneses of asthma attack. We applied continuous electrical field stimulation (c-EFS) or continuous electrical stimulation (c-ES) of low frequency to the vagal nerve of the rat in vitro and in vivo to determine the role of cholinergic nerve activation in inducing airway constriction.. Fifty-seven male Wistar rats were used. In an in vitro study we examined whether tetrodotoxin (TTX), an Na(+)-channel blocker, 4-DAMP, a muscarinic M(3) receptor antagonist, or neostigmine could affect c-EFS-induced contraction of the tracheal ring. In an in vivo study, we examined whether c-ES of the vagal nerve could increase maximum airway pressure (P (max)) and whether neostigmine could potentiate c-ES-induced P (max).. TTX and 4-DAMP completely inhibited c-EFS-induced contraction whereas neostigmine potentiated c-EFS-induced contraction dose-dependently. P (max) was not increased by neostigmine. P (max) was not increased by 2-Hz c-ES, but was increased by the addition of neostigmine. P (max) was increased by 5-Hz c-ES, and further increased by the addition of neostigmine.. The contractile response of the tracheal ring to c-EFS is potentiated by neostigmine. P (max) is increased by c-ES of the vagal nerve, and is potentiated by neostigmine. These data suggest that increased activity of the cholinergic nerve could be involved in asthma attack.

Topics: Animals; Asthma; Bronchoconstriction; Electric Stimulation; Male; Neostigmine; Parasympathomimetics; Piperidines; Rats; Rats, Wistar; Tetrodotoxin; Trachea

Crotonis Fructus is the mature fruit of Croton tiglium L. (Euphorbiaceae), which has been used in traditional Chinese medicine for the treatment of gastrointestinal (GI) diseases, such as constipation, abdominal pain, peptic ulcer, and intestinal inflammation for thousands of years. The aim of this study was to investigate the pharmacological effect of extracts and fractions from Crotonis Fructus on GI tract.. The activities of methanol extract and fractions from Crotonis Fructus on the smooth muscle contractions were evaluated using isolated rabbit jejunum model.. The results suggest that the n-BuOH and H(2)O fractions showed spasmolytic activity, while the MeOH extract, PE and EtOAc fractions exerted spasmogenic effect. Moreover, bioassay-guided fractionation verified that the EtOAc fraction was more potent than others, followed by PE fraction and methanol extract. Additionally, atropine (10μM), 4-DAMP (10μM) and verapamil (0.1μM) produced a significant inhibition of contractions caused by EtOAc fraction, while either hexamethonium (10μM) or methoctramine (10μM) was inactive. Additionally, a HPLC fingerprint of EtOAc fraction was appraised to ensure its chemical consistency and the main component has been identified as phorbol 12-acetate-13-tiglate.. These data indicate that the regulatory effect of EtOAc fraction on GI motility are medicated via the activation of M3 muscarinic receptor and Ca(2+) influx through L-type Ca(2+) channel. These provide a scientific basis for the traditional use of Crotonis Fructus in GI disorders.

Topics: Animals; Atropine; Calcium; Calcium Channel Blockers; Croton; Female; Fruit; In Vitro Techniques; Jejunum; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Plant Extracts; Rabbits; Receptor, Muscarinic M3; Solvents; Verapamil

The presence of M3 cholinoreceptors and their role in mediation of action potential waveform modulation were determined by immunolabeling of receptor proteins and standard microelectrode technique, respectively. The sinoatrial node (SAN), which was determined as a connexin 43 negative area within the intercaval region, the surrounding atrial tissue, and the working ventricular myocardium exhibited labeling of both M3 and M2 receptors. However, the density of M3 and M2 labeling was about twofold higher in the SAN compared to working myocardium. The stimulation of M3 receptors was obtained by application of nonselective M1 and M3 muscarinic agonist pilocarpine (10(-5) M) in the presence of selective M2 blocker methoctramine (10(-7) M). Stimulation of M3 receptors provoked marked shortening of action potential duration in atrial and ventricular working myocardium. In the SAN, M3 stimulation leads to a significant reduction of sinus rhythm rate accompanied with slowing of diastolic depolarization and increase of action potential upstroke velocity. All electrophysiological effects of selective M3 stimulation were suppressed by specific blocker of M3 receptors 4-DAMP (10(-8) M). We conclude that M3 cholinoreceptors are present in pacemaker and working myocardium of murine heart, where they mediate negative cholinergic effects: slowing of sinus rhythm and shortening of action potentials.

Topics: Action Potentials; Animals; Diamines; Male; Mice; Muscarinic Agonists; Muscarinic Antagonists; Myocardium; Pilocarpine; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Sinoatrial Node

To determine whether muscarinic receptor antagonism affects stretching-induced release of ATP.. Mucosal strips, dissected from guinea pig (male, 450g; n = 10) urinary bladders, were placed in horizontal organ baths and superfused with Ca(2+) -free Tyrode's solution. Superfusate samples were taken pre- and post- intervention (rapid stretching or relaxation) and ATP concentration was quantified using a luciferin-luciferase assay. The effect of muscarinic acetylcholine receptor antagonism on ATP release was assessed by addition of methoctramine (1 µM) and 4-DAMP (10 nM).. Rapid stretching (0 to 13.3 ± 1.2 mN; no. strips = 20) increased ATP in the superfusate to a median threefold increase over basal levels. After a period of equilibration, tension in the mucosal strips relaxed until it had reached a new steady-state after 60 min and stretching was repeated. In the presence of 4-DAMP (10 nM) or methoctramine (1 µM), ATP concentrations after stretching reduced to 61% or 20%, respectively. By contrast, ATP concentrations in mucosa-matched controls, perfused with vehicle, increased in response to stretching by 391% and 1500%, respectively. Rapid relaxation also stimulated ATP release. This release did not appear to be sensitive to 4-DAMP or methoctramine.. An alteration of resting mucosal tension is the key determinant of ATP release, as ATP is released from the mucosa in response to both stretching and relaxation. Muscarinic receptor antagonism inhibits stretching-evoked ATP release from bladder mucosa, suggesting that anticholinergic agents used to treat human lower urinary tract pathologies act on urothelial muscarinic receptors.

Topics: Adenosine Triphosphate; Animals; Biomechanical Phenomena; Diamines; Guinea Pigs; In Vitro Techniques; Male; Mucous Membrane; Muscarinic Antagonists; Piperidines; Urinary Bladder; Urothelium

We investigated the role of muscarinic receptor subtypes in calcium sensitization and their contribution to rho-kinase (ROK) and protein kinase C (PKC) pathways in carbachol (CCh)-induced contraction of human detrusor smooth muscle (DSM).. α-toxin-permeabilized human DSM strips were prepared and mounted horizontally to record isometric force. The roles of M(2) and M(3) muscarinic receptors in Ca(2+) sensitization were studied using selective antagonists of M(2) (AF-DX116) and M(3) (4-DAMP) receptor subtypes. The effects of a selective inhibitor of ROK, Y-27632, and a selective inhibitor of PKC, bisindolylmaleimide I (GF-109203X), were also studied on contraction induced by 10 μM CCh with 100 μM guanosine triphosphate at a fixed 1 μM [Ca(2+)](i) after preincubation with 1 μM AF-DX116 or 1 μM 4-DAMP.. Carbachol-induced Ca(2+) sensitization was predominantly inhibited by 4-DAMP compared with AF-DX116. Four-DAMP equivalently inhibited the relaxation effect of 5 μM GF-109203X as well as that of 5 μM Y-27632 on CCh-induced Ca(2+) sensitization. AF-DX116 reduced the relaxation effect of Y-27632 to a greater degree than GF-109203X.. The results of the present study have demonstrated the predominant role of M(3) receptor subtype in Ca(2+) sensitization and the relative contribution to ROK and PKC pathways. Our study also shows that the ROK pathway is dominant compared with the PKC pathway after M(2) receptor activation, which in turn is inferior, but not negligible, in producing Ca(2+) sensitization.

Topics: Amides; Calcium; Carbachol; Enzyme Inhibitors; Humans; In Vitro Techniques; Indoles; Maleimides; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxants, Central; Muscle Relaxation; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Protein Kinase C; Pyridines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; rho-Associated Kinases; Signal Transduction; Urinary Bladder

Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction.

Topics: Animals; Carbachol; Cyclophosphamide; Cystitis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Male; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Synthase; Nitroarginine; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Urinary Bladder; Urothelium

The experiments employing high-frequency ultrasonic technique and selective blockers of M1, M3, and M4 muscarinic cholinergic receptors pirenzepine, 4-DAMP, and tropicamide, respectively, revealed individual roles of these receptors in the development of severe posthemorrhagic hypotension in rats with low or high individual resistance to circulatory hypoxia. The study showed that M1 and M4 muscarinic receptors are involved in shock-limiting and shock-activating processes, respectively, while M3 receptors exert no effect on the course of posthemorrhagic abnormalities in systemic and hepatic portal circulation and on the posthemorrhagic lifespan. Poor resistance of the cardiovascular system to circulatory hypoxia during shock development is considered to be dysregulatory pathology.

Topics: Animals; Blood Circulation; Blood Pressure; Hemorrhage; Hypotension; Male; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Tropicamide

We tested the potential role of the mAChR in lipopolysaccharide (LPS)-induced inflammatory response in in vivo and in vitro models and a possible signaling pathway involved in the inflammatory process.. Anesthetized mice were challenged with intratracheal LPS to induce acute lung injury. The cytology and histopathology changes, expression of cytokines and pulmonary vascular permeability were used to evaluate the effects of the cholinergic agent. Alveolar macrophage cell line NR8383 was also used to confirm the role of mAChRs and the molecular mechanisms underlying the LPS-induced events.. LPS-induced acute lung injury (ALI) was significantly improved by atropine (a non-selective mAChR antagonist) and 4-DAMP (a M3 mAChR antagonist), as indicated by the diminution of neutrophil infiltration, pulmonary vascular permeability and IL-6 and TNF-α production. LPS-induced TNF-α production from the alveolar macrophage was significantly inhibited by atropine and 4-DAMP, but not pirenzepine (a M1 mAChR antagonist) and methoctramine (a M2 mAChR antagonist). Interestingly, LPS-induced TNF-α production was enhanced by the muscarinic receptor agonist pilocarpine, and treatment with pilocarpine alone was able to trigger TNF-α production from the alveolar macrophage, which was effectively attenuated by 4-DAMP. Western blot analysis showed that LPS-induced degradation of IκBα was strongly blocked by atropine/4-DAMP both in vivo and in vitro, indicating that M3 mAChR was involved in LPS-induced lung inflammation by mediating the NF-κB signaling pathway.. Our findings bring the evidence that the blockage of mAChR exerts anti-inflammatory properties, in which the M3 mAChR plays an important role in the LPS-induced lung inflammation.

Topics: Acute Lung Injury; Animals; Atropine; Cell Line; Lipopolysaccharides; Macrophages, Alveolar; Male; Mice; Muscarinic Antagonists; NF-kappa B; Piperidines; Pneumonia; Receptor, Muscarinic M3

The effects of muscarinic M(1), M(2), and M(3) cholinergic receptor blockade on the regulation of chronotropic function of the heart were studied in vivo in 7-day-old rat pups. Intravenous injection of M(2) receptor blocker gallamine produced no changes in cardiac chronotropy. In contrast, M(1) receptor blocker pirenzepine and M(3) receptor blocker 4DAMP provoked bradycardia. These data attest to the involvement of M(1) and especially M(3) cholinergic receptors in the regulation of cardiac chronotropy in rat pups, which confirms the view on pronounced species-specific and age-related peculiarities in the heart control mechanisms.

Topics: Animals; Animals, Newborn; Gallamine Triethiodide; Heart Rate; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Stimulation, Chemical; Vagus Nerve

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca(2+) channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1μM reversibly inhibited T-currents by ~26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca(2+) channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.

Topics: Animals; Calcium Channels, T-Type; Calcium Signaling; Cholera Toxin; Cobra Neurotoxin Proteins; Estrenes; Ganglia, Spinal; Indoles; Maleimides; Mice; Mice, Inbred ICR; Muscarinic Antagonists; Neurons; Pain Measurement; Pertussis Toxin; Piperidines; Protein Kinase C; Pyrrolidinones; Receptor, Muscarinic M3; Type C Phospholipases

The purpose of this study was to examine the pharmacologic plasticity of cholinergic, non-adrenergic non-cholinergic (NANC), and purinergic contractions in neurogenic bladder strips from spinal cord injured (SCI) rats. Bladder strips were harvested from female rats three to four weeks after T(9)-T(10) spinal cord transection. The strips were electrically stimulated using two experimental protocols to compare the contribution of muscarinic and NANC/purinergic contractions in the presence and the absence of carbachol or muscarine. The endpoints of the study were: (1) percent NANC contraction that was unmasked by the muscarinic antagonist 4-DAMP, and (2) P2X purinergic contraction that was evoked by α,β-methylene ATP. NANC contraction accounted for 78.5% of the neurally evoked contraction in SCI bladders. When SCI bladder strips were treated with carbachol (10 μM) prior to 4-DAMP (500 nM), the percent NANC contraction decreased dramatically to only 13.1% of the neurally evoked contraction (P=0.041). This was accompanied by a substantial decrease in α,β-methylene ATP evoked P2X contraction, and desensitization of purinergic receptors (the ratio of subsequent over initial P2X contraction decreased from 97.2% to 42.1%, P=0.0017). Sequential activation of the cholinergic receptors with carbachol (or with muscarine in neurally intact bladders) and unmasking of the NANC response with 4-DAMP switched the neurally evoked bladder contraction from predominantly NANC to predominantly cholinergic. We conclude that activation of muscarinic receptors (with carbachol or muscarine) blocks NANC and purinergic contractions in neurally intact or in SCI rat bladders. The carbachol-induced inhibition of the NANC contraction is expressed more in SCI bladders compared to neurally intact bladders. Along with receptor plasticity, this change in bladder function may involve P2X-independent mechanisms.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Carbachol; Cholinergic Agonists; Female; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neuronal Plasticity; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Receptors, Purinergic; Spinal Cord Injuries; Urinary Bladder

Interstitial cells of Cajal (ICC) provide pacemaker activity and functional bridges between enteric motor nerve terminals and gastrointestinal smooth muscle cells. The ionic conductance(s) in ICC that are activated by excitatory neural inputs are unknown. Transgenic mice (Kit(copGFP/+)) with constitutive expression of a bright green fluorescent protein were used to investigate cellular responses of ICC to cholinergic stimulation. ICC displayed spontaneous transient inward currents (STICs) under voltage clamp that corresponded to spontaneous transient depolarizations (STDs) under current clamp. STICs reversed at 0 mV when E(Cl) = 0 mV and at -40 mV when E(Cl) was -40 mV, suggesting the STICs were due to a chloride conductance. Carbachol (CCh, 100 nm and 1 μm) induced a sustained inward current (depolarization in current clamp) and increased the amplitude and frequency of STICs and STDs. CCh responses were blocked by atropine (10 μm) or 4-DAMP (100 nm), an M(3) receptor antagonist. STDs were blocked by niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (both 100 μm), and CCh had no effect in the presence of these drugs. The responses of intact circular muscles to CCh and stimulation of intrinsic excitatory nerves by electrical field stimulation (EFS) were also compared. CCh (1 μm) caused atropine-sensitive depolarization and increased the maximum depolarization of slow waves. Similar atropine-sensitive responses were elicited by stimulation of intrinsic excitatory neurons. Niflumic acid (100 μm) blocked responses to EFS but had minor effect on responses to exogenous CCh. These data suggest that different ionic conductances are responsible for electrical responses elicited by bath-applied CCh and cholinergic nerve stimulation.

Topics: Animals; Atropine; Carbachol; Chloride Channels; Green Fluorescent Proteins; Interstitial Cells of Cajal; Intestine, Small; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Muscarinic Agonists; Muscarinic Antagonists; Patch-Clamp Techniques; Piperidines; Receptors, Muscarinic

To investigate the effects of tissue stretch and muscarinic receptor stimulation on the spontaneous activity of the urothelium/lamina propria and identify the specific receptor subtype mediating these responses.. Isolated strips of porcine urothelium with lamina propria were set up for in vitro recording of contractile activity. Muscarinic receptor subtype-selective antagonists were used to identify the receptors influencing the contractile rate responses to stretch and stimulation with carbachol.. Isolated strips of urothelium with lamina propria developed spontaneous contractions (3.7 cycles/min) that were unaffected by tetrodotoxin, Nω-nitro-L-arginine, or indomethacin. Carbachol (1 μM) increased the spontaneous contractile rate of these tissue strips by 122% ± 27% (P < .001). These responses were significantly depressed in the presence of the M3-selective muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (10-30 nM) but were not affected by the M1-selective antagonist pirenzepine (30-100 nM) or the M2-selective antagonist methoctramine (0.1-1 μM). Stretching of the tissue also caused an increase in the spontaneous contractile rate, and these responses were abolished by atropine (1 μM) and low concentrations of 4-diphenylacetoxy-N-methylpiperidine methiodide (10 nM). Darifenacin, oxybutynin, tolterodine, and solifenacin (1 μM) all significantly depressed the frequency responses to carbachol (1 μM).. The urothelium with the lamina propria exhibits a spontaneous contractile activity that is increased during stretch. The mechanism appears to involve endogenous acetylcholine release acting on M3 muscarinic receptors. Anticholinergic drugs used clinically depress the responses of these tissues, and this mechanism might represent an additional site of action for these drugs in the treatment of bladder overactivity.

Topics: Animals; Atropine; Benzhydryl Compounds; Benzofurans; Carbachol; Cresols; Diamines; Mandelic Acids; Mucous Membrane; Muscarinic Antagonists; Muscle Contraction; Phenylpropanolamine; Piperidines; Pirenzepine; Pyrrolidines; Quinuclidines; Receptor, Muscarinic M3; Solifenacin Succinate; Stress, Mechanical; Swine; Tetrahydroisoquinolines; Tolterodine Tartrate; Urinary Bladder; Urothelium

Cerebral insulin can regulate glucose homeostasis via activation of the parasympathetic nervous system, which results in the reduction of hepatic glucose output. However, the precise mechanism(s) through which cerebral insulin directly exerts an effect on insulin secretion remains unclear. In the present study, we found that cerebral administration of insulin caused an increase of plasma insulin concentration and a concomitant decrease in plasma glucose levels within one hour. These effects were blocked by vagotomy or intraperitoneal injection of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide, a specific M (3) antagonist. The mediating influence of parasympathetic activation can thus be considered. The adenosine triphosphate-sensitive potassium (K-ATP) channel is a key mediator of the cerebral action of insulin. The plasma glucose-lowering action of insulin was abolished by cerebral administration of glibenclamide or repaglinide at concentrations sufficient to block K-ATP channels. In conclusion, our findings suggest that cerebral insulin may induce insulin release by stimulating the opening of K-ATP channels, which in turn activate parasympathetic tone in pancreatic tissue.

Topics: Adenosine Triphosphate; Animals; Blood Glucose; Brain; Glyburide; Hypoglycemic Agents; Injections, Intravenous; Injections, Intraventricular; Insulin; KATP Channels; Male; Muscarinic Antagonists; Parasympathetic Nervous System; Piperidines; Potassium Channel Blockers; Rats; Rats, Wistar; Vagotomy

Cholinergic receptors are expressed in small cell lung cancer (SCLC); however, the distinct functions of muscarinic cholinergic receptor 3 (mAChR3) and the nicotinic cholinergic receptor (nAChR) in SCLC have not yet been completely elucidated.. RT-PCR and Western blotting were used to investigate the expression of cholinergic receptors. Flow cytometry was used to detect the integrin expression. Cell proliferation, adhesion and migration assays were carried out in vitro to determine the roles of the cholinergic receptors in SBC3 human SCLC cells.. Both mAChR3 and nAChR were expressed in the SBC3 cells. Acetylcholine iodide (Ach) stimulated SBC3 cell proliferation, adhesion and migration toward fibronectin (Fn). The mAChR3 antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), or the nAChR antagonist, mecamylamine hydrochloride (Meca), inhibited SBC3 cell proliferation in the presence or the absence of exogenous Ach. 4-DAMP abrogated cell adhesion and migration toward Fn induced by Ach, while Meca had no effect. Interestingly, Ach did not alter Fn receptor (alphavbeta1 or alpha5beta1 integrin) expression, while anti-beta1 integrin antibody or anti-alphav and anti-alpha5 integrin antibody completely abrogated cell adhesion to Fn induced by Ach.. Both mAChR3 and nAChR are expressed in SCLC. SBC3 cell proliferation is regulated in vitro through both cholinergic receptors. In contrast, SBC3 cell migration and adhesion toward Fn are modulated only by mAChR. Moreover, the stimulatory effects of Ach on cell adhesion and migration through mAChR3 are presumably modulated by functional alteration of alphavbeta1 and alpha5beta1 integrin, but not by any variation in their expression. The mAChR3 antagonist may therefore be a beneficial therapeutic modality for SCLC patients, especially those with chronic obstructive pulmonary disease (COPD) as a comorbidity.

Topics: Blotting, Western; Carcinoma, Small Cell; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cholinergic Agonists; Drugs, Chinese Herbal; Humans; Lung Neoplasms; Muscarinic Antagonists; Piperidines; Polysaccharides; Receptor, Muscarinic M3; Receptors, Nicotinic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We demonstrate that patients with primary Sjögren's syndrome (pSS) produce functional IgG autoantibodies that interact with the glandular M(3) muscarinic acetylcholine receptors (mAChRs). These autoantibodies act as a partial muscarinic agonist, increasing prostaglandin E(2) (PGE(2)) and cyclic AMP production through modifying Na(+)/K(+)-ATPase activity, but also interfere with the secretory effect of the parasympathetic neurotransmitter. The IgG from patients with pSS has two effects on the submandibular gland. On the one hand, it may act as an inducer of the proinflammatory molecule (PGE(2)) that, in turn, inhibits Na(+)/K(+)-ATPase activity. On the other hand, it plays a role in the pathogenesis of dry mouth, abolishing the Na(+)/K(+)-ATPase inhibition and the net K(+) efflux stimulation of the salivary gland in response to the authentic agonist pilocarpine, decreasing salivary fluid production.

Topics: Adult; Animals; Autoantibodies; Cells, Cultured; Cyclic AMP; Dinoprostone; Female; Humans; Immunoglobulin G; Immunologic Factors; Inflammation Mediators; Keratoconjunctivitis Sicca; Male; Middle Aged; Muscarinic Agonists; Muscarinic Antagonists; Pilocarpine; Piperidines; Pirenzepine; Potassium; Rats; Rats, Wistar; Receptor, Muscarinic M3; Sjogren's Syndrome; Sodium-Potassium-Exchanging ATPase; Submandibular Gland; Tropicamide; Xerostomia

Information about the muscarinic receptor subtype(s) mediating pulmonary circulatory vasodilator responses to acetylcholine (ACh) is limited. The aim of this study was to pharmacologically characterize the muscarinic receptors associated with ACh-induced pulmonary vasodilation in a pulmonary hypertension model. Vasodilation of rabbit isolated buffer-perfused lungs in which pulmonary hypertension was induced with the thromboxane A₂ analogue U-46619 was evoked by ACh at a just maximally effective concentration (2 x 10⁻⁷ M). The effects of cumulative concentrations of three specific muscarinic receptor subtype antagonists [pirenzepine (M₁), methoctramine (M₂), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M₃] on ACh-induced pulmonary vasodilation were determined. Double vascular occlusion pressure was recorded to locate the muscarinic receptors within the pulmonary vasculature. Based on the 50% inhibitory concentrations (IC₅₀), the rank of order of antagonist potency was 4-DAMP >> pirenzepine > methoctramine. The vascular effects of all three inhibitors were localized to the precapillary segment. These findings suggest that the vasodilator action of ACh on rabbit isolated perfused U-46619 pretreated lungs is mediated by M₃ muscarinic receptors located in the pulmonary arterial bed.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Diamines; Hypertension, Pulmonary; Lung; Piperidines; Pirenzepine; Rabbits; Receptor, Muscarinic M3; Vasodilation

This study was performed to determine whether spinal cholinergic systems mediate the relieving effects of electroacupuncture (EA) on cold and warm allodynia in a rat model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the injury, the intrathecal (i.t.) catheter was implanted. Five days after the catheterization, the rats were injected with atropine (non-selective muscarinic antagonist, 30 microg), mecamylamine (non-selective nicotinic antagonist, 50 microg), pirenzepine (M(1) muscarinic antagonist, 10 microg), methoctramine (M(2) antagonist, 10 microg) or 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (M(3) antagonist, 10 microg). Ten minutes after the injection, EA was applied to the ST36 acupoint for 30 min. The cold and warm allodynia were assessed by the tail immersion test [i.e., immersing the tail in cold (4 degrees C) or warm (40 degrees C) water and measuring the latency of an abrupt tail movement] before and after the treatments. The i.t. atropine, but not mecamylamine, blocked the relieving effects of EA on cold and warm allodynia. Furthermore, i.t. pirenzepine attenuated the antiallodynic effects of EA, whereas methoctramine and 4-DAMP did not. These results suggest that spinal muscarinic receptors, especially M(1) subtype, mediate the EA-induced antiallodynia in neuropathic rats.

Topics: Animals; Atropine; Cholinergic Agents; Cholinergic Fibers; Cold Temperature; Diamines; Disease Models, Animal; Electroacupuncture; Hot Temperature; Hyperesthesia; Male; Mecamylamine; Neuralgia; Pain Threshold; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Spinal Nerves

1 In the present study we have investigated the expression of muscarinic receptors in K562 erythroleukaemic cells and the effects of muscarinic agonist and antagonists on extracellular citrulline levels in these cells, as a marker of nitric oxide (NO) generation. 2 Muscarinic acetylcholine receptors (M(1)-M(5)) play key roles in regulating many diverse physiological processes. Recent studies suggest that muscarinic receptors mediate some cellular events in haematopoietic cells. Multiple subtypes of muscarinic receptors are expressed in different human cells. NO, a free radical and a signaling molecule, is involved in the regulation of many physiological functions and derived from certain nitric oxide synthases (NOS), which are related to muscarinic receptors. 3 In this study, the presence of M(2), M(3) and M(4) subtypes in K562, an erythroleukaemic cell line, was demonstrated by using the reverse transcriptase-polymerase chain reaction. Moreover, the generation of NO induced by carbachol, a non-selective muscarinic agonist, was investigated by using high-performance liquid chromatography to measure changes in extracellular l-citrulline levels. 4 We found that carbachol enhanced l-citrulline production in K562 erythroleukaemic cells. The effect of carbachol on l-citrulline production was antagonized by atropine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), while tropicamide had little effect. These results suggest that the muscarinic receptor M(3) subtype may mediate NO signaling in K562 erythroleukaemic cells.

Topics: Arginine; Carbachol; Chromatography, High Pressure Liquid; Citrulline; Dose-Response Relationship, Drug; Humans; K562 Cells; Microdialysis; Muscarinic Agonists; Muscarinic Antagonists; Nitric Oxide; Nitric Oxide Synthase; Piperidines; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tropicamide

The role of cholinergic neurotransmission in male rat sexual behavior at the brain level has been studied by several researchers. However, little is known about its role at the spinal cord level. In this study, the effects of the intrathecal (IT) administration of the muscarinic receptor antagonist subtypes (MRAs) methoctramine (Meth), tropicamide (Trop) and 4-DAMP on male rat sexual behavior were evaluated during three ejaculatory series. Meth and Trop are preferring antagonists for the M2/M4 receptor subtypes, and 4-DAMP is a preferring antagonist for the M3 receptor subtype. All the MRAs tested noticeably inhibited male rat copulatory behavior, reflected by a reduction in the number of animals engaging in sexual behavior and a gradual decrease in the number of animals able to ejaculate. Significant increases in intromission latency (IL), ejaculation latency (EL) and post-ejaculatory interval (PEI) were observed. The ranking of inhibitory potency in all recorded parameters was Meth>/=4-DAMP>Trop. In theory, the effects of Meth and Trop could be a result of interaction with M2/M4 receptors. However, given that the M2 receptor constitutes the greatest population of muscarinic receptors at all spinal cord sites and given the high affinity for Meth on M2 receptors, the high potency in the inhibitory effects of Meth is indicative of the special role of M2 spinal receptors in the implementation of this behavior. The weaker effects of Trop could be linked to the smaller population of M4 receptors in the spinal cord, but some interaction with M2 receptors is probable. Since some differences in the pattern of inhibitory response between Meth and 4-DAMP were observed in this and a previous study, a possible role for M3 receptors must be considered. The data obtained in this study confirm the facilitating effect of acetylcholine (ACh) at the spinal cord level on male rat sexual behavior through muscarinic mechanisms, with an important influence on ejaculatory processes. These data support the hypothesis of the modulating role of ACh on male rat sexual behavior at the spinal cord level.

Topics: Animals; Diamines; Injections, Spinal; Male; Muscarinic Antagonists; Piperidines; Rats; Rats, Wistar; Rotarod Performance Test; Sexual Behavior, Animal; Spinal Cord; Tropicamide

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca2+-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of-70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M(3) receptor antagonist, but not by methotramine, a muscarinic M(2) receptor antagonist. Intracellular GDP-beta-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na+-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca2+. In recording of intracellular Ca2+ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca2+ concentrations with increasing of Ca2+ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M(3) receptors by a G-protein dependent intracellular Ca2+ release mechanism.

Topics: Animals; Anti-Inflammatory Agents; Biological Clocks; Calcium; Calcium-Transporting ATPases; Carbachol; Cation Transport Proteins; Cells, Cultured; Diamines; Flufenamic Acid; Guanosine Diphosphate; Intestine, Small; Membrane Potentials; Mice; Mice, Inbred BALB C; Neuromuscular Depolarizing Agents; Patch-Clamp Techniques; Piperidines; Plant Roots; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Thapsia; Thapsigargin; Thionucleotides

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca(2+) dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and alpha,beta methylene ATP (alpha,beta-MeATP) (1 microM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 microM) elicited Ca(2+) transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 microM). Ca(2+) transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 microM). Stimulation produced a marked increase in the frequency and number of Ca(2+) transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The alpha(2)-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca(2+) transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional alpha(2)-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca(2+) events, with no effect on Ca(2+) transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca(2+) transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M(3) muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca(2+) dynamics in this tissue.

Topics: Acetylcholine; Adrenergic alpha-2 Receptor Antagonists; Age Factors; Animals; Calcium; Cholinesterase Inhibitors; Cyclopentolate; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Muscle Contraction; Muscle, Smooth; Neostigmine; Neuroeffector Junction; Neurons; Norepinephrine; Piperidines; Receptor, Muscarinic M3; Receptors, Adrenergic, alpha-2; Vas Deferens; Yohimbine

Ca(2+) in the central nervous system plays important roles in brain physiology, including neuronal survival and regeneration in rats with injured facial motoneurons. The present research was to study the modulations of intracellular free Ca(2+) concentrations by cholinergic receptors in rat facial nucleus, and the mechanisms of the modulations.. The fluorescence intensity of facial nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca(2+) measurement technique via confocal laser scanning microscope. The changes of fluorescence intensity of facial nucleus indicate the average changes of intracellular free Ca(2+) levels of the neurons.. Acetylcholine was effective at increasing the fluorescence intensity of facial nucleus. Muscarine chloride induced a marked increase of fluorescence intensity in a concentration dependent fashion. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (depletor of intracellular Ca(2+) store; P < 0.01), rather than Ca(2+) free artifical cerebrospinal fluid or EGTA (free Ca(2+) chelator; P > 0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine (M(1) subtype selective antagonist; P < 0.01) and 4-DAMP (M(3) subtype selective antagonist; P < 0.01). In addition, fluorescence intensity was markedly increased by nicotine. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine (L-type voltage-gated Ca(2+) channel blocker), dihydro-beta-erythroidine (alpha4beta2 subtype selective antagonist), and in Ca(2+) free artificial cerebrospinal fluid (P < 0.01), but not in the presence of mibefradil (M-type voltage-gated Ca(2+) channel blocker) or thapsigargin (P > 0.05).. The data provide the evidence that muscarinic receptors may induce the increase of intracellular free Ca(2+) levels through the Ca(2+) release of intracellular Ca(2+) stores, in a manner related to M(1) and M(3) subtypes of muscarinic receptors in rat facial nucleus. Nicotine may increase intracellular free Ca(2+) concentrations via the influx of extracellular Ca(2+)+ mainly across L-type voltage-gated Ca(2+) channels, in a manner related to the alpha4beta2 subtype of nicotinic receptors.

Topics: Acetylcholine; Aniline Compounds; Animals; Brain Stem; Calcium; Diamines; Facial Nerve; Female; Fluorescent Dyes; In Vitro Techniques; Male; Microscopy, Confocal; Motor Neurons; Muscarinic Agonists; Nicotine; Nicotinic Agonists; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Receptors, Muscarinic; Receptors, Nicotinic; Tropicamide; Xanthenes

1. Using rats, we examined the muscarinic receptor subtype mediating pilocarpine-induced parotid salivary secretion and the contributions of ion transporter systems (effluxes of K+ and Cl(-)) and aquaporin-5 (AQP5) translocation to this response in parotid glands in irradiated-induced xerostomia. 2. Salivary secretion was significantly lower in irradiated compared with sham-irradiated (normal) rats. In xerostomia rats, 0.4 and 0.8 mg/kg pilocarpine significantly increased parotid salivary secretion, although the salivary volume was still significantly less than in normal rats after the same dose of pilocarpine. 3. Pirenzepine (1 x 10(-6) to 1 x 10(-1) mol/L), AF-DX 116 (3 x 10(-6) to 3 x 10(-2) mol/L) and N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP; 1 x 10(-8) to 1 x 10(-2) mol/L) dose-dependently displaced radioligand binding to M(1), M(2) and M(3) receptors, respectively, in parotid membranes from both normal and irradiated rats. In each group of rats, 4-DAMP had the highest binding affinity. Pretreatment with 4-DAMP or pirenzepine dose-dependently inhibited pilocarpine-induced parotid secretion in both normal and irradiated rats, with 4-DAMP being markedly more potent than pirenzepine. 4. Normal and irradiated-rat parotid cells did not differ significantly in terms of pilocarpine-induced changes in [Ca2+](i), [K+](i) and [Cl(-)](i). Pilocarpine markedly increased the amount of AQP5 in the apical plasma membrane of parotid cells isolated from normal but not irradiated rats. 5. Thus, pilocarpine induces parotid salivary secretion mainly via the M(3) receptor subtype in both irradiated and normal rats. The reduction in this pilocarpine-induced secretion seen in irradiated rats is due not to disturbances of intracellular Ca2+ mobilization or ion transporter systems, but rather to a disturbance of AQP5 translocation, which may be involved in the pathogenesis of X-ray irradiation-induced xerostomia.

Topics: Animals; Aquaporin 5; Calcium; Male; Muscarinic Agonists; Muscarinic Antagonists; Parotid Gland; Pilocarpine; Piperidines; Protein Transport; Radiation Injuries, Experimental; Radioligand Assay; Rats; Rats, Sprague-Dawley; Saliva; Salivation; Whole-Body Irradiation; Xerostomia

This study was designed to evaluate the effects of pilocarpine and explore the underlying ionic mechanism, using both aconitine-induced rat and ouabain-induced guinea pig arrhythmia models. Confocal microscopy was used to measure intracellular free-calcium concentrations ([Ca(2+)](i)) in isolated myocytes. The current data showed that pilocarpine significantly delayed onset of arrhythmias, decreased the time course of ventricular tachycardia and fibrillation, reduced arrhythmia score, and increased the survival time of arrhythmic rats and guinea pigs. [Ca(2+)](i) overload induced by aconitine or ouabain was reduced in isolated myocytes pretreated with pilocarpine. Moreover, M(3)-muscarinic acetylcholine receptor (mAChR) antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) partially abolished the beneficial effects of pilocarpine. These data suggest that pilocarpine produced antiarrhythmic actions on arrhythmic rat and guinea pig models induced by aconitine or ouabain via stimulating the cardiac M(3)-mAChR. The mechanism may be related to the improvement of Ca(2+) handling.

Topics: Aconitine; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Calcium; Disease Models, Animal; Female; Guinea Pigs; Male; Microscopy, Confocal; Muscarinic Agonists; Muscarinic Antagonists; Myocytes, Cardiac; Ouabain; Pilocarpine; Piperidines; Rats; Rats, Wistar; Receptor, Muscarinic M3; Time Factors

Systemic sclerosis (SSc) IgGs affecting the M(3)-muscarinic receptor (M(3)-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of M(3)-R and IgG-M(3)-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 +/- 1.2% caused by 10(-4) M BeCh was significantly attenuated to 8.3 +/- 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on K(+) depolarization and alpha(1)-adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-M(3)-R complex. SSc IgGs attenuated M(3)-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies.

Topics: Adrenergic alpha-Agonists; Adult; Aged; Aged, 80 and over; Anal Canal; Animals; Autoantibodies; Benzofurans; Bethanechol; Case-Control Studies; Dose-Response Relationship, Drug; Female; Humans; Immunoglobulins; In Vitro Techniques; Male; Middle Aged; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Myocytes, Smooth Muscle; Phenylephrine; Piperidines; Potassium Chloride; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Scleroderma, Systemic

Pancreatic neuronal changes associated with beta cell loss in type 1 diabetes mellitus are complex, involving, in part, parasympathetic mechanisms to compensate for preclinical hyperglycemia. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor has been shown to be a useful marker of cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[(18)F]fluorobenzyltrozamicol ([(18)F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. The compound 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), available in a tritiated form, binds to M3 muscarinic receptors on beta cells and is a potential target for assessing pancreatic beta cell mass. In this study, we investigate the feasibility of dual radiotracer analysis in identifying neurofunctional changes that may signify type 1 diabetes mellitus in its early preclinical state.. Ex vivo determinations of pancreatic uptake were performed in prediabetic nonobese diabetic mice and controls after intravenous injection of [(18)F]FBT or 4-[(3)H]DAMP. Beta cell loss in prediabetic mice was confirmed using immunohistochemical methods.. [(18)F]FBT uptake was significantly higher in prediabetic pancreata than controls: 3.22 +/- 0.81 and 2.51 +/- 1.04, respectively (P < 0.03). 4-[(3)H]DAMP uptake was significantly lower in prediabetic pancreata than controls: 0.612 +/- 0.161 and 0.968 +/- 0.364, respectively (P = 0.01).. These data suggest that a combination of radiotracer imaging agents that bind to neuronal elements intimately involved in insulin production may be an effective method of evaluating changes associated with early beta cell loss using PET.

Topics: Animals; Fluorine Radioisotopes; Fluorobenzenes; Insulin-Secreting Cells; Mice; Pancreas; Parasympatholytics; Piperidines; Prediabetic State; Radiography; Tritium

Previous reports suggested that there is no significant difference in the binding affinity of pilocarpine and cevimeline on muscarinic receptors (1). However, in vivo studies from our laboratory suggested that pilocarpine-induced salivation from the parotid gland is greater than that induced by cevimeline in rats. Therefore, in the present study, sialogogue-induced intracellular Ca(2+) mobilization was investigated in isolated parotid gland cells in vitro. Pilocarpine and cevimeline increased the intracellular Ca(2+) concentration of parotid gland acinar and duct cells over 1 microM in a dose-dependent manner. Pilocarpine-induced responses were higher than cevimeline-induced responses. Ca(2+) responses to both agents were completely blocked by 1 microM 4-DAMP, an M3 muscarinic receptor antagonist. In the absence of extracellular Ca(2+), both sialogogues induced transient Ca(2+) increase in parotid gland acinar cells. These results suggest that the sialogogues stimulate some common routes via the Ca(2+) signaling in parotid gland acinar cells. We also report a significant difference of Ca(2+) responses in concentration between pilocarpine and cevimeline in parotid gland acinar cells. The different Ca(2+) responses between the sialogogues would explain the different saliva volumes from the parotid gland between them that we have observed in previous in vivo studies.

Topics: Animals; Calcium; Calcium Signaling; Muscarinic Agonists; Muscarinic Antagonists; Parotid Gland; Pilocarpine; Piperidines; Quinuclidines; Rats; Receptors, Muscarinic; Thiophenes

Methylmercury (MeHg) is an environmental toxicant that is known to induce lymphocyte apoptosis; however, little is known about the molecular mechanism involved. Data showed that MOLT-3 cells were more sensitive to MeHg-induced cytotoxic effects than Jurkat clone E6-1 cells, suggesting that the lymphocytic muscarinic cholinergic system may be involved since the expressions of five subtypes (M1-M5) of muscarinic acetylcholine receptor (mAChR) in MOLT-3 cells are higher than in Jurkat cells. The role of mAChR-linked pathways in MeHg-induced apoptosis in human leukemic T cells was examined in this study. Treatment of the MOLT-3 cells with 1 microM MeHg produced induction of c-Fos expression, apoptotic cell death, and downregulation of mAChR. MeHg-induced c-Fos expression was significantly reduced by pretreatment with atropine (a nonselective mAChR antagonist), or 4-DAMP (a selective M1/M3 mAChR antagonist), whereas pirenzipine (a selective M1 mAChR antagonist) or himbazine (a selective M2/M4 mAChR antagonist) did not reduce this induction, suggesting that MeHg-induced c-Fos expression through the activation of the mAChR, at least M3 subtype, is involved. Pretreatment with 4-DAMP or SB 203580 (a specific p38 inhibitor) resulted in decreases in the level of phosphorylated p38, c-Fos expression, and apoptotic cell death induced by MeHg. Taken together, these data suggest that the mAChR-p38-dependent pathway participates in the increase of c-Fos expression, which is involved in MeHg-induced lymphocyte apoptosis. In addition, a noncytotoxic concentration of MeHg (0.1 microM) inhibited PHA/PMA-stimulated interleukin (IL)-2 production, and this inhibition was reversed by pretreatment with atropine or 4-DAMP. Overall, this study provides initial evidence that MeHg may alter the immune system by targeting the lymphocytic mAChR.

Topics: Apoptosis; Drug Interactions; Genes, fos; Humans; Jurkat Cells; Methylmercury Compounds; Muscarinic Antagonists; Piperidines; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tumor Cells, Cultured

The cardiomyocytes in the superior vena cava (SVC) myocardial sleeve have distinct action potentials and ionic current profiles, but the refractoriness of these cells has not been reported. Using standard intracellular microelectrode techniques, we demonstrated in sheep that the effective refractory period (ERP) of the cardiomyocytes in the SVC (114.7 +/- 6.5 ms) is shorter than that in the inferior vena cava (IVC) (166.7 +/- 6.2 ms), right atrial free wall (RAFW) (201.0 +/- 6.0 ms) and right atrial appendage (RAA) (203.1 +/- 5.8 ms) (P < 0.05). The right atrial cardiomyocyte ERP was heterogeneously shortened by acetylcholine, a muscarinic type 2 receptor (M(2)R) agonist. After perfusion with 15 microM acetylcholine, the shortest ERP occurred in the SVC (the ERP in the SVC, IVC, RAFW and RAA was 53.6 +/- 2.7, 98.9 +/- 2.2, 121.8 +/- 6.0 and 109.7 +/- 5.1 ms, respectively; P < 0.05). Carbachol (1 microM), another M(2)R agonist, produced a similar effect as acetylcholine. Furthermore, we used methoctramine, a M(2)R blocker, 4-DAMP, a muscarinic type 3 receptor (M(3)R) blocker, and tropicamide, a muscarinic type 4 receptor (M(4)R) blocker to inhibit the acetylcholine-induced ERP shortening of SVC cardiomyocytes, and found that the 50% inhibitory concentration for methoctramine, 4-DAMP and tropicamide was 5.91, 45.72 and 80.34 nM, respectively. Therefore, we conclude that the sheep SVC myocardial sleeve is a unique electrophysiological region of the right atrium with the shortest ERP both under physiological condition and under cholinergic agonist stimulation. M(2)R might play a major role in the response of the SVC myocardial sleeve to parasympathetic nerve tone. The association between the distinct refractoriness in SVC and atrial fibrillation originating from the region deserves further investigation.

Topics: Acetylcholine; Animals; Carbachol; Cholinergic Agonists; Diamines; Electrophysiologic Techniques, Cardiac; In Vitro Techniques; Microelectrodes; Muscarinic Antagonists; Myocardium; Myocytes, Cardiac; Piperidines; Refractometry; Sheep; Tropicamide; Vasodilator Agents; Vena Cava, Superior

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 microM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to alpha,beta-methylene ATP (alpha,beta-mATP) (10 microM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 microM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100 microM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with alpha,beta-mATP) decreased the NANC contractions and the direct contractile response to alpha,beta-mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Antineoplastic Agents; Atropine; Calcium Channel Agonists; Carbachol; Cholinergic Agonists; Cholinesterase Inhibitors; Electric Stimulation; Female; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Physostigmine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Suramin; Urinary Bladder

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. In the present study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-PP-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP, in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta2 proteins, enhanced P70S6K and MAPK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Topics: Animals; Animals, Newborn; Antigens, Neoplasm; Biomarkers, Tumor; Carbachol; Carrier Proteins; Immunoblotting; In Vitro Techniques; Insulin; Islets of Langerhans; Lectins, C-Type; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Pancreatitis-Associated Proteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Piperidines; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases

Decreased muscarinic M1 receptor (CHRM1) mRNA has been reported in Brodmann's area (BA) 6 from subjects with schizophrenia. We have extended this study by measuring levels of CHRM1 ([(3)H]pirenzepine binding), CHRM3 ([(3)H]4-DAMP binding), the transcription factor SP1 and the CHRM1 downstream target beta-site APP-cleaving enzyme 1 (BACE1) in BA 6 from 19 subjects with schizophrenia and 19 control subjects. Radioligand binding was quantified using either in situ radioligand binding with autoradiography or, in cohorts of 10 control subjects and 10 subjects with schizophrenia, membrane enriched fraction (MEF) CNS ([(3)H]pirenzepine binding only). Levels of SP1 and BACE1 were measured by Western blotting. [(3)H]pirenzepine binding to tissue sections was in two layers, binding to tissue sections (Binding layer 1: p<0.01; Binding layer 2: p<0.001) and MEF (p<0.05) were decreased in schizophrenia. Levels of [(3)H]4-DAMP binding, SP1 and BACE1 were not altered in subjects with the disorder. This study shows a decrease in levels of CHRM1 in BA 6 from subjects with schizophrenia; as CHRM1 and BA 6 are important in maintaining normal cognitive function, these data support the hypothesis that decreased levels of cortical CHRM1 may contribute to the cognitive deficits associated with schizophrenia. Our findings on BACE1 suggest that the schizophrenia phenotype reported in BACE(-/-) mice is not simply due to lack of that protein in the cortex.

Topics: Adult; Aged; Autoradiography; Binding Sites; Cathepsin B; Cerebral Cortex; Female; Humans; Male; Middle Aged; Piperidines; Pirenzepine; Radioligand Assay; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Schizophrenia; Sp1 Transcription Factor

Recent evidence has suggested that pilocarpine (ACh receptor agonist) injected peripherally may act centrally producing salivation and hypertension. In this study, we investigated the effects of specific M(1) (pirenzepine), M(2)/M(4) (methoctramine), M(1)/M(3) (4-DAMP) and M(4) (tropicamide) muscarinic receptor subtype antagonists injected into the lateral cerebral ventricle (LV) on salivation, water intake and pressor responses to peripheral pilocarpine.. Male Holtzman rats with stainless steel cannulae implanted in the LV were used. Salivation was measured in rats anaesthetized with ketamine (100 mg per kg body weight) and arterial pressure was recorded in unanaesthetized rats.. Salivation induced by i.p. pilocarpine (4 micromol per kg body weight) was reduced only by 4-DAMP (25-250 nmol) injected into the LV, not by pirenzepine, methoctramine or tropicamide at the dose of 500 nmol. Pirenzepine (0.1 and 1 nmol) and 4-DAMP (5 and 10 nmol) injected into the LV reduced i.p. pilocarpine-induced water intake, whereas metoctramine (50 nmol) produced nonspecific effects on ingestive behaviours. Injection of pirenzepine (100 nmol) or 4-DAMP (25 and 50 nmol) into the LV reduced i.v. pilocarpine-induced pressor responses. Tropicamide (500 nmol) injected into the LV had no effect on pilocarpine-induced salivation, pressor responses or water intake.. The results suggest that central M(3) receptors are involved in peripheral pilocarpine-induced salivation and M(1) receptors in water intake and pressor responses. The involvement of M(3) receptors in water intake and pressor responses is not clear because 4-DAMP blocks both M(1) and M(3) receptors.

Topics: Animals; Blood Pressure; Diamines; Drinking Behavior; Heart Rate; Hypertension; Injections, Intraventricular; Male; Muscarinic Antagonists; Pilocarpine; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Saliva; Tropicamide

To detect the function and expression of ventricular M3 receptor (M3R) in cerebral-cardiac syndrome (CCS) model rats and to explore the relationship between the expression of M3R and the arrhythmia resulted from CCS, CCS model rats were induced by occluding right middle cerebral artery. ECG was monitored. Intracellular calcium ([Ca2+]i) changes after agitating M3R were recorded by laser scanning confocal microscope. Changes of M3R expression in the ventricular tissue were detected by Western blotting. QRS and QT intervals in CCS group were remarkably longer than that in sham group. According to the results of Western blotting, the level of M3R expression was remarkably lower in CCS group compared with that in the normal group. KCl induced [Ca2+]i increasing in CCS group could be depressed by choline and the effect of choline could be blocked by 4-DAMP. The lower expression of M3R in CCS group may be one of important reasons of arrhythmia resulted from CCS. M3R that depressed the [Ca2+]i increasing agitated by choline may become a new target to cure arrhythmia resulted from CCS.

Topics: Animals; Arrhythmias, Cardiac; Calcium; Choline; Electrocardiography; Heart Ventricles; Infarction, Middle Cerebral Artery; Male; Muscarinic Antagonists; Myocardium; Myocytes, Cardiac; Piperidines; Potassium Chloride; Random Allocation; Rats; Rats, Wistar; Receptor, Muscarinic M3

It is well known that choline has protective effects on ischemic arrhythmias. We designed the present study to evaluate the antiarrhythmic effects of choline and to detect its related mechanisms in aconitine-induced rat and ouabain-induced guinea pig models of arrhythmia. Laser scanning confocal microscopy and patch-clamp technique were utilized to study the action of choline on intracellular calcium concentration and L-type calcium current (ICa-L) of cardiac myocytes. M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) was applied preliminarily to evaluate the role of the M3 receptor. Choline significantly increased the survival time of arrhythmic rats and guinea pigs, delayed the onset of arrhythmias and ventricular tachycardia, and decreased the arrhythmia score. The overload of intracellular Ca2+ induced by aconitine or ouabain was reduced in isolated myocytes pretreated with choline. Choline reduced the increased density of ICa-L induced by aconitine or ouabain. Moreover, the beneficial effects of choline were reversed by 4-DAMP. Choline produced antiarrhythmic actions on arrhythmia models by stimulating the cardiac M3 receptor. The mechanism may be related to the improvement of Ca2+ handling.

Topics: Aconitine; Animals; Arrhythmias, Cardiac; Calcium; Choline; Disease Models, Animal; Female; Guinea Pigs; Male; Myocytes, Cardiac; Ouabain; Piperidines; Rats; Rats, Wistar; Receptor, Muscarinic M3

Taste bud cells (TBCs) express various neurotransmitter receptors assumed to facilitate or modify taste information processing within taste buds. We investigated the functional expression of muscarinic acetylcholine receptor (mAChR) subtypes, M1-M5, in mouse fungiform TBCs. ACh applied to the basolateral membrane of TBCs elevates the intracellular Ca(2+) level in a concentration-dependent manner with the 50% effective concentration (EC(50)) of 0.6 microM. The Ca(2+) responses occur in the absence of extracellular Ca(2+) and are inhibited by atropine, a selective antagonist against mAChRs. The order of 50% inhibitory concentration (IC(50)) examined with a series of antagonists selective to mAChR subtypes shows the expression of M3 on TBCs. Perforated whole-cell voltage clamp studies show that 1 microM ACh blocks an outwardly rectifying current and that 100 nM atropine reverses the block. Reverse transcriptase-mediated polymerase chain reaction studies suggest the expression of M3 but not the other mAChR subtypes. Immunohistochemical studies show that phospholipase Cbeta-immunoreactive TBCs and synaptosome-associated protein of 25 kDa-immunoreactive nerve endings are immunoreactive to a transporter that packs ACh molecules into synaptic vesicles (vesicular acetylcholine transporter). These results show that M3 occurs on a few fungiform TBCs and suggest that a few nerve endings, and probably a few TBCs, release ACh by exocytosis. The role of ACh in taste responses is discussed.

Topics: Acetylcholine; Animals; Atropine; Calcium Signaling; Cells, Cultured; Diamines; Electrophysiology; Gene Expression; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mice, Inbred Strains; Muscarinic Antagonists; Patch-Clamp Techniques; Phospholipase C beta; Piperidines; Pirenzepine; Receptor, Muscarinic M3; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; Synaptosomal-Associated Protein 25; Taste Buds; Tropicamide; Vesicular Acetylcholine Transport Proteins

This study was designed to determine if chronotropic responses induced by neurally released acetylcholine are modified by subtype-selective blockade of cardiac muscarinic cholinoreceptors. In anesthetized cats, a single burst of vagal stimulation was generated with an incremental time delay after the P wave of the atrial electrogram (P-Stimulus interval). The slope of the relationships between P-Stimulus and P-P intervals was used to assess changes in responsiveness of cardiac pacemaker to vagal effects throughout the cardiac cycle. An increase in P-Stimulus interval over the initial portion (approximately 120 ms) of the cardiac cycle produced a significant increment in lengthening of the P-P interval. Once the maximal negative chronotropic response was achieved, a further increase in P-Stimulus interval by only approximately 25 ms resulted in profound (by 80-90%) reductions in vagal effects, thus yielding a bimodal vagal phase response curve. Antagonists of M1 (pirenzepine), M2 (methoctramine and gallamine), and M3 (4-DAMP) muscarinic cholinoreceptors produced a reduction in the magnitude of maximal lengthening of cardiac cycle as well as an increase in latency of vagal effects. However, the increment in prolongation of P-P interval induced by a given change in timing of vagal stimulation during cardiac cycle was reduced by M1 and M2 muscarinic receptor blockers, but was unaffected by 4-DAMP. None of the antagonists modified the range of P-Stimulus intervals over which the maximum-to-minimum change of vagal responses occurred. Taken together, these data suggest different contribution of various subtypes of cardiac muscarinic receptors into the negative chronotropic responses induced by brief bursts of vagal stimulation.

Topics: Animals; Cats; Diamines; Electrocardiography; Female; Gallamine Triethiodide; Heart; Heart Rate; Male; Muscarinic Antagonists; Nicotinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Vagus Nerve

1. In cyclophosphamide-induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM-54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2. The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3. Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration-dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 +/- 6%. Pre-incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M(1) receptor selective), methoctramine (M(2)/M(4) receptor selective) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M(3)/M(1)/M(5) receptor selective)), gave rise to a concentration-dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4. Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M(1)-M(5)) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M(2) receptors was decreased, whereas the expression of muscarinic M(3) receptors was increased. 5. In conclusion, the urothelial cell line TBM-54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide-induced cystitis.

Topics: Acrolein; Animals; Blotting, Western; Bufo marinus; Carbachol; Cell Line; Diamines; Dose-Response Relationship, Drug; Extracellular Fluid; Hydrogen-Ion Concentration; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptors, Muscarinic; Urinary Bladder; Urothelium

Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M(1), M(2), M(3) receptors in human bronchial epithelial cell line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-alpha phosphorilation (pIkBalpha) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M(3), M(2) and M(1) receptors with levels of muscarinic M(3) receptor>muscarinic M(1) receptor>muscarinic M(2) receptor. Acetylcholine (10 microM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.

Topics: Acetylcholine; Bronchi; Cell Line, Transformed; Chemotaxis, Leukocyte; Epithelial Cells; Flavonoids; Gallamine Triethiodide; Humans; Indoles; Interleukin-8; Maleimides; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscarinic Antagonists; Neutrophils; NF-kappa B; Nitriles; Piperidines; Pirenzepine; Protein Kinase C; Receptors, Muscarinic; RNA, Messenger; Scopolamine Derivatives; Sulfones; Tiotropium Bromide

The present study is designed to screen the effect of syringin, an active principle purified from the rhizome and root parts of Eleutherococcus senticosus (Araliaceae), on the plasma glucose and investigate the possible mechanisms. Plasma glucose decreased in a dose-dependent manner 60 min after intravenous injection of syringin into fasting Wistar rats. In parallel to the decrease of plasma glucose, increases of plasma insulin level as well as the plasma C-peptide was also observed in rats receiving same treatment. Both the plasma glucose lowering action and the raised plasma levels of insulin and C-peptide induced by syringin were also inhibited by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP), the antagonist of the muscarinic M3 receptors, but not affected by the ganglionic nicotinic antagonist, pentolinium or hexamethonium. Moreover, disruption of synaptic available acetylcholine (ACh) using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished these actions of syringin. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the actions of syringin. Mediation of ACh release from the nerve terminals to enhance insulin secretion by syringin can thus be considered. The results suggest that syringin has an ability to raise the release of ACh from nerve terminals, which in turn to stimulate muscarinic M3 receptors in pancreatic cells and augment the insulin release to result in plasma glucose lowering action.

Topics: Acetylcholine; Animals; Blood Glucose; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Eleutherococcus; Glucosides; Insulin; Insulin Secretion; Male; Muscarinic Antagonists; Phenylpropionates; Phytotherapy; Piperidines; Rats; Receptor, Muscarinic M3

In the presence of neostigmine (0.1 microM), S-isopetasin competitively antagonized cumulative acetylcholine-induced contractions in guinea pig trachealis, because the slope [1.18+/-0.15 (n=6)] of Schild's plot did not significantly differ from unity. The pA2 value of S-isopetasin was calculated to be 4.62+/-0.05 (n=18). The receptor binding assay for muscarinic receptors of cultured human tracheal smooth muscle cells (HTSMCs) was performed using [3H]-N-methylscopolamine ([3H]-NMS). Saturation binding assays were carried out with [3H]-NMS in the presence (non-specific binding) and absence (total binding) of atropine (1 microM). Analysis of the Scatchard plot (y=0.247-1.306x, r2=0.95) revealed that the muscarinic receptor binding sites in cultured HTSMCs constituted a single population (n(H)=1.00). The equilibrium dissociation constant (Kd) and the maximal receptor density (B(max)) for [3H]-NMS binding were 766 pM and 0.189 pmol/mg of protein, respectively. The -logIC50 values of S-isopetasin, methoctramine, and 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) for displacing 0.4 nM [3H]-NMS-specific binding were 5.05, 6.25, and 8.56, respectively, which suggests that [3H]-NMS binding is predominantly on muscarinic M3 receptors of cultured HTSMCs. The inhibitory effects of S-isopetasin on enhanced pause (P(enh)) value were similar to that of ipratropium bromide, a reference drug. The duration of action of S-isopetasin (20 microM), also similar to that of ipratropium bromide (20 microM), was 3 h. In contrast to ipratropium bromide, which non-selectively acts on muscarinic receptors, S-isopetasin preferentially acts on muscarinic M3 receptors. In conclusion, S-isopetasin may be beneficial as a bronchodilator in the treatment of chronic obstructive pulmonary disease and asthma exacerbations.

Topics: Acetylcholine; Animals; Atropine; Binding, Competitive; Bronchial Hyperreactivity; Bronchoconstrictor Agents; Bronchodilator Agents; Cells, Cultured; Cholinesterase Inhibitors; Diamines; Dose-Response Relationship, Drug; Guinea Pigs; Humans; Ipratropium; Male; Muscarinic Antagonists; Muscle, Smooth; N-Methylscopolamine; Neostigmine; Petasites; Piperidines; Protein Binding; Receptor, Muscarinic M3; Sesquiterpenes; Time Factors; Trachea

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.

Topics: Animals; Atropine; Capillary Permeability; Diamines; Female; Ganglionic Blockers; Gastroesophageal Reflux; Guinea Pigs; Hexamethonium; In Vitro Techniques; Male; Muscarinic Antagonists; Nicotinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M4; Receptors, Muscarinic; Receptors, Nicotinic; Regional Blood Flow; Respiratory System

Neuropsychiatric behaviours in Alzheimer's disease (AD) patients have been associated with neocortical alterations of presynaptic cholinergic and muscarinic M2 receptor markers. In contrast, it is unclear whether non-M2 muscarinic receptors have a role to play in AD behavioural symptoms.. To correlate the alterations of neocortical postsynaptic muscarinic receptors with clinical features of AD.. [(3)H]4-DAMP were used in binding assays with lysates of Chinese hamster ovary (CHO) cells stably transfected with M1-M5 receptors. [(3)H]4-DAMP was further used to measure muscarinic receptors in the postmortem orbitofrontal cortex of aged controls and AD patients longitudinally assessed for cognitive decline and behavioural symptoms.. [(3)H]4-DAMP binds to human postmortem brain homogenates and M1-, M3-, M4- and M5-transfected CHO lysates with subnanomolar affinity. Compared to the controls, the [(3)H]4-DAMP binding density is reduced only in AD patients with significant psychotic symptoms. The association between reduced [(3)H]4-DAMP binding and psychosis is independent of the effects of dementia severity or neurofibrillary tangle burden.. This study suggests that the loss of non-M2 muscarinic receptors in the orbitofrontal cortex may be a neurochemical substrate of psychosis in AD and provides a rationale for further development of muscarinic receptor ligands in AD pharmacotherapy.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; CHO Cells; Cohort Studies; Cricetinae; Cricetulus; Female; Humans; Longitudinal Studies; Male; Muscarinic Antagonists; Neocortex; Neurofibrillary Tangles; Neuropsychological Tests; Piperidines; Psychotic Disorders; Radioligand Assay; Receptor, Muscarinic M2; Receptors, Muscarinic; Synapses; Transfection

Functional roles of muscarinic acetylcholine receptors in the regulation of mouse stomach motility were examined using mice genetically lacking muscarinic M(2) receptor and/or M(3) receptor and their corresponding wild-type (WT) mice. Single application of carbachol (1 nM-30 microM) produced concentration-dependent contraction in antral and fundus strips from muscarinic M(2) receptor knockout (M(2)R-KO) and M(3) receptor knockout (M(3)R-KO) mice but not in those from M(2) and M(3) receptors double knockout (M(2)/M(3)R-KO) mice. A comparison of the concentration-response curves with those for WT mice showed a significant decrease in the negative logarithm of EC(50) (pEC(50)) value (M(2)R-KO) or amplitude of maximum contraction (M(3)R-KO) in the muscarinic receptor-deficient mice. The tonic phase of carbachol-induced contraction was decreased in gastric strips from M(3)R-KO mice. Antagonistic affinity for 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) indicated that the contractile responses in M(2)R-KO and M(3)R-KO mice were mediated by muscarinic M(3) and M(2) receptors, respectively. Electrical field stimulation (EFS, 0.5-32 Hz) elicited frequency-dependent contraction in physostigmine- and N(omega)-nitro-L-arginine methylester (l-NAME)-treated fundic and antral strips from M(2)R-KO and M(3)R-KO mice, but the cholinergic contractile components decreased significantly compared with those in WT mice. In gastric strips from M(2)/M(3)R-KO mice, cholinergic contractions elicited by EFS were not observed but atropine-resistant contractions were more conspicuous than those in gastric strips from WT mice. Gastric emptying in WT mice and that in M(2)/M(3)R-KO mice were comparable, suggesting that motor function of the stomach in the KO mice did not differ from that in the WT mice. The results indicate that both muscarinic M(2) and M(3) receptors but not other subtypes mediate carbachol- or EFS-induced contraction in the mouse stomach but that the contribution of each receptor to concentration-response relationships is distinguishable. Although there was impairment of nerve-mediated cholinergic responses in the stomach of KO mice, gastric emptying in KO mice was the same as that in WT mice probably due to the compensatory enhancement of the non-cholinergic contraction pathway.

Topics: Animals; Carbachol; Cholinergic Agonists; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Female; Gastric Emptying; Gastric Fundus; Genotype; In Vitro Techniques; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Physostigmine; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Stomach

This study examines whether M(2) receptors contribute to direct contraction of the detrusor in human neurogenic and idiopathic overactive bladders.. Control detrusor muscle was obtained from patients undergoing cystectomy for bladder cancer, whilst overactive detrusor muscle was obtained from patients undergoing clam cystoplasty for idiopathic or neurogenic detrusor overactivity. The affinities of a range of subtype selective antagonists (DAMP, darifenacin, methoctramine R0-320-6206, and pirenzepine) were obtained in tissue bath experiments by using carbachol as the agonist. These affinity values were then compared with the known affinities for these antagonists at the muscarinic receptor subtypes.. An increased sensitivity to carbachol was observed in both the neurogenic and idiopathic overactive detrusors compared with the control human detrusor. The M(2)-selective antagonists (methoctramine, R0-320-6206) and M(1)-selective antagonist (pirenzepine) had low affinities, whilst the M(3)-selective antagonists (4-DAMP and darifenacin) had high affinities for the human detrusor muscarinic receptor in all three groups of tissues. The affinities (pK(B) values) for the five antagonists were consistent with antagonisms at the M(3) receptor in all three groups; Schild plot analysis indicated an action at this single receptor subtype.. Contraction mediated by muscarinic receptors is enhanced in idiopathic and neurogenic overactive detrusors compared with control detrusor. The direct contractile response to carbachol is mediated by the M(3) receptor in both human normal and overactive bladders, indicating no change in receptor subtype contribution to contraction in the disease state.

Topics: Adult; Aged; Benzofurans; Carbachol; Diamines; Dose-Response Relationship, Drug; Female; Humans; In Vitro Techniques; Male; Middle Aged; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Pyrrolidines; Receptors, Muscarinic; Urinary Bladder; Urinary Bladder, Neurogenic; Urinary Bladder, Overactive

The inotropic response to muscarinic receptor stimulation of isolated chick ventricular myocardium was examined at various developmental stages, and the receptor subtype involved was pharmacologically characterized. In embryonic chick ventricles, carbachol (CCh) produced positive inotropy at micromolar concentrations. In hatched chick ventricles, CCh produced negative inotropy at nanomolar concentrations. Neither positive nor negative inotropy was observed in the 19 - 21-day-old embryos. Both positive and negative inotropy were also observed with acetylcholine and oxotremoline-M. The CCh-induced positive inotropy in 7 - 9-day-old embryonic ventricles and the negative inotropy in 1 - 3-day-old hatched chick ventricles were antagonized by muscarinic receptor antagonists; pA(2) values for the positive and negative responses of pirenzepine were 7.5 and 7.2, those of AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one) were 6.8 and 6.9, those of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were 9.0 and 8.5, and those of himbacine were 7.0 and 8.0, respectively. CCh had no effect on action potential configuration. In conclusion, the positive inotropy is most likely mediated by muscarinic M(1) receptors and the negative inotropy is mostly likely mediated by muscarinic M(4) receptors.

