piperidines and methoctramine

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

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

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

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

We investigated the effects of nonselective muscarinic antagonist (atropine) and of selective muscarinic subtype 1 (M1), 2 (M2), 3 (M3) antagonists (VU0255035, methoctramine, pFHHSiD, respectively) on the contractions evoked by electrical field stimulation (EFS) or by exogenous ACh in isolated horse bronchial muscle. Atropine completely inhibited neurogenic contractions in a concentration-dependent fashion, whereas selective muscarinic antagonists induced relevant modifications only at the highest concentration tested. Experiments with selective muscarinic antagonists in combination showed that only the simultaneous blockade of M1 /M3 or M2 /M3 receptors was able to induce a nearly complete suppression of contractions. The contractions induced by exogenous ACh were competitively antagonized only by atropine (pA2 = 9.01 ± 0.05). M3 selective antagonist, up to 10(-6) m, caused a moderate concentration-dependent rightward shift of ACh curve (pA2 = 7.96 ± 0.10). These data show that M3 muscarinic receptors possess a central role in mediating cholinergic contraction of horse bronchi, while M1 and M2 receptors seem to have a cooperative role. Selective muscarinic antagonists seem unlikely to be useful against bronchoconstriction associated with airway diseases in horses. Conversely, compounds with selectivity for both M1 and M3 receptors could be as effective as traditional anticholinergics and induce fewer cardiac side effects.

Topics: Animals; Bronchi; Bronchial Spasm; Diamines; Gene Expression Regulation; Horses; Male; Parasympatholytics; Piperidines; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Sulfonamides; Thiadiazoles

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 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

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

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

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

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

Acetylcholine interacts with endothelial muscarinic receptors releasing nitric oxide and causing vasodilatation. To identify the receptor subtype responsible for acetylcholine-induced relaxation in canine uterine artery, the usual organ bath method for in vitro investigation on isolated blood vessels was applied. Using a range of muscarinic receptor antagonists such as atropine (nonselective), pirenzepine (M(1)-selective), methoctramine (M(2)-selective) and p-fluoro-hexahydro-sila-difenidol (p-FHHSiD) (M(1)/M(3)) and determining pA2 value of those antagonists through Shild analysis, we aimed at establishing a precise receptor mechanism underlying acetylcholine-induced relaxation in isolated canine uterine artery. The relaxation of uterine arterial rings in response to acetylcholine in the presence or absence of selective muscarinic receptors antagonists was calculated using concentration response curves. Acetylcholine induced concentration-dependent and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC(50) = 6.90 +/- 0.02). Muscarinic receptors antagonists atropine, pirenzepine, methoctramine and p-FHHSiD competitively antagonized the response to acetylcholine and obtained pA(2) values were 9.91 +/- 0.06, 6.60 +/- 0.04, 6.21 +/- 0.08 and 8.05 +/- 0.1, respectively. This study showed that acetylcholine induced endothelium-dependent relaxation of canine uterine artery by stimulation of muscarinic receptors localized on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the acetylcholine-induced relaxation of canine uterine artery are predominantly of M(3) subtype.

Topics: Acetylcholine; Animals; Arteries; Atropine; Cholinergic Agents; Diamines; Dogs; Endothelium, Vascular; Female; Hysterectomy; In Vitro Techniques; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Uterus

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

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

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

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

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

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

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

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

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 role of aging on contraction or relaxation through muscarinic or alpha-adrenergic receptors, respectively, was studied in isolated rat jejunum. Furthermore, the influence of extracellular calcium was analyzed, through functional and radioligand binding assays. The rank order of potency for selective muscarinic antagonists for M(1), M(2), and M(3) receptor subtypes, measured from affinity (pA(2)) values, was p-fluorohexahydrosiladifenidol (pFHHSiD) (M(3)) > pirenzepine (M(1)) > methoctramine (M(2)), indicating a predominance of M(3) subtype. This order was unchanged with age. Contractions by muscarinic agonist methacholine (MCh) were diminished in aged rats, resulting in lower apparent affinity (pD(2)) values, compared with adult controls. A larger decrease of MCh contractions occurred in aged rats after Ca(2+) withdrawal or after the calcium channel blocker isradipine. Changes were not detected for relaxation by adrenergic agonists. In conclusion, aging caused a decrease of MCh potency, which is probably related to the reduction of calcium sensitivity in jejunum.

Topics: Adrenergic Agonists; Aging; Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Diamines; Isradipine; Jejunum; Male; Methacholine Chloride; Muscarinic Agonists; Muscarinic Antagonists; Peristalsis; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Adrenergic, alpha; Receptors, Muscarinic

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

The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration-response curves were constructed for acetylcholine (ACh) (pEC50: 6.16+/-0.04; maximum response 80.00+/-1.98% of the responses induced by serotonin 10 microM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33+/-0.03; double inhibition: pEC50 6.57+/-0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction.In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. The M1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN-A-343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this tissue.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Atropine; Diamines; Humans; In Vitro Techniques; Piperidines; Pirenzepine; Receptors, Muscarinic; Umbilical Veins; Vasoconstriction

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

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 Fallopian tubes are scarcely innervated with cholinergic nerve fibers. Acetylcholine released from these nerves contracts smooth muscles of the tubes. The aim of our study was to investigate the effect of acetylcholine on the Fallopian tubes using selective antagonists in different hormonal settings. We have investigated effects of acetylcholine on isolated ampulla of Fallopian tubes taken from 83 patients during abdominal hysterectomy with adnexectomy. Twenty-eight patients were in follicular, 36 were in luteal phase of menstrual cycle, and 19 patients were in menopause. Selective and non-selective muscarinic and nicotinic receptor blockers were used for investigation of the effects. Acetylcholine (from 1.8 to 658.6 microM/l) produced concentration-dependent tonic contraction of ampulla taken from the patients in follicular phase, luteal phase and menopause. The nicotinic receptor blocker mecamylamine (6.5 microM/l) and local anesthetic lidocaine (230.8 microM/l) did not affect the effect of acetylcholine. While M(1)- and M(2)-selective muscarinic receptor blockers pirenzepine (1.6 microM/l) and methoctramine (0.9 microM/l) did not show specific effect, atropine (0.01 microM/l) and selective M(3)-receptor blocker p-fluoro-hexahydro-sila-difenidol (pFHHSiD, 0.2 microM/l) effectively blocked contractions caused by acetylcholine (maximal pA(2) values 9.74 and 7.54, respectively). The affinity of pFHHSiD for muscarinic receptors was highest in the follicular phase. The results of our study suggest existence of functional M(3) muscarinic receptors in ampulla of Fallopian tubes, located on the smooth muscle cells.

