Characterization of postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle

The present study was designed to characterize the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle by means of a series of muscarinic agonists and subtype-preferring key muscarinic antagonists. Cumulative addition of muscarinic agonists elicited concentration-dependent contractions with the following rank order of potency (pD₂ values): (+)-muscarine (6.36) ≥ oxotremorine M (6.21) ≥ arecaidine propargyl ester (APE) (6.18) > carbachol (5.68)=(±)-methacholine (5.65) > 4-(4-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride (4-Cl-McN-A-343) (4.28) > 4-(3-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) (3.89). (+)-Muscarine, oxotremorine M, carbachol and (±)-methacholine behaved as full agonists, whereas APE, 4-Cl-McN-A-343 and McN-A-343 displayed partial agonism. The contractile responses of the rat anococcygeus muscle to (±)-methacholine were competitively antagonized by pirenzepine (pA₂=6.92), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl] 5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one (AQ-RA 741; pA₂=6.75), himbacine (pA₂=7.11), (±)-p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD; pA₂=7.68) and the (R)- and (S)-enantiomers of hexahydro-difenidol [(R)-HHD: pA₂=8.52; (S)-HHD: pA₂=6.06]. A comparison of the pA₂ values derived from studies of contraction in rat anococcygeus muscle with literature binding (pK_i values) and functional affinities (pA₂ values) obtained at native M₁-M₄ receptors strongly suggests that the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle are of the M₃ subtype. Received: 18 April / Accepted: 18 July 1997     

Autoradiographic analyses of agonist binding to muscarinic receptor subtypes

The binding of four muscarinic receptor agonists to regions of rat brain was examined through quantitative autoradiographic techniques. Oxotremorine, arecoline, pilocarpine and bethanechol were chosen based on their different potencies and efficacies in muscarinic second messenger systems. Overall, the order of potency for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain slices was oxotremorine greater than pilocarpine = arecoline much greater than bethanechol. Regional assays of agonist potency indicated that all agonists were more selective for brainstem and thalamic regions than for hippocampal and cortical regions. The high selectivity of agonists for areas such as the paraventricular thalamus and the superior colliculus, which also display low affinity for pirenzepine, suggests that muscarinic agonists bind with higher affinity to M2 receptors. Of the four agonists examined, pilocarpine displayed the lowest selectivity for M2 receptors in that IC50 values for pilocarpine were only 3-fold higher in the hippocampal and striatal regions (e.g. CA3: 40.6 +/- 9.4 microM) than in thalamic and brainstem regions (e.g. paraventricular thalamus: 14.9 +/- 6.2 microM). Oxotremorine was 8-fold more potent in the brainstem and thalamus, while arecoline and bethanechol were, respectively, 19- and 100-fold more selective for brainstem and thalamic receptors. Scatchard analyses revealed heterogeneous binding profiles for some agonists within single brain regions, suggesting that multiple agonist sites exist even within regions of predominantly M1 or M2 receptors. For example, arecoline displayed curved Scatchard plots within the external layers of the cerebral cortex, layer CA1 of the hippocampus (predominantly M1 subtype), and the paraventricular thalamus (predominantly M2 subtype). The ability of agonists to recognize multiple sites within a single region may reflect the ability to recognize receptors coupled or uncoupled to second messenger systems through G-proteins.     

Comparative pharmacological profile of muscarinic agonists in the isolated ileum, the pithed rat, and the mouse charcoal meal transit test.

1. Several reportedly selective (McN-A-343, M1; RS-86, M2; pilocarpine, M3) and non-selective (oxotremorine, acetylcholine, cis-dioxalone, arecoline, muscarine) muscarinic agonists were examined for comparative pharmacological potency in three diverse models: the guinea pig ileum, the pithed rat, and the mouse charcoal meal transit test. 2. In the guinea pig ileum, all of the compounds examined were associated with concentration-dependent contractions. 3. The apparent order of potency in the isolated ileum was cis-dioxalone greater than acetylcholine greater than oxotremorine greater than arecoline greater than RS-86 greater than pilocarpine greater than McN-A-343. 4. The pA2 values for atropine and pirenzepine in the ileum ranged from 8.4 to 9.4 and 6.1 to 7.7, respectively, indicative of a single receptor, most likely M3. 5. In the mouse charcoal meal transit test, non-selective muscarinic agonists produced dose-dependent increases in gastrointestinal transit, while selective agonists failed to produce any significant changes. 6. Scopolamine methylbromide, a peripherally acting non-selective muscarinic antagonist, significantly reduced the ability of muscarine to increase transit. 7. The compounds were further examined for dose-dependent pressor effects in the pithed rat, which are known to be mediated by stimulation of M1-receptors in sympathetic ganglia. 8. McN-A-343 produced the greatest pressor response, as measured by the percent increase in mean pressure, followed by pilocarpine. 9. Pirenzepine antagonized the pressor response of McN-A-343 and pilocarpine in a dose-dependent manner.     

