piperidines and 4-fluorohexahydrosiladifenidol

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

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

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

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

Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by approximately 40% (P<0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively (P<0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n=25) whereas half of colon cancer specimens (n=24) exhibited moderate to strong staining (P<0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.

Topics: Acetylcholine; Acetylcholinesterase; Atropine; Autocrine Communication; Caco-2 Cells; Cell Proliferation; Choline; Choline O-Acetyltransferase; Cholinergic Agents; Cholinesterase Inhibitors; Chromatography, High Pressure Liquid; Colonic Neoplasms; Dose-Response Relationship, Drug; Electrochemistry; Hemicholinium 3; HT29 Cells; Humans; Immunohistochemistry; Membrane Transport Proteins; Muscarinic Antagonists; Physostigmine; Piperidines; Polymerase Chain Reaction; Receptor, Muscarinic M3; RNA, Messenger; Tacrine

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

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.

Topics: Animals; Atropine; Chorda Tympani Nerve; Electric Stimulation; Female; Immunohistochemistry; Muscarinic Antagonists; Piperidines; Pirenzepine; Receptor, Muscarinic M1; Receptor, Muscarinic M4; Receptor, Muscarinic M5; Salivation; Sheep; Submandibular Gland; Time Factors; Vasodilation

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

1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague-Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p-F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominantly mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions.

Topics: Age Factors; Animals; Benzofurans; Carbachol; Denervation; Disease Models, Animal; Electric Stimulation; Female; Gene Expression Regulation; Humans; Hypertrophy; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Pyrrolidines; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; RNA, Messenger; Urinary Bladder; Urinary Bladder Neck Obstruction; Urinary Bladder, Neurogenic

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

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

Laterodorsal (LDT) and pedunculopontine (PPT) tegmental nuclei in the mesopontine project cholinergic inputs to the midbrain ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), respectively, to directly and indirectly influence the activity of dopamine neuronal cells via actions on muscarinic and nicotinic receptors. The present study investigated the role of midbrain muscarinic receptors in the functional modulation of VTA and SNc dopamine cell activity as reflected by alterations in, respectively, nucleus accumbens (NAc) and striataldopamine efflux. In vivo chronoamperometry was used to measure changes in basal dopamine efflux via stearate-graphite paste electrodes implanted unilaterally in the NAc or striatum of urethane-anaesthetized rats, following blockade or activation of, respectively, VTA or SNc muscarinic receptors. Intra-VTA or -SNc infusion of the muscarinic antagonist scopolamine (200 microg/microL) reduced, respectively, NAc and striatal dopamine efflux while infusion of the muscarinic and nicotinic agonist carbachol (0.5 microg/microL) or the prototypical muscarinic agonist muscarine (0.5 microg/microL) increased NAc and striatal dopamine efflux. Transient decreases in dopamine efflux preceded these increases selectively in the striatum, suggesting a reduction in excitatory or increase in inhibitory drive to the SNc by preferential activation of M3 muscarinic receptors on GABA interneurons and glutamatergic inputs. This was confirmed by showing that selective blockade of M3 receptors with p-F-HHSiD (0.5 microg/microL) increased striatal, but not NAc, dopamine efflux. Together, these findings suggest that midbrain muscarinic receptors, probably M5 subtypes on VTA and SNc dopamine neurons, contribute to the tonic excitatory regulation of forebrain basal dopamine transmission whereas presynaptic M3 receptors serve to counter excessive excitation of nigral dopamine cell activity.

Topics: Animals; Carbachol; Cholinergic Agonists; Dopamine; Drug Interactions; Electrochemistry; Electrodes; Male; Muscarinic Antagonists; Nucleus Accumbens; Piperidines; Rats; Rats, Wistar; Receptors, Muscarinic; Scopolamine; Substantia Nigra; Synaptic Transmission; Ventral Tegmental Area

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

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

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

M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.