Topics: Acetylcholine; Adult; Aged; Diamines; Fallopian Tubes; Female; Follicular Phase; Humans; Lidocaine; Luteal Phase; Mecamylamine; Menopause; Middle Aged; Muscle Contraction; Muscle, Smooth; Nifedipine; Piperidines; Pirenzepine; Verapamil

In an attempt to examine whether the muscarinic receptor-activated intestinal function is altered by aging, we studied the changes in (1) contractile responses to acetylcholine (Ach), (2) muscarinic cholinoceptors and (3) cholinesterase (ChE) activities, in jejunum and colon of the young (2-3 months) and aged (24-28 months) Fischer 344 rats. In the physiological contraction experiments of jejunum and colon, Ach concentration-dependently increased the force of contraction, and the contractile responses to Ach were not affected by aging. In addition, the true- and pseudo-ChE activities were not significantly changed by aging. The Ach-induced contraction was competitively inhibited by muscarinic M3-selective antagonist hexahydro-sila-difenidolhydrochloride p-fluoroanalog (p-F-HHSiD), suggesting that the contractile responses in the rat jejunum and colon were mediated through M3-cholinoceptor. Age-related changes in muscarinic cholinoceptors of jejunum and colon were determined with the use of specific muscarinic radioligand [3H]-quinuclidinylbenzilate (QNB). The [3H]QNB saturation binding experiments revealed that the maximal binding (B(max)) was increased only in aged jejunum without changes in K(D) values. These results suggest that aging may not attenuate the Ach-induced intestinal contraction via muscarinic M3 receptor, although the expression of muscarinic cholinoceptor is differentially modulated in jejunum and colon.

Topics: Acetylcholine; Aging; Animals; Atropine; Cholinesterases; Colon; Diamines; Dose-Response Relationship, Drug; Gastrointestinal Motility; In Vitro Techniques; Intestine, Large; Jejunum; Male; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Pirenzepine; Rats; Rats, Inbred F344

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

The Fallopian tubes are sparsely innervated with cholinergic nerve fibers. Acetylcholine is released from these nerves and contracts the smooth muscles of the tubes. The aim of our study was to investigate the effect of acetylcholine on the isthmic segment of the Fallopian tubes using selective antagonists in different hormonal settings. We investigated the effects of acetylcholine on the isolated isthmus of Fallopian tubes taken from 83 patients during abdominal hysterectomy with adnexectomy. Twenty-eight patients were in the follicular phase, 36 were in the luteal phase of the menstrual cycle, and 19 patients were in menopause. Selective and non-selective muscarinic and nicotinic receptor antagonists were used. Acetylcholine (1.8-658.6 micro M) produced concentration-dependent tonic contraction of isthmus taken from the patients in the follicular phase, the luteal phase and menopause. The nicotinic receptor antagonist mecamylamine (6.5 micro M) and local anesthetic lidocaine (230.8 micro M) did not alter the effect of acetylcholine. While M(1) and M(2)-selective muscarinic receptor antagonists pirenzepine (1.6 micro M) and methoctramine (0.9 micro M) did not show specific effect, atropine (0.01 micro M) and the selective M(3)-receptor antagonist p-fluoro-hexahydro-sila-difenidol (pFHHSiD; 0.2 micro M) effectively blocked contractions caused by acetylcholine (maximal pA(2) values 9.74 and 7.54, respectively). The affinity of pFHHSiD for muscarinic receptors was highest in the follicular phase. The results of our study suggest the existence of functional M(3) muscarinic receptors in the isthmus of the Fallopian tubes, located on the smooth muscle cells.

Topics: Acetylcholine; Adult; Aged; Atropine; Diamines; Dose-Response Relationship, Drug; Fallopian Tubes; Female; Follicular Phase; Humans; Lidocaine; Luteal Phase; Mecamylamine; Middle Aged; Muscle Contraction; Muscle, Smooth; Nifedipine; Piperidines; Pirenzepine; Postmenopause; Receptors, Muscarinic

We studied the functional role of individual subtypes of muscarinic cholinoceptors in the pathogenesis of neuroleptic parkinsonism in rats. Blockade of M4 receptors prevented the development of extrapyramidal disorders, which was abolished by simultaneous blockade of M2 receptors. The data suggest that various subtypes of muscarinic receptors are involved in the regulation of dopamine concentration.

Topics: Animals; Catalepsy; Cyclopentanes; Diamines; Dopamine; Haloperidol; Male; Mandelic Acids; Muscarinic Antagonists; Piperidines; Receptor, Muscarinic M2; Syndrome; Trihexyphenidyl

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

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

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

The effect of the cholinomimetic agent, bethanechol on macroscopic membrane currents was studied in dispersed cat atrial myocytes, using the whole-cell patch-clamp technique. Bethanechol activated an inward rectifying potassium current similar to I(K(ACh)), and a delayed rectifying-like outward current, similar to I(KM3) activated by pilocarpine, choline, and tetramethylammonium, and I(KM4) activated by 4-aminopyridine. The relatively specific muscarinic receptors subtype antagonists methoctramine (M(2)), and tropicamide (M(4)) inhibited both current components induced by bethanechol, suggesting a lack of specificity of these antagonists on cat atrial myocytes. The specific antagonist of M(3) receptors, para-fluoro-hexahydro-siladifenidol did not significantly inhibit the bethanechol-induced currents. In addition, pretreatment with PTX prevented activation of the bethanechol-induced inward and outward currents, suggesting that M(3) receptors are probably not involved in the bethanechol action. The I(K(ACh)) specific blocker tertiapin inhibited both inward rectifying- and delayed rectifying-like currents. These results suggest that both current components result from activation of a single channel type, likely I(K(ACh)).

Topics: Animals; Barium; Bee Venoms; Benzodiazepines; Bethanechol; Cats; Cholinergic Agents; Diamines; Heart Atria; In Vitro Techniques; Membrane Potentials; Muscarinic Agonists; Muscarinic Antagonists; Muscle Cells; Phenethylamines; Piperidines; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Sulfonamides; Tropicamide

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

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

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal beta cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1--m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 microM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 microM of methoctramine (M2 antagonist) increased ACh (100 microM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.

Topics: Acetylcholine; Animals; Cell Line; Diamines; Insulin; Insulin Secretion; Islets of Langerhans; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The effect of acetylcholine (ACh) on the isolated, nonprecontracted perforating branch of the human internal mammary artery (HIMA) was investigated. ACh induced concentration-dependent contractions of nonprecontracted rings with denuded endothelium (pEC(50) = 6.72 +/- 0.02, E(max) = 88.8% of contractions induced by phenylephrine, 10(-5) mol/l) and was without effect on arterial segments with intact endothelium. An inhibitor of nitric oxide synthase, N(G)-monomethyl-L-arginine (L-NMMA), or indometacin, a cyclooxygenase inhibitor, had no effect on acetylcholine-induced contractions of rings of the perforating branch of HIMA with denuded endothelium (pEC(50) = 6.76 +/- 0.03 and 6.62 +/- 0.05, respectively). In the presence of indometacin, ACh did not evoke contractions of arterial segments with intact endothelium. In contrast, in the same type of preparations ACh induced contractions in the presence of L-NMMA (E(max) = 34%). The muscarinic receptor antagonists atropine (no selectivity), pirenzepine (M(1)), methoctramine (M(2)), and p-fluoro-hexahydro-sila-difenidol (M(1)/M(3)) competitively antagonized the response to ACh. The pA(2) values were 9.60 +/- 0.10, 6.99 +/- 0.02, 6.37 +/- 0.17, and 8.02 +/- 0.06, respectively. In conclusion, the results obtained indicate that secretion of nitric oxide from vascular endothelium may protect the perforating branch of HIMA against the contractile effects of ACh. On the basis of differential antagonist affinity, it can be suggested that the muscarinic receptors involved in the ACh-induced contractions of the isolated perforating branch of the HIMA are predominantly of the M(3) subtype.