Topics: Action Potentials; Alkaloids; Animals; Carbachol; Chick Embryo; Dose-Response Relationship, Drug; Furans; Heart Ventricles; Myocardial Contraction; Naphthalenes; Piperidines; Pirenzepine; Receptors, Muscarinic

Exposure to a high-carbohydrate (HC) milk formula during the suckling period results in permanent metabolic programming of hyperinsulinemia in HC rats. Previous studies have shown that hyperinsulinemia in HC rats involves a programmed hyperresponsiveness to glucose. However, the immediate onset and persistence of enhanced insulin secretion throughout life suggests a role for numerous factors that control insulin secretion. Present in vivo and in vitro studies have shown a role for altered autonomic activity, including increased parasympathetic and decreased sympathetic activities, in the maintenance of hyperinsulinemia in 100-day-old HC rats. HC rats were shown to be more sensitive to cholinergic-induced potentiation of glucose-stimulated insulin secretion (GSIS) in response to acetylcholine and showed increased sensitivity to blockade of cholinergic-induced insulin secretion by the muscarinic-type 3 receptor-specific antagonist 4-diphenylacetoxy-N-methylpiperidine. In addition, HC rats were less sensitive to adrenergic-induced inhibition of insulin secretion by oxymetazoline, whereas treatment with yohimbine resulted in increased GSIS. Furthermore, HC rats showed greater reductions in plasma insulin levels after vagotomy, as well as an attenuation of yohimbine-induced potentiation of GSIS, suggesting that yohimbine-mediated changes are mediated by parasympathetic activity. Changes in autonomic regulation of GSIS are supported by increased mRNA levels of the parasympathetic signaling molecules muscarinic-type 3 receptor, phospholipase Cbeta1, and protein kinase C-alpha and decreased levels of alpha(2a)-adrenergic receptors in islets from adult HC rats. In conclusion, metabolic programming of hyperinsulinemia throughout adulthood of HC rats involves changes in autonomic activity in response to the HC dietary intervention in the suckling period.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Animals, Newborn; Autonomic Nervous System; Cholinergic Agents; Dietary Carbohydrates; Female; Glucose; Glucose Tolerance Test; Hyperinsulinism; Insulin; Muscarinic Antagonists; Oxymetazoline; Piperidines; Pregnancy; Random Allocation; Rats; Rats, Sprague-Dawley; Vagotomy; Yohimbine

In cyclophosphamide-induced cystitis in the rat, detrusor function is impaired and the expression and effects of muscarinic receptors altered. Whether or not the neuronal transmission may be affected by cystitis was presently investigated. Responses of urinary strip preparations from control and cyclophosphamide-pretreated rats to electrical field stimulation and to agonists were assessed in the absence and presence of muscarinic, adrenergic and purinergic receptor antagonists. Generally, atropine reduced contractions, but in contrast to controls, it also reduced responses to low electrical field stimulation intensity (1-5 Hz) in inflamed preparations. In both types, purinoceptor desensitization with alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) caused further reductions at low frequencies (<10 Hz). The muscarinic receptor antagonists atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) ('M(1)/M(3)/M(5)-selective'), methoctramine ('M(2)-selective') and pirenzepine ('M(1)-selective') antagonized the tonic component of the electrical field stimulation-evoked contractile response more potently than the phasic component. 4-DAMP inhibited the tonic contractions in controls more potently than methoctramine and pirenzepine. In inflamed preparations, the muscarinic receptor antagonism on the phasic component of the electrical field stimulation-evoked contraction was decreased and the pirenzepine and 4-DAMP antagonism on the tonic component was much less efficient than in controls. In contrast to controls, methoctramine increased -- instead of decreased -- the tonic responses at high frequencies. While contractions to carbachol and ATP were the same in inflamed and in control strips when related to a reference potassium response, isoprenaline-induced relaxations were smaller in inflamed strips. Thus, in cystitis substantial changes of the efferent functional responses occur. While postjunctional beta-adrenoceptor-mediated relaxations are reduced, effects by prejunctional inhibitory muscarinic receptors may be increased.

Topics: Adenosine Triphosphate; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Animals; Cyclophosphamide; Cystitis; Diamines; Electric Stimulation; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Parasympatholytics; Phentolamine; Piperidines; Pirenzepine; Propranolol; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Suramin; Sympatholytics; Urinary Bladder

The bed nucleus of stria terminalis (BST) has been reported to be involved in central cardiovascular control in rat. We presently report on the cardiovascular effects of carbachol (CBH) microinjection into the BST as well as on local receptor and peripheral mechanisms involved in their mediation. Microinjection of CBH (0.1 to 3 nmol/100 nL) into the BST of anesthetized rats caused dose-related pressor and bradycardiac responses. The cardiovascular response evoked by 1 nmol of CBH was blocked by local microinjection of the nonselective muscarinic receptor antagonist atropine (3 nmol) or the selective M(2)-muscarinic receptor antagonist 4-DAMP (2 nmol). Microinjection of the selective M(1)-muscarinic receptor antagonist pirenzepine (6 nmol) did not affect cardiovascular responses to CBH, suggesting their mediation by local BST M(2)-muscarinic receptors. Cardiovascular responses to CBH microinjected in the BST were markedly reduced in urethane-anesthetized rats. The pressor response was potentiated by i.v. pretreatment with the ganglion blocker pentolinium (10 mg/kg) and blocked by i.v. pretreatment with the vasopressin antagonist dTyr(CH2)5(Me)AVP (50 microg/kg), suggesting involvement of circulating vasopressin in response mediation. In conclusion, results suggest that microinjection of CBH in the BST activates local M(2)-muscarinic receptor evoking pressor and bradycardiac responses, which are mediated by acute vasopressin release into circulation.

Topics: Animals; Arginine Vasopressin; Atropine; Blood Pressure; Carbachol; Cholinergic Agonists; Dose-Response Relationship, Drug; Drug Interactions; Heart Rate; Male; Microinjections; Muscarinic Antagonists; Nicotinic Antagonists; Pentolinium Tartrate; Piperidines; Rats; Rats, Wistar; Septal Nuclei

To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction.. Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows.. Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (A(max)), and area under the curve (AUC) were evaluated.. Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M(3) antagonists (more commonly for basal tone than for A(max) and AUC). The M(2) receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M(3) receptor antagonists were generally more potent than the M(2) receptor antagonists. In a protection experiment, an M(3) receptor antagonist was less potent than when used in combination with an M(2) receptor antagonist. Receptor antagonists for M(1) and M(4) did not affect contractility variables.. Bethanechol acting on muscarinic receptor sub-types M(2) and M(3) may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows.

Topics: Animals; Bethanechol; Cattle; Dairying; Dose-Response Relationship, Drug; Duodenum; Female; In Vitro Techniques; Jejunum; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Tropicamide

Verrucotoxin is the major component of venom from the stonefish (Synanceia verrucosa). Stings from the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system, occasionally leading to death. It has been reported that verrucotoxin might modulate ATP-sensitive K+ (KATP) current in frog atrial fibers. However, the mechanism by which verrucotoxin acts on KATP current remains unclear. In this study, we examined whether verrucotoxin inhibited KATP current in guinea pig ventricular myocytes, using the patch clamp method. Verrucotoxin suppressed KATP current induced by pinacidil (KATP channel opener) in a concentration-dependent manner, with a half maximum concentration of 16.3 microg/ml. The effect of verrucotoxin on KATP current was suppressed by atropine (1 microM), a muscarinic receptor antagonist, or by 4-diphenylacetoxy-N-methylpiperidine (100 nM), a muscarinic M3 receptor antagonist. Furthermore, the effect of verrucotoxin on KATP current was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (10 microM) and calphostin C (10 microM), yet not by the cAMP-dependent protein kinase (PKA) inhibitor H-89 (0.5 microM). These results suggest that verrucotoxin inhibits KATP current through the muscarinic M3 receptor-PKC pathway. These findings enhance our understanding of the toxic effects of verrucotoxin from the stonefish.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Atropine; Benzophenanthridines; Dose-Response Relationship, Drug; Female; Fish Venoms; Glycoproteins; Guinea Pigs; Heart Ventricles; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Muscarinic Antagonists; Myocytes, Cardiac; Patch-Clamp Techniques; Pinacidil; Piperidines; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Protein Kinase Inhibitors; Receptor, Muscarinic M3; Signal Transduction; Time Factors

Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB(1) cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M(3) muscarinic acetylcholine (ACh) receptors. N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB(1) antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB(1) receptor agonist, mimics M(3) activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action-potential-evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP-dependent protein kinase. 2-Arachidonoylglycerol (2-AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of 2-AG, significantly limit muscarine-induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby 2-AG mediates the muscarine-induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide prevents synaptic inhibition induced by 20 min of 1-Hz stimulation.

Topics: Animals; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Inhibitory Postsynaptic Potentials; Lizards; Models, Biological; Morpholines; Muscarine; Muscarinic Agonists; Neuromuscular Junction; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1

As inhibitors of acetylcholinesterase, clinical presentations of poisoning from organophosphate compounds are generally believed to entail overstimulation by the accumulated acetylcholine on muscarinic receptors at peripheral and central synapses. That some patients still yielded to acute organophosphate poisoning despite repeated dosing of atropine suggests that cellular mechanisms that are independent of muscarinic receptor activation may also be engaged in organophosphate poisoning. The present study was undertaken to test the hypothesis that muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in rostral ventrolateral medulla (RVLM), a medullary site where sympathetic vasomotor tone originates and where the organophosphate poison mevinphos (Mev) acts, is involved in the cardiovascular responses exhibited during organophosphate intoxication. In Sprague-Dawley rats, microinjection bilaterally of Mev (10 nmol) into the RVLM significantly augmented PKA activity in ventrolateral medulla that was not antagonized by coadministration of an equimolar concentration (1 nmol) of atropine or selective muscarinic receptor type M1 (pirenzepine), M2 (methoctramine), M3 (4-diphenyl-acetoxy-N-dimethylpiperidinium), or M4 (tropicamide) inhibitor. Comicroinjection of two selective PKA antagonists (100 pmol), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolol[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-1][1,6]benzodiazocine-10-carboxylic acid, significantly blunted the initial sympathoexcitatory cardiovascular response and the accompanying augmentation of nitric oxide synthase (NOS I) expression in the ventrolateral medulla exhibited during Mev intoxication; the secondary sympathoinhibitory phase and associated elevation in NOS II expression were unaffected. We conclude that whereas a muscarinic receptor-independent augmentation of PKA activity in the ventrolateral medulla was manifested throughout acute Mev intoxication, this activation was preferentially involved in the sympathoexcitatory phase by an upregulation of NOS I expression.

Topics: Animals; Atropine; Blotting, Western; Cardiovascular System; Cholinesterase Inhibitors; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Intracellular Signaling Peptides and Proteins; Male; Medulla Oblongata; Mevinphos; Muscarinic Antagonists; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Time Factors; Tropicamide

Administration of abdominal radiotherapy results in small intestinal motor dysfunction. We have developed a rat radiation enteritis model that, after exposure in vivo, shows high-amplitude, long-duration (HALD) pressure waves in ex vivo ileal segments. These resemble in vivo dysmotility where giant contractions migrate both antegradely and retrogradely. Mediation of these motor patterns is unclear, although enteric neural components are implicated. After the induction of acute radiation enteritis in vivo, ileal segments were isolated and arterially perfused. TTX, hexamethonium, atropine, or the selective muscarinic antagonists pirenzepine (M(1)), methoctramine (M(2)), and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; M(3)) were added to the perfusate. The baseline mean rate per minute per channel of HALD pressure waves was 0.35 +/- 0.047. This was significantly reduced by TTX (83.3%, P < 0.01), hexamethonium (90.3%, P < 0.03), and atropine (98.4%, P < 0.01). The HALD pressure wave mean rate per minute per channel was significantly reduced by pirenzepine (81.1%, P < 0.03), methoctramine (96.8%, P < 0.001), and 4-DAMP (93.1%, P < 0.03) compared with predrug baseline data. As an indicator of normal motility patterns, the frequency of low-amplitude, short-duration pressure waves was also assessed. The mean rate per minute per channel of 5.15 +/- 0.98 was significantly increased by TTX (19%, P < 0.05) but significantly reduced by pirenzepine (35.1%, P < 0.02) and methoctramine (75%, P < 0.0003). However, the rate of small-amplitude pressure waves was not affected by hexamethonium, atropine, or the M(3) antagonist 4-DAMP. The data indicate a role for neuronal mechanisms and the specific involvement of cholinergic receptors in generating dysmotility in acute radiation enteritis. The effect of selective M(3) receptor antagonism suggests that M(3) receptors may provide specific therapeutic targets in acute radiation enteritis.

Topics: Animals; Diamines; Enteritis; Gastrointestinal Motility; Ileum; Male; Manometry; Piperidines; Pirenzepine; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic

Human and animal studies have shown that toluene can cause hearing loss. In the rat, the outer hair cells are first disrupted by the ototoxicant. Because of their particular sensitivity to toluene, the cochlear microphonic potential (CMP) was used for monitoring the cochlea activity of anesthetized rats exposed to both noise (band noise centered at 4 kHz) and toluene. In the present experiment, the conditions were specifically designed to study the toluene effects on CMP and not those of its metabolites. To this end, 100-microL injections of a vehicle containing different concentrations of solvent were made into the carotid artery connected to the tested cochlea. Interestingly, an injection of 116.2-mM toluene dramatically increased in the CMP amplitude (approximately 4 dB) in response to an 85-dB SPL noise. Moreover, the rise in CMP magnitude was intensity dependent at this concentration suggesting that toluene could inhibit the auditory efferent system involved in the inner-ear or/and middle-ear acoustic reflexes. Because acetylcholine is the neurotransmitter mediated by the auditory efferent bundles, injections of antagonists of cholinergic receptors (AchRs) such as atropine, 4-diphenylacetoxy-N-methylpiperidine-methiodide (mAchR antagonist) and dihydro-beta-erythroidine (nAchR antagonist) were also tested in this investigation. They all provoked rises in CMP having amplitudes as large as those obtained with toluene. The results showed for the first time in an in vivo study that toluene mimics the effects of AchR antagonists. It is likely that toluene might modify the response of protective acoustic reflexes.

Topics: Acoustic Stimulation; Animals; Atropine; Cholinergic Antagonists; Cochlea; Cochlear Microphonic Potentials; Dihydro-beta-Erythroidine; Dose-Response Relationship, Drug; Gentamicins; Neurons, Efferent; Noise; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Reflex, Acoustic; Solvents; Toluene

To analyse pressure changes induced by muscarinic agonists on the isolated bladder in order to examine whether there are different responses representing different components of a motor/sensory system within the bladder wall.. Whole isolated bladders from 19 female guinea-pigs (280-400 g) were used. A cannula was inserted into the urethra to monitor intravesical pressure and the bladder was suspended in a heated chamber containing carboxygenated physiological solution at 33-36 degrees C. Initially, the responses to the cholinergic agonists, arecaidine but-2-ynyl ester tosylate and carbachol were assessed. Then, in an attempt to identify the muscarinic receptor subtypes involved, the effects of selective muscarinic antagonists on the arecaidine-induced bladder responses were assessed. The antagonists used were the relatively M(3)-selective 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and darifenicin, and relatively M(2)-selective AFDX-116. All drugs were added to the solution bathing the ablumenal surface of the bladder.. The whole bladders exposed to cholinergic agonists respond with complex changes in intravesical pressure. Immediately after application of the agonist there was a burst of high frequency transient contractions. During continued application of agonist the frequency of the transients decreased and their amplitude increased. Thus, there appear to be two components to the response: an initial fast phase and a later slow component. The maximum frequency of the initial burst increased with increasing concentrations of agonist. By contrast, the frequency of the transients in the steady state showed little dependence on agonist concentration. There were quantitative differences between the responses to arecaidine and carbachol. Arecaidine was less effective in generating the initial burst of high-frequency activity and the transients were significantly larger. At low dose, arecaidine was more effective in producing the large transients in the steady state. Pre-exposure of the bladder to 4-DAMP (0.1-10 nM) or darifenicin (0.1-10 nM) significantly reduced the frequency of the initial burst of activity; 0.3 nM 4-DAMP reduced the frequency by half. In this concentration range, 4-DAMP reduced the amplitude of the initial transients but did not affect the frequency of the transients in the steady state. There were similar results with darifenicin. However, darifenicin was less effective in reducing the amplitude of the initial transients. By contrast, ADFX-116 had little effect on the frequency of the initial transients but did reduce amplitude; 300 nM AFDX-116 was needed to reduce the frequency of the initial burst by half.. This analysis suggests that there are different but interrelated mechanisms in the isolated bladder contributing to complex contractile activity. Three components can be identified: a mechanism operating during voiding to produce a global contraction of the whole bladder and two mechanisms, pacemaker and conductive, involved in generating and propagating local contractions in the bladder wall. The pacemaker component is more sensitive to darifenicin and 4-DAMP than to AFDX-116 suggesting that the underlying processes rely predominantly on M(3) receptors and less so on M(2) (M(3) > M(2)). The phasic activity in the later stages is less affected by M(3) antagonists and might therefore involve predominantly M(2) receptors (M(2) > M(3)). The potential importance of these results in terms of the general physiology and pharmacology of the bladder is discussed.

Topics: Animals; Arecoline; Benzofurans; Carbachol; Cholinergic Agonists; Female; Guinea Pigs; Muscarinic Agonists; Piperidines; Pyrrolidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Urinary Bladder; Urination

The presence of circulating antibodies from primary Sjögren Syndrome (pSS) patients enable to interact with rat cerebral frontal cortex by activating muscarinic acetylcholine receptors (mAChR). ELISA assay for PGE2 generation, nitric oxide synthase (NOS) activity was measured in cerebral frontal cortex slices by production of [U-14C]-citruline and mRNA isolation/quantitative PCR for COX-1 and COX-2 gene expression were carried out. By ELISA assay, it was shown that IgG from pSS patients reacted to cerebral frontal cortex cell surface and with human M1 and M3 mAChR. Beside pSS IgG displayed an agonistic-like activity stimulating NOS activity and PGE2 production associated with an increased COX-1 mRNA gene expression, without affecting COX-2 mRNA levels. Inhibition of phospholipase A2 (PLA2) and NOS prevented pSS IgG effects upon both PGE2 production and COX-1 mRNA levels. The results support the notion that serum IgG auto antibodies in pSS patients target cerebral mAChR may have pathogenic role in immune neuroinflammation and on cognitive dysfunction present in pSS patients.

Topics: Adult; Animals; Autoantibodies; Cerebral Cortex; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Gene Expression; Humans; Immunoglobulin G; Male; Membrane Proteins; Middle Aged; Muscarinic Antagonists; Nitric Oxide; Nitric Oxide Synthase; Phospholipase A2 Inhibitors; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Sjogren's Syndrome

Recently, a cholinergic neurogenic component of contraction has been characterised in the aganglionic mouse vas deferens. In this paper, a cholinergic component of contraction in the guinea-pig vas deferens is characterised pharmacologically. A residual, tetrodotoxin-sensitive (TTX, 0.3 microM), neurogenic contraction was revealed after prolonged exposure (5 h) to the adrenergic neurone blocker bretylium (20 microM) or in the presence of prazosin (100 nM) and alpha,beta-methylene ATP (1 microM), a purinergic agonist which desensitizes P2X receptors. The bretylium-resistant component was potentiated by the acetylcholinesterase (AChE) inhibitor neostigmine (10 microM) and inhibited by the muscarinic-receptor (mAChR) antagonist cyclopentolate (1 microM). Nicotine (30 microM) enhanced the bretylium-resistant component. Neostigmine increased the second component of contraction in the presence of prazosin and alpha,beta-methylene ATP, whilst yohimbine (1 microM), an alpha(2) adrenergic receptor antagonist, enhanced both the first and second components of the electrically evoked contraction. These enhanced contractions were blocked by cyclopentolate in both cases. Nicotine enhanced the cholinergic component of contraction revealed by neostigmine but failed to have any detectable effects in the presence of cyclopentolate. Neostigmine alone increased the slow component of contraction which was reversed by cyclopentolate to control levels. The M(3) receptor-antagonist 4-DAMP (10 nM) markedly inhibited the cholinergic component of contraction to a level comparable with cyclopentolate. Laser microscopy has shown that neostigmine also increased the frequency of spontaneous Ca(2+) transients remaining in smooth muscle cells after perfusion with prazosin and alpha,beta-methylene ATP, an effect blocked by 4-DAMP. These experimental data show that there is a functional cholinergic innervation in the guinea-pig vas deferens whose action is limited by acetylcholinesterase, blocked by cyclopentolate and mediated through M3 receptors. Moreover, by blocking the cholinesterase, the increased amount of ACh generates spontaneous Ca(2+) transients in smooth muscle cells.

Topics: Acetylcholine; Adrenergic alpha-Antagonists; Animals; Bretylium Compounds; Calcium; Cholinesterase Inhibitors; Cyclopentolate; Guinea Pigs; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Myocytes, Smooth Muscle; Neostigmine; Nicotine; Nicotinic Agonists; Piperidines; Receptors, Muscarinic; Vas Deferens; Yohimbine

We studied the role of M3 cholinergic receptors in the regulation of cardiac activity in rats during early postnatal ontogeny in vivo. Blockade of M3 cholinergic receptors in 20-week-old animals increased heart rate and decreased blood pressure. In rats aging 8, 6, and 3 weeks, blockade of M3 cholinergic receptors had little effect on R-R interval, but unexpectedly increased it in 1-week-old animals. It can be hypothesized that tonic inhibitory effect of the vagus nerve in adult rats is realized through M3 cholinergic receptors of the heart. The decrease in heart rate during blockade of M3 cholinergic receptors in 1-week-old rats was probably related to specific innervation of the heart in animals of this age.

Topics: Animals; Blood Pressure; Cholinergic Fibers; Heart; Heart Rate; Muscarinic Antagonists; Piperidines; Rats; Receptor, Muscarinic M3; Vagus Nerve

Gastric peristaltic contractions are driven by electrical slow waves modulated by neural and humoral inputs. Excitatory neural input comes primarily from cholinergic motor neurons, but ACh causes depolarization and chronotropic effects that might disrupt the normal proximal-to-distal spread of gastric slow waves. We used intracellular electrical recording techniques to study cholinergic responses in stomach tissues from wild-type and W/W(V) mice. Electrical field stimulation (5 Hz) enhanced slow-wave frequency. These effects were abolished by atropine and the muscarinic M(3)-receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. ACh released from nerves did not depolarize antral muscles. At higher rates of stimulation (10 Hz), chronotropic effects were mediated by ACh and a noncholinergic transmitter and blocked by muscarinic antagonists and neurokinin (NK(1) and NK(2))-receptor antagonists. Neostigmine enhanced slow-wave frequency, suggesting that the frequency of antral pacemakers is kept low by efficient metabolism of ACh. Neostigmine had no effect on slow-wave frequency in muscles of W/W(v) mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). These muscles also showed no significant chronotropic response to 5-Hz electrical field stimulation or the cholinergic agonist carbachol. The data suggest that the chronotropic effects of cholinergic nerve stimulation occur via ICC-IM in the murine stomach. The capacity of gastric muscles to metabolize ACh released from enteric motor neurons contributes to the maintenance of the proximal-to-distal slow-wave frequency gradient in the murine stomach. ICC-IM play a critical role in neural regulation of gastric motility, and ICC-IM become the dominant pacemaker cells during sustained cholinergic drive.

Topics: Acetylcholine; Androstenes; Animals; Atropine; Benzamides; Benzimidazoles; Carbachol; Electric Stimulation; Electrophysiology; Enteric Nervous System; Female; Gastric Emptying; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Motor Neurons; Muscle Contraction; Myenteric Plexus; Neostigmine; Piperidines; Pyloric Antrum; Tetrodotoxin

In mouse intestinal smooth muscle cells held at -50 mV, carbachol evoked an atropine-sensitive inward current in the intracellular presence of Cs(+). The current response consisted of an initial peak followed by a smaller plateau component on which oscillatory currents frequently arose. Results from various experimental procedures indicated that the inward current is a muscarinic receptor-operated cationic current (mI(cat)) sensitive to cytosolic Ca(2+) concentration ([Ca(2+)](i)) and that the initial peak and oscillatory components are contaminated by Ca(2+)-activated Cl(-) currents. Under conditions of [Ca(2+)](i) buffered to 100 nM, the mI(cat) response to cumulative carbachol applications was inhibited competitively by an M(2)-selective antagonist but non-competitively by an M(3)-selective one. Also it was severely reduced by pertussis toxin (PTX) treatment or a phospholipase C (PLC) inhibitor. Comparative analysis of mI(cat) in mouse and guinea-pig intestinal myocytes indicated that the underlying channels resemble between those myocytes in agonist sensitivity, current-voltage relationship, and unitary conductance. The results suggest that in mouse intestinal myocytes, mI(cat) arises mainly via an M(2)/M(3) synergistic mechanism involving PTX-sensitive G-proteins and PLC activity in the absence of current modulation by [Ca(2+)](i) changes, as described for guinea-pig ileal mI(cat). The channels underlying mI(cat) are also indistinguishable in gating properties between both types of myocytes.

Topics: Animals; Carbachol; Cations; Cholinergic Agonists; Diamines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Guinea Pigs; In Vitro Techniques; Intestine, Small; Ion Channel Gating; Ion Channels; Male; Membrane Potentials; Mice; Muscarinic Antagonists; Myocytes, Smooth Muscle; Pertussis Toxin; Piperidines; Pyrrolidinones; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Type C Phospholipases

In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in bladders from 1- to 2-wk-old Sprague-Dawley rats were not affected by M2 or M3 receptor antagonists. However, administration of 0.5 microM physostigmine, an anticholinesterase agent that increases the levels of endogenous acetylcholine, or 50-100 nM carbachol, a cholinergic agonist at low concentrations, which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M2 receptors with 0.1 microM AF-DX 116 or 1 microM methoctramine or blockade of M3 receptors with 50 nM 4-diphenylacetoxy-N-methylpiperidine methiodide or 0.1 microM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP mustard) reversed the physostigmine and carbachol responses. M2 and M3 receptor blockade did not alter the facilitation of spontaneous contractions induced by 10 nM BAY K 8644, an L-type Ca2+ channel opener, or 0.1 microM iberiotoxin, a large-conductance Ca2+-activated K+ channel blocker. NS-1619 (30 microM), a large-conductance Ca2+-activated K+ channel opener, decreased carbachol-augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of M2 and M3 receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Animals, Newborn; Calcium Channel Agonists; Carbachol; Cholinergic Agonists; Cholinesterase Inhibitors; Diamines; Electric Stimulation; Muscle Contraction; Muscle Hypertonia; Muscle, Smooth; Parasympatholytics; Peptides; Physostigmine; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Urinary Bladder

It has been shown that mature neurons in adult vertebrates can co-express glutamate and acetylcholine. Furthermore, interactions at the synaptic level have been demonstrated. In a previous study we found that also motoneurons at early embryonic stages, thus well prior to synapse formation, release acetylcholine, and that glutamate increases this release. We now report the existence of a glutamate release from embryonic motoneurons and the increase of glutamate release by acetylcholine. This effect is mediated by nicotinic and muscarinic cholinergic receptors present on embryonic motoneurons. Using conditions of partial or total depletion of calcium, we show that the glutamate release has two components: one is calcium-dependent and the other calcium-independent. Furthermore, we show that extracellular glutamate can be taken up by motoneurons, probably via the neuronal glutamate transporter EAAC1, which we find to be expressed at this stage. Monitoring of the glutamate release kinetics showed that extracellular glutamate concentration reached a steady-state level, strongly suggesting the establishment of equilibrium between glutamate release and uptake. Altogether, these results support the idea that glutamate can act as a neurotransmitter in embryonic motoneurons. We hypothesise that, glutamate acts as a regulator of motoneuron maturation and spinal cord development.

Topics: Acetylcholine; Animals; Buffers; Calcium; Excitatory Amino Acid Transporter 3; Female; Glutamic Acid; Immunohistochemistry; Motor Neurons; Piperidines; Pregnancy; Rats; Rats, Wistar; Receptors, Muscarinic; Receptors, Nicotinic; Spinal Cord

1 In the bovine ciliary muscle, stimulation of muscarinic receptors with carbachol (CCh) opens two types of non-selective cation channels (NSCCS and NSCCL) with widely different unitary conductances (100 fS and 35 pS). Here we examined the dependence of the activity of NSCCS on the agonist (CCh) concentration by whole-cell voltage clamp in freshly isolated bovine ciliary muscle cells. We also examined the sensitivity of CCh-evoked NSCCS currents to several muscarinic receptor antagonists. 2 The voltage clamp experiments were carried out using Ba2+ as the charge carrier, as this divalent cation is the most permeant for NSCCS of the alkali and alkaline earth metal ions hitherto examined, whereas it is relatively impermeant to NSCCL. For the dose-activation relationship obtained, the apparent dissociation constant K was estimated to be 0.5 +/- 0.2 microm (n = 31), a value of an order of magnitude smaller than the one reported for CCh-evoked NSCCL currents in our previous experiments. 3 In the dose-inhibition experiments we observed that the CCh-evoked NSCCS currents were inhibited by the muscarinic antagonists with the following potency sequence: atropine approximately 4-DAMP >> pirenzepine > AF-DX116, indicating that the activation of NSCCS by CCh is mediated by an M3 muscarinic receptor. 4 We have previously shown by reverse transcriptase-polymerase chain reaction that the bovine ciliary muscle contains mRNAs for several transient receptor potential channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6) which are attracting attention as molecular candidates for receptor-operated NSCCs. In the present experiments, we succeeded in visually identifying these TRPCs in the plasma membrane of cultured bovine ciliary muscle cells by immunofluorescence microscopy.

Topics: Animals; Atropine; Barium; Carbachol; Cattle; Cells, Cultured; Ciliary Body; Dose-Response Relationship, Drug; Membrane Potentials; Microscopy, Fluorescence; Muscarinic Agonists; Muscarinic Antagonists; Muscle Cells; Patch-Clamp Techniques; Piperidines; Pirenzepine; Receptor, Muscarinic M3; TRPC Cation Channels

Propiverine is a commonly used antimuscarinic drug used as therapy for symptoms of an overactive bladder. Propiverine is extensively biotransformed into several metabolites that could contribute to its spasmolytic action. In fact, three propiverine metabolites (M-5, M-6 and M-14) have been shown to affect various detrusor functions, including contractile responses and L-type calcium-currents, in humans, pigs and mice, albeit with different potency. The aim of our study was to provide experimental evidence for the relationship between the binding of propiverine and its metabolites to human muscarinic receptor subtypes (hM(1)-hM(5)) expressed in chinese hamster ovary cells, and to examine the effects of these compounds on muscarinic receptor-mediated detrusor function. Propiverine, M-5, M-6 and M-14 bound to hM(1)-hM(5) receptors with the same order of affinity for all five subtypes: M-6 > propiverine > M-14 > M-5. In HEK-293 cells expressing hM(3), carbachol-induced release of intracellular Ca(2+) ([Ca(2+)](i)) was suppressed by propiverine and its metabolites; the respective concentration-response curves for carbachol-induced Ca(2+)-responses were shifted to the right. At higher concentrations, propiverine and M-14, but not M-5 and M-6, directly elevated [Ca(2+)](i). These results were confirmed for propiverine in human detrusor smooth muscle cells (hDSMC). Propiverine and the three metabolites decreased detrusor contractions evoked by electric field stimulation in a concentration-dependent manner, the order of potency being the same as the order of binding affinity. We conclude that, in comparison with the parent compound, loss of the aliphatic side chain in propiverine metabolites is associated with higher binding affinity to hM(1)-hM(5) receptors and higher functional potency. Change from a tertiary to a secondary amine (M-14) results in lower binding affinity and reduced potency. Oxidation of the nitrogen (M-5) further lowers binding affinity as well as functional potency.

Topics: Animals; Benzilates; Binding, Competitive; Calcium; Carbachol; Cell Line; Cells, Cultured; CHO Cells; Cholinergic Antagonists; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Electric Stimulation; Epithelial Cells; Humans; Molecular Structure; Muscle Contraction; Muscle, Smooth; N-Methylscopolamine; Parasympatholytics; Piperidines; Potassium Chloride; Receptors, Muscarinic; Urinary Bladder

Cardiac activity and blood pressure in adult rats were recorded during selective blockade of cholinoceptors. Blockade of muscarinic M1 and M2-cholinoceptors had little effect on cardiac activity. Blockade of muscarinic M3-cholinoceptors was followed by heart acceleration. The data suggest that the tonic inhibitory influence of the vagus nerve is mediated via cardiac muscarinic M3-cholinoceptors. Electrostimulation of the right vagus nerve during selective blockade of various subtypes of muscarinic cholinoceptors was followed by the decrease in heart rate. Our results indicate that muscarinic cholinoceptors play a role in the immediate inhibition of cardiac activity upon vagus nerve stimulation.

Topics: Animals; Blood Pressure; Electric Stimulation; Electrocardiography; Gallamine Triethiodide; Heart Rate; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Time Factors; Vagus Nerve

We investigated the ability of the muscarinic antagonist p-fluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea. We also investigated the ability of p-fluorohexahydrosiladifenidol to affect the inhibition of contraction caused by the known competitive muscarinic antagonist atropine. Using resultant analysis to analyze this interaction, we found that the true dissociation constant of p-fluorohexahydrosiladifenidol for competitively antagonizing oxotremorine-M-induced contractions in the ileum exhibited significantly lower potency than when calculated assuming a simple competitive model. In contrast, resultant analysis showed little difference between the true and observed potencies of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced contractions in the trachea. Using a simple competitive model, we found little difference in the observed dissociation constant of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced phosphoinositide hydrolysis in guinea pig ileum and bovine trachea. We also noted that p-fluorohexahydrosiladifenidol (0.3-1.0 microM) moderately inhibited histamine-induced contractions of ileum but not of trachea. Our results suggest that p-fluorohexahydrosiladifenidol does not discriminate markedly between M(3) muscarinic receptors in the ileum and trachea and that it may posses a more potent, nonmuscarinic inhibitory effect on contraction in the ileum.

Topics: Animals; Atropine; Cattle; Guinea Pigs; Histamine Antagonists; Hydrolysis; Ileum; In Vitro Techniques; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Oxotremorine; Phosphatidylinositols; Piperidines; Receptor, Muscarinic M3; Trachea

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.

Topics: Acetylcholine; Animals; Cats; Electrophysiology; Esophagus; Membrane Potentials; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Pirenzepine; Potassium Channels; Receptor, Muscarinic M3

In this paper we have determined the different signal pathways involved in M(1) and M(3) muscarinic acetylcholine receptor (mAChR) dependent stimulation of cyclo-oxygenase 1 (cox-1) mRNA gene expression and PGE(2) production on rat cerebral frontal cortex. Carbachol stimulation of M(1) and M(3) mAChR exerts an increase in cox-1 mRNA gene expression without affecting cox-2 mRNA expression and increased PGE(2) generation. Besides, increased phosphoinositide (PI) turnover and stimulation of nitric oxide synthase (NOS) and cyclic GMP (cGMP) production. Inhibitors of phospholipase A(2) (PLA(2)), COX and phospholipase C (PLC), calcium/calmodulin (CaM), NOS and soluble guanylate cyclase prevent the carbachol effect. These results suggest that carbachol-activation of M(1) and M(3) mAChR increased PGE(2) release associated with an increased expression of cox-1 and NO-cGMP production. The mechanism appears to occur directly to PLC stimulation and indirectly to PLA(2) activation. These results may contribute to understand the effects and side effect of non-steroidal anti-inflammatory drugs in patients with cerebral degenerative diseases.

Topics: Animals; Carbachol; Cholinergic Agonists; Cyclic GMP; Cyclooxygenase 1; Dinoprostone; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Frontal Lobe; Gene Expression Regulation; Male; Membrane Proteins; Models, Biological; Muscarinic Antagonists; Nitric Oxide Synthase; Phosphatidylinositols; Piperidines; Pirenzepine; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

To investigate the relationship between M3-R/IK(M3) and arrhythmia in order to find a new target for antiarrhythmic agents.. Using the acute ischemic model of rats and patch-clamp techniques, the effects of the M3 receptor on the occurrence of arrhythmias and its possible mechanisms were studied.. In acute ischemic model of rats, the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine-methiodide (4DAMP) increased the occurrence of arrhythmias, and the M3 receptor agonist choline suppressed the onset and the development of arrhythmias (P < 0. 01). No change was observed after treatment with other receptor antagonists (M1, M2, and M4). With patch-clamp techniques, it was found that choline induced K+ current could be inhibited by 4DAMP. Antagonists toward M1, M2, and M4 receptors all failed to alter the current.. Choline modulates the cellular electrical properties of the heart, probably by activating a K+ current via stimulation of the M3 receptor. M3-R/IK(M3) may act as a new target for antiarrhythmic agents.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Cell Separation; Choline; Guinea Pigs; Heart Ventricles; Male; Myocytes, Cardiac; Piperidines; Rats; Rats, Wistar; Receptor, Muscarinic M3

Topics: Animals; Cats; Electrocardiography; Heart Conduction System; Heart Rate; Myocardial Contraction; Parasympatholytics; Piperidines; Receptors, Cholinergic; Vagus Nerve

The anti-inflammatory effect (AI) induced by peripheral injection of diluted bee venom (dBV) involves activation of spinal cord circuits and is mediated by catecholamine release from adrenal medulla, but the precise neuronal mechanisms involved are not fully understood. In a recent study, we demonstrated that an increase in spinal acetylcholine is involved in mediating the anti-inflammatory effect of dBV and that this mediation also involves adrenomedullary activation. The present study utilized the mouse air pouch inflammation model to evaluate the involvement of spinal acetylcholine receptors and sympathetic preganglionic neurons (SPNs) in dBV's anti-inflammatory effect (dBVAI). Intrathecal (IT) pretreatment with atropine (muscarinic cholinergic antagonist) but not hexamethonium (nicotinic cholinergic antagonist) significantly suppressed dBVAI on zymosan-evoked leukocyte migration. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic receptor type 2; M(2) antagonist), but not pirenzepine (an M(1) antagonist) or 4-DAMP (an M(3) antagonist), suppressed the dBVAI. In addition, dBV stimulation specifically increased Fos expression in SPNs of the T7-T11, but not the T1-T6 or T12-L2 spinal cord segments, in animals with zymosan-induced inflammation. Moreover, IT methoctramine pretreatment suppressed this dBV-induced Fos expression specifically in SPNs of T7-T11 level. Peripheral sympathetic denervation using 6-hydroxydopamine (6-OHDA) treatment (which spares sympathetic adrenal medullary innervation) did not alter dBVAI. Collectively these results indicate that dBV stimulation leads to spinal cord acetylcholine release that in turn acts on spinal M(2) receptors, which via a hypothesized disinhibition mechanism activates SPNs that project to the adrenal medulla. This activation ultimately leads to the release of adrenal catecholamines that contribute to dBVAI.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Atropine; Autonomic Fibers, Preganglionic; Bee Venoms; Catecholamines; Cell Count; Choline O-Acetyltransferase; Disease Models, Animal; Drug Interactions; Exudates and Transudates; Hexamethonium; Immunohistochemistry; Inflammation; Leukocytes; Male; Mice; Mice, Inbred ICR; Muscarinic Antagonists; Neurons; Nicotinic Antagonists; Oncogene Proteins v-fos; Oxidopamine; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Spinal Cord; Sympathectomy, Chemical

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.

Topics: Animals; Carbachol; Cholinergic Agonists; Cyclic GMP; Dose-Response Relationship, Drug; In Vitro Techniques; Inositol Phosphates; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Nitric Oxide Synthase Type I; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Tritium; Tropicamide; Urinary Bladder

1. The aim of the present study was to examine the modulator influence of muscarinic M(2) receptors on responses of rat urinary bladder detrusor muscle evoked by endogenous stimuli, i.e. by stimulation of the bladder innervation. 2. Responses were evoked by electrical field stimulation (EFS; 2-20 Hz, 0.8 ms, 60 V) of isolated strip preparations mounted in organ baths. The tension of the muscle strips was recorded digitally. EFS was performed by applying stimulation with either a short duration (5 s) or a longer duration (to reach peak response; approximately 20 s). 3. Effects of muscarinic receptor antagonists (muscarinic M(1)/M(3) receptor selective: 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP); muscarinic M(2) receptor selective: methoctramine), a beta-adrenergic antagonist (propranolol) and an adenosine receptor antagonist (8-p-sulfophenyltheophylline) were assessed on contractile activity and on poststimulatory relaxations. 4. Low concentrations of methoctramine (10(-8) m) reduced or tended to reduce the EFS-induced contraction, e.g. at 2 Hz by 12% while methoctramine at 10(-7) m had no significant effect. In addition, in the presence of 4-DAMP (10(-9) m), which tended to inhibit contractions at all frequencies (2-20 Hz; -17 to -25%), methoctramine at 10(-8) and 10(-7) m induced a further reduction of the contractile responses (-5 to -10%; 2-20 Hz). 5. The beta-adrenergic receptor antagonist propranolol (10(-6) m) and the adenosine receptor antagonist 8-p-sulfophenyltheophylline (10(-6) m) both increased contractile responses by 9-21% (2-10 Hz, long duration; P < 0.05-0.001) as a consequence of antagonizing relaxatory stimuli. Neither antagonist affected the contractile responses to EFS with the short duration stimulation. Poststimulatory relaxations were reduced by 30-60% (P < 0.05) by propranolol and by 40-60% (P < 0.001) by 8-p-sulfophenyltheophylline, but for 8-p-sulfophenyltheophylline only after stimulation with the short duration. 6. In the presence of methoctramine (10(-7) m), the 8-p-sulfophenyltheophylline-induced increases of the contractile response to long duration EFS were significantly enhanced at 10 Hz (+12 +/- 4%; P < 0.05), whereas no such enhancement of the propranolol inhibitory effect occurred in the presence of methoctramine. However, poststimulatory beta-adrenoceptor-evoked relaxations after short duration EFS were increased by about 35% in the presence of methoctramine, but not those after long duration. 7. Thus,

Topics: Animals; Diamines; Electric Stimulation; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Parasympatholytics; Piperidines; Propranolol; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Theophylline; Time Factors; Urinary Bladder; Vasodilator Agents

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M(2) or M(3) muscarinic receptors, in order to characterize the mechanisms involved in M(2) and M(3) receptor-mediated contractile responses. Single applications of carbachol (0.1-100 microM) produced concentration-dependent contractions in preparations from M(2)-knockout (KO) and M(3)-KO mice, mediated via M(3) and M(2) receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M(2)-preferring antagonist methoctramine (300 nM) or the M(3)-preferring antagonist 4-DAMP (30 nM). The M(2)-mediated contractions were mimicked in shape by submaximal stimulation with high K(+) concentrations (up to 35 mM), almost abolished by voltage-dependent Ca(2+) channel (VDCC) antagonists or depolarization with 140 mM K(+) medium, and greatly reduced by pertussis toxin (PTX) treatment. The M(3)-mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K(+)-depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K(+) depolarization consisted of different components, either sensitive or insensitive to extracellular Ca(2+). The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M(2) receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca(2+) entry, especially associated with action potential discharge, and that the M(3) receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca(2+) entry and intracellular Ca(2+) release. In intact tissues coexpressing M(2) and M(3) receptors, M(2) receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Topics: Animals; Calcium Channel Blockers; Carbachol; Cholinergic Agonists; Diamines; Female; Ileum; In Vitro Techniques; Male; Mice; Mice, Knockout; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Nicardipine; Pertussis Toxin; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3

A recent model of acute incisional pain has been characterized that strongly parallels the postoperative period in patients experiencing evoked pain. In that setting, abundant literature has revealed antihypersensitive effects produced by intrathecally administered alpha2-adrenergic receptor agonists, such as clonidine, in both animals and humans. Recent reports have suggested an obligatory role of spinal acetylcholine receptors in the analgesic action of intrathecal clonidine. The authors sought to determine the involvement of spinal muscarinic and nicotinic receptor subpopulations in the antihypersensitivity effect of intrathecal clonidine in a rodent model for human postoperative pain.. After intrathecal catheterization, rats underwent superficial plantar incision. Clonidine or a combination of clonidine and muscarinic receptor subtype antagonists (M1, M2, M3, and M4) or nicotinic receptor subtype antagonists (alpha4beta2 and alpha7) were intrathecally administered, and withdrawal thresholds to mechanical stimuli were examined.. Spinal clonidine maximally reduced hypersensitivity adjacent to the wound 30 min after its injection. When animals were intrathecally pretreated with the M1 muscarinic antagonist toxin MT-7, the M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine, and the M4 muscarinic antagonist toxin MT-3, clonidine lost its antihypersensitive action. When animals were intrathecally pretreated with the alpha4beta2 nicotinic receptor antagonist dihydro-beta-erythroidine, but not with the alpha7 nicotinic receptor antagonist methyllycaconitine, the antihypersensitivity action of clonidine was abolished.. These data indicate for the first time that the clonidine-induced increase in punctuate mechanical threshold is mediated via the activation of all but M2 muscarinic receptor subtypes, and via the activation of alpha4beta2 but not alpha7 nicotinic receptor subtypes in a rodent model for human postoperative pain.

Topics: Aconitine; Adrenergic alpha-Agonists; Animals; Behavior, Animal; Clonidine; Diamines; Dihydro-beta-Erythroidine; Injections, Spinal; Male; Muscarinic Agonists; Nicotinic Agonists; Pain; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptors, Muscarinic; Receptors, Nicotinic; Spine

The importance of muscarinic receptors in proliferation of different cell types and in insulin secretion from pancreatic beta cells has been extensively studied. However, the role of pancreatic muscarinic receptors during pancreatic regeneration has not yet been studied. For the first time, the functional status of the muscarinic M1 and M3 receptors in regeneration of the pancreas is investigated here. It is observed that the number and affinity of high-affinity muscarinic M3 receptors increased at the time of regeneration. The low-affinity M3 receptors also showed a similar trend. In the case of muscarinic M1 receptors, the receptor number increased with a decrease in affinity. We also observed an increase in the circulating insulin levels at the time of active regeneration. The in vitro studies confirmed that muscarinic receptors are stimulatory to insulin secretion. Our results suggest that the increased muscarinic M1 and M3 receptor subtypes stimulate insulin secretion and islet cell proliferation during the regeneration of pancreas.

Topics: Animals; Atropine; Carbachol; Cholinergic Agonists; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Male; Muscarinic Antagonists; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Regeneration

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Topics: Acetylcholine; Animals; Barium; Calcium Channels, L-Type; Carbazoles; Cells, Cultured; Chromones; Class Ib Phosphatidylinositol 3-Kinase; Diamines; Enzyme Inhibitors; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Indoles; Ion Channel Gating; Ion Transport; Isoenzymes; Male; Morpholines; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthalenes; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperidines; Portal Vein; Protein Kinase C; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Rabbits; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Signal Transduction

Central dopamine neurones are involved in regulating cognitive and motor processes. Most of these neurones are located in the ventral mesencephalon where they receive abundant intrinsic and extrinsic GABAergic input. Cholinergic neurones, originating from mesopontine nuclei, project profusely in the mesencephalon where they preferentially synapse onto local GABAergic neurones. The physiological role of this cholinergic innervation of GABAergic neurones remains to be determined, but these observations raise the hypothesis that ACh may regulate dopamine neurones indirectly through GABAergic interneurones. Using a mesencephalic primary culture model, we studied the impact of cholinergic agonists on mesencephalic GABAergic neurones. ACh increased the frequency of spontaneous IPSCs (151 +/- 49%). Selective activation of muscarinic receptors increased the firing rate of isolated GABAergic neurones by 67 +/- 13%. The enhancement in firing rate was Ca(2+) dependent since inclusion of BAPTA in the pipette blocked it, actually revealing a decrease in firing rate accompanied by membrane hyperpolarization. This inhibitory action was prevented by tertiapin, a blocker of GIRK-type K(+) channels. In addition to its excitatory somatodendritic effect, activation of muscarinic receptors also acted presynaptically, inhibiting the amplitude of unitary GABAergic synaptic currents. Both the enhancement in spontaneous IPSC frequency and presynaptic inhibition were abolished by 4-DAMP (100 nm), a preferential M3 muscarinic receptor antagonist. The presence of M3-like receptors on mesencephalic GABAergic neurones was confirmed by immunocytochemistry. Taken together, these results demonstrate that mesencephalic GABAergic neurones can be regulated directly through muscarinic receptors. Our findings provide new data that should be helpful in better understanding the influence of local GABAergic neurones during cholinergic activation of mesencephalic circuits.

Topics: Acetylcholine; Action Potentials; Animals; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; gamma-Aminobutyric Acid; Mesencephalon; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Neural Inhibition; Neurons; Piperidines; Potassium Channels, Voltage-Gated; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Synapses

Although metoclopramide, often used as an antiemetic, is reported to have an anticholinesterase action, the effect on airway smooth muscle remains unclear. We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M(3) receptors in rat trachea preparations. Male Wistar rats were anesthetized and their tracheas excised and chopped into 3-mm-wide rings, 1-mm-wide slices, or frozen 10- microm-thick sections. Contraction was induced with 0.55 microM carbachol (CCh) and, 30 min later, metoclopramide (10 microM to 1 mM) was added. The slices were incubated with (3)[H]myo-inositol, 0.55 microM CCh, and metoclopramide, and the formation of (3)[H] inositol monophosphate was measured. A radioligand binding study was conducted to examine the effects of metoclopramide using [(3)H] 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), a muscarinic M(3) receptor antagonist, in sections of the trachea. Metoclopramide concentration dependently attenuated CCh-induced contraction and inositol monophosphate accumulation, and also attenuated the binding affinity of 4-DAMP to muscarinic M(3) receptors. The 50% inhibitory concentration of metoclopramide against the binding affinity of 4-DAMP to muscarinic M(3) receptors of rat trachea was 24 micro M. These findings suggest that the attenuation by metoclopramide of CCh-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M(3) receptors.. We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M(3) receptors in rat trachea preparations. Our findings suggest that the attenuation by metoclopramide of carbachol-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M(3) receptors.

Topics: Animals; Antiemetics; Carbachol; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Metoclopramide; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Phosphatidylinositols; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptor, Muscarinic M3; Trachea

The C57BLKS/J db/db mouse develops hyperglycemia and has delayed gastric emptying that is improved with tegaserod, a partial 5-HT4 agonist. Our aims here were to determine regional gastric contractility alterations in C57BLKS/J db/db mice and to determine the effects of serotonin and tegaserod. The contractile effects of bethanechol, serotonin, and tegaserod in fundic, antral, and pyloric circular muscle were compared in C57BLKS/J db/db mice and normal littermates. The effects of tetrodotoxin, atropine, and 5-HT receptor antagonists were studied. Contractions in response to bethanechol were decreased in the fundus, similar in the antrum, but increased in the pylorus in diabetic mice compared with controls. Serotonin and, to a lesser extent, tegaserod caused contractions that were more pronounced in the fundus than in the antrum and pylorus in both diabetic and normal mice. Serotonin-induced contractions were partially inhibited by atropine, the 5-HT4 antagonist GR113808, and the 5-HT2 antagonist cinanseron but not tetrodotoxin. Regional gastric contractility alterations are present in this diabetic gastroparesis mouse model. Fundic contractility was decreased, but pyloric contractility was increased in the pylorus to cholinergic stimulation in diabetic mice. Serotonin's contractile effect is mediated, in part, through muscarinic, 5-HT2, and 5-HT4 receptors. This study suggests that fundic hypomotility and pyloric hypercontractility, rather than antral hypomotility, play important roles for the gastric dysmotility that occurs in diabetes.

Topics: Animals; Bethanechol; Cholinergic Agonists; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Electric Stimulation; Enteric Nervous System; Female; Gastroparesis; In Vitro Techniques; Indoles; Mice; Mice, Inbred C57BL; Muscarinic Agonists; Muscle Contraction; Muscle, Smooth; Piperidines; Potassium; Receptors, Muscarinic; Serotonin; Serotonin Receptor Agonists; Stomach

The effects of anticholinergic drugs selective for muscarinic receptor subtypes on prepulse inhibition of acoustic startle response were determined in mice. The prepulse inhibition is associated with sensorimotor information processing in the brain. The anticholinergic agent scopolamine (0.3 mg/kg, s.c.) significantly attenuated prepulse inhibition, while the drug (1-10 mg/kg, s.c.) had no effects on startle amplitude as an indicator of startle response. The muscarinic M(1) receptor antagonist pirenzepine (0.1-10 microg/mouse, i.c.v.) and the muscarinic M(2) receptor antagonist AF-DX116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (0.1-10 microg/mouse, i.c.v.) had no effects on prepulse inhibition or startle amplitude. The muscarinic M(3) receptor antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxy-piperidinium iodide) (30 microg/mouse, i.c.v.) and the muscarinic M(4) receptor antagonist tropicamide (0.1 microg/mouse, i.c.v.) significantly attenuated prepulse inhibition, while tropicamide (0.01 microg/mouse, i.c.v.) but not 4-DAMP (10 and 30 microg/mouse, i.c.v.) produced a significant increase in startle amplitude. These results suggest that the blockade of muscarinic M(3) and M(4) receptors leads to the disruption of prepulse inhibition.

Topics: Animals; Cholinergic Antagonists; Injections, Intraventricular; Male; Mice; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Receptors, Muscarinic; Reflex, Startle; Scopolamine; Tropicamide

In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents.. The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion.. Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Topics: Adenylyl Cyclase Inhibitors; Animals; Arecoline; Atropine; Carbachol; Colforsin; Cyclic AMP; Cytoplasmic Granules; Light; Muscarinic Agonists; Muscarinic Antagonists; Perciformes; Pigment Epithelium of Eye; Piperidines; Pirenzepine; Quaternary Ammonium Compounds; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M4; Receptors, Muscarinic; Retinal Pigments; Second Messenger Systems; Tropicamide

Acetylcholine (ACh) stimulates ion secretion in the small intestine and colon. The purpose of the present study was to characterize the ACh-induced electrogenic ion transport in human duodenum and determine the muscarinic receptor subtypes functionally involved.. Biopsies from the second part of duodenum were obtained from 28 patients during endoscopy. Biopsies were mounted in modified Ussing chambers with air-suction for measurements of short-circuit current by a previously validated technique. Short-circuit current was measured after application of chloride/bicarbonate transport inhibitors bumetanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), diphenylamine-2-carboxylate (DPC), and acetazolamide. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and two mamba toxins MT3 and MT7 were used to characterize the mAChR receptor subtypes involved. The effects of transport inhibitors and receptor antagonists were measured by comparing two consecutive responses of ACh on short-circuit current in the same biopsy specimen.. Bumetanide and 4-DAMP significantly inhibited ACh-induced short-circuit current, whereas SITS, DPC, acetazolamide, mamba toxin MT3, and mamba toxin MT7 all failed to show any significant effect.. In conclusion, our results indicate that muscarinic receptor subtype M3 acts as the main mediator of bumetanide-sensitive ACh-induced secretion in human duodenum.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Acetylcholine; Bumetanide; Calcium Channel Blockers; Diuretics; Duodenum; Elapid Venoms; Humans; Intercellular Signaling Peptides and Proteins; Ion Transport; Muscarinic Antagonists; Neurotoxins; ortho-Aminobenzoates; Peptides; Piperidines; Receptors, Muscarinic

To explore the effects of M3 receptor on myocyte apoptosis induced by acute myocardial infarction in rats.. Rat model was induced by ligation of the anterior branch of the left coronary artery. All animals were divided into four groups: sham-operated group, occlusion group, choline group (10 mg x kg(-1), iv), and 4DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) group (0.12 mg x kg(-1), iv). The serum malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. The infarct size areas on the myocardium were identified by TTC staining. The apoptosis in cardiomyocyte was detected by TUNEL assay and apoptosis-related proteins in Bcl-2 and Fas expression were measured by immunohistochemistry assay.. M3 receptor agonist choline reduced serum MDA content and increased SOD activity. The myocardial expression of Bcl-2 was increased, whereas the expression of Fas was decreased by choline. However, blockade of M3 receptor by 4DAMP completely inhibited these effects of choline on cardiac myocytes.. Activation of M3 receptor has protective effect on myocyte apoptosis induced by acute myocardial infarction in rat, and this effect might be related to modulating the expression of some immediateearly genes including Bcl-2 and Fas.

Topics: Animals; Apoptosis; Choline; fas Receptor; Male; Malondialdehyde; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Piperidines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Tumor Necrosis Factor; Superoxide Dismutase

Muscarinic M1 and M3 receptor changes in the brain stem during pancreatic regeneration were investigated. Brain stem acetylcholine esterase activity decreased at the time of regeneration. Sympathetic activity also decreased as indicated by the norepinephrine (NE) and epinephrine (EPI) content of adrenals and also in the plasma. Muscarinic M1 and M3 receptors showed reciprocal changes in the brain stem during regeneration. Muscarinic M1 receptor number decreased at time of regeneration without any change in the affinity. High affinity M3 receptors showed an increase in the number. The affinity did not show any change. The number of low affinity receptors decreased with decreased Kd at 72 hours after partial pancreatectomy. The Kd reversed to control value with a reversal of the number of receptors to near control value. Gene expression studies also showed a similar change in the mRNA level of M1 and M3 receptors. These alterations in the muscarinic receptors regulate sympathetic activity and maintain glucose level during pancreatic regeneration. Central muscarinic M1 and M3 receptor subtypes functional balance is suggested to regulate sympathetic and parasympathetic activity, which in turn control the islet cell proliferation and glucose homeostasis.

Topics: Acetylcholinesterase; Adrenal Glands; Animals; Blood Glucose; Body Weight; Brain Stem; Chromatography, High Pressure Liquid; DNA Primers; Electrochemistry; Epinephrine; Insulin; Kinetics; Muscarinic Antagonists; Norepinephrine; Pancreas; Pancreatectomy; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; Thymidine

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.

Topics: Animals; Arecoline; Atropine; Colon; Dose-Response Relationship, Drug; Female; Ganglionic Stimulants; Gastrointestinal Motility; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Piperidines; Rabbits; Receptor, Muscarinic M2; Receptor, Muscarinic M3

This study characterized the functional effects of a novel gastroprokinetic agent, N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1, 3-thiazole-4-carboxyamide monohydrochloride trihydrate (Z338), on the muscarinic M1, M2, and M3 receptors expressed in Xenopus oocytes using the two-electrode voltage clamp method. Z-338 did not produce by itself any currents in oocytes expressing muscarinic M1, M3 receptors or muscarinic M2 receptors/G protein-gated inward rectifying K+ channels (Kir3.1 channels). In oocytes expressing muscarinic M1 receptors, Z-338 inhibited the acetylcholine-induced Ca2+ -activated Cl- current with an IC50 of 1.8 microM. In oocytes expressing muscarinic M2 receptors/Kir3.1 channels, Z-338 inhibited the acetylcholine-induced K+ currents with an IC50 of 10.1 microM, whereas in oocytes expressing muscarinic M3 receptors, Z-338 did not inhibit the acetylcholine-induced Ca2+ -activated Cl- current in a concentration-dependent manner. These results indicate that Z-338 is a potent antagonist not for muscarinic M3 receptor but for both muscarinic M1 and M2 receptors. Thus, Z-338 is a gastrokinetic agent with a unique profile.

Topics: Acetylcholine; Animals; Benzamides; Dose-Response Relationship, Drug; Female; Gastrointestinal Agents; Gene Expression; Humans; Membrane Potentials; Muscarinic Antagonists; Oocytes; Piperidines; Pirenzepine; Potassium; Rats; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Reproducibility of Results; Thiazoles; Xenopus

We examined the involvement of the spinal muscarinic receptors in the clonidine-induced antiallodynic effects. Mechanical sensitivity was assessed by stimulating the hind paw with von Frey filaments. In streptozotocin-treated (200 mg/kg, i.v.) diabetic mice, hypersensitivity to mechanical stimulation appeared 3 days after streptozotocin administration, and persisted for 11 days. This mechanical hypersensitivity (allodynia) was inhibited by the intrathecal (i.t.) injection of clonidine. The muscarinic receptor antagonist atropine (i.t.) and alpha2-adrenoreceptor antagonist yohimbine (i.t. or subcutaneous injection) abolished the antiallodynic effect of clonidine. The effect was mimicked by the muscarinic M1 receptor antagonist pirenzepine, but not by the muscarinic M2 receptor antagonist methoctoramine or the muscarinic M3 receptor antagonist 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine methiodide). In addition, the mechanical hypersensitivity in diabetic mice was reduced by the selective muscarinic M1 receptor agonist McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride) (i.t.). These results suggest that spinal muscarinic M1 receptors participate in the antiallodynic effect of clonidine in diabetic mice.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Analgesics; Animals; Atropine; Blood Glucose; Clonidine; Diabetes Mellitus, Experimental; Diamines; Injections, Spinal; Male; Mice; Motor Activity; Muscarinic Agonists; Muscarinic Antagonists; Pain; Pain Threshold; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Spinal Cord; Streptozocin; Stress, Mechanical; Time Factors

This study was designed to investigate the effects of the cholinesterase inhibitors soman and pyridostigmine bromide (PB) on synaptic transmission in the CA1 field of rat hippocampal slices. Soman (1-100 nM, 10-15 min) decreased the amplitude of GABAergic postsynaptic currents (IPSCs) evoked by stimulation of Schaffer collaterals and recorded from CA1 pyramidal neurons. It also decreased the amplitude and frequency of spontaneous IPSCs recorded from pyramidal neurons. Whereas the maximal effect of soman on evoked GABAergic transmission was observed at 10 nM, full cholinesterase inhibition was induced by 1 nM soman. After 10-15-min exposure of hippocampal slices to 100 nM PB, GABAergic transmission was facilitated and cholinesterase activity was not significantly affected. At nanomolar concentrations, soman and PB have no direct effect on GABA(A) receptors. The effects of soman and PB on GABAergic transmission were inhibited by the m2 receptor antagonist 11-[[[2-diethylamino-O-methyl]-1-piperidinyl] acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6- one (1 nM) and the m3 receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine (100 nM), respectively, and by the nonselective muscarinic receptor antagonist atropine (1 microM). Thus, changes in GABAergic transmission are likely to result from direct interactions of soman and PB with m2 and m3 receptors, respectively, located on GABAergic fibers/neurons synapsing onto the neurons under study. Although the effects of 1 nM soman and 100 nM PB were diametrically opposed, they only canceled one another when PB was applied to the neurons before soman. Therefore, PB, acting via m3 receptors, can effectively counteract effects arising from the interactions of soman with m2 receptors in the brain.

Topics: Animals; Atropine; Central Nervous System; Cholinesterase Inhibitors; Cholinesterases; Electric Stimulation; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Membrane Potentials; Muscarinic Antagonists; Neurons; Patch-Clamp Techniques; Piperidines; Pirenzepine; Pyramidal Cells; Pyridostigmine Bromide; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Receptors, Nicotinic; Soman; Synaptic Transmission

Previously, we demonstrated that the rostral part of the ventral zone of the lateral septal area (LSV) was involved in the restraint stress-induced pressor response. It is suggested that there exist acetylcholine receptors responsible for blood pressure increase in the caudal part of the lateral septal area. In this study, we examined whether acetylcholine receptors responsible for pressor responses also exist in the rostral part of the LSV and whether these acetylcholine receptors are involved in the stress-induced pressor response in rats. Microinjection of either carbachol (10-100pmol) or physostigmine (0.46 and 1.5nmol) into the LSV caused a dose-dependent increase in blood pressure. The pressor response to carbachol (30pmol) was inhibited by the M1 antagonist pirenzepine and the M3 antagonist 4-DAMP mustard but not by the M2 antagonist methoctramine injected into the LSV. Bilateral microinjections of the M1/M3 antagonist 4-DAMP (1nmol) inhibited the restraint stress-induced pressor response. These findings suggest that M1/M3 muscarinic receptors responsible for blood pressure increase exist in the rostral part of the LSV and they are partly involved in the stress-induced pressor response.

Topics: Animals; Blood Pressure; Carbachol; Cholinergic Agonists; Cholinesterase Inhibitors; Epinephrine; GABA Agonists; Injections, Intraventricular; Male; Muscarinic Antagonists; Muscimol; Norepinephrine; Physostigmine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Restraint, Physical; Septal Nuclei; Stress, Physiological

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [(3)H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M(3)-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 microM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1-10 microM), but only in cells having functional muscarinic M(3)-receptors. This synergism could be suppressed significantly by the selective M(3)-receptor antagonists DAU 5884 (0.1 microM) and 4-DAMP (10 nM), but not at all by the M(2)-subtype selective antagonist gallamine (10 microM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M(3)-receptors in bovine tracheal smooth muscle cells.

Topics: Animals; Cattle; Cells, Cultured; Drug Synergism; Inositol Phosphates; Methacholine Chloride; Mitosis; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Platelet-Derived Growth Factor; Receptor, Muscarinic M3; Receptors, Muscarinic; Thymidine; Trachea

This study examined the upstream signaling pathways initiated by muscarinic m2 and m3 receptors that mediate sustained ERK1/2- and p38 MAP kinase-dependent phosphorylation and activation of the 85-kDa cytosolic phospholipase (cPL)A(2) in smooth muscle. The pathway initiated by m2 receptors involved sequential activation of Gbetagamma(i3), phosphatidylinositol (PI)3-kinase, Cdc42, and Rac1, p21-activated kinase (PAK1), p38 mitogen-activated protein (MAP) kinase, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (61 +/- 5 to 72 +/- 4%) by the m2 antagonist methoctramine, Gbeta antibody, pertussis toxin, the PI3-kinase inhibitor LY 294002, PAK1 antibody, the p38 MAP kinase inhibitor SB-203580, and a Cdc42/Rac1 GEF (Vav2) antibody and by coexpression of dominant-negative Cdc42 and Rac1 mutants. The pathway initiated by m3 receptors involved sequential activation of Galpha(q), PLC-beta1, PKC, ERK1/2, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (35 +/- 3 to 41 +/- 5%) by the m3 antagonist 4-diphenylacetoxy-N-methylpiperdine (4-DAMP), the phosphoinositide hydrolysis inhibitor U-73122, the PKC inhibitor bisindolylmaleimide, and the ERK1/2 inhibitor PD 98059. cPLA(2) activity was not affected in cells coexpressing dominant-negative RhoA and PLC-delta1 mutants, implying that PKC was not derived from phosphatidylcholine hydrolysis. The effects of ERK1/2 and p38 MAP kinase on cPLA(2) activity were additive and accounted fully for activation and phosphorylation of cPLA(2).

Topics: Acetylcholine; Animals; cdc42 GTP-Binding Protein; Cells, Cultured; Enzyme Activation; GTP-Binding Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth; p21-Activated Kinases; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phospholipase D; Phospholipases A; Phosphorylation; Piperidines; Protein Serine-Threonine Kinases; Rabbits; rac1 GTP-Binding Protein; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Serine; Signal Transduction

Muscarinic facilitation of 14C-ACh release from post-ganglionic parasympathetic nerve terminals was studied in bladder strips prepared from spinal intact (SI) and spinal cord transected (SCT) rats. The spinal cord was transected at the lower thoracic spinal segments 3 weeks prior to the experiments. Using non-facilitatory stimulation (2 Hz) the release of ACh in spinal intact rats did not change in the presence of a non-specific muscarinic antagonist, atropine (100 nM), an M(1) specific antagonist (pirenzepine, 50 nM) or an M(1)-M(3) specific antagonist (4-DAMP, 5 nM). However, during a facilitatory stimulation paradigm (10 Hz or 40 Hz, 100 shocks) atropine and pirenzepine, but not 4-DAMP inhibited the release of ACh in bladders from spinal intact rats, indicating an M(1) receptor-mediated facilitation. In spinal cord transected rats, 2 Hz stimulation-induced release was significantly inhibited by atropine or 4-DAMP but not by pirenzepine indicating that a pre-junctional facilitatory mechanism mediated via M(3) muscarinic receptors could be induced by a non-facilitatory stimulation paradigm after spinal injury. In bladders of spinal cord transected rats, 10 Hz stimulation-evoked release of ACh was also inhibited by atropine and 4-DAMP (5 nM) but not by pirenzepine (50 nM). These results indicate that pre-junctional muscarinic receptors at cholinergic nerve endings in the bladder change after chronic spinal cord injury. It appears that low affinity M(1) muscarinic receptors are replaced by high affinity M(3) receptors. This change in modulation of ACh release may partly explain the bladder hyperactivity after chronic spinal cord injury.