Topics: Animals; Carbachol; Cholinergic Agents; Denervation; Female; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic; Second Messenger Systems; Signal Transduction; Urinary Bladder; Urinary Incontinence

We previously observed that noradrenaline (NA)-induced contraction of the portal vein of rabbit was relaxed by the antimuscarinic drugs of atropine sulfate, but not scopolamine hydrobromide. In the present study we examined the possible effect of the antimuscarinic drugs of atropine sulfate, scopolamine hydrobromide, p-fluoro-hexa-hydro-sila-difenidol ( p-F-HHSiD, the M(3)-receptor antagonist) and pirenzepine (the M(1)-receptor antagonist) on alpha(1)-adrenoceptor (AR). Atropine and p-F-HHSiD relaxed the alpha(1)-AR agonist methoxamine-induced contraction of the rabbit portal vein in a concentration-dependent manner; however, scopolamine and pirenzepine had no such inhibitory effect. Radioligand binding studies with the alpha(1)-AR ligand 2-[2-(4-hydroxy-3-[(125)I]iodo-phenyl)ethylaminomethyl]-alpha-tetralone ([(125)I]HEAT) in membrane preparations from mouse whole brain showed that atropine (p K(i)=5.33) and p-F-HHSiD (p K(i)=5.88) had higher affinities than scopolamine (p K(i)=3.17) and pirenzepine (p K(i)<2.70). Furthermore, atropine and p-F-HHSiD had higher affinities for all human cloned alpha(1)-ARs than scopolamine and pirenzepine. The results show that the antimuscarinic drugs atropine and p-F-HHSiD have a direct but weak antagonistic activity against alpha(1)-ARs.

Topics: Adrenergic alpha-1 Receptor Antagonists; Animals; Atropine; Brain; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Muscarinic Antagonists; Piperidines; Rabbits; Receptors, Adrenergic, alpha-1; Vasodilation

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

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

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 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

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

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

Isolated human bronchi and rat tracheae were incubated in organ baths to measure histamine release. The calcium ionophore A23187, 3 micromol/L in rat trachea and 10 micromol/L in human bronchi, stimulated histamine release by 145 +/- 50% (n = 6) and 270 +/- 48% (n = 7) above the prestimulation level, respectively. Acetylcholine (100 pmol/L; human bronchi) or oxotremorine (1, 100, 10,000 nmol/L; rat trachea) did not affect the spontaneous histamine release. In rat tracheae neither acetylcholine nor oxotremorine inhibited A23187-evoked histamine release, whereas 100 pmol/L acetylcholine significantly suppressed the evoked histamine release in human bronchi by 86%. For receptor characterization the following subtype-specific muscarinic receptor antagonists were applied: pirenzepine (M1 subtype), para-fluorohexahydrosiladifendiol (pFHHSiD; similar affinities at human cloned M1-, M3-, and M4-receptors), AF-DX 116 (M2 subtype), and clozapine (antagonist at cloned M1-, M2-, M3-receptors; agonist at cloned M4-receptors). Pirenzepine, pFHHSiD, AF-DX 116, and clozapine (100 nmol/L each) antagonized the inhibitory effect of 100 pmol/L acetylcholine by 83 +/- 20% (n = 6), 83 +/- 9% (n = 8), 50 +/- 14% (n = 6), and 35 +/- 7% (6). In conclusion, a species difference exists in the cholinergic control of histamine release between human and rat airways. In human airways muscarinic receptors most likely of the M1 subtype are involved in the inhibitory control of mast cell function, whereas such an inhibitory pathway does not exist in the rat trachea.

Topics: Acetylcholine; Animals; Bronchi; Calcimycin; Clozapine; Female; Histamine Release; Humans; Ionophores; Male; Mast Cells; Muscarinic Antagonists; Organ Culture Techniques; Oxotremorine; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Species Specificity; Trachea

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 the present study was to investigate whether muscarinic M1 receptor activation induces intestinal relaxation via nerve-dependent nitric oxide formation. Mechanical activity in longitudinal segments of rat jejunum was recorded isotonically in organ baths. The muscarinic M1 receptor agonist 4-[[[(3-Chlorophenyl)amino]carbonyl]oxy]-N,N,N,-trimethyl-2-butyn- 1-ammonium chloride (McN-A-343, 10(-7)-10(-4) M) induced a concentration-dependent relaxation of rat jejunum. Relaxations induced by McN-A-343 (10(-5) M) were inhibited by the M1 receptor antagonist telenzepine (10(-8) M), and enhanced by the M3 receptor antagonist para-fluorohexahydrosiladifenidol (p-F-HHSiD; 3x10(-7) M). The inhibitory responses induced by McN-A-343 were abolished by the nitric oxide synthase inhibitors Nomega-nitro-L-arginine (L-NOARG; 10(-4) M) and Nomega-monomethyl-L-arginine (L-NMMA; 3x10(-5) M), the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 10(-5) M), and by tetrodotoxin (TTX; 3x10(-7) M). Guanethidine or hexamethonium did not affect inhibitory responses induced by McN-A-343. In conclusion, McN-A-343 induces nerve-dependent, nitrergic relaxations in rat jejunum, via activation of muscarinic M1 receptors. Hence, selective muscarinic M1 receptor agonists or antagonists might offer possibilities for pharmacological manipulation of the NO system.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Enzyme Inhibitors; In Vitro Techniques; Isotonic Contraction; Jejunum; Muscarinic Agonists; Muscarinic Antagonists; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Receptors, Muscarinic