Topics: Acetylcholine; Atropine; Diamines; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Female; Humans; In Vitro Techniques; Indomethacin; Mammary Arteries; Middle Aged; Muscarinic Antagonists; Nitric Oxide Synthase; omega-N-Methylarginine; Phenylephrine; Piperidines; Pirenzepine; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

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

To clarify the supraspinal and spinal actions of a cholinergic agonist, carbachol, and an opioid, oxycodone, we studied their antinociceptive and behavioral effects when administered into brainstem medial pontine reticular formation (mPRF) or spinal subarachnoid space with or without pretreatment of muscarinic receptor subtype antagonist. Sprague-Dawley rats were implanted with a 24-gauge stainless steel guide cannula into the mPRF and chronically implanted with a lumbar intrathecal catheter. Antinociception was tested using tail flick latency, motor coordination was evaluated by the rotarod test, and overt sedation was assessed using a behavioral checklist. Carbachol (0.5-4.0 microg) administered into the mPRF produced significant dose- and time-dependent antinociception, sedation, and motor dysfunction. These were completely blocked by pretreatment with atropine and the M(2) muscarinic antagonist, methoctramine, and partially blocked by pretreatment with M(1) pirenzepine but not with M(3) p-fHHSID: Oxycodone administered into the mPRF did not produce such effects. Spinal carbachol and oxycodone produced antinociception without any behavioral effects; their antinociceptive effects were completely blocked by pretreatment with atropine and M(2) antagonist. These results suggest that the antinociceptive action of carbachol is mediated by muscarinic cholinergic receptor activation, especially by M(2) receptor subtype in mPRF and spinal cord, and that although oxycodone seems unlikely to affect the cholinergic transmission of mPRF, spinal oxycodone-induced analgesia is at least partly mediated via the activation of M(2) receptor subtype at the spinal cord.. Carbachol-induced antinociception and sedation is mediated with the activation of M(2) muscarinic receptors. Oxycodone administered into brainstem medial pontine reticular formation did not cause any antinociceptive or behavioral effects, but its spinal administration produced a significant antinociception via M(2) muscarinic receptor activation

Topics: Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Atropine; Behavior, Animal; Carbachol; Cholinergic Agonists; Conscious Sedation; Diamines; Injections, Spinal; Male; Microinjections; Motor Activity; Muscarinic Antagonists; Pain Threshold; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Reticular Formation; Subarachnoid Space

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 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

K+ secretion in strial marginal cells (SMC) of stria vascularis (SV) is stimulated by beta1-adrenergic receptors. The aim of the present study was to determine, whether SMC from the gerbil inner ear contain muscarinic receptors that inhibit K+ secretion. Receptors were identified with pharmacological tools in functional studies where K+ secretion was monitored as transepithelial current (Isc). The cytosolic Ca2+ concentration ([Ca2+]i) was measured as fluo-4 fluorescence and cAMP production with a colorimetric immunoassay. Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. The cholinergic receptor agonist carbachol (CCh) caused a transient increase in [Ca2+]i with a half-maximal concentration value (EC50) of (5 +/- 6) x 10(-6) m (n = 29) and a decrease in basal and stimulated cAMP production. Apical CCh had no effect on Isc but basolateral CCh caused a transient increase in Isc with an EC50 of (3 +/- 1) x 10(-6) m and a sustained decrease of Isc with an EC50 of (1.2 +/- 0.2) x 10(-5) m (n = 129). The effects of CCh on Isc and [Ca2+]i were inhibited in the presence of muscarinic antagonist 10(-6) m atropine. Further, the muscarinic antagonists pirenzipine, methoctramine and para-fluoro-hexahydo-sila-defenidol (pFHHSiD) inhibited the CCh-induced transient increase of Isc with affinity constants (KDB) of 3 x 10(-8) m (pKDB = 7.54 +/- 0.19, n = 17), 2 x 10(-6) m (pKDB = 5.71 +/- 0.26, n = 19) and 2 x 10(-8) m (pKDB = 7.65 +/- 0.28, n = 19) and the sustained decrease of Isc with KDB of 7 x 10(-8) m (pKDB = 7.05 +/- 0.09, n = 33), 6 x 10(-6) m (pKDB = 5.21 +/- 0.13, n = 23), 5 x 10(-8) m (pKDB = 7.34 +/- 0.13, n = 31), respectively. RT-PCR of total RNA isolated from SV using primers specific for the M1-M5 muscarinic receptors revealed products of the predicted sizes for the M3- and M4- but not the M1-, M2- and M5-muscarinic receptor subtypes. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results suggest that K+ secretion in SMC is under the control of M3- and M4-muscarinic receptors that may be located in the basolateral membrane of strial marginal cells.

Topics: Adrenergic beta-Agonists; Animals; Atropine; Calcium; Carbachol; Cholinergic Agonists; Cloning, Molecular; Cyclic AMP; Diamines; Dose-Response Relationship, Drug; Gerbillinae; Isoproterenol; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Potassium; Receptors, Muscarinic; Reverse Transcriptase Polymerase Chain Reaction; Stria Vascularis

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 effect of acetylcholine (ACh) on the isolated perforating branch of the human internal mammary artery (HIMA) was investigated. ACh induced concentration- and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC(50) = 6.93 +/- 0.01). The muscarinic receptor antagonist atropine (no selectivity), pirenzepine (M(1)), methoctramine (M(2)), and p-fluoro-hexahydro-siladifenidol (M(1)/M(3)) competitively antagonized the response to ACh. The pA(2) values were 9.81 +/- 0.15, 7.74 +/- 0.08, 6.27 +/- 0.08, and 7.88 +/- 0.04, respectively. In conclusion, this study has shown that ACh induced an endothelium-dependent relaxation of the perforating branch of the HIMA by stimulation of muscarinic receptors on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the ACh-induced relaxation of the isolated perforating branch of HIMA are predominantly of M(1) subtype.