Topics: Animals; Atropine; Electric Stimulation; Female; Muscarinic Antagonists; Neurotransmitter Agents; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Receptors, Muscarinic; Spinal Cord Injuries; Urinary Bladder; Urinary Bladder, Neurogenic

We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility. To determine muscarinic effects on lateral migration of KC, we used an agarose gel keratinocyte outgrowth system (AGKOS) which provides for measurements of the response of large cell populations (> 10(4) cells). Muscarine produced a dose-dependent stimulatory effect on cell migration (p < 0.05). This activity was abolished by atropine, which decreased migration distance when given alone. To identify the mAChR subtype(s) mediating these muscarinic effects, we substituted atropine with subtype-selective antagonists. Tropicamide (M(4)-selective) was more effective at decreasing the migration distance than pirenzepine and 4-DAMP at nanomolar concentrations. We then compared lateral migration of KC obtained from M(4) mAChR knockout mice with that of wild-type murine KC, using AGKOS. In the absence of M(4) mAChR, the migration distance of KC was significantly (p < 0.05) decreased. These results indicate that the M(4) mAChR plays a central role in mediating cholinergic control of keratinocyte migration by endogenous acetylcholine produced by these cells.

Topics: Animals; Atropine; Cell Movement; Humans; Infant, Newborn; Keratinocytes; Mice; Mice, Knockout; Muscarine; Muscarinic Agonists; Piperidines; Pirenzepine; Receptor, Muscarinic M4; Receptors, Muscarinic; Stimulation, Chemical

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (I(CCh)) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited I(CCh) dose-dependently with IC(50) of 1.1 0.1 nM (n = 6). GTPgS-induced current, however, was not inhibited by 10 nM 4-DAMP. I(CCh) was not recorded in pertussis-toxin (PTX)-pretreated smooth muscle cells of gastric antrum. I(CCh) values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 3 pA (n = 6) in the control and PTX-treated cells, respectively (P 0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that G(o) protein is important for activation of ACh-activated NSC channels, our results suggest that I(CCh) might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.

Topics: Animals; Base Sequence; Carbachol; Cations; Cholinergic Agonists; Dose-Response Relationship, Drug; Drug Interactions; Gastric Mucosa; Guinea Pigs; Ion Channels; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Receptors, Muscarinic; Stomach

The brain regions and receptor subtypes involved in water intake following central cholinergic stimulation have been incompletely characterized.. To examine whether drinking and brain Fos-immunoreactivity (ir) induced in rats by central administration of bethanecol is reversed by either the preferential M1 antagonist pirenzepine, the M3 antagonist 4-DAMP, or their combination.. Male Sprague-Dawley rats were surgically implanted with cerebroventricular cannulae. The muscarinic agonist, bethanecol was used as the dipsogenic agent. Either nonselective (atropine) or selective muscarinic receptor antagonists were injected together with bethanecol to determine blockade of drinking. In parallel studies, Fos-ir was assessed in discrete brain regions.. Bethanecol-induced drinking was completely blocked by atropine or by a combination of pirenzepine and 4-DAMP; these latter antagonists alone produced sub-total inhibition of drinking. In contrast, water intake induced by angiotensin II was unaffected by combination of pirenzepine and 4-DAMP. Fos-ir was induced by bethanecol in many brain regions previously implicated in body fluid regulation, including subfornical organ and the magnocellular supraoptic and paraventricular hypothalamic nuclei. Induced Fos-ir was substantially but not completely prevented by co-injection of either pirenzepine or 4-DAMP, but their combination did not seem markedly more effective than either alone.. Drinking induced by brain muscarinic receptor stimulation seems to proceed by a combination of M1 and M3 receptor subtypes. Drinking induced by angiotensin II occurs independently of this mechanism. Fos-ir induced in fluid-related brain regions by bethanecol either uses additional receptor type(s) or is less easily blocked than drinking behavior.

Topics: Animals; Atropine; Bethanechol; Dose-Response Relationship, Drug; Drinking Behavior; Injections, Intraventricular; Male; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Water

A safe and effective ultra-short-acting nondepolarizing neuromuscular blocking agent is required to block nicotinic receptors to facilitate intubation. Rapacuronium, which sought to fulfill these criteria, was withdrawn from clinical use due to a high incidence of bronchospasm resulting in death. Understanding the mechanism by which rapacuronium induces fatal bronchospasm is imperative so that newly synthesized neuromuscular blocking agents that share this mechanism will not be introduced clinically. Selective inhibition of M2 muscarinic receptors by muscle relaxants during periods of parasympathetic nerve stimulation (e.g., intubation) can result in the massive release of acetylcholine to act on unopposed M3 muscarinic receptors in airway smooth muscle, thereby facilitating bronchoconstriction.. Competitive radioligand binding determined the binding affinities of rapacuronium, vecuronium, cisatracurium, methoctramine (selective M2 antagonist), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; selective M3 antagonist) for M2 and M3 muscarinic receptors.. Rapacuronium competitively displaced 3H-QNB from the M2 muscarinic receptors but not from the M3 muscarinic receptors within clinically relevant concentrations. Fifty percent inhibitory concentrations (mean +/- SE) for rapacuronium were as follows: M2 muscarinic receptor, 5.10 +/- 1.5 microm (n = 6); M3 muscarinic receptor, 77.9 +/- 11 microm (n = 8). Cisatracurium and vecuronium competitively displaced 3H-QNB from both M2 and M3 muscarinic receptors but had affinities at greater than clinically achieved concentrations for these relaxants.. Rapacuronium in clinically significant doses has a higher affinity for M2 muscarinic receptors as compared with M3 muscarinic receptors. A potential mechanism by which rapacuronium may potentiate bronchoconstriction is by blockade of M2 muscarinic receptors on prejunctional parasympathetic nerves, leading to increased release of acetylcholine and thereby resulting in M3 muscarinic receptor-mediated airway smooth muscle constriction.

Topics: Animals; Atracurium; Binding, Competitive; Bronchial Spasm; Cell Membrane; CHO Cells; Cricetinae; Diamines; Indicators and Reagents; Muscarinic Antagonists; Neuromuscular Nondepolarizing Agents; Piperidines; Quinuclidinyl Benzilate; Radioligand Assay; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Vecuronium Bromide

Different routes of administration of CCK-33 and blockage of CCK-A and muscarinic (m3) receptors are used in this study to evaluate the mechanisms by which cholecystokinin can stimulate the exocrine pancreas.. The experiment was performed on eight anaesthetized pigs during control conditions and after administration of the CCK-A and m3 receptor antagonists, Tarazepide and 4-DAMP, respectively. Catheters were surgically implanted in the pancreatic duct for juice collection and in the gastric and right gastro-epipoic arteries and in the jugular vein, so that infusions of CCK-33 could be made exclusively to the duodenum/stomach, duodenum/pancreas or general circulation, respectively.. Infusion of a low dose of CCK-33 (13 pmol kg(-1)) to the general circulation did not affect pancreatic protein or trypsin output. When the same dose was given directly to the duodenum/stomach or the duodenum/pancreas, pancreatic output increased during both control conditions and after Tarazepide and/or 4-DAMP treatment, though the increase in trypsin output was lower after Tarazepide and/or 4-DAMP blockade. A high dose of CCK-33 (130 pmol kg(-1)) given peripherally stimulated the pancreatic secretion, but this response was totally abolished in Tarazepide and 4-Damp treated animals.. Pancreatic enzyme secretion due to CCK-33 stimulation depends on the presence of short duodenal-pancreatic peptidergic reflexes evoked mainly via low sensitive, probably CCK-B, receptors located in the duodenum/stomach. Pancreatic secretion evoked by peripheral CCK-33 in pharmacological doses was independent of m3 receptors blockade but depended on CCK-A receptors located elsewhere than in the duodenum/pancreas.

Topics: Animals; Benzodiazepines; Cholecystokinin; Duodenum; Muscarinic Antagonists; Pancreas; Pancreatic Juice; Piperidines; Receptor, Cholecystokinin A; Receptor, Muscarinic M3; Receptors, Cholecystokinin; Receptors, Muscarinic; Reflex; Swine; Trypsin

The present study investigated the role of muscarinic receptor subtypes in the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha of the rat rostral ventrolateral medulla in morphine-induced antinociception. The antinociceptive effects of morphine were evoked by systemic administration or microinjection into the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha. Administration of morphine produced antinociception for hot plate and tail immersion responses to noxious heat stimuli. These effects were antagonized by prior exposure to naloxone and inhibited by mecamylamine pretreatment. Morphine-induced antinociception was significantly inhibited by atropine in a dose-dependent manner. Muscarinic toxin-1 and pirenzepine inhibited morphine-induced antinociception for both the hot plate and tail immersion tests. At a dose of 5 nmol/site, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) also inhibited morphine-induced antinociception, although low doses of this drug did not significantly affect hot plate test response latency when morphine was systemically administered. These results suggest that the antinociceptive effects induced by morphine in part involve the muscarinic M(1) and M(3) receptors of the rat nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha.

Topics: Analgesics, Opioid; Animals; Atropine; Diamines; Male; Mecamylamine; Medulla Oblongata; Microinjections; Morphine; Naloxone; Narcotic Antagonists; Pain Measurement; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Time Factors

To compare activation of the p42/p44 mitogen-activated protein kinase (MAPK) by cholinergic agonists and epidermal growth factor (EGF) in cultured human and rat goblet cells.. . Conjunctiva was removed from either humans during ocular surgery or male Sprague-Dawley rats and cultured in RPMI medium. These cells were incubated with the cholinergic agonist carbachol (10(-4) M) or EGF (10(-8) M) for various times. Before stimulation, cells were incubated with the EGF receptor (EGFR) inhibitor, AG1478 (10(-7) M) or the muscarinic M(3) receptor inhibitor, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP; 10(-5) M) for 10 minutes. Proteins were analyzed by Western blot analysis, using antibodies specific to phosphorylated (activated) p42/44-MAPK or total p42-MAPK. Immunoreactive bands were quantified, and data were expressed as percentage of increase over basal.. Carbachol (10(-4) M) increased MAPK activity in human and rat cultured goblet cells in a time-dependent manner, increasing pMAPK with a maximum at 10 minutes. EGF (10(-8) M) activated MAPK in human and rat goblet cells in a time-dependent manner with a maximum at 5 minutes. Carbachol- and EGF-induced activation of pMAPK was completely inhibited by AG1478 in cultured conjunctival goblet cells from both species. Carbachol-induced MAPK activity was also completely inhibited by 4-DAMP in both species.. In human and rat cultured conjunctival goblet cells, cholinergic agonists and EGF activate MAPK with a similar time dependency, this activation is receptor mediated, and cholinergic agonists transactivate the EGF receptor. Thus, rat cultured conjunctival goblet cells can be used as a model to study human conjunctival goblet cells.

Topics: Adult; Aged; Aged, 80 and over; Animals; Blotting, Western; Carbachol; Cells, Cultured; Cholinergic Agonists; Conjunctiva; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Goblet Cells; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinases; Muscarinic Antagonists; Piperidines; Quinazolines; Rats; Rats, Sprague-Dawley; Time Factors; Tyrphostins

1 The mechanism and receptor subtypes involved in carbachol-stimulated amylase release and its changes after castration were studied in parotid slices from male rats. 2 Carbachol induced both amylase release and inositol phosphate (IP) accumulation in parotid slices from control and castrated rats, but castration induced a decrease of carbachol maximal effect. The effect of castration was reverted by testosterone replacement. 3 The selective M(1) and M(3) muscarinic receptor antagonists, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, respectively, inhibited carbachol-stimulated amylase release and IP accumulation in a dose-dependent manner in parotid slices from control and castrated rats. 4 A diminution of binding sites of muscarinic receptor in parotid membrane from castrated rats was observed. Competition binding assays showed that both, M(1) and M(3) muscarinic receptor subtypes are expressed in membranes of parotid glands from control and castrated rats, M(3) being the greater population. 5 These results suggest that amylase release induced by carbachol in parotid slices is mediated by phosphoinositide accumulation. This mechanism appears to be triggered by the activation of M(1) and M(3) muscarinic receptor subtypes. Castration induced a decrease of the maximal effect of carbachol evoked amylase release and IP accumulation followed by a diminution in the number of parotid gland muscarinic acetylcholine receptors.

Topics: Amylases; Animals; Binding, Competitive; Carbachol; Cholinergic Agonists; Down-Regulation; Inositol Phosphates; Male; Orchiectomy; Parotid Gland; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptors, Muscarinic

Cholinergic muscarinic inputs to subfornical organ (SFO) neurones in rats were studied using histochemical, molecular-biological and electrophysiological techniques. Neurones in the medial septum and the diagonal band (MS-DBB) were retrogradely labelled by a tracer wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex injected into the SFO. Some in the MS-DBB were double-labelled by choline acetyltransferase (ChAT) antibody. Many ChAT-immunoreactive fibres were observed in the SFO. M3 muscarinic receptor subtype-like immunoreactivity, detected using a polyclonal antiserum, was observed in the SFO. In slice preparations, muscarine induced inward currents in a dose-related manner. The inward currents were suppressed by the relatively M3 muscarinic receptor selective antagonist 4-diphenylacetoxy-N-methylpiredine methiodide. In the whole-cell current mode, muscarine depolarized the membrane with increased frequency of action potentials. Reverse transcriptase-polymerase chain reaction showed the presence of M2-M5 receptor mRNA in the SFO tissues. These results suggest that the SFO receives cholinergic muscarinic synaptic inputs from the MS-DBB. Acetylcholine postsynaptically activates and depolarizes neurones in the SFO partly through specific muscarinic receptors, including M3 receptor subtypes.

Topics: Acetylcholine; Animals; Choline O-Acetyltransferase; Gold Colloid; Immunohistochemistry; Male; Membrane Potentials; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Neural Pathways; Neurons; Patch-Clamp Techniques; Piperidines; Rats; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; Subfornical Organ; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

Activation of muscarinic receptors leads to proliferation of astroglial cells and this effect is inhibited by ethanol. Among the intracellular pathways involved in the mitogenic action of muscarinic agonists, activation of the atypical protein kinase C zeta (PKC zeta) appears to be of most importance, and is also affected by low ethanol concentrations. PKC zeta has been reported to activate nuclear factor kappaB (NF-kappaB), a transcription factor that has been shown to play an important role in cell proliferation. The aim of this study was, therefore, to determine whether muscarinic receptors would activate NF-kappaB in astroglial cells, whether such activation would play a role in the mitogenic action of muscarinic agonists, and whether it would represent a possible target for ethanol. Carbachol activated NF-kappaB in human 1321N1 astrocytoma cells, as evidenced by translocation of the p65 subunit of NF-kappaB to the nucleus, phosphorylation and degradation of IkappaBalpha in the cytosol, and increase NF-kappaB binding to DNA. Carbachol also induced translocation of p65 to the nucleus in primary rat astrocytes. Carbachol-induced NF-kappaB activation was mediated by the M3 subtype of muscarinic receptors and appeared to involve Ca(2+) mobilization and activation of PKC epsilon and PKC zeta, but not PI3-kinase and mitogen-activated protein kinase. The NF-kappaB peptide inhibitor SN50, but not the inactive peptide SN50M, strongly inhibited carbachol-induced astrocytoma cells proliferation and p65 translocation to the nucleus. Increased DNA synthesis was also antagonized by the IkappaBalpha kinase inhibitor BAY 11-7082. Ethanol (25-100 mM) inhibited the translocation of p65 and the binding of NF-kappaB to DNA in both 1321N1 astrocytoma cells and primary rat cortical astrocytes. Together, these results suggest that activation of NF-kappaB by muscarinic receptors in astroglial cells is important for carbachol-induced DNA synthesis and that ethanol-mediated inhibition of cell proliferation may be due in part to inhibition of NF-kappaB activation.

Topics: Astrocytes; Astrocytoma; Atropine; Blotting, Western; Carbachol; Cell Line; Cellular Structures; Central Nervous System Depressants; Chelating Agents; Cholinergic Agonists; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Ethanol; Gallamine Triethiodide; Humans; Muscarinic Antagonists; NF-kappa B; Nicotinic Antagonists; Pertussis Toxin; Piperidines; Receptors, Muscarinic; Thymidine; Time Factors; Tritium

We previously demonstrated that restraint stress-induced pressor responses were inhibited by bilateral microinjection of muscimol into the rostral part of the ventral zone of the lateral septal area (LSV). The caudal part of the lateral septal area is also reported to be involved in blood pressure regulation. In this study, we examined whether the LSV receives projections from the caudal part of the dorsal zone of the lateral septal area (LSD) in rats. Injections of a fluorescent tracer into the LSV produced maximal retrograde labeling within the LSD. Microinjection of carbachol (10-100 pmol) into the LSD produced a dose-dependent pressor response. The pressor response to carbachol was inhibited by microinjection of muscimol (80 pmol) or 4-DAMP (1 nmol) into the ipsilateral side of the LSV. Microinjection of muscimol (80 pmol) into the LSD also inhibited the pressor response induced by restraint stress. Repeated injections of carbachol (30 pmol) into the LSD produced Fos immunoreactivity in the ipsilateral side of the LSV. These findings suggest that the LSD projects to the LSV and that these projections may be involved in blood pressure increase.

Topics: Animals; Blood Pressure; Carbachol; Cholinergic Agonists; Dose-Response Relationship, Drug; Fluorescent Dyes; GABA Agonists; Hypertension; Male; Microinjections; Muscarinic Antagonists; Muscimol; Neural Pathways; Neurons; Piperidines; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Reaction Time; Septum of Brain; Stress, Physiological

This study examined enteric neural reflexes activating submucosal cholinergic vasodilator motoneurons, which innervate the final resistance vessels regulating mucosal blood flow. Videomicroscopy was employed to monitor dilatation of submucosal arterioles in in vitro preparations from guinea-pig ileum. Balloon distension of intact lumen evoked reflex vasodilatation and flat sheet preparations were employed to separate mucosal mechanical stimulation from intestinal distension. Mucosal stroking and balloon distension of the orad segment evoked vasodilatations > 1.5 cm from the stimulating site. Mucosal stimulation was blocked by combined 5-HT3/5HT4 antagonists but distension-evoked responses were unaffected. Distension-evoked responses were also unaffected by nifedipine (5 microM) or nifedipine (1 microM) and wortmannin (300 nM), suggesting stretch activation rather than stretch-activated contraction was involved. Mucosal and distension-evoked responses were completely blocked when the myenteric plexus was surgically lesioned and were significantly inhibited by hexamethonium. The muscarinic antagonist 4-DAMP, which inhibits vasodilatations evoked by submucosal cholinergic vasodilator neurons, blocked dilatations elicited by mucosal stimulation and balloon distension. Maximal dilatations evoked with either sensory modality could be further enhanced when stimulated with the second modality. Dilatations evoked by stimulation of the aborad segment were similar to those elicited in the orad segment. In conclusion, sensory mechanisms in the mucosa and muscularis propria activate vasodilator pathways in the myenteric plexus which project for significant distances in both ascending and descending directions before innervating submucosal arterioles. These reflexes could co-ordinate mucosal blood flow during multiple motor events such as peristalsis and intestinal mixing between propulsive events.

Topics: Androstadienes; Animals; Arterioles; Catheterization; Denervation; Female; Guinea Pigs; Ileum; Intestinal Mucosa; Male; Microvilli; Myenteric Plexus; Neurons; Parasympatholytics; Piperidines; Reflex; Tetrodotoxin; Vasodilation; Wortmannin

Acetylcholine (ACh) mimics ischemic preconditioning (PC) and therefore protects the heart against lethal ischemia. Steps common to both ischemic and drug-induced PC are opening of mitochondrial K(ATP) channels (mito K(ATP)) and generation of reactive oxygen species (ROS). The aim of this study was to test whether ACh-induced ROS production could be seen in a vascular smooth muscle cell line, and, if so, to investigate the underlying signaling pathway.. Mitochondrial ROS generation was quantified by measuring changes in fluorescence of ROS-sensitive intracellular markers in vascular smooth muscle cells (A7r5).. Fluorescence, and, therefore, ROS production, was increased to 197.5+/-8.5% of baseline after 45 min of exposure of cells to 2 mM ACh (P<0.001 vs. untreated controls). This effect was blocked by co-treatment with a muscarinic receptor antagonist (atropine 102.8+/-2.9%, 4-DAMP 92.6+/-7.4%) or by inhibition of G(i) with pertussis toxin (PTX) (90.5+/-4.4%), implicating a receptor-mediated rather than non-specific effect of ACh. The increased fluorescence induced by ACh was also abrogated by the free radical scavenger N-(2-mercaptopropionyl) glycine (104.2+/-10.1%), documenting that ROS were indeed the cause of the enhanced fluorescence. Both diazoxide, a K(ATP) channel opener, and valinomycin, a potassium ionophore, also significantly increased ROS production, and these effects were not blocked by PTX, while the K(ATP) channel closer 5-hydroxydecanoate blocked ACh-induced ROS production (92.3+/-3.8%). These results suggest ROS production is directly influenced by K(ATP) activity and K(+) movements in the cell. The tyrosine kinase inhibitor genistein (102.8+/-6.6%) and the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (90.7+/-4.1%) also inhibited the ability of ACh to increase ROS production.. The signaling pathway by which ACh leads to ROS generation in A7r5 cells involves a muscarinic surface receptor, a pertussis toxin-sensitive G protein, PI3-kinase, at least one tyrosine kinase, and a 5-hydroxydecanoate (5-HD)-dependent K(ATP) (presumably that in mitochondria).

Topics: Acetylcholine; Analysis of Variance; Androstadienes; Animals; Aorta; Atropine; Cell Line; Decanoic Acids; Diazoxide; Enzyme Inhibitors; Female; Free Radical Scavengers; Genistein; GTP-Binding Protein alpha Subunits, Gi-Go; Hydroxy Acids; Ionophores; Ischemic Preconditioning; Male; Methacholine Chloride; Microscopy, Fluorescence; Muscarinic Agonists; Muscle, Smooth, Vascular; Perfusion; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperidines; Potassium Channel Blockers; Potassium Channels; Protein-Tyrosine Kinases; Rabbits; Rats; Reactive Oxygen Species; Receptors, Cholinergic; Signal Transduction; Tiopronin; Valinomycin; Wortmannin

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.

Topics: Acetylcholine; Bronchi; Colforsin; Cyclic AMP; Humans; In Vitro Techniques; Isoproterenol; Lung Neoplasms; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Piperidines; Receptor, Muscarinic M2; Receptors, Muscarinic

The in vivo study aimed to examine whether muscarinic receptor subtypes other than muscarinic M3 receptors exert exocrine functional roles in the rat salivary glands. The effects of pirenzepine, methoctramine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were examined on secretion from the major salivary glands evoked by acetylcholine (0.001-10 micromol kg(-1) i.v.) in pentobarbitone-anaesthetized rats. Observations were occasionally made on glandular blood flow. 4-DAMP (0.1-100 nmol kg(-1) i.v.) markedly and equipotently inhibited the acetylcholine-evoked fluid responses in all glands. Pirenzepine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) showed significantly lower inhibitory potency than 4-DAMP, most conspicuously in the parotid, while methoctramine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) exerted an even lesser inhibitory effect. Also against acetylcholine-evoked blood flow increases, 4-DAMP showed a conspicuous potency. At 1 and 10 micromol kg(-1) i.v. of pirenzepine, the antagonist reduced the protein concentration in the submandibular saliva, but not in the parotid saliva. While 4-DAMP (1 and 10 nmol kg(-1) i.v.) significantly inhibited acetylcholine-evoked protein secretory responses in the submandibular glands, methoctramine (below 10 micromol kg(-1) i.v.) affected the responses in neither gland. The reduction of the protein concentration in submandibular saliva caused by 4-DAMP and pirenzepine was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME; 30 mg kg(-1) i.p.), while L-NAME had no or only minute effects on the parotid protein secretion. Thus, in addition to muscarinic M3 receptors, other muscarinic receptors contribute to in vivo functional responses in rat submandibular and sublingual glands. While these other receptors are muscarinic M1 receptors in the sublingual gland, they may be a different subtype, possibly muscarinic M5 receptors, in the submandibular gland. However, muscarinic M1 receptors may induce indirect effects via nitric oxide in the submandibular gland.

Topics: Acetylcholine; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Analysis of Variance; Anesthesia; Animals; Blood Pressure; Diamines; Dose-Response Relationship, Drug; Drug Interactions; Female; Muscarinic Antagonists; Parotid Gland; Phentolamine; Piperidines; Pirenzepine; Propranolol; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Receptors, Muscarinic; Saliva; Salivary Glands; Sublingual Gland; Submandibular Gland; Vasodilator Agents

We have recently observed that Die-Huang-Wan has an ability to stimulate the secretion of insulin to decrease the plasma glucose levels in normal rats. In the present study, this effect of Die-Huang-Wan was reversed by the general muscarinic antagonists atropine and scopolamine, but not affected by the ganglionic nicotinic antagonist pentolinium or hexamethonium. Moreover, disruption of synaptically available acetylcholine using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished the actions induced by Die-Huang-Wan. Mediation of acetylcholine released from nerve terminals by this product can thus be considered. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the effect of Die-Huang-Wan. Blockade of the increase of plasma insulin and plasma glucose lowering action of Die-Huang-Wan by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP) indicated the mediation of muscarinic M3 receptors. The results suggest that Die-Huang-Wan may enhance the release of acetylcholine from nerve terminals to stimulate the muscarinic M3 receptors for augmenting insulin release to produce plasma glucose lowering action.

Topics: Acetylcholine; Administration, Oral; Animals; Blood Glucose; Cholinergic Agents; Cholinesterase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Drugs, Chinese Herbal; Hemicholinium 3; Insulin; Insulin Secretion; Male; Muscarinic Antagonists; Neuromuscular Depolarizing Agents; Physostigmine; Piperidines; Rats; Rats, Wistar

1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.

Topics: Acetylcholine; Animals; Atropine; Calcium; Calcium Signaling; CHO Cells; Colon; Cricetinae; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Quaternary Ammonium Compounds; Receptor, Muscarinic M3; Receptors, Muscarinic; Time Factors; Vasodilator Agents

In the bladder body M2 muscarinic receptors predominate but a smaller population of M3 receptors mediates direct detrusor contraction. M2 receptors have an indirect role by inhibiting cyclic adenosine monophosphate mediated relaxation in the bladder body. We investigated whether a similar mechanism also exists in the bladder base.. Experiments were performed on pig detrusor muscle. In receptor binding studies using l-quinuclidinyl [phenyl-4-(3)H] benzilate ([(3)H]QNB) (NEN Life Science Products, Inc., Boston, Massachusetts) displacement experiments were performed with subtype selective antagonists to determine the M2-to-M3 receptor ratio. In functional studies the affinity of these antagonists against carbachol induced contractions was calculated in normal tissues and in tissues after cyclic adenosine monophosphate elevation by pre-contraction with KCl and relaxation with isoprenaline, and/or selective M3 inactivation by incubation with 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) mustard (Sigma Chemical Co., St. Louis, Missouri) in the presence of methoctramine (Sigma Chemical Co.) to protect M2 receptors.. In saturation binding studies receptor density was 130.5 of fmol./mg. protein and the dissociation constant for [(3)H]QNB was 0.27 nM. Displacement of [(3)H]QNB by the M3 selective antagonist 4-DAMP and the M2 antagonist methoctramine indicated an M2-to-M3 ratio of about 3:1. In functional studies 4-DAMP and methoctramine caused competitive antagonism of responses with affinity values of 9.5 and 6.3 in normal tissues, and 9.3 and 6.1, respectively, in cyclic adenosine monophosphate elevated tissues, suggesting the involvement of M3 receptors only. In tissues in which M3 receptors were inactivated and cyclic adenosine monophosphate levels were elevated the affinity of 4-DAMP was significantly reduced to 8.5 but that of methoctramine was significantly increased to 6.5, suggesting the involvement of M2 receptors.. The M3 subtype appears to mediate contraction of the normal and cyclic adenosine monophosphate elevated tissues of the bladder base. Involvement of M2 receptors was only noted after selective M3 inactivation and cyclic adenosine monophosphate elevation.

Topics: Animals; Cyclic AMP; Diamines; Female; In Vitro Techniques; Isoproterenol; Muscle Contraction; Muscle, Smooth; Piperidines; Potassium Chloride; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Swine; Urinary Bladder

The purpose of this study was to determine whether Src tyrosine kinases are one of the signaling intermediaries linking M(2) receptor stimulation to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in cultures of canine colonic smooth muscle cells (CSMC). RT-PCR studies demonstrate expression of multiple Src tyrosine kinases, including Src, Fyn, and Yes, in CSMC. Muscarinic stimulation of CSMC with 10 microM ACh results in a twofold increase in Src activity within 10 min but does not increase the activity of Fyn. Treatment with the M(2) antagonist AF-DX 116 (10 microM) blocks ACh-stimulated Src activation in primary CSMC cultures that express both M(2) and M(3) receptors and in first-passage CSMC cultures that express predominantly M(2) receptors. Alkylation of M(3) receptors with 100 nM N,N-dimethyl-4-piperidinyl diphenylacetate mustard has no effect on Src activity. Treatment with the pyrazolopyrimidine Src inhibitor PP1 (10 microM) or AF-DX 116 (10 microM) blocks ACh-stimulated ERK phosphorylation. Together these results indicate that M(2) receptors are coupled to Src tyrosine kinase and subsequent activation of ERK in cultured CSMC.

Topics: Animals; Atropine; Cells, Cultured; Colon; Dogs; Female; Gene Expression Regulation, Enzymologic; Genistein; Male; Mitogen-Activated Protein Kinases; Muscarinic Antagonists; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Pyrazoles; Pyrimidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; src-Family Kinases; Teprotide

The purpose of the study was to examine the importance of the cholinergic system in triggering the conditioned NK cell response. The fact that serotonergic system can modulate cholinergic functions suggested that it might be involved in conditioned NK cell response. To evaluate the potential pathways, cholinergic and serotonergic antagonists were applied centrally at either the conditioned association or recall stage, to interfere with the conditioned NK cell response. The results showed that both the cholinergic and serotonergic systems were necessary for eliciting the conditioned enhancement of NK cell activity. Involvements of the two systems were found to be critical for establishing the conditioned association and recall of the conditioned response. The blocks are believed to be receptor mediated. The receptors identified to be involved in the regulation of the conditioned NK cell response were: M(1), M(2) and M(3) muscarinic; nicotinic; 5 HT(1) and 5 HT(2) receptors.

Topics: Acetylcholine; Animals; Female; Gallamine Triethiodide; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Parasympathetic Nervous System; Piperidines; Poly I-C; Receptors, Muscarinic; Receptors, Serotonin; Serotonin

Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) activity was found coupled to muscarinic receptor activation as well as to nitric oxide-mediated amylase secretion elicited by carbachol. Parotid glands presented a predominant M(3) and a minor muscarinic M(1) acetylcholine receptor population, though carbachol stimulated NOS activity only through muscarinic M(3) receptors as revealed in the presence of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and pirenzepine. Amylase secretion induced by carbachol appeared to be partly mediated by nitric oxide and nitric oxide-induced signaling since N-nitro-L-arginine methyl ester (L-NAME) inhibited the effect as well as did methylene blue. A negative regulation of NOS by protein kinase C activation in the presence of a high concentration of carbachol was seen in parotid glands and this inhibition was paralleled by amylase secretion.

Topics: Amylases; Animals; Atropine; Binding, Competitive; Carbachol; Cell Membrane; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Male; Muscarinic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Parotid Gland; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptors, Muscarinic; Specific Pathogen-Free Organisms

Pontine micuturition center and storage center are controlled by upper brain. Pharmacological and molecular biological data indicate the distribution of muscarinic receptor subtypes in rat brain, however, the effect of central muscarinic cholinergic mechanisms on the bladder activity has not been evaluated. We investigated the diversity of effect of each subtype in rat brain.. The muscarinic agents such as muscarine (non-specific agonist), atropine (non-specific antagonist), pirenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist) and 4-DAMP (M3 receptor antagonist) were administrated with intracerebroventricular injection (I.C.V. group) or intravenous injection (i.v. group). The drug effects on rhythmic bladder contraction were investigated.. In I.C.V. group, the bladder contraction pressure were reduced by atropine, pirenzepine and 4-DAMP. The contraction time were shortened by pirenzepine and 4-DAMP but extended by AF-DX116. The contraction frequency was decreased by AF-DX116. Whereas AF-DX116 in i.v. group reduced the contraction pressure but did not cause any changes of the contraction time and the frequency. Other agents in i.v. group had same tendency with those of I.C.V. group except for intensity.. That is to say AF-DX116 in I.C.V. group facilitate both micuturition center and storage center. In other words, these data indicate M2 receptor in rat brain inhibits both micurition and storage center. In addition, the effect of AF-DX116 in brain suggests that the development of new drug which proceed to central neuron system might provide a new treatment of neurogenic bladder.

Topics: Animals; Atropine; Brain; Female; Muscarine; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Urinary Bladder; Urination

To study the activation of choline on M3-R in heart and observe the hemodynamic changes of rat and rabbit.. A cardiac catheter was inserted into the left ventricular cavity via the right carotid artery, then the HR, LVSP, LVEDP, and +/- dp/dt were measured using a polygraph system.. Choline was shown to decrease the hemodynamic assessments, such as HR, + dp/dt, LVSP and LVEDP. while the M3-R antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) showed little effect on these assessments. It was found to reverse the hemodynamic effects of choline.. M3 receptor agonist can produce negative inotropic and chronotropic effects on the heart of rat and rabbit.

Topics: Animals; Choline; Female; Heart; Heart Rate; Hemodynamics; Male; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Rabbits; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Muscarinic

G protein-gated inwardly rectifier K+ current in atrial myocytes (I(K(ACh))) upon stimulation with acetylcholine (ACh) shows a fast desensitizing component (t(1/2) approximately 5 s). After washout of ACh, I(K(ACh)) recovers from fast desensitization within < 30 s. A recent hypothesis suggests that fast desensitization is caused by depletion of phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)), resulting from costimulation of phospholipase C (PLC)-coupled M3 receptors (M3AChR). The effects of stimulating two established PLC-coupled receptors, alpha-adrenergic and endothelin (ET(A)), on I(K(ACh)) were studied in rat atrial myocytes. Stimulation of these receptors caused activation of I(K(ACh)) and inhibition of the M2AChR-activated current. In myocytes loaded with GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), causing stable activation of I(K(ACh)), inhibition via alpha-agonists and ET-1 was studied in isolation. Stimulation of either type of receptor under this condition, via G(q/11), caused a slow inhibition (t(1/2) approximately 50 s) by about 70%. No comparable effect on GTPgammaS-activated I(K(ACh)) was induced by ACh, suggesting that PLC-coupled M3AChRs are not functionally expressed in rat myocytes, which was supported by the finding that M3AChR transcripts were not detected by reverse transcriptase-polymerase chain reaction in identified atrial myocytes. Supplementing the pipette solution with PtIns(4,5)P(2) significantly reduced inhibition of I(K(ACh)) but had no effect on fast desensitization. From these data it is concluded that stimulation of PLC-coupled receptors causes slow inhibition of I(K(ACh)) by depletion of PtIns(4,5)P(2), whereas fast desensitization of I(K(ACh)) is not related to PtIns(4,5)P(2) depletion. As muscarinic stimulation by ACh does not exert inhibition of I(K(ACh)) comparable to stimulation of alpha(1)- and ET(A) receptors, expression of functional PLC-coupled muscarinic receptors in rat atrial myocytes is unlikely.

Topics: Acetylcholine; Electric Conductivity; Endothelin-1; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Heart Atria; Ion Channel Gating; Muscarinic Antagonists; Phenylephrine; Phosphatidylinositol 4,5-Diphosphate; Piperidines; Potassium Channels; Potassium Channels, Inwardly Rectifying; Receptors, Cell Surface; Receptors, Endothelin; Receptors, Muscarinic; Type C Phospholipases

Submucosal glands were isolated within 4 h of death from tracheae and bronchi obtained from autopsied lungs, and the secretory response of secretory leukocyte protease inhibitor (SLPI) was examined with ELISA and a secretory index. Although human neutrophil elastase (HNE) at low concentrations increased SLPI secretion above the control level (i.e., 149% of control level at 10(-11) M), HNE at high concentrations significantly decreased it below the control level (i.e., 16% of control level at 10(-7) M). The decrease in SLPI concentration was shown to result from the degradation of SLPI by excessive HNE. Methacholine induced significant secretion (i.e., 363% of control level at 10(-5) M) that was abolished by both M(1) and M(3) receptor antagonists. A semiquantitative analysis of SLPI mRNA by RT-PCR and Southern blot showed that compared with the superficial epithelium, submucosal glands had a 30-fold or higher level of SLPI mRNA. Both HNE and methacholine significantly increased the level of SLPI mRNA in submucosal glands in a dose-dependent manner (i.e., 357% of control level at 10(-7) M and 175% of control level at 10(-5) M, respectively). These findings indicate that human airway submucosal glands can transcribe 30-fold or more SLPI mRNA than the superficial epithelium and that SLPI mRNA transcription and secretion are regulated by both HNE and muscarinic receptors.

Topics: Adult; Aged; Aged, 80 and over; Atropine; Bronchi; Dexamethasone; Dose-Response Relationship, Drug; Exocrine Glands; Female; Gene Expression; Glucocorticoids; Humans; Leukocyte Elastase; Male; Methacholine Chloride; Middle Aged; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Proteinase Inhibitory Proteins, Secretory; Proteins; Respiratory Mucosa; RNA, Messenger; Secretory Leukocyte Peptidase Inhibitor; Trachea

We investigated whether anticholinesterase drugs in large doses inhibit muscarinic receptors of airway smooth muscle. In vitro measurements of isometric tension and [(3)H]inositol monophosphate (IP(1)) that formed were conducted by using rat tracheal rings or slices. Neostigmine and pyridostigmine caused muscular contraction and IP(1) accumulation in small doses (10 microM and < or = 100 microM, respectively), but they attenuated muscular contraction and IP(1) accumulation in larger doses (1000 microM). Edrophonium did not affect the smooth muscle tone and IP(1) levels. Neostigmine, pyridostigmine, and edrophonium attenuated the carbachol (5.5 microM)-induced smooth muscle contraction and IP(1) accumulation, when administered in large doses (1000 microM). The attenuation of contraction by neostigmine at large doses was not affected by methoctramine, an M(2) muscarinic receptor antagonist, but was reversed by washing with fresh Krebs-Henseleit solution. The results suggest that anticholinesterase drugs have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.. Neostigmine and pyridostigmine, but not edrophonium, have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.

Topics: Animals; Carbachol; Cholinergic Agents; Cholinesterase Inhibitors; Dose-Response Relationship, Drug; Edrophonium; In Vitro Techniques; Inositol Phosphates; Isometric Contraction; Male; Muscarinic Antagonists; Muscle, Smooth; Neostigmine; Phosphatidylinositols; Piperidines; Pyridostigmine Bromide; Rats; Rats, Wistar; Stimulation, Chemical; Trachea

To characterize the mechanisms of acetylcholine (ACh)-induced vasorelaxation in rabbit renal arteries precontracted with high K+ (100 mM), muscle tension and cytosolic free Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded arterial strips. In the artery with endothelium, high K+ increased both [Ca2+]i and muscle tension. Addition of ACh (10 microM) during high-K+ induced contraction significantly relaxed the muscle and induced additional increase in [Ca2+]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM). ACh increased [Ca2+]i without relaxing the muscle. In the artery without endothelium, high K+ increased both [Ca2+]i and muscle tension although ACh was ineffective, suggesting that ACh acts selectively on endothelium to increase [Ca2+]i. 4-DAMP (10 nM) or atropine (0.1 microM) abolished the ACh-induced increase in [Ca2+]i and relaxation. However, pirenzepine (0.1 microM), AF-DX 116 (1 microM) and tropicamide (1 microM) were ineffective. The ACh-induced increase of [Ca2+li and vasorelaxation was significantly reduced by 3 microM gadolinium, 10 microM lanthanum or 10 microM SKF 96365. These results suggest that, in rabbit renal artery, ACh-evoked relaxation of 100 mM K+-induced contractions is mediated by the release of endothelial NO. ACh may stimulates the M3 subtype of muscarinic receptor in the endothelial cells, resulting in the opening of the nonselective cation channels followed by an increase of [Ca2+]i and stimulation of NO synthase.

Topics: Acetylcholine; Animals; Atropine; Calcium; Calcium Channel Blockers; Cations, Monovalent; Endothelium, Vascular; Gadolinium; Imidazoles; In Vitro Techniques; Lanthanum; Male; Muscle Contraction; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Parasympatholytics; Piperidines; Pirenzepine; Potassium; Rabbits; Renal Artery; Tropicamide; Vasoconstriction; Vasodilation

1. Muscarinic cholinoceptors (MChR) in freshly dispersed rat salivary gland (RSG) cells were characterized using microphysiometry to measure changes in acidification rates. Several non-selective and selective muscarinic antagonists were used to elucidate the nature of the subtypes mediating the response to carbachol. 2. The effects of carbachol (pEC(50) = 5.74 +/- 0.02 s.e.mean; n = 53) were highly reproducible and most antagonists acted in a surmountable, reversible fashion. The following antagonist rank order, with apparent affinity constants in parentheses, was noted: 4-DAMP (8.9)= atropine (8.9) > tolterodine (8.5) > oxybutynin (7.9) > S-secoverine (7.2) > pirenzepine (6.9) > himbacine (6.8) > AQ-RA 741 (6.6) > methoctramine (5.9). 3. These studies validate the use of primary isolated RSG cells in microphysiometry for pharmacological analysis. These data are consistent with, and extend, previous studies using alternative functional methods, which reported a lack of differential receptor pharmacology between bladder and salivary gland tissue. 4. The antagonist affinity profile significantly correlated with the profile at human recombinant muscarinic M(3) and M(5) receptors. Given a lack of antagonists that discriminate between M(3) and M(5), definitive conclusion of which subtype(s) is present within RSG cells cannot be determined.

Topics: Alkaloids; Animals; Atropine; Benzhydryl Compounds; Benzodiazepinones; Binding, Competitive; Biosensing Techniques; Carbachol; Cholinergic Agonists; Cresols; Diamines; Dose-Response Relationship, Drug; Furans; Male; Mandelic Acids; Muscarinic Antagonists; Naphthalenes; Phenethylamines; Phenylpropanolamine; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Submandibular Gland; Tolterodine Tartrate

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.

Topics: Animals; Bronchial Hyperreactivity; Carbachol; Diamines; Gene Expression; Guinea Pigs; Lung; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trachea

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3'5'(c)AMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3'5' cyclic adenosine monophosphate (3'5'(c)AMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M(1) and M(3), which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Arginine Vasopressin; Atropine; Biological Transport; Bufo marinus; Calcium; Calcium Channel Blockers; Carbachol; Cholinergic Agonists; Cholinergic Antagonists; In Vitro Techniques; Isotonic Solutions; Lanthanum; Osmolar Concentration; Piperidines; Pirenzepine; Urinary Bladder; Vasoconstrictor Agents; Water

The aim of this study was to investigate the role of the parasympathetic (cholinergic and peptidergic) nervous system in the regulation of exocrine pancreas function in piglets during their early postnatal development. The cholinergic and peptidergic regulatory pathways of exocrine pancreatic function were tested by the specific muscarinic receptor blocker 4-diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP) and bombesin, respectively. At the age of 2 weeks, piglets were surgically fitted with a chronic pancreatic duct catheter, a duodenal re-entrant cannula and a jugular vein catheter. The experiments comprised a pre-weaning period, and a post-weaning period that commenced at the beginning of the 5th week of age. Intravenous infusion of 4-DAMP (100 pmol x kg(-1) x h(-1)) reduced the outflow of pancreatic juice, the output of total protein and the activity of trypsin, chymotrypsin, carboxyl ester hydrolase and amylase during preprandial and postprandial pancreatic secretion, in both the pre- and post-weaning periods. However, the inhibitory effect of 4-DAMP during postprandial secretion was significantly greater (P < 0.05) in suckling piglets. The infusion of bombesin (10, 100 and 1000 pmol x kg(-1) x h(-1)) stimulated exocrine pancreatic secretion in a dose-dependent manner during both the pre- and post-weaning periods. However, the stimulatory effect of 1000 pmol x kg(-1) x h(-1) bombesin on total protein output and the activities of trypsin, chymotrypsin and amylase were significantly higher (P < 0.05) in suckling piglets. In summary, our study showed that cholinergic and peptidergic mechanisms are involved in the regulation of exocrine pancreas function in piglets in both the pre- and post-weaning stages. 4-DAMP had a greater inhibitory effect on exocrine pancreatic secretion in piglets during the pre-weaning period. Thus, these observations suggest that the parasympathetic nervous system plays a dominant role in the functioning of the exocrine pancreas at this time. The action of bombesin suggests that it is a potent secretagogue for the exocrine pancreas in pigs during their postnatal development.

Topics: Amylases; Animals; Animals, Suckling; Bombesin; Carboxylesterase; Carboxylic Ester Hydrolases; Cholinergic Fibers; Chymotrypsin; Eating; Milk; Muscarinic Antagonists; Pancreas; Pancreatic Juice; Parasympathetic Fibers, Postganglionic; Piperidines; Postprandial Period; Swine; Trypsin; Weaning

The pharmacological characteristics of muscarinic receptors in the male mice urinary bladder smooth muscle were studied. (+)-Cis-dioxolane, oxotremorine-M, acetylcholine, carbachol and pilocarpine induced concentration-dependent contractions of the urinary bladder smooth muscle (pEC(50)=6.6+/-0.1, 6.9+/-0.1, 6.7+/-0.1, 5.8+/-0.1 and 5.8+/-0.1, E(Max)=3.2+/-0.8 g, 2.7+/-0.4 g, 1.0+/-0.1 g, 2.7+/-0.3 and 0.9+/-0.2 g, respectively, n=4). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK(B) values): atropine (9.22+/-0.09), pirenzepine (6.85+/-0.08), 4-DAMP (8.42+/-0.14), methoctramine (5.96+/-0.05), p-F-HHSiD (7.48+/-0.09), tolterodine (8.89+/-0.13), AQ-RA 741 (7.04+/-0.12), s-secoverine (8.21+/-0.09), zamifenacin (8.30+/-0.17) and darifenacin (8.70+/-0.09). In this tissue, the pK(B) values correlated most favourably with pK(i) values for these compounds at human recombinant muscarinic M(3) receptors. A significant correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between M(3) and m5 receptors. In recontraction studies, in which the muscarinic M(3) receptor population was decreased, and conditions optimized to study M(2) receptor activation, methoctramine exhibited an affinity estimate consistent with muscarinic M(3) receptors (pK(B)=6.23+/-0.14; pA(2)=6.16+/-0.03). Overall, these data study suggest that muscarinic M(3) receptors are the predominant, if not the exclusive, subtype mediating contractile responses to muscarinic agonists in male mouse urinary bladder smooth muscle.

Topics: Acetylcholine; Animals; Carbachol; Diamines; Dioxolanes; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Oxotremorine; Pilocarpine; Piperidines; Receptors, Muscarinic; Urinary Bladder

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M2 receptors, and strychnine did so also at the M4 receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M2) and scopolamine (M2), between strychnine and butylscopolamine (M4), L-hyoscyamine (M2 and M4) and scopolamine (M4), and between brucine and scopolamine (M2). Positive effects of alcuronium, strychnine and brucine on the affinity of the M2 receptors for 4-DAMP have been confirmed by direct measurements of the binding of [3H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M2 receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.

Topics: Alcuronium; Allosteric Regulation; Animals; CHO Cells; Cricetinae; Muscarinic Antagonists; N-Methylscopolamine; Piperidines; Radioligand Assay; Strychnine; Tritium

The aim of the present study was to characterize the muscarinic acetylcholine receptor subtypes present in the caput and cauda of rat epididymis. The specific binding of [3H]quinuclidinyl benzilate ([3H]QNB) to epididymal membranes was time dependent, temperature dependent, and saturable. The cauda epididymis showed higher affinity to [3H]QNB and higher muscarinic receptor density when compared to the caput region. The [3H]QNB binding was tested in competition studies with different muscarinic receptor antagonists. Each antagonist tested displaced [3H]QNB bound to caput and cauda epididymal membrane with similar affinity. Correlation among the negative logarithm of inhibition constant values (pK(i)) for these antagonists obtained in the epididymis with their correspondent published pK(i) values obtained in tissues that expressed each receptor subtype (M1, M2, M3, and M4) indicated that the muscarinic receptors present in caput and cauda epididymis belong to the muscarinic M2 receptor subtype. When reverse transcription-polymerase chain reaction was used to identify muscarinic receptor mRNA subtypes in the epididymis, only m2 transcripts were detected in the caput region, while both m2 and m3 mRNA subtypes were observed in the cauda region. In conclusion, these results demonstrate that muscarinic receptors are present in the rat epididymis, with expression levels dependent on the region of the epididymis analyzed. Thus, the cholinergic neurotransmitter in the epididymis may be a factor controlling contractility and/or the luminal fluid microenvironment.

Topics: Animals; Atropine; Cell Membrane; Diamines; Epididymis; Male; Muscarinic Antagonists; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tritium

The functional effects of muscarinic receptor and purinoceptor agonists and antagonists were studied on isolated strip preparations of the rat urinary bladder. The muscarinic "M3/M1-selective" receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) most conspicuously inhibited the carbachol-evoked contractile responses (pA2=9.8), while the muscarinic "M1-selective" receptor antagonist pirenzepine and the muscarinic "M2-selective" receptor antagonist methoctramine were less potent (pA2=7.0 and 6.5, respectively). Administration of 4-DAMP in combination with methoctramine in selective dosages gave no significant additional reduction of carbachol-evoked contractile responses. Adenosine 5'-triphosphate (ATP) elicited transient dose-dependent contractile responses and it caused relaxation of the carbachol-contracted detrusor strips. The relaxatory response was enhanced in the presence of methoctramine and furthermore, was attenuated by the adenosine receptor antagonist 8-p-sulfophenyltheophylline. Administration of 2-chloro-adenosine to pre-contracted strips tended to cause dose-dependent relaxations, which were significantly increased in the presence of methoctramine. The purinergic contractile response, on the other hand, was not affected by methoctramine. Thus, the results are consistent with the cholinergic contractile response in the rat urinary bladder being exerted via activation of muscarinic M3 receptors, while the muscarinic M2 receptors exerted a modulator effect on purine-evoked relaxations in the rat urinary bladder.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Triphosphate; Animals; Carbachol; Diamines; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Piperidines; Pirenzepine; Purinergic Agonists; Purinergic Antagonists; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptors, Muscarinic; Receptors, Purinergic; Theophylline; Urinary Bladder

Increases in cortical cerebral blood flow are induced by stimulation of basal forebrain cholinergic neurons. This response is mediated in part by nitric oxide (NO) and reportedly involves both nicotinic and muscarinic receptors, some of which are possibly located in the vessel wall. In the present study, the vasomotor response(s) elicited by acetylcholine (ACh) on isolated and pressurized bovine and/or human intracortical penetrating arterioles were investigated, and pharmacological characterization of the receptor involved in this response was carried out. Acetylcholine (10(-11) to 10(-4) mol/L) dose dependently dilated bovine and human intracortical arterioles at spontaneous tone (respective pD2 values of 6.4+/-0.3 and 7.2+/-0.3 and E(Amax) of 65.0+/-26.8 and 43.2+/-30.1% of the maximal dilation obtained with papaverine) and bovine arterioles after preconstriction with serotonin (pD2 = 6.3+/-0.1, E(Amax) = 80.0+/-17.9% of induced tone). In contrast, nicotine (10(-8) to 10(-4) mol/L) failed to induce any vasomotor response in bovine vessels whether at spontaneous or at pharmacologically induced tone. Application of the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-5) mol/L) elicited a gradual constriction (approximately 20%) of the arterioles, indicating the presence of constitutive NO release in these vessels. Nomega-Nitro-L-argigine (10(-5) to 10(-4) mol/L) also significantly blocked the dilation induced by ACh. The muscarinic ACh receptor (mAChR) antagonists pirenzepine, 4-DAMP, and AF-DX 384 dose dependently inhibited the dilatation induced by ACh (10(-5) mol/L) with the following rank order of potency: 4-DAMP (pIC50 = 9.2+/-0.3) >> pirenzepine (pIC50 = 6.7+/-0.4) > AF-DX 384 (pIC50 = 5.9+/-0.2). These results suggest that ACh can induce a potent, dose-dependent, and NO-mediated dilation of bovine and/or human intracortical arterioles via interaction with an mAChR that best corresponds to the M5 subtype.

Topics: Acetylcholine; Animals; Arterioles; Cattle; Cerebral Cortex; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Humans; Microcirculation; Muscarinic Antagonists; Nitric Oxide; Organ Culture Techniques; Parasympatholytics; Piperidines; Pirenzepine; Receptor, Muscarinic M5; Receptors, Muscarinic; Receptors, Nicotinic; Vasodilation; Vasodilator Agents

Perturbations of cholinergic neurotransmission in the brain stem affect respiratory motor pattern both in vivo and in vitro; the underlying cellular mechanisms are unclear. Using a medullary slice preparation from neonatal rat that spontaneously generates respiratory rhythm, we patch-clamped inspiratory neurons in the preBötzinger complex (preBötC), the hypothesized site for respiratory rhythm generation, and simultaneously recorded respiratory-related motor output from the hypoglossal nerve (XIIn). Most (88%) of the inspiratory neurons tested responded to local application of acetylcholine (ACh) or carbachol (CCh) or bath application of muscarine. Bath application of 50 microM muscarine increased the frequency, amplitude, and duration of XIIn inspiratory bursts. At the cellular level, muscarine induced a tonic inward current, increased the duration, and decreased the amplitude of the phasic inspiratory inward currents in preBötC inspiratory neurons recorded under voltage clamp at -60 mV. Muscarine also induced seizure-like activity evident during expiratory periods in XIIn activity; these effects were blocked by atropine. In the presence of tetrodotoxin (TTX), local ejection of 2 mM CCh or ACh onto preBötC inspiratory neurons induced an inward current along with an increase in membrane conductance under voltage clamp and induced a depolarization under current clamp. This response was blocked by atropine in a concentration-dependent manner. Bath application of 1 microM pirenzepine, 10 microM gallamine, or 10 microM himbacine had little effect on the CCh-induced current, whereas 10 microM 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the current. The current-voltage (I-V) relationship of the CCh-induced response was linear in the range of -110 to -20 mV and reversed at -11.4 mV. Similar responses were found in both pacemaker and nonpacemaker inspiratory neurons. The response to CCh was unaffected when patch electrodes contained a high concentration of EGTA (11 mM) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (10 mM). The response to CCh was reduced greatly by substitution of 128 mM Tris-Cl for NaCl in the bath solution; the I-V curve shifted to the left and the reversal potential shifted to -47 mV. Lowering extracellular Cl(-) concentration from 140 to 70 mM had no effect on the reversal potential. These results suggest that in preBötC inspiratory neurons, ACh acts on M3-like ACh receptors on the postsynaptic neurons to open a channel perme

Topics: Acetylcholine; Animals; Animals, Newborn; Carbachol; Electrophysiology; In Vitro Techniques; Membrane Potentials; Motor Neurons; Muscarinic Agonists; Muscarinic Antagonists; Neurons; Patch-Clamp Techniques; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Receptors, Muscarinic; Respiratory Mechanics; Synapses; Tetrodotoxin

Activation of the parasympathetic ciliary ganglion input to the choroid causes increases in choroidal blood flow. We examined the role and the type of muscarinic receptors within the choroid that are involved in these increases in choroidal blood flow, using electrical stimulation of the nucleus of Edinger-Westphal (EW) to activate the ciliary ganglion input to choroid in ketamine anesthetized pigeons. Baseline choroidal blood flow and its EW-evoked increases measured as peak and total (area under the curve) responses were determined using laser Doppler flowmetry. The EW-evoked responses were reduced dose-dependently after administration of 4-diphenyl-acetoxy-N-methylpiperedine (4-DAMP), a relatively selective antagonist of M3 type muscarinic receptors, with a maximal mean decrease of 86% (peak response) and 93% (total response) at a dose of 10 microg kg(-1)i.v. without a significant effect on baseline choroidal blood flow, heart rate or systemic arterial blood pressure. Atropine, a non-selective antagonist of muscarinic receptors, decreased the EW-evoked responses to a lesser extent than 4-DAMP after intravenous administration of 1 mg kg(-1)(by 67% for peak response and by 53% for total response) or topical administration of a 5% solution (by 41% for peak response and by 62% for total response), both of which increased heart rate and systemic arterial blood pressure without a consistent effect on baseline choroidal blood flow. In contrast, himbacine (i.p. 10 microg kg(-1)), a relatively selective antagonist of M2 type muscarinic receptors, increased the EW-evoked parasympathetic cholinergic vasodilation (by 93% for the peak response and by 142% for the total response) without a significant effect on heart rate, systemic arterial blood pressure or baseline choroidal blood flow. The results of our study suggest a major role of M3 type muscarinic receptors in the EW-evoked increases in choroidal blood flow. Based on findings that the ciliary ganglion input to choroid does not synthesize nitric oxide but inhibitors of NO production do block EW-evoked choroidal vasodilation, it seems likely that the M3 receptors acted on by 4-DAMP are present on choroidal endothelial cells and mediate choroidal vasodilation via stimulation of endothelial release of nitric oxide. In contrast, M2 muscarinic receptors may play a presynaptic role in downregulating EW-evoked parasympathetic cholinergic vasodilation in avian choroid.

Topics: Alkaloids; Animals; Atropine; Blood Pressure; Choroid; Columbidae; Electric Stimulation; Furans; Heart Rate; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Receptors, Muscarinic; Regional Blood Flow; Vasodilation

Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase (1-5 min) and the second phase (7-30 min) after the injection. The muscarinic antagonist atropine (0.1-10 ng, i.t.) and the M(3) receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.1-20 ng, i.t.) inhibited the second phase of this response, whereas higher doses of atropine (20-100 ng, i.t.) did not cause inhibition. The M(1) muscarinic receptor antagonist pirenzepine (10-100 ng, i.t.) did not inhibit either the first or the second phase response, but a high dose of pirenzepine (1000 ng, i.t.) tended to inhibit the second phase response. On the other hand, the M(2) muscarinic receptor antagonist 11-¿(2-[(diethylamino)methyl]-1-piperidinyl¿acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one (AF-DX116; 10-1000 ng, i.t.) had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine (25 ng) significantly inhibited only the second phase. The acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 microg, i.t.) completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP (10 ng, i.t.). These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M(3) muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.

Topics: Acetylcholine; Animals; Atropine; Cholinergic Agents; Cholinesterase Inhibitors; Formaldehyde; Hemicholinium 3; Male; Mice; Mice, Inbred Strains; Muscarinic Antagonists; Naloxone; Narcotic Antagonists; Neostigmine; Nociceptors; Pain; Pain Measurement; Piperidines; Receptor, Muscarinic M3; Receptors, Muscarinic; Spinal Cord

We previously demonstrated that after several days of serum deprivation about one-sixth of confluent cultured canine tracheal myocytes acquire an elongated, structurally and functionally contractile phenotype. These myocytes demonstrated significant shortening on ACh exposure. To evaluate the mechanism by which these myocytes acquire responsiveness to ACh, we assessed receptor-Ca(2+) coupling using fura 2-AM fluorescence imaging and muscarinic receptor expression using Western analysis. Cells were grown to confluence in 10% fetal bovine serum and then maintained for 7-13 days in serum-free medium. A fraction of serum-deprived cells exhibited reproducible intracellular Ca(2+) mobilization in response to ACh that was uniformly absent from airway myocytes before serum deprivation. The Ca(2+) response to 10(-4) M ACh was ablated by inositol 1,4,5-trisphosphate (IP(3)) receptor blockade using 10(-6) M xestospongin C but not by removal of extracellular Ca(2+). Also, 10(-7) M atropine or 10(-7) M 4-diphenylacetoxy-N-methylpiperidine completely blocked the response to ACh, but intracellular Ca(2+) mobilization was not ablated by 10(-6) M pirenzepine or 10(-6) M methoctramine. In contrast, 10(-5) M bradykinin (BK) was without effect in these ACh-responsive myocytes. Interestingly, myocytes that did not respond to ACh demonstrated robust increases in intracellular Ca(2+) on exposure to 10(-5) M BK that were blocked by removal of extracellular Ca(2+) and were only modestly affected by IP(3) receptor blockade. Serum deprivation increased the abundance of M(3) receptor protein and of BK(2) receptor protein by two- to threefold in whole cell lysates within 2 days of serum deprivation, whereas M(2) receptor protein fell by >75%. An increase in M(3) receptor abundance and restoration of M(3) receptor-mediated Ca(2+) mobilization occur concomitant with reacquisition of a contractile phenotype during prolonged serum deprivation. These data demonstrate plasticity in muscarinic surface receptor expression and function in a subpopulation of airway myocytes that show mutually exclusive physiological and pharmacological diversity with other cells in the same culture.

Topics: Acetylcholine; Animals; Atropine; Blood Proteins; Blotting, Western; Bradykinin; Calcium; Calcium Channels; Cells, Cultured; Diamines; Dogs; Dose-Response Relationship, Drug; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Muscarinic Antagonists; Muscle, Smooth; Oxazoles; Parasympatholytics; Phenotype; Piperidines; Pirenzepine; Receptor, Muscarinic M3; Receptors, Bradykinin; Receptors, Cytoplasmic and Nuclear; Receptors, Muscarinic; Trachea; Vasodilator Agents

Acetylcholine outflow can be modulated through inhibitory presynaptic muscarinic autoreceptors. This study was to identify which subtype is involved in mouse cortex. Five muscarinic antagonists and their ability to elevate stimulation-induced (S-I) acetylcholine outflow were tested in the presence of neostigmine, which decreased S-I outflow. The potency of each antagonist was determined, expressed as a ratio of the potency of each other antagonist and compared with the potency ratios of the antagonists for each of the defined muscarinic receptors (M(1)-M(4)), as recorded in the literature. Linear regression analysis revealed that the data fitted the M(2) (r(2)>0.97) and M(4) (r(2)>0.85) subtypes best, with no correlation for the M(1) and M(3) subtypes.

Topics: Acetylcholine; Alkaloids; Animals; Atropine; Autoreceptors; Binding, Competitive; Cerebral Cortex; Furans; Male; Mice; Muscarinic Antagonists; Naphthalenes; Neostigmine; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptor, Muscarinic M4; Receptors, Muscarinic

The effect of sympathectomy on parasympathetic regulation of ocular perfusion was investigated. Uveal blood flow through the vortex veins was measured by laser Doppler flowmetry during electrical stimulation of the superior salivatory nucleus, which activates ocular parasympathetic nerves, in adult rats with intact innervation and 2 days or 6 weeks after excision of the ipsilateral superior cervical ganglion. In all groups, parasympathetic stimulation produced comparable increases in flux, which were abolished by the selective neuronal nitric-oxide synthetase inhibitor, 1-(2-trifluoromethylphenyl) imidazole. Atropine had no effect in control and acutely sympathectomized rats but abolished the flux increase in four of six chronically sympathectomized animals, and 1-(2-trifluoromethylphenyl) imidazole eliminated the residual response. The muscarinic receptor agonist bethanechol did not affect basal flow in control or sympathectomized rats. However, bethanechol enhanced parasympathetically mediated vasodilation, but only in rats studied at 6 weeks after sympathectomy, a finding consistent with the appearance of muscarinic prejunctional facilitation of nitrergic transmission. In chronically sympathectomized rats, the M(2) and M(4) receptor antagonists methoctramine and tropicamide did not affect choroidal flow during parasympathetic activation. However, pirenzepine increased flux, implying the presence of M(1) inhibitory autoreceptors on these nerves. Parasympathetically mediated increased flux was partially blocked by the M(3) antagonist 4-diphenylacetoxy-N-methylpiperdine, and the remaining vasodilation was blocked by atropine. We conclude that parasympathetic prejunctional facilitatory M(3) and probably M(5) receptors adopt a crucial role after chronic sympathectomy in maintaining nitrergic vasodilatory ocular neurotransmission in the face of down-regulated nitric oxide transmitter mechanisms.

Topics: Animals; Bethanechol; Choroid; Electric Stimulation; Enzyme Inhibitors; Female; Ganglionectomy; Imidazoles; Muscarinic Agonists; Muscarinic Antagonists; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Parasympathetic Nervous System; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Receptors, Presynaptic; Regional Blood Flow; Salivary Glands; Superior Cervical Ganglion; Synaptic Transmission; Time Factors

This study examined whether myenteric neurons activate submucosal vasodilator pathways in in vitro combined submucosal-myenteric plexus preparations from guinea pig ileum. Exposed myenteric ganglia were electrically stimulated, and changes in the outside diameter of submucosal arterioles were monitored in adjoining tissue by videomicroscopy. Stimulation up to 18 mm from the recording site evoked large TTX-sensitive vasodilations in both orad and aborad directions. In double-chamber baths, which isolated the stimulating myenteric chamber from the recording submucosal chamber, hexamethonium or the muscarinic antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperdine hydrochloride (4-DAMP) almost completely blocked dilations when superfused in the submucosal chamber. When hexamethonium was placed in the myenteric chamber approximately 50% of responses were hexamethonium sensitive in both orad and aboard orientations. The addition of 4-DAMP or substitution of Ca(2+)-free, 12 mM Mg(2+) solution did not cause further inhibition. These results demonstrate that polysynaptic pathways in the myenteric plexus projecting orad and aborad can activate submucosal vasodilator neurons. These pathways could coordinate intestinal blood flow and motility.

Topics: Animals; Arterioles; Calcium; Ganglionic Blockers; Guinea Pigs; Hexamethonium; Ileum; Intestinal Mucosa; Magnesium; Myenteric Plexus; Neurons; Parasympatholytics; Piperidines; Reflex; Serotonin; Tetrodotoxin; Vasodilation

The resting membrane potential (RMP) of denervated muscle fibres of rat diaphragm muscle is depolarized by approximately 8-10 mV during the first 3 h after nerve section and this early postdenervation depolarization is reduced substantially by the presence of 5x10(-8) M acetylcholine (ACh) or carbachol (CB). The muscarinic antagonist atropine (Atr; 5x10(-9) to 5x10(-6) M) reduced the effect of CB in a dose-dependent manner (K(i)=7x10(-8) M) and increased the rate of the early postdenervation depolarization. In lower doses (5x10(-7) M), Atr acted only in the presence of an allosteric stabilizator hexamethylene-bis-[dimethyl-(3-phtalimidopropyl)ammonium] (W-84). Also pirenzepine, a specific inhibitor of the M1 subtype of muscarinic receptor, blocked the action of CB in a dose-dependent manner with an apparent inhibition constant K(i)=1x10(-7) microM. DAMP, a specific M3 antagonist, was without effect on the muscle hyperpolarization induced by CB. CB also hyperpolarized the membrane potentials of muscles which were denervated for 1-3 days. It is concluded that ACh and CB protect the muscle fibres from early depolarization through M1-cholinergic receptors on the muscle membrane. These particular receptors can apparently mediate the 'trophic', non-impulse regulation of RMP in skeletal muscles when they are activated by acetylcholine released non-quantally.