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

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

Quantitative effects of some muscarinic agonists on evoked surface-negative field potentials recorded from the guinea-pig olfactory cortex slice

1. The effects of muscarinic receptor agonists on the electrically-evoked surface-negative field potential (N-wave) were measured in the guinea-pig olfactory cortex slice maintained in vitro. 2. Bath-superfusion of (+/-)-muscarine, acetylcholine (ACh), carbachol (CCh), or methacholine (MCh) (10-200 microM) produced reversible, dose-dependent depressions of the N-wave (ACh and MCh effects were observed in the presence of 10 microM neostigmine). The order of potencies (based on agonist dose causing 50% field depression: IC50) was: ACh greater than or equal to muscarine greater than CCh greater than MCh. All four agonists depressed the field potential by 100% at doses greater than 500 microM. 3. Pilocarpine and bethanechol were weak agonists and only produced measurable effects at high doses (1-2 mM). Neither agonist evoked a maximum response at doses up to 10 mM. 4. The muscarinic ganglion stimulant, McN-A-343 yielded inconsistent results, depressing the field potential in some slices, but having no effect in others. Pre-application of a conditioning dose (100 microM) of McN-A-343 reduced subsequent responses to CCh, suggesting possible partial agonist properties. 5. Oxotremorine (up to 100 microM) did not depress the field potential, but it reversibly antagonized the effects of CCh. 6. It is concluded that reproducible, quantifiable responses to muscarinic agonists can be evoked in the olfactory cortex slice. We suggest this preparation may be useful for conducting pharmacological studies of 'intact' central muscarinic receptors.     

Central effects of muscarinic agonists and antagonists on hippocampal theta rhythm and blood pressure in the anaesthetised rat

The in vivo central effects of a range of full and partial muscarinic receptor agonists have been investigated on hippocampal theta rhythm and blood pressure. In the isoflurane-anaesthetised rat, pretreated with N-methylscopolamine, i.v. administration of arecoline, oxotremorine, arecaidine propargyl ester, aceclidine and pilocarpine produced dose-dependent increases in the frequency of hippocampal theta rhythm and blood pressure, with an order of potency of arecoline = oxotremorine = arecaidine propargyl ester greater than aceclidine greater than or equal to pilocarpine. To increase theta wave frequency, pilocarpine showed a low maximum response and possessed antagonist activity against arecoline, indicating that pilocarpine was acting as a partial agonist. AF102B failed to alter blood pressure or theta rhythm. Intraventricular injections of scopolamine and the M1 receptor-selective antagonist, pirenzepine, produced dose-dependent antagonism of the enhanced theta wave frequency and hypertensive response produced by arecoline. The differences in antagonist potency for the two responses was less than 6-fold, which indicated that both the increase in hippocampal theta wave activity and increase in blood pressure may have been mediated through muscarinic receptors of the M1 subtype. Further studies using a wider range of antagonists will be required to confirm this conclusion.     

Synthesis and muscarinic activity of a series of tertiary and quaternary N-substituted guvacine esters structurally related to arecoline and arecaidine propargyl ester

A series of tertiary and quaternary N-substituted guvacine (1,2,5,6-tetrahydro-3-carboxy-pyridine) methyl and propargyl esters have been synthesized and tested for muscarinic/antimuscarinic activity on rat ileum and electrically paced left atria. Arecoline and arecaidine propargyl ester (APE) as well as their corresponding N-demethyl derivatives, guvacoline (norarecoline) and guvacine propargyl ester, acted as full agonists at both atrial and ileal muscarinic receptors (range of pD2-values 6.09-8.07). However, in both preparations arecoline and APE were clearly more potent (up to 15-fold) than their N-demethyl analogues. Replacement of the N-methyl group in arecoline and APE by larger substituents (ethyl, n-propyl, n-butyl, benzyl, phenylethyl) as well as N-methylation resulted in a decrease or even a complete loss of agonistic activity. In both organs, the propargyl esters usually showed higher potency than the corresponding methyl ester analogues. N-Ethylguvacine propargyl ester and APE methiodide displayed pronounced agonistic activity in the atria (pD2 approximately 6.5; intrinsic activity = 0.79 and 0.67, respectively) but behaved as competitive antagonists in the ileum (pA2 = 6.06 and 5.62, respectively). Beside the lower sensitivity to muscarinic agonists of the rat ileum as compared to rat atria, the cardioselective stimulant action of both agents may also be due to their ability to recognize structural differences between atrial M2 alpha and ileal M2 beta muscarinic receptor subtypes.     

Characterization of muscarinic receptors mediating vasodilation in guinea-pig ileum submucosal arterioles by the use of computer-assisted videomicroscopy.