Topics: Acetylcholine; Adult; Aged; Atropine; Diamines; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Humans; In Vitro Techniques; Mammary Arteries; Middle Aged; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptors, Muscarinic; Vasodilation; Vasodilator Agents

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

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

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

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

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

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

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

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

The effect of muscarinic cholinoceptor antagonists was investigated on the ascending neural pathways activated by electrical stimulation in the guinea-pig ileum. For comparison, prejunctional and postjunctional effects of muscarinic cholinoceptor antagonists were also studied on circular smooth muscle. A two-compartment (oral and anal compartments) bath was used to study the ascending neural pathways. These were activated by electrical field stimulation in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded isotonically. Pirenzepine (10-300 nM), a muscarinic M1 cholinoceptor antagonist, reduced the ascending neural contractions in a concentration-dependent fashion when applied either to the oral or anal compartments (11-52% and 13-55% inhibition, respectively, P < 0.05). Pirenzepine inhibited (31+/-7%, P < 0.05) the acetylcholine (100 nM)-induced contractions at a higher non-selective concentration (300 nM), while its effect on the electrically-induced contractions was biphasic (10 and 30nM: 8-15% increase, P<0.05; 100 and 300 nM: 16-28% inhibition, P<0.05). The muscarinic M2 cholinoceptor antagonist methoctramine (3-100 nM) did not modify the contractions produced by 100 nM acetylcholine, electrically-induced contractions and the ascending neural contractions (when applied to either compartment). Parafluorohexahydrosiladifenidol (3-100 nM), a muscarinic M3 cholinoceptor antagonist, inhibited the contractions produced by 100 nM acetylcholine (19-81% and 15-69%), electrically-induced contractions (11-71% and 12-72%) and the ascending neural contractions (13-76% and 866%) when applied to the oral compartment, but it was without effect when applied to the anal compartment. These studies suggest that in the enteric ascending neural pathway, muscarinic M1 receptors are involved in neuroneuronal transmission, muscle contraction is mediated by muscarinic M3 cholinergic receptors, whereas muscarinic M2 receptors do not seem to participate.

Topics: Acetylcholine; Afferent Pathways; Anal Canal; Animals; Diamines; Electric Stimulation; Guinea Pigs; Ileum; In Vitro Techniques; Male; Mouth; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Pirenzepine

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

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

The effect of acetylcholine on the isolated, non-precontracted, porcine internal mammary artery (IMA) was investigated. Acetylcholine induced concentration-dependent contractions of non-precontracted IMA rings with denuded endothelium (pEC50 = 5.80 +/- 0.04) and was without effect on arterial segments with intact endothelium. The muscarinic receptor antagonists atropine, pirenzepine, methoctramine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) antagonized the response to acetylcholine. The constrained pA2 values were 10.14, 7.74, 7.34 and 10.5, respectively. It is concluded that acetylcholine induces concentration-dependent contractions of porcine internal mammary artery rings on basal tone and that this contractile effect is probably due to direct cholinergic stimulation of smooth muscle cells, maybe including activation of muscarinic M1 receptors.

Topics: Acetylcholine; Animals; Diamines; Endothelium, Vascular; In Vitro Techniques; Mammary Arteries; Muscle Contraction; Muscle, Smooth, Vascular; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Swine

1. Blind patch clamp recordings were made from substantia gelatinosa (SG) neurones in the adult rat spinal cord slice to study the mechanisms of cholinergic modulation of GABAergic inhibition. 2. In the majority of SG neurones tested, carbachol (10 microM) increased the frequency (677 % of control) of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). A portion of these events appeared to result from the generation of spikes by GABAergic interneurones, since large amplitude IPSCs were eliminated by tetrodotoxin (1 microM). 3. The effect of carbachol on spontaneous IPSCs was mimicked by neostigmine, suggesting that GABAergic interneurones are under tonic regulation by cholinergic systems. 4. The frequency of GABAergic miniature IPSCs in the presence of tetrodotoxin (1 microM) was also increased by carbachol without affecting amplitude distribution, indicating that acetylcholine facilitates quantal release of GABA through presynaptic mechanisms. 5. Neither the M1 receptor agonist McN-A-343 (10-300 microM) nor the M2 receptor agonist, arecaidine (10-100 microM), mimicked the effects of carbachol. All effects of carbachol and neostigmine were antagonized by atropine (1 muM), while pirenzepine (100 nM), methoctramine (1 microM) and hexahydrosiladifenidol hydrochloride, p-fluoro-analog (100 nM) had no effect. 6. Focal stimulation of deep dorsal horn, but not dorsolateral funiculus, evoked a similar increase in IPSC frequency to that evoked by carbachol and neostigmine. The stimulation-induced facilitation of GABAergic transmission lasted for 2-3 min post stimulation, and the effect was antagonized by atropine (100 nM). 7. Our observations suggest that GABAergic interneurones possess muscarinic receptors on both axon terminals and somatodendritic sites, that the activation of these receptors increases the excitability of inhibitory interneurones and enhances GABA release in SG and that the GABAergic inhibitory system is further controlled by cholinergic neurones located in the deep dorsal horn. Those effects may be responsible for the antinociceptive action produced by the intrathecal administration of muscarinic agonists and acetylcholinesterase inhibitors.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Atropine; Carbachol; Diamines; Evoked Potentials; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Muscarinic Agonists; Neostigmine; Neurons; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptor, Muscarinic M1; Receptors, Muscarinic; Substantia Gelatinosa; Synaptic Transmission; Tetrodotoxin

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

The localisation of M1-M4 muscarinic cholinergic receptor subtypes was investigated in sections of normal human term placenta by light microscope autoradiography. Muscarinic cholinergic receptor subtypes were found almost exclusively in syncytiotrophoblast. Neither other cellular components of placenta, nor blood vessels were labelled. Quantitative analysis of the density of silver grains developed in sections incubated with the different protocols for labelling M1-M4 receptor subtypes, revealed that syncytiotrophoblast expresses all subtypes of muscarinic cholinergic receptor investigated. A higher density of binding sites was found in the apical than in the basal portion of syncytiotrophoblast. The demonstration of muscarinic cholinergic receptors in syncytiotrophoblast suggests that a cholinergic system may have a role in regulating transport of compounds from maternal to foetal interface.

Topics: Adult; Binding, Competitive; Diamines; Female; Humans; Microvilli; Muscarinic Antagonists; Piperidines; Pirenzepine; Placenta; Pregnancy; Receptors, Muscarinic; Trophoblasts; Tropicamide

The cardiovascular effects of three different acetylcholinesterase inhibitors: physostigmine, tacrine and rivastigmine injected by intravenous (i.v.) route were compared in freely moving Wistar rats. The three drugs significantly increased both systolic and diastolic blood pressure and decreased heart rate. Compared to physostigmine, a 20-fold higher dose of tacrine and a 40-fold higher dose of rivastigmine was necessary to induce a comparable pressor effect. Tacrine was chosen as a model to study the mechanisms underlying the cardiovascular effects of i.v. cholinesterase inhibitors. Atropine totally abolished while methylatropine did not affect tacrine pressor effects. Conversely, both drugs abolished tacrine-induced bradycardia. The alpha1-adrenoceptor antagonist prazosin or the vasopressin V1 receptor antagonist, [beta-mercapto-beta,beta-cyclopenta-methylenepropionyl1, O-Me-Tyr2, Arg8] vasopressin partially but significantly reduced tacrine pressor effect and mostly abolished it when administered concomitantly. The tacrine pressor response was inhibited in a dose-dependent manner by the i.c.v. administration of the non-selective muscarinic receptor antagonist atropine (ID50 = 1.45 microg), the muscarinic M1 receptor antagonist pirenzepine (ID50 = 4.33 microg), the muscarinic M2 receptor antagonist methoctramine (ID50 = 1.39 microg) and the muscarinic M3 receptor antagonist para-fluoro-hexahydro-sila-difenidol (ID50 = 31.19 microg). Central injection of such muscarinic receptor antagonists did not affect tacrine-induced bradycardia. Our results show that acetylcholinesterase inhibitors induce significant cardiovascular effects with a pressor response mediated mainly by the stimulation of central muscarinic M2 receptors inducing a secondary increase in sympathetic outflow and vasopressin release. Conversely, acetylcholinesterase inhibitor-induced bradycardia appears to be mediated by peripheral muscarinic mechanisms.