Topics: Acetylcholine; Animals; Arecoline; Atropine; Carbachol; Culture Techniques; Diaphragm; Male; Membrane Potentials; Muscarinic Agonists; Muscarinic Antagonists; Muscle Denervation; Muscle Fibers, Skeletal; Oxotremorine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptors, Muscarinic

Spontaneous waves of excitation in the developing mammalian retina are mediated, to a large extent, by neurotransmission. However, it is unclear how the underlying neurotransmitter systems interact with each other to play specific roles in the formation of retinal waves at various developmental stages. In particular, it is puzzling why the waves maintain a similar propagation pattern even after underlying neurotransmitter systems have undergone drastic developmental changes. Using Ca(2+) imaging and patch clamp in a whole-mount preparation of the developing rabbit retina, we discovered two dramatic and coordinated transitions in the excitatory drive for retinal waves: one from a nicotinic to a muscarinic system, and the other from a fast cholinergic to a fast glutamatergic input. Retinal waves before the age of postnatal day 1 (P1) were blocked by nicotinic antagonists, but not by muscarinic or glutamatergic antagonists. After P3, however, the spontaneous wave, whose basic spatiotemporal pattern remained similar, was completely inhibited by muscarinic or glutamate antagonists, but not by nicotinic antagonists. We also found that the muscarinic drive, mediated primarily by M1 and M3 receptors, was particularly important for wave propagation, whereas the glutamatergic drive seemed more important for local excitation. Our results suggest (1) a novel mechanism by which a neurotransmitter system changes its functional role via a switch between two completely different classes of receptors for the same transmitter, (2) the cholinergic system plays a critical role in not only early but also late spontaneous waves, and (3) the continued participation of the cholinergic system may provide a network basis for the consistency in the overall propagation pattern of spontaneous retinal waves.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Animals, Newborn; Atropine; Bungarotoxins; Calcium; Curare; Dimethylphenylpiperazinium Iodide; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Fura-2; Hexamethonium; Membrane Potentials; Muscarine; Muscarinic Antagonists; Neurotransmitter Agents; Nicotinic Antagonists; Patch-Clamp Techniques; Piperidines; Rabbits; Retina; Retinal Ganglion Cells

1. Experiments were done to determine the influence of gender and the oestrous cycle on rat urinary bladder contractility in response to cholinergic stimulation. 2. Bladder strips from female rats responded to high frequency stimulation with smaller contractile responses than did strips from males, and to low concentrations of carbachol with greater responses. The decreased responsiveness of bladder strips from female rats to electrical field stimulation can be primarily attributed to the rats in the oestrous stage of the oestrous cycle. 3. Bladder strips from female rats in all stages of the oestrous cycle were more sensitive to carbachol than those from males, but there were no differences in sensitivity to electrical field stimulation. 4. The contractile responses of strips from both male and female rats to carbachol were antagonized by muscarinic antagonists with the following rank order of affinity (pA(2)) estimates: 4-DAMP>>pirenzepine>methoctramine, suggesting that the receptor mediating contraction was the M3 subtype. There were no differences in pA(2) values between bladder strips from male and female rats. 5. The data indicate that responsiveness of bladder strips to electrical field stimulation and carbachol is altered in female rats in the oestrous stage of the oestrous cycle. Furthermore, gender influences the sensitivity of rat bladder to muscarinic stimulation.

Topics: Animals; Carbachol; Cholinergic Agonists; Diamines; Electric Stimulation; Estrus; Female; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Sex Characteristics; Time Factors; Urinary Bladder; Uterine Contraction

We have compared the efficacy of cromakalim and nifedipine to inhibit acetylcholine (ACh) and pilocarpine-induced tonic contractions in control preparations and in tissues where a fraction of the muscarinic receptor population had been removed by alkylation with phenoxybenzamine (PBZ). Both agonists induced contractions by stimulating pharmacologically similar receptors, probably of the M3 muscarinic subtype. The receptor reserve was larger, and the coupling between stimulation and contraction (E-C coupling) more efficient when ACh was the stimulating agonist. For stimulations that produced equal levels of muscle response, cromakalim was more efficacious in inhibiting contractions induced by pilocarpine. The efficacy of cromakalim in relaxing contractions induced by ACh increased when the number of functional receptors decreased. Cromakalim and nifedipine decreased the efficiency of E-C coupling for ACh and pilocarpine. Cromakalim efficacy decreased in a sigmoid manner when stimulating concentrations of ACh (and receptor occupancy) increased, and there was an inverse relationship between receptor occupancy by ACh and cromakalim efficacy. In the presence of TEA, a K+ channel blocker, nifedipine almost completely inhibited contractions induced by the M3 muscarinic agonist bethanechol. These data indicate that in bovine tracheal smooth muscle, electro-mechanical coupling is an inherent part of muscarinic E-C coupling, but its functional expression is dependent upon the efficacy of stimulation. The data also suggest that the M3 receptor is coupled to a cellular pathway linked with the activation of K+ channels that exerts a potent functional antagonism against activation of voltage-dependent Ca2+ entry.

Topics: Acetylcholine; Animals; Calcium; Cattle; Cromakalim; Diamines; Dose-Response Relationship, Drug; Drug Interactions; In Vitro Techniques; Muscarinic Agonists; Muscle Contraction; Muscle, Smooth; Nifedipine; Phenoxybenzamine; Pilocarpine; Piperidines; Pirenzepine; Receptors, Muscarinic; Stress, Mechanical; Tetraethylammonium; Trachea

This study examined the neural pathways innervating Brunner's glands using a novel in vitro model of acinar secretion from Brunner's glands in submucosal preparations from the guinea pig duodenum. Neural pathways were activated by focal electrical stimulation and excitatory agonists, and videomicroscopy was used to monitor dilation of acinar lumen. Electrical stimulation of perivascular nerves evoked large dilations that were blocked by TTX (1 microM) or the muscarinic receptor antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (1 microM). The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (100 microM) had no effect, and the nerve-evoked responses were not inhibited by hexamethonium (200 microM). Dilations were abolished in preparations from chronically vagotomized animals. Activation of submucosal ganglia significantly dilated submucosal arterioles but not Brunner's glands. Effects of electrical stimulation of perivascular and submucosal nerves were not altered by guanethidine. Capsaicin and substance P also dilated arterioles but had no effect on Brunner's glands. Cholinergic (choline acetyltransferase-immunoreactive) nerve fibers were found in Brunner's glands. These findings demonstrate that Brunner's glands are innervated by cholinergic vagal fibers but not by capsaicin-sensitive or intrinsic enteric nerves.

Topics: Animals; Brunner Glands; Capsaicin; Carbachol; Choline O-Acetyltransferase; Cholinergic Agonists; Cholinergic Fibers; Dimethylphenylpiperazinium Iodide; Duodenum; Electric Stimulation; Female; Ganglia, Sympathetic; Guanethidine; Guinea Pigs; Hexamethonium; In Vitro Techniques; Male; Muscarinic Antagonists; Neural Pathways; Neurotransmitter Agents; Nicotinic Agonists; Nicotinic Antagonists; Piperidines; Submucous Plexus; Substance P; Sympatholytics; Tetrodotoxin; Vagotomy; Vagus Nerve

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10-14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific alpha-actin. mRNA encoding muscarinic receptor subtypes M(1)-M(5) was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura 2 fluorescence. Basal [Ca(2+)](i), which was 135 +/- 22 nM, increased transiently to 543 +/- 29 nM in response to 10 microM ACh in CM cells (n = 8). This response was decreased <95% by 0.01 microM 4-diphenylacetoxy-N-methylpiperidine, a M(1)/M(3)-selective antagonist, whereas 0.1 microM methoctramine, a M(2)/M(4)-selective antagonist, and 0.1 microM pirenzepine, a M(1)-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca(2+)](i) mediated primarily by the M(3) receptor and involving release of Ca(2+) from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Acetylcholine; Blotting, Western; Calcium; Calcium Channel Agonists; Calcium Signaling; Cells, Cultured; Diamines; DNA Primers; Esophagus; Humans; Immunohistochemistry; Muscarinic Antagonists; Muscle Fibers, Skeletal; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Receptor, Muscarinic M5; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasodilator Agents

1. In urinary bladder, M(2)-muscarinic receptors predominate, but it is the smaller population of M(3)-receptors which mediate detrusor contraction. This study examines the M(2) : M(3) ratio and the role of M(2)-receptors in contraction of pig urinary bladder. 2. Competition experiments with [(3)H]-QNB determined the ratio of M(2) : M(3). In functional studies, affinity values (pK(B)) for 4-DAMP, darifenacin and methoctramine were calculated. Similar experiments were performed on tissues following selective M(3)-inactivation (incubation with 40 nM 4-DAMP mustard in the presence of 1 microM methoctramine to protect M(2)-receptors), precontraction with 50 mM KCl and relaxation with isoprenaline (30 microM) or forskolin (1 microM). 3. In competition binding, displacement of [(3)H]-QNB by 4-DAMP, darifenacin and methoctramine best fitted a two-site model suggesting a predominant (70 - 80%) population of M(2)-receptors. 4. On normal detrusor in vitro, 4-DAMP and methoctramine caused surmountable antagonism of responses to carbachol with pK(B) values of 9.37+/-0.07 and 6.05+/-0.05 respectively. Darifenacin caused unsurmountable antagonism, the apparent pK(B) value being 8.61+/-0.10. 5. In tissues where the M(3)-receptors had been inactivated and cyclic AMP levels elevated, 4-DAMP and darifenacin were less potent, with apparent pK(B) values of 8.72+/-0.08 and 6.74+/-0.07. In contrast, methoctramine was more potent, the apparent pK(B) value increasing significantly to 6.86+/-0.06. 6. se data suggest that the pig bladder possesses a similar muscarinic receptor population to the human bladder and that the M(3)-receptor subtype mediates contraction of the normal detrusor muscle. However an involvement of M(2)-receptors in contraction can be observed following pharmacological manipulation of the receptor population.

Topics: Animals; Benzofurans; Binding, Competitive; Carbachol; Diamines; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Membranes; Muscarinic Antagonists; Muscle Contraction; Piperidines; Pyrrolidines; Quinuclidinyl Benzilate; Radioligand Assay; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Swine; Tritium; Urinary Bladder

1. The objective of the study was to determine the role of muscarinic receptor subtypes in mediating contraction of the porcine detrusor smooth muscle in vitro. 2. Strips of pig detrusor muscle were set up in physiological salt solution and the tensions developed by the tissues were recorded. Responses to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (4-DAMP, methoctramine, darifenacin, oxybutynin, tolterodine and pirenzepine). Antagonist affinity values (pKB values) were calculated and compared with those quoted in the literature for these antagonists at each of the muscarinic receptor subtypes. 3. The M3-selective antagonists, 4-DAMP and darifenacin had high affinities (pKB values of 9.4 and 8.6, respectively). Oxybutynin, tolterodine and pirenzepine had affinities of 8.2, 8.1 and 6.8, respectively, whilst the M2-selective agent methoctramine had a relatively low affinity (pKB = 6.1). The rank order of affinities was, therefore, 4-DAMP > darifenacin > oxybutynin > tolterodine > pirenzepine > methoctramine for the pig detrusor. Correlation of the antagonist affinities obtained on the bladder with those published for these antagonists at the five muscarinic receptor subtypes identified the M3(m3)-receptor as the muscarinic subtype mediating detrusor contractile responses in vitro. 4. These data suggest that a small population of M3-muscarinic receptors must mediate direct contractile responses of the pig detrusor muscle to muscarinic receptor stimulation in vitro.

Topics: Animals; Carbachol; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Swine; Urinary Bladder

The effects of acute exercise on receptor-mediated endothelium-dependent vasodilation and its possible mechanisms were investigated in the presence of indomethacin. Male Wistar rats (16-20 weeks old) were divided into control and exercise groups. The exercise group ran on a drum exerciser until exhaustion, followed by immediate decapitation. Acetylcholine (ACh)- or clonidine (CLO)-induced vasodilating responses in thoracic aortae of the control and exercise groups were compared. Receptor-binding assays were performed to determine whether there were any upregulations of endothelial receptors after acute exercise. Our results indicated that acute exercise induced the following effects: (1) the dose-response curves of ACh and CLO shifted to the left; (2) the high-affinity M3 binding sites increased in number but not in affinity; (3) the alpha2 binding sites decreased in number but increased in affinity. We conclude that acute exercise enhances receptor-mediated vasodilation responses, at least in part, by regulating either endothelial receptor number or receptor affinity.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Animals; Clonidine; Endothelium, Vascular; In Vitro Techniques; Male; Muscarinic Antagonists; Physical Conditioning, Animal; Piperidines; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Adrenergic, alpha-2; Receptors, Muscarinic; Thoracic Arteries; Tritium; Up-Regulation; Vasodilation

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats which had their major pelvic ganglion bilaterally removed (denervation, DEN) or from rats in which the spinal cord was injured (SCI) via compression. DEN induced both hypertrophy (505+/-51 mg bladder weight) and a supersensitivity of the bladders to carbachol (EC50=0.7+/-0.1 uM). Some of the SCI rats regained the ability to void spontaneously (SPV). The bladders of these animals weighed 184+/-17 mg, significantly less than the bladders of non voiding rats (NV, 644+/-92 mg). The potency of carbachol was greater in bladder strips from NV SCI animals (EC50=0.54+/-0.1 uM) than either bladder strips from SPV SCI (EC50=0.93+/-0.3 microM), DEN or control (EC50=1.2+/-0.1 microM) animals. Antagonist affinities in control bladders for antagonism of carbachol induced contractions were consistent with M3 mediated contractions. Antagonist affinities in DEN bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.5) and para fluoro hexahydrosilodifenidol (p-F-HHSiD, 6.6); were consistent with M2 mediated contractions, although the methoctramine affinity (6.5) was consistent with M3 mediated contractions. p-F-HHSiD inhibited carbachol induced contraction with an affinity consistent with M2 receptors in bladders from NV SCI (pKb=6.4) animals and M3 receptors in bladders from SPV SCI animals (pKb=7.9). Subtype selective immunoprecipitation of muscarinic receptors revealed an increase in total and an increase in M2 receptor density with no change in M3 receptor density in bladders from DEN and NV SCI animals compared to normal or sham operated controls. M3 receptor density was lower in bladders from SPV SCI animals while the M2 receptor density was not different from control. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediate smooth muscle contraction in bladders from DEN and NV SCI rats.

Topics: Animals; Binding Sites; Carbachol; Diamines; Dose-Response Relationship, Drug; Female; Hypertrophy; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle Denervation; Muscle, Smooth; Organ Size; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptors, Muscarinic; Spinal Cord Injuries; Urinary Bladder; Urinary Bladder, Neurogenic

Arteriolar relaxation caused by the application of muscarinic agonists is mediated by multiple factors. One factor causes dilation only at the point of drug microapplication (local response), and a second factor causes responses remote (500 microm away) from the site of application (conducted response). This study was performed to determine if different muscarinic subtypes mediate the two responses. Arterioles of anesthetized hamster cheek pouch were studied with videomicroscopy. Muscarinic antagonists methscopolamine, scopolamine, pirenzepine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), and AFDX-116 [(11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepin-6-one)] were cumulatively applied, and the K(B) for each antagonist was determined for the local and conducted responses caused by methacholine microapplication (10(-4) M, 5 s). The pK(B) (local, conducted) were not significantly different for the two responses when using scopolamine (10.5, 10.4). When the antagonist AFDX-116 (5.6, 6.3), selective for muscarinic receptor (m2) subtype was applied, the K(B) was greater for the conducted response. The pK(B) was greater, however, for the local response when the m1 subtype-selective pirenzepine (7.7, 6.9) or m3 subtype-selective 4-DAMP (10.1, 9.8) was applied. Thus the antagonist pK(B) ratio for on the local and conducted responses depends on the subtype selectivity of the antagonist. These data strongly suggest that different receptors are involved in the two responses.

Topics: Animals; Arterioles; Cheek; Cricetinae; Dose-Response Relationship, Drug; Microcirculation; Mouth Mucosa; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptors, Muscarinic; Reference Values; Scopolamine; Vasodilation

The aim of this study was to characterize the modulation of synaptic transmission in the glutamatergic corticostriatal pathway by cholinergic and adrenergic receptors. In coronal slices of mouse brain, negative-going field potentials were recorded in the dorsal striatum in response to stimulation of the overlying white matter, and their susceptibility to various pharmacological manipulations was studied. The responses were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors, since they were augmented by aniracetam (0.5-1.5 mM), a positive modulator of AMPA-type glutamate receptors, and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (> or = 10 microM), a selective antagonist of AMPA receptors. Carbachol (10 microM), a muscarinic agonist, reduced the size of responses and abolished paired-pulse depression; these effects being consistent with previous studies indicating that muscarinic activation inhibits release of glutamate in the corticostriatal pathway. Muscarinic antagonists could block the effect of carbachol. Their rank order was: 10 microM scopolamine (a non-selective muscarinic antagonist) > or = 1 microM 4-diphenylacetoxy-N-methyl-piperidine (M3/M1 antagonist)>1 microM pirenzepine (M1 antagonist)>10 microM methoctramine (M2 antagonist). McN-A-343 (1-10 microM), an M1 muscarinic agonist, was ineffective in this preparation. In contrast, isoproterenol (10-30 microM), a beta-adrenergic agonist, slightly increased the synaptic responses, but it did not affect paired-pulse depression. None of alpha-adrenergic agents (30 nM-1.0 microM dexmedetomidine, an alpha2-adrenergic agonist, 0.3 microM atipamezole, an alpha2-adrenergic antagonist or 30 microM phenylephrine, an alpha1-adrenergic agonist) influenced the size of the responses; neither did these drugs alter paired-pulse depression. These results indicate that the activation of striatal M3-like muscarinic receptors and beta-adrenoceptors, but not M2-like muscarinic receptors and alpha-adrenoceptors, modulates directly corticostriatal glutamatergic neurotransmission.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Adrenergic beta-Agonists; Adrenergic Fibers; Animals; Carbachol; Cerebral Cortex; Cholinergic Fibers; Corpus Striatum; Diamines; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Imidazoles; Isoproterenol; Male; Medetomidine; Mice; Mice, Inbred DBA; Muscarinic Agonists; Muscarinic Antagonists; Phenylephrine; Piperidines; Pirenzepine; Pyrrolidinones; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Receptors, AMPA; Receptors, Muscarinic; Scopolamine; Synaptic Transmission

Partial outlet obstruction of the rat urinary bladder leads to hypertrophy and alteration in contractility of the detrusor muscle involving changes in muscarinic receptors. m3 muscarinic receptor subtype has been known to play a predominant role in contractility of normal urinary bladder. The purpose of the present study was to assess the role of m3 receptors in contractility of the obstructed bladder.. In male rats, partial outlet obstruction of the urinary bladder was performed by surgically tying a 6-0 suture around the bladder neck, reducing the diameter of it by 2/3 of the original size. Four weeks after the surgery, the bladders were removed and thin strips were microdissected. Similarly, bladder strips from age matched unoperated normal rats were obtained. Sets of four strips from four normal or four obstructed rats were mounted in an in vitro multi-muscle chamber containing normal physiological solution at 37C. The tension responses evoked by optimal electrical field stimulation at 1, 10, 30, 50, and 100 Hz, and the contracture responses evoked by 120 mM potassium and 0.01 to 300.0 microM carbachol were recorded using a Nicolet digital oscilloscope. Similar responses were recorded in different sets of four strips following exposure to 10 and 100 nM 4-DAMP, which is a muscarinic antagonist with a high affinity for m3 and m1 receptor subtypes.. The obstructed bladders showed 119% increase in weight. In control physiological solution, the obstructed bladder strips did not show significant difference in electrically-evoked tension or carbachol contractures, but showed significantly lower potassium contractures compared with normal bladder strips. 4-DAMP at 10 to 100 nM significantly reduced the electrically evoked tension responses by about the same degree in normal and obstructed bladders, without affecting the potassium contractures. It significantly increased the EC50 values for carbachol contractures in normal bladder, and to a significantly lesser extent in obstructed bladder. Schild plots using the Hill transformed EC50 values showed that the pA2 value for 4-DAMP was not significantly different in normal and obstructed bladders.. Significantly smaller potassium contracture in the obstructed bladder indicates that depolarizability of the detrusor muscle membrane, and consequently the activity of voltage-gated Ca2+ channels may be reduced in the detrusor after partial outlet obstruction. Lack of a significant difference in the effect of 4-DAMP on the electrically evoked tension responses and in the pA2 values for 4-DAMP assessed by carbachol contractures, in normal and obstructed bladder strips, indicates that m3 muscarinic receptors still play a predominant role in causing detrusor contractility in the obstructed bladder, as in the normal bladder.

Topics: Animals; Carbachol; Dose-Response Relationship, Drug; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Urinary Bladder Neck Obstruction

Choline is a necessary substrate of the lipid membrane and for acetylcholine synthesis. Accumulating evidence indicates that besides being a structural component, choline is also a functional modulator of the membrane. It has been shown to be a muscarinic acetylcholine receptor (mAChR) agonist and can induce a novel K+ current in cardiac cells. However, the potential role of choline in modulating cardiac functions remained unstudied despite that mAChRs are known to be important in regulating heart functions. With microelectrode techniques, we found that choline produced concentration-dependent (0.1 approximately 10 mm) decreases in sinus rhythm and action potential duration in isolated guinea pig atria. The effects were reversed by 2 nm 4DAMP (an M3-selective antagonist). Whole-cell patch-clamp recordings in dispersed myocytes from guinea pig and canine atria revealed that choline is able to induce a K+ current with delayed rectifying properties. The choline-induced current was suppressed by low concentrations of 4DAMP (2 approximately 10 nm). Antagonists toward other subtypes (M1, M2 or M4) all failed to alter the current. The affinity of choline (Kd) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 0.9 mm, consistent with the concentration needed for the current induction and for the HR and APD modulation. Our data indicate that choline modulates the cellular electrical properties of the hearts, likely by activating a K+ current via stimulation of M3 receptors.

Topics: Animals; Choline; Dogs; Guinea Pigs; Heart; Heart Atria; In Vitro Techniques; Membrane Potentials; Muscarinic Antagonists; Myocardium; Patch-Clamp Techniques; Piperidines; Potassium Channels; Receptor, Muscarinic M3; Receptors, Muscarinic

It is well known that chronic topical administration of cholinergic agonists results in a subsensitization in ciliary muscle-mediated increases in outflow facility and accommodation in monkey eyes in vivo. These physiologic changes are apparently mediated by the M3 subtype receptor. However, the nature of this subsensitization remains unclear. This study investigated the effect of the continuous presence of carbachol, a muscarinic agonist, on the expression of the muscarinic receptor subtype m3 and the binding of [3H]4-DAMP in cultured human ciliary muscle cells (H7CM). The H7CM cell line, derived from the ciliary muscle of a one-day-old human infant, was used in this study. Confluent monolayers were treated individually with 1 mM carbachol for 2, 6, 24 and 48 hours. The level of mRNA encoding muscarinic receptor subtype m3 was measured by RNase protection. For confirmation, a receptor binding assay was done using [3H]4-DAMP, a radioligand selective for M3 subtype receptors. At each timepoint, results were compared with untreated controls. Treatment with carbachol resulted in a down regulation ranging from 23.4% to 34.8% of m3 mRNA expression at all time points. All [3H]4-DAMP binding assay results also decreased, ranging from 24.5% to 31.0%.

Topics: Carbachol; Cells, Cultured; Cholinergic Agonists; Ciliary Body; Humans; Muscarinic Antagonists; Piperidines; Receptor, Muscarinic M3; Receptors, Muscarinic; RNA, Messenger

The aim of this study was to determine the functional role of muscarinic receptor subtypes regulating gallbladder cholinergic contractions. Electrical field stimulation (EFS; 16 Hz) produced contractile responses of guinea pig gallbladder muscle strips in vitro that were inhibited by 1 microM tetrodotoxin (2 +/- 2% of control) and 1 microM atropine (1 +/- 1% of control), indicating activation of intrinsic cholinergic nerves. Exogenous ACh (5 microM)-induced contractions were inhibited by atropine (1 +/- 1% of control) but not tetrodotoxin (102 +/- 1% of control), indicating a direct effect on smooth muscle. The M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh-induced contractions but inhibited EFS-induced contractions by 11 +/- 3%. The M2 antagonist methoctramine (10 nM) had no effect on ACh-induced contractions but augmented EFS-induced contractions by 5 +/- 2%. The M3 antagonist 4-DAMP (10 nM) inhibited ACh-induced contractions by 14 +/- 4% and EFS-induced contractions by 22 +/- 5%. In conclusion, specific M1, M2, and M3 receptors modulate gallbladder muscle contractions by regulating ACh release from cholinergic nerves and mediating the contraction. Cholinergic contractions are mediated by M3 receptors directly on the smooth muscle. M2 receptors are on cholinergic nerves and function as prejunctional inhibitory autoreceptors. M1 receptors are on cholinergic nerves and function as prejunctional facilitatory autoreceptors.

Topics: Acetylcholine; Animals; Atropine; Diamines; Electric Stimulation; Gallbladder; Guinea Pigs; Hexamethonium; Muscarinic Antagonists; Muscle Contraction; Parasympatholytics; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Tetrodotoxin; Tropicamide

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.

Topics: Alkylation; Animals; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Rats; Receptors, Muscarinic; Signal Transduction; Urinary Bladder

Growing body of evidence indicates that the functional responses of cells to muscarinic acetylcholine receptors (mAChRs) are mediated by multiple receptor subtypes. It is commonly thought that the M2 receptor is the only functional mAChR subtype in the heart and little data regarding the potential roles of other subtypes in cardiac tissues has been reported. In the present study, we provide functional evidence for the presence and physiological function of an M3 receptor in canine atrial myocytes. Using whole-cell patch-clamp techniques, we consistently found that pilocarpine, an mAChR agonist, induced a K+ current similar to but distinct from the classical delayed rectifier K+ current. Same observations were obtained when choline or tetramethylammonium (TMA) was applied to the bath. The currents were abolished by 1 microM atropine. Antagonists selective to M1 (pirenzepine, 100 nM), M2 (methoctramine 100 nM), or M4 (tropicamide 200 nM) receptors failed to alter the currents. Conversely, three different M3-selective inhibitors, p-F-HHSiD (20-200 nM), 4-DAMP methiodide (2-10 nM) and 4-DAMP mustard (4-20 nM), all produced concentration-dependent suppression of the currents. A cDNA fragment representing the M3 receptor was isolated from dog atrial RNA and the mRNA level of this construct was 0.7 +/- 0.1 pg/microg total RNA, as quantified by the competitive RT-PCR methods. Our data strongly suggested that an M3 receptor exists and is coupled to a K+ channel in the heart.

Topics: Animals; Dogs; Heart; Membrane Potentials; Piperidines; Potassium Channels; Receptor, Muscarinic M3; Receptors, Muscarinic; Tetraethylammonium Compounds

The effects of carbachol (0.01-30 microM) and muscarine (10-30 microM) on the excitatory synaptic potentials were studied using conventional intracellular recordings from dopaminergic neurons in rat mesencephalic slices. Both muscarinic agonists reversibly reduced the excitatory synaptic potentials, evoked by local electrical stimulation. The EC50 for carbachol was determined to be 4.5 microM. The maximal degree of the excitatory synaptic potentials suppression caused by carbachol and muscarine was around 40% of control. This suppression was completely blocked by the non-specific muscarinic antagonist atropine (1 microM) and the selective M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). Other antagonists, preferentially acting at M1, M2 and M4 receptors, were not effective. Furthermore, the acetylcholinesterase inhibitor, physostigmine (50 microM), decreased the amplitude of the excitatory synaptic potentials, indicating that ambient acetylcholine can depress this potential. Direct depolarizing responses to glutamate were not changed by muscarine. In addition, muscarine facilitated the second excitatory synaptic potentials during a paired-pulse protocol. Thus, the effect of the muscarinic agonists is attributable to a presynaptic locus of action. The action of muscarine was not mediated by an N-ethylmaleimide-sensitive G-protein since it was not modified by a treatment of the slices with this agent. The calcium channels blockers, omega-conotoxin GIVA, omega-agatoxin IVA and omega-conotoxin MVIIC did not affect the action of muscarine on the excitatory synaptic potentials. When the potassium currents were reduced by extracellular barium and 4-aminopyridine, the muscarinic agonists still depressed the excitatory synaptic potentials. Our data indicate that presynaptically located M3 receptors modulate the excitatory transmission to midbrain dopaminergic neurons via a N-ethylmaleimide-insensitive G-protein which activates mechanisms neither linked to N-, P-, Q-type calcium channels nor to barium- and 4-aminopyridine-sensitive potassium channels.

Topics: 4-Aminopyridine; Acetylcholine; Animals; Atropine; Carbachol; Diamines; Dicyclomine; Dopamine; Electric Stimulation; Excitatory Postsynaptic Potentials; Mesencephalon; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Neurons; Parasympatholytics; Physostigmine; Piperidines; Pirenzepine; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Rats, Wistar; Reaction Time; Receptor, Muscarinic M3; Receptors, Muscarinic; Synaptic Transmission; Tetrodotoxin

1. Pilocarpine, a muscarinic acetylcholine receptor (mAChR) agonist, is widely used for treatment of xerostomia and glaucoma. It can also cause many other cellular responses by activating different subtypes of mAChRs in different tissues. However, the potential role of pilocarpine in modulating cardiac function remained unstudied. 2. We found that pilocarpine produced concentration-dependent (0.1-10 microM) decrease in sinus rhythm and action potential duration, and hyperpolarization of membrane potential in guinea-pig hearts. The effects were nearly completely reversed by 1 microM atropine or 2 nM 4DAMP methiodide (an M3-selective antagonist). 3. Patch-clamp recordings in dispersed myocytes from guinea-pig and canine atria revealed that pilocarpine induces a novel K+ current with delayed rectifying properties. The current was suppressed by low concentrations of M3-selective antagonists 4DAMP methiodide (2-10 nM), 4DAMP mustard (4-20 nM, an ackylating agent) and p-F-HHSiD (20-200 nM). Antagonists towards other subtypes (M1, M2 or M4) all failed to alter the current. 4. The affinity of pilocarpine (KD) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 2.2 microM (65% of the total binding) and that of 4DAMP methiodide was 2.8 nM (70% of total binding), consistent with the concentration of pilocarpine needed for the current induction and for the modulation of the cardiac electrical activity and the concentration of 4DAMP to block pilocarpine effects. 5. Our data indicate, for the first time, that pilocarpine modulates the cellular electrical properties of the hearts, likely by activating a K+ current mediated by M3 receptors.

Topics: Action Potentials; Animals; Atrial Function; Binding, Competitive; Dogs; Electrophysiology; Female; Guinea Pigs; Heart; Heart Atria; Heart Rate; Male; Membrane Potentials; Microelectrodes; Muscarinic Agonists; Muscarinic Antagonists; Myocardium; N-Methylscopolamine; Parasympatholytics; Pilocarpine; Piperidines; Potassium Channels; Receptor, Muscarinic M3; Receptors, Muscarinic; Time Factors

The effects of the muscarinic cholinoceptor antagonists atropine (non-selective), pirenzepine (M1-selective), methoctramine (M2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M3-selective) were examined on the responsiveness of guinea pig and rat tracheal tissue to acetylcholine and carbachol. Results indicate that smooth muscle contraction in isolated tracheal tissue from both species was mediated primarily by muscarinic M3 cholinoceptors. The effects of atropine, pirenzepine and 4-DAMP were similar against the contractile actions of acetylcholine and carbachol in both species and in epithelium-intact and epithelium-denuded tissue. In contrast, differences in the effects of methoctramine in antagonising contractile responses to acetylcholine and carbachol were observed between the two species and following epithelium removal in the guinea pig. Thus, whilst this study has found that tracheal smooth muscle contraction in the guinea pig and rat is mediated primarily by muscarinic M3 cholinoceptors, anomalies in the functional inositol phosphate generation results obtained with the muscarinic cholinoceptor antagonists highlight species differences in the actions of acetylcholine and carbachol in eliciting smooth muscle contraction suggesting the possible existence of functional non-M3 muscarinic cholinoceptors.

Topics: Animals; Atropine; Diamines; Guinea Pigs; In Vitro Techniques; Inositol Phosphates; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Trachea

Injection of acetylcholine (ACh) (2.5-60 nmol) into the anterior cingulate cortex caused dose-dependent hypotensive responses (Emax = -25.3 mmHg) and no change in the heart rate. The hypotensive response to 30 nmol of ACh was blocked by local pretreatment with atropine (3 nmol) or 4-DAMP (6.7 nmol), a non-tropine muscarinic antagonist. When the same dose of atropine was injected i.v., no changes were observed in the hypotensive response to intracortical ACh. This observation rules out the possible leakage of ACh into the peripheral circulation and favors the idea of a cortical site of action. The injection of the same dose of ACh into the corpus callosum or the occipital cortex did not cause changes in the cardiovascular system. The present results confirm earlier evidence that the cingulate cortex is involved in the control of the autonomic system and indicate that cholinergic muscarinic receptors in the cingulate cortex mediate a hypotensive response without a change in heart rate.

Topics: Acetylcholine; Animals; Atropine; Blood Pressure; Dose-Response Relationship, Drug; Gyrus Cinguli; Hemodynamics; Injections; Male; Muscarinic Antagonists; Occipital Lobe; Piperidines; Prefrontal Cortex; Rats; Rats, Wistar

We tested the effect of cholinergic drugs on the concentration of intracellular free calcium in rat melanotropes. Acetylcholine, muscarine, carbachol, and nicotine resulted in a significant rise in this parameter. Effect of acetylcholine was reduced by atropine (non-selective muscarinic antagonist), pirenzepine (M1 muscarinic antagonist), and 4-DAMP (M3 > M1 muscarinic antagonist), but exposure to the M1 muscarinic agonist McN-A 343 resulted in a significantly smaller calcium-response than that seen in response to acetylcholine or to muscarine. This suggests the involvement of both M1 and M3 muscarinic receptors in the acetylcholine-induced calcium-rise. On the other hand, in the presence of atropine the acetylcholine-induced calcium-rise was not eliminated: this fact indicates that nicotinic receptors are also involved in the acetylcholine-induced intracellular calcium-rise. The acetylcholine-, and nicotine-induced calcium-rise was significantly reduced in presence of the neuronal-type nicotinic antagonist, mecamylamine. This suggests the involvement of a neuronal-type nicotinic receptor in the acetylcholine-induced intracellular calcium-response. Moreover, because in a further experiment almost 80% of the cells investigated responded to muscarine as well as nicotine, we conclude that both functionally active muscarinic and nicotinic receptors are present on the same cell.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Age Factors; Animals; Atropine; Calcium; Carbachol; Cells, Cultured; Cholinergic Agonists; Cytosol; Female; Male; Melanocytes; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Nicotine; Nicotinic Agonists; Piperidines; Pirenzepine; Pituitary Gland; Rats; Rats, Wistar; Vasodilator Agents

Rapid eye movement (REM) sleep is generated, in part, by activating muscarinic cholinergic receptors (mAChRs) in the medial pontine reticular formation (mPRF). Molecular cloning has identified five mAChR subtypes, and this study tested the hypothesis that the M2 subtype in the mPRF modulates the amount of REM sleep. This hypothesis cannot be tested directly, due to lack of subtype selective muscarinic agonists. However, the amount of REM sleep can be enhanced by mPRF microinjection of a muscarinic agonist, and the relative potencies of muscarinic antagonists to block the REM sleep enhancement can be determined. Two muscarinic antagonists, methoctramine and 4-DAMP, were studied. Six concentrations of each antagonist were microinjected into the mPRF of conscious cat 15 min prior to the agonist bethanechol. Nonlinear regression analysis was used to calculate the dose of antagonist that caused a 50% inhibition (ID50) of bethanechol-induced REM sleep. Bethanechol significantly increased (442%) the amount of time spent in REM sleep. Both methoctramine and 4-DAMP significantly blocked the bethanechol-induced REM sleep increase, with an ID50 of 1.8 microM and 0.6 microM, respectively. The ID50 ratio for methoctramine-to-4-DAMP (3.0) was similar to the affinity ratio of methoctramine-to-4-DAMP only at the M2 subtype (3.5), suggesting that the M2 subtype in the mPRF modulates the amount of REM sleep. This study also tested the null hypothesis that sleep-dependent respiratory depression evoked by mPRF cholinomimetics would not be antagonized by pretreatment of the mPRF with muscarinic antagonists. Neither methoctramine nor 4-DAMP antagonized the bethanechol-induced decrease in respiratory rate.

Topics: Animals; Bethanechol; Cats; Diamines; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pons; Receptors, Muscarinic; Respiration; Reticular Formation; Sleep, REM; Time Factors; Wakefulness

1. Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs. 2. Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half-maximal effective concentrations (EC50) were 6.6 +/- 0.4 and 784 +/- 4 nM, respectively. 3. Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 +/- 0.07 nM, whilst the IC50 for IK,cAMP inhibition was 5.9 +/- 0.2 microM. 4. Oxotremorine, a muscarinic agonist, preferentially stimulated IK, cAMP inhibition (EC50 = 15.8 +/- 1.4 microM), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 +/- 0.7 microM. 5. Fin elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling. 6. Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition. 7. In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype and the other the M3 subtype. These receptors are coupled to distinct membrane mechanisms leading to independent regulation of two membrane conductances.

Topics: Acetylcholine; Animals; Carbachol; Cholinergic Agonists; Dose-Response Relationship, Drug; Female; Follicle Stimulating Hormone; Gene Expression; Membrane Potentials; Muscarinic Antagonists; Oocytes; Ovarian Follicle; Oxotremorine; Patch-Clamp Techniques; Piperidines; Pirenzepine; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tropicamide; Xenopus laevis

Nonselective nitric oxide synthase (NOS) inhibition has detrimental effects in sepsis because of inhibition of the physiologically important endothelial NOS (eNOS). The authors hypothesized that selective inducible NOS (iNOS) inhibition would maintain eNOS vasodilation but prevent acetylcholine- and bradykinin-mediated vasoconstriction caused by lipopolysaccharide-induced endothelial dysfunction.. Rats were administered intraperitoneal lipopolysaccharide (15 mg/kg) with and without the selective iNOS inhibitors L-N6-(1-iminoethyl)-lysine (L-NIL, 3 mg/kg), dexamethasone (1 mg/kg), or the nonselective NOS inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 5 mg/kg). Six hours later, the lungs were isolated and pulmonary vasoreactivity was assessed with hypoxic vasoconstrictions (3% O2), acetylcholine (1 microg), Biochemical Engineering, and bradykinin (3 microg). In additional lipopolysaccharide experiments, L-NIL (10 microM) or 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 100 microM), a selective muscarinic M3 antagonist, was added into the perfusate.. Exhaled nitric oxide was higher in the lipopolysaccharide group (37.7+/-17.8 ppb) compared with the control group (0.4+/-0.7 ppb). L-NIL and dexamethasone decreased exhaled nitric oxide in lipopolysaccharide rats by 83 and 79%, respectively, whereas L-NAME had no effect. In control lungs, L-NAME significantly decreased acetylcholine- and bradykinin-induced vasodilation by 75% and increased hypoxic vasoconstrictions, whereas L-NIL and dexamethasone had no effect. In lipopolysaccharide lungs, acetylcholine and bradykinin both transiently increased the pulmonary artery pressure by 8.4+/-2.0 mmHg and 35.3+/-11.7 mmHg, respectively, immediately after vasodilation. L-NIL and dexamethasone both attenuated this vasoconstriction by 70%, whereas L-NAME did not. The acetylcholine vasoconstriction was dose-dependent (0.01-1.0 microg), unaffected by L-NIL added to the perfusate, and abolished by 4-DAMP.. In isolated perfused lungs, acetylcholine and bradykinin caused vasoconstriction in lipopolysaccharide-treated rats. This vasoconstriction was attenuated by administration of the iNOS inhibitor L-NIL but not with L-NAME. Furthermore, L-NIL administered with lipopolysaccharide preserved endothelium nitric oxide-dependent vasodilation, whereas L-NAME did not.

Topics: Acetylcholine; Animals; Bradykinin; Dexamethasone; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Lipopolysaccharides; Lung; Lysine; Male; Muscarinic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroprusside; Piperidines; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Salmonella typhimurium; Vasoconstriction

The contractile roles of the M2 and M3 muscarinic receptors were investigated in guinea pig longitudinal colonic smooth muscle. Prior treatment of the colon with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) (40 nM) in combination with [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116) (1.0 microM) caused a subsequent, irreversible inhibition of oxotremorine-M-induced contractions when measured after extensive washing. The estimate of the degree of receptor inactivation after 2 hr (97%) was not much greater than that measured after 1 hr (95%), which suggests that both 4-DAMP mustard-sensitive and -insensitive muscarinic subtypes contribute to the contractile response. Pertussis toxin treatment had no significant inhibitory effect on the control contractile response to oxotremorine-M, but caused an 8.8-fold increase in the EC50 value measured after a 2-hr treatment with 4-DAMP mustard. These results suggest that, after elimination of most of the M3 receptors with 4-DAMP mustard, the contractile response can be mediated by the pertussis toxin-sensitive M2 receptor. After pertussis toxin treatment, the kinetics of alkylation of muscarinic receptors in the colon were consistent with a single, 4-DAMP mustard-sensitive, M3 receptor subtype mediating the contractile response. When measured after a 2-hr treatment with 4-DAMP mustard and in the presence of histamine (0.30 microM) and either forskolin (10 microM) or isoproterenol (0.60 microM), the contractile responses to oxotremorine-M were pertussis toxin-sensitive and potently antagonized by the M2 selective antagonist, AF-DX 116. Collectively, our results indicate that the M2 receptor elicits contraction through two mechanisms, a direct contraction and an indirect contraction by preventing the relaxant effects of cAMP-generating agents.

Topics: Animals; Colforsin; Colon; Cyclic AMP; Guinea Pigs; Histamine; In Vitro Techniques; Isoproterenol; Male; Muscle Contraction; Oxotremorine; Pertussis Toxin; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Virulence Factors, Bordetella

Gastrin-releasing peptide (GRP) stimulates insulin secretion by a direct islet effect. In this study, we initially demonstrated, by immunocytochemistry of the mouse pancreas, GRP immunoreactive nerve fibers within exocrine tissue, islets, and intrapancreatic ganglia. A more pronounced GRP innervation was found in ganglia compared with in islets. We therefore studied whether indirect cholinergic mechanisms contribute to the insulinotropic action of GRP. In mice, the insulinotropic response to GRP (4.25 nmol/kg i.v.) was inhibited by the m3-selective, muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP, 0.21 mol/kg; by 68%, P < 0.05) and by the ganglionic blocker hexamethonium (28 mol/kg; by 98%, P < 0.05). In contrast, in isolated islets, 4-DAMP or hexamethonium (10 or 100 microM) did not inhibit GRP (100 nM)-induced insulin secretion. Furthermore, afferent denervation by neonatal capsaicin did not affect the insulin response to GRP. We conclude that the insulinotropic effect of GRP in the mouse is mediated by both direct islet effects and through activation, at the ganglionic level, of postganglionic cholinergic nerves. In vivo, the indirect cholinergic mechanism predominates.

Topics: Animals; Blood Glucose; Capsaicin; Cells, Cultured; Female; Ganglia, Sympathetic; Gastrin-Releasing Peptide; Glucagon; Hexamethonium; Insulin; Insulin Secretion; Islets of Langerhans; Mice; Mice, Inbred Strains; Muscarinic Antagonists; Pancreas; Piperidines; Receptor, Muscarinic M3; Receptors, Muscarinic

Membrane-bound guanylyl cyclase (GC) is regulated by muscarinic receptors (mAChRs). Carbamylcholine (CC) induces a "dual" biological response on GC activity. Thus, an activation is observed at 0.1 nM and a maximal response at 1 nM CC. However, at higher agonist concentration (> 100 nM), there is an agonist-dependent inhibition of GC. This CC dual response is affected by 4-DAMP and HDD (M3 antagonists), which produce a right-shift of the CC curve; the maximal CC dose response with 4-DAMP is more potent than that with HDD. Moreover, AFDX-DS (an M2 antagonist) increases basal activity and decreases the agonist-dependent inhibition. Neither the CC response nor the CC maximal dose responses are affected by pirenzepine (PZ, M1 antagonist). The agonist-dependent stimulation of GC activity is inhibited by 4-DAMP showing a -log IC50 = 8.4 +/- 0.4, while AFDX116 DS poorly inhibits such activity with a -log IC50 = 5.0 +/- 0.2. The agonist-independent (basal) GC activity also was inhibited by 4-DAMP, in a dose-dependent manner, with an IC50 = 8.5 +/- 0.2. Nonetheless, other muscarinic antagonists (PZ and HDD) were not able to inhibit this basal GC. Pertussis toxin treatment produces a complete blockade of the agonist-dependent inhibition of GC with a full expression of the agonist-dependent activation of membrane-bound GC. These results indicate that membrane-bound GC is regulated by muscarinic agents through two opposite signaling pathways; one involves the activation of GC via an M3 mAchR coupled to a PTX-insensitive G protein, while the GC inhibition is mediated through a PTX-sensitive Gi/o protein possibly coupled to an M2 mAChR.

Topics: Animals; Carbachol; Cattle; Cell Membrane; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanylate Cyclase; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Pertussis Toxin; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Signal Transduction; Trachea; Virulence Factors, Bordetella

Racemic albuterol has been one of the most widely used beta2-adrenoceptor agonists for the relief of the symptoms of asthma, yet the use of beta2 agonists has been known to induce bronchial hyperresponsiveness. To probe a possible role of the S-enantiomer for hyperresponsiveness, we determined the effects of (S)-albuterol on intracellular Ca2+ concentration ([Ca2+]i) in dissociated bovine tracheal smooth muscle cells. Both (S)-and (R,S)-albuterol increased [Ca2+]i at concentrations of >10 pM and 1 nM, respectively, with a maximal response by 150 and 100 nM, respectively. (S)-Albuterol (1 and 10 muM) induced Ca2+ oscillations, reaching 1-2 muM [Ca2+]i. This response is in a stark contrast to that of (R)-albuterol, which decreased [Ca2+]i. The increase in [Ca2+]i was blocked by 100 nM atropine or 500 nM 4-diphenylacetoxy-N-methylpiperidine but was insensitive to the beta2 antagonist ICI 118,551 (10 muM). (S)-Albuterol (10 muM) increased inositol-1,4,5-trisphosphate levels by 213 +/- 34.4% (p < 0.05, four experiments) in cells exposed for 30 sec. The sustained phase of the Ca2+ increase was absent in Ca2+-free solution, suggesting that Ca2+ influx was responsible for the sustained Ca2+ response. The results also suggest that (S)-albuterol may cross-react with muscarinic receptors. As a Ca2+ agonist in airway smooth muscle, (S)-albuterol may have profound clinical implications because 50% of prescribed racemic albuterol is composed of (S)-albuterol.

Topics: Adrenergic beta-Agonists; Albuterol; Animals; Calcium; Cattle; Estrenes; Piperidines; Propanolamines; Pyrrolidinones; Receptors, Muscarinic; Stereoisomerism; Trachea; Type C Phospholipases

A series of tropinyl and piperidinyl esters was synthesized and evaluated for inhibitory activities on the endothelial muscarinic receptors of rat (M3) and rabbit (M2) aorta. Some of the esters (cyclohexylphenylglycolates and cyclohexylphenylpropionates) were found to be better antimuscarinic compounds than standard M2 and M3 inhibitors such as AFDX116 and 4-diphenylacetoxy-N-methylpiperidine (DAMP), with pKEC50 values in the range of 8-9. A few esters were found to be more selective M3 than M2 inhibitors, but these tended to have low activities. The hydrophobic, electronic and steric characteristics of these esters were correlated with antimuscarinic activity by using appropriate parameters representing hydrophobicity (HPLC capacity factor, log kw), size (molecular volume) and electronic character (Taft's polar substituent constant sigma * and 13C chemical shift difference delta delta). Finally, 92% of the M2-inhibitory activities of the esters could be accounted for by the size and electronic character sigma * of the side chain. In contrast, the M3-inhibitory activities of these esters were mainly attributed to the electronic nature (sigma *, delta delta) of the side chain, with good activity being associated with electron-withdrawing groups. Visualization of the comparative molecular field analysis (CoMFA) steric and electrostatic fields provided further confirmation of the structure-activity relationship (SAR) derived from traditional quantitative structure activity relationship (QSAR) approaches.

Topics: Animals; Esters; In Vitro Techniques; Kinetics; Male; Models, Molecular; Muscarinic Antagonists; Piperidines; Pirenzepine; Rabbits; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Tropanes

We have characterized in vitro the muscarinic receptors mediating the contraction of the detrusor muscle in Cynomolgus monkeys and guinea pigs using carbachol as the agonist and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M3-selective), methoctramine (M2-selective) and pirenzepine (M1-selective) as the antagonists. Carbachol induced a concentration-dependent contraction of the detrusor muscle of monkey and guinea pig yielding similar pD2 values of 6.67+/-0.03 (n=50) and 6.77+/-0.06 (n=36), respectively. In the detrusor muscle of Cynomolgus monkey, all antagonists produced a concentration-dependent inhibition of carbachol-induced contractions, without decreasing the maximal response. Schild plot analysis yielded slopes not different from unity for all antagonists. The order of antagonist potency was: 4-DAMP (pA2=8.96)>pirenzepine (pA2=6.66)>methoctramine (pA2=6.03), suggesting that M3 receptors have a dominant role in mediating detrusor contraction. In the detrusor muscle of the guinea pig, 4-DAMP and pirenzepine, but not methoctramine, produced a concentration-dependent inhibition of the carbachol-induced contractions, without decreasing the maximal response. Schild plot analysis yielded a slope not different from unity for 4-DAMP and pirenzepine. 4-DAMP (pA2=9.07) had a higher potency than pirenzepine (pA2=6.66), a finding consistent with previously published data. The present study shows that in Cynomolgus monkey stimulation of the M3 subtype is dominant in mediating detrusor contraction upon carbachol stimulation.

Topics: Animals; Carbachol; Diamines; Female; Guinea Pigs; Macaca fascicularis; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptors, Muscarinic; Urinary Bladder

Smooth muscle cells of guinea pig ileum express both M2 and M3 subtypes of muscarinic receptors. Under voltage clamp, activation of the muscarinic receptors with carbachol (CCh) induces Ca2+-activated K+ current (I[K-Ca]) and nonselective cationic current (Icat). Receptor subtypes mediating the current responses were characterized by using pirenzepine, AF-DX116, 4-DAMP and atropine, which have different profiles of the affinity constants for muscarinic receptor subtypes. The muscarinic antagonists inhibited either CCh-evoked I(K-Ca) or Icat with different potencies. Their relative potencies for I(K-Ca) and Icat inhibition resembled the relative affinity constants for M3 and M2 subtypes, respectively. Thus, the I(K-Ca) is mediated via the M3 subtype and the Icat via the M2 subtype.

Topics: Animals; Atropine; Calcium; Carbachol; Guinea Pigs; Male; Muscarinic Agonists; Muscle, Smooth; Piperidines; Pirenzepine; Potassium Channels; Receptors, Muscarinic

The muscarinic receptor subtypes that mediate cholinergic responses in cat esophageal smooth muscle were examined. Antagonist effects on carbachol-induced and nerve-evoked contractions were studied in vitro using muscle strips from the distal esophagus. Antagonists displayed similar relative selectivities in suppressing carbachol and nerve-mediated responses as follows: 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > zamifenacin > para-fluoro-hexahydrosiladiphenidol > pirenzepine > AF-DX 116 > methoctramine, indicating that these responses are mediated by the same receptor subtype. 4-DAMP, pirenzepine and methoctramine effects on carbachol responses gave pA2 values characteristic of the M3 receptor in both the circular muscle (9.25 +/- 0.12, 6.79 +/- 0.09 and 6.04 +/- 0.11, respectively) and longitudinal muscle (9.46 +/- 0.14, 7.25 +/- 0.07 and 6.10 +/- 0.06, respectively). Reverse transcription-polymerase chain reaction analysis was done using primer sequences based on the cloned human muscarinic receptor subtypes. Messenger RNA for the m3 receptor was readily identified, whereas m2 was not detected in esophageal muscle, but was present in cardiac muscle. Sequence homology between the amplified products from cat tissue and the corresponding human m2 and m3 receptors genes were 93% and 89%, respectively. In the cat esophagus, the M3 receptor mediates functional responses and messenger RNA for the corresponding molecular form of this receptor is abundant in this tissue.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carbachol; Cats; Dioxoles; Electric Stimulation; Esophagus; Female; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Piperidines; Polymerase Chain Reaction; Receptors, Muscarinic; RNA, Messenger

Basal forebrain (BF) cholinergic regulation of cataplexy was investigated in narcoleptic canines. Specific cholinergic agonists and antagonists, and excitatory or inhibitory amino acid neurotransmitter receptor agonists, were perfused through microdialysis probes implanted bilaterally in the BF of narcoleptic canines. Cataplexy was monitored using the food-elicited cataplexy test (FECT) and recordings of electroencephalogram, electrooculogram, and electromyogram. In narcoleptic canines, carbachol and oxotremorine (10(-5)-10(-3) M), but not McN-A-343 or nicotine (10(-4)-10(-3) M), produced a dose-dependent increase in cataplexy. In addition, N-methyl-d-aspartate (10(-4)-10(-3) M) and kainic acid (10(-5)-10(-4) M) did not have any effects, while muscimol (10(-3) M) produced a weak (P < 0.10) increase in cataplexy. In control canines, carbachol (10(-5)-10(-3) M), but not oxotremorine (10(-4)-10(-3) M), produced muscle atonia after the highest concentration in one of three animals. Carbachol (10(-3) M)-induced cataplexy in narcoleptic canines was blocked by equimolar perfusion with the muscarinic antagonists atropine, gallamine, and 4-DAMP but not pirenzepine. These findings indicate that carbachol-stimulated cataplexy in the BF of narcoleptic canines is mediated by M2, and perhaps M3, muscarinic receptors. The release of acetylcholine in the BF was also examined during FECT and non-FECT behavioral stimulation in narcoleptic and control canines. A significant increase in acetylcholine release was found in both narcoleptic and control BF during FECT stimulation. In contrast, simple motor activity and feeding, approximating that which occurs during an FECT, did not affect acetylcholine release in the BF of narcoleptic canines. These findings indicate that BF acetylcholine release is enhanced during learned emotion/reward associated behaviors in canines.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Atropine; Behavior, Animal; Carbachol; Cataplexy; Cholinergic Fibers; Dogs; Feeding Behavior; Female; GABA Agonists; Gallamine Triethiodide; Male; Microdialysis; Muscarinic Agonists; Muscarinic Antagonists; Muscimol; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Oxotremorine; Piperidines; Pirenzepine; Prosencephalon

We examined the effects of the muscarinic agonist carbachol on ion secretion induced by substance P (SP) in piglet jejunal tissues mounted in Ussing chambers. Tetrodotoxin was present in all solutions to inhibit neural activity. Carbachol added 10 min prior to 0.75 microM SP dose dependently inhibited subsequent SP responses, with 90% inhibition at 10 microM carbachol. Addition of an equipotent dose of SP (7.5 microM) had no effect on subsequent carbachol-induced secretion. Carbachol's inhibition of SP-induced secretion was evident for at least 45 min and was abolished by prior addition of the M3 receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), but remained intact in the presence of the M2 antagonist gallamine or the nicotinic antagonist mecamylamine. Atropine added 10 min after carbachol restored subsequent SP responses toward control levels. Carbachol also reduced secretory responses to histamine and, to a lesser extent, prostaglandin E2 (PGE2). SP-induced secretion was not affected by prior addition of histamine and was reduced by PGE2 only at the highest PGE2 concentration. The results suggest that activation of the epithelial M3 receptor by carbachol inhibits subsequent secretory responses to the calcium-mediated agonists SP and histamine in piglet jejunum. This may reflect muscarinic activation of a negative messenger in epithelial cells that limits Cl- secretion.

Topics: Animals; Atropine; Carbachol; Cholinergic Antagonists; Dose-Response Relationship, Drug; Drug Interactions; Gallamine Triethiodide; Histamine; Ion Transport; Jejunum; Mecamylamine; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Substance P; Swine; Time Factors

This study consists of two parts, first to compare the pharmacological profile of atropine and CEB-1957 substance toward muscarinic receptor subtypes. In various rat brain structures, binding properties were determined by competition experiments of [3H]pirenzepine, [3H]AF-DX 384, and [3H]4-DAMP in quantitative autoradiography of M1, M2, and M3 muscarinic receptor subtypes, respectively. Competition curves have shown that atropine presents similar nanomolar inhibition constants toward each subtype, while CEB-1957 has distinct affinities (Ki from 0.26 to 73 nM) with the following range order: M3 > or = M2 > M1. The second part is to compare atropine and CEB-1957 (in combination with pralidoxime) for their ability to protect against the lethality induced by 2 x LD50 of the acetylcholinesterase inhibitor sarin. CEB-1957 reduced the mortality at doses 10 times lower than atropine. Finally, from these results, it is proposed that a selective blockade of M2 and M3 receptor subtypes could play a pivotal role in the protective effect against sarin poisoning.

Topics: Animals; Atropine; Autoradiography; Binding, Competitive; Brain; Cholinesterase Inhibitors; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Poisoning; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Sarin; Thiophenes

The secretion of alphaMSH from the intermediate lobe of the frog pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh), acting via muscarinic receptors, stimulates alphaMSH release from frog neurointermediate lobes (NILs) in vitro. The aim of the present study was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope cells. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated alphaMSH release from perifused frog NILs, whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced alphaMSH release. Administration of a brief pulse of muscarine in the vicinity of cultured melanotrope cells provoked a 4-fold increase in the cytosolic calcium concentration ([Ca2+]i). Suppression of Ca2+ in the culture medium or addition of 3 mM Ni2+ abrogated the stimulatory effect of muscarine on [Ca2+]i and alphaMSH release. In contrast, omega-conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca2+]i and alphaMSH secretion. Exposure of NILs to muscarine provoked an increase in inositol phosphate formation, and this effect was dependent on extracellular Ca2+. The inhibitor of polyphosphoinositide turnover neomycin significantly attenuated the muscarine-evoked alphaMSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the stimulatory effect of muscarine on alphaMSH release was not affected by the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin 25. Muscarine at a high concentration (10(-4) M) only produced a 40% increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of alphaMSH release. These results indicate that frog melanotrope cells express a muscarinic receptor subtype pharmacologically related to the mammalian M3 receptor. Activation of this receptor causes calcium influx through Ni2+-sensitive Ca2+ channels and subsequent activation of the phopholipase C/protein kinase C transduction pathway.

Topics: Acetylcholine; alpha-MSH; Animals; Calcium; Carbachol; Cyclic AMP; Enzyme Inhibitors; Inositol Phosphates; Male; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Pertussis Toxin; Piperidines; Pituitary Gland; Protein-Tyrosine Kinases; Rana ridibunda; Receptors, Muscarinic; Signal Transduction; Virulence Factors, Bordetella

Vagal stimuli increase duodenal mucosal HCO-3 secretion and may provide anticipatory protection against acid injury, but duodenal enterocyte (duodenocyte) responses and cholinoceptor selectivity have not been defined. We therefore developed a stable primary culture model of duodenocytes from rats and humans. Brief digestion of scraped rat duodenal mucosa or human biopsies with collagenase/dispase yielded cells that attached to the extracellular matrix Matrigel within a few hours of plating. Columnar cells with villus enterocyte morphology that exhibited spontaneous active movement were evident between 1 and 3 days of culture. Rat duodenocytes loaded with fura 2 responded to carbachol with a transient increase in intracellular calcium concentration ([Ca2+]i), with an apparent EC50 of approximately 3 microM. In a first type of signaling pattern, [Ca2+]i returned to basal or near basal values within 3-5 min. In a second type, observed in cells with enlarged vacuoles characteristic of crypt cell morphology, the initial transient increase was followed by rhythmic oscillations. Human duodenocytes responded with a more sustained increase in [Ca2+]i, and oscillations were not observed. Rat as well as human duodenocytes also responded to CCK-octapeptide but not to vasoactive intestinal polypeptide. Equimolar concentrations (100 nM) of the subtype-independent muscarinic antagonist atropine and the M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the response to 10 microM carbachol, whereas the M1 antagonist pirenzepine and the M2 antagonists methoctramine and AF-DX 116BS had no effect at similar concentrations. Responses in rat and human duodenocytes were similar. A new agonist-sensitive primary culture model for rat and human duodenocytes has thus been established and the presence of enterocyte CCK and muscarinic M3 receptors demonstrated.

Topics: Animals; Calcium; Carbachol; Cell Adhesion; Cells, Cultured; Dose-Response Relationship, Drug; Duodenum; Extracellular Matrix; Fluorescent Dyes; Fura-2; Humans; Intestinal Mucosa; Male; Parasympatholytics; Piperidines; Rats; Rats, Inbred Lew; Signal Transduction; Sincalide; Vacuoles

Acetylcholine-induced calcium signaling dynamics have been described in cell monolayers derived from colonic mucosa, but not in intact colonic crypts. The aim of this study was to characterize the spatiotemporal characteristics of calcium signaling induced by acetylcholine in isolated intact rat colonic crypts and to identify the muscarinic receptor subtype coupled to this signaling pathway.. Isolated crypts from the distal colon of male Wistar rats were loaded with the calcium-sensitive dye Fura-2 and imaged with a charge-coupled device video camera.. Acetylcholine mobilized intracellular calcium with an EC50 of 3.9 micromol/L. The response was initiated at the base of the crypt and progressed toward the surface. The velocity of propagation was dose dependent. Addition of muscarinic antagonists inhibited the response (pKb values calculated for pirenzepine and 4-DAMP, 6.08 and 8.65, respectively). Microperfusion of acetylcholine initiated a calcium signal throughout the lower half of the crypt. Microinjection of inositol 1,4,5-triphosphate induced a propagation of a calcium signal along the crypt axis. Heptanol inhibited the velocity of acetylcholine-induced wave propagation by 33%.. M3 muscarinic receptors are coupled to the mobilization of calcium from intracellular stores of intact, isolated rat colonic crypts. Intercellular communication potentiates the propagation of the acetylcholine-induced calcium signal along the crypt axis.

Topics: Acetylcholine; Animals; Biological Transport; Calcium; Colon; Dose-Response Relationship, Drug; Heptanol; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar; Signal Transduction; Tissue Distribution

Previous studies have demonstrated that vascular responses to acetylcholine (ACh), an endothelium-dependent vasodilator, are enhanced in exercise-trained animals. In order to see if chronic exercise upregulates endothelial muscarinic (M) receptor, the subtype of M receptors responsible for ACh-induced vasorelaxation in the thoracic aorta of male Wistar rats was characterized first, then a receptor assay was performed. These animals were divided into exercise and control groups. The trained rats ran on a treadmill with a moderate intensity for 60 min per day, 5 days per week. After 10 weeks of training, rats were decapitated and their thoracic aortae were isolated. The subclass of M receptor in endothelium was pharmacologically identified on the basis of selective affinity of antagonists; ie, pirenzepine for M1, gallamine for M2, and 4-diphenylacetoxy-N-methylpiperidine methiodide for M3. Our results showed that in the thoracic aorta of Wistar rats, 1) ACh-induced vasorelaxation was mediated by M3 receptor; 2) chronic exercise enhanced ACh-evoked vasodilating responses. However, this alteration was not caused by receptor upregulation, as maximal binding sites and affinity of M3 receptor were not changed by chronic exercise. Other possible mechanisms need to be further studied.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Blood Pressure; Dose-Response Relationship, Drug; Endothelium, Vascular; Gallamine Triethiodide; Heart Rate; Male; Muscarinic Antagonists; Nicotinic Antagonists; Nitric Oxide; Physical Conditioning, Animal; Physical Exertion; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Muscarinic; Tritium; Up-Regulation; Vasodilation; Vasodilator Agents

We investigated the presence and subtypes of functionally prejunctional receptors in cholinergic nerve endings of rabbit detrusor smooth muscle strips using high-performance liquid chromatography coupled with a microdialysis procedure. The effects of pretreatment with various drugs on acetylcholine (ACh) release and contractile responses induced by electrical field stimulation were evaluated. Although atropine (a muscarinic nonselective antagonist) and 4-DAMP (a muscarinic M3 antagonist) did not influence the ACh release, they markedly reduced the contractile responses. Pirenzepine (M1 antagonist) decreased ACh release and contractile responses. Methoctramine (M2 antagonist) increased the ACh release, but did not influence to the contractile responses. These results suggest that the muscarinic receptors in the rabbit detrusor smooth muscle are heterogeneous, prejunctional facilitatory (M1 receptors), and inhibitory (M2 receptors) for ACh release and postjunctional M3 receptors mediating contractile responses.

Topics: Acetylcholine; Animals; Chromatography, High Pressure Liquid; Culture Techniques; Diamines; Female; Muscarinic Antagonists; Muscle Contraction; Muscle, Skeletal; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Reference Values

Some anticholinesterases (anti-ChE) such as neostigmine and pyridostigmine but not edrophonium, stimulate phosphatidylinositol (PI) response. Although a direct relationship was suggested between the increase in PI response and airway smooth muscle contraction, there are no data regarding the effects of anti-ChE drugs on airway smooth muscle. Thus, we examined the contractile properties and PI responses produced by anti-ChE drugs.. Contractile response. Rat tracheal ring was suspended between two stainless hooks in Krebs-Henseleit (K-H) solution. (1) Carbachol (CCh), anti-ChE drugs (neostigmine, pyridostigmine, edrophonium) or DMPP (a selective ganglionic nicotinic agonist) were added to induce active contraction. (2) The effects of 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), an M3 muscarinic receptor antagonist, on neostigmine- or pyridostigmine-induced contraction of rat tracheal ring were examined. (3) Tetrodotoxin (TTX) was tested on the anti-ChE drugs-induced responses. PI response. The tracheal slices were incubated in K-H solution containing LiCl and 3[H]myo-inositol in the presence of neostigmine or pyridostigmine with or without 4-DAMP, an M3 muscarinic receptor antagonist. 3[H]inositol monophosphate (IP1) formed was counted with a liquid scintillation counter.. Carbachol (0.1 microM), neostigmine (1 microM), pyridostigmine (10 microM) but not edrophonium or DMPP, caused tracheal ring contraction. 4-DAMP, but not tetrodotoxin, inhibited neostigmine and pyridostigmine-induced contraction. Neostigmine- or pyridostigmine-induced IP1, accumulation was inhibited by 4-DAMP.. The data suggest that anti-ChE drugs activate the M3 receptors at the tracheal effector site.