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

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

The 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 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

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

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

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

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

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

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

The laryngeal chemoresponse (LCR), comprising laryngeal adductor spasm, central apnea, and subsequent cardiovascular instability, is thought to be a factor in sudden infant death syndrome. A muscarinic subtype receptor, M3, appears to be involved in central respiratory drive and control. Both the duration of the LCR apnea and levels of M3 receptor messenger RNA in the brain stem change according to postnatal age. This study examined the effect of central nervous system antagonism at M3 receptors on the LCR with respect to animal age and dose of antagonist. Ten piglets in each of three age groups (group 1, 5 to 8 days; group 2, 18 to 21 days; and group 3, 40 to 43 days) received a series of four increasing doses of an M3 antagonist (p-fluoro-hexahydro-sila-diphenidol) by intracerebral ventricle injection. The LCR was evoked at baseline and after each dose of antagonist. An effect on susceptible animals (groups 1 and 2) was evident by the second antagonist dose, and persisted for the remainder of the experiment (2 hours). At completion of the experiment, mean apnea duration had decreased in group 1 (61%, p < .05), and group 2 (57%, p < .05), but was unchanged in group 3 (<10%, p not significant). Length of mean baseline apneas correlated directly with degree of apnea shortening. The reduction is not attributable to changes in arterial PO2 or PCO2 or baseline respiratory rate. These results support an age-related influence on the LCR by M3 receptors in younger animals that decreases with maturation.

Topics: Age Factors; Animals; Animals, Newborn; Apnea; Chemoreceptor Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Hemodynamics; Humans; Infant, Newborn; Laryngismus; Larynx; Muscarinic Antagonists; Piperidines; Receptor, Muscarinic M3; Receptors, Muscarinic; Sudden Infant Death; Swine; Time Factors

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

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

To clarify which muscarinic receptor subtype(s) mediate changes in sleep and cortical temperature (Tcort) induced by carbachol microinjections into the medial preoptic area (MPA), pirenzepine, tripitramine and +/- p < > -fluorohexahydro-sila-difenidol (p-F-HHSiD), which are highly selective muscarinic M1, M2 and M3 antagonists, respectively, were microinjected into the MPA of rats. Whereas pirenzepine (3.45 and 7.08 nmol) and p-F-HHSiD (3.90 and 7.80 nmol) were without effect, tripitramine (0.67 and 3.37 nmol) enhanced wakefulness, decreased slow wave and desynchronized sleep, and raised Tcort with the higher dose. The data suggest that in the MPA only M2 muscarinic subtypes may be functionally important in mediating the cholinergic effects on sleep and thermoregulation.

Topics: Animals; Benzodiazepines; Body Temperature Regulation; Male; Microinjections; Muscarinic Antagonists; Piperidines; Pirenzepine; Preoptic Area; Rats; Receptors, Muscarinic; Sleep; Sleep, REM; Wakefulness

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

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

The 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

1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H-compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with [14C]-prostaglandin E2([14C]-PGE2) and [3H]-PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]-arachidonic acid remained unaffected. 4. Using an acetonitril gradient separation it was shown that [3H]-PGE2, [3H]-PGD2 and [3H]-PGF2alpha are released spontaneously, but [3H]-PGE2 represented the major fraction of 3H-prostaglandins. Acetylcholine enhanced the release of all three 3H-prostaglandins, but the effect on PGE2 was most pronounced and most consistent. 5. After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H-prostaglandins ([3H]-PGE2/PGD2 peak) was abolished. 6. Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa-dependent manner. 7. After inhibition of cyclo-oxygenase by 3 microM indomethacin, the outflow of 3H-prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release [3H]-arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 microM) also blocked the effect of acetylcholine on the outflow of 3H-prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]-arachidonic acid. 8. In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.

Topics: Acetonitriles; Acetylcholine; Animals; Arachidonic Acid; Dose-Response Relationship, Drug; Drug Interactions; Female; In Vitro Techniques; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Prostaglandins; Rats; Rats, Sprague-Dawley; Trachea

We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I gamma promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I gamma promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays.