Topics: Adrenergic Antagonists; Animals; Antidiuretic Hormone Receptor Antagonists; Atropine; Atropine Derivatives; Blood Pressure; Carbamates; Cardiovascular Agents; Chlorisondamine; Cholinergic Antagonists; Cholinesterase Inhibitors; Diamines; Diastole; Dose-Response Relationship, Drug; Heart Rate; Male; Phenylcarbamates; Physostigmine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Rivastigmine; Systole; Tacrine

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

The role of four muscarinic receptor subtypes M1, M2, M3 and M4 which have been characterized pharmacologically was examined in motility control of isolated rat gastric fundus. Acetylcholine produced concentration-dependent tonic contraction of isolated rat fundus (EC50 = 9.64 +/- 0.14 x 10(-8)M). These contractions were concentration-dependently antagonized by atropine (KB = 2.45 x 10(-11)M), M1 selective blockers telenzepine (KB = 6.64 x 10(-11)M) and pirenzepine (KB = 2.3 x 10(-8)M), and hexocyclium (KB = 2.82 x 10(-10)M). M3-selective blocker p-fluoro-hexahydro-sila-difenidol (pFHHSiD) was a less potent antagonist (KB = 2.3 x 10(-8)M), while M2 and M4-selective methoctramine produced only weak blockade of tonic contractions caused by acetylcholine (KB = 4.68 x 10(-6)M). These results suggest that only M1 and M3 muscarinic receptors have functional roles in motility control of rat gastric fundus, M1 receptors being more important.

Topics: Acetylcholine; Animals; Atropine; Diamines; Female; Gastric Fundus; Gastrointestinal Motility; Male; Muscarinic Antagonists; Piperazines; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic

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

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 primary goal of this study was to determine the extent that selective muscarinic receptor antagonists could discriminate between the chronotropic and coronary vasoconstrictor responses to acetylcholine in isolated rat hearts perfused at constant flow rate. Bolus injections of acetylcholine caused dose-dependent decreases in heart rate and increases in perfusion pressure. The ED50 (95% confidence) of acetylcholine for decreasing rate was 0.463 (0.336-0.640) nmol and the dose that increased perfusion pressure by 30 mm Hg (ED30 mmHg) was 3.19 (2.00-5.08) nmol. The M2 selective antagonist methoctramine (3.16 microM) produced a 307-fold increase in the ED50 for bradycardia but had no significant effect on the pressor response to acetylcholine. In marked contrast, the M3 antagonist hexahydrosiladifenidol displayed a distinct preference for inhibiting coronary vasoconstrictor responses to acetylcholine. When present at 316 nM, this drug produced a 66-fold increase in the ED30 mmHg but only a 6-fold increase in the ED50 for bradycardia. The M1 selective antagonist pirenzepine (316 nM) produced a 5- to 7-fold increase in both parameters. Pretreatment with pertussis toxin (25 microg/kg, i.p.) essentially eliminated acetylcholine-evoked bradycardia although pressor responses persisted with some reduction. These observations demonstrate that cardiac and coronary vascular effects of acetylcholine can be clearly discriminated with specific muscarinic antagonists. Furthermore, they provide evidence that the M3 receptor subtype mediates the vasoconstrictor effect of acetylcholine on resistance vessels in rat heart.

Topics: Acetylcholine; Animals; Atropine; Coronary Vessels; Depression, Chemical; Diamines; Heart Rate; In Vitro Techniques; Male; Pertussis Toxin; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Vasoconstriction; Virulence Factors, Bordetella

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

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

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

Muscarinic M2 receptors account for more than half the muscarinic receptor population in smooth muscles of a number of species and yet it is the smaller M3 receptor population that mediates contraction of many of these tissues. The role of the majority of M2 receptors in the control of smooth muscle tone is unclear. In guinea-pig ileal smooth muscle, an indirect contractile role (re-contraction) for M2 receptors has been demonstrated in tissues subjected to M3 receptor alkylation and stimulation of adenylyl cyclase. The present studies have employed the technique of irreversible receptor alkylation in order to investigate the role of muscarinic M2 and M3 receptors in the control of guinea-pig tracheal smooth muscle tone. Experiments were performed to determine (i) whether an indirect contractile role for M2 receptors can be demonstrated in tracheal smooth muscle as described for ileum, and (ii) whether stimulation of M2 receptors can inhibit isoprenaline-induced relaxations of histamine pre-contracted trachea after selective M3 receptor alkylation. Our results suggest (i) that there is no evidence of M2 receptor-mediated re-contraction of tracheal smooth muscle after M3 receptor alkylation and stimulation of adenylyl cyclase, but (ii) that activation of M2 receptors, after M3 receptor alkylation, has a small inhibitory effect on relaxant responses to isoprenaline in guinea-pig tracheal smooth muscle. Therefore, it appears that the major role of postjunctional muscarinic M2 receptors in guinea-pig trachea remains to be determined.

Topics: Alkylation; Animals; Diamines; Dioxolanes; Diphenylacetic Acids; Drug Interactions; Guinea Pigs; Histamine; Isoproterenol; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Oxotremorine; Piperidines; Receptors, Muscarinic; Stimulation, Chemical; Trachea

1. Methoctramine, AF-DX 116 and hexahydrosiladifenidol (HHSiD) are the muscarinic antagonists most widely used to study muscarinic receptor subtypes. 2. The present study was undertaken to examine the mode of antagonism of these compounds in guinea-pig left atrium and ileum by comparison of the Schild and resultant analysis. With this method the effect of various concentrations of the test antagonist on the antagonism produced by specific concentrations of a reference antagonist was measured and the equilibrium dissociation constant of the test antagonist-receptor complex estimated. Atropine was used for comparative purposes and scopolamine as the reference antagonist. 3. At the cardiac level the affinity values obtained by Schild and resultant analysis for methoctramine and AF-DX 116, as for atropine, are very similar: these results indicate that the two cardio-selective antagonists and the non-selective antagonist, atropine, bind at a common site with the reference antagonist scopolamine. The resultant plot for the ileo-selective HHSiD has a slope considerably less than unity: this finding might indicate that this antagonist binds to a site different from that of scopolamine and it should be considered like an allosteric antagonist. 4. At the ileal level the affinity values obtained by Schild and resultant analysis are identical for the ileo-selective antagonist HHSiD as for atropine but not for methoctramine and AF-DX 116. This indicates a mutual binding site with scopolamine for HHSiD and atropine but not for the two cardio-selective antagonists. However, it is worth emphasizing that the difference between affinity values obtained by Schild and resultant analysis is seen when relatively high concentrations are required: a dual mode of interaction (both competitive and allosteric) could be involved.