Topics: Animals; Carbachol; Cholinergic Agonists; Cholinesterase Inhibitors; Dimethylphenylpiperazinium Iodide; Edrophonium; Glucose; Inositol; Inositol Phosphates; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neostigmine; Neuromuscular Blocking Agents; Nicotinic Agonists; Organ Preservation Solutions; Phosphatidylinositols; Piperidines; Pyridostigmine Bromide; Radiopharmaceuticals; Rats; Rats, Wistar; Tetrodotoxin; Trachea; Tritium; Tromethamine

The purpose of this study was to characterize the role of M2 muscarinic receptors in inhibiting relaxant effects of drugs that stimulate cyclic AMP (cAMP) accumulation in the guinea pig ileum. We investigated the ability of oxotremorine-M (oxo-M) to inhibit cAMP accumulation in the presence of agonists that stimulate adenylyl cyclase in other cells and tissues. Appreciable stimulation of cAMP (> 50% over basal levels) was achieved with forskolin and maximally effective concentrations of isoproterenol, cicaprost, prostaglandin E1, prostaglandin E2 and prostaglandin I2, with the stimulation over basal levels of cAMP being 14.9-, 2.51-, 2.45-, 2.27-, 2.28- and 1.52-fold, respectively. Moderate or no cAMP stimulation was observed with dopamine, 5-hydroxytryptamine, 5-methoxytryptamine, dimaprit, vasoactive intestinal peptide, SKF-38393, 2-chloroadenosine, CGS-21680, prostaglandin D2, secretin and vasopressin. Oxo-M (1 microM) inhibited cAMP accumulation by 35% under basal conditions. Oxo-M inhibited specific agonist-stimulated cAMP levels by 20 to 70%. However, oxo-M caused little or no inhibition of specific prostaglandin I2- and cicaprost-stimulated cAMP levels (5 and 0%, respectively). In general, there was a correlation between the abilities of the various agonists to stimulate cAMP accumulation and to cause relaxation of the isolated ileum, with an exception being cicaprost. Experiments were carried out with isolated ileum to determine whether activation of M2 receptors inhibited the relaxant effects of the various agonists. In these experiments, the ileum was first treated with N-(2-chloroethyl)-4-piperidinyl diphenylacetate to selectively inactivate M3 receptors. After this treatment phase, contractile responses to oxotremorine-M were measured in the presence of histamine and a given relaxant agent. These measurements were repeated in the presence of the M2-selective antagonist AF-DX 116. Analysis of the data showed that part of the contractile response to oxotremorine-M could be attributed to an M2-mediated inhibition of the relaxation. This M2 component of the contractile response was greatest when forskolin or isoproterenol was used as the relaxant agent. In contrast, little or no M2 response was measured in the presence of dopamine and cicaprost.

Topics: Animals; Cyclic AMP; Guinea Pigs; Histamine; Ileum; In Vitro Techniques; Male; Muscarinic Agonists; Muscle Relaxation; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptors, Muscarinic

There is conflicting evidence concerning whether muscarinic regulation of gastrin release in humans is a direct or indirect effect on antral G cells. The present experiments were designed to resolve this question using an isolated human G-cell preparation.. The ability of muscarinic agonists to stimulate or inhibit gastrin release was assessed with or without an immunoneutralizing somatostatin antibody or an m3 receptor antagonist. The effect of secretogogues on G and D cells was monitored by intracellular calcium imaging.. Muscarinic agonists failed to stimulate gastrin release, even after the removal of somatostatin-induced inhibition. Our group has previously shown that muscarinic agonists stimulated somatostatin release from antral D cells. Methacholine (100 mumol/L) increased intracellular calcium levels in cocultured D cells; this increase was inhibited by the m3 receptor antagonist 4-diphenylacetoxy-n-methylpiperidine methiodide. Gastrin cells in the same field of view lacked a response to methacholine but showed a clear response to 10 nmol/L bombesin.. The experiments indicate that vagal control of gastrin release in humans is indirect; stimulation would be achieved by the activation of intrinsic gastrin-releasing peptide neurons, and inhibition would be via the paracrine action of somatostatin released from adjacent D cells.

Topics: Adult; Antibodies; Bombesin; Calcium; Carbachol; Cells, Cultured; Female; Gastrins; Humans; Male; Methacholine Chloride; Middle Aged; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pyloric Antrum; Somatostatin

In the cat, microdialysis application of 200 microM carbachol to the peri-locus coeruleus alpha (peri-LC alpha) of the mediodorsal pontine tegmentum produced a marked (< or = 5-fold) increase in paradoxical sleep. This effect was blocked by 5-50 microM 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an M1/M3-selective muscarinic receptor antagonist. In contrast, the effect was not reversed by methoctramine, an M2-selective antagonist, or pirenzepine, an M1-selective antagonist, even at concentrations as high as 500 microM. In addition, unilateral application of 5 microM 4-DAMP alone to the peri-LC alpha induced both a > 60% decrease in paradoxical sleep and a state of paradoxical sleep without atonia, whereas 50 microM pirenzepine and 500 microM methoctramine had no effect. Our findings are further evidence for the important role played by the peri-LC alpha and demonstrate a critical role for M3 muscarinic cholinergic receptors in the generation of paradoxical sleep.

Topics: Animals; Carbachol; Cats; Muscarinic Antagonists; Piperidines; Receptors, Muscarinic; Sleep

Several studies have shown that the nervous (and hormonal) system controls immune functions. In the present study, we examined the presence of muscarinic acetylcholine receptors and the effect of carbachol on DNA fragmentation in adult rat thymocytes. Rat thymocytes possessed high affinity binding sites for the muscarinic antagonist [3H]3-quinuclidinyl benzilate (QNB). The average number of binding sites per cells was 3000, and the equilibrium dissociation constant of [3H]QNB on intact cell was approximately 80 nM. The binding was inhibited by an M1- and M3-selective antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodine (4-DAMP). Hydrocortisone (100 mg/kg, s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of [3H]QNB binding sites on thymocytes by 82 +/- 33%. The gel electrophoresis of DNA extracted from carbachol-treated thymocytes revealed a ladder pattern typical of intranucleosomal fragmentation. The addition of oxotremorine-M also induced DNA fragmentation and the effects of muscarinic agonists were inhibited by the addition of atropine or 4-DAMP. The results suggest the existence of muscarinic receptors and the possible involvement in apoptosis in thymocytes.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Atropine; Binding, Competitive; Carbachol; DNA Fragmentation; Electrophoresis, Agar Gel; Hydrocortisone; Injections, Subcutaneous; Isotope Labeling; Male; Muscarinic Agonists; Muscarinic Antagonists; Nucleosomes; Oxotremorine; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Thymus Gland

The role of Ca2+/calmodulin-dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+ concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3 mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne). Carbamylcholine (100 microM) elicited an initial transient peak in [Ca2+]i due to mobilization of Ca2+ from internal stores, followed by a sustained elevation in [Ca2+]i that depended on the influx of extracellular Ca2+ and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 microM KN-62. KN-62 inhibited mAChR-induced Ca2+ influx and mobilization from internal stores by about 25-30%, producing a half-maximal effect at approximately 1 microM. In contrast, KN-62 (25 microM) almost completely abolished carbamylcholine-stimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+ influx induced by depolarization of the cells with 25 mM K+ (IC50 = 3 microM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+ and the stimulation of Ca2+ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+ occurs through Ca2+ channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Enzyme Inhibitors; Humans; Neuroblastoma; Piperidines; Receptor, Muscarinic M3; Receptors, Muscarinic; Signal Transduction; Tumor Cells, Cultured

1. Contractile responses of smooth muscle from the Wistar rat urinary bladder were studied with the use of muscarinic agonists and antagonists. 2. McN-A-343 induced only weak contractile responses of the bladder muscle. In contrast, oxotremorine showed higher potency than either acetylcholine or bethanechol in inducing a contractile response (the respective pD2 values were 6.38 +/- 0.25, 4.82 +/- 0.24 and 4.42 +/- 0.14). 3. The M2 antagonists, methoctramine (10(-9) M to 10(-5) M) and gallamine (10(-9) M to 10(-5) M), did not reduce acetylcholine-induced (10(-5) M) contractions of the bladder muscle strip. On the other hand, 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP, 10(-10) M to 10(-7) M), an M3 receptor blocker, effectively antagonized the acetylcholine-induced contractions in a concentration-dependent manner. 4-DAMP had a similar pA2 value to those of the non-selective antagonists, atropine and scopolamine (pA2 values were 8.26 +/- 0.05, 8.36 +/- 0.05 and 8.41 +/- 0.11, respectively). Pirenzepine, and M1 blocker, antagonized the contractions at higher concentrations (10(-8) M to 10(-5) M, pA2 = 6.23 +/- 0.04). 4. It is concluded that (1) the dominant muscarinic receptor subtype responsible for smooth muscle contraction in the rat urinary bladder is M3; and (2) the muscarinic agonist oxotremorine was more potent than acetylcholine and bethanechol in inducing a contractile response.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Analysis of Variance; Animals; Bethanechol; Diamines; Dose-Response Relationship, Drug; Female; Gallamine Triethiodide; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Nicotinic Antagonists; Oxotremorine; Parasympatholytics; Piperidines; Rats; Rats, Wistar; Receptors, Muscarinic; Structure-Activity Relationship; Urinary Bladder

The role of muscarinic receptor subtype(s) in the control of gastric phase III contractions and motilin release was determined in dogs. Pirenzepine (226 nmol/kg/h) shortened the phase III cycle, causing an early increase in plasma motilin concentrations. Both AF-DX116BS and 4-DAMP inhibited the occurrence of spontaneous and motilin-induced phase III contractions, and 4-DAMP also inhibited the spontaneous increase in plasma motilin concentration. Only 4-DAMP inhibited carbachol-induced motilin release in perifused duodenal mucosal cells. In conclusion, M1 receptors are involved in the indirect inhibition of motilin release. Motilin-induced contractions are mediated mainly by M3 receptors and partly by M2 receptors, and M3 receptors are present in motilin-producing cells.

Topics: Animals; Cell Separation; Dogs; Duodenum; Female; Infusions, Intravenous; Intestinal Mucosa; Male; Motilin; Muscarinic Antagonists; Piperidines; Pirenzepine; Stomach

This investigation was undertaken to characterize the muscarinic receptor subtypes involved in methacholine-induced vasodilation, vagal bradycardia, neurally-evoked sudomotor responses and sympathetic muscarinic ganglionic transmission in anesthetized cats. Dose-response curves were constructed using the putatively selective antagonists pirenzepine (M1), gallamine (M2) and 4-DAMP (M3: 4-diphenyl-acetoxy-N-methylpiperidine) and compared with the non-selective blocker, atropine. Methacholine hypotension and evoked sudomotor responses exhibited an M3 muscarinic receptor profile with the following potency relationships: atropine > or = 4-DAMP > pirenzepine >> gallamine. Vagal bradycardia (M2) was antagonized by gallamine and exhibited a lower relative sensitivity to 4-DAMP when corrected for atropine effect. Pirenzepine was inactive in inhibition of bradycardia but was highly potent against transmission in the sympathetic ganglion (M1) with the following potency relationships: atropine > or = pirenzepine > 4-DAMP >> gallamine. In comparison with atropine, 4-DAMP exhibited a significantly lower potency for M1 and M2 muscarinic receptors as compared to its effect on the M3 muscarinic receptor subtypes.

Topics: Animals; Atropine; Blood Pressure; Cats; Gallamine Triethiodide; Galvanic Skin Response; Heart Rate; Methacholine Chloride; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Superior Cervical Ganglion; Synaptic Transmission; Vagus Nerve; Vasodilation

Acetylcholine acting via muscarinic receptors located in the intestinal mucosa controls ion and fluid transport. This study examined the pathway(s) by which cholinergic receptors mediate secretion in rat isolated duodenum, jejunum and ileum using the short-circuit current (Isc) as an index of electrogenic CL- secretion. Carbachol and bethanechol induced electrogenic CL- transport which was insensitive to the neural blocker tetrodotoxin, indicating their direct action on the enterocytes. Functional characterization of electrogenic secretion activated via muscarinic receptors on jejunal and ileal enterocytes was achieved by use of selective muscarinic antagonists in the presence of tetrodotoxin. In both regions the rank order of potency of these compounds (atropine > 4-diphenylacetoxy-N-piperidine methiodide (4-DAMP) > hexahydro-sila-difenidol (HHSiD) > pirenzepine > methoctramine) indicated the M3 receptor subtype. Secretion activated by the muscarinic agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N, N-trimethyl-2-butyn-1-ammonium chloride (McN-A-343) was sensitive to tetrodotoxin and pirenzepine but not to the ganglionic blocker, hexamethonium, indicating the M1 receptor subtype on post ganglionic neurons. Regional differences for bethanechol-activated secretion showed an increasing gradient in secretory capacity (Isc max) in a proximal-to-distal direction along the small intestine. Responses to McN-A-343 also showed regional differences but these were unlike those of bethanechol. These results show that cholinomimetic-induced electrogenic CL- secretion in rat isolated small intestine appears to be mediated by two dissimilar populations of muscarinic receptor: M3 muscarinic receptors positioned on enterocytes and M1 muscarinic receptors sited on submucosal neurons.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Analysis of Variance; Animals; Atropine; Bethanechol; Carbachol; Chlorides; Diamines; Duodenum; Ileum; Intestinal Mucosa; Ion Transport; Jejunum; Male; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Muscarinic; Structure-Activity Relationship; Tetrodotoxin

Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 microM) alone did not affect the basal release of adenosine. However, carbachol (100 microM) induced a 253% increase in NMDA (20 microM)-evoked adenosine release in the presence of Mg2+. In the absence of Mg2+, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 microM) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (1 microM). Neither an M1-selective dose of pirenzepine (50 nM) nor the M2-selective antagonist methoctramine (1 microM) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.

Topics: Adenosine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Atropine; Carbachol; Cerebral Cortex; Diamines; Drug Synergism; In Vitro Techniques; Kinetics; Magnesium; Male; Muscarinic Agonists; Muscarinic Antagonists; N-Methylaspartate; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Muscarinic

The effects of ACh on [3H]D-aspartate efflux and on calcium levels ([Ca2+]i) were studied at the same time in sister cultures of rat cerebellar granule cells stimulated with electrical pulses (5-20 Hz) or depolarized with KCl (15-40 mM). ACh, 0.3-1000 nM, greatly facilitated the 10-Hz-evoked tritium efflux while its effect on 20 mM KCl-evoked efflux was significantly smaller. ACh, 10-1000 nM, enhanced [Ca2+]i levels to a limited extent under both experimental conditions. Therefore, ACh facilitation was evident above all on the electrically evoked [3H]D-aspartate efflux. The ACh-mediated responses depended on the activation of M3-muscarinic receptors since these responses were blocked by 4-DAMP. ACh, 50 microM, reduced the [Ca2+]i plateau, determined by prolonged electrical or KCl stimulation. This effect was due to its action of M2-receptors being blocked by AF-DX 116. In conclusion, at very low concentrations, ACh greatly facilitated the electrically evoked [3H]D-aspartate efflux through M3-receptors, while at a higher concentrations, it inhibited, through M2-receptors, the rise in [Ca2+]i caused by prolonged cell depolarization.

Topics: Acetylcholine; Animals; Aspartic Acid; Calcium; Cells, Cultured; Cerebellum; Electric Stimulation; Muscarinic Antagonists; Neurons; Piperidines; Potassium Chloride; Rats; Receptors, Muscarinic

In vibratome-cut slices from rat striatum and in the presence of 10 mM LiCl, the cholinergic agonist carbachol stimulated the accumulation of total [3H]inositol phosphates (EC50 11+/-1 microM and maximum effect 546+/-36% of basal). The response to 100 microM carbachol (497+/-24% of basal) was inhibited by muscarinic antagonists (1 microM), the rank order of efficacy being 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 100% inhibition) approximately pirenzepine (98+/-3%) > p-fluoro analog of hexahydro-sila-difenidol (pFHHSiD; 90+/-3%) >> methoctramine (32+/-7%) approximately tropicamide (30+/-10%). Antagonist inhibition curves best fit to a single-site model for 4-DAMP (pKi 8.9+/-0.2) whereas, for both pirenzepine and pFHHSiD, the best fit was to the two-site model. The pKi values for the high-affinity (8.3+/-0.2) and low-affinity (6.9+/-0.2) components for pirenzepine-mediated inhibition corresponded to those reported for M1 and M3 receptors, respectively. The pKi values for the high-affinity (8.2+/-0.3) and low-affinity (7.0+/-0.2) components for pFHHSiD inhibition were in good agreement with those reported for M3 and M1 receptors, respectively. Altogether these results indicate that carbachol-induced [3H]inositol phosphate formation in rat striatal slices is mediated by both M1 and M3 muscarinic receptors.

Topics: Animals; Carbachol; Corpus Striatum; Drug Evaluation, Preclinical; In Vitro Techniques; Inositol Phosphates; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. 2. Ascending concentrations of carbachol (3-300 microM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4+/-1.4 nS and a mean -log EC50 of 5.12+/-0.03 (mean+/-s.e.mean) (n=114). 3. Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. 4. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. 5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPgammaS to the pipette (without application of carbachol) was unaffected. 6. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M2 receptors are disabled.

Topics: Animals; Carbachol; Dioxoles; Dose-Response Relationship, Drug; Electric Conductivity; Guinea Pigs; Ileum; In Vitro Techniques; Ion Channels; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Patch-Clamp Techniques; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic

A comparison of muscarinic acetylcholine receptor (mAChR) antagonist binding properties was made between intact cerebellar granule cell cultures and membranes prepared from these cells. [3H]quinuclidinyl benzylate (QNB) binding displacement by four mAChR antagonists was measured and the selectivities for m2- or m3-mAChRs estimated by curve fitting. For each antagonist, the preparation of membranes caused a subtype selective decrease in receptor affinity, as compared to intact cell binding. The m2-selective antagonists had lower affinities in membranes for m2- but not for m3-mAChR, while the m3-selective antagonists had lower affinities for m3- but not for m2-mAChR. As a result, the m2-mAChR selectivity of AF-DX 116 and methoctramine in membranes was 66- and 1.7-fold less than in intact cells, and the m3-mAChR selectivity of 4-DAMP and pFHHSiD was 2.4- and 3.9-fold less in membranes than in intact cells. The m3-mAChR selectivity of 4-DAMP in intact cells was unaffected by cytoskeletal depolymerization with cytochalasins and colchicine. We suggest that the changes in selectivity seen with cell disruption may be due to a loss of cellular factors which regulate receptor properties. Antagonists binding to receptors on intact cells may cause subtype-specific changes in the interaction of the mAChR with these factors. These data suggest that mAChR antagonist binding selectivity needs to be re-examined in intact cell systems.

Topics: Animals; Binding, Competitive; Cell Membrane; Cells, Cultured; Cerebellum; Dose-Response Relationship, Drug; Muscarinic Antagonists; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Time Factors

In sublingual mucous acini, membrane depolarization induces a threefold transient increase in cytosolic free Ca(2+) concentration [(Ca(2+))i]. The underlying mechanism was examined by using the Ca(2+) sensitive fluorescent indicator fura-2. Membrane depolarization with high K+ induced a transient [Ca(2+)]i increase in acini, but not in single acinar cells. Atropine, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the[Ca(2)+]i increase, suggesting the involvement of muscarinic receptor activation. Inhibition of the inositol trisphosphate (IP3)-sensitive Ca(2+) release pathway with S-(diethylamino)-octyl-3,4,5-trimethoxybenzoate prevented the depolarization-induced increase in [Ca(2+)]i. Blockade of nicotinic receptors and L-, N-, and P-type voltage-dependent Ca(2+) channels (hexamethonium, nifedipine, diltiazem, (omega-conotoxin GVIA and omega-agatoxin IVA) did not inhibit the increase in [Ca(2+)]i. However, Cd(2)+ (0.2 mM) blocked >85 percent of the [Ca2+]i increase. The depolarization-induced [Ca(2+)]i increase was also extracellular Ca(2+)-dependent. These results suggest that the membrane depolarization-induced Ca(2+) increase in sublingual acini is mediated by activating Cd(2+)-sensitive, voltage-dependent Ca(2+) channels in nerve terminals associated with the dispersed acini and stimulating release of acetylcholine, which then triggers the [Ca(2+)]i increase in acinar cells.

Topics: Acetylcholine; Animals; Atropine; Cadmium; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Membrane; Cytosol; Fluorescent Dyes; Fura-2; Gallic Acid; Inositol 1,4,5-Trisphosphate; Male; Muscarinic Antagonists; Nerve Endings; Nicotinic Antagonists; Piperidines; Pirenzepine; Potassium; Rats; Rats, Wistar; Receptors, Nicotinic; Sublingual Gland

1. Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35SO4 as a label for secreted mucus, and the muscarinic receptor antagonists telenzepine for the M1 receptor subtype, methoctramine for the M2 subtype and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) for the M3 receptor. We also performed receptor binding and mapping studies. 2. Each muscarinic antagonist displaced [N-methyl-3H]scopolamine binding with high-affinity binding constant (KH) values of 1.9, 2.7 and 5.0 nM for telenzepine, methoctramine and 4-DAMP, respectively. Muscarinic M1 and M3 receptors localized to submucosal glands, whereas M2 receptors did not. 3. In vitro, electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 160%. Telenzepine did not inhibit the neurogenic secretory response at concentrations two-or twentyfold its KH value, nor did it inhibit secretion induced by acetylcholine (ACh). 4-DAMP inhibited neurogenic secretion by 80 and 95%, respectively, at concentrations two-and twentyfold its KH value, and also inhibited ACh-induced secretion. Methoctramine potentiated neurogenic secretion induced at 2.5 Hz (50 V, 0.5 ms for 5 min) in a dose-related (5.4-100 nM) manner with increases of 33-451% above electrically stimulated values. Methoctramine did not potentiate secretion induced at 10 Hz and did not have any effect on ACh-induced secretion. 4. In vivo, vagal stimulation (10 V, 10 Hz, 2 ms for 8 min) increased output of 35SO4 by approximately 120%. Telenzepine had no significant effect on neurogenic secretion. Methoctramine approximately doubled the stimulated response, whereas 4-DAMP abolished the stimulated secretory response. 5. We conclude that in ferret trachea, cholinergic nerve stimulation increases mucus secretion via muscarinic M3 receptors on the submucosal glands. The magnitude of the secretory response is regulated by neuronal M2 muscarinic receptors. The muscarinic M1 receptors localized to the submucosal glands do not appear to be involved with mucus secretion.

Topics: Animals; Diamines; Electric Stimulation; Ferrets; In Vitro Techniques; Male; Mucus; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Radioligand Assay; Receptors, Muscarinic; Scopolamine; Sulfates; Sulfur Radioisotopes; Trachea; Tritium

In cross-chopped slices from rat thalamus and in the presence of 10 mM LiC1, the cholinergic agonist carbachol stimulated the accumulation of total [3H]inositol phosphates ([3H]IP2 = [3H]IP1 + [3H]IP2 + [3H]IP3). Best-fit values for the concentration-response curve for carbachol after 60 min incubation yielded an EC50 of 44 +/- 6 microM, maximum effect of 199 +/- 6% of basal accumulation and Hill coefficient (nH) of 1.1 +/- 0.1. Carbachol-induced [3H]IPs accumulation was inhibited by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; pKi 9.1) and the p-fluoro analogue of hexahydro-sila-difenidol (pF-HHSiD; pKi 8.1). Concentration-response curves for carbachol were shifted to the right in a parallel fashion by pirenzepine (100, 300 and 100 nM). A Schild plot of the data was linear (slope 0.95 +/- 0.04) and yielded a log KD for pirenzepine of -6.8 +/- 0.1. Taken together, these results suggest that carbachol-induced inositol phosphate accumulation in rat thalamus is mediated by muscarinic M3-receptors.

Topics: Animals; Binding, Competitive; Carbachol; Dose-Response Relationship, Drug; Inositol Phosphates; Male; Muscarinic Agonists; Muscarinic Antagonists; Organ Culture Techniques; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Thalamus; Tritium

The ability of anticholinergic agents microinjected into the subthalamic nucleus to reduce reserpine-induced muscular rigidity was assessed in rats. The electromyographical activity of the gastrocnemius-soleus muscle was used as a parameter of muscular rigidity. Reserpine (5 mg/kg i.p.) produced the appearance of electromyographical activity. The muscarinic antagonists M3 (1.27 nmol of 4-DAMP) and M1 (2.36 nmol of pirenzepine) markedly reduced the reserpine-induced electromyographical activity, whereas the M2 antagonist AFDX-116 (2.37 nmol) had no effect. These results suggest that a high cholinergic tone in the subthalamic nucleus is associated with the reserpine-induced muscular rigidity. Moreover, the M3 muscarinic antagonist is more effective than the M1 muscarinic antagonist in reducing the muscular rigidity in reserpinized rats, a model of Parkinson's disease, by blocking the high cholinergic tone in the subthalamic nucleus.

Topics: Anesthetics, Intravenous; Animals; Chloral Hydrate; Cholinergic Antagonists; Electromyography; Locomotion; Male; Microinjections; Muscarinic Antagonists; Muscle Rigidity; Muscle, Skeletal; Piperidines; Pirenzepine; Rats; Rats, Wistar; Reserpine; Sympatholytics; Thalamic Nuclei

1. Using the cannula insertion method, muscarinic receptor subtypes were analysed in isolated, perfused canine lingual arteries preconstricted with phenylephrine. 2. Both acetylcholine and McN-A-343 induced a profound vasodilation in a dose-related manner. Acetylcholine-induced dilations were approximately 1000-times more potent than McN-A-343-induced dilation. 3. Acetylcholine-induced dilations were abolished after removal of the endothelium by intraluminal treatment with 1 mg saponin. 4. Acetylcholine-induced dilations were markedly inhibited by an M1/M3 receptor antagonist, 4-DAMP. Moreover, they were slightly, but significantly, inhibited by an M1 antagonist, pirenzepine, but never influenced by an M2 antagonist, AF-DX 116. Mc-N-A-343-induced vasodilations were inhibited by both 4-DAMP or pirenzepine. 5. These results suggest that there are abundant functional M3 and a few M1 receptors in the canine lingual artery that mediate vasodilation and that this vasodilation is dependent on the presence of an intact endothelium.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Analysis of Variance; Animals; Dogs; Dose-Response Relationship, Drug; Female; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth, Vascular; Piperidines; Pirenzepine; Receptors, Muscarinic; Saponins; Tongue; Vasoconstriction; Vasodilation

Muscarinic receptors are believed to play an important role in modulation of ciliary action in respiratory system. We studied the in vitro effect of methacholine, a beta-methyl ester of acetylcholine, on the ciliary beat frequency (CBF). Adenoid explants were cultured in Minimum Essential Medium Eagle (MEM). CBF was determined using microphotometry. Methacholine (10(-6) M) increased CBF a maximum of 10.34 +/- 0.42% (p < 0.001). The non-selective muscarinic antagonist atropine (10(-6) M) significantly inhibited the ciliostimulatory effects of methacholine (p < 0.001). To characterize the muscarinic receptor subtypes in nasal mucosa, the selective M1-, M2- and M3-muscarinic antagonists pirenzepine dihydrochloride (PZ), gallamine triethiodide (gallamine), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were used prior to addition of methacholine, PZ and 4-DAMP, at concentrations of 10(-6) to 10(-8) M, significantly inhibited the ciliostimulatory effects of methacholine (p < 0.0001). There was no significant inhibition of methacholine-induced ciliostimulation by gallamine (p > 0.3). Our study showed that ciliostimulation by methacholine in human upper airway mucosa involves M1- and M3- muscarinic receptor subtypes, but not the M2-receptor subtype. The identification of the muscarinic receptor subtypes and intracellular signalling mechanisms involved in CBF modulation will permit the selection appropriate of pharmacological agents for treating the cholinergic symptoms of rhinitis.

Topics: Atropine; Cilia; Gallamine Triethiodide; Humans; In Vitro Techniques; Methacholine Chloride; Mucociliary Clearance; Muscarinic Agonists; Muscarinic Antagonists; Nasal Mucosa; Piperidines; Pirenzepine; Receptors, Muscarinic

The direct effect of acetylcholine on pituitary growth hormone secretion during the postnatal period of the rat was studied using a superfusion system. Acetylcholine elicited a dose related stimulatory effect on growth hormone (GH) secretion in the pituitaries from 2-day old rats. M1 muscarinic agonist McN A343 mimicked the GH releasing effect of acetylcholine, nicotine was ineffective. The GH release elicited by acetylcholine diminished with postnatal development, it was small by the end of the third postnatal week and was not demonstrable in the adult pituitaries. The effect of acetylcholine was potently antagonized by pirenzepine (M1 antagonist) and 4-DAMP (M3 and M1 antagonist) but not by methoctramine (M2 antagonist). It is concluded that unlike in the adult, in the newborn rat the cholinergic regulation of pituitary GH secretion plays a prominent role directly at pituitary level most likely via M1 receptors.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Age Factors; Animals; Animals, Newborn; Atropine; Diamines; Dose-Response Relationship, Drug; Female; Growth Hormone; Male; Muscarinic Agonists; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Pituitary Gland; Rats; Rats, Wistar

The present study was designed to investigate which muscarinic receptors the indoline derivatives interact with and also their pharmacological properties. Compounds I and II contracted guinea-pig ileum in a concentration-dependent manner, whereas compounds III-IX behaved as antagonists and Schild plots gave straight lines. 4-DAMP antagonized the contractile responses to compounds I and II, and the pA2 values for 4-DAMP were 8.96 +/- 0.23 and 9.09 +/- 0.06, respectively. In guinea-pig left atrium, compound I partly inhibited twitch responses, whereas compound II did not have any effect. In rabbit vas deferens, compounds I and II produced inhibitory effects on twitch responses evoked by field stimulation. Pirenzepine antagonized the inhibitory responses of compounds I and II, and the pA2 values for pirenzepine were 7.90 +/- 0.13 and 8.12 +/- 0.06, respectively. Compound I has about 150-fold higher affinity to M1 receptors than to M3 receptors, while compound II has about 360-fold higher affinity to M1 receptors. Our results indicate that compounds I and II show 7- and 16-fold higher M1 receptor selectivity than McN-A-343, respectively. Therefore, compound II is selective for M1 receptors over M2 or M3 receptors.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Electric Stimulation; Female; Guinea Pigs; Heart Atria; Ileum; In Vitro Techniques; Indoles; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Myocardial Contraction; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Vas Deferens

We investigated the role of M1 and M3 receptors in regulating exocrine secretion from acini isolated from rat sublingual glands. In secretion experiments, we derived affinity values (KB) from Schild regression analysis for the antagonists pirenzepine (61.0 nM) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 1.06 nM). The KB for 4-DAMP is similar to its affinity value [equilibrium dissociation constant from competition studies (Ki); 1.81 nM] determined from radioligand competition experiments. In contrast, the KB for pirenzepine is between its high-affinity (17.6 nM) and low-affinity (404 nM) Ki values. In separate secretion experiments, we found that the M1 receptor antagonist, M1-toxin, induces a rightward shift in the concentration-response curve to muscarinic agonist and inhibits maximal secretion by 40%. The inhibitory effect of M1-toxin appears specific for M1 receptor blockade, since the toxin abolishes acinar high-affinity pirenzepine-binding sites and does not inhibit secretion induced by nonmuscarinic agents. Additional pharmacological studies indicate muscarinic receptors do not function through putative neural elements within isolated acini. Our combined results are consistent with both M1 and M3 receptors directly regulating mucous acinar exocrine secretion and indicate M3 receptors alone are insufficient to induce a maximal muscarinic response.

Topics: Animals; Male; Mucus; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Signal Transduction; Sublingual Gland

In this study we demonstrate that IgG present in the sera of patients with primary Sjögren's syndrome (PSS) could bind and activate muscarinic acetylcholine receptors (mAChRs) of rat parotid gland. These antibodies were able to inhibit in a non-competitive manner the binding of 3H-quinuclidinyl benzilate (QNB) to mAChRs of purified rat parotid gland membranes. Moreover, IgG from PSS could modify biological effects mediated by mAChR activation; i.e. decrease cAMP, increase phosphoinositide turnover without affecting cGMP. Atropine and 4-DAMP blocked all of these effects, and carbachol mimicked them, confirming the M3 subtype mAChRs mediated PSS IgG action. Neither binding nor biological effect were obtained with IgG from sera of normal women. The prevalence of cholinergic antibody was 100% in PSS, and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against mAChRs may be another serum factor to be considered in the pathophysiology of the development of PSS.

Topics: Adolescent; Adrenergic beta-Agonists; Adult; Animals; Antibodies, Blocking; Atropine; Carbachol; Carbamates; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Female; Humans; Immunoglobulin G; Isoproterenol; Muscarinic Antagonists; Parasympatholytics; Parotid Gland; Phenylcarbamates; Phosphatidylinositols; Piperidines; Pirenzepine; Protease Inhibitors; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptors, Muscarinic; Sjogren's Syndrome

We investigated the roles played by three muscarinic receptors (M1, M2, and M3) in the pressor response with bradycardia that followed the injection of neostigmine (5 x 10(-8) mol) into the hippocampus of anesthetized rats. These changes were blocked by the co-administration of methylatropine (5 x 10(-8) mol). The intrahippocampal injection of pirenzepine (M1 antagonist) (5 x 10(-9) - 5 x 10(-7) mol) suppressed the neostigmine-induced pressor response dose-dependently. However injection of gallamine (M2 antagonist) (5 x 10(-8) - 5 x 10(-7) mol) and of 4-DAMP (M1 and M3 antagonist) (5 x 10(-8) - 5 x 10(-7) mol) did not suppress this hypertensive response. These findings suggest that the neostigmine-induced pressor response with bradycardia is mediated through the M1 muscarinic receptor subtype.

Topics: Animals; Atropine Derivatives; Blood Pressure; Drug Interactions; Gallamine Triethiodide; Heart Rate; Hippocampus; Male; Muscarinic Antagonists; Neostigmine; Parasympathomimetics; Piperidines; Pirenzepine; Pressoreceptors; Rats; Rats, Wistar; Receptors, Muscarinic

Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [125I]alpha-bungarotoxin revealed a KD of 62 +/- 2 nM, Bmax = 7.2 +/- 1.8 x 10(7) binding sites/OHC, and a slowly reversible dissociation rate constant, kappa-1 = 2.2 +/- 0.01 x 10(-4) min-1. L-[3H]Nicotine bound reversibly (estimated KD approximately 230 nM and Bmax approximately 5 x 10(7)) with kinetic rate constants of association kappa-1 = 6.2 +/- 0.06 x 10(4) min-1 nM-1 and dissociation kappa-1 = 0.23 +/- 0.003 min-1. [3H]Strychnine bound to OHC with a KD of 35 +/- 6 nM and Bmax = 2.6 +/- 0.5 x 10(7), and binding increased 3-4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > nM concentrations, of [3H]3-quinuclidinyl benzilate (KD = 11.5 +/- 5 nM; Bmax = 2.5 +/- 0.6 x 10(6)) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 +/- 0.5 and 6.5 +/- 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.

Topics: Animals; Atropine; Binding, Competitive; Bungarotoxins; Cochlea; Female; Guinea Pigs; Hair Cells, Auditory, Outer; In Vitro Techniques; Ligands; Male; Muscarinic Antagonists; Nicotine; Parasympatholytics; Piperidines; Quinuclidinyl Benzilate; Radioligand Assay; Receptors, Nicotinic; Strychnine

Previous studies demonstrated neurally mediated recurrent increases in short-circuit current (Isc) suggestive of anion secretion in guinea pig distal colon. To determine the neural pathways involved, segments of distal colon from guinea pigs were mounted in flux chambers. In muscle-stripped or whole thickness preparations, serosal addition of the histamine H2 receptor agonist, dimaprit, caused cyclical increases in Isc, which were reduced by the chloride channel blocker, N-phenylanthranilic acid, but not by the sodium channel blocker amiloride. Dimaprit stimulated release of [3H]acetylcholine and vasoactive intestinal polypeptide (VIP) from submucosal/mucosal sheets. Dimaprit caused recurrent increases in Isc, which were significantly decreased by mecamylamine, a nicotinic receptor antagonist, and nearly abolished by the muscarinic antagonist, atropine (M3 > M1 = M2). The muscarinic antagonist, 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP, M3 > M1), was more potent than pirenzepine (M1 > M3) in reducing recurrent increases in Isc. Dimaprit- and electrically evoked secretion were inhibited by the VIP antagonists [4Cl-D-Phe6, Leu17]VIP and VIP hybrid. The results suggest the involvement of VIP-ergic and cholinergic neurons utilizing nicotinic and muscarinic synapses in mediating secretion.

Topics: Acetylcholine; Amiloride; Animals; Atropine; Choline; Colon; Dimaprit; Electric Conductivity; Guinea Pigs; Histamine Release; Intestinal Mucosa; Male; ortho-Aminobenzoates; Piperidines; Pirenzepine; Receptors, Histamine H2; Vasoactive Intestinal Peptide

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Alkaloids; Animals; Arecoline; Bethanechol; Carbachol; Diamines; Dose-Response Relationship, Drug; Furans; Ganglia, Parasympathetic; Ganglionectomy; Iris; Male; Methacholine Chloride; Muscarinic Agonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Parasympatholytics; Pilocarpine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic

Acetylcholine receptor agonists and antagonists were used in a pharmacological analysis to identify which muscarinic receptor(s) may be involved in cholinergic regulation of Cl- secretion across rat colonic mucosa in vitro. A comparative ligand binding analysis for each of the antagonists was carried out in parallel. Both studies elicited identical rank order potencies (atropine > or = 4-diphenyl-acetoxy-N-piperidine methiodide (4-DAMP) > pirenzepine > 11-[[2[(diethylamino)methyl]-1-pipiridinyl]acetyl[5,11- dihydro-6H-pyrido[2,3-b]]1,4]benzodiazepine-6-one (AF-DX 116). Cholinomimetic-induced Cl- secretion was predominantly mediated by activation of muscarinic receptors in rat isolated colonic mucosa, with only a modest contribution from nicotinic receptors. Short circuit current responses evoked by the selective muscarinic M1 receptor agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-a minium chloride (McN-A-343) suggest that this receptor subtype, which is thought to be neuronally sited, also plays a minor role in regulation of intestinal ion transport. The principal epithelial cell receptors responsible for acetylcholine receptor-mediated Cl- secretion appear to belong to the M3 class.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Binding, Competitive; Chlorides; Cholinergic Agonists; Cholinergic Antagonists; Colon; Dose-Response Relationship, Drug; Epithelium; Hexamethonium; In Vitro Techniques; Ion Transport; Male; Muscarinic Agonists; Parasympatholytics; Piperidines; Pirenzepine; Radioligand Assay; Rats; Rats, Wistar

While retrograde regulation of neuronal development by target-derived factors in the autonomic nervous system is well established, the importance of anterograde influences on target development is unclear. Previous studies suggest that sympathetic innervation of sweat glands plays a critical role in the acquisition and maintenance of their secretory function. To define the signal(s) responsible, we disrupted muscarinic cholinergic transmission in developing and adult rats. Treatment of young rats with the nonselective antagonist, atropine, or an antagonist selective for the glandular muscarinic subtype, 4-DAMP, delayed the development of secretory responsiveness. Treatment of adult animals with atropine caused its loss. Further, following denervation, treatment with the muscarinic agonist, pilocarpine, largely preserved responsiveness while untreated animals lost function. Thus, acetylcholine, whose presence in sweat gland innervation is retrogradely specified by developmental interactions with the target tissue, in turn plays an important role in inducing and maintaining target tissue responsiveness through muscarinic receptor activation.

Topics: Acetylcholine; Age Factors; Animals; Atropine; Parasympatholytics; Pilocarpine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Sweat Glands; Time Factors

The densities of M1, M2 and M3 muscarinic receptors in human detrusor muscle were measured using 3H-pirenzepine (3H-PZP), 3H-AFDX-116 (3H-AFDX) and 3H-4-diphenyl-acetoxy-N-methyl-piperidine methidide (3H-4DAMP). The affinities of PZP, AFDX and 4DAMP for human detrusor were determined in inhibition experiments with 3H-quinuclidinyl benzilate (3H-QNB). Saturation experiments with 3H-PZP, 3H-AFDX and 3H-4DAMP revealed the presence of M1, M2 and M3 receptors in human detrusor. The KD values (nM) and the Bmax values (fmol/mg protein) (mean +/- SD, n = 6) were 0.84 +/- 0.15 and 13.04 +/- 1.54 for 3H-PZP, 0.68 +/- 0.21 and 9.30 +/- 1.10 for 3H-AFDX, and 0.25 +/- 0.13 and 102.1 +/- 7.40 for 3H-4DAMP. These data indicate that the bladder muscarinic receptors consist mainly of the M3 subtype. Nonlabeled PZP, AFDX and 4DAMP inhibited the 3H-QNB binding to human detrusor with Ki values (nM) (mean +/- SD, n = 6) of 243 +/- 62.5, 59.7 +/- 15.3, 2.69 +/- 0.96, respectively. Human detrusor was found to have a high affinity for 4DAMP. These data suggest that M3 muscarinic receptors are biochemically predominant in human detrusor muscle.

Topics: Aged; Cystectomy; Female; Humans; Male; Muscle, Smooth; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Urinary Bladder Neoplasms

A number of compounds structurally related to 4-DAMP (1) were synthesized and a single crystal X-ray structural study on a representative member of this series was carried out. All the compounds were tested for the antagonist activity in isolated guinea pig atria (M2 muscarinic receptors) and ileum (M3 muscarinic receptors). Affinity values (pA2) for the muscarinic receptor subtypes ranged from 5.39 to 9.71 (M2) and from 5.68 to 9.92 (M3), depending on different structural features of the compounds. A molecular modeling study was performed, with the aim of rationalizing the affinity data for both M2 and M3 muscarinic receptor subtypes. The presence in the series of two highly active, structurally constrained derivatives allowed us to define two different pharmacophoric frames on which all the compounds could be fitted in a satisfactory manner.

Topics: Animals; Crystallography, X-Ray; Guinea Pigs; Ileum; Models, Molecular; Molecular Conformation; Molecular Structure; Muscarinic Antagonists; Myocardium; Piperidines; Receptors, Muscarinic

1. In order to determine an involvement of muscarinic M2 receptors in the regulation of systemic arterial blood pressure, we investigated the cardiovascular effects of the M2-selective antagonist methoctramine and other agents in anaesthetized guinea-pigs. 2. Intravenous injection of methoctramine, atropine, pirenzepine (an M1-selective muscarinic antagonist) or 4-DAMP (an M3-selective muscarinic antagonist) each significantly increased heart rate in comparison to vehicle controls. 3. Methoctramine produced significant, dose-dependent decreases in mean arterial blood pressure, with an ED50 of 0.1 mg kg-1. Atropine decreased blood pressure only at high doses. Pirenzepine and 4-DAMP did not alter blood pressure, indicating that M1 or M3 receptor antagonism was not responsible for the cardiovascular effects of methoctramine. 4. The hypotensive effect of methoctramine was unaltered by indomethacin pretreatment, ruling out an alteration in arachidonic acid metabolism as the mechanism of action. 5. In contrast to methoctramine, mecamylamine (a nicotinic ganglionic receptor antagonist) greatly decreased heart rate and slightly decreased blood pressure, suggesting that ganglionic blockade was not the mechanism for the cardiovascular effects of methoctramine. 6. Methoctramine (0.3 mg kg-1) pretreatment did not alter the hypertensive effect of intravenous noradrenaline, demonstrating that methoctramine did not directly inhibit vascular reactivity and indicating an indirect hypotensive of action of methoctramine. 7. In summary, the results suggest that the hypotensive action of methoctramine resulted from selective M2 receptor antagonism. Therefore, muscarinic M2 receptors appear to play a role in the regulation of systemic arterial blood pressure in guinea-pigs. However, the anatomical site(s) of action of methoctramine remains to be determined.

Topics: Animals; Atropine; Blood Pressure; Depression, Chemical; Diamines; Guinea Pigs; Heart Rate; Male; Muscarinic Antagonists; Norepinephrine; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic

This report documents slow changes in cochlear responses produced by electrical stimulation of the olivocochlear bundle (OCB), which provides efferent innervation to the hair cells of the cochlea. These slow changes have time constants of 25-50 sec, three orders of magnitude slower than those reported previously. Such "slow effects" are similar to classically described "fast effects" in that (1) they comprise a suppression of the compound action potential (CAP) of the auditory nerve mirrored by an enhancement of the cochlear microphonic potential (CM) generated largely by the outer hair cells; (2) the magnitude of suppression decreases as the intensity of the acoustic stimulus increases; (3) they share the same dependence on OCB stimulation rate; (4) both are extinguished upon cutting the OCB; and (5) both are blocked with similar concentrations of a variety of cholinergic antagonists as well as with strychnine and bicuculline. These observations suggest that both fast and slow effects are mediated by the same receptor and are produced by conductance changes in outer hair cells. Slow effects differ from fast effects in that (1) fast effects are greatest for acoustic stimulus frequencies between 6 and 10 kHz, whereas slow effects peak for frequencies from 12 to 16 kHz, and (2) fast effects persist over long periods of OCB stimulation, whereas slow effects diminish after 60 sec of stimulation. The time course of the slow effects can be described mathematically by assuming that each shock-burst produces, in addition to a fast effect, a small decrease in CAP amplitude that decays exponentially with a time constant that is long relative to the intershock interval. The long time constant of the slow effect compared to the fast effect suggests that it may arise from a distinct intracellular mechanism, possibly mediated by second-messenger systems.

Topics: Acoustic Stimulation; Animals; Atropine; Bicuculline; Cochlea; Decamethonium Compounds; Dose-Response Relationship, Drug; Efferent Pathways; Electric Stimulation; Guinea Pigs; Hair Cells, Auditory, Outer; Hexamethonium; Membrane Potentials; Models, Neurological; Nerve Fibers; Piperidines; Strychnine; Time Factors; Vestibulocochlear Nerve

1. The effects of pertussis toxin (PTX) on contraction and/or relaxation induced by agonists or transmural nerve stimulation (TNS) were examined in the rat iris dilator and sphincter muscles. 2. TNS in the presence of phentolamine induced an atropine-sensitive biphasic response: initial contraction followed by relaxation in dilator muscles. Exogenously applied acetylcholine (ACh) elicited a large relaxation at low doses (3 microM or less) and a concentration at high doses. 3. Only the ACh-induced relaxation was affected by injection of PTX (10 ng) into the anterior eye chamber. Relaxation was decreased 12 h after injection and had completely disappeared after 24 h. Relaxation recovered in part 3 weeks and almost completely 8 weeks after PTX treatment. A gradual decrease in muscarinic relaxation in a dilator muscle was also observed in vitro after addition of PTX to the bathing solution. 4. The pA2 values of muscarinic blockers, pirenzepine, AF-DX 116, 4-DAMP, and himbacine for competitive antagonism to ACh-induced contraction were 7.14, 6.53, 9.03, and 6.80, respectively, in PTX-pretreated dilator muscles. These values are comparable to those obtained in parasympathectomized dilator muscles and may indicate involvement of M3 or M3-like receptors in muscle contraction. 5. Pretreatment with PTX did not significantly affect contraction induced by noradrenaline or 5-hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles. 6. In conclusion, among several agonist-induced responses in the rat iris dilator and sphincter muscles, only muscarinic relaxation in dilator muscle occurs via activation of PTX-sensitive GTP binding proteins.

Topics: Acetylcholine; Alkaloids; Animals; Atropine; Diamines; Dose-Response Relationship, Drug; Furans; GTP-Binding Proteins; In Vitro Techniques; Iris; Male; Microinjections; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Parasympatholytics; Pertussis Toxin; Phentolamine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Virulence Factors, Bordetella

The pirenzepine-related analogue, nuvenzepine, and the antagonists selective for the three muscarinic receptor subtypes 4-DAMP (M1 and M3 receptors), pirenzepine (M1 receptors), methoctramine (M2 receptors) have been tested on rabbit isolated small and large intestinal regions provided with spontaneous motor activity. The employed drugs differently affected intestinal motility patterns. The ileum pendular movements as well as the proximal colon and taenia coli tone, spike amplitude and frequency were concentration-dependently inhibited by the compounds here employed. Their pIC50 values followed the rank order of potency generally reported for the involvement of the M3 muscarinic receptors (4-DAMP > or = atropine > nuvenzepine > or = pirenzepine > methoctramine). Unlike nuvenzepine and the other antimuscarinics assayed, the M1 selective antagonist pirenzepine, at nanomolar concentrations, was able to enhance the proximal taenia coli motility patterns suggesting that a M1-inhibitory pathway might operate in the physiological modulation of taenia coli motility. At variance with longitudinal ileum and colon contractile activity, proximal circular colon motility was resistant to muscarinic as well as to alpha 1-, H1-, 5-HT-blockade indicating that NANC neuronal mechanisms could act at this level. In summary, these data provide evidence that, at intestinal level, nuvenzepine is almost completely devoid of reliable M1-linked effect thus possessing a different pharmacological selectivity at muscarinic receptor subtypes with respect to pirenzepine. Furthermore, it emerges that rabbit spontaneous small and large intestinal motility is probably driven by different physiological mechanisms regional-related. The peculiar circular colon refractoriness deserves further studies to be extended to the human tissue.

Topics: Animals; Atropine; Benzodiazepinones; Diamines; Dose-Response Relationship, Drug; Gastrointestinal Motility; Intestine, Large; Intestine, Small; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic

The functional role of the cholinergic nervous system in regulating gastrin release was investigated using enriched canine antral G cells. Gastrin content was 30.1 +/- 2.9 pmol per well and basal gastrin release was 900 +/- 27 fmol per well (n = 45). Carbachol (10(-8) to 10(-5) M) dose-dependently stimulated gastrin release with a maximal stimulatory response achieved at a concentration of 10(-5) M (330% over basal). To characterize the muscarinic receptor which mediates gastrin release from antral G cells, we examined the effect of three muscarinic receptor antagonists on carbachol-stimulated gastrin release; atropine (nonselective muscarinic receptor antagonist), pirenzepine (M1 muscarinic receptor antagonist) and 4-DAMP (M3 muscarinic receptor antagonist). Atropine (10(-9) to 10(-6) M), pirenzepine (10(-8) to 10(-5) M) and 4-DAMP (10(-9) to 10(-6) M) had no effect on the basal gastrin release. However, carbachol (10(-5) M)-stimulated gastrin release was effectively inhibited by atropine and 4-DAMP with Ki values of 0.48 and 0.66 nM, respectively. Pirenzepine at a high concentration (10(-5) M) also inhibited carbachol-stimulated gastrin release with a Ki value of 46.3 nM. These results suggest that the cholinergic nervous system directly stimulates gastrin release via M3 muscarinic receptors located on antral G cells.

Topics: Animals; Atropine; Carbachol; Cells, Cultured; Dogs; Dose-Response Relationship, Drug; Enterochromaffin Cells; Gastrins; Muscarinic Antagonists; Parasympathetic Nervous System; Parasympatholytics; Piperidines; Pirenzepine; Pyloric Antrum; Receptor, Muscarinic M3; Receptors, Muscarinic; Stimulation, Chemical

The effect of carbachol on the excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of carbachol (0.01-3 microM) reversibly decreases the EPSP amplitude in a concentration-dependent manner and with an estimated IC50 of 0.3 microM. While, neither the N-methyl-D-aspartate (NMDA, 100 microM)- nor (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 100 microM)-induced response was affected by carbachol (0.1 microM). In addition, the inhibitory effect induced by carbachol at a low concentration of 0.1 microM was attenuated by 4-diphenylacetoxy-N,N-methyl-piperidine (4-DAMP), a selective M3 muscarinic receptor antagonist. However, other muscarinic subtype (M1 or M2) antagonists could also block the inhibitory effect by carbachol 0.1 microM. The rank order of antagonist potency was: 4-DAMP (M3 antagonist) > methoctramine (M2 antagonist) > pirenzepine (M1 antagonist). Based on these findings, we conclude that carbachol at a low concentration (< or = 0.1 microM) reduced the excitatory response of neostriatal neurons following cortical stimulation via presynaptic M3 muscarinic receptors located on the terminals of corticostriatal neurons.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Carbachol; Depression, Chemical; Electric Stimulation; Evoked Potentials; In Vitro Techniques; N-Methylaspartate; Neostriatum; Neurons; Patch-Clamp Techniques; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Receptors, Presynaptic; Synaptic Transmission

1. The effects of seven muscarinic receptor antagonists were used to characterize the receptors which mediate carbachol-evoked contractions of intertaenial circular and taenial longitudinal muscle in human isolated colon. The effects of these antagonists were studied upon colon contractions induced by cumulatively added carbachol which had mean EC50 values of 11.7 +/- 2.3 microM (n = 8) and 12.6 +/- 2.3 microM (n = 8) respectively upon circular and longitudinal smooth muscle. 2. All antagonists displaced concentration-response curves to carbachol to the right in a parallel manner. The maximum concentration of each antagonist added (30 nM-10 microM) did not significantly suppress the maximum response. 3. In circular muscle, the M3 muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladiphenidol (HHSiD) and para-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) inhibited responses with pA2 values of 9.41 +/- 0.23, 7.17 +/- 0.07, 6.94 +/- 0.18 respectively. The M2 muscarinic receptor antagonist, AF-DX 116, the M2/M4 muscarinic receptor antagonist, himbacine, and the M1 muscarinic receptor antagonist, pirenzepine, yielded pA2 values of 7.36 +/- 0.43, 7.47 +/- 0.14 and 7.23 +/- 0.48 respectively. The non-selective antagonist, atropine, had a pA2 of 8.72 +/- 0.28. 4. In longitudinal muscle 4-DAMP, HHSiD, p-F-HHSiD, AF-DX 116, himbacine and pirenzepine gave pA2 values of 9.09 +/- 0.16, 7.45 +/- 0.43, 7.44 +/- 0.21, 6.44 +/- 0.1, 7.54 +/- 0.40, 6.87 +/- 0.38 respectively. Atropine yielded a pA2 value of 8.60 +/- 0.08. 5. The pharmacological profile of antagonist affinities at the muscarinic receptor population responding to muscarinic agonist-evoked contraction is similar to that widely accepted as characterizing the activation of an M3 muscarinic receptor subtype, although pA2 values of some antagonists are lower than that seen in other investigations.

Topics: Alkaloids; Atropine; Carbachol; Carcinoma; Colon; Colonic Neoplasms; Dose-Response Relationship, Drug; Furans; Humans; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Naphthalenes; Piperidines; Pirenzepine

The influence of slaframine (SF), a parasympathomimetic compound isolated from the fungus Rizoctonia leguminicola, on circulating metabolic hormone concentrations was investigated in goats. In Exp. 1, SF was administered i.v. at 0 (CONT), 50 (LSF), 100 (MSF), or 150 (HSF) microgram/kg.75 BW in four mature Spanish-cross does (average BW 36 +/- 7 kg) fitted with indwelling jugular vein catheters in a 4 x 4 Latin square design. Plasma glucose peaked (P < .06) at 120 min with LSF and at 180 min with HSF and was higher (P <.06) than the CONT at these times. Glucose exhibited a quadratic response (P < .03) to SF. Area under the response curve for glucose differed (P < .02) in HSF from CONT and MSF. Insulin peaked (P < .01) at 240 min with MSF and at 180 min with HSF. Plasma triiodothyronine was maintained at a higher level (P < .03) with HSF. Thyroxine peaked (P < .06) at 120 min with MSF and 300 min with HSF. Plasma NEFA and somatotropin concentrations were not affected (P > .10) by SF. In Exp. 2, four mature Spanish-cross wethers (average BW 27 +/- 2 kg) fitted with jugular vein catheters were administered SF (0 and 114 micrograms/kg.75 BW) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4DAMP; 0 and 258 micrograms/kg.75 BW), a M3-muscarinic receptor antagonist, i.v. in a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. With SF, glucose peaked (P < .06) at 60 min and insulin peaked (P < .05) at 180 min. Plasma triiodothyronine levels were maintained (P < .05) with SF but declined with other treatments. Plasma NEFA and thyroxine concentrations remained unchanged regardless of treatment. Slaframine administration induced hyperglycemia and hyperinsulinemia in goats; however, these changes were blocked by preadministration of isomolar quantities of the M3-muscarinic receptor antagonist, 4DAMP.

Topics: Alkaloids; Animals; Blood Glucose; Endocrine Glands; Fatty Acids, Nonesterified; Female; Goat Diseases; Goats; Growth Hormone; Hyperglycemia; Hyperinsulinism; Injections, Intravenous; Insulin; Male; Muscarinic Agonists; Muscarinic Antagonists; Parasympathomimetics; Piperidines; Receptors, Muscarinic; Thyroxine; Triiodothyronine

Muscarinic receptor subtypes mediating catecholamine secretion and increase in the intracellular concentration of Ca2+ ([Ca2+]i) were examined using muscarinic agonists and antagonists in dispersed adrenal chromaffin cells of the guinea pig. All muscarinic agonists (1-1,000 microM) tested caused increases in adrenaline secretion in a dose-dependent manner. Muscarine and methacholine were more effective than bethanechol, oxotremorine and pilocarpine. Muscarine and oxotremorine caused a small increase in adrenaline secretion even in the absence of extracellular Ca2+. Both 4-DAMP (0.1 microM) and pirenzepine (0.1 microM), but not methoctramine (0.1 microM), shifted the dose-response curve for muscarine-induced adrenaline secretion to the right. These muscarinic agonists also caused increase in [Ca2+]i in the presence of extracellular Ca2+. Muscarine-induced [Ca2+]i rises were reduced, but not abolished, by removal of extracellular Ca2+. These results suggest that catecholamine secretion induced by muscarinic agonists is mediated through M1, or M1 and M3 muscarinic receptor subtypes in adrenal chromaffin cells of the guinea pig.

Topics: Adrenal Medulla; Animals; Calcium; Cells, Cultured; Cholinergic Agents; Chromatography, High Pressure Liquid; Diamines; Dose-Response Relationship, Drug; Epinephrine; Female; Guinea Pigs; Kinetics; Male; Methacholine Chloride; Muscarine; Norepinephrine; Oxotremorine; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic

1. The muscarinic receptor subtype mediating contraction of the canine saphenous vein has been characterized using a range of muscarinic agonists and subtype-selective antagonists. 2. Oxotremorine M and (+)-cis-dioxolane behaved as full agonists, while in comparison L-660,863 ((+/-)-3-(3-amino-1,2,4-oxadiazole-5-yl)quinuclidine) acted as a partial agonist. SDZ ENS 163 (thiopilocarpine), pilocarpine and McN-A-343 (0.1 microM-0.3 mM) did not elicit a response. The profile of agonist potencies suggests a low receptor reserve for contraction. 3. The rank order of antagonist apparent affinities was 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide; 8.41) > pirenzepine (8.10) > himbacine (7.34) > or = p-F-HHSiD (para-fluoro-hexahydrosiladifenidol; 7.15) > methoctramine (6.23). This antagonist apparent affinity profile is consistent with the activation of muscarinic M1 receptors.

Topics: Animals; Dioxolanes; Dogs; Female; In Vitro Techniques; Muscarinic Agonists; Muscle Contraction; Muscle, Smooth, Vascular; Oxotremorine; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptors, Muscarinic; Saphenous Vein

1. In pentobarbitone-anaesthetized rabbits, the inhibitory effects of muscarinic receptor antagonists with different selectivity profiles were examined on carbachol-evoked submandibular secretion and urinary bladder contractions, and on parasympathetically nerve-evoked secretion. On isolated submandibular gland fragments, the inhibitory effects of the antagonists were studied on carbachol-evoked release of potassium and on the overflow of tritium in response to electrical field stimulation. 2. In vivo, 4-DAMP equipotently inhibited simultaneously carbachol-evoked submandibular secretory and contractile responses of the urinary bladder, while pirenzepine was found to be four times as potent in inhibiting the secretory response compared with the contractile response. 3. The inhibition of carbachol-evoked salivation caused by atropine, 4-DAMP and pirenzepine was as great as their inhibition of parasympathetic nerve-evoked salivation. Methoctramine exerted less inhibitory effect on nerve-evoked salivation than on carbachol-evoked, thus seemingly causing greater presynaptic inhibition. 4. In vitro, pirenzepine was only 30 times less potent in inhibiting carbachol-evoked potassium release than 4-DAMP (pA2, 9.58 vs 8.10). Whereas atropine, 4-DAMP and pirenzepine abolished the overflow of tritium from isolated glands in response to electrical field stimulation, methoctramine increased it. 5. It is concluded that the muscarinic secretory response in the rabbit submandibular gland is exerted via both muscarinic M1 and M3 receptors, while the contractile response of the urinary bladder to muscarinic agonists is exerted via muscarinic M3 receptors. The release of acetylcholine from nerve terminals in the gland can be inhibited via M2 autoreceptors in rabbits.

Topics: Animals; Atropine; Carbachol; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Muscarinic Antagonists; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Submandibular Gland; Urinary Bladder

ACh regulates the gene encoding phenylethanolamine N-methyltransferase (PNMT) in bovine adrenal chromaffin cells. In addition to stimulating catecholamine release from these cells, cholinergic agents elevate transcription of the PNMT gene. Carbachol, which activates both nicotinic and muscarinic receptors, produces 12-19-fold increases in PNMT mRNA and a 22-fold increase in epinephrine release. Selective nicotinic and muscarinic antagonists (hexamethonium and atropine) each partially reduce carbachol-stimulated increases in PNMT mRNA while a combination of both eliminates > 90% of the carbachol response, thus indicating that separable nicotinic and muscarinic components contribute to the cholinergic increase in PNMT mRNA. Muscarine alone produces a dose-dependent increase (mean sixfold) in steady state PNMT mRNA levels and stimulates the rate of transcription fivefold. Only atropine and the m3-m4-selective muscarinic antagonist 4-diphenylacetoxy-4-methyl-piperidine (4-DAMP) reduce the response to muscarine, strongly suggesting that the m4 receptor is crucial for PNMT mRNA activation. In these chromaffin cells, muscarine inhibits adenylate cyclase, antagonist bind with affinities characteristic of m4 receptors, and cDNA hybridization detects only m4 mRNAs (Fernando et al., 1991). Nicotine also induces a dose-dependent increase (mean of 8.5-fold) in PNMT mRNA levels. The importance of voltage-gated Ca2+ channels in the nicotine effect is demonstrated by the stimulatory effects of calcium ionophores on PNMT mRNA levels (two-to fivefold increase) and the ability of the L- and N-type channel blockers nifedipine and omega-conotoxin to decrease the nicotine response (by 60% and 40%, respectively). Nuclear "run-on" assays further reveal that nicotine enhances transcription of the PNMT gene (approximately fourfold). Thus, this study provides the first demonstration that both nicotinic and muscarinic stimulation modify genomic responses of bovine adrenergic chromaffin cells and identifies possible mechanisms.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Acetylcholine; Adrenal Medulla; Animals; Atropine; Binding, Competitive; Calcimycin; Calcium; Carbachol; Cattle; Cells, Cultured; Colforsin; Epinephrine; Gene Expression Regulation, Enzymologic; Hexamethonium; Hexamethonium Compounds; Kinetics; Muscarine; Nicotine; Phenylethanolamine N-Methyltransferase; Piperidines; Quinuclidinyl Benzilate; Receptors, Muscarinic; Receptors, Nicotinic; RNA, Messenger

Muscarinic acetylcholine receptors (mAChRs) on the cell body of the fast coxal depressor motor neurone (Df) in the metathoracic ganglion of the cockroach Periplaneta americana were investigated using electrophysiological methods. Muscarinic agonists, arecoline and oxotremorine, induced dose-dependent depolarizations on motor neurone Df. McN-A-343, a vertebrate mAChR M1 subtype-selective agonist, failed to induce any responses when tested on the same neurone at concentrations of up to 1.0 x 10(-4) M. The order of effectiveness of a series of muscarinic antagonists on the mAChRs of motor neurone Df is as follows: scopolamine > atropine > pirenzepine. 4-DAMP (1.0 x 10(-5) M) had only a weak blocking effect and AF-DX 116 (1.0 x 10(-5) M) was completely inactive. The pharmacological profile of muscarinic responses on motor neurone Df reveals a novel type of insect mAChR.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Arecoline; Atropine; Dose-Response Relationship, Drug; Evoked Potentials; Ganglia, Invertebrate; In Vitro Techniques; Male; Motor Neurons; Muscarinic Agonists; Muscarinic Antagonists; Oxotremorine; Periplaneta; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine; Vertebrates

There are ultrastructural and histochemical differences between the longitudinal (putatively more relevant to outflow facility) and circular (putatively more relevant to accommodation) portions of the primate ciliary muscle. Oxotremorine, a muscarinic agonist putatively somewhat selective for the M2 receptor subtype, binds preferentially to the longitudinal rather than the circular portion. Aceclidine, a putatively non-subtype selective muscarinic agonist, can dissociate accommodative and outflow facility responses in monkeys and humans. We used the muscarinic receptor subtype antagonists 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), 11-([2-(diethylamino)methyl]-1-piperidinyl)acetyl]- 5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116), and pirenzepine to inhibit contractile responses to the muscarinic agonists carbachol, aceclidine and oxotremorine in the longitudinal and circular vectors of the rhesus monkey ciliary muscle in vitro. Oxotremorine generated dose-response curves that were similar in both the circular and longitudinal vectors and intermediate to those previously reported for carbachol and aceclidine. 4-DAMP (M3 selective) was the most potent inhibitor of contractile responses to all three agonists, with IC50 values ranging from 33 to 68 nM for the circular and from 27 to 63 nM for the longitudinal vector, depending on the agonist used to elicit contraction. Pirenzepine (M1 selective) was > or = 25-fold less potent and AF-DX 116 (M2 selective) was > or = 108-fold less potent at inhibiting contractile responses to all three agonists in either vector, indicating that M3 is the predominant receptor subtype mediating ciliary muscle contraction in both vectors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbachol; Ciliary Body; Dose-Response Relationship, Drug; In Vitro Techniques; Macaca mulatta; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Oxotremorine; Piperidines; Pirenzepine; Quinuclidines

Muscarinic receptor subtype in type-1 and type-2 astrocytes from rat neonalal cerebral cortex was examined for carbachol-elicited inositol phosphate (IP) formation. The formation of carbachol-elicited IP was inhibited by various muscarinic antagonists in the following relative order of potency: 4-DAMP > or = atropine >> pirenzepine > AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the stimulation of IP formation in both astrocytes.

Topics: Animals; Animals, Newborn; Astrocytes; Atropine; Carbachol; Cells, Cultured; Cerebral Cortex; Inositol Phosphates; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Signal Transduction; Tubocurarine

The purpose of this study was to determine the regulation of pancreatic polypeptide (PP) release by using pirenzepine (a specific M1 muscarinic receptor antagonist), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP, a specific M3 muscarinic receptor antagonist), atropine (a nonspecific muscarinic receptor antagonist), and loxiglumide (cholecystokinin, CCK, receptor antagonist) in dogs. In conscious dogs with chronic gastric and duodenal fistulas, release of PP and exocrine pancreatic secretion were stimulated by constant intravenous infusion of CCK-8 (200 ng/kg/h). Graded doses of pirenzepine (0.18-4.7 mmol/kg/h), 4-DAMP (6.7-180 nmol/kg/h), or atropine (0.89-24 nmol/kg/h) dose-dependently reduced plasma PP responses to CCK-8 without influence on exocrine pancreatic secretion. ID50 calculated from these results were 492 +/- 150 nmol/kg/h for pirenzepine, 10.7 +/- 1.8 nmol/kg/h for 4-DAMP and 19.4 +/- 5.2 nmol/kg/h for atropine. A similar sequence in the inhibitory potency was observed in 2-deoxy-D-glucose (2-DG, 100 mg/kg)-stimulated PP release, exocrine pancreatic, and gastric secretions. On the other hand, loxiglumide, a CCK receptor antagonist, did not influence PP release stimulated by 2-DG. These findings suggest that both CCK- and 2-DG-stimulated PP releases are mainly under cholinergic nerve control mediated by M3 muscarinic receptor in dogs.