Topics: Amino Acid Sequence; Animals; Base Sequence; Benzodiazepinones; Cell Membrane; Dictyostelium; Discoidins; Fungal Proteins; Gene Expression; Humans; Lectins; Molecular Sequence Data; Muscarinic Antagonists; Parasympatholytics; Piperidines; Promoter Regions, Genetic; Protein Sorting Signals; Protozoan Proteins; Receptors, Muscarinic

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

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

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

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

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

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

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

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

Muscarinic receptor subtypes have been identified on airways of several mammalian species, including humans. They are distributed in the lung over submucosal glands, airway ganglia and over the nerves; M1-receptors are found in airway ganglia, M2-receptors (autoreceptors) are present in cholinergic postganglionic nerves at the prejunctional level, whereas the muscarinic receptor subtypes found in airway smooth muscle are of the M3-receptor subtype. We had previously reported the presence of M1-receptors on the smooth muscle of large and small airways of patients suffering from chronic bronchitis, but only is the peripheral airways of healthy subjects; one possible explanation of these results is that the bronchial epithelium may play a role in bronchial responsiveness to relaxant muscarinic antagonists. To clarify this hypothesis we have studied in vitro the responses to acetylcholine (ACh), and some muscarinic selective (pirenzepine, PZ; p-fluorohexahydrosiladyphenidol, pFHHSiD) and non-selective (atropine, ATR) antagonists, on human bronchial smooth muscle in the presence and in the absence of epithelium. The absence of epithelium did not modify the responsiveness to ACh to any great extent and did not change the responsiveness to relaxant antagonists. Our results show that the M1-receptors may play a role in mediating contraction of airway smooth muscle; research is in progress regarding their function in disease.

Topics: Acetylcholine; Atropine; Bronchi; Epithelium; Humans; In Vitro Techniques; Muscarinic Antagonists; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic

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

The "antimuscarinic effect" of fentanyl and its dependence on subtypes of receptors were characterized in isolated porcine coronary arteries. Left anterior descending coronary arteries were dissected from the hearts of 60 adult pigs obtained at a slaughterhouse and prepared for isometric tension studies. The effects of fentanyl on the cumulative concentration-response curve for acetylcholine were obtained in the presence and absence of muscarinic blockade by atropine. Fentanyl shifted the concentration-response curve to the right in a concentration-dependent fashion. Atropine shifted the concentration-response curve to the right, and no further shift was caused by fentanyl. To investigate the dependence on muscarinic receptor subtypes, the effect of fentanyl on acetylcholine-induced contraction was examined in the presence of specific M1-, M2-, and M3-muscarinic antagonists. The pA2 values for fentanyl decreased significantly in the presence of atropine (a nonspecific antagonist) and also in the presence of p-F-HHSiD (an M3-antagonist). In contrast, no significant change of pA2 value for fentanyl was observed in the presence of both pirenzepine (an M1-antagonist) and methoctramine (an M2-antagonist). We conclude that fentanyl has an antimuscarinic effect, and that this antagonism occurs in a competitive manner. Furthermore, the significant decrease of the pA2 value for fentanyl in the presence of M3-, but not in the presence of M1 + M2-antagonists, suggests that the attenuation of cholinergic contraction of porcine coronary arteries by fentanyl is mediated through the M3-muscarinic receptor subtype.

Topics: Acetylcholine; Animals; Atropine; Coronary Vessels; Depression, Chemical; Female; Fentanyl; In Vitro Techniques; Male; Piperidines; Pirenzepine; Receptors, Muscarinic; Swine; Vasoconstriction