Topics: Animals; Diamines; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Myocardial Contraction; Parasympatholytics; Piperidines; Pirenzepine

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

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

1. Using a guinea-pig model of allergic asthma, in which the animals display early (0-5 h) and late phase (8-23 h after antigen challenge) bronchoconstrictor reactions, the function of prejunctional inhibitory M2 and postjunctional M3 receptors in isolated tracheal preparations have been investigated. In addition, cardiac M2 receptor function in vitro and bronchial responsiveness to histamine in vivo were evaluated. 2. Sensitivity to inhaled histamine was increased 3.1 fold and 1.6 fold after the early and late allergic reactions (i.e. at 5 h and 23 h after a single ovalbumin challenge), respectively. At 23 h after the last of four allergen challenges, executed on four consecutive days, bronchial hyperresponsiveness to histamine was diminished to 1.3 fold. 3. After the early response, there was no change in cardiac muscarinic M2 receptor function, since in left atria pD2 (-log EC50) and Emax values of pilocarpine and pKB values of AQ-RA 741, a selective M2 receptor antagonist, were not significantly different from controls (unchallenged sensitized animals), and this also applied to methacholine pD2 values for muscarinic M3 receptors in tracheal smooth muscle. 4. Prejunctional inhibitory muscarinic M2 autoreceptors in airway smooth muscle were markedly dysfunctional after the early allergic response, since potentiation of electrically evoked twitch contractions of tracheal preparations by low concentrations of the M2-selective muscarinic receptor antagonists, gallamine, methoctramine, AQ-RA 741 and AF-DX 116, which is the result of M2 receptor blockade, was clearly and significantly diminished compared to controls. However, after the late response, both in single and repeatedly challenged animals, twitch potentiation was not significantly different from and similar to controls, indicating restoration of M2 receptor function during the late allergic reaction.5. It is concluded that dysfunction of muscarinic M2 autoreceptors in the airways of sensitized and challenged guinea-pigs is already present after the early allergic reaction, and that it has recovered after the late response. Since histamine-induced bronchoconstriction involves vagal pathways, the present results suggest that bronchial hyperresponsiveness to histamine is partly due to M2 auto receptor dysfunction, leading to increased release of acetylcholine.

Topics: Administration, Inhalation; Allergens; Animals; Asthma; Benzodiazepinones; Bronchial Hyperreactivity; Diamines; Disease Models, Animal; Electric Stimulation; Female; Gallamine Triethiodide; Guinea Pigs; Heart; Histamine; Hypersensitivity; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle, Smooth; Neuromuscular Junction; Parasympatholytics; Pilocarpine; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

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 effects of muscarinic receptor antagonists were examined in vivo and in vitro on the rabbit urinary bladder. Inhibitory effects on carbachol-evoked contractions of detrusor strips were pronounced for 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; -logIC50: 8.64), p-fluoro-hexahydro-sila-diphenidol (pFHHSiD; 7.84) and atropine (8.27), while they were less pronounced for pirenzepine (6.62) and methoctramine (5.36). 4-DAMP and methoctramine increased 3H overflow from [3H]choline-labelled strips in response to electrical stimulation, contrary to pirenzepine, which decreased the overflow. Concomitant contractions were markedly reduced by 4-DAMP and by pirenzepine, but not by methoctramine. The -logIC50 estimations for atropine-sensitive electrically evoked contractions revealed methoctramine (4.85) to be less potent on nerve-evoked contractions than on carbachol-evoked contractions, in contrast to pirenzepine (7.15) and 4-DAMP (9.15). The effects of the antagonists in anaesthetized rabbits resembled those in vitro. Thus, muscarinic receptors in the rabbit urinary bladder are heterogeneous; prejunctional facilitatory (M1) and inhibitory (M2) for acetylcholine release, and postjunctional muscarinic M3 receptors mediating contractile responses.

Topics: Acetylcholine; Animals; Carbachol; Diamines; Female; Gallamine Triethiodide; Muscarinic Antagonists; Muscle Contraction; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Tritium; Urinary Bladder

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

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.

Topics: Animals; Atrial Function; Benzodiazepines; Benzodiazepinones; Diamines; Drug Design; Guinea Pigs; Heart Atria; Ileum; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Structure-Activity Relationship; Submandibular Gland; Trachea

Human muscarinic receptors comprise a family of five separate gene products, three of which (designated as M1, M2 and M3 subtypes) can be distinguished pharmacologically. Previous work indicates that sympathetic nerve terminals in the anterior uvea contain prejunctional muscarinic receptors that, upon activation by agonists, inhibit the neural release of norepinephrine. The aim of this study was to characterize the prejunctional effects of muscarinic agents on the electrically-evoked secretion of 3H-norepinephrine in isolated, superfused human iris-ciliary body tissue segments. Stimulation-evoked 3H-norepinephrine release was inhibited > 80% by carbachol and muscarine, but was unaffected by the M1-selective agonist McN A-343. Pilocarpine behaved as a partial agonist in this system, producing less than 40% of the maximum inhibition. The rank order of potency of selective antagonists at prejunctional muscarinic receptors was methoctramine (M2) > AF-DX 116 (M2) > pirenzepine (M1) > or = para-fluoro hexahydro-sila-definidol (M3). These data suggest that prejunctional muscarinic receptors in the human iris-ciliary body correspond to the M2 subtype. No evidence for age-related differences in prejunctional muscarinic receptor activity was seen in tissues obtained from 13 human donors, aged 10-83 years. Prejunctional muscarinic receptors may play a role in mediating the inhibitory effects of parasympathetic nerve stimulation or cholinomimetic drugs on ocular sympathetic neurotransmission in vivo.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Carbachol; Child; Ciliary Body; Diamines; Dose-Response Relationship, Drug; Electric Stimulation; Humans; In Vitro Techniques; Iris; Middle Aged; Muscarine; Norepinephrine; Pilocarpine; Piperidines; Pirenzepine; Receptors, Muscarinic

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 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