Topics: Animals; Atropine; Cholecystokinin; Dogs; Female; Gastric Acid; Male; Pancreatic Juice; Pancreatic Polypeptide; Parasympatholytics; Piperidines; Pirenzepine; Proglumide; Receptors, Muscarinic; Vagus Nerve

Injections of acetylcholine (ACh) into the lateral septal area (LSA) caused blood pressure increases in unanesthetized freely moving rats. ACh was injected in the dose range of 0.1-54 nmol/500 nl using regular metal needles (200 microns o.d.). In the LSA, injections of carbachol or ACh (2.5 nmol/500 nl) were equipotent (+22 +/- 2 and +19 +/- 3 mmHg, respectively) suggesting the existence of an ACh-sensitive pressor site in the LSA. Maximum responses to ACh injected either intracerebroventricularly (i.c.v.) or into the LSA were not significantly different (+23 +/- 1 and +21 +/- 2 mmHg, respectively). However, the ED50 for the injection into the LSA (0.24 nmol) was significantly lower than that observed after i.c.v. injection (2.6 nmol), ruling out a possible leakage of ACh from the LSA and into the ventricular space. This idea is supported by data showing that the effect of the intraseptal injection of 30 nmol of ACh was blocked by pretreatment with 3 nmol of atropine either i.c.v. or into the LSA, whereas the effects of i.c.v. ACh were completely blocked by i.c.v. atropine, but only partially (42%) when atropine was injected into the LSA. The idea of the existence of an ACh-sensitive site in the LSA is further supported by the more direct observation that injections of 30 nmol/100 nl of ACh into the LSA using glass needles (50-70 microns o.d.) caused similar pressor responses. Neither the i.v. pretreatment with pentolinium or adrenalectomy affected the response to 30 nmol/500 nl of ACh injected into the LSA, ruling out the involvement of the sympathetic nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Adrenal Glands; Animals; Atropine; Blood Pressure; Ganglionic Blockers; Injections, Intraventricular; Male; Parasympatholytics; Piperidines; Pirenzepine; Pituitary Gland; Rats; Rats, Wistar; Septal Nuclei; Stimulation, Chemical; Vasopressins

Cholinergic agents used to lower intraocular pressure in the medical management of glaucoma act on muscarinic receptors in the eye. Recently, subtypes of muscarinic receptors have been recognized in many tissues including the eye. To localize the M3 muscarinic receptor subtype and m3 messenger RNA (mRNA) in the human eye, we used in vitro ligand binding and in situ hybridization techniques on post-mortem sections. We used an M3 antagonist, [3H]-4-DAMP ([3H]-4-diphenylacetoxy-N-methylpiperidine methiodide) to identify M3-binding sites and a [35S]-labelled oligonucleotide probe to detect and localize m3 mRNA. M3 and m3 mRNA were both localized in the ciliary muscle, ciliary epithelium, iris, corneal epithelium and anterior lens epithelium. The m3 transcript was also detected in the trabecular meshwork and corneal endothelium.

Topics: Acetylcholine; Adult; Aged; Anterior Eye Segment; Autoradiography; Blotting, Northern; Ciliary Body; Female; Humans; In Situ Hybridization; Iris; Male; Middle Aged; Parasympatholytics; Piperidines; Receptors, Muscarinic; RNA, Messenger

We investigated the effects of subtype-selective muscarinic receptor antagonists upon aerosol antigen-induced bronchoconstriction in anesthetized guinea pigs. Neither pirenzepine (muscarinic M1 receptor-selective), 4-methylpiperidine methiodide (4-DAMP, muscarinic M3 receptor-selective), [N-iminomethyl-N'-[(2-hydroxy-2-phenyl-2-cyclohexyl)-ethyl] piperazine HCl (DAC-5945, muscarinic M3 receptor-selective), ipratropium or atropine inhibited bronchoconstriction, but methoctramine (muscarinic M2 receptor-selective) produced a dose-dependent increase in bronchoconstriction (up to 46%). Methoctramine also produced increases in bronchoconstriction induced by aerosols of histamine (up to 45%) and platelet activating factor (up to 118%), demonstrating nonspecific airway hyperresponsiveness. This effect of methoctramine was not inhibited by atropine, DAC-5945 or vagotomy and could not be attributed to altered arachidonic acid metabolism or beta-adrenergic antagonism. However, propranolol prevented methoctramine-induced airway hyperresponsiveness, suggesting that this effect resulted from the reported ganglionic blocking activity of methoctramine. In conclusion, muscarinic receptors do not appear to play an important role in antigen-induced bronchoconstriction in anesthetized guinea pigs. Furthermore, caution should be exercised in using methoctramine to characterize the roles of muscarinic receptors in airway inflammatory responses in vivo.

Topics: Aerosols; Albuterol; Animals; Arachidonic Acid; Atropine; Bronchial Hyperreactivity; Bronchoconstriction; Diamines; Drug Synergism; Gallamine Triethiodide; Guinea Pigs; Histamine; Male; Muscarinic Antagonists; Ovalbumin; Parasympatholytics; Piperazines; Piperidines; Platelet Activating Factor; Propranolol; Receptors, Muscarinic

1. The antimuscarinic properties of liriodenine, isolated from Fissistigma glaucescens, were compared with methoctramine (cardioselective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, smooth muscle selective M3 antagonist) by radioligand binding tests, functional tests and measurements of second messenger generation in canine cultured tracheal smooth muscle cells. 2. Liriodenine, pirenzepine, methoctramine and 4-DAMP displaced [3H]-N-methyl scopolamine ([3H]-NMS) binding in a concentration-dependent manner with Ki values of 2.2 +/- 0.4 x 10(-6), 3.3 +/- 0.7 x 10(-7), 8.9 +/- 2.3 x 10(-8) and 2.3 +/- 0.6 x 10(-9) M, respectively. The curves for competitive inhibition of [3H]-NMS with liriodenine, methoctramine and 4-DAMP were best fitted according to a two site model of binding, but pirenzepine was best fitted according to a model with one site. 3. Liriodenine and 4-DAMP displayed a high affinity for blocking tracheal contraction (pKB = 5.9 and 9.1, respectively) and inositol phosphate formation (pKB = 6.0 and 8.9, respectively), but a low affinity for antagonism of cyclic AMP inhibition (pKB = 4.7 and 7.8, respectively). 4. Methoctramine blocked cyclic AMP inhibition with a high affinity (pKB = 7.4), but it antagonized tracheal contraction and inositol phosphate formation with a low affinity (pKB = 6.1 and 6.0, respectively). 5. In conclusion, both M2 and M3 muscarinic receptor subtypes coexist in canine tracheal smooth muscle and are coupled to the inhibition of cyclic AMP formation and phosphoinositide breakdown, respectively. The antimuscarinic characteristics of liriodenine are similar to those of 4-DAMP. It may act as a selective M3 receptor antagonist in canine tracheal smooth muscle.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Antineoplastic Agents, Phytogenic; Aporphines; Binding, Competitive; Carbachol; Cells, Cultured; Cyclic AMP; Diamines; Dogs; Female; Hydrolysis; Inositol Phosphates; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Phosphatidylinositols; Piperidines; Plants, Medicinal; Receptors, Muscarinic; Trachea

The present studies were conducted to determine whether [3H]quinuclidinyl benzilate binding in rat colonic membranes and/or carbachol-mediated stimulation of particulate guanylate cyclase were altered by changes in vitamin D status. EC50 values for the stimulation of colonic guanylate cyclase by carbachol were found to be significantly greater in vitamin D-deficient rats compared to their D-sufficient counterparts. Concomitantly, the density of receptors (Bmax) were significantly lower, and dissociation constants (Kd) were significantly higher in D-deficient colonic membranes. In vitamin D-repleted animals, moreover, all of these aforementioned alterations were at least partially corrected.

Topics: Animals; Atropine; Colon; Diamines; Enzyme Activation; Guanylate Cyclase; Male; Muscarinic Antagonists; Nutritional Status; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Vitamin D

The aim was to determine the presynaptic modulation of noradrenaline (NA) release from the sympathetic nerve terminals in human isolated papillary muscle.. Papillary muscle and the right atrial appendage were obtained from operations on 22 patients (10 men and 12 women). The papillary muscle preparations were preincubated with [3H]NA and the release of [3H] at rest and in response to field stimulation was measured.. Using an immunohistochemical method dopamine-beta-hydroxylase-positive neurones were found in the papillary muscle and right atrial appendage sample. The release of noradrenaline from the papillary muscle, associated with axonal activity, was enhanced by 7,8(methylenedioxy)-14-alpha-hydroxyalloberbane HCl (CH-38083), a selective alpha 2 adrenoceptor antagonist, and inhibited by xylazine, an alpha 2 adrenoceptor agonist, indicating that negative feedback modulation was functioning. In addition, the release of [3H]NA was enhanced by atropine, pancuronium, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a selective M3 muscarinic receptor antagonist, and reduced by oxotremorine, a selective muscarinic receptor agonist, indicating that acetylcholine released from the parasympathetic nerve ending was able to reach the varicose noradrenergic axon terminals that are equipped with inhibitory M3 muscarinic receptors.. These findings, obtained for the first time in human papillary muscle, indicate that the release of noradrenaline is modulated by alpha 2 autoreceptors activated by noradrenaline and M3 muscarinic heteroreceptors. Thus during parasympathetic stimulation the release of noradrenaline from the sympathetic axon terminals is presynaptically controlled through muscarinic receptors.

Topics: Adrenergic beta-Antagonists; Adult; Aged; Atropine; Berberine; Chromatography, High Pressure Liquid; Culture Techniques; Dopamine beta-Hydroxylase; Electric Stimulation; Feedback; Female; Heart Atria; Humans; Immunohistochemistry; Male; Middle Aged; Norepinephrine; Oxotremorine; Pancuronium; Papillary Muscles; Piperidines; Presynaptic Terminals; Receptors, Muscarinic; Sympathetic Nervous System; Tritium; Xylazine

Aceclidine can dissociate accommodative and outflow facility responses in monkeys and humans. We sought to determine if different muscarinic receptor subtypes control outflow facility, accommodative and pupillary responses to aceclidine in the living rhesus monkey eye, as a possible basis for this separation. Each eye was cannulated with one branched and one unbranched needle. Baseline measurements (of outflow facility by two-level constant pressure perfusion; refraction by Hartinger coincidence refractometry and pupil diameter by vernier calipers) were recorded after anterior chamber exchange with (one eye) or without (opposite eye), muscarinic receptor subtype antagonist (pirenzepine, AF-DX 116 or 4-DAMP). The eyes were then exchanged a second time with these solutions plus added aceclidine. The response to aceclidine (the differences from baseline) in the presence or absence of antagonist were compared. The M3 muscarinic receptor subtype antagonist 4-DAMP was the most potent inhibitor of all three responses to aceclidine. The dissociation of accommodative, outflow facility and miotic responses to aceclidine in rhesus monkeys does not appear to be due to differences in the muscarinic receptor subtypes that can currently be distinguished pharmacologically.

Topics: Accommodation, Ocular; Animals; Aqueous Humor; Dose-Response Relationship, Drug; Female; Macaca mulatta; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Pupil; Quinuclidines

The role of the muscarinic cholinergic system in the generation of LTP in the medial perforant path-dentate granule cell synapses in vivo was investigated using anesthetized rats. Cholinergic denervation with AF64A, a cholinergic toxin, did not significantly affect LTP induced by a strong tetanus (100 pulses at 100 Hz), but attenuated the LTP induced by a weak tetanus (30 pulses at 60 Hz). The i.c.v. injection of scopolamine (1.5-50 nmol) did not significantly affect the LTP induced by the strong tetanus but attenuated the magnitude of LTP produced by the weak tetanus in a concentration-dependent manner. These results suggest that the cholinergic system is not essential for induction of LTP by strong stimuli but plays a role in facilitating the generation of LTP by weak stimuli. Furthermore, the induction of LTP by a weak tetanus was blocked by pirenzepine but affected by neither AF-DX116 nor 4-diphenylacetoxy-N-methylpiperidine. The LTP-facilitatory action of the cholinergic system is probably mediated by muscarinic M1 receptors.

Topics: Animals; Evoked Potentials; Hippocampus; Injections, Intraventricular; Long-Term Potentiation; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Scopolamine

1. Muscarinic receptors mediating contraction of rabbit endothelium-denuded aorta have been characterized functionally, in vitro, using a range of antagonists (atropine, pirenzepine, methoctramine, himbacine, 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP) and para-fluoro-hexahydro-siladifenidol (p-F-HHSiD). 2. The non-selective muscarinic agonist, (+)cis-dioxolane, induced concentration-dependent contractions of endothelium-denuded aortic rings. The potency (EC50) of (+)cis-dioxolane was 1.0 +/- 0.4 microM and the maximal increase in isometric tension was 944 +/- 98 mg (mean +/- SEM, n = 25). The concentration-effect curves to (+)cis-dioxolane were shifted to the right in the presence of antagonists, in a concentration-dependent manner. The following affinities (-log KB) were calculated; atropine, 9.4; pirenzepine, 6.6; methoctramine, 5.9; himbacine, 7.1; 4-DAMP, 9.2; and p-F-HHSiD, 7.7. 3. It is concluded that muscarinic M3 receptors mediate contractions of endothelium-denuded aorta. The low potency of (+)cis-dioxolane, when compared to its potency in other M3 receptor assays, suggests that the efficiency of receptor coupling, associated with contraction of this tissue, is poor.

Topics: Animals; Aorta, Thoracic; Dioxolanes; Endothelium, Vascular; Female; In Vitro Techniques; Piperidines; Rabbits; Receptors, Muscarinic; Vasoconstriction

Three Holstein steers (345 +/- 22 kg) surgically fitted with a pancreatic cannula were used in two 3 x 3 Latin square design experiments to examine the effects of slaframine (SF), a muscarinic agonist, or 4-diphenylacetoxy-N-methylpiperidine methiodide (4DAMP), an M3 muscarinic glandular receptor antagonist, on pancreatic exocrine secretion. Pancreatic exocrine secretion was collected for 8 h postdosing at 30-min intervals beginning 1 h postfeeding. In experiment 1, steers were dosed with 0, 25, or 50 micrograms.kg-1 body weight (BW) of SF. Secretion of pancreatic juice and the pH of the secreted juice increased linearly (p < 0.05) with SF; however, secretion rate showed a time by treatment interaction (p < 0.05), as treatments converged 7 h postdosing. Trypsin secretion tended (p < 0.10) to show a quadratic response to SF administration, with the 25 micrograms SF.kg-1 BW dose having the lowest value. In experiment 2, steers received 50 micrograms.kg-1 BW of SF (positive control), 113 micrograms.kg-1 BW of 4DAMP (isosmolar with SF), or both. SF caused a greater pancreatic fluid secretion (p < 0.10) than 4DAMP, with SF plus 4DAMP intermediate. A time by treatment interaction (p < 0.04) was found, since treatments converged 8 h postdosing. Trypsin secretion was higher (p < 0.05) for SF than the other treatments. Chymotrypsin, alpha-amylase, and protein secretion were not affected. SF and 4DAMP alter pancreatic fluid secretion in the steer but have minimal effects on enzyme secretions.

Topics: Alkaloids; alpha-Amylases; Animals; Cattle; Chymotrypsin; Hydrogen-Ion Concentration; Male; Muscarinic Antagonists; Pancreas; Pancreatic Juice; Parasympatholytics; Parasympathomimetics; Piperidines; Proteins

The aim of the present study was to elucidate the role of the non-M1 muscarinic receptors, in the extrinsic and intrinsic nerve control of in vivo colonic motility. Experiments were performed on the proximal colon of anaesthetized rabbits. In this species, the parasympathetic innervation of the proximal colon originates from the vagus nerves. The action of methoctramine and 4-diphenyl-acetoxy-N-methylpiperidine methobromide (4-DAMP) was studied on excitatory junction potentials (EJPs), and on inhibitory junction potentials (IJPs) elicited in smooth muscle cells by stimulating parasympathetic efferents. The effects of the same drugs on spontaneous spiking activity were also investigated. The EJPs either decreased or disappeared after intra-arterial (i.a.) administration of 4-DAMP (45 pg to 450 ng). In the presence of 4-DAMP, further intravenous (i.v.) administration of pirenzepine (0.1 mg.kg-1) had facilitatory effects on the inhibitory pathway, i.e., after abolition of the EJPs, vagal stimulation elicited IJPs. With the highest dose of 4-DAMP, vagal stimulation immediately elicited IJPs the amplitude of which still increased after pirenzepine. In the presence of 4-DAMP, the spontaneous spike discharge was not noticeably altered. Methoctramine (0.37 to 75 micrograms, i.a. or 50 micrograms to 0.2 mg.kg-1, i.v.) increased the amplitude of the EJPs, whereas it decreased that of the IJPs. In addition, at the same doses, it either initiated or increased spike discharges that were not altered by pirenzepine up to 0.2 mg.kg-1, i.v. The so-called rebound excitation occurring after IJPs was not affected by methoctramine. No change in the EJP or IJP amplitude was observed with gallamine at sufficiently high doses to paralyse striated muscles (up to 3 mg.kg-1.h-1). It is concluded that the parasympathetic excitatory pathway to smooth muscle is blocked by 4-DAMP, whereas it is facilitated by methoctramine. 4-DAMP has no effect on the inhibitory pathway which is strongly depressed by methoctramine; however, the fact that these two drugs have opposite effects indicates that 4-DAMP and methoctramine may act on different muscarinic receptor subtypes. In addition, the facilitatory effects of pirenzepine on IJPs observed in animals pre-treated with 4-DAMP, indicates that the latter drug may act on non-M1 and non-M2 (presumably M3) muscarinic receptors. Methoctramine acts on non-M1 and non-M3 (presumably M2) receptors. The spike discharge induced by methoctramine is presumab

Topics: Action Potentials; Animals; Colon; Diamines; Electric Stimulation; Gallamine Triethiodide; Gastrointestinal Motility; Male; Muscle, Smooth; Parasympathetic Nervous System; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Vagus Nerve

We have used the pilocarpine-induced seizure model in mice and i.c.v. injection of subtype-specific receptor antagonists to investigate the muscarinic receptor subtype specificity of cholinergically-activated seizures. The rank order potencies of antagonists for inhibition of pilocarpine-induced seizures are atropine = telenzepine > 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP) > pirenzepine with ID50's of 8.6, 12.0, 29.9, and 83.0 nmol/mouse, respectively. The M3-specific antagonists hexahydrosila-difenidol and its p-fluoro analog showed no effect on pilocarpine-induced seizures. The M2-specific antagonists gallamine and methoctramine cause seizures in mice in the absence of a pilocarpine injection. These seizures could be inhibited by coinjection of methoctramine with the M1-specific antagonist, pirenzepine. These data suggest a role of muscarinic M1 receptors in mediating pilocarpine-induced seizures and a role of the muscarinic M2 receptors in modulating neuronal activity.

Topics: Animals; Atropine; Cerebral Ventricles; Injections, Intraventricular; Male; Mice; Parasympatholytics; Pilocarpine; Piperidines; Pirenzepine; Receptors, Muscarinic; Seizures

The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP3) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140-280% of baseline at 10(-3) M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30-50% of the maximal stimulation) obtained at low carbachol concentrations (< 10(-5) M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP3 accumulation induced by 10(-7) M (endothelium-dependent submaximal effect) and 10(-4) M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP3 production by 10(-4) M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC50): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H- dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11- dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M1 receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbachol; Cats; Cerebral Arteries; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Endothelium, Vascular; In Vitro Techniques; Inositol Phosphates; Muscarinic Antagonists; Muscle, Smooth, Vascular; Parasympatholytics; Phosphatidylinositols; Pia Mater; Piperidines; Pirenzepine; Receptors, Muscarinic; Vasoconstriction; Vasodilation

Acetylcholine (ACh) is the major neurotransmitter released from the efferent fibers in the cochlea onto the outer hair cells (OHCs). The type of ACh receptor on OHCs and the events subsequent to receptor activation are unclear. Therefore we studied the effect of agonists and antagonists of the ACh receptor on isolated OHCs from the guinea pig. OHCs were recorded from in whole cell voltage and current clamp configuration. ACh induced an increase in outward K+ current (IACh) which hyperpolarized the OHCs. No desensitization to ACh application was observed. Cs+ replaced K+ in carrying the IACh. The IACh is Ca(2+)-dependent, time and voltage sensitive, and different from the IKCa induced by depolarization of the membrane potential. When tested at 100 microM, several agonists also induced outward current responses (acetylcholine > suberyldicholine > or = carbachol > DMPP) whereas nicotine, cytisine and muscarine did not. The IACh response to 10 microM ACh was blocked by low concentrations of traditional and non-traditional-nicotinic antagonists (strychnine > curare > bicuculline > alpha-bungarotoxin > thimethaphan) and by higher concentrations of muscarinic antagonists (atropine > 4-DAMP > AF-DX 116 > pirenzepine). Pharmacologically, the ACh receptor on OHCs is nicotinic.

Topics: Acetylcholine; Alkaloids; Animals; Atropine; Azocines; Bungarotoxins; Carbachol; Choline; Cholinergic Antagonists; Curare; Dimethylphenylpiperazinium Iodide; Guinea Pigs; Hair Cells, Auditory, Outer; In Vitro Techniques; Membrane Potentials; Nicotine; Piperidines; Pirenzepine; Quinolizines; Receptors, Cholinergic; Strychnine; Trimethaphan

Muscarinic cholinergic receptors were identified and characterized by radioligand receptor binding assay using [3H]quinuclidinyl benzilate (QNB) in rat vas deferens membrane particulates of three experimental groups: 1) 8-week diabetic, 2) 8-week diabetic insulin-treated and 3) age-matched control. Diabetes was induced by the intravenous injection of 65 mg./kg. streptozotocin (STZ). The density of muscarinic receptors (Bmax values), as determined by saturation experiments with [3H]QNB, was demonstrated to be higher in the vas deferens of diabetic rats than in the vas deferens of control and diabetic insulin-treated rats. The equilibrium dissociation constants (KD values), however, were similar in all three groups. Muscarinic cholinergic antagonists competed with [3H]QNB binding sites in the vas deferens membrane particulates with the following rank order of Ki values: atropine < methoctramine < or = 4-DAMP < AF-DX 116 < HHSiD < pirenzepine = pfHHSiD. The pharmacological profile of muscarinic receptors was similar in all three groups. Additional pharmacological studies showed a similar rank order of Ki values for vas deferens, bladder dome and heart, but this rank order was significantly different in cerebral cortex and prostate. This is consistent with the predominance of the M2 muscarinic cholinergic receptor subtype in the rat vas deferens. It is concluded that STZ-induced diabetes causes an upregulation of muscarinic cholinergic receptor density in the rat vas deferens that can be prevented by the administration of insulin.

Topics: Animals; Atropine; Binding, Competitive; Diabetes Mellitus, Experimental; Diamines; Insulin; Male; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Up-Regulation; Vas Deferens

The N-terminal segment of the third intracellular loop (i3) of muscarinic acetylcholine receptors and other G protein-coupled receptors has been shown to largely determine the G-protein coupling selectivity displayed by a given receptor subtype. Based on secondary-structure prediction algorithms, we have tested the hypothesis that this region adopts an alpha-helical secondary structure. Using the rat m3 muscarinic receptor as a model system, a series of five mutant receptors, m3(+1A) to m3(+5A) were created in which one to five additional alanine residues were inserted between the end of the fifth transmembrane domain and the beginning of i3. We speculated that this manipulation should lead to a rotation of the N-terminal segment of the i3 domain (if it is in fact alpha-helically arranged), thus producing pronounced effects on receptor/G protein coupling. Pharmacological analysis of the various mutant receptors expressed in COS-7 cells showed that m3(+1A), m3(+3A), and m3(+4A) retained strong functional activity, whereas m3(+2A) and m3(+5A) proved to be virtually inactive. Helical wheel models show that this pattern is fully consistent with the notion that the N-terminal portion of i3 forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents the G-protein recognition surface.

Topics: Acetylcholine; Amino Acid Sequence; Animals; Binding Sites; Carbachol; Cell Line; Chlorocebus aethiops; GTP-Binding Proteins; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Insertional; N-Methylscopolamine; Phosphatidylinositols; Piperidines; Protein Structure, Secondary; Rats; Receptors, Muscarinic; Recombinant Proteins; Scopolamine Derivatives; Transfection

Receptor autoradiographic methods specific for M3 and M1 muscarinic acetylcholine receptors were used to investigate the development and input-dependent laminar redistribution of these receptor populations during the critical period for kitten visual cortex plasticity. Analysis of the binding curves of [3H]4-diphenylacetyl-N-methyl-piperidine (4-DAMP) and [3H]pirenzepine (PZ) indicated that these two ligands bound heterogeneously to muscarinic acetylcholine receptors with different affinities. While [3H]4-DAMP showed a high affinity for M3 receptors and much lower affinities for M1 and M2 receptors, [3H]PZ displayed higher affinity for M1 receptors. By carefully choosing concentrations of labelled and unlabeled ligands, the patterns of laminar distribution for both receptor subtypes within visual cortex were obtained. Both receptors were most concentrated in cortical layer IV immediately after birth and during the most sensitive period of visual cortex plasticity. The binding density for both receptor subtypes thinned out progressively in this layer to concentrate in more superficial layers as plasticity waned with age. Moreover, interruption of visual or spontaneous input to visual cortex induced either by lesion or by tetrodotoxin infusion into lateral geniculate nucleus prevented the developmental redistribution of these receptors from layer IV to superficial layers, that is, the pattern of laminar distribution remained that of the age at which the lesion or tetrodotoxin infusion into the lateral geniculate nucleus was performed. The results indicate that the developmental expression of M3 and M1 muscarinic acetylcholine receptors in kitten visual cortex depends on cortical inputs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Autoradiography; Cats; Geniculate Bodies; Ligands; Neuronal Plasticity; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Tetrodotoxin; Visual Cortex

Duodenal or jejunal motility (monitored as pressure changes in a saline-perfused intraluminal catheter) was studied in anaesthetized rats, vagotomized and pretreated with adrenergic blocking agents. In the duodenum (but not the jejunum), atropine or the selective muscarinic M1 and M3 receptor antagonists, pirenzepine and 4-diphenyl-acetoxy-N-methylpiperidine (4-DAMP), respectively, augmented the spontaneous contractile activity. This effect could be abolished either by nicotinic ganglionic receptor antagonism with hexamethonium, or with morphine. Moreover, blockade of the synthesis of nitric oxide by N omega-nitro-L-arginine elicited hypermotility both in the duodenum and the jejunum, and also this response was abolished by hexamethonium. It is proposed from the present results that the rat small is controlled by non-adrenergic, non-cholinergic inhibitory as well as excitatory motor neurons. The latter motor neurons seem to be modulated by muscarinic, nitroxergic or opioidergic mechanisms.

Topics: Animals; Arginine; Duodenum; Gastrointestinal Motility; Jejunum; Male; Morphine; Naloxone; NG-Nitroarginine Methyl Ester; Nitric Oxide; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Sympatholytics; Vagotomy

Muscarinic receptors (mAChRs) in the pontine reticular formation comprise a critical part of the REM sleep-generating system. Although the role of specific mAChR subtypes remains unclear, data from in vivo microinjection studies suggest that in the pons the M2 subtype is important for REM sleep generation. The present study tested the hypothesis that M2 antagonists would show a greater binding potency in feline pons than M1 or M3 antagonists. Competition binding assays showed 4-DAMP to be more potent than pirenzepine or AF-DX 116 in its ability to displace tritiated quinuclidinyl benzilate, and linear regression analyses indicated that 4-DAMP and pirenzepine each interacted with more than one binding site. These data demonstrate the presence of a mixture of mAChR subtypes in the feline pons, and are consistent with the view that REM sleep is mediated by more than one mAChR subtype.

Topics: Animals; Atropine; Binding, Competitive; Cats; Cerebral Cortex; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Pons; Receptors, Muscarinic

Previous studies from our laboratory demonstrated that 8 weeks of streptozocin (STZ)-induced diabetes and sucrose-fed diuresis resulted in increases in the density of muscarinic receptors in rat bladder dome and that early insulin treatment (started 3 days after the onset of diabetes) prevented the diabetes-induced upregulation (J Pharmacol Exp Ther 248:81-88, 1989; Diabetes 40: 1150-1156, 1991; J Urol 147:760-763, 1992). To determine whether diabetes- and diuresis-induced alterations in muscarinic receptors in rat bladder dome are reversible, we administered insulin (beginning 8 weeks after the onset of diabetes) or removed sucrose from drinking water of diuretic rats (beginning 8 weeks after the onset of diuresis). Five groups of rats were maintained for 16 weeks: 1) STZ-induced diabetic rats (65 mg/kg intravenously); 2) insulin-treated diabetic rats (5-8 U/day insulin subcutaneously beginning 8 weeks after the onset of diabetes); 3) sucrose-fed diuretic rats (5% sucrose in drinking water throughout 16 weeks); 4) sucrose-removed rats (sucrose withdrawn from drinking water after 8 weeks of the sucrose-induced diuretic state); and 5) age-matched control rats. Radioligand receptor binding experiments with [3H]quinuclidinyl benzilate showed an increase in the density of muscarinic receptors in bladder dome of diabetic and sucrose-fed rats compared with age-matched control rats. Removing the 5% sucrose from the drinking water of diuretic rats reversed the increased water intake and urine output, decreased the bladder hypertrophy that accompanied the diuretic state, and corrected the upregulation of the muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Body Weight; Cell Membrane; Diabetes Mellitus, Experimental; Diamines; Diuresis; Drinking Behavior; Insulin; Kinetics; Male; Myocardium; Organ Size; Parasympatholytics; Piperidines; Pirenzepine; Prostate; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Sucrose; Testosterone; Up-Regulation; Urinary Bladder

The biodistribution of [3H]4-DAMP (a M3-selective cholinoceptor antagonist) was studied in rats which had received either saline or saline containing atropine (to block cholinoceptors). Specific binding of the radioligand was observed in the urinary bladder, ileum, pancreas, stomach, submandibular gland and trachea. Maximal ratios of total-to-non-specific uptake reached values of 1.8 (trachea), 3.2 (bladder), 4.0 (stomach), 4.8 (ileum), 6.6 (pancreas) and 6.9 (submandibular gland) at 5-10 min post-injection; this rank order reflects the tissue densities of M3 cholinoceptors, 4-DAMP did not bind to blood cells and it was rapidly cleared from the circulation (> 90% with a half-life of 0.2 min, the remainder with a half-life of 9.4 min). Labelled metabolites appeared within 5 min in plasma, but metabolite uptake by the target organs was low (< 15% of total radioactivity 40 min post-injection). Although 4-DAMP binds to M3-cholinoceptors in vivo, its potential use as a radiopharmaceutical appears limited since the compound does not cross the blood-brain barrier and it does not show measurable specific binding in airways.

Topics: Animals; Male; Muscarinic Antagonists; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Tissue Distribution; Tritium

Using the cannula insertion method, we investigated the vascular responses of isolated simian coronary artery to acetylcholine (ACh). When the preparation was partially precontracted by 20 mM KCl, ACh and carbachol induced vasodilation dose dependently in coronary artery with endothelium, but ACh and carbachol contracted the coronary artery after removal of the endothelium by 1 mg saponin. A selective M1 receptor agonist 4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium++ + chloride (McN-A-343) did not affect the perfusion pressure of the precontracted coronary arteries significantly. Both these responses to ACh were inhibited by the M3 receptor antagonist 4-dipheny-lacetoxy N-methylpiperidine methobromide (4-DAMP) in a dose-dependent manner, but not by a selective M2 receptor antagonist AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzo-diazepine-6-one). A selective M1 receptor antagonist pirenzepine did not affect ACh-induced vasoconstriction significantly and inhibited the vasodilation partially only at the highest dose (100 nmol). The effects of three antagonists on the vasodilative responses to carbachol were also studied and almost the same results were observed. Removal of the endothelium did not affect sodium nitroprusside (SNP)-induced vasodilation significantly. Pirenzepine, AF-DX 116, and 4-DAMP did not affect the action of isoproterenol. These data suggest that the vasodilation elicited by ACh is mediated by release of endothelium-derived relaxing factors (EDRF) consequent to the activation of M3 receptors on endothelial cells, and the constriction is mediated by stimulation of M3 receptors on smooth muscle cells in isolated simian coronary arteries.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Coronary Vessels; Endothelium, Vascular; Female; In Vitro Techniques; Macaca; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth, Vascular; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Vasoconstriction; Vasodilation

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.

Topics: Acetylcholine; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Blotting, Northern; Carbachol; Cell Line; Codon; GTP-Binding Proteins; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; N-Methylscopolamine; Parasympatholytics; Piperidines; Proline; Protein Conformation; Rats; Receptors, Muscarinic; Recombinant Proteins; Restriction Mapping; RNA; Scopolamine Derivatives; Transfection; Tryptophan

Bath application of the muscarinic receptor agonist, muscarine, produced a concentration-dependent depression of synaptic activity in the dentate gyrus of hippocampal slices. A concentration of 10 microM muscarine produced a reversible depression that could be competitively antagonized by the muscarinic receptor antagonist pirenzepine. However, other muscarinic receptor subtype (M1-M3) antagonists could also block the effects of muscarine. The rank order of antagonist potency was: 4-diphenylacetoxy-N-methyl-piperidine methiodide (M3/M1 antagonist) > pirenzepine (M1) > AFDX-116 (M2). The depression produced by 10 microM muscarine was not affected by in vivo pretreatment with pertussis toxin, and therefore was not mediated by a pertussis toxin-sensitive G-protein. In addition, high concentrations of muscarine did not affect either basal or isoproterenol-stimulated accumulation of cyclic AMP from slices of dentate gyrus. Muscarine also produced a concentration-dependent blockade of the induction of norepinephrine-induced long-lasting potentiation in the dentate gyrus. Norepinephrine-induced long-lasting potentiation is a form of long-lasting plasticity induced in medial perforant path synapses by beta-adrenergic agonists such as isoproterenol. The muscarinic blockade of norepinephrine-induced long-lasting potentiation was also prevented by pretreatment with pirenzepine. Based on these pharmacological data, we conclude that muscarinic depression of evoked responses, as well as blockade of norepinephrine-induced long-lasting potentiation, involves activation of either M3 or M1, but not M2, muscarinic receptors. These data also demonstrate that in addition to modulating normal synaptic transmission, muscarinic receptors may also play an important role in modulating synaptic plasticity.

Topics: 1-Methyl-3-isobutylxanthine; Analysis of Variance; Animals; Cyclic AMP; Electric Stimulation; Evoked Potentials; Hippocampus; In Vitro Techniques; Male; Muscarine; Norepinephrine; Parasympatholytics; Pertussis Toxin; Piperidines; Pirenzepine; Pyramidal Tracts; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Synapses; Synaptic Transmission; Virulence Factors, Bordetella

We measured endogenous overflow of acetylcholine (ACh) from a vascularly perfused rat stomach in vitro with modified Krebs-Ringer solution containing 100 microM physostigmine. Evoked ACh overflow by vagal stimulation at 2.5 Hz for 2 min was abolished by tetrodotoxin (3 x 10(-7) M) or Ca++ removal and reduced by hexamethonium (10(-4) M). The evoked overflow was inhibited by oxotremorine (10(-7) to 10(-5) M), but not attenuated by 4-(N-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammonium chloride (10(-6) and 10(-5) M). The evoked ACh overflow was enhanced by atropine (10(-9) to 10(-6) M), 4-diphenylacetoxy-N-methylpiperidine (10(-8) to 10(-6) M), methoctramine (10(-8) to 10(-6) M) and pirenzepine (10(-8) to 10(-6) M) in a concentration-dependent manner with the following potency; atropine > 4-diphenylacetoxy-N-methylpiperidine > methoctramine > pirenzepine. In the presence of 10(-6) M atropine, clonidine (10(-8) and 10(-7) M), but not phenylephrine (10(-7) and 10(-6) M), concentration-dependently decreased the evoked overflow of ACh at 1 Hz. Electrical stimulation of periarterial gastric sympathetic nerves (5 and 10 Hz for 2 min) frequency-dependently inhibited the evoked ACh overflow at 1 Hz in the presence of atropine and sympathetic inhibition mediated at 5 Hz was abolished by 10(-6) M rauwolscine, but not by 10(-6) M prazosin. These results indicate that the ACh release from gastric parasympathetic neurons is inhibited by M3 muscarinic autoreceptors and alpha-2 adrenoceptors.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Adrenergic alpha-Antagonists; Animals; Atropine; Calcium; Clonidine; Diamines; Drug Interactions; Electric Stimulation; Gastric Mucosa; Hexamethonium; Hexamethonium Compounds; Male; Muscarinic Antagonists; Oxotremorine; Parasympatholytics; Perfusion; Phenylephrine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Receptors, Muscarinic; Stomach; Sympathetic Nervous System; Tetrodotoxin; Vagus Nerve

Systemic and local responses mediated by chemonociceptive receptors located in the mucosa of the canine distal esophagus were examined following stimulation with capsaicin (8-methyl-N-vanillyl-6-nonenamide). The neural pathways and neurotransmitters mediating these sensory responses were also investigated. Topical application of capsaicin solution to the distal esophageal mucosa produced significant increases in lower esophageal sphincter pressure (LESP), mean arterial pressure (MAP), pulse rate (PR), and respiratory rate (RR) (P < 0.01). Pretreatment with tetrodotoxin completely abolished this reflex activity. Following truncal vagotomy and pyloroplasty, topical capsaicin application produced an increase in LESP, but the increases in MAP, PR, and RR were blocked. The initial increase in LESP was blocked by hexamethonium, atropine, and 4-diphenylacetoxy-N-methylpiperidine, but was not inhibited by phentolamine. Excitatory cardiovascular responses were inhibited by hexamethonium. Administration of a Substance P antagonist attenuated both local and systemic responses. These studies suggest that the vagus nerves serve as the primary afferent pathways through which chemonociceptive esophageal stimuli can induce cardiovascular and respiratory reflex excitation. The increase in lower esophageal sphincter pressure in response to mucosal capsaicin stimulation is mediated via an intrinsic neural pathway that functions independently of vagal innervation, but is dependent on both cholinergic ganglionic neurotransmission and muscarinic type 2 smooth muscle receptor excitation. Substance P appears to play a role in primary sensory afferents as a chemonociceptive neurotransmitter in the canine distal esophagus.

Topics: Afferent Pathways; Animals; Atropine; Blood Pressure; Capsaicin; Chemoreceptor Cells; Dogs; Esophagus; Hexamethonium; Hexamethonium Compounds; Mucous Membrane; Nociceptors; Piperidines; Pressure; Pulse; Respiration; Substance P; Tetrodotoxin; Vagotomy

Muscarinic receptor agonist and antagonist effects on acetylcholine release evoked by electrical or dimethylphenylpiperazinium stimulation from guinea pig ileum were evaluated by measuring acetylcholine with a high performance liquid chromatography-electrochemical detector system. AF102B (cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine), a muscarinic M1 receptor agonist, increased markedly the evoked release of acetylcholine. In contrast, pirenzepine decreased the evoked acetylcholine release. 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine methiodide) and p-F-HHSiD (p-fluoro-hexahydrosiladifenidol), muscarinic M3 antagonists, increased the release of acetylcholine. Atropine enhanced acetylcholine release to a similar extent while bethanechol reduced the electrically evoked acetylcholine release. This reduction was virtually unaffected by methoctramine, but was antagonized by 4-DAMP or p-F-HHSiD. The results from direct determination of acetylcholine suggest that, in contrast to autoinhibition by stimulation of muscarinic M3 receptors, stimulation of presynaptic muscarinic M1 receptors is predominantly involved in enhancement of the acetylcholine release from guinea pig ileal nerves, and that AF102B functions as a muscarinic M1 agonist in this peripheral neuron.

Topics: Acetylcholine; Analysis of Variance; Animals; Bethanechol; Bethanechol Compounds; Chromatography, High Pressure Liquid; Dimethylphenylpiperazinium Iodide; Electric Stimulation; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle, Smooth; Parasympatholytics; Parasympathomimetics; Piperidines; Quinuclidines; Receptors, Muscarinic; Thiophenes

Nicotinic and muscarinic mediated synaptic mechanisms were investigated in isolated, canine intracardiac ganglia taken from the right atrial fat pad. Using conventional intracellular microelectrode recording techniques on 216 neurons, fast and slow synaptic potentials were evoked by single or trains of stimulation of presynaptic fibers in interganglionic nerves. By varying the stimulus intensity, single or multiple fast excitatory postsynaptic potentials (f-EPSPs) were evoked, indicating the convergence of synaptic inputs on these cells. These f-EPSPs often reached the action potential threshold, were enhanced by the acetylcholinesterase inhibitor physostigmine and were blocked by the nicotinic antagonist hexamethonium. The f-EPSPs were accompanied by a decreased input resistance and had an extrapolated reversal potential of -7.1 mV, suggesting increased conductances to more than one cation. Repetitive presynaptic stimulation evoked slow excitatory postsynaptic potentials (s-EPSPs) in 41% of the cells while slow inhibitory postsynaptic potentials (s-IPSPs) or s-IPSPs followed by s-EPSPs were evoked in 19% of the cells. All slow potentials were abolished by atropine and low Ca2+/high Mg2+ solutions and enhanced by physostigmine. Hexamethonium and adrenergic receptor antagonists had no effects on s-EPSP and s-IPSP. The M1 receptor antagonist pirenzepine reversibly blocked the s-EPSP but not the s-IPSP. On the other hand, the M2 receptor blocker 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP) had no effects on the s-EPSP. These observations suggest that s-EPSPs and s-EPSPs are mediated by distinct muscarinic receptors. The amplitude of the s-EPSP and the depolarization evoked by the muscarinic agonist, bethanechol were accompanied by increased input resistance. These responses were decreased in amplitude by membrane hyperpolarization and either reversed polarity or declined to zero amplitude at about -80 mV, suggesting the inhibition of a potassium conductance.

Topics: Acetylcholine; Animals; Bethanechol Compounds; Dogs; Electric Stimulation; Evoked Potentials; Female; Ganglia, Autonomic; Heart; Hexamethonium Compounds; Male; Membrane Potentials; Neurons; Neurotransmitter Agents; Parasympatholytics; Piperidines; Receptors, Muscarinic; Receptors, Nicotinic; Sinoatrial Node; Synapses; Synaptic Transmission

Preliminary evidence of muscarinic acetylcholine receptors (AchRs) in rabbit and human peritoneal mesothelial cells grown in tissue culture is reported. Atropine displaceable binding of L-quinuclidinyl[phenyl-4-3H]-benzilate (QNB), an antagonist that binds to all AchR subtypes, and [N-methyl-3H]-4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), an antagonist specific for AchR subtypes 1 and 3, to homogenates of mesothelial cells was approximately 400 and 120 fmol ligand bound/milligram cell protein, respectively. A similar value for specific [3H]-QNB binding was observed in living cells exposed to ligand and atropine for 1 hour at 37C. Guanosine 5'-triphosphate (GTP)-binding proteins measured as beta,gamma-imido[8-3H] guanosine 5'-triphosphate binding to mesothelial cell membranes was approximately 9 pmol/mg cell protein. Our finding of both muscarinic receptors and GTP-binding proteins in mesothelial cells suggests first, the presence of a functional muscarinic receptor system(s) in peritoneal mesothelial cells, and second, the potential susceptibility of these cells to direct pharmacological manipulation by muscarinic drugs.

Topics: Animals; Atropine; Binding, Competitive; Cells, Cultured; Epithelium; GTP-Binding Proteins; Humans; Peritoneum; Piperidines; Quinuclidinyl Benzilate; Rabbits; Receptors, Muscarinic

The nature of the muscarinic (M) receptor subtype mediating endothelium-dependent vasodilation was investigated in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).. Characterization of the muscarinic receptor mediating vasodilation and the possible hypertension-induced effects on the nature of this receptor, which have both received little attention in resistance vessels of the SHR.. After a methoxamine-induced vasoconstriction, the vessels were dilated with acetyl-beta-metacholine (MCh). The MCh-induced vasodilation was analysed by means of the M1-selective antagonist pirenzepine, the M2-selective antagonists AF-DX116 and AQ-RA 741 and the M3-selective antagonists 4-DAMP and p-FHHSiD. The potency of these compounds was quantified by means of pA2 values. Atropine, a non-selective muscarinic antagonist, was used for comparison.. The rank order of potency for the muscarinic receptor antagonists in preparations taken from SHR and WKY rats appears to be atropine > 4-DAMP > p-FHHSiD > pirenzepine > AQ-RA 741 > AF-DX 116. This rank order corresponds to that found in isolated conduit arteries.. The pA2 values for the various compounds were not significantly different in SHR and WKY rat preparations, indicating that the nature of this receptor is not influenced by hypertension. The high potency of the M3-selective drugs and the weak activity of pirenzepine and the M2-selective antagonists suggest a major role of M3-receptors in the cholinergic vasodilation in the perfused mesenteric vascular bed both in SHR and WKY rat preparations.

Topics: Animals; Atropine; Benzodiazepinones; Endothelium, Vascular; Hypertension; Male; Mesenteric Artery, Superior; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Muscarinic; Vasodilation

Previous studies in this laboratory have demonstrated that prior intracerebroventricular (i.c.v.) administration of the muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) in morphine dependent rats significantly attenuates the development of cardiovascular and certain behavioral responses precipitated by the opiate antagonist, naloxone. The purpose of this study was to determine whether both supraspinal and spinal cholinergic neurons are involved in the expression of withdrawal symptoms. Employing localized (i.c.v. or intrathecal, i.t.) infusions of muscarinic antagonists, it was determined that a significant antiwithdrawal action could be produced through both an inhibition of supraspinal and spinal cholinergic neurons. Pharmacological difference emerged regarding the antiwithdrawal potential of 4-DAMP and the partially M1 selective antagonist, pirenzepine. While our previous studies had revealed that pirenzepine had essentially no antiwithdrawal activity when administered by the i.c.v. route, in the present study, pirenzepine evoked a marked antiwithdrawal action by the i.t. route, significantly inhibiting both cardiovascular and behavioral signs of withdrawal. In contrast, 4-DAMP which was effective by the i.c.v. route (especially for the cardiovascular symptoms), elicited no antiwithdrawal action by the i.t. route. As a muscarinic antagonist (ability to block the pressor response to central injection of carbachol) 4-DAMP was equally active by i.c.v. or i.t. injection. However, pirenzepine was clearly more effective in this regard by the i.t. route. These results are consistent with ability of muscarinic antagonists to offer significant anti-morphine withdrawal activity at both supraspinal and spinal locations. They also suggest that different muscarinic systems, possibly different receptor subtypes, mediate the expression of morphine withdrawal symptoms within the two regions of the CNS.

Topics: Animals; Blood Pressure; Cerebral Ventricles; Heart Rate; Injections, Intraventricular; Injections, Spinal; Male; Morphine; Muscarinic Antagonists; Naloxone; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Spinal Cord; Substance Withdrawal Syndrome; Time Factors

[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cyclic AMP; Inositol Phosphates; Kinetics; Muscarine; Muscarinic Antagonists; Myocardium; N-Methylscopolamine; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Scopolamine Derivatives

Cerebellar granule cells express m2- and m3-muscarinic acetylcholine receptors (mAChRs) and their corresponding mRNA with m3-mAChR being the predominant receptor subtype. After stimulation with the mAChR agonist, carbachol, m2- and m3-mAChR mRNA levels were decreased in a time- and concentration-dependent manner with the maximal down-regulation at 2 and 8 hr, respectively. Immunoprecipitation studies revealed that amounts of m2- and m3-mAChR protein also decreased at 8 and 24 hr, respectively. The carbachol-induced down-regulation of m3-mAChR mRNA was associated with a decrease in the transcription rate, but a substantial enhancement of the mRNA stability. Upon removal of carbachol after treatment for 8 hr, the levels of m3-mAChR mRNA and mAChR binding sites returned to their original values with a t1/2 of approximately 80 min and 6 hr, respectively. The carbachol-elicited loss of m2- and m3-mAChR mRNA was blocked by their corresponding receptor subtype-specific antagonists, AF-DX 116 (m2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (m3-selective), and was concurrent with an increase in c-fos mRNA levels. Exposure of granule cells to the nonselective mAChR antagonist, atropine, caused a time- and concentration-dependent increase in the level of both m2- and m3-mAChR mRNA and mAChR binding sites. At 24 hr, immunoprecipitable m3-mAChR protein was predominantly increased. The atropine-induced up-regulation of m3-mAChR mRNA was concurrent with a marked enhancement of the mRNA stability and its transcription rate. The elevated levels of m3-mAChR mRNA and binding sites declined to their untreated values after the removal of atropine. Treatment with AF-DX 116 and 4-DAMP also produced an increase in the level of m2- and m3-mAChR mRNA and their corresponding immunoprecipitable receptor protein. These results demonstrate that the mAChR agonist and antagonist induce a down- and up-regulation of mAChR expression, respectively, through receptor-mediated mechanisms in cerebellar granule cells. Moreover, at least for m3-mAChR mRNA, the agonist- and antagonist-induced effects are reversible and associated with corresponding changes in the transcription rate of this receptor mRNA species.

Topics: Animals; Atropine; Carbachol; Cells, Cultured; Cerebellum; Down-Regulation; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; RNA, Messenger; Up-Regulation

Vasomotor neurons in the enteric nervous system release acetylcholine to dilate submucosal arterioles, but it is not known whether sensory nerves that project to these vessels also can provide a vasodilator innervation. This possibility was examined by determining the mechanism of action of capsaicin on guinea pig ileal submucosal arterioles in vitro. Capsaicin dilated all vessels that had been preconstricted with prostaglandin F2 alpha; mean effective concentration was 11 nM, and maximal dilation occurred at 60-200 nM. The vasodilation showed marked desensitization upon repeated applications of capsaicin. Tetrodotoxin blocked the capsaicin-induced vasodilation but not the desensitization observed upon repeated application. Muscarinic receptor antagonists did not affect the actions of capsaicin. Capsaicin did not dilate arterioles whose extrinsic sensory afferent fibers had been surgically removed. Substance P and human calcitonin gene-related peptide II dilated arterioles; these dilations were not inhibited after desensitization of the capsaicin-induced vasodilation. Thus capsaicin dilates submucosal arterioles by selectively activating extrinsic afferent fibers that release vasodilator transmitter substances onto these vessels.

Topics: Animals; Arterioles; Capsaicin; Guinea Pigs; Ileum; Intestinal Mucosa; Parasympatholytics; Perfusion; Piperidines; Tetrodotoxin; Vasodilation; Vasodilator Agents

Biochemical and pharmacological characteristics of muscarinic cholinergic receptors in isolated guinea pig pancreatic ducts were determined in the present study. Duct homogenates bound 6.82 +/- 0.69 fmol of [3H]N-methylscopolamine ([3H]NMS)/micrograms of DNA with a Kd of 0.73 +/- 0.05 nM. The density of [3H]NMS binding sites in the excretory ducts was seven times greater than that in acini from the same pancreases. Competition binding studies with atropine, pirenzepine, 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b] [1,4]benzodiazepine-6-one (AF-DX 116), and 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) indicated that both M2 and M3 subtypes of muscarinic receptors are present in these preparations of isolated pancreatic ducts. Electrophoretic analysis of [3H]propylbenzilylcholine mustard-labeled unreduced and reduced duct muscarinic receptors provided molecular mass estimates of 62.6 +/- 2.5 and 58.0 +/- 1.6 kDa, respectively. Deglycosylation of ductal muscarinic receptors with N-glycanase decreased their apparent molecular mass by approximately 4 kDa. These results demonstrate that isolated pancreatic ducts express both M2 and M3 muscarinic receptors, with the former subtype predominating.

Topics: Amidohydrolases; Animals; Binding, Competitive; Guinea Pigs; In Vitro Techniques; Male; Molecular Weight; N-Methylscopolamine; Pancreatic Ducts; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Piperidines; Propylbenzilylcholine Mustard; Receptors, Muscarinic; Scopolamine Derivatives; Tritium

Muscarinic receptors present in guinea-pig lung were characterized using the M3 selective radioligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([3H]4-DAMP). In saturation studies, [3H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (Kd = 0.21 nM and Bmax = 10 fmol/mg prot.) while the remaining sites were low affinity ones (Kd = 18.11 nM and Bmax = 269 fmol/mg prot.). In competition studies with [3H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M2 and the M3 receptors, respectively. No evidence was found for high affinity [3H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[3H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [3H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M1 receptors and reflected more the presence of M4 or a mixture of M3 and M4 receptors. The low affinity pirenzepine binding sites may represent M2 receptors. These results provide further evidence for the occurrence of M2 and M3 receptors and suggest the presence of the M4 muscarinic receptor subtype in guinea-pig lung.

Topics: Animals; Binding, Competitive; Cerebral Cortex; Guinea Pigs; Kinetics; Lung; Male; Myocardium; Piperidines; Rabbits; Radioligand Assay; Receptors, Muscarinic; Tritium

The goal of this study was to elucidate mechanisms responsible for age-related reductions in responsiveness to cholinergic muscarinic stimulation in guinea pigs, by examining the binding properties of muscarinic receptors and their coupling to guanine nucleotide regulatory proteins as a function of animal age. In addition, the binding constants of three selective muscarinic receptor antagonists pirenzepine, [11-((2-[(diethylamino)methyl]-1-piperidinyl)-acetyl)-5, 11-dihydro-6H-pyrido(2,3)(1,4)benzodiazepine-6-on], and 4-diphenylacetoxy-N-methylpiperidine methobromide were examined. We found that there were no changes in either the receptor density or the affinity of the muscarinic receptor with age. There was a significant reduction in the affinity constant for the high-affinity agonist binding site in the old tissues (7.63 +/- 0.08) compared with the young tissues (8.31 +/- 0.10). Guanine nucleotides lowered agonist affinity for the receptor in young lungs, however, they had no effect on agonist binding in old tissues. Antagonist competition binding curves in young tissues revealed that 73% of the receptors are of the M2 type, with 27% being of the M3 subtype. In contrast, antagonist competition binding curves in the old tissues revealed that 37% of the receptors were of the M2 subtype, 30% were M3, and 33% were of the M1 subtype. Our studies provide evidence that the loss of sensitivity to cholinergic muscarinic stimulation in the senescent lung may be due to changes in both muscarinic receptor subtypes and receptor coupling to G proteins.

Topics: Aging; Animals; Binding, Competitive; Guinea Pigs; Lung; Male; Muscarine; Parasympathomimetics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic

We have tested the hypothesis that guanine-nucleotide-binding-protein-coupled receptors may be able to interact with each other at a molecular level. To address this question, we have initially created two chimeric receptors, alpha 2/m3 and m3/alpha 2, in which the C-terminal receptor portions (containing transmembrane domains VI and VII) were exchanged between the alpha 2C-adrenergic and the m3 muscarinic receptor. Transfection of COS-7 cells with either of the two chimeric constructs alone did not result in any detectable binding activity for the muscarinic ligand N-[3H]methylscopolamine or the adrenergic ligand [3H]rauwolscine. However, cotransfection with alpha 2/m3 and m3/alpha 2 resulted in the appearance of specific binding sites (30-35 fmol/mg of membrane protein) for both radioligands. These sites displayed ligand binding properties similar to those of the two wild-type receptors. Furthermore, COS-7 cells cotransfected with alpha 2/m3 and m3/alpha 2 were able to mediate a pronounced stimulation of phosphatidylinositol hydrolysis upon stimulation with the muscarinic agonist carbachol (Emax approximately 40-50% of wild-type m3). A mutant m3 receptor (containing 16 amino acids of m2 receptor sequence at the N terminus of the third cytoplasmic loop) that was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis was also used in various cotransfection experiments. Coexpression of this chimeric receptor with other functionally impaired mutant muscarinic receptors (e.g., with an m3 receptor containing a Pro-->Ala point mutation in transmembrane region VII) resulted in a considerable stimulation of phosphatidylinositol breakdown after carbachol treatment (Emax approximately 40-50% of wild-type m3). Thus, these data suggest that guanine-nucleotide-binding-protein-coupled receptors can interact with each other at a molecular level. One may speculate that the formation of receptor dimers involving the intermolecular exchange of N- and C-terminal receptor domains (containing transmembrane domains I-V and VI and VII, respectively) may underlie this phenomenon.

Topics: Acetylcholine; Animals; Carbachol; Cell Line; GTP-Binding Proteins; Kinetics; Models, Structural; N-Methylscopolamine; Parasympatholytics; Phosphatidylinositols; Piperidines; Protein Structure, Secondary; Rats; Receptors, Adrenergic, alpha; Receptors, Muscarinic; Recombinant Fusion Proteins; Scopolamine Derivatives; Signal Transduction; Transfection; Yohimbine

Muscarinic receptors present in cultured human iris sphincter and ciliary smooth muscle cells were characterized by both ligand ([3H]QNB binding) and functional (phosphoinositide hydrolysis) studies. Ligand binding studies showed that [3H]QNB represented a single population of binding sites with KD values of 4.02 x 10(-11) M in the ciliary and 5.6 x 10(-11) M in the iris sphincter cells. In competition studies, the selective antagonist, 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP) was the most potent in displacing [3H]QNB with selectivity of 150-350-fold over pirenzepine (M1) and 450-1700-fold over AF-DX 116 (M2). 4-DAMP recognized one site in the iris sphincter cells (Ki = 0.34 nM) but two sites in the ciliary cells (KH = 0.9 nM and KL = 49 nM). 4-DAMP was also the most potent in inhibiting carbachol-induced hydrolysis of inositol phospholipids (PI) in both cell types. However, the IC50 values for PI hydrolysis were several fold lower than those for [3H]QNB binding. Using these selective antagonists, our data supports the presence of functional muscarinic receptors of M3 subtype in human iris sphincter and ciliary cells. It also shows the presence of a second low affinity site in the ciliary smooth muscle cells that is recognized by 4-DAMP.

Topics: Binding, Competitive; Cells, Cultured; Ciliary Body; Humans; Iris; Muscle, Smooth; Phosphatidylinositols; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic

The regional distribution of putative M3 receptors was studied by quantitative autoradiography of rat brain sections labeled with 4-diphenylacetoxy-N-methyl-piperidine methioxide. The radioligand appears to label multiple muscarinic receptor subtypes in direct binding assays, as evidenced by competition experiments with unlabeled pirenzepine and 11-([2-[(2-(diethylamino)methyl]-1- piperidinyl]acetyl-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodi azepine-6-on. Conditions favoring the selective labeling of M3 receptors were determined on the basis of the competition studies and were subsequently applied to regional mapping of M3 receptor binding. Under the conditions used, autoradiographic images may be obtained with both 9-fold M3-to-M1 selectivity and 12-fold M3-to-M2 selectivity. Although distributed widely at relatively low prevalence throughout the neuraxis, M3 receptors are apparently enriched relative to M1 and M2 receptors in several diencephalic and brain stem regions including thalamic and hypothalamic nuclei, the substantia nigra, superior colliculus, periaqueductal gray and the pons. The present results suggest that the binding of 4-diphenylacetoxy-N-methyl-piperidine methioxide, under appropriate conditions, may permit direct estimation of M3 receptors in mammalian brain.

Topics: Animals; Autoradiography; Binding, Competitive; Brain; Male; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Tritium

We measured endogenous noradrenaline (NA) overflow from a vascularly perfused rat stomach in vitro. The stomach was perfused with Krebs-Ringer solution containing 10 microM pargyline. Periarterial nerves, which contain postganglionic sympathetic nerves, around the left gastric artery were stimulated for 1 min with square-wave pulses of 2 msec duration, 2.5 to 5.0 Hz, supramaximal intensity (10 mA). Oxotremorine (10(-8) to 10(-6) M) concentration-dependently inhibited the periarterial nerve stimulation-evoked NA overflow under the presence of 10(-6) M phentolamine. Bilateral vagus nerve stimulation (5 Hz, 2 msec duration, 10 mA, for 1 min) reduced the evoked NA overflow. Oxotremorine (10(-7) M)-induced inhibition of NA overflow was attenuated by atropine, methoctramine (muscarinic M2 receptor antagonist), 4-diphenylacetoxy-N-methylpiperidine (M3 receptor antagonist) and pirenzepine (M1 receptor antagonist) with the following potency; atropine > methoctramine > 4-diphenylacetoxy-N-methylpiperidine >> pirenzepine. The oxotremorine-induced inhibition was attenuated by N-ethylmaleimide (3 x 10(-5) M for 50 min), but was not affected by pertussis toxin pretreatment (10 micrograms/rat, for 4 days). However, this pretreatment with pertussis toxin abolished completely negative chronotropic and inotropic effects of oxotremorine in rat atria. These results suggest that NA release from gastric sympathetic nerve terminals is inhibited by activation of muscarinic M2 receptor, and this receptor-mediated inhibitory mechanisms are insensitive to pertussis toxin.

Topics: Animals; Arteries; Atrial Function; Atropine; Depression, Chemical; Diamines; Electric Stimulation; Ethylmaleimide; Heart Atria; Male; Myocardial Contraction; Nerve Endings; Norepinephrine; Oxotremorine; Parasympatholytics; Perfusion; Pertussis Toxin; Phentolamine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Stomach; Sympathetic Nervous System; Vagus Nerve; Virulence Factors, Bordetella

In this study we investigated the effects of antimuscarinics with different selectivity on the intestinal migrating myoelectric complex (MMC) in five fasting, conscious dogs, chronically fitted with electrodes along the small bowel. Furthermore, we evaluated the chronotropic and mydriatic effects to assess the in vivo selectivity of the agents tested. Dose-response studies were performed with the following drugs administered i.v.: atropine, telenzepine (M1 antagonist), AF-DX 116 [11,2-(diethylamino)methyl-1-piperidinyl-acetyl-5,11-dihydro-6H-pyrido-2 ,3b- 1,4-benzodiazepine-6-one (M2 antagonist)] and 4-diphenylacetoxy-N- methylpiperidine methiodide (4-DAMP) (M3 antagonist). All the antimuscarinics tested dose-dependently increased the duration of the MMC period and inhibited spike activity, except low-dose telenzepine (3-10 nmol/kg), which shortened the MMC period and stimulated spike activity. High-dose telenzepine (> 100 nmol/kg) mimicked the inhibitory effect of atropine on the intestine. ED50 values for delay of MMC onset were 87,232, > 10,000 and 129 nmol/kg for atropine, telenzepine, AF-DX 116 and 4-DAMP, respectively. At doses lengthening the MMC period, atropine and 4-DAMP also induced tachycardia and mydriasis. At doses shortening the MMC period, telenzepine had no effect on pupil diameter or heart rate, except at the dose of 10 nmol/kg, which reduced heart rate. Finally, AF-DX 116, at doses inducing marked tachycardia, had a minor intestinal effect and no mydriatic effect. The present data are consistent with the hypothesis that both M1 and M3 receptors are involved in the regulation of the MMC: M1 receptors are probably located on an inhibitory pathway, whereas M3 receptors mediate excitatory stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Dogs; Female; Gastrointestinal Motility; Heart Rate; Male; Parasympatholytics; Piperidines; Pirenzepine; Pupil; Receptors, Muscarinic

The binding of the muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), which has been suggested as an M3-selective antagonist in peripheral tissues, was examined through quantitative autoradiographic techniques in brain. The ability of 4-DAMP to displace [3H](R)-quinuclindinyl benzilate (QNB) binding to rat brain sections was compared with the known distribution of M1 and M2 muscarinic receptor subtypes as measured previously with pirenzepine and AF-DX 116 (Messer et al., 1989a). 4-DAMP displayed a high affinity for [3H](R)-QNB binding sites in rat brain sections. Analysis of 4-DAMP binding to various brain regions revealed heterogeneous binding profiles, suggesting an interaction with multiple receptor sites. Quantification of the autoradiograms indicated that 4-DAMP bound with the highest affinity to muscarinic receptors in the midline thalamus (IC50 values < 30 nM), and had a slightly lower affinity for hippocampal receptors (IC50 values between 30 and 46 nM). 4-DAMP also displayed a lower affinity for cortical receptors with IC50 values between 30 and 50 nM. The binding profile of the putative M3 muscarinic antagonist did not exhibit a marked selectivity for any single region of brain. The data suggest that whereas 4-DAMP may be selective for M3 receptors in peripheral tissues, it has limited selectivity in the CNS. Minimum energy conformations for 4-DAMP were calculated using molecular mechanics calculations. 4-DAMP displayed two global minimum energy conformations, differing in the relative position of the piperidine ring with respect to the aromatic rings. The minimum energy conformations of 4-DAMP were compared with conformations generated for pirenzepine (Messer et al., 1989a). The lowest energy conformation of 4-DAMP was superimposable on the lowest energy conformation of pirenzepine (RMS = 0.297 A). It is suggested that the conformations available to 4-DAMP permit binding to several muscarinic receptors in the CNS.

Topics: Animals; Autoradiography; Binding, Competitive; Brain; Image Processing, Computer-Assisted; In Vitro Techniques; Molecular Conformation; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Receptors, Muscarinic; Tissue Distribution

Primary cultures of dissociated cerebral cortex cells were used to characterize the muscarinic acetylcholinergic receptors (mAChR) present and to study receptor down-regulation and receptor mediated 2nd messenger responses induced by muscarinic agonists. Binding of the hydrophilic antagonist [3H]N-methyl scopolamine ([3H]NMS) to the cultured cells was saturated after one hour at 4 degrees C with a Kd of 93 pM and a Bmax of 958 fmol/mg protein. Competition binding studies with several antagonists and agonists indicated that the mAChR present in the culture were of a mixed M1/M3 subtype. The number of muscarinic receptors at the cell surface decreased by 60% after one hour pre-incubation of the cultures with 10 microM carbachol or oxotremorine. After down-regulation with carbachol affinity for pirenzepine was decreased, while low affinity sites for 4-DAMP were lost, indicating that especially M1 subtypes are sensitive to this type of regulation. Carbachol and oxotremorine-M induced a 2-3 fold increase in phosphatidyl inositide (PI) turnover, which was blocked with high affinity by both pirenzepine and 4-DAMP. Down-regulation of the mAChR and stimulation of PI-turnover by agonists with different potency and intrinsic activity appeared highly correlated. These data suggest that activation of the PI second-messenger system is involved in the desensitization and down-regulation of the muscarinic acetylcholine receptor.

Topics: Animals; Binding, Competitive; Carbachol; Cells, Cultured; Cerebral Cortex; Down-Regulation; Oxotremorine; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Second Messenger Systems

The muscarinic receptors in the aorta of the normo- and hypertensive rats were characterised with tritiated acetylcholine (3H-ACh) and various muscarinic receptor antagonists. The binding of 3H-ACh to the endothelial membranes of the normotensive Wistar Kyoto rats (WKY) and the spontaneously hypertensive rats (SHR) was displaceable by nanomolar range of scopolamine but only by micromolar range of atropine and homatropine. The reverse was observed with the muscle binding sites, i.e. the 3H-ACh was displaceable by nanomolar range of atropine and homatropine but only by micromolar range of scopolamine. Pirenzepine and 4-diphenyl-acetoxy-N-methyl-piperidine metobromide (4-DAMP) displaced the binding of 3H-ACh from both tissues in the nano to micromolar range, with the displacement from the endothelial binding sites occurring at lower concentration range of the ligands. The apparent IC50 values of both compounds for the smooth muscle were 9 and 16 times greater than those for the endothelial binding sites respectively. When saturated with guanylyl-imididiphosphate (GppNHp), conversion of high to low-affinity binding site occurred in both tissues of the WKY but only in the smooth muscle of the SHR. GppNHp had no apparent effect on the binding of 3H-ACh to the endothelial binding sites confirming that the high-affinity site for 3H-ACh was missing in the endothelium of the SHR.