Muscarinic cholinergic receptor function in rat brain cortex was characterized by performing binding assays with [3H](-)quinuclidinyl benzilate ([3H]QNB) in parallel with assays of phospholipase C (PLC) activation by carbachol using membrane preparations and exogenous [3H]-phosphatidylinositol 4,5-bisphosphate ([3H]PIP2). Competitive binding studies revealed high- and low-affinity binding sites for the receptor antagonists, pirenzepine, methoctramine and the p-fluoro analog of hexahydro-sila-difenidol (p-F-HHSiD). Carbachol-stimulated [3H]-phosphatidylinositol 4,5-biphosphate breakdown was specifically inhibited by pirenzepine and p-F-HHiSD. The inhibition curves for these antagonists were best described by interactions at two sites. There was quantitative agreement between the antagonist affinity constants and the proportion of high- and low-affinity sites derived in functional and binding studies. The characteristics of the putative subtypes of muscarinic receptors and their stimulation of phospholipase C was examined after treatment with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and propylbenzilylcholine mustard. Loss of receptors was closely correlated with loss of PLC activation by carbachol, without alteration of the EC50 value (21 microM) of this agonist, clearly demonstrating a lack of receptor reserve. When both alkylating treatments were adjusted to induce a decrease of 60% in the maximal number of [3H]QNB binding sites, a similar (60%) reduction in the maximal effect of carbachol on PLC activation was found. However, the characteristics of the remaining receptors after the treatment with the two alkylating agents differ markedly as determined by competition of pirenzepine, p-F-HHSiD and methoctramine for [3H]QNB binding, and for inhibition of carbachol-stimulated phospholipase C by pirenzepine and p-F-HHSiD.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkylating Agents; Animals; Carbachol; Cerebral Cortex; Enzyme Activation; In Vitro Techniques; Male; Mustard Compounds; Piperidines; Pirenzepine; Propylbenzilylcholine Mustard; Quinolines; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Type C Phospholipases

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

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

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

P-fluoro-hexahydro-sila-difenidol hydrochloride (p-F-HHSiD) (15, 30 micrograms) and pirenzepine (7.5, 15, 30 micrograms), which are highly selective M3 and M1 muscarinic antagonists, respectively, were injected intracerebroventricularly into freely moving rats. p-F-HHSiD (30 micrograms) reduced wakefulness (W) (from 34.7 +/- 3.1 to 24.9 +/- 1.3 min) and increased slow wave sleep (SWS) (from 56.7 +/- 2.4 to 67.2 +/- 1.5 min); however, it did not modify desynchronized sleep (DS) latency and percentage in 6 h recordings. W and SWS were not affected by pirenzepine (7.5, 15, 30 micrograms) which decreased significantly DS amount but left unaffected DS latency. The results suggest that each muscarinic receptor subtype may induce different and specific changes in sleep phases and cortical desynchronization processes.

Topics: Animals; Injections, Intraventricular; Male; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Sleep; Wakefulness

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

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

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

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

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

In this study the effects of muscarinic antagonists and agonists on M1 muscarinic receptors in the isolated rat superior cervical ganglion and the rat hippocampal slice were investigated. Oxotremorine and APE but not pilocarpine, McN-A-343 or 4-Cl-McN-A-343 induced small M2 muscarinic receptor-mediated hyperpolarizations in the rat superior cervical ganglion. Nevertheless, for all the agonists investigated the pA2 values of the muscarinic antagonists pirenzepine, AF-DX 116 and p-fluoro-hexahydro-sila-difenidol indicated the presence of only M1 muscarinic receptors in the rat superior cervical ganglion and hippocampal slice. Full agonistic behaviour with respect to depolarization of the rat superior cervical ganglion was observed for pilocarpine, McN-A-343 and 4-Cl-McN-A-343. Oxotremorine and arecaidine propargyl ester were partial agonists in this preparation, with maximal effects of 35 and 46% of the maximum obtained with pilocarpine, respectively. Pilocarpine, oxotremorine and arecaidin propargyl ester displayed full agonistic behaviour on the increase in firing rate of pyramidal cells in rat hippocampal slices. Whereas 4-Cl-McN-A-343 was a partial agonist (maximal effect of 63% of the maximum obtained with pilocarpine), McN-A-343 displayed no agonistic or antagonistic activity in rat hippocampal slices. It remains to be established whether the heterogeneous behaviour of the agonists in both preparations reflects as yet unknown differences in the M1 receptor protein or results from differences in the coupling of receptor to second messenger.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Arecoline; Ganglia, Sympathetic; Hippocampus; In Vitro Techniques; Male; Oxotremorine; Parasympathomimetics; Pilocarpine; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains

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

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

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

The M3-selective antagonist, p-fluorohexahydro-sila-difenidol was used to characterize muscarinic receptors in two vascular preparations, the rabbit ear artery with an endothelium-dependent relaxation and the bovine coronary artery with an endothelium-independent contractile response. pKB values were consistent with the presence of M3 receptors, 7.9 and 7.5 in coronary and ear arteries, respectively. These findings confirm that muscarinic receptors of the rabbit ear artery and bovine coronary artery have similar characteristics and belong to the M3 subtype.