1. In guinea-pig and canine airway smooth muscle, there is reduced beta-adrenoceptor agonist sensitivity in tissues pre-contracted with muscarinic agonists when compared to tissues pre-contracted with other spasmogens, such as histamine or leukotriene D4. This reduced sensitivity may be the result of an interaction between muscarinic receptors and beta-adrenoceptors. In this study the effects of M2 receptor antagonism and stimulation have been investigated on the relaxant potency of isoprenaline in guinea-pig isolated tracheal smooth muscle. 2. (+)-cis-Dioxolane contracted isolated tracheal strips in a concentration-dependent manner (EC50 = 11.5 +/- 0.9 nM). The rank order of antagonist apparent affinities (with pA2 values in parentheses) was atropine (9.4 +/- 0.1) > zamifenacin (8.2 +/- 0.1) > para-fluoro-hexahydro-siladiphenidol (p-F-HHSiD, 7.2 +/- 0.1) > pirenzepine (6.5 +/- 0.1) > methoctramine (5.5 +/- 0.1). Schild slopes were not significantly different from unity. This was consistent with a role of muscarinic M3 receptors in mediating contraction. 3. In tissues pre-contracted to 3 g isometric tension using (+)-cis-dioxolane (0.2 microM, approximately EC80), the relaxant potency of isoprenaline was significantly (P < 0.05) increased by 0.3 microM methoctramine (control EC50 = 32.2 +/- 4.3 nM, plus methoctramine EC50 = 19.1 +/- 4.5 nM). This concentration of methoctramine had no effect on contractile responses to (+)-cis-dioxolane (control, EC50 = 17.6 +/- 3.2 nM, plus methoctramine, EC50 = 21.0 +/- 4.4 nM). 4 When acetylcholine (non-selective), (+)-cis-dioxolane (non-selective), L-660,863 ((+/- )-3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine, M2-selective) or SDZ ENS 163 (thiopilocarpine, mixed M2 antagonist,partial M3 agonist) were used to achieve isometric tensions of 3 g, the relaxant potency of isoprenaline ranged from 3.7 +/- 0.3 nM (SDZ ENS 163) to 49.4 +/- 3.2 nM ((+)-cis-dioxolane). Reducing the concentration of these agonists (and therefore the level of developed tension to 2 g), significantly(P<0.05) increased the relaxant potency of isoprenaline. In contrast, when histamine was used to pre-contract tissues to either 2 or 3 g (EC50 = 4.2 +/- 0.6 and 3.8 +/- 1.1 nM, respectively), there was no significant effect on the relaxant potency of isoprenaline.5. There was a slight but significant (P<0.05) reduction in the relaxant potency of isoprenaline, in tissues pre-contracted to 3 g using histamine in combination with (+ )-cis-dioxolane (30 n

Topics: Animals; Diamines; Dioxolanes; Guinea Pigs; Histamine; In Vitro Techniques; Isometric Contraction; Isoproterenol; Male; Muscarinic Antagonists; Muscle Relaxation; Muscle, Smooth; Parasympatholytics; Piperidines; Receptors, Muscarinic; Trachea

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

Changes induced in the sleep-wake cycle by pontine microinjections of muscarinic antagonists were studied in freely moving rats, instrumented for chronic polygraphic recordings. Pirenzepine (PIR), methoctramine (MET) and p-fluoro-hexahydro-siladifenidol (p-F-HHSiD), which are highly selective M1, M2 and M3 antagonists, respectively, were dissolved in 0.1 microliter of sterile isotonic saline (0.2 microliter of distilled water for p-F-HHSiD) and injected into the pontine reticular nucleus, where the administration of 0.5 microgram carbachol (a mixed muscarinic agonist) induced a 52% increase in the amount of desynchronized sleep (DS) over a 6 h recording period. The blockade of M2 receptors was shown to (i) antagonize DS, by increasing its latency and decreasing its percentage, (ii) decrease slow wave sleep, and (iii) enhance wakefulness. These effects were dose-dependent. No changes in the sleep-wake cycle were observed following microinjection of M1 or M3 antagonists. The results support the hypothesis that at the brain stem level only M2 receptors are involved in sleep mechanisms and, particularly, in the generation and maintenance of DS.

Topics: Animals; Brain Stem; Carbachol; Diamines; Electroencephalography; Male; Microinjections; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Polysomnography; Rats; Reticular Formation; Sleep; Wakefulness

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

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

1. The role of muscarinic M2 and M3 receptors in ileal smooth muscle has been evaluated by use of selective receptor alkylation. The alkylating agents, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP mustard) was studied for effects against (+)-cis-dioxolane, at muscarinic M2 and M3 receptors in guinea-pig atria or ileum, respectively. 4-DAMP mustard (10 nM, 40 min exposure) did not discriminate between these muscarinic receptors. In ileum, 4-DAMP mustard, at 100 nM, resulted in a large dextral shift (197 fold) and depression in maxima. In atria there was a smaller dextral shift (14 fold) but no depression in maxima. 2. The muscarinic antagonists, atropine (non-selective), methoctramine (M2-selective) and para-fluorohexahydro-siladiphenidol (pFHHSiD; M3 selective) were studied in protection studies against alkylation by phenoxybenzamine. Washout studies following equilibration of the tissues with atropine (30 nM), methoctramine (0.3 microM) or pFHHSiD (3 microM), showed the compounds to be reversible. No temporal changes in sensitivity to (+)-cis-dioxolane were observed. 3. Exposure, for 20 min, of atria and ileum to phenoxybenzamine (3 and 10 microM respectively) caused dextral shifts and depressions in the maxima of the concentration-response curve to (+)-cis-dioxolane. These effects were inhibited by prior equilibration with atropine (30 nM) and methoctramine (0.1 microM) in atria or atropine (30 nM) and pFHHSiD (3 microM) in ileum. Similar results in ileum were obtained when pilocarpine was used as the agonist. 4. These data were consistent with muscarinic M2 receptors mediating responses in atria and M3 receptors mediating responses in ileum. No evidence was provided for a direct role of muscarinic M2 receptors in ileal contraction.5. It is concluded that receptor protection by reversible antagonists for muscarinic M2 or M3 receptors provides a means to isolate pharmacologically a single subtype in a tissue possessing heterogeneous populations. This technique may prove useful in defining the role of the respective subtypes in smooth muscle contraction.

Topics: Alkylating Agents; Animals; Atropine; Diamines; Dioxolanes; Diphenylacetic Acids; Guinea Pigs; Heart; Heart Atria; Ileum; In Vitro Techniques; Isometric Contraction; Male; Muscarinic Antagonists; Muscle, Smooth; Parasympatholytics; Phenoxybenzamine; Pilocarpine; Piperidines; Receptors, Muscarinic

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

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Astrocytoma; Atropine; Carbachol; Diamines; Humans; Hydrolysis; Parasympathomimetics; Phosphatidylinositols; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine; Tumor Cells, Cultured

Overflow of endogenous noradrenaline (NA) from guinea-pig isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses). The muscarinic receptor agonist oxotremorine inhibited the evoked overflow of NA in a concentration-dependent manner (EC50 84 nM). Methoctramine, pirenzepine and p-fluoro-hexahydrosiladiphenidol (each 1 microM) shifted the concentration-response curves of oxotremorine to the right with apparent pA2 values of 7.60, 6.74 and 6.18, respectively. It is concluded that sympathetic nerve terminals in the guinea-pig trachea are endowed with inhibitory muscarinic M2 receptors.