Topics: Acetylcholine; Animals; Aorta; Atropine; Binding, Competitive; Endothelium, Vascular; Guanylyl Imidodiphosphate; Hypertension; In Vitro Techniques; Kinetics; Muscle, Smooth, Vascular; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Muscarinic; Scopolamine; Tropanes

1. Carbachol produced a relaxation of dilator muscle at a concentration lower than 1 microM and a contraction at a concentration higher than 1 microM. 2. We studied the effects of the M1-selective antagonist, pirenzepine, the M2-selective antagonist, himbacine, the M3-selective antagonist, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (4-DAMP) and the non-selective antagonist, atropine, on carbachol-induced relaxation and contraction of the rat iris dilator smooth muscle. All the antagonists competitively inhibited both the responses to carbachol. 3. In relaxation and contraction, the low affinity of pirenzepine and himbacine suggest that the rat iris dilator smooth muscle receptors are not of the M1 and M2 subtypes. In contrast, 4-DAMP potently inhibited the carbachol-induced relaxation and contraction with affinities similar to those reported for the M3 subtype. 4. Carbachol-induced relaxation and contraction of the rat iris dilator appears to be mediated through a homogeneous population of M3 subtype.

Topics: Alkaloids; Animals; Atropine; Carbachol; Furans; In Vitro Techniques; Iris; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic

1. The effects of muscarinic receptor antagonists considered to be selective for M1 receptors (pirenzepine; PZ), M2 receptors (AFDX-116), and for M3 receptors (4-diphenyl acetoxy N-methyl-piperidine (4-DAMP)) were used to investigate the existence of muscarinic receptors subtypes in murine airways. Atropine was used as a nonselective antagonist. The effects of these antagonists were studied upon tracheal contractions induced either by EFS (electric field stimulation) or by application of an exogenous cholinoceptor agonist (arecoline). 2. The muscarinic receptor antagonists tested inhibited arecoline-induced tracheal contractions with the following rank order of potency: 4-DAMP = atropine > pirenzepine = AFDX-116. The rank order of potency of the muscarinic antagonists used in inhibiting EFS-induced tracheal contractions was: 4-DAMP = atropine > PZ > AFDX-116. The pA2 values for these antagonists were similar when compared to the pA2 values determined in guinea-pig and bovine airway smooth muscle. 3. In addition to in vitro studies, the effects of inhalation of the different muscarinic antagonists on lung function parameters in vivo were investigated. Inhalation of 4-DAMP induced a decrease in airway resistance and an increase in lung compliance. In contrast, inhalation of AFDX-116 induced an increase in airway resistance and almost no change in lung compliance. Apart from some minor effects of atropine on airway resistance, atropine, PZ, and pilocarpine failed to induce changes in lung mechanics as determined by in vivo lung function measurements. 4. The results provide evidence for the existence of M3 receptors on murine tracheae that are involved in the contraction of tracheal smooth muscle. This is in agreement with other animal species such as the guinea-pig and bovine. In vivo experiments also demonstrated that in the mouse, M3 receptors play an important role in bronchial smooth muscle contraction and thus in bronchoconstriction. Interestingly we have also demonstrated that M2 receptors can play a role in bronchodilatation. Inhalation of an M2 receptor antagonist induced an increase in airway resistance whereas inhalation of an M3 receptor antagonist induced a decrease in airway resistance. It is therefore likely that an M3/M2 receptor balance plays an important role in the regulation of airway function.

Topics: Administration, Inhalation; Airway Resistance; Animals; Atropine; Bronchi; Electric Stimulation; In Vitro Techniques; Isometric Contraction; Lung Compliance; Male; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

In order to identify subtypes of muscarinic receptor on the rat pancreas, the effects of new muscarinic receptor antagonists, [11-[[2-(diethylamino)-methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) and 4-diphenylacetoxy-N-methylpiperadine-methiodide (4-DAMP), on amylase secretion stimulated by carbachol and binding of [3H]quinuclidinyl benzilate (QNB) were evaluated using isolated rat pancreatic acini. Atropine, pirenzepine, AF-DX 116 and 4-DAMP inhibited carbachol-stimulated amylase release in a dose-dependent manner. All these antagonists caused a concentration-dependent rightward shift of the dose-response curve for carbachol-stimulated amylase release without altering the maximal response. Schild plots revealed that pA2 values for atropine, pirenzepine, AF-DX 116 and 4-DAMP were 9.15, 6.78, 6.09 and 8.79, respectively. Every slope of Schild plots was not different from unity, suggesting that these antagonists act as competitive inhibitors. These antagonists also inhibited the binding of [3H]QNB in a dose-dependent manner. The inhibition constants were 1.21 x 10(-9) M (atropine), 1.26 x 10(-7) M (pirenzepine), 0.57 x 10(-6) M (AF-DX 116) and 2.75 x 10(-9) M (4-DAMP). Thus, the order of inhibitory potencies was atropine > or = 4-DAMP > pirenzepine > AF-DX 116. These findings suggest that 4-DAMP-sensitive M3 receptor may play an important role in the pancreatic exocrine functions.

Topics: Amylases; Animals; Atropine; Carbachol; Dose-Response Relationship, Drug; Male; Pancreas; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptors, Muscarinic; Sincalide

Topics: Animals; Chemical Phenomena; Chemistry, Physical; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Muscle, Smooth, Vascular; Piperidines; Receptors, Muscarinic; Sympatholytics

To determine what muscarinic receptor subtype regulates the rise of intracellular calcium concentration ([Ca2+]i) and resultant airway submucosal gland secretion on muscarinic receptor stimulation, we examined the effects of atropine (ART), pirenzepine (PZ), 11([2-(diethylamino) methyl-1-piperidinyl] acetyl)-5,11-dihydro-6H-pirido (2,3-b)(1,4)-benzodiazepine-6-one (AF-DX116) and 4-diphenylacetoxy-N-methylpiperidine methiodode (4-DAMP) on methacholine (MCh)-evoked [Ca2+]i rise in acinar cells, mucus glycoprotein (MGP) secretion and electrolyte secretion from submucosal glands isolated from feline tracheae. [Ca2+]i was measured with the Ca(2+)-sensitive fluorescent dye, fura2. We determined MGP secretion by measuring trichloroacetic acid (TCA)-precipitable 3H-labeled glycoconjugates and electrolyte secretion by the change in the rate constant of 22Na-efflux from isolated glands. Half maximal inhibitory concentrations (IC50) of PZ, AF-DX116, 4-DAMP and ATR against MCh (10(-5) M)-evoked [Ca2+]i rise were 10(-7) M, 6 x 10(-6) M, 8 x 10(-9) M and 6 x 10(-9) M, respectively. IC50 values of these antagonists against MCh (10(-5) M)-evoked MGP secretion were 10(-6) M, 2 x 10(-5) M, 8 x 10(-9) M and 6 x 10(-9) M, respectively. MCh (10(-5) M)-evoked 22Na-efflux was significantly inhibited by 10(-7) M 4-DAMP and 10(-7) M ATR (p < 0.01, respectively), but the inhibitory effect of PZ (10(-7) M) was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Calcium; Cats; Electrolytes; Glycoproteins; In Vitro Techniques; Mucous Membrane; Piperidines; Pirenzepine; Receptors, Muscarinic; Second Messenger Systems; Trachea

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Binding Sites; Carbachol; Cyclic AMP; Hydrolysis; Male; Paraoxon; Phosphatidylinositols; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Regression Analysis; Submandibular Gland

Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with receptor binding, Ca++ mobilization and Northern blotting. Displacement of [3H]NMS with pirenzepine in IMR-32 cells revealed apparent binding sites with Kd values of 5 (41%) and 237 nM (59%). With 4-diphenylacetoxy-N-metylpiperidine metiodid, a similar proportion of apparent high- and low-affinity binding was obtained: 36 (Kd = 0.26 nM) and 64% (Kd = 6.3 nM), respectively. In SH-SY5Y cells, two different affinities with apparent Kd of 40 (24%) and 460 nM (76%) could be distinguished with pirenzepine, even though the Kd of the apparent high-affinity site varied markedly (variation = 8.7-96.8 nM). Inhibition of carbachol-induced Ca++ mobilization displayed high sensitivity to 4-diphenylacetoxy-N-methylpiperidine metiodid in both cell lines. IMR-32 cells displayed high sensitivity to pirenzepine, whereas the sensitivity varied between different batches of SH-SY5Y cells. DNA fragments (approximately 1000 base pairs) from SH-SY5Y DNA amplified with polymerase chain reaction were used as probes for muscarinic receptor mRNA. Northern blotting with the Hm1-specific probe gave a stronger signal for SH-SY5Y than for IMR-32, whereas the result obtained with the Hm2-probe was the opposite. Also, the Hm3 mRNA was detected in SH-SY5Y cells. The Hm4 and Hm5 transcripts were not detected in either of these cell lines.

Topics: Blotting, Northern; Calcium; Humans; N-Methylscopolamine; Neuroblastoma; Phosphatidylinositols; Piperidines; Pirenzepine; Radioligand Assay; Receptors, Muscarinic; RNA, Messenger; Scopolamine Derivatives; Tumor Cells, Cultured

A newly developed, primary culture of human antral epithelial cells has been utilized to examine the effect of parasympathomimetics on somatostatin release. The cholinergic agonists, carbachol and methacholine, stimulated somatostatin secretion in a concentration-dependent manner. Maximal release in response to carbachol was observed at 0.1 mmol/l. Methacholine was 10 times more potent with a significant release being observed at 1 mumol/l, maximal secretion was observed at 10 mumol/l. Somatostatin release, stimulated by the mixed nicotinic and muscarinic agonist, carbachol, was attenuated by the addition of atropine at 0.1 mumol/l but was unaffected by the same concentration of pirenzepine. Methacholine-stimulated release was attenuated by addition of 0.1 mumol/l atropine and unaffected by the same concentration of pirenzepine. The response to methacholine was reversed by the addition of 0.1 mumol/l 4-diphenylacetoxy-n-methylpiperidine methiodide (4-DAMP) and attenuated by 1 nmol/l 4-DAMP indicating that the effect was mediated by an M3 receptor. In conclusion, human antral D cells are stimulated by parasympathomimetics acting at an M3 receptor.

Topics: Adult; Carbachol; Cells, Cultured; Epithelium; Female; Humans; Male; Methacholine Chloride; Parasympatholytics; Piperidines; Pyloric Antrum; Radioimmunoassay; Receptors, Muscarinic; Somatostatin

1. Significant differences were observed between the hamster and the rat in terms of the secretion of fluid and protein from submandibular glands in response to pilocarpine, phenylephrine and isoproterenol. 2. In both the rat and the hamster the secretory responses induced by pilocarpine, phenylephrine and isoproterenol were inhibited by pretreatment with 4-DAMP, prazosin and metoprolol, respectively. 3. These results suggest that the submandibular glands of the hamster and the rat have M3-cholinoreceptors, as well as alpha 1- and beta 1-adrenoceptors, and that these receptors play different roles in the secretion of fluid and protein from hamster and rat submandibular glands.

Topics: Animals; Cricetinae; Isoproterenol; Male; Mesocricetus; Metoprolol; Parasympathomimetics; Phenylephrine; Pilocarpine; Piperidines; Prazosin; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Receptors, Cholinergic; Saliva; Salivary Proteins and Peptides; Submandibular Gland; Sympathomimetics

1. 4-Diphenylacetoxy-1:1-dimethyl cyclohexane (carbo-4-DAMP) is the carbon analogue of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide. The compounds differ only in that the quaternary nitrogen atom in 4-DAMP methiodide is replaced by a quaternary carbon atom, which is uncharged. 2. Carbo-4-DAMP appears to act competitively at functional (M3) muscarinic receptors in guinea-pig ileum. Estimates of log affinity constant are 6.0 at 30 degrees C and 5.9 at 37 degrees C, i.e. the compound has 0.1% of the affinity of 4-DAMP methobromide. 3. The absence of charge makes little difference to the conformation as determined by X-ray crystallography. The bond lengths and angles are very similar, though the bonds in the cyclohexane ring of carbo-4-DAMP are consistently slightly longer than those in the piperidinium ring of 4-DAMP methiodide, and the presence of the charge slightly reduces the space between molecules. 4. The difference between the affinities of 4-DAMP methobromide and carbo-4-DAMP indicates that the contribution of coulombic forces to the binding between 4-DAMP methiodide and muscarinic (M3) receptors is at least 17 kJ mol-1 (4.1 kcal mol-1) at 37 degrees C. How much this is an underestimate depends upon how much hydrophobic binding is greater with the uncharged compound.

Topics: Animals; Cyclohexanes; Guinea Pigs; Ileum; In Vitro Techniques; Piperidines; Receptors, Muscarinic; Structure-Activity Relationship

1. Airways derived from different levels of the lung exhibit a difference in the reactivity and sensitivity to agonists. We have evaluated the effect of acetylcholine and cholinergic selective (pirenzepine, gallamine and 4-dipherylacetoxymethyl piperidine [4-DAMP]) and non-selective (atropine) antagonists on bovine proximal and distal smooth muscle preparations. 2. The distal preparations are more sensitive to acetylcholine than proximal bronchi. The relaxant effect of three selective antagonists on the distal and proximal tissues was the same when the results for each drug were compared. 3. Atropine and 4-DAMP were more potent than pirenzepine and gallamine in relaxing both proximal and distal bovine smooth muscle preparations. 4. These data suggest that the muscarinic sites on the smooth muscle of bovine airways are of the M3 subtype.

Topics: Acetylcholine; Animals; Atropine; Bronchi; Cattle; Gallamine Triethiodide; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine

Acetylcholine, acting on muscarinic receptors, has a powerful modulatory effect on neurons in the cerebral cortex. Recent evidence that cortical tissue contains at least four different muscarinic receptor subtypes having different pharmacological properties indicates the value of new investigations of the receptor subtypes that mediate electrophysiological responses. Here we studied the ionic nature and pharmacology of the depolarizing response to acetylcholine in identified corticocallosal neurons in primary culture. This response is the result of a non-rectifying 'leak' K+ conductance which is reduced in the presence of acetylcholine. The relative sensitivity of this conductance change to the antagonists pirenzepine and 4-DAMP suggests that it is mediated by the M3 subtype of muscarinic receptor.

Topics: Acetylcholine; Animals; Cells, Cultured; Cerebral Cortex; Corpus Callosum; Electrophysiology; Microspheres; Neurons; Piperidines; Pirenzepine; Potassium Channels; Rats; Receptors, Muscarinic

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of different selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60-80%) correspond to M2 muscarinic subtypes, and the rest is constituted by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.

Topics: Adrenal Medulla; Animals; Atropine; Binding Sites; Binding, Competitive; Cattle; Cell Membrane; Dicyclomine; Muscarine; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic

Selective muscarinic receptor antagonists were used to identify muscarinic receptor subtypes in equine trachealis strips. The M1 receptor antagonist pirenzepine (10(-7) mol/L to 3 x 10(-5) mol/L) and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10(-9) mol/L to 3 x 10(-7) mol/L3) dose dependently inhibited the contractile responses to electrical field stimulation (EFS) and exogenous acetylcholine (ACh). Schild plots yielded a pA2 value for pirenzepine vs ACh of 6.75 +/- 0.09, which is consistent with the affinity for M2 or M3 receptors, and a pA2 value for 4-DAMP vs ACh of 8.47 +/- 0.09, which is in agreement with the affinity for M3 receptors. The M2 receptor antagonist gallamine (10(-5) mol/L and 10(-4) mol/L) did not affect the response of trachealis to exogenous ACh and low-frequency EFS (0.1-2 Hz) but decreased the responses to high-frequency EFS (4-16 Hz). These results suggest that the muscarinic receptors mediating contractions induced by ACh in equine tracheal smooth muscle are of the M3 subtype. The lack of an increase in the response to EFS following gallamine suggests that functional prejunctional inhibitory M2 receptors are not present on the cholinergic nerves innervating equine tracheal smooth muscle.

Topics: Acetylcholine; Animals; Electric Stimulation; Gallamine Triethiodide; Horses; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

The muscarinic receptor subtype antagonists 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), 11-[(2-[diethylamino)methyl]-1- piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one (AF-DX 116) and pirenzepine were used to inhibit the outflow facility and accommodative and miotic responses to near-maximal intracameral doses of pilocarpine in the living rhesus monkey eye. The pharmacologic M3 antagonist 4-DAMP was the most potent inhibitor of all three responses, with IC50 values of 41.7 nM for outflow facility (vs. 40.9 microM pilocarpine), 19.8 nM for accommodation (vs. 40.9 or 81.8 microM pilocarpine) and 3.2 nM for miosis (vs. 4.1 microM pilocarpine). The M1 antagonist pirenzepine was at least 30-fold less potent, with IC50 values of 2.2 microM for outflow facility, 1.4 microM for accommodation and 0.1 microM for miosis. The M2 antagonist AF-DX 116 was the least potent by far, with IC50 values of 15.5 microM for outflow facility, 14.2 microM for accommodation and 1.5 microM for miosis. The results suggest that these three functional responses to pilocarpine are all mediated through an M3 receptor subtype.

Topics: Accommodation, Ocular; Animals; Aqueous Humor; Female; Intraocular Pressure; Macaca mulatta; Male; Miosis; Muscarinic Antagonists; Parasympatholytics; Phenylephrine; Pilocarpine; Piperidines; Pirenzepine; Pupil

Prior studies have suggested the presence of muscarinic acetylcholine receptor (MAChR) subtypes within the mammalian central nervous system on the bases of functional ligand binding or molecular biologic evidence. Autoradiographic differentiation of MAChR subtypes in ligand binding assays has previously relied on the use of agonists, results of homogenate binding assays to determine relative subtype binding affinities or both. In the present study, the binding of [3H]scopolamine to intact slide-mounted tissue sections is characterized. The rapid binding kinetics of the ligand permit autoradiographic saturation experiments. Autoradiographic competition assays utilizing [3H]scopolamine and the unlabeled subtype-selective ligands pirenzepine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5,11,-dihydro-6H-pyr ido (2,3-b)(1,4)benzodiazepin-6-on (AF-DX 116) and 4-diphenylacetoxy-N-methylpiperidine methiodide reveal evidence for the presence of multiple MAChR subtypes distributed heterogeneously throughout the brain. High-affinity pirenzepine MAChR (putative M1 subtype) predominate in the telencephalon. High-affinity AF-DX 116 MAChR (putative M2 subtype) are widely distributed, but are quantitatively minor populations, with the exception of motor cranial nerve nuclei and the basal pons, where they represent the dominant MAChR fractions. Evidence for relative enrichment of M3 receptors was obtained in the thalamus, the superficial layer of the superior colliculus, the periqueductal region, the substantia nigra pars reticulata and the pons. The autoradiographic assays developed in this work may assist in defining altered receptor populations arising in pathologic conditions or resulting from drug therapy.

Topics: Animals; Autoradiography; Binding, Competitive; Brain Chemistry; Male; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Scopolamine

Ca2+ mobilizations in SH-SY5Y and IMR-32 human neuroblastoma cell lines were measured using the fluorescent Ca2+ indicator fura-2. A variety of antagonists (atropine, pirenzepine, 4-DAMP and N-methyl-scopolamine) inhibited carbamyl choline-induced transient Ca2+ mobilization both in a competitive and a noncompetitive manner. The apparent noncompetitive inhibition constants were lower in IMR-32 than in SH-SY5Y cells even when the competitive inhibition constants were similar. This may relate to the previously reported differential expression of muscarinic receptor subtypes in these cell lines.

Topics: Atropine; Calcium; Fura-2; Humans; Kinetics; Muscarinic Antagonists; N-Methylscopolamine; Neuroblastoma; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine Derivatives; Spectrometry, Fluorescence; Tumor Cells, Cultured

1. The nature of the muscarinic receptor subtype mediating endothelium-dependent vascular relaxation was investigated in the perfused mesenteric vascular bed preparation which is a model for resistance vessels. 2. After methoxamine-induced vasoconstriction the vessels were dilated with acetyl-beta-metacholine (MCh). 3. The potency of the M1-selective antagonist pirenzepine, the M2-selective antagonists AF-DX 116 and AQ-RA 741, the M3-selective antagonists 4-DAMP and p-FHHSiD to block the MCh-induced vasodilation was quantified by means of pA2-values. Atropine was used for comparison. 4. The rank order of potency for the various muscarinic receptor antagonists appears to be: atropine > 4-DAMP > p-FHHSiD > pirenzepine > AQ-RA 741 > AF-DX 116 which is similar to findings in conduit arteries. 5. The high potency of the M3-selective antagonists 4-DAMP and p-FHHSiD and the low potency of the M1- and M2-selective antagonists suggest a major role of M3-receptors in the cholinergic vasodilatation in the perfused mesenteric vascular bed.

Topics: Animals; Atropine; Benzodiazepinones; Dose-Response Relationship, Drug; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Arteries; Mesenteric Veins; Methacholine Chloride; Parasympatholytics; Perfusion; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Vasodilation

Muscarinic receptors of resistance vessels (submucosal arterioles, outside diameter 50-75 microns) from the guinea-pig small intestine were investigated in vitro using a computer-assisted videomicroscopy system (Diamtrak). The muscarinic receptor which mediates vasodilation of precontracted [U-46619 (300 nM) or (-)-noradrenaline (10 microM)] arterioles was characterized with several muscarinic agonists and subtype-selective antagonists. The following agonists all produced equivalent maximum vasodilation (given in rank order of potency): acetylcholine = arecaidine propargyl ester (APE) greater than oxotremorine = (+/-)-muscarine = (+/-)-methacholine greater than carbachol greater than 4-[[N-(4-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). 4-[[N-(3-Chlorophenyl)-carbamoyl]oxy]-2-butynyltrimethylammonium chloride (McN-A-343) and N-ethyl-guvacine propargyl ester (NEN-APE) produced minimal or no arteriolar vasodilation. The muscarinic antagonists pirenzepine, (+-)-5,11-dihydro-11-[[[2-[2-((dipropylamino)methyl)-1-piperidinyl] ethyl]amino]-carbonyl]-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one (AF-DX 384), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one (AQ-RA 741), p-fluorohexahydro-sila-difenidol (p-F-HHSiD), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and (R)- and (S)-hexahydro-difenidol [(R)-HHD, (S)-HHD] shifted the muscarine, methacholine or carbachol dose-response curve to the right in a competitive manner. Schild analysis of the data yielded pA2 values for pirenzepine (6.74/6.9), AF-DX 384 (6.72), AQ-RA 741 (6.58), p-F-HHSiD (7.53/7.57), 4-DAMP (9.06), (R)-HHD (7.88/8.32) and (S)-HHD (5.52/5.88). Thus, it can be concluded that submucosal arterioles possess only the M3 functional muscarinic receptor, the activation of which causes blood vessel dilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Arterioles; Benzodiazepinones; Carbachol; Guinea Pigs; Ileum; Methacholine Chloride; Muscarine; Muscarinic Antagonists; Oxotremorine; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Prostaglandin Endoperoxides, Synthetic; Receptors, Muscarinic; Vasodilation

Spontaneous and electrically evoked endogenous acetylcholine release and [3H]-choline efflux from slices of guinea pig nucleus basalis magnocellularis (nbM) were studied. Tetrodotoxin reduced the spontaneous endogenous release by 55%, while the Ca(2+)-free medium reduced it by about 30%. Evoked [3H]-choline efflux was Na+ and Ca2+ dependent and frequency related. Physostigmine, 30 microM, nearly halved the stimulation-evoked efflux; atropine, 0.15 microM, not only antagonized, but even reversed this effect into facilitation. Pirenzepine, 1 microM, and AFDX 116, 1 microM, were less effective than atropine, and reversed the inhibitory effect of physostigmine only when applied together. 4-DAMP, 0.01 microM, was ineffective. These findings indicate that acetylcholine release in guinea pig nbM slices is inhibited by the cooperation of muscarinic autoreceptors, possibly belonging to the M1 and M2 subclasses.

Topics: Acetylcholine; Animals; Atropine; Calcium; Choline; Female; Guinea Pigs; Male; Parasympatholytics; Physostigmine; Piperidines; Pirenzepine; Receptors, Muscarinic; Substantia Innominata; Tetrodotoxin

Unilateral microinjection of the acetylcholine receptor agonist carbachol into the posterior hypothalamic nucleus evokes a pressor response in the conscious, freely moving rat. To further localize this response 3.3 or 5.5 nmol of carbachol was microinjected in a volume of 50 nl directly into and outside the region of the posterior hypothalamic nucleus. Administration of carbachol outside the posterior hypothalamic nucleus failed to evoke a change in blood pressure indicating that the carbachol-induced pressor response is mediated from the posterior hypothalamic nucleus. Since posterior hypothalamic administration of atropine completely blocks the carbachol-induced increase in blood pressure and atropine blocks the three pharmacologically identified muscarinic receptor subtypes, methylatropine and progressively more selective muscarinic antagonists were administered into the posterior hypothalamic nucleus prior to 5.5 nmol of carbachol. Microinjection of the M1/M2/M3 muscarinic antagonist methylatropine (0.19-12.5 nmol), the M1/M3 antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine; 0.9-3.6 nmol), the M1 antagonist pirenzepine (9.5-38 nmol), the M2 antagonist methoctramine (5.5-44 nmol), or the M3 antagonist p-F-HHSiD (para-fluoro-hexahydro-sila-difenidol; 2.1-8.3 nmol) inhibited the peak increase in mean arterial pressure and the area under the curve of the change in mean arterial pressure versus time plot in a dose-dependent manner. Log ID50s calculated for the antagonists from the dose-response curves were found to correlate significantly with the log Kis of the antagonists for the muscarinic M3 receptor subtype. These results demonstrate that the increase in mean arterial pressure evoked by microinjection of carbachol into the posterior hypothalamic nucleus is mediated by the muscarinic M3 receptor.

Topics: Animals; Atropine Derivatives; Blood Pressure; Carbachol; Diamines; Dose-Response Relationship, Drug; Hypothalamus, Posterior; Kinetics; Male; Microinjections; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic

To enhance our understanding of cholinergic mechanisms and muscarinic receptors in bronchoconstriction, we have characterized the muscarinic receptor subtypes in rabbit tracheal smooth muscle using radioligand binding and functional assays. The Kd for [3H]quinuclidinyl benzilate ([3H](-)QNB) binding determined from saturation isotherms was 12.6 x/divided by 1.1 pM (geometric mean x/divided by SEM), and the Bmax was 269 +/- 7 fmol/mg protein (arithmetic mean +/- SEM). Competitive inhibition studies with the muscarinic antagonists pirenzepine (PZ), 11[[2-[(diethylamino)-methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), and hexahydrosiladifenidol (HHSiD) demonstrated heterogeneity of muscarinic receptor subtypes in rabbit tracheal smooth muscle. PZ bound with low affinity to a single receptor site, indicative of an absence of M1 receptors. AF-DX116 (M2 selective) bound with high affinity to approximately 83% of muscarinic binding sites, and 4-DAMP and HHSiD (M3 antagonists) bound with high affinity to approximately 24 and 28% of muscarinic binding sites, respectively. Additionally, direct binding studies with [3H]4-DAMP demonstrated high-affinity binding with 23% of muscarinic binding sites. Thus, the majority of muscarinic receptors in rabbit tracheal smooth muscle bound with high affinity to an M2-selective antagonist, and the remaining receptor sites bound with high affinity to M3 antagonists. The inhibitory effects of atropine, PZ, AF-DX116, and 4-DAMP on methacholine-induced contraction of rabbit tracheal rings were compared. 4-DAMP was a potent inhibitor of methacholine-induced contraction, but PZ and AF-DX116 demonstrated low potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Female; Male; Methacholine Chloride; Muscle Contraction; Muscle, Smooth; Myocardium; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rabbits; Radioligand Assay; Receptors, Muscarinic; Trachea

1. We compared the binding properties of 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and nine analogues of this compound on muscarinic receptors of human neuroblastoma NB-OK1 cells (M1 subtype), rat heart (M2 subtype), rat pancreas (M3 subtype) and to the putative M4 subtype in striatum. 2. The requirements for high affinity binding were somewhat different for the four receptor subtypes. In general, the requirements of M3 receptors were more stringent than for M1, M2 or putative M4 receptors. 3. The abilities of the compounds to discriminate muscarinic receptor subtypes were not correlated with their affinities at any subtype. 4. The temperature-dependence of binding of 4-DAMP analogues to M2 receptors varied with the drug structure. In particular, the increased affinity of the alpha-methyl derivative of 4-DAMP could be ascribed to van der Waals interactions. 5. The affinities of most 4-DAMP analogues for M2 and M3 receptors were similar to their pharmacological potencies on atrial and ileum preparations, respectively. 6. At concentrations above 1 microM, all 4-DAMP analogues as well as atropine, reduced the [3H]-N-methyl scopolamine ([3H]-NMS) dissociation rate from cardiac muscarinic receptors, with no obvious structure-activity relationship.

Topics: Allosteric Regulation; Animals; Corpus Striatum; In Vitro Techniques; Male; Myocardium; N-Methylscopolamine; Neuroblastoma; Pancreas; Piperidines; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Structure-Activity Relationship; Temperature; Tumor Cells, Cultured

GTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 microM oxotremorine and 100 microM carbachol. (4-Hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity. 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) inhibition of oxotremorine stimulation demonstrated the presence of two populations of receptors, a low-affinity site (IC50 +/- SEM, 0.63 +/- 0.18 microM) which accounted for 63% of the inhibition and a high-affinity site (IC50 less than 1 nM) which accounted for the remaining 37%. When carbachol-stimulated GTPase activity was assayed, a single 4-DAMP inhibitory site was apparent (IC50 +/- SEM, 2.0 +/- 0.9 microM). Pirenzepine inhibited GTPase activity at a single site (IC50 values +/- SEM, 46.9 +/- 11 and 25.4 +/- 6.5 microM against oxotremorine and carbachol, respectively). Methoctramine was equipotent against carbachol and oxotremorine stimulation (IC50 values, 4.2 +/- 1.8 and 6.2 +/- 1.5 microM). Inhibition of maximal carbachol and oxotremorine stimulation by muscarinic antagonists at the major site had a rank order of potency of 4-DAMP = methoctramine greater than pirenzepine. Thus, the major site for muscarinic stimulation of GTPase activity in bovine retinal membranes is pharmacologically similar to M2 receptors.

Topics: Animals; Cattle; Chromatography, Thin Layer; Diamines; Dose-Response Relationship, Drug; GTP Phosphohydrolases; Guanosine Triphosphate; Muscarinic Antagonists; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Receptors, Muscarinic; Retina; Stimulation, Chemical; Synaptic Membranes

1. Isolated rings of rabbit external jugular vein (RbJV) and rat thoracic aorta (RA) were used to study the effect of the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) on muscarinic and 5-hydroxytryptamine (5-HT) receptor-stimulated, endothelium-dependent vascular relaxations. 2. In RbJV relaxations produced by the endothelial 5-HT receptor agonist alpha-methyl-5-HT were potently and non-surmountably inhibited by L-NAME (10 microM), whereas acetylcholine relaxations in this tissue were unaffected by this concentration of inhibitor. By contrast, acetylcholine relaxations in RA were virtually abolished by 10 microM L-NAME. In each case an equivalent concentration of D-NAME was without effect on agonist-induced relaxations. 3. The different effect of L-NAME on acetylcholine relaxations in RbJV and RA was not due to muscarinic receptor differences. Affinity estimates for acetylcholine (pKA = 6.12 +/- 0.09; 6.09 +/- 0.08 respectively) and for 4-diphenyl-acetoxy-N-methylpiperidine methobromide (4-DAMP, pKB = 9.01 +2- 0.012; 9.24 +/- 0.16 respectively) indicated that the receptors in both tissues belong to the same M3 class. Tissue differences resulting from the release of a cyclo-oxygenase product or a glibenclamide-sensitive K(+)-channel-linked hyperpolarizing factor were also ruled out by selective inhibition of these pathways. 4. When phenoxybenzamine was used to reduce the efficacy of acetylcholine in RbJV so that it behaved as a partial agonist in this tissue, L-NAME (10 microM) now produced non-surmountable inhibition of relaxation responses. In untreated tissues the same concentration of L-NAME also profoundly inhibited responses produced by butyrylcholine and pilocarpine, both of which behave as partial agonists at the M3 receptor in RbJV. 5. A simple model was developed which describes the theoretical behaviour of receptor-stimulated synthesis and release of NO. The model predicts that competitive inhibition of NO formation results in parallel displacements of the agonist response curve in the case of high efficacy agonist, but right-shift with concomitant depression of the curve maximum in the case of low efficacy agonists. Simulations based on the model showed reasonable agreement with the experimental data. 6. It is concluded that analogues of L-arginine demonstrate tissue- and agonist-dependence in terms of their ability to inhibit receptor-mediated events involving the liberation of NO. This behaviour can reflect differences in ag

Topics: Acetylcholine; Alkylation; Animals; Aorta, Thoracic; Arginine; Glyburide; In Vitro Techniques; Indomethacin; Jugular Veins; Male; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroglycerin; Parasympatholytics; Piperidines; Rabbits; Receptors, Muscarinic; Receptors, Serotonin; Serotonin Antagonists

We demonstrated in this study that 4-DAMP [4-diphenylacetoxy-1-(2- chloroethyl) piperidine] mustard, which cyclizes to the aziridinium ion, behaved as a non-selective, non-competitive inhibitor of muscarinic receptors in rat brain cortex. It inactivated to the same extent the M1, M2 and M4 muscarinic receptors present in this tissue, as well as receptors accessible or not accessible to quaternary antimuscarinic drugs. Under mild incubation conditions, the muscarinic receptors in a state with super high affinity for agonists (SH receptors) were less affected by preactivated 4-DAMP mustard than the receptors in the states with lower affinity for agonists (H and L receptors).

Topics: Animals; Carbachol; Cerebral Cortex; Diphenylacetic Acids; Muscarinic Antagonists; Myocardium; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

Topics: Acetylcholine; Adenoma, Islet Cell; Atropine; Calcimycin; Calcium; Carbachol; Chromogranin A; Gastrin-Releasing Peptide; Humans; Pancreatic Hormones; Pancreatic Neoplasms; Parasympatholytics; Peptides; Piperidines; Pirenzepine; Receptors, Muscarinic; Sincalide; Tumor Cells, Cultured

We previously showed that M1 and M2 muscarinic receptors in dissociated cells of the adult rat cortex couple to phosphoinositide (Pl) and cyclic AMP (cAMP) metabolism, respectively. To further classify these receptors according to probable subtype, we have employed a group of selective muscarinic antagonists to obtain pharmacological profiles of the cortical M1 and M2 receptors, and to compare them with the muscarinic receptors in N1E-115 cells, which contain M1 receptors mediating cyclic GMP elevation and M4 receptors inhibiting cAMP levels. The M2-mediated inhibition of cAMP levels in cortex was blocked by 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) with higher potency (0.29 nM) than for reported potency in cardiac tissue (approximately 10 nM), indicating that this cortical response is probably not mediated by the m2 gene product. Similarly, the potency of hexahydrosiladiphenidol (HSD) at the cortical M2 receptor (159 nM) was somewhat greater than the reported potency in cardiac tissue (295 nM). The cardioselective drugs AF-DX 116 and methoctramine blocked the cortical M2 response less potently (135 nM and 229 nM, respectively) than would be expected for involvement of the m2 gene product. Thus, the potencies of AF-DX 116, methoctramine, 4-DAMP and HSD suggest that the cortical M2 response, like the striatal M2 receptor, is mediated by a noncardiac M2 receptor, perhaps by the m4 gene product. This postulate was supported by the significant correlations between cortical and striatal M2 receptors as compared to the M4 receptor in N1E-115 cells (r = 0.92 and 0.99, respectively, P less than .025).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cerebral Cortex; Cyclic AMP; Diamines; In Vitro Techniques; Muscarinic Antagonists; Parasympatholytics; Phenethylamines; Phosphatidylinositols; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Tumor Cells, Cultured

Muscarinic receptors in N1E 115 mouse neuroblastoma cells were characterized by competition binding experiments using three agonists and five antagonists, including 4-DAMP and AF-DX 116, and by studying the effect of agonist stimulation on the cellular cAMP and cGMP content. The results of the binding studies with the antagonists suggest that only one single homogeneous binding site of the M1 muscarinic receptor subtype is present. For the binding with the agonists, two binding sites were detected, one with high affinity for the ligand (between 53 and 77% of the total binding sites depending on the agonist) and one with low affinity. In contrast to the results obtained with the binding experiments using antagonists, the study of the cellular cyclic nucleotide response upon carbachol stimulation suggested the presence of both the M1 and M2 subtypes as there was an increase in cyclic GMP concentration while at the same time, the prostaglandin-stimulated synthesis of cyclic AMP was inhibited. Considering both binding and functional data we suggest that in N1E 115 cells a majority of M1 and a minority of M2 muscarinic receptors are present; there is no evidence for the presence of M3 muscarinic receptors.

Topics: Animals; Carbachol; Cyclic AMP; Cyclic GMP; Indicators and Reagents; Ligands; Mice; Neuroblastoma; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Tumor Cells, Cultured

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.

Topics: Animals; Animals, Newborn; Binding, Competitive; Carbachol; Cerebellum; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Male; Parasympatholytics; Phosphatidylinositols; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Carbachol; Cells, Cultured; Cerebellum; Cycloleucine; Cytoplasm; Ibotenic Acid; Kinetics; N-Methylaspartate; Neurotoxins; Piperidines; Quisqualic Acid; Receptors, Neurotransmitter

Dose-dependent depression of the electrically evoked surface-negative field potential (N-wave) produced by bath-superfusion of carbachol was measured in guinea-pig olfactory cortex slices maintained in vitro. The possible involvement of M3 (smooth muscle/glandular) type muscarinic receptors in partly mediating this response was investigated by testing the effectiveness of the muscarinic M3 receptor antagonists hexahydro-sila-difenidol (HHSiD) and p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD). Low doses of HHSiD (10-100 nM) or p-F-HHSiD (up to 1 microM), pre-applied for 30 min, produced no obvious antagonism of carbachol responses. However, a clear competitive-type inhibition of carbachol effects was observed in 250 nM-1 microM HHSiD or 10-50 microM p-F-HHSiD respectively. Schild plot analysis (regression slope constrained to unity) of pooled data yielded pA2 values of 6.6 for HHSiD (n = 6 slices) and 5.5 for p-F-HHSiD (n = 6 slices) respectively, suggesting a weak competitive antagonism by both compounds. In addition, combination experiments using either HHSiD or p-F-HHSiD with atropine, produced dose-ratio shifts close to those predicted for two antagonists competing for a common receptor site. By comparison, another suggested M3-receptor antagonist, 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) was a potent competitive blocker of carbachol responses. Schild analysis for 4-DAMP versus carbachol gave a pA2 of 7.9 (n = 6 slices). It is concluded that the muscarinic receptors involved in the suppression of the olfactory cortical N-wave possess a low affinity for HHSiD and particularly for p-F-HHSiD, but not 4-DAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Action Potentials; Animals; Carbachol; Cerebral Cortex; Evoked Potentials; Guinea Pigs; In Vitro Techniques; Olfactory Bulb; Parasympatholytics; Piperidines; Receptors, Muscarinic; Synaptic Transmission

In order to investigate whether treatment with terbutaline (1 mg subcutaneously) in patients with bronchial obstruction is accompanied by changes in the densities of receptors of the autonomic nervous system, beta-adrenergic receptors or m-cholinergic receptors were measured in membrance preparations of human peripheral lung tissue from patients undergoing lung resection for bronchial carcinoma. The density of beta-adrenergic receptors and mononuclear leukocytes (MNLs) of treated and untreated patients and beta-adrenergic receptors and m-cholinergic receptors in lung strips from the same patients exposed to terbutaline in vitro were studied for comparison. In patients, treatment with terbutaline did not have any effect on human lung beta-adrenergic receptors and m-cholinergic receptors, whereas a 57 percent decline was measured in the number of beta-receptors on MNLs of the very same patients. In contrast, in vitro exposure of human peripheral lung strips to terbutaline (100 mumol/L for 36 hours) evoked a time-dependent and concentration-dependent decline of 46 percent in beta-adrenergic receptors. Again, there was no change in the number of m-cholinergic receptors. The antagonist affinities, as judged from the KD values, did not differ under either condition. We concluded that lung beta-adrenergic receptors are subject to down-regulation when exposed to agonists in vitro. This down-regulation in the human lung is not accompanied by alterations in m-cholinergic receptors. Down-regulation of beta-adrenergic receptors or up-regulation of m-cholinergic receptors appears not to play a role in the proposed tolerance to beta-adrenergic receptor agonist treatment in clinical situations. The reduction of beta-adrenergic receptors in MNLs provides evidence that treatment with terbutaline was sufficient to affect beta-adrenergic receptors in vivo in certain cell types but also shows that alterations in blood cells do not necessarily reflect the situation in the lung.

Topics: Aged; Aged, 80 and over; Airway Obstruction; Dose-Response Relationship, Drug; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Humans; In Vitro Techniques; Leukocytes, Mononuclear; Lung; Middle Aged; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Adrenergic, beta; Receptors, Cholinergic; Terbutaline

The cGMP response and the accumulation of inositol monophosphate (IP) induced by carbachol were compared in slices of different rat brain structures. Basal cGMP and the responses of cGMP to carbachol appeared dependent on the concentration of added Ca2+, suggesting that distinct Ca(2+)-mediated and Ca(2+)-sensitive muscarinic receptor-mediated mechanisms stimulate guanylate cyclase. Regional responses of cGMP to carbachol or to direct stimulation of guanylate cyclase with sodium nitroprusside were markedly distinct, indicating that a major proportion of guanylate cyclase in the cortex, an intermediate proportion in other forebrain regions, and only a minor proportion in the brainstem is sensitive to muscarinic receptor stimulation. The regional patterns of IP and cGMP responses to carbachol were different in the forebrain. Maximal IP accumulation was found in the cortex, whereas cGMP responses were highest in the hippocampus. Moreover, IP and cGMP formation in the hippocampus were differently antagonized by atropine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), the M2-receptor subtype-preferring antagonist AF-DX 116 and the M1-selective antagonist pirenzepine. These data support the notion that the IP formation induced by carbachol in the forebrain predominantly is mediated by muscarinic receptors of the M1 subtype, and indicate the involvement of muscarinic receptors of the M3 subtype in the carbachol-induced cGMP formation.

Topics: Animals; Atropine; Brain; Carbachol; Cyclic GMP; Guanylate Cyclase; Hippocampus; Inositol Phosphates; Male; Nitroprusside; Phosphatidylinositols; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Succinimides

Serosal addition of muscarinic agonists elicited rapid changes in electrical parameters across the isolated canine esophageal epithelium set up in vitro. Both carbachol and the M1-selective agonist, McNeil A343 (McN), increased transmucosal potential differences (PDs), decreased transmucosal resistances (R), and increased short-circuit currents (Isc). Carbachol was more potent and more effective than McN. Muscarinic antagonists were used to define the muscarinic receptor involved. The pA2 values obtained with Schild plots were as follows: atropine 9.14, 4-DAMP 8.98, AFDX-116 6.71, and pirenzepine 7.12. Low concentrations of pirenzepine (10(-8) M), produced a rightward shift in the dose-response curve to McN, without inhibiting responses to carbachol. Thus the receptor subtype is clearly not an M2. As in other glandular systems, M3 receptors are present. Whether M1 receptors also exist requires better definition of receptor densities-reserves in this tissue. Carbachol induced net secretion of Na and Cl and converted a predominantly absorptive tissue to a secretory one.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Amiloride; Animals; Atropine; Carbachol; Chlorides; Dogs; Electric Conductivity; Esophagus; Female; In Vitro Techniques; Ion Channels; Male; Membrane Potentials; Mucous Membrane; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Sodium

The muscarinic (M) receptors involved in the vasodilation elicited by acetylcholine (ACh) and in the carbachol inhibition in electrically induced [3H]noradrenaline (NA) release in cat cerebral arteries was investigated. For this, atropine, pirenzepine, AF-DX 116, 4-DAMP, non-specific, M1, M2 and M3 receptor antagonists, respectively, were used. ACh elicited concentration-dependent relaxations up to 10(-6) M which were attenuated by these antagonists; the order of potency (pA2 values) to inhibit the ACh-induced relaxation was: atropine (10.1) 4-DAMP (8.9) greater than pirenzepine (7.6) greater than AF-DX 116 (5.9). The electrical stimulation (200 mA, 0.3 ms, 2 Hz, during 1 min) of these arteries preincubated with [3H]NA caused tritium release which was inhibited by carbachol (10(-6) M). The 4 antagonists attenuated the action of the M agonist; the order of potency (pIC50 values) was: atropine (8.7) greater than 4-DAMP (8.1) greater than AF-DX 116 (7.9) greater than pirenzepine (5.8). The action of McN-A-343, a putative M1 agonist, was also investigated. This agent produced small vasodilator responses and elevated concentrations (5 x 10(-5) M) inhibited the stimulated NA release, which was partially antagonized by atropine (10(-7) M) and pirenzepine (10(-8) and 10(-7) M). These results suggest the existence of M3 and M2 receptors mediating the relaxation induced by ACh and the NA release inhibition evoked by carbachol, respectively.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Atropine; Carbachol; Cats; Cerebral Arteries; Endothelium, Vascular; Female; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Norepinephrine; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Receptors, Muscarinic; Vasodilation

1 The affinity of some muscarinic antagonists for muscarinic receptors was determined in functional isolated tissue studies in order to compare the muscarinic receptor subtypes in the rabbit. 2 Our attention was specially focused on the question of whether the muscarinic receptors mediating vasodilatation in the aorta resemble or not the ones present on the jejunum of the gastrointestinal tract. 3 Isolated aorta, jejunum, stimulated left atrium and vas deferens preparations of rabbit were investigated with the following muscarinic antagonists: atropine, pirenzepine, methoctramine (N,N'-bis[6-92-methoxybenzyl)amino hexyl]-1,8-octane-diamine tetrahydrochloride) and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide). 4 The results demonstrate that the receptors on aorta are unlike those on the other rabbit tissues: pirenzepine pA2 was 6.4 on aorta but 8.1 on vas deferens; methoctramine pA2 was 5.9 on aorta but 7.1 on heart; 4-DAMP pA2 was 8.7 on aorta and 8.0 on jejunum. This raises the question: what subtype might be involved?

Topics: Animals; Aorta, Thoracic; Atropine; Diamines; Jejunum; Male; Muscarinic Antagonists; Muscle Contraction; Myocardium; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Vas Deferens; Vasodilation

The parasympathetic neurotransmitter acetylcholine, acting postsynaptically at the smooth muscle muscarinic receptor, is a principle determinant of colonic motility. In order to elucidate the receptor signal-transduction events responsible for muscarinic receptor-induced contraction of colonic circular smooth muscle, we present here and in the accompanying work studies designed to characterize the muscarinic receptors present in colon and to determine their biochemical coupling. Muscarinic receptor subtypes in canine colonic circular smooth muscle were characterized using radioligand binding techniques. The nonselective muscarinic receptor antagonist radioligand [3H]quinuclidinyl benzilate ([3H]QNB) binds rapidly and reversibly to a single class of saturable sites in colon circular smooth muscle membranes, with an affinity (KD) for the antagonist radioligand of 79.8 +/- 12.6 pM and a density of 123.3 +/- 18.7 fmol/mg of protein. Experiments using membranes prepared from isolated cells purified from the circular smooth muscle layer of canine colon (KD = 102.4 +/- 13.5 pM) confirm the smooth muscle origin of the binding and yield a receptor density of 124,340 receptors/cell. The order of potencies of selective muscarinic receptor antagonists in competition with [3H]QNB for binding to colonic receptors is 4-diphenylacetoxy-N-methylpiperidine methobromide greater than methoctramine greater than AF-DX 116 greater than pirenzepine. Unlike other antagonists tested, pirenzepine competition of [3H]QNB binding is biphasic. The high and low affinities deduced from nonlinear fit of the binding data in colon correlate very well with affinities determined for pirenzepine in mixtures of both submandibular gland (M3) and atrium (M2), indicating the presence of two muscarinic receptor subtypes (82% M2, 18% M3) in colon circular smooth muscle. The muscarinic agonist carbachol binds to both high and low affinity sites in colon, and addition of guanine nucleotide (100 microM GTP gamma S) shifts the agonist competition curve to the right, without eliminating high affinity binding sites. Agonist competition studies with a known ratio of M2 and M3 receptors, obtained by mixing pure M2 and M3 populations, predict the result obtained in colon. cDNA probes specific for each of the muscarinic receptors m1 through m4 were hybridized to colon RNA in a Northern blot analysis. Only m2 and m3 probes hybridized to colon RNA, suggesting the presence of both M2 and M3 receptors. Our data d

Topics: Animals; Binding, Competitive; Cells, Cultured; Colon; Dogs; Female; GTP-Binding Proteins; Male; Membranes; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Radioligand Assay; Receptors, Muscarinic; Tritium

To examine muscarinic receptor subtypes involved in cholinergically mediated contractions of the airway, we studied the effects of the M1-selective antagonist, pirenzepine, the M2-selective antagonist, AF-DX 116, the M3-selective antagonist, 4-diphenyl-acetoxy-N-methylpiperidine (4-DAMP) methiodide, and the non-selective antagonist, atropine, on acetylcholine (ACh)- and electrically induced contractions in dog bronchi and bronchioles. The relative potencies of the antagonists based on IC50 values of each antagonist for contractions induced by the two concentrations of ACh that produced 50% of the maximum (ED50) and the maximum (EDmax) contractions and the pA2 values were atropine greater than or equal to 4-DAMP methiodide greater than pirenzepine = AF-DX 116 in both the bronchi and bronchioles. The IC50 and pA2 values of each antagonist did not differ significantly between the bronchi and bronchioles. 4-DAMP methiodide significantly inhibited the contractile response to electrical field stimulation (EFS) at 5 Hz at concentrations that did not alter the contractile responses to exogenous ACh in both the bronchi and bronchioles, whereas pirenzepine, AF-DX 116 and atropine inhibited the EFS-induced contraction only at the concentrations that reduced the contraction induced by exogenous ACh. The present results suggest that the cholinergic contraction is mediated via the postsynaptic receptor M3, based on functional potencies of muscarinic antagonists and presynaptic receptor auto-facilitatory M3, based on the suppression of the contractile response to EFS by 4-DAMP methiodide in central and peripheral airways.

Topics: Acetylcholine; Animals; Atropine; Bronchi; Dogs; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic

Autoradiography of [3H] quinuclidinyl benzilate was used to demonstrate the distribution of muscarinic acetylcholine binding in the spinal cord of sheep. Binding was confined to the grey matter of the cord, and was most densely distributed in the substantia gelatinosa region of the dorsal horn, the lamina X region around the central canal, the intermediolateral columns and in various regions of the ventral horn. The use of specific M1 and M2 receptor subtype ligands, pirenzipine and 4-DAMP indicated that both receptor subtypes were present in most regions of dense binding.

Topics: Animals; Autoradiography; Ligands; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Scopolamine; Sheep; Spinal Cord

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.

Topics: Animals; Autoradiography; Binding Sites; Brain; Ligands; Male; Membranes; Piperidines; Pirenzepine; Prosencephalon; Rats; Receptors, Muscarinic; Tissue Distribution; Tritium

The effect of alinidine, a bradycardic agent, on the vasodilator responses to acetylcholine was examined in isolated and perfused dog coronary arteries. Single injections of acetylcholine (10(-12)-10(-6) mol) and carbachol (10(-10)-10(-6) mol) produced dose-dependent vasodilations. The endothelial removal by a bolus injection of saponin (1 mg) inhibited those vasodilations. Alinidine (10(-6) M) shifted the dose-response curves of acetylcholine and carbachol to the right, but it did not affect those for isosorbide dinitrate, isoproterenol and adenosine. The rank order of potency of muscarinic antagonists for inhibiting the acetylcholine-induced vasodilation was 4-DAMP greater than or equal to atropine greater than AF-DX 116 greater than or equal to pirenzepine greater than alinidine. Alinidine was approximately 100 times less potent than atropine. Single injection of alinidine (10(-8)-10(-6) mol) dilated the dog coronary artery in a dose-related manner. The vasodilation was not affected by the pretreatment with phentolamine (10(-6) M), pindolol (10(-6) M), atropine (10(-6) M), chlorpheniramine (10(-6) M), cimetidine (10(-6) M) or methysergide (10(-6) M). These results suggest that alinidine has a weak anti-muscarinic effect on the endothelium-dependent vasodilation of the dog coronary artery.

Topics: Acetylcholine; Animals; Carbachol; Clonidine; Coronary Vessels; Dogs; Female; In Vitro Techniques; Male; Parasympatholytics; Piperidines; Pirenzepine; Vasodilation

We investigated the effect of selective muscarinic antagonists on atrial natriuretic factor (ANF) release induced by intracerebroventricular (i.c.v) injection of carbachol in the rat. The muscarinic antagonists were given by i.c.v. injection 1 min before carbachol, 1 micrograms/rat. 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a rather selective M1 and M3 receptor antagonist, was the most potent inhibitor of carbachol-induced ANF release, its ID50 being 0.18 nmol/rat. Pirenzepine, a selective M1 antagonist, also potently inhibited the effect of carbachol, its ID50 being 2.74 nmol/rat. The M3-selective antagonist, p-fluoro-hexahydro-sila-diphenidol, was much weaker than pirenzepine, with an ID50 of 57.52 nmol/rat. The selective M2 receptor antagonist, methoctramine, on the other hand, was a very weak inhibitor of carbachol-induced ANF release. The rank order of potency as well as the - log ID50 of the antagonists tested were consistent with their pA2 values for muscarinic M1 receptors, suggesting that this receptor subtype may mediate the central effect of cholinergic mechanisms in the control of ANF release.

Topics: Animals; Atrial Natriuretic Factor; Brain; Carbachol; Diamines; Dose-Response Relationship, Drug; Injections, Intraventricular; Injections, Subcutaneous; Male; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic

The muscarinic cholinergic receptor of rat eccrine sweat gland was characterized using quantitative autoradiography and [3H]QNB as radioligand. The distribution of radioligand was maximal in the secretory coil. Autoradiographic competition binding studies were performed using selective antagonists to M1 (pirenzepine), M2 (AF-DX 116), and M3 (4-DAMP) and the classical nonselective antagonist atropine. pKi for pirenzepine, AF-DX 116, 4-DAMP, and atropine was 6.58, 5.47, 8.50, and 8.66 respectively indicating that the eccrine sweat gland muscarinic receptor was predominantly M3.

Topics: Animals; Atropine; Autoradiography; Binding, Competitive; Eccrine Glands; Kinetics; Male; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Tritium

Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (greater than 30 nM), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca]i transient. Displacement of 3H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca]i response. Elevation of steady-state [Ca]i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca]i is necessary but not sufficient for acid secretion.

Topics: Aminopyrine; Animals; Arachidonic Acids; Calcium; Carbachol; Glucose; Image Processing, Computer-Assisted; Ionomycin; N-Methylscopolamine; Parasympatholytics; Parietal Cells, Gastric; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Scopolamine Derivatives; Tritium

To study the role of muscarinic receptor subtypes in sleep control, methoctramine (25, 50, 75 micrograms), a highly selective M2 antagonist, was injected intra-cerebroventricularly into freely moving rats. Methoctramine induced a dose-dependent increase in desynchronized sleep (DS) latency (from 62.7 +/- 10 min following saline to 122.4 +/- 13.8 min with the lowest dose) and a 75% decrease in the amount of DS in 6 h recordings. 4DAMP (a M3/M1 selective antagonist) did not significantly change DS latency and percentage time, but it reduced wakefulness (from 38 +/- 2.8% following saline to 25.3 +/- 3.7% with a dose of 2.5), and increased slow wave sleep. The results suggest that M2 muscarinic receptors play a selective role in DS physiology.

Topics: Animals; Cerebral Ventricles; Diamines; Dose-Response Relationship, Drug; Injections, Intraventricular; Male; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Reference Values; Sleep; Sympatholytics; Wakefulness

Muscarinic agonists evoke a voltage-dependent inward current in motoneurons of the lobster cardiac ganglion. In this study, a number of drugs, known to show muscarinic receptor subtype selectivity in mammals, were used to determine the pharmacological profile of the muscarinic receptor on lobster motoneurons. The neurons were held under voltage-clamp, and various concentrations of the antagonists were applied in the presence of 1 mM methacholine. From competition curves plotting agonist-induced current against antagonist concentration, the inhibitor affinity constant and the slope factor were determined. The rank order of potencies of antagonists having an effect was: atropine greater than pirenzepine greater than 4-DAMP greater than methoctramine greater than HHSiD = (R)-HHD greater than (S)-HHD. Neither AF-DX 116 nor gallamine were effective at concentrations as high as 10 mM. The M1-selective agonist McN-A-343 had no effect. Although this crustacean muscarinic receptor resembles the mammalian M1 muscarinic receptor because of its relatively high affinity for pirenzepine, the rank order of other subtype-specific antagonists does not otherwise resemble that of any of the pharmacologically defined muscarinic receptors in mammals. It may be preferable, therefore, to use a term such as 'pirenzepine-sensitive' muscarinic receptor rather than M1 or 'M1-like' for invertebrate muscarinic receptors with pharmacological characteristics like those reported here.

Topics: Acetylcholine; Animals; Atropine; Binding, Competitive; Ganglia; In Vitro Techniques; Mammals; Methacholine Chloride; Motor Neurons; Nephropidae; Parasympathomimetics; Piperidines; Pirenzepine; Receptors, Muscarinic

The distribution of cholinergic nerve fibres, as well as the characterization of the muscarinic receptors responsible for the contraction, were determined in the detrusor smooth muscle of the sheep. The results obtained demonstrated a rich presence of acetylcholinesterase (AChE)-positive fibres distributed throughout the bladder body forming dense neuromuscular, subepithelial and perivascular plexuses. Furthermore, intramural ganglia containing AChE-positive cell bodies were identified. However, acetylcholine and carbachol induced a dose-dependent contraction of detrusor smooth muscle. The effect observed with carbachol was competitively antagonized by atropine (pA2: 8.94), pirenzepine (pA2: 7.38), AF-DX 116 (pA2: 7.35), 4-DAMP (pA2: 9.26) and hexahydroxiladifenidol (HHSiD) (pA2: 8.49). The pA2 value for pirenzepine is intermediate between M1- and M2-receptors which suggests that this antagonist does not act on M1- or M2-receptors, but that it does on M3-receptors. The pA2 value for AF-DX 116 is consistent with the presence of M2-receptors in this tissue. Moreover, the pA2 values obtained for both 4-DAMP and HHSiD are in agreement with the presence of M3-receptors, due to the lack of effect of pirenzepine on M1-muscarinic receptors. These results indicate the existence of a rich parasympathetic innervation in the sheep detrusor muscle and suggest that its contraction could be mediated by the stimulation of muscarinic receptors belonging to both M3- and M2-subtypes.

Topics: Acetylcholine; Acetylcholinesterase; Animals; Atropine; Carbachol; Cholinesterase Inhibitors; Ganglia, Parasympathetic; Male; Muscle Contraction; Muscle, Smooth; Nerve Tissue Proteins; Piperidines; Pirenzepine; Receptors, Muscarinic; Sheep; Tetraisopropylpyrophosphamide; Urinary Bladder

Arecoline produces a biphasic response in rat left atria, i.e., a depression of basal inotropy at low doses and a positive inotropic effect at higher doses. These present studies were designed to determine whether it can be shown that the two separate responses to arecoline are mediated by two distinct cell surface muscarinic receptors. The antagonists scopolamine, 4-DAMP and AF-DX 116 produced apparent simple competitive antagonism of the negative responses to arecoline. Schild analysis was used to measure the equilibrium dissociation constant of the antagonist-receptor complex for antagonism of this response to arecoline by these antagonists. In atria from rats treated with pertussis toxin, the negative inotropy to arecoline was abolished and only the positive inotropic effects were observed. The antagonism of the positive inotropic response to arecoline by these antagonists was studied separately in atria from rats treated with pertussis toxin by the Schild technique. The pKB estimates made from the Schild regressions indicated no evidence to suggest that the two responses to arecoline (negative and positive inotropy) were mediated by two separate receptors in rat left atria. These data are discussed in terms of a single muscarinic receptor in this tissue mediating these two responses by interaction with two G-proteins in the same cell membrane. These data also are discussed in terms of the use of agonist potency ratios for the classification of receptors.

Topics: Animals; Arecoline; Dose-Response Relationship, Drug; GTP-Binding Proteins; Heart Atria; In Vitro Techniques; Male; Myocardial Contraction; Pertussis Toxin; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Virulence Factors, Bordetella

The effects of hypothalamic microinfusions of cholecystokinin octapeptide and its antagonist L364,718 on cecocolonic myoelectrical activity were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the cecum and proximal colon and cannulas placed bilaterally in either the ventromedial or lateral hypothalamus. In fasted rats, microinfusion of cholecystokinin octapeptide (10 ng/kg) into the ventromedial hypothalamus increased the spike-burst frequency of the cecum and the colon by 45.6% and 43.7%, respectively, during the 30-minute period after treatment. The injection of cholecystokinin octapeptide (10 ng/kg) into the lateral hypothalamus had no effect on either cecal or colonic motility. Feeding increased the frequency of cecal and colonic spike bursts by 52.1% and 50.1% for 30 minutes postprandially. When infused bilaterally into the ventromedial hypothalamus 10 minutes before feeding, L364,718 (1 or 5 micrograms/kg) abolished the increase of the frequency of cecal and colonic contractions induced by the meal. Infused into the lateral hypothalamus at similar dosages, L364,718 had no effect on the postprandial enhancement of cecocolonic motility. Increase of cecocolonic spike-burst frequency induced by feeding or by cholecystokinin octapeptide injected into the ventromedial hypothalamus was abolished by previous intracerebroventricular but not intraperitoneal administration of atropine (1 microgram) and 4-diphenylacetoxy-N-methylpiperidine (1 microgram), a selective muscarinic M2-receptor antagonist. In contrast, pirenzepine (1 microgram, intracerebroventricularly) did not significantly reduce the meal- or cholecystokin octapeptide-induced increase in cecal and colonic motility. These results suggest that, in rats, (a) cholecystokinin octapeptide is involved in the generation of the cecocolonic motor response to a meal and these effects are mediated through cholecystokinin octapeptide receptors located in the ventromedial hypothalamic nuclei, and (b) these postprandial colonic motor changes involve central cholinergic activation through muscarinic M2 receptors.

Topics: Animals; Atropine; Benzodiazepinones; Cholecystokinin; Colon; Devazepide; Electromyography; Food; Gastrointestinal Motility; Hypothalamic Area, Lateral; Hypothalamus; Male; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Cholecystokinin; Receptors, Muscarinic; Sincalide; Ventromedial Hypothalamic Nucleus

It has been recognized for many years that central cholinergic neurons are susceptible to inhibition by opiates and that during withdrawal their firing rates are enhanced. Nevertheless, classical nonselective muscarinic receptor antagonists have not been demonstrated to provide consistent inhibition of withdrawal symptoms in humans or in animal models. The purpose of this study was to determine whether selective blockade of central M1 or M2 muscarinic receptor subtypes could provide inhibition of naloxone precipitated withdrawal symptoms in morphine dependent rats. As with earlier human studies, both cardiovascular and behavioral measures of withdrawal were quantitated. The selective M2 receptor antagonist 4-DAMP was significantly more effective than the M1 antagonist pirenzepine in reducing both cardiovascular and behavioral symptoms. These results are consistent with a role for cholinergic neurons in the expression of certain morphine withdrawal symptoms and suggest that future therapies might be targeted towards central M2 receptors.

Topics: Animals; Hemodynamics; Morphine; Muscarinic Antagonists; Naloxone; Parasympatholytics; Piperidines; Pirenzepine; Rats; Substance Withdrawal Syndrome

1. The tritiated muscarinic antagonist N-methyl scopolamine, [3H]-NMS, was used to characterize the muscarinic receptors associated with the intact dog tracheal smooth muscle cells. Based on receptor binding assays, the intact tracheal smooth muscle cells had specific, saturable, high-affinity binding sites for [3H]-NMS. 2. Specific binding was cell concentration- and time-dependent. The specific binding of [3H]-NMS was increased linearly with increasing cell concentrations. The equilibrium for association of [3H]-NMS with the muscarinic receptors was attained within 30 min at 37 degrees C. 3. Binding was saturable with respect to [3H]-NMS concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 320 +/- 20 pM and a maximum receptor density (Bmax) of 13.7 +/- 1.4 fmole per 5 x 10(4) cells. The Hill coefficient for [3H]-NMS binding was 1.00 +/- 0.01. The association (K1) and dissociation (K-1) rate constants were determined to be (1.19 +/- 0.23) x 10(8) M-1 min-1 and 0.034 +/- 0.09 min-1, respectively. KD, calculated from the ratio of K-1 and K1, was 286 +/- 65 pM; this value is close to the value of KD calculated from Scatchard plots of binding isotherms. 4. The non-selective muscarinic antagonist atropine and M1 selective antagonist pirenzepine did not reveal any selectivity of these muscarinic receptors. Pirenzepine competed with [3H]-NMS for a single binding site with a Ki value of (6.02 +/- 0.69) x 10(-7) M which is close to the value of M2 or M3 receptors, indicating that the M1 receptor subtype did not exist in the intact tracheal smooth muscle cells. 5. Competition with cardioselective antagonist (M2), methoctramine; smooth muscle selective antagonists (M3), hexahydrodifenidol and hexahydrosiladifenidol; as well as carbachol, were best fit by a two-binding site model. The results suggest that both M2 and M3 receptor subtypes exist at the cell surface of tracheal smooth muscle cells.

Topics: Animals; Atropine; Carbachol; Diamines; Dogs; In Vitro Techniques; Kinetics; Muscle, Smooth; N-Methylscopolamine; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine Derivatives; Trachea

1. The muscarine receptor mediating electrically-stimulated acid secretion in the mouse isolated stomach was characterized using a variety of muscarine receptor antagonists confirming the M1 nature of the antagonist effect of telenzepine. 2. Field stimulation (7 V, 10 Hz, 0.5 ms) resulted in a plateau acid secretion over at least 90 min which was completely blocked by either 1 mumol/l TTX or H2 receptor antagonists (100 mumol/l cimetidine or 10 mumol/l lupitidine). Ranitidine, which is known to potently inhibit mucosal acetylcholine esterase, was ineffective. Compound 48/80 at 100 mumol/l, which depletes mucosal histamine stores, initially mimicked electrical stimulation but subsequently prevented it from inducing acid secretion. 3. 10 muscarine receptor antagonists with differing relative affinities for M1, M2 and M3 receptors were introduced at 1 mumol/l to inhibit electrically-stimulated acid secretion. The percentages inhibition were plotted against binding affinities of the antagonists at either M1, M2 or M3 binding sites. A statistically significant correlation between functional and binding data was detected only when based on M1 affinities. 4. It is concluded that field stimulation, which probably mimicks vagal drive, results in muscarinic M1 receptor activation on paracrine cells to release histamine. Histamine then stimulates parietal cells to secrete acid. Hence, according to the present and our previous data, telenzepine inhibits acid secretion under these conditions by blocking M1 receptors at least partially located on histamine-releasing paracrine cells.