Topics: Animals; Arteries; Cattle; Coronary Vessels; Ear, External; Female; Heart; In Vitro Techniques; Isometric Contraction; Male; Methacholine Compounds; Muscle, Smooth, Vascular; Piperidines; Rabbits; Receptors, Muscarinic

1. para-Fluoro-hexahydrosila-difenidol (p-F-HHSiD) has been proposed as an M3 selective antagonist. However, the M3 selectivity is variable in that it exhibits a high pA2 value for M3 muscarinic receptors in guinea-pig ileum but a low value at muscarinic receptors in guinea-pig trachea. 2. The pA2 value in the trachea was found to be agonist independent since similar pA2 values were found when acetylcholine, carbachol, (+)-cis-dioxolane or OXA-22 were used (7.13, 7.03, 6.85 and 6.97, respectively). The pA2 value was not meaningfully increased when the equilibrium period was increased from 60 to 180 min. The pA2 value was unaffected by blockade of M1 or M2 receptors, using 0.1 microM pirenzepine or methoctramine (7.03 and 7.14, respectively). p-F-HHSiD and atropine appeared to act at the same site, as adjudged by combination concentration-ratio studies. 3. The pA2 values for p-F-HHSiD vary by 10 fold between ileal (8.0) and tracheal M3 receptors (7.0). The precise reason for this is unknown, but appears to be unrelated to conditions of disequilibrium that could be detected. The antagonist should therefore only be employed to distinguish M3 or M1 from M2 receptors. In this respect, although the M1/M3 vs M2 discrimination is relatively large (68 fold), p-F-HHSiD exhibits similar properties to other putative M3 selective antagonists such as 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) or the parent compound, hexahydrosiladifenidol (HHSiD).

Topics: Animals; Guinea Pigs; In Vitro Techniques; Isometric Contraction; Male; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Parasympathomimetics; Piperidines; Receptors, Muscarinic; Trachea

1. The antagonistic actions of parafluorohexahydrosiladiphenidol (pFHHSiD) at muscarinic receptors has been studied in cardiac muscle, smooth muscle and cell culture preparations. In this paper, the classification scheme of Doods et al. (1987) is employed. This scheme is based upon differential affinities of muscarinic antagonists. pFHHSiD exhibited high pA2 values at M3 receptors mediating contractions of guinea-pig ileum and oesophageal muscularis mucosae (7.8 and 8.2 respectively) whereas low values were determined at M2 receptors mediating negative inotropic responses in guinea-pig atria (6.0). Intermediate pA2 values were determined at M1 receptors mediating contractions of the canine femoral and saphenous veins. 2. The pA2 values of pFHHSiD at receptors mediating endothelial-dependent relaxation of rat aortic rings, rabbit jugular vein and canine femoral artery (7.6-7.9) were similar to those determined on the ileum. However, the pA2 values of pFHHSiD at receptors mediating contractions of the guinea-pig trachea (7.1), which has been previously shown to possess M3 receptors, were different from those determined in the ileum. 3. The similarity in pA2 values of pFHHSiD between the M3 receptors in guinea-pig ileum and the receptors mediating endothelial-dependent relaxations provide further evidence for the role of M3 receptors in this vascular response. Taken together, pA2 values for pFHHSiD range from 7.1 to 8.2, depending upon the M3 preparation used. The selectivity of the compound therefore for the M3 versus the M2 muscarinic receptor ranged from 13 to 163 fold. 4. At muscarinic receptors mediating stimulation of phosphatidylinositol hydrolysis, pFHHSiD paradoxically displayed a high affinity for the M1 receptor in the SH-SY5Y cell line (pA2 = 7.9) as well as for the M3 receptor in the human astrocytoma (1321 NI cell line (pA2 = 7.6). The value at the M1 receptor in SH-SY5Y cells was greater than was observed at M1 receptors mediating contractions of both the canine saphenous and femoral veins (7.1). 5. pFHHSiD, therefore, clearly delineated M3 from M2 muscarinic receptors, whilst the separation between M1 and M3 receptors was variable. The reason for the anomalous affinity estimates in some functional studies remains unclear. These data indicate that the pA2 values for pFHHSiD appear to be tissue-dependent since the M3 selectivity varies according to the preparations studied. As a result the utility of pFHHSiD in muscarinic receptor classificatio

Topics: Animals; Astrocytoma; Cells, Cultured; Dogs; Guinea Pigs; Heart; Humans; In Vitro Techniques; Isometric Contraction; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Muscle, Smooth, Vascular; Parasympatholytics; Phosphatidylinositols; Piperidines; Rabbits; Rats; Rats, Inbred Strains; 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

Topics: Animals; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Myocardium; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Vas Deferens