Topics: Animals; Diamines; Dose-Response Relationship, Drug; Female; Guinea Pigs; In Vitro Techniques; Male; Muscarinic Antagonists; Norepinephrine; Oxotremorine; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

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

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

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

Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE-induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro-sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1-piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Antihypertensive Agents; Aorta, Abdominal; Arecoline; Atropine; Diamines; Endothelium, Vascular; Hexamethonium; Hexamethonium Compounds; Indomethacin; Male; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Norepinephrine; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic

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

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 antimuscarinic properties of the methoctramine with high selectivity for cardiac muscarinic M2 receptors were investigated on cholinergically induced changes in prostaglandin (PG) synthesis and mechanical function in the isolated perfused rabbit heart. Acetylcholine (ACh)- and arecaidine propargyl ester (APE)-induced increases in PG synthesis were significantly attenuated by methoctramine in a concentration-dependent manner. Methoctramine at a low concentration of 0.1 microM potentiated ACh-induced PG synthesis, which was blocked by simultaneous infusion of hexahydro-sila-difenidol (HHSiD), a M3 receptor antagonist. Methoctramine produced an additive effect with HHSiD in diminishing the ACh- or APE-induced PG synthesis. Methoctramine displayed a potent antagonistic activity at M2 receptors that mediate the decrease in heart rate and increase in coronary perfusion pressure in isolated perfused rabbit heart. Methoctramine also minimized ACh- and APE-induced decrease in developed tension. In contrast, at 0.1-0.75 microM it exhibited no antagonistic activity at vascular muscarinic receptors (M3) mediating vasodilation in response to ACh or APE. These data suggest that methoctramine has a high affinity for cardiac M2 receptors mediating increases in PG output and coronary perfusion pressure as well as decrease in heart rate and developed tension and has a low affinity for M3 receptors mediating coronary vasodilator response.

Topics: 6-Ketoprostaglandin F1 alpha; Acetylcholine; Animals; Arachidonic Acid; Arachidonic Acids; Arecoline; Coronary Circulation; Diamines; Heart; Heart Rate; In Vitro Techniques; Isoproterenol; Male; Myocardium; Parasympatholytics; Piperidines; Prostaglandins; Rabbits; Receptors, Muscarinic

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

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

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

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

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

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

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

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

The possibility of an allosteric interaction by himbacine, a cardioselective antagonist, with rat cardiac muscarinic receptors was studied. Himbacine allosterically decelerated the dissociation of bound [3H]N-methylscopolamine [( 3H]NMS) in a concentration-dependent manner with an IC50 value of 103.7 microM. When compared to the IC50 values of other cardioselective antagonists, the rank order of potencies was: methoctramine greater than gallamine greater than himbacine greater than AF-DX 116. In contrast, the potencies of these compounds to displace [3H]NMS binding were: himbacine greater than methoctramine greater than AF-DX 116 greater than gallamine. The allosteric potencies were found not to be correlated with binding potencies (correlation coefficient = -0.15). A striking common feature of the cardioselective antagonists is their ability to bind to an allosteric site on cardiac muscarinic receptors.

Topics: Alkaloids; Allosteric Regulation; Animals; Atropine; Binding, Competitive; Diamines; Dose-Response Relationship, Drug; Furans; Gallamine Triethiodide; Male; Myocardium; N-Methylscopolamine; Naphthalenes; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.

Topics: Alkaloids; Animals; Brain; Diamines; Furans; Humans; Hybrid Cells; N-Methylscopolamine; Naphthalenes; Parasympatholytics; Piperidines; Rats; Receptors, Muscarinic; Scopolamine Derivatives; Tumor Cells, Cultured

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

In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a series of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M1-(rabbit vas deferens), M2- (guinea-pig atria) and M3- (guinea-pig ileum) receptors. All compounds were competitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenyl ring with a methoxy group or a chlorine atom as well as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 approximately M3 greater than M2. A different selectivity pattern was observed for p-fluoro-hexahydro-sila-difenidol: M3 greater than M1 greater than M2. This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA2 = 6.68) and lowest affinity for the M2-receptors in guinea-pig atria (pA2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2) and ileum (M3) of the rat. Furthermore, dose ratios obtained with either pirenzepine (M1) or hexahydrosila-difenidol (M2 and M3) and the p-fluoro analogue used in combination suggested that the antagonism was additive, implying mutual competition with a single population of muscarinic receptor subtypes. These results indicate that p-fluoro-hexahydro-sila-difenidol represents a valuable tool for characterization of muscarinic receptor subtypes.

Topics: Animals; Diamines; Female; Ganglia, Sympathetic; Guinea Pigs; Heart; Ileum; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Piperidines; Pirenzepine; Rabbits; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Vas Deferens

Two novel muscarinic antagonists, methoctramine and hexahydrodifenidol, have been assessed for their action against two muscarinic agonist-induced responses on the rat superior cervical ganglion in vitro. DC recordings were made between the desheathed ganglion and its internal carotid nerve using the grease-gap technique. Hexahydrodifenidol and methoctramine antagonised the muscarine-induced M1-mediated depolarisation of this preparation with estimated pA2 values of 7.5 and 6.5, respectively. In 0.3 microM pirenzepine and 0.1 mM CaCl2, 1 microM muscarine evoked a hyperpolarisation mediated by cardiac-like M2 receptors. Hexahydrodifenidol and methoctramine antagonised this response with pIC50 values (-log10IC50) of 5.7 and 7.4, respectively. The selectivity of methoctramine for cardiac-like M2 receptors over M1 receptors is therefore confirmed and extended to these two neuronal responses. The selectivity of hexahydrodifenidol was opposite to, and greater than, that seen with methoctramine.

Topics: Animals; Diamines; Ganglia, Sympathetic; In Vitro Techniques; Male; Membrane Potentials; Muscarine; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic

1. The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations. 2. Methoctramine (1 x 10(-6)-3.2 x 10(-4) M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10(-6) M) but were antagonized by propranolol (1 x 10(-6) M). 3. Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10(-6) M, the pKB value for methoctramine was 7.88 +/- 0.15 (mean +/- s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 +/- 0.06 (n = 3). 4. At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 +/- 0.05 and 6.03 +/- 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 +/- 0.05 and 7.57 +/- 0.04 respectively, n = 4). 5. Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 +/- 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria. The pA2 values for himbacine at endothelial muscarinic receptors were approximately 0.5 pA2 units lower than those observed at muscarinic receptors in smooth muscle (6.92 + 0.80, n = 6). In addition, the Schild slopes for methoctramine and himbacine at these receptors were significantly (P < 0.05) less than unity. 6. Methoctramine, and to a lesser extent himbacine, are potent and selective antagonists for cardiac muscarinic receptors. However, caution should be used in interpretation of the data with methoctramine in view of the inhibitory allosteric properties and direct inotropic actions of this compound.

Topics: Alkaloids; Animals; Diamines; Drug Interactions; Endothelium; Furans; Guinea Pigs; Heart Atria; In Vitro Techniques; Male; Muscle, Smooth; Naphthalenes; Piperidines; 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