Topics: Acetylcholine; Alkaloids; Animals; Anti-Ulcer Agents; Electric Stimulation; Furans; Gastric Acid; Gastric Mucosa; Histamine; Mice; Muscarinic Antagonists; Naphthalenes; p-Methoxy-N-methylphenethylamine; Parasympatholytics; Piperidines; Pirenzepine; Stomach; Thiazepines; Vagus Nerve

Prejunctional inhibitory muscarinic receptors in guinea pig tracheal strips were investigated by electrical field stimulation. Pilocarpine and methacholine caused, in a similar way, a dose-dependent increase in baseline with a concomitant decrease in twitch response. We showed by using selective muscarinic antagonists, such as pirenzepine (M1-selective), methoctramine (M2-selective), AF-DX 116 (11-[[2-[diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro- 6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one, M2-selective), gallamine (M2-selective) and 4-DAMP (4-diphenylacetoxy-N- methylpiperidinemethiodide, M3-selective), that the prejunctional inhibitory muscarinic receptor is of the M2 subtype.

Topics: Animals; Diamines; Electric Stimulation; Gallamine Triethiodide; Guinea Pigs; In Vitro Techniques; Male; Parasympathetic Nervous System; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

We have recently described two types of muscarinic responses in native Xenopus oocytes of different donors (common and variant) that display qualitative and quantitative differences (Lupu-Meiri et al., 1990). Here we characterized the muscarinic receptors mediating these two types. The anti-muscarinic toxins from Dentroaspis significantly inhibited responses in oocytes of common donors, but had little effect on responses in oocytes of variant donors, possibly indicating expression of different receptor subtypes. Using specific muscarinic antagonists, we found that oocytes of common donors exhibit a pattern compatible with the M3 subtype of muscarinic receptors, while oocytes of variant donors appear to possess receptors of the M1 subtype. To more directly determine the subtypes of muscarinic receptors in oocytes of both populations of donors, we have microinjected antisense oligonucleotides into native oocytes. Antisense oligonucleotides to unique sequences in the N-terminal and the third cytoplasmic loop of M3 muscarinic receptors caused a significant inhibition of the response of common oocytes, but had virtually no effect on responses in oocytes of variant donors. Conversely, oligonucleotides complementary to the unique sequences of the m1 muscarinic receptors inhibited the response in variant oocytes, but not in oocytes of common donors. We conclude that native Xenopus oocytes of different donors phenotypically express either M3-like (majority) or M1-like (minority) muscarinic receptor subtypes. The differences in receptor subtype expression may explain the different characteristics of responses in the two populations.

Topics: Animals; Atropine; Elapid Venoms; Female; Oligonucleotide Probes; Oligonucleotides, Antisense; Oocytes; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Xenopus laevis

The present study investigated the effect of selective muscarinic antagonists on natriuresis, kaliuresis and antidiuresis induced by intracerebroventricular (i.c.v.) injection of carbachol in the rat. The muscarinic antagonists were given by i.c.v. injection 1 min before carbachol (1 microgram/rat). 4-Diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a rather selective M1 and M3 receptor antagonist, was the most potent inhibitor of carbachol-induced natriuresis, kaliuresis and antidiuresis, its ID50 being respectively 0.12, 0.04 and 0.56 nmol/rat. Pirenzepine, a selective M1 antagonist, potently inhibited the above mentioned carbachol effects, its ID50 being 1.85, 3.25 and 1.49 nmol/rat, respectively. On the other hand, the M2-selective antagonist methoctramine and the M3-selective antagonist p-fluoro-hexahydro-sila-difenidol were very weak inhibitors. Methoctramine at doses up to 60 nmol/rat produced non statistically significant inhibition of carbachol-induced natriuresis, kaliuresis and antidiuresis. Para-fluoro-hexahydro-sila-diphenidol showed an ID50 of 64.4 nmol/rat on carbachol-induced natriuresis, while at the maximum dose employed, 100 nmol/rat, the inhibition of carbachol-induced kaliuresis and antidiuresis was lower than 50%. The rank order of potency of the antagonists tested proved to be related to their pA2 values for muscarinic M1 receptors, suggesting that this receptor subtype mediates the central effects of cholinergic mechanisms on water and electrolyte excretion.

Topics: Animals; Carbachol; Diamines; Diuresis; Dose-Response Relationship, Drug; Injections, Intraventricular; Male; Muscarinic Antagonists; Natriuresis; Piperidines; Potassium; Rats; Rats, Inbred Strains; Receptors, Muscarinic

We have attempted to characterize a muscarinic receptor subtype involved in Cl- secretion in isolated epithelium of hen trachea, taking advantage of drugs developed in the last 15 yr. Hen trachea can be stimulated to secrete Cl- equal to approximately 80-90 microA/cm2 by application of acetylcholine (ACh) to the serosal side. The process has an apparent dissociation constant (Kd) for ACh of 740 nM. The Cl- secretion is completely inhibited by 20 microM bumetanide at the serosal side. Of five selective antagonists for muscarinic receptors, pirenzepine, hexahydrosiladifenidol, dicyclomine, 11-([2-[(diethylamino)-methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, and 4 diphenyl acetoxy-N-methylpiperidine methobromide, only the latter had a high affinity for the functional receptor with an apparent Kd of 3 nM. The receptor may be classified as an M4-muscarinic receptor subtype and probably belongs to a group of muscarinic receptors on exocrine glands and mucosal cells involved in ion transport. All the functional responses caused by muscarinic agonists and antagonists tested exhibited exponents (apparent Hill coefficients) in the range from 1.3 to 2.4, indicating a gain in the stimulus secretion coupling mechanism, an aspect of muscarinic receptor function that is not revealed in radioligand binding studies.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Atropine; Bethanechol; Bethanechol Compounds; Bumetanide; Calcium Channels; Chickens; Chlorides; Dicyclomine; Female; In Vitro Techniques; Ion Channels; Kinetics; Mucous Membrane; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

The muscarinic receptors responsible for two effects elicited by McN-A-343, i.e. the relaxation of the rat duodenum and the inhibition of the twitch contraction of rabbit vas deferens, were investigated by use of derivatives of 4-diphenyl acetoxy-N-methyl piperidine methobromide (4-DAMP). Both receptors had been previously identified as M1 on the basis of the high affinity shown towards pirenzepine. Schild analysis of antagonism revealed that the affinities of 4-DAMP and three of its analogues in the rat duodenum were significantly different from those estimated in rabbit vas deferens. These data indicate that distinct receptor subtypes mediate duodenal relaxation and vas deferens inhibition of twitch contraction and suggest that receptors classified as M1 by means of pirenzepine affinity constitute a heterogeneous population.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Duodenum; In Vitro Techniques; Isotonic Contraction; Male; Muscle Relaxation; Muscle, Smooth; Parasympathomimetics; Piperidines; Pirenzepine; Rabbits; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Vas Deferens

The guinea-pig oesophageal muscularis mucosae was used to determine the affinity for muscarinic receptors of two new tricyclic compounds, DF 545 and DF 594, which are structurally related to pirenzepine. Both acetylcholine and bethanechol induced a concentration-dependent contraction of the muscularis mucosae. This contraction was competitively antagonized by DF 545 and DF 594 over the dose range 10(-7)-10(-5) M, while at higher concentrations both antagonists caused a depression of the maximal response to the cholinomimetics. The potency of DF 545 and DF 594 appeared to be comparable to that of pirenzepine and approximately 50 times lower than that of atropine. By comparing the affinities of DF 545 and DF 594 with those of selective antagonists (methoctramine and 4-DAMP) which discriminate between M2/M3 muscarinic receptor subtypes, it emerged that pirenzepine as well as DF 545 and DF 594 might act on M3 receptors, which seem to be predominant in the guinea-pig oesophageal muscularis mucosae. McN-A-343 exhibited no agonist activity while it acted as a competitive antagonist against acetylcholine and bethanechol. None of the compounds exhibited calcium antagonist properties. DF 545 inhibited the contractile responses to histamine, but DF 594 and pirenzepine did not.

Topics: Acetylcholine; Animals; Atropine; Benzodiazepinones; Bethanechol; Bethanechol Compounds; Esophagus; Guinea Pigs; Histamine; Histamine Antagonists; Male; Muscle Contraction; Piperidines; Pirenzepine; Potassium; Pyrilamine; Receptors, Muscarinic

4-Diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (I) cyclizes at neutral pH to form an aziridinium salt. The formation and breakdown of the salt depend on the temperature (in the range 25 to 37 degrees C). In solution at 30 degrees C, peak levels, corresponding to 60-80% conversion, are reached after around 60 min and the half-life exceeds 100 min. In the presence of 0.9% NaCl conversion was reduced to 45-60%. I blocks muscarinic receptors in guinea-pig ileum and atria irreversibly and it is possible to produce dose-ratios on ileum with 10 nM I which are about 100 times those on atria. After about 30 min exposure to solutions of I (prepared 15-20 min previously so that formation of aziridinium ions is well-established) the graph of log (dose-ratio) against time is linear and similar plots were obtained with two different agonists, carbachol and ethoxyethyltrimethylammonium. With results for the ileum, extrapolation of the line suggests that it does not start from zero (dose-ratio = 1): this is because of an initial relatively rapid reversible block. This early phase is similar to that seen on ileum with 10 nM 4DAMP methobromide, which is a competitive antagonist, so is probably caused by competitive block by the aziridinium ion, which closely resembles 4DAMP metho-salts. The subsequent irreversible phase should be caused by alkylation of the receptors. I is easy to make and should be a valuable tool for the study of muscarinic receptors.

Topics: Animals; Aziridines; Cyclization; Diphenylacetic Acids; Guinea Pigs; Heart; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Parasympatholytics; Piperidines; Temperature

Using the cannula insertion method, we investigated the vascular response to acetylcholine (ACh) and other vasoactive substances. ACh consistently induced only vasoconstriction, whereas isoproterenol and norepinephrine usually induced dilatation. Vasoconstriction induced by phenylephrine was less potent than that induced by ACh. Clonidine and xylazine did not induce significant vascular responses. ACh-induced constrictions were readily inhibited by atropine and slightly potentiated by physostigmine. They were slightly but significantly inhibited by pirenzepine (a muscarinic M1-receptor antagonist), but not influenced by AF-DX 116 (a M2-receptor antagonist). 4-DAMP (4-diphenylacetoxy N-methylpiperidine; a M3-receptor antagonist), strongly inhibited the ACh-induced constrictions. They were not modified by bunazosin but slightly suppressed by diltiazem. Removal of the endothelium did not significantly modify the ACh-induced constrictions. From our results, we conclude that the simian facial vein has many constrictory muscarinic receptors, especially of the M3 subtype.

Topics: Acetylcholine; Adrenergic alpha-Antagonists; Animals; Atropine; Blood Pressure; Diltiazem; Face; Female; In Vitro Techniques; Macaca; Male; Muscle, Smooth, Vascular; Parasympatholytics; Physostigmine; Piperidines; Pirenzepine; Potassium Chloride; Quinazolines; Receptors, Muscarinic; Saponins; Vasoconstriction; Veins

Acetylcholine has a variety of actions on hippocampal neurons, possibly through the activation of different muscarinic receptor subtypes. We have used several competitive muscarinic antagonists and the hippocampal slice preparation to test the hypothesis that a particular muscarinic response can be ascribed to a specific subtype. Initially we performed experiments to determine the relative ability of 3 antagonists to inhibit the cholinergic slow EPSP. Dose-response curves were constructed, and the IC50s determined for 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), pirenzepine (PRZ) and 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11,-dihydro- 6H-pyrido[2,3-b] [1,4]benzodiazepin-6-one (AF-DX 116) were 72 nM, 385 nM, and 24 microM, respectively. While this pattern of antagonism argues against the contribution of the M2 receptor subtype, which would be expected to be relatively more sensitive to antagonism by AF-DX 116, this evidence does not adequately discriminate between other muscarinic receptor subtypes. We therefore performed studies designed to determine the antagonist binding affinities that can be diagnostic for determining different pharmacologically defined receptor subtypes. Using Schild plot analysis we examined the carbachol-induced block of the potassium (K)-dependent leak current, which is the muscarinic response thought to underlie the generation of the synaptically evoked slow excitatory postsynaptic potential (EPSP) in hippocampal neurons. Estimated affinity constants (KBs) for 4-DAMP, PRZ and AF-DX 116 were 7.9, 278 and 1364 nM, respectively. These values are in good quantitative agreement with data from binding studies and strongly suggest a physiological role for the pharmacologically defined 'M3' receptor in the block of the leak conductance and, hence, in the slow EPSP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbachol; Evoked Potentials; Hippocampus; In Vitro Techniques; Kinetics; Parasympatholytics; Piperidines; Pirenzepine; Pyramidal Tracts; Rats; Receptors, Muscarinic

Bilateral 10-nl injections of the muscarinic (M1) receptor antagonist pirenzepine (PZ) and the M3-antagonist 4-diphenylacetoxy-N-methylpiperidine (DAMP) just below the surface of the rostral ventrolateral medulla (VLM) in anesthetized, paralyzed, vagotomized, glomectomized, servo-ventilated cats decreased base-line phrenic activity and CO2 sensitivity but not blood pressure. Unilateral 10-nl injections of PZ, DAMP, or the M2-antagonist AFDX-116 resulted in the following. AFDX-116 greater than PZ greater than DAMP decreased blood pressure. DAMP greater than PZ greater than AFDX-116 decreased base-line phrenic activity and the CO2 response slope. Many injections had no effect, and some affected only one variable. The injection sites were within 700 microns of the VLM surface extending from the caudal aspect of the superior olive through a region ventromedial to the facial nucleus to the rostral aspect of the inferior olive. In a small number of experiments the response to carotid sinus nerve stimulation was affected by some injections of each antagonist. In the rostral VLM cardiovascular regulation involves M2-receptors, whereas respiratory regulation involves predominantly the M3-subtype. Neurons with these receptors appear to be closely intermingled.

Topics: Animals; Carbon Dioxide; Cardiovascular Physiological Phenomena; Cardiovascular System; Cats; Chemoreceptor Cells; Female; Male; Medulla Oblongata; Parasympatholytics; Phrenic Nerve; Piperidines; Pirenzepine; Receptors, Muscarinic; Respiratory Physiological Phenomena; Respiratory System

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA2-values characteristic of M3 (smooth muscle/glandular) muscarinic receptors. Since these pA2-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M3 subtype-selective muscarinic antagonists to be used as bronchodilators.

Topics: Bronchi; Humans; In Vitro Techniques; Ipratropium; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

In the guinea pig myometrium, muscarinic receptor activation leads to contraction and elicits two biochemical responses viz. an increased formation of inositol phosphates (via a guanine nucleotide regulatory protein, distinct from the stimulatory and inhibitory G proteins of the adenylate cyclase system and a decreased synthesis of cyclic AMP involving inhibitory G protein activation. We now describe two major differences in the effects of muscarinic agonists. First, the greater potency of carbachol in inhibiting cyclic AMP formation (EC50 = 8 nM) than in stimulating the accumulation of inositol phosphates and tension (EC50 = 15 and 2 microM, respectively). Second, carbachol, oxotremorine and pilocarpine were equally effective in eliciting cyclic AMP inhibition but the order of potency for inositol phosphate formation was carbachol greater than oxotremorine and pilocarpine was without effect. The partial agonists, pilocarpine and oxotremorine, inhibited carbachol-mediated inositol phosphate formation. Pirenzepine, selective for muscarinic M1 receptor subtype, displayed a low affinity for antagonizing cyclic AMP inhibition, inositol phosphate generation and tension due to carbachol (Ki = 286, 92 and 110 nM, respectively). AF-DX116 (11-[( 2-[(diethylamino)methyl]-1- piperidinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine- 6-one), selective for cardiac M2 receptors blocked cyclic AMP inhibition with high affinity (Ki = 1.14 nM) while it antagonized inositol phosphate formation with low affinity (Ki = 346 nM). Both high (Ki = 1 nM) and low (Ki = 100 nM) affinities were displayed by AF-DX116 in antagonizing contractions due to carbachol (24 and 76% inhibition, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclase Inhibitors; Animals; Carbachol; Cyclic AMP; Female; Guinea Pigs; In Vitro Techniques; Inositol Phosphates; Myometrium; Pilocarpine; Piperidines; Pirenzepine; Receptors, Muscarinic; Type C Phospholipases; Uterine Contraction

The receptor subtypes involved in muscarinic-induced phosphoinositide hydrolysis and adenylate cyclase inhibition in rat submandibular acinar cells were characterized by comparing the inhibitory potencies of four muscarinic antagonists on the two signal transduction responses. Carbachol-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was inhibited by all antagonists with a potency rank order of 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP) = atropine much greater than pirenzepine much greater than AF-DX 116 (P less than 0.01). The same rank order was observed in antagonist-reversal of the reduction of cAMP caused by carbachol in the model. These findings suggest that muscarinic effects are mediated by M3 receptors in both the phosphoinositide and adenylate cyclase pathways in the submandibular gland.

Topics: Animals; Atropine; Carbachol; Cyclic AMP; Parasympatholytics; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Signal Transduction; Submandibular Gland

1. The muscarinic receptor subtype involved in the methacholine-induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2-selective antagonist AF-DX 116 and the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide, in addition to the M1-selective antagonist pirenzepine, in a classical Schild analysis. 2. All the antagonists shifted the methacholine dose-response curve to the right in a parallel and concentration-dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF-DX 116 and 4-DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3. The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.

Topics: Animals; Cattle; In Vitro Techniques; Inositol Phosphates; Methacholine Compounds; Muscle Contraction; Muscle, Smooth; Phosphatidylinositols; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

In an experimental model of bleeding-induced hemorrhagic shock causing the death of all saline-treated rats within 30 min, the intravenous injection of ACTH-(1-24) at the dose of 160 micrograms/kg induced a sustained reversal of the shock condition, with almost complete recovery of blood pressure, pulse amplitude, respiratory rate, heart rate, and 100% survival, at least for the 2 h of observation. This effect of ACTH-(1-24) was prevented by the intracerebroventricular injection of 4-DAMP (a highly selective antagonist for M1 and M3 muscarinic receptors), but unaffected by the intracerebroventricular injection of pirenzepine (a highly selective antagonist for M1 muscarinic receptors). These data indicate that an essential step in the complex mechanism of the ACTH-induced shock reversal may be the activation of brain M3 muscarinic receptors.

Topics: Adrenocorticotropic Hormone; Animals; Blood Pressure; Brain Chemistry; Female; Heart Rate; Injections, Intraventricular; Male; Piperidines; Pirenzepine; Pulse; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Shock, Hemorrhagic

The muscarinic agonist oxotremorine-M produced a concentration-dependent increase in phosphoinositide hydrolysis in bovine pial arteries. The maximal effect was 5.9 +/- 0.89 fold over basal levels, and the EC50 for oxotremorine-M was 8.9 x 10(-6) M. The phosphoinositide response in arteries with the luminal endothelium removed was similar to the response in intact arteries. The specific muscarinic antagonists pirenzepine, 4-DAMP and methoctramine produced parallel shifts of the concentration-response curve to oxotremorine-M, with the following order of potency (pKB): 4-DAMP (8.59 +/- 0.10) greater than pirenzepine (8.12 +/- 0.11) greater than methoctramine (6.77 +/- 0.20). These results indicate that muscarinic stimulation activates phosphoinositide hydrolysis in cerebral arteries, and that the muscarinic receptors mediating this increase are similar to the M1 subtype.

Topics: Animals; Cattle; Cerebral Arteries; Diamines; Endothelium, Vascular; Hydrolysis; Inositol Phosphates; Kinetics; Oxotremorine; Phosphatidylinositols; Piperidines; Pirenzepine; Receptors, Muscarinic; Signal Transduction

Topics: Affinity Labels; Animals; Atropine; Binding, Competitive; Male; Muscarinic Antagonists; Myocardium; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Muscarinic receptors present in longitudinal ileum were characterized using the non-selective radioligand [3H]N-methylscopolamine [( 3H]NMS) and the M3 selective radioligand [3H]4-diphenylacetoxy-N-methylpiperidine methiodide [( 3H]4DAMP). In saturation studies, [3H]4DAMP, but not [3H]NMS, identified two populations of binding sites with 17% of the sites (155 fmol/mg protein) displaying high affinity (Kd = 0.39 nM) for [3H]4DAMP and the remaining sites displaying low affinity for the radioligand (Kd = 4.43 nM). In competition studies gallamine and methoctramine, but not AF-DX 116, identified two populations of [3H]NMS binding sites. Affinity estimates for gallamine and methoctramine indicated that 80% of the [3H]NMS binding sites were of the M2 subtype. The minor population of [3H]NMS binding sites could not be readily characterized, due partly to the low selectivity of the competing ligands and also to the relatively low density of the sites. In studies using the M3 muscarinic receptor selective radioligand [3H]4DAMP, the minor population of sites could be preferentially labeled by using a low concentration (0.4 nM) of [3H]4DAMP. Under these conditions, [3H]4DAMP labeled approximately equal levels of the two muscarinic receptor binding sites present in the ileum. Competition studies with AF-DX 116, gallamine and methoctramine indicated that the two [3H]4DAMP binding sites displayed the pharmacology expected of the M2 and M3 receptors, respectively. These results provide additional evidence that longitudinal ileal smooth muscle membranes contain both M2 and M3 muscarinic receptors and indicate that [3H]4DAMP is a useful ligand for identifying heterogeneity of muscarinic receptor subtypes.

Topics: Animals; Binding, Competitive; Diamines; Gallamine Triethiodide; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Piperidines; Pirenzepine; Receptors, Muscarinic

1. Negative chrono- and inotropic responses to both carbachol (CCh) into the sinus node artery and electrical stimulation of vagal nerve fibres (ES) were studied in the isolated, blood-perfused canine right atrium, using four muscarinic receptor antagonists, atropine, 4-DAMP, AF-DX 116 and pirenzepine. 2. ES and CCh evoked negative chrono- and inotropic responses in a frequency-dependent manner and in a dose-related manner, respectively. 3. Each antagonist inhibited these negative chrono- and inotropic responses in a dose-dependent manner. The ranking order of blocking potency (ID50) was atropine greater than or equal to 4-DAMP greater than AF-DX 116 greater than pirenzepine. 4. The ID50 of atropine, 4-DAMP or AF-DX 116 against sinus rate decreases induced by CCh or ES was not significantly different from that against the atrial tension decreases. In contrast, the ID50 of pirenzepine against sinus rate decreases evoked by CCh (17 nmol) or ES (20 nmol) was significantly smaller than that for the atrial tension decrease (CCh, 200 and ES, 53 nmol, respectively). 5. These results suggest that, in the isolated dog atrium, M2-receptor-mediated-responses are predominant. However, M1-receptor activation may also be involved in sinus rate response.

Topics: Animals; Atropine; Carbachol; Cardiotonic Agents; Dogs; Electric Stimulation; Female; Heart Atria; Male; Myocardial Contraction; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Vagus Nerve

1. In the portal vein of permanently cannulated, freely moving, unanaesthetized rats, methacholine (MCh) is able to inhibit the electrically-evoked endogenous noradrenaline (NA) overflow. This inhibition is mediated by presynaptic inhibitory muscarinic heteroreceptors. 2. By use of pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and AF-DX 116 as M1-, M3-, and M2-selective antagonists respectively, the MCh (0.1 microM)-induced inhibition of the electrically-evoked NA overflow could be reversed to the control stimulation value dose-dependently. 3. The potency order of the antagonists was: 4-DAMP greater than AF-DX 116 greater than pirenzepine, pIC50 values being 8.50, 7.96 and 7.01, respectively. 4. From these results it was concluded that the inhibitory presynaptic heteroreceptors in the portal vein of conscious unrestrained rats are of the cardiac M2-subtype.

Topics: Animals; Catecholamines; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Electrochemistry; Male; Muscle, Smooth, Vascular; Norepinephrine; Piperidines; Pirenzepine; Portal Vein; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Synapses

1. Lengthening the chain in diphenylacetylcholine decreases affinity for muscarinic cholinoceptors in guinea-pig ileum. Diphenylacetoxypropyldimethylamine and its quaternary trimethylammonium salt are roughly equiactive: the dimethylamine and the piperidine have some selectivity for ileum compared with atria, but are not as active nor as selective as 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide (MeBr). With the weaker diphenylacetoxybutyl compounds the base is more active than the quaternary salt. 2. The diphenylacetoxybutyl-, cis-butenyl and trans-butenyl compounds have similar affinities. The quaternary salts are less active than the tertiary bases, but they are less selective than the butynyl analogues studied in earlier work. 3. 1,1-Diphenyl-1-hydroxy-2,4-hexadiynyl dimethylamine and its trimethylammonium salt are inactive in concentrations below 100 microM, as are the (+)-camphor-sulphonyl ester of 4-hydroxy-N-methyl piperidine and its methiodide. The (+/-)-phenylcyclopentylacetyl ester of 4-hydroxy-N-methylpiperidine methobromide is more active than its cyclohexyl analogue and than 4-DAMP MeBr but it is less selective than 4-DAMP MeBr. 4. The high selectivity of p-fluoro-hexahydrosila-diphenidol is confirmed but this compound has relatively low affinity (for ileum log K = 7.8). 5. The results indicate steric constraints to binding at muscarinic receptors which could be used to check molecular modelling of the receptor based on its known amino acid sequence. The group binding the charged nitrogen is probably at the mouth of a cavity which can accommodate two large rings (as in 4-DAMP MeBr) but with a depth less than about 7 A so that the rod-like hexadiynes cannot fit. Differences between types of receptor may only involve small changes in geometry secondary to differences in amino acids not directly involved in binding and the production of selectivity depends upon finding substituents which interfere with binding more at one type of receptor than at another.

Topics: Animals; Chemical Phenomena; Chemistry; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Myocardial Contraction; Parasympathomimetics; Piperidines; Receptors, Muscarinic; Structure-Activity Relationship

Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using [3H](-)quinuclidinyl benzilate [( 3H]QNB) and selective muscarinic antagonists. [3H]QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with [3H]pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies.

Topics: Animals; Autoradiography; Binding Sites; Diamines; Guinea Pigs; Humans; Lung; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Radioligand Assay; Receptors, Muscarinic; Tritium

1. The effects of muscarine upon intracardiac neurones cultured from ganglia within the atria and interatrial septum of the newborn guinea-pig heart were studied using intracellular recording techniques. 2. Muscarine applied to the neuronal soma typically produced a biphasic change in membrane potential which consisted of a small hyperpolarization followed by a depolarization. In addition, muscarine (0.01-10 microM) inhibited the calcium-dependent, after-hyperpolarization (AHP) and greatly increased the number of action potentials that could be evoked by a given depolarizing current. 3. The hyperpolarization was associated with a decrease in input resistance and it reversed to become a depolarization at a potential of -86.5 mV. This response was antagonized by 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP; 100 nM) and AF-DX 116 (500 nM), but was unaffected by pirenzepine (0.1-5 microM). 4. Two types of slow depolarization were observed in the presence of muscarine. The most common was associated with an increase in input resistance in the potential range -70 to -40 mV. Pirenzepine (100 nM) selectively antagonized this response, 4-DAMP (100 nM) similarly antagonized the response, but was non-selective. AF-DX 116 (0.5-5 microM) showed no antagonist effect. The less common depolarization (5% of cells) had a long latency and was associated with a decrease in input resistance. 5. Muscarine reduced the duration of the action potential and inhibited the AHP. Cadmium chloride (100 microM) mimicked these actions of muscarine. Application of muscarine immediately following a train of action potentials did not inhibit the AHP, suggesting that muscarine did not directly inhibit the calcium-activated potassium current (IK(Ca)). Muscarine-induced depression of the slow AHP was antagonized by 4-DAMP (100 nM) but was not antagonized by either pirenzepine (0.1-0.5 microM) or AF-DX 116 (0.5-5 microM). 6. It is concluded that the muscarine-induced depolarization of guinea-pig intracardiac neurones results from reduction of a potassium conductance similar to the M-conductance, through activation of M1 muscarinic receptors. The hyperpolarization results from an increase in potassium conductance, through activation of M2 muscarinic receptors.

Topics: Action Potentials; Animals; Cells, Cultured; Ganglia; Guinea Pigs; Heart; Membrane Potentials; Muscarine; Neural Inhibition; Neurons; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.

Topics: Animals; Atropine; Binding, Competitive; Cell Survival; Cells, Cultured; Cerebellum; Diamines; Granulocytes; N-Methylscopolamine; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine Derivatives; Tritium

Prejunctional and postjunctional muscarinic receptor subtypes were characterized in canine trachealis muscle strips. In vitro contractile responses of muscle strips to acetylcholine or electric field stimulation were determined in the absence and the presence of gallamine, pirenzepine, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP). Gallamine had no effect on the contractile response to acetylcholine but enhanced the contractile response to electric field stimulation. Pirenzepine and 4-DAMP reduced the contractile response to acetylcholine and electric field stimulation. The pA2 value for pirenzepine vs. acetylcholine [7.18 +/- 0.59 (SD)] was consistent with the affinity of pirenzepine for M2 or M3-receptors; whereas the pA2 value for 4-DAMP vs. acetylcholine (8.92 +/- 0.42) and the extremely low affinity of gallamine indicated postjunctional muscarinic receptors of the M3 subtype. The enhancement of the contractile response to electric field stimulation by gallamine suggested the presence of M2-prejunctional receptors.

Topics: Acetylcholine; Animals; Dogs; Electric Stimulation; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Receptors, Muscarinic; Regression Analysis; Trachea

Selective muscarinic antagonists were used in an attempt to characterize the muscarinic autoreceptor modulating the release of acetylcholine in the striatum of the rat. In vivo microdialysis was applied to infuse atropine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), pirenzepine or AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro[2,3-b][1,4]benzodiazepine-6-one), leading to a dose-dependent increase in the overflow of acetylcholine, the order of potency being: atropine greater than 4-DAMP greater than pirenzepine greater than AF-DX 116. We conclude from these data that the muscarinic receptor modulating release in the striatum is of the M3 type.

Topics: Acetylcholine; Animals; Atropine; Chromatography, High Pressure Liquid; Corpus Striatum; Dialysis; Dose-Response Relationship, Drug; Male; Neostigmine; Oxotremorine; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Time Factors

The possibility that differences in beta-adrenergic sensitivity among canine trachealis muscles contracted with different contractile agonists are related to differences in the receptor-occupancy characteristics of the contractile agonists was investigated. Relaxation to isoproterenol was compared in muscles contracted with the muscarinic agonists McN-A-343 and acetylcholine (ACh). The apparent dissociation constant (pKB) values for the M1-antagonist, pirenzepine, against ACh (6.96 +/- 0.18) and McN-A-343 (6.84 +/- 0.08) were similar. The pKB values for the M3-antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) against ACh (8.76 +/- 0.13) and McN-A-343 (8.71 +/- 0.10) were also similar, suggesting that these agonists were activating the same subtype of muscarinic receptor, probably M3. However, the contractile response to ACh was associated with a greater receptor reserve than that for McN-A-343. Isoproterenol relaxed muscles contracted with McN-A-343 much more effectively than those contracted with an equieffective concentration of ACh. The results suggest that the relative resistance of ACh-induced contractions to relaxation by isoproterenol may not be an inherent quality of muscarinic receptor stimulation. The large receptor reserve available to ACh may act to buffer the contractile response from the inhibitory effects of beta-adrenergic stimulation. Alternatively, ACh may be able to initiate subcellular mechanisms that are unavailable to agonists of lower efficacy.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Dogs; In Vitro Techniques; Isoproterenol; Muscle Contraction; Muscle, Smooth; Parasympathomimetics; Piperidines; Pirenzepine; Receptors, Adrenergic, beta; Receptors, Muscarinic; Trachea

Pharmacological profiles of the striatal and brainstem M2 receptors were developed with a group of selective muscarinic antagonists. The striatal M2 muscarinic receptor was identified by its inhibition of [3H]cyclic AMP levels, whereas the brainstem M2 receptor was characterized using competition with [3H]quinuclidinyl benzilate binding. The potency of pirenzepine does not differentiate clearly between the striatal M2 receptor (Ki approximately 300 nM) and the brainstem M2 receptor (Ki = 219 nM) or peripheral M2 receptors. In the present study, we used 4-diphenylacetoxy-N-methylpiperidine methbromide, hexahydrosiladifenidol, AF-DX 116 and methoctramine to characterize the striatal and brainstem M2 receptors in more pharmacological detail. For comparison, the potencies of these antagonists were also measured at cortical M2 receptors (using competition with [3H]pirenzepine binding). The potencies of 4-diphenylacetoxy-N-methylpiperidine methbromide (KB = 0.19 nM) and hexahydrosiladifenidol (KB = 14 nM) in blocking the striatal M2 receptor suggested similarity to those M2 receptors localized in certain smooth muscles or in glands. However, AF-DX 116 (KB = 155 nM) and methoctramine (KB = 47 nM) were considerably more potent in blocking the striatal M2 receptor than as reported in functional studies in smooth muscle or glands. Thus, the profile of the striatal M2 receptor obtained with these antagonists did not match in all respects with either glandular (probable M4 gene product) or cardiac (probable M2 gene product) muscarinic receptors. In contrast, our data with the brainstem M2 receptor was highly correlated (r = 0.93) with literature data regarding the cardiac muscarinic system.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain Stem; Corpus Striatum; Cyclic AMP; Diamines; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic

The cardioselective muscarinic antagonist, AF-DX 116 [11[2-[(diethyl-amino)-methyl]-O-1-piperidinyl]-5,11-dihydro-6H-pyrido- [2,3-b][1,4]-benzodiazepine-6-one), was weak at blocking the M2 muscarinic receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) formation in mouse neuroblastoma cells (clone N1E-115). In contrast, the glandular-selective antagonists, hexahydro-sila-difenidol (HHSiD) and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), were quite potent at inhibiting this response, being 14- and 318-fold more potent than AF-DX 116 in this regard, respectively. According to the rank order of potency of these two classes of antagonists, these data provide the first pharmacological evidence that inhibition of cAMP formation in a neuronal tissue is mediated by a non-cardiac M2 muscarinic receptor subtype.

Topics: Animals; Binding, Competitive; Cyclic AMP; Mice; Muscarinic Antagonists; N-Methylscopolamine; Neuroblastoma; Piperidines; Receptors, Muscarinic; Scopolamine Derivatives; Tumor Cells, Cultured

Since there have been only a few studies on muscarinic receptor subtypes in airway smooth muscle, the effect was investigated of pirenzepine on airways of patients with chronic obstructive pulmonary disease (COPD) and the functional responses compared from these patients with those from healthy subjects. Our data demonstrated that the therapy with pirenzepine significantly improved ventilatory function in patients with COPD. The data also suggested that this drug exerts its action on small airways, but not larger airways in normal subjects. It is possible that in healthy human beings pirenzepine produces mild bronchodilation by means of a vagal efferent blockade, while in patients with COPD, it may be effective because it not only decreases the activity of the vagal efferent pathway, but also decreases the sensitivity of vagal sensory endings and causes a vagal afferent blockade.

Topics: Atropine; Forced Expiratory Volume; Humans; Lung Diseases, Obstructive; Muscle, Smooth; Piperidines; Pirenzepine; Receptors, Muscarinic; Respiratory Muscles

Characterization of muscarinic receptor subtypes on the dog pancreas in situ was examined by using specific muscarinic receptor antagonists to study pancreatic exocrine secretion. Bethanechol caused an increase in pancreatic exocrine secretion, with a high concentration of protein and little effect on bicarbonate concentration. Thus bethanechol may mainly affect the muscarinic receptors of the acinar cells. Bethanechol-stimulated secretion was inhibited by pirenzepine (a specific M1 muscarinic antagonist), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP, a specific M3 muscarinic antagonist), and atropine (a mixed muscarinic receptor antagonist). However, [11-[[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11- dihydro-6H-pyrido[2,3-b][1,4]benzo-diazepine-6-one (AF-DX 116, a specific cardioselective M2 antagonist) did not have any effects on bethanechol-stimulated secretion. Increased protein secretion in pancreatic juice stimulated by bethanechol was significantly inhibited by 4-DAMP and atropine and was suppressed by pirenzepine, but was not modified by AF-DX 116. Bicarbonate concentration was not modified by these antagonists. 4-DAMP, atropine, and pirenzepine caused a progressive parallel rightward shift in the dose-response curve of pancreatic secretion for bethanechol. Schild analysis of the data indicated a pA2 value of 8.7 for 4-DAMP, 7.9 for atropine, and 6.2 for pirenzepine, respectively. Thus 4-DAMP has 7.5 times and 440 times greater potency than atropine and pirenzepine to inhibit bethanechol-stimulated secretion. The slope of the Schild regression line was not different from 1. These results suggest that inhibitions of bethanechol-stimulated pancreatic secretion are competitive for 4-DAMP, atropine, and pirenzepine and that bethanechol-stimulated pancreatic secretion is mediated by M3 muscarinic receptors in dogs.

Topics: Animals; Atropine; Bethanechol Compounds; Dogs; Female; Kinetics; Male; Pancreas; Pancreatic Juice; Parasympathomimetics; Piperidines; Pirenzepine; Receptors, Muscarinic; Reference Values

1. Muscarinic agonist acetylcholine (ACh) (non-selective) and arecaidine propargyl ester (APE) (M2 selective agonist) produced a increase in the output of 6-keto PGF1 alpha and a decrease in the heart rate and myocardial developed tension in a dose-dependent manner. 2. Lower doses of ACh (1.0-5.0 nmol) caused coronary vasodilation, whereas higher doses of ACh (10.0 nmol) and lower as well as higher doses of APE produced a biphasic effect--an initial vasodilation followed by vasoconstriction. 3. The increase in 6-keto PGF1 alpha output elicited by 3 nmol of ACh or APE was inhibited by 10 nM of classical muscarinic receptor antagonist atropine or by the selective M2 beta muscarinic receptor antagonist 4-(diphenylacetoxy-N-methyl piperidine) methiodide (4-DAMP). 4. The decrease in heart rate and myocardial developed tension produced by ACh and APE was attenuated by atropine and 4-DAMP. The coronary vasodilator effect of ACh and APE and the vasoconstrictor effect of APE were also attenuated by both of these muscarinic antagonists.

Topics: Animals; Coronary Circulation; Drug Interactions; Heart; Heart Rate; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Parasympatholytics; Parasympathomimetics; Piperidines; Prostaglandins; Rabbits; Receptors, Muscarinic

The present study examines the muscarinic receptor binding characteristics of parent human neuroblastoma (SK-N-SH) and its neuroblast (SH-SY5Y) and epithelial-like (SH-EP1) clones using [3H]methylscopolamine [( 3H]NMS). Specific [3H]NMS binding to intact SK-N-SH and SH-SY5Y cells was saturable with a Kd of 0.2 nM and Bmax of 100-150 fmol/mg protein. Specific [3H]NMS binding to whole cell preparations of SH-EP 1 could not be detected. Pharmacological analysis of the binding site both in whole cells and membranes of SK-N-SH are indicative of an homogeneous receptor population possessing low affinity for the M1-selective antagonist pirenzepine. The muscarinic receptors expressed by the neuroblast clone, SH-SY5Y were further characterized and shown to have the properties of an homogeneous M3 subtype with low affinity for the M1-selective antagonist pirenzepine and the M2-cardioselective AFDX-116 but high affinity for 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP). In conclusion the SH-SY5Y neuroblastoma should provide an important human neuronal cell model with which to define the regulation of post-receptor events driven by a single receptor population.

Topics: Arecoline; Atropine; Carbachol; Cell Membrane; Humans; Neuroblastoma; Piperidines; Pirenzepine; Receptors, Muscarinic; Tumor Cells, Cultured

1. Crystals of 4-diphenylcarbamyl-N-methylpiperidine methobromide contain 2 conformers, one with the ester group equatorial and the other with the ester group axial to the piperidine ring. 2. In both conformers and diphenylcarbamyl nitrogen is trigonally substituted and flat compared with the tetrahedral substitution of the corresponding carbon atom in 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide. 3. The low activity and selectivity of the carbamate at muscarinic receptors is comparable with that of compounds containing only one phenyl ring, e.g. phenylacetyl-N-methyl piperidine methiodide or phenylacetylcholine. It is suggested that the high affinity of 4-DAMP metho-salts and their selectivity depend upon interactions with groups arranged in three dimensions and that the trigonal arrangement about the nitrogen atom in the carbamate makes it impossible for both benzene rings to interact at once.

Topics: Crystallization; Molecular Conformation; Piperidines; X-Ray Diffraction

1. Under voltage-clamp dissociated adult and foetal rat superior cervical ganglion (s.c.g.) cells exhibited a non-inactivating voltage- and time-dependent component of K+ current termed the M-current (IM). IM was detected and measured from the current decay during hyperpolarizing voltage steps applied from potentials where IM was pre-activated. 2. Neither the resting membrane current nor the amplitude of these current decay relaxations were reduced by omitting Ca from the bathing fluid, showing that the M-current was not a 'Ca-activated' K-current dependent on a primary Ca-influx. Concentrations of (+)-tubocurarine sufficient to block the slow Ca-activated K-current IAHP did not inhibit IM or antagonize the effect of muscarinic agonists on IM, showing that IM was not contaminated by IAHP. Tetraethylammonium (1 mM), which blocks the fast Ca-activated K-current IC, produced a small inhibition of IM. This was not due to contamination of IM by IC since muscarinic agonists did not consistently block IC. 3. The muscarinic agonists muscarine, oxotremorine, McN-A-343 and methacholine reversibly suppressed IM, resulting in an inward (depolarizing) current. The rank order of potency was: oxotremorine greater than or equal to muscarine greater than McN-A-343 greater than methacholine. 4. The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5. IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 +/- 0.13 (n = 3) and 6.02 +/- 0.13 (n = 4) respectively. 6. The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 microM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nM. 7. Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 +/- 0.03 (n = 8). 8. These results suggest that the receptors mediating suppression of the M-current accord with those designated pharmacologically as M1 and that these receptors reach maturity at a very early stage in the development of the rat s.c.g.

Topics: Animals; Calcium; Gallamine Triethiodide; Ganglia, Spinal; Ganglia, Sympathetic; Ileum; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; N-Methylscopolamine; Piperidines; Pirenzepine; Potassium Channels; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives

1. A range of muscarinic receptor antagonists were examined for affinity at the M1 muscarinic binding site, present in rat cerebrocortical membranes and the M2 muscarinic binding sites of rat cardiac and submaxillary gland membranes. 2. The results obtained were consistent with the presence of three classes of muscarinic binding site. 3. Both the M1 binding site, labelled by [3H]-pirenzepine ([3H]-Pir) in rat cerebrocortical membranes, and the M2 gland binding site, labelled by [3H]-N-methyl scopolamine ([3H]-NMS) in rat submaxillary gland membranes, displayed higher affinity for pirenzepine, dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and cyclohexylphenyl (2-piperidinoethyl) silanol (CPPS) than did the M2 binding sites of cardiac membranes labelled by [3H]-NMS. 4. The M2 cardiac sites displayed higher affinity for methoctramine, himbacine and AF-DX 116 than did either the M1 binding site of cerebrocortical membranes or the M2 gland binding site present in rat submaxillary gland membranes. 5. The M1 and M2 gland binding sites could only be distinguished by considering the absolute affinity of compounds for these two sites. Thus, all compounds, with the exception of 4-DAMP, displayed between a 2 and 8 fold higher affinity for the M1 than for the M2 gland binding site. There were no antagonists with higher M2 gland than M1 affinity.

Topics: Animals; Buffers; Cerebral Cortex; In Vitro Techniques; Male; Myocardium; N-Methylscopolamine; Parasympatholytics; Piperidines; Pirenzepine; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Submandibular Gland

We used binding of [N-methyl-3H]scopolamine ( [3H]-NMS) to tissue homogenates and isometric contraction of muscle strips to characterize perinatal changes in the muscarinic receptor on rabbit gastric smooth muscle. In homogenates from fetal (28 days of gestation), 1-, 3-, and 7-day, 4- and 11-wk-old rabbits, specific binding was saturable and temperature dependent, achieved equilibrium by 10 min at 30 degrees C, and was linearly related to tissue concentration. Specific binding was 80 +/- 2% of total binding at 0.2 nM [3H]NMS. The number of binding sites was 120,000 receptors/cell, maximal during the first week of life compared with the fetus or older animals. Affinity of [3H]NMS was highest in the first week of life (Kd = 345 +/- 24 pM, 1 day old). Age did not affect Hill coefficients or Ki values; secoverine and 4-diphenylacetoxy-N-methylpiperidine methiodide were 50-fold more potent than pirenzepine. In muscle strips, bethanechol stimulated dose-dependent atropine-inhibitable isometric contraction. The doses required for half-maximal contraction were similar in both age groups (5-6 microM), but maximal contraction was fivefold greater in weanlings compared with neonates. Increasing extracellular potassium concentration resulted in similar differences, suggesting that the differences were not receptor related. These results suggest that well-differentiated M2-muscarinic receptors are functional on rabbit gastric smooth muscle during the perinatal period.

Topics: Animals; Animals, Newborn; Atropine; Bethanechol; Bethanechol Compounds; Dose-Response Relationship, Drug; Kinetics; Muscle Contraction; Muscle Development; Muscle, Smooth; N-Methylscopolamine; Phenethylamines; Piperidines; Rabbits; Receptors, Muscarinic; Scopolamine Derivatives; Stomach

Displacement of [3H]oxotremorine-M [( 3H]oxo-M) binding by muscarinic antagonists that are functionally non-selective (atropine), ileoselective (4-DAMP) or cardioselective (gallamine, pancuronium, vecuronium, himbacine) was investigated in guinea-pig atrial and ileal longitudinal muscle membranes. [3H]Oxo-M bound to a single population of high affinity sites in atrial (KD = 11.40 nM) and ileal (KD = 6.15 nM) membranes. Atropine displaced [3H]oxo-M binding sites in a competitive manner, showing similar affinities in the two tissues. 4-DAMP showed two binding sites in ileum but not in atria. The dissociation constant at the high affinity site in ileum was ca 5-fold lower than the value observed in atria, indicating ileoselectivity. Vecuronium also displaced [3H]oxo-M binding in a competitive manner and exhibited similar affinities in both tissues. Gallamine, pancuronium and himbacine displayed two binding sites in each of the two tissues with the majority of sites (ca. 60-80%) showing high affinity. Overall the cardioselective antagonists do not exhibit any consistent correlation between the affinities found in functional experiments and those determined in binding experiments.

Topics: Animals; Atropine; Binding, Competitive; Female; Gallamine Triethiodide; Guinea Pigs; Heart Atria; Ileum; In Vitro Techniques; Male; Oxotremorine; Parasympatholytics; Piperidines

1. The replacement of 4-hydroxy-N-methyl piperidine (HO NMe) in 4-diphenylacetoxy-N-methyl piperidine (4-DAMP) metho-bromide by 4-hydroxy-but-2-ynylamines (HOCH2C=CCH2NR2) reduces the affinity for muscarine-sensitive acetylcholine receptors in guinea-pig ileum and atria. It does not abolish selectivity. The tertiary amines are more active and more selective than the corresponding quaternary salts. 2. Analogous derivatives of 4-hydroxy-but-2-ynylamines which lack the ester group (i.e. substituted 4-hydroxymethyl-propynyl amines) are less active and less selective. The quaternary compounds are more active than the tertiary bases. 3. The diphenylcarbamyl ester of 4-hydroxy-N-methylpiperidine methobromide has less than one-thousandth of the activity of the diphenylacetyl ester (4-DAMP methobromide) and is not selective. 4. Although 4-diphenylacetoxy-butynyl dimethylamine is only about one-hundredth as active as 4-DAMP methobromide it appears to have comparable selectivity. It is an interesting compound because it is a tertiary amine and should cross membranes.

Topics: Animals; Guinea Pigs; Heart; Ileum; In Vitro Techniques; Piperidines; Receptors, Muscarinic; Structure-Activity Relationship

The muscarinic acetylcholine receptor of the Xenopus oocyte was characterized electrophysiologically. Iontophoretic responses to acetylcholine were inhibited by the muscarinic antagonists 4-DAMP, pirenzepine and AF-DX 116 at respective IC50 values of 7 nM, 15 microM and 2 microM. This antagonist sensitivity order indicates that the receptor is of the M3 muscarinic subtype.

Topics: Acetylcholine; Animals; Female; In Vitro Techniques; Membrane Potentials; Oocytes; Piperidines; Pirenzepine; Receptors, Cholinergic; Xenopus laevis

Injection of neostigmine into the lateral cerebral ventricle of urethane-anesthetized rats increases arterial blood pressure. Prior injection of atropine or the muscarinic M2 antagonist 4-DAMP into the posterior hypothalamic nuclei inhibited the pressor response to neostigmine by up to approximately 56%. The same maximum degree of inhibition was elicited by bilateral electrical lesions of the posterior hypothalamic nuclei. The response was not modified by intrahypothalamic injection of pirenzepine or intraventricular injection of hexamethonium, but was prevented by intraventricular injection of 4-DAMP. The results indicate that about half of the pressor response to intraventricular injection of neostigmine was mediated through M2 muscarinic receptors in the posterior hypothalamic nuclei, and the remainder through M2 muscarinic receptors in other regions of the brain.

Topics: Animals; Atropine; Blood Pressure; Heart Rate; Hypothalamus; Injections, Intraventricular; Male; Neostigmine; Parasympathomimetics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Binding studies in several species have demonstrated a high proportion of M1 muscarinic receptors in the lung but their localization is uncertain. Using [3H]quinuclidinyl benzylate we have confirmed that binding sites with high affinity for pirenzepine account for 50% of muscarinic receptors in the rat lung. Our functional studies using the muscarinic antagonists 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), methoctramine and pirenzepine have demonstrated that the muscarinic receptor on the rat pulmonary artery endothelium which mediates vasodilation is of the M3 subtype and cannot account for the high proportion of M1 receptors identified in lung homogenates.

Topics: Animals; Binding Sites; Cell Membrane; Diamines; Endothelium, Vascular; Lung; Male; Piperidines; Pirenzepine; Pulmonary Artery; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Vasodilation

Injections of the M2 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1.5-40 micrograms) into the cerebral ventricles of urethane-anesthetized rats caused a dose-related inhibition of the pressor response to intravenously injected physostigmine. A similar reduction was obtained with 1/80th the dose of methylatropine, but not with the selective M1 antagonist pirenzepine. Intraventricular injection of 4-DAMP (6.25-25 micrograms) caused a dose-related reduction in blood pressure in unanesthetized spontaneously hypertensive rats (SHR), but not in normotensive controls. Systolic pressure fell 42 +/- 6 mm Hg at the 25-micrograms dose. Pirenzepine did not lower blood pressure in SHR and inhibited the antihypertensive effect of 4-DAMP.

Topics: Animals; Atropine Derivatives; Blood Pressure; Brain; Male; Parasympatholytics; Parasympathomimetics; Physostigmine; Piperidines; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY

In order to characterize the muscarinic binding site on coronary smooth muscle, we investigated the binding properties of (3H)quinuclidinyl benzilate (QNB) in membrane preparations of pig coronary arteries and atria. Scatchard analysis and Hill plot showed that (3H)QNB binds to a single population of sites in both tissues. The binding profiles of the muscarine receptor antagonists atropine, 11-[2-[dimethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrid o(2,3- b) (1,4)benzodiazepine-6-one (AF-DX 116), pirenzepine, and 4-diphenylacetoxy-N-methylpiperidine methiobromide (4-DAMP) in both tissues were compared with binding data from other tissues, representative for different muscarinic binding site subtypes. It is concluded that the pig coronary smooth muscle muscarinic binding site is different from M1 and M2 binding sites investigated so far.

Topics: Animals; Atropine; Carbachol; Coronary Vessels; Heart Atria; In Vitro Techniques; Muscle, Smooth, Vascular; Piperazines; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Swine; Tritium

The binding of hexahydrosiladifenidol, procyclidine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) and AF-DX 116 to muscarinic receptors in the heart, ileum, urinary bladder, parotid gland and cerebral cortex from guinea pig was studied in competition experiments with (-)-[3H]QNB. The affinity of AF-DX 116 was higher in the heart than in the cortex and it was extremely low in the parotid gland. The affinities of hexahydrosiladefinidol, procyclidine and 4-DAMP were higher in the cortex and parotid gland than in the heart, bladder and ileum. Hexahydrosiladifenidol and 4-DAMP recognized two classes of muscarinic binding sites in the cortex. However, in contrast to functional data, binding results showed that 4-DAMP hexahydrosiladifenidol and procyclidine did not distinguish between the sites in the smooth muscles and those in the heart. Nevertheless, the present data support the view that the putative M2-receptors are heterogeneous, since the four drugs examined were found to distinguish between the muscarinic binding sites in the parotid gland and those in smooth muscles and heart.

Topics: Animals; Cerebral Cortex; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Myocardial Contraction; Parasympatholytics; Parotid Gland; Piperidines; Pirenzepine; Procyclidine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Urinary Bladder

The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (3H)N-methylscopolamine [3)NMS) (KD values of 140 and 280 nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-([(2-[diethylamino)-methyl)-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on) (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptors also showed different (3H)NMS association and dissociation rates. These results support the concept of M2 receptor heterogeneity and confirm that M2 receptor subtypes have different binding kinetic properties.

Topics: Animals; Binding, Competitive; GTP-Binding Proteins; Kinetics; Male; Myocardium; N-Methylscopolamine; Pancreas; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Type C Phospholipases

In the present study we investigated the nature of the muscarinic receptors present in the hippocampus, sympathetic ganglia, atria and salivary glands of the rat. The heterogeneity of the muscarinic receptors was examined both in vivo and in radioligand binding experiments. To study whether the receptors present in the investigated tissues are indeed distinct subtypes we determined the potencies of antagonists in both systems. It is proposed that there are three different binding sites present in hippocampal, atrial and submandibular membranes and we suggest to classify them as M1, M2 and M3, respectively. Both in vivo and in vitro pirenzepine appears to possess high affinity for M1 receptors, whereas 4-diphenylacetoxy-N-methylpiperidine methobromide and dicyclomine show high affinity for both M1 and M3 receptors. AF-DX 116 (11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one) displayed high affinity for M2 receptors.

Topics: Animals; Dicyclomine; Ganglia, Sympathetic; Hippocampus; Male; Myocardium; Parasympathomimetics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Submandibular Gland

Muscle cells were isolated from the longitudinal muscle layer of guinea pig and human jejunum and used to identify the muscarinic receptor subtype (M1 or M2) that mediates contraction. Single muscle cells were anchored to the ceiling of a minichamber and their contraction was measured in response to acetylcholine, alone and in combination with three muscarinic antagonists: atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and pirenzepine. Estimates of the inhibitory dissociation constants (Ki) were closely similar in human and guinea pig muscle cells (atropine 2.5-5 X 10(-11) M, 4-DAMP 1.9-2.9 X 10(10) M, and pirenzepine 8.2-9.5 X 10(-8) M). Thus, pirenzepine, a preferential M1 antagonist, was 1900-3280 times less potent than atropine and 279-500 times less potent than 4-DAMP. Comparative measurements on longitudinal muscle strips from guinea pig jejunum confirmed the greater potency of atropine and 4-DAMP relative to pirenzepine. Inactivation of muscarinic receptors on single muscle cells with dibenamine showed that only a small fraction of receptors was responsible for the response to acetylcholine. It was concluded that intestinal muscle cells contain a large reservoir of muscarinic M2 receptors that exhibit considerable spareness and heterogeneity.

Topics: Acetylcholine; Animals; Atropine; Guinea Pigs; Humans; Jejunum; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine; Receptors, Muscarinic

Guinea-pig olfactory cortical neurones in vitro were voltage clamped by means of a single intracellular microelectrode technique. Hyperpolarizing voltage commands from holding potentials between -40 to -50 mV produced slow inward current relaxations reflecting deactivation of the M-current (IM). IM was reversibly suppressed by 30 microM muscarine or carbachol; this suppression was insensitive to pirenzepine (up to 300 nM) but was inhibited by gallamine (10-20 microM) or 4-diphenyl-acetoxy-N-methylpiperidine (100, 500 nM), suggesting the involvement of the M2-type muscarinic receptor.

Topics: Animals; Cerebral Cortex; Gallamine Triethiodide; Guinea Pigs; In Vitro Techniques; Membrane Potentials; Muscarine; Piperidines; Receptors, Muscarinic

Characterization of muscarinic receptor subtypes on rat pancreatic acinar cells was examined by using specific muscarinic receptor antagonists to study amylase secretion and binding of [N-methyl-3H]scopolamine ([3H]NMS). Rat pancreatic acini were dispersed in HEPES-Ringer buffer and incubated with acetylcholine +/- 4-diphenylacetoxy-N-methylpiperadine-methiodide (4-DAMP, a specific M2 muscarinic receptor antagonist) or +/- pirenzepine (a specific M1 muscarinic receptor antagonist). 4-DAMP (10(-9) to 10(-6) M) caused a progressive parallel rightward shift in the acetylcholine dose-response curve without a change in maximal amylase release. Only high concentrations of pirenzepine (10(-6) to 10(-4) M) caused a rightward shift in the dose-response curve to acetylcholine. Schild analysis of the data indicated an inhibitory constant (Ki) of 200 pM for 4-DAMP and 183 nM for pirenzepine. The slope of the Schild regression lines was not different from unity, suggesting competitive inhibition. Binding of 50 pM [3H]NMS was specific, rapid, and saturable. [3H]NMS binding was displaced by increasing concentrations of 4-DAMP or pirenzepine with apparent Ki's of 102 pM and 330 nM, respectively, and similar maximal binding levels of 60 fmol/mg prot. We have demonstrated that 4-DAMP has an approximately 1,000-fold greater potency than pirenzepine to inhibit amylase release and binding, indicating that cholinergic-stimulated amylase release from pancreatic acini is mediated by M2 muscarinic receptors.

Topics: Acetylcholine; Amylases; Animals; Benzodiazepinones; Binding Sites; Male; N-Methylscopolamine; Pancreas; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Tritium

In this study we examined the role of M1- and M2-muscarinic receptors in the mediation of circular smooth muscle esophageal contractions elicited by pharmacological cholinergic stimulation and during peristalsis in anesthetized opossums. Esophageal-body contractions were induced by bethanechol administration, whereas peristalsis was elicited by pharyngeal stroking or cervical vagal stimulation. Contractions were measured by a low-compliance manometric recording system. The incidence and amplitude of bethanechol-induced contractions were antagonized by 4-diphenylacetoxy-n-methylpiperidine (4-DAMP) and atropine but not pirenzepine. 4-DAMP and atropine caused an increased velocity, decreased amplitude, and preferential reduction of the incidence of primary peristaltic contractions in the proximal smooth muscle esophagus. During long-train vagal stimulation, intra-stimulus A-waves had a velocity similar to primary peristalsis, whereas poststimulus B-waves showed a velocity considerably faster than primary peristalsis. Short-train vagal stimulation produced a contraction sequence, termed an "S-wave," that had a velocity similar to that of the A-wave. At low doses 4-DAMP increased the velocity and decreased the amplitude of A-wave and S-wave contractions, and at high doses 4-DAMP abolished both the A-wave and S-wave contractions. B-wave contractions were minimally affected by 4-DAMP. Pirenzepine had no effect on contractions induced by swallows or vagal stimulation. We conclude that M2-muscarinic receptors mediate esophageal contractions in the circular smooth muscle during primary peristalsis and during A-waves and S-waves induced by vagal stimulation, and M1-receptors do not have any important role in the excitatory neural pathway to the esophagus.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Benzodiazepinones; Bethanechol; Bethanechol Compounds; Dose-Response Relationship, Drug; Electric Stimulation; Esophagus; Female; Male; Manometry; Muscle Contraction; Muscle, Smooth; Opossums; Peristalsis; Piperidines; Pirenzepine; Receptors, Muscarinic; Vagus Nerve

To determine the muscarinic receptor subtype (M1 or M2 or both) responsible for cholinergic coronary vasodilatation and alterations in the transmural distribution (endocardial to epicardial blood flow ratio) of coronary blood flow, systemic and coronary haemodynamic indices and regional myocardial blood flow (radioactive microspheres) were measured in anaesthetised dogs. Submaximal vasodilative doses of the mixed muscarinic agonist acetylcholine were given by intracoronary infusion to avoid peripheral haemodynamic effects. Acetylcholine produced significant increases in myocardial perfusion and selectively redistributed flow to the subendocardium (increased endocardial to epicardial blood flow ratio). Pirenzepine (160 nmol X kg-1 iv), a selective M1 antagonist, produced no change in endocardial to epicardial blood flow ratio or myocardial blood flow but blocked the increase in endocardial to epicardial blood flow ratio and attenuated the transmural increase in myocardial perfusion during acetylcholine infusion. 4-Diphenylacetoxy-N-methylpiperidine methyl bromide (4-DAMP) (17 nmol X kg-1 iv), a selective M2 antagonist, also attenuated the transmural increase in myocardial blood flow but had little effect on the increase in endocardial to epicardial blood flow ratio produced by acetylcholine. These results support the hypothesis that M1 muscarinic coronary receptors are responsible for the redistribution of blood flow to the subendocardium (increased endocardial to epicardial blood flow ratio) during cholinergic coronary vasodilatation, whereas both M1 and M2 receptors are involved in increasing myocardial perfusion.

Topics: Acetylcholine; Animals; Coronary Circulation; Dogs; Endocardium; Female; Male; Myocardium; Perfusion; Pericardium; Piperidines; Pirenzepine; Receptors, Muscarinic

In an attempt to obtain more selective antagonists acting at muscarinic M2-receptors, analogues of 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP methobromide) have been synthesized. These were tested, along with silabenzhexol, procyclidine, sila-procyclidine and AFDX-116, in dose-ratio experiments with guinea-pig isolated atria at 30 degrees C and ileum at 30 degrees C and 37 degrees C. The agonist was carbachol and the selectivity was assessed from the difference between log K for receptors in ileum and log K for receptors in atria. The selectivity was not related to the affinity and some weakly active compounds retained appreciable selectivity but no compound had greater selectivity than 4-DAMP methobromide or pentamethylene bis-(4-diphenylacetoxy-N-methylpiperidinium) bromide. Structure-activity relations are discussed. There seem to be steric limits to affinity but there are no obvious indications of the structural features associated with selectivity. It is suggested that more selective drugs may be obtained by introducing groups which may reduce affinity.

Topics: Animals; Guinea Pigs; Ileum; In Vitro Techniques; Muscle, Smooth; Myocardial Contraction; Myocardium; Parasympathomimetics; Piperidines; Receptors, Muscarinic

The muscarinic receptor mediating vasodilatation of the rabbit aorta and dog femoral artery has been assessed using muscarinic antagonists. With the exception of pirenzepine, the antagonist affinities were similar to those reported for the ileal receptors and dissimilar to those reported for the atrial receptors. Pirenzepine exhibited an affinity (7.54) intermediate between that reported for the CNS receptors (8.4) and that reported for the ileal receptors (6.77). This value for pirenzepine was confirmed using acetylcholine as the agonist and using the dog femoral artery as the vascular tissue. It is concluded that the muscarinic receptor profile mediating vasodilatation is not easily accommodated into the current receptor classification.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Atropine; Benzodiazepinones; Carbachol; Dogs; Female; Femoral Artery; Gallamine Triethiodide; In Vitro Techniques; Male; Pancuronium; Parasympatholytics; Phenethylamines; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Vasodilation

Isolated preparations of guinea-pig ileum and atria have been used to estimate the dose-ratios produced by antagonists at muscarinic receptors. Experiments with 4-diphenyl-acetoxy-N-methylpiperidine (4DAMP) metho-salts and with its isomer, 3DAMP methiodide, indicate that these are only slightly affected by the choice of physiological salt solution, the choice of agonist and the presence or absence of hexamethonium. Methyl or chloro groups in the p-position of the two benzene rings in 4DAMP metho-salts markedly reduce affinity and selectivity. When the two benzene rings are linked together, as in the fluorene-9-carboxylic ester, the affinity for the receptors in the atria is comparable with that of 4DAMP methobromide but that for the ileum is about half, so the selectivity is reduced. When the rings are linked as in the xanthene-9-carboxylic ester, the affinity for receptors in both tissues is greater than that of 4DAMP methobromide but there is less selectivity. When two molecules of 4DAMP are linked together by a polymethylene chain of from 4 to 12 carbon atoms the effects on affinity for muscarinic receptors in the guinea-pig ileum are different from those on affinity for muscarinic receptors in guinea-pig atria. The pentamethylene compound is the most selective: compared with 4DAMP methobromide it has slightly less affinity for receptors in the ileum but much less affinity for receptors in the atria. The effects of the compounds in antagonizing the actions of carbachol on atrial rate are not markedly different from their effects in antagonizing its actions on the force of the atrial contractions.

Topics: Animals; Atrial Function; Carbachol; Chemical Phenomena; Chemistry; Guinea Pigs; Heart Rate; Ileum; Muscle Contraction; Myocardial Contraction; Piperidines; Receptors, Muscarinic; Structure-Activity Relationship

Lower esophageal sphincter pressures were monitored with water-filled catheters in anesthetized opossums. Muscarinic agonists McN-A-343 and bethanechol were administered in the arterial supply of the sphincter. McN-A-343 caused relaxation after a brief contraction of the sphincter. Bethanechol caused a dose-dependent contraction. Tetrodotoxin antagonized the inhibitory effect of McN-A-343 but did not antagonized sphincter contraction caused by McN-A-343 or bethanechol. The mean ED50 values were 6.9 nmol/kg i.a. for McN-A-343-induced relaxation, 10.5 nmol/kg i.a. for McN-A-343-induced contraction and 0.4 nmol/kg i.a. for bethanechol-induced contraction. Atropine caused a dose-dependent rightward shift in the dose-response curves of inhibitory and excitatory effects of the two muscarinic agonists. Pirenzepine caused a dose-dependent rightward shift in the dose-response curves of McN-A-343-induced relaxation. Pirenzepine did not modify sphincter contraction caused by the muscarinic agonists. 4-Diphenylacetoxy-N-methylpiperidine methiodide, on the other hand, did not modify McN-A-343-induced sphincter relaxation but caused dose-dependent rightward shifts in the dose-response curves of sphincter contraction caused by McN-A-343 or bethanechol. These studies suggest that there are two distinct types of muscarine receptors in the opossum lower esophageal sphincter. The M1 muscarine receptors are present on the inhibitory neurons and participate in the synaptic transmission between vagal preganglionic and intramural postganglionic inhibitory neurons. They are activated by McN-A-343 and antagonized by pirenzepine. The M2 muscarine receptors are located directly on the sphincter muscle. They are also activated by McN-A-343, but are selectively activated by bethanechol and are antagonized by 4-diphenylacetoxy-N-methylpiperidine methiodide.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Atropine; Benzodiazepinones; Bethanechol; Bethanechol Compounds; Esophagogastric Junction; Hexamethonium Compounds; Opossums; Piperidines; Pirenzepine; Receptors, Muscarinic; Tetrodotoxin; Vagus Nerve