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

Action of some ganglion-stimulating substances on the secretion of saliva from the submandibular gland

An attempt has been made to determine whether the ganglionic actions of pilocarpine and of 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) contribute to their effect on salivary secretion. Salivary flow was measured from the submandibular glands of spinal cats. Destruction of the superior cervical ganglion and adrenalectomy failed to reduce the stimulant effect of pilocarpine and McN-A-343. Substances known to interfere with the ganglionic actions of pilocarpine (cocaine, methadone and choline 2:6-xylyl ether bromide) likewise failed to modify the response. It is concluded that stimulation of autonomic ganglia and of the adrenal medulla does not contribute to the salivary secretion observed after intravenous injections of pilocarpine and of McN-A-343. Dimethylphenylpiperazinium, a nicotine-like ganglion-stimulating substance, causes salivary flow by stimulating the adrenal medulla as well as parasympathetic ganglion cells; stimulation of the superior cervical ganglion by this substance does not contribute to the salivary response.     

Effect of muscarinic cholinergic drugs on ischemia-induced decreases in glucose uptake and CA1 field potentials in rat hippocampus slices.

To clarify the role of muscarinic acetylcholine receptors in the hypoxia/hypoglycemia (ischemia)-induced functional deficit in hippocampal neurons, we examined the effect of cholinergic drugs on ischemia-induced impairments of glucose uptake and CA1 field potentials in hippocampus slices. Muscarinic receptors were subdivided into M1 (high affinity for pirenzepine) and M2 (low affinity for pirenzepine) subtypes. The M1 receptor subtype is coupled to an increase in phosphoinositide hydrolysis and the M2 receptor subtype is associated with inhibition of adenylate cyclase. The greater potency of carbachol in stimulating phosphoinositide hydrolysis resulted in exacerbated ischemia-induced deficits. Treatment with the muscarinic receptor antagonists scopolamine and pirenzepine (M1 receptor-selective antagonist) had a strong dose-dependent protective effect against ischemia-induced deficits. Oxotremorine and McN-A-343, weak stimulators of phosphoinositide hydrolysis and strong inhibitors of adenylate cyclase, had a weak neuroprotective action against ischemia-induced deficits. These results suggest that stimulation of M1 muscarinic receptors coupled with an increase in phosphoinositide hydrolysis may play a facilitatory role in ischemia-induced deficits. Stimulation of M2 muscarinic receptors may play an inhibitory role in ischemia-induced neuronal deficits.     

Effects of several muscarinic agonists on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart

1. The effects of several muscarinic agonists on atrial tension development, ventricular rate and noradrenaline release from terminal sympathetic fibres evoked by electrical nerve stimulation (SNS) and 1,1-dimethyl-4-phenylpiperazinium (DMPP) were measured in isolated perfused rabbit hearts.2. Hexamethonium, in a concentration which almost abolished the release of noradrenaline by DMPP, had no effect on the release produced by SNS, confirming that the stimulation was postganglionic.3. The order of potency for inhibition of atrial tension development was N-methyl-1,2,5,6, tetrahydro-nicotinic acid prop-2-yne ester (MH-1)>oxotremorine > acetylcholine > methacholine > carbachol > furtrethonium > pilocarpine>4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343)>N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602). All effects were abolished by atropine (1.4 x 10(-6)M).4. Each compound was more potent relative to acetylcholine in inhibiting ventricular rate than atrial tension. With the exception of carbachol, the order of potency was the same.5. Both AHR 602 and McN-A-343 facilitated the release of noradrenaline by SNS and inhibited that by DMPP. The effects were atropine-resistant and hence non-muscarinic.6. The muscarinic compounds (except AHR 602 and McN-A-343) each produce atropine-sensitive inhibition of noradrenaline release evoked both by SNS and DMPP although it is likely that furtrethonium and pilocarpine have additional non-muscarinic inhibitory activity against DMPP. The order of potency on both parameters and the potencies relative to acetylcholine were in good agreement with those for inhibition of atrial tension.7. The results suggest that similar muscarinic receptors mediate inhibition of atrial tension development, ventricular rate and neuronal noradrenaline release caused by SNS and DMPP.8. In terms of the two muscarinic sites known to be present in the superior cervical ganglion, the receptors of the terminal fibres mediating inhibition of noradrenaline release are more likely to correspond to those mediating hyperpolarization than to those mediating depolarization, for which AHR 602 and McN-A-343 show specificity.     

Lesion of septal-hippocampal neurons with 192 IgG-saporin alters function of M1 muscarinic receptors

Cholinergic neurons projecting from the medial septum to the hippocampus were lesioned with the selective neurotoxin 192 IgG-saporin. Injection of 300 ng of 192 IgG-saporin into the medial septum produced a 60% decrease in choline acetyltransferase activity. M1 muscarinic receptor function was examined by measuring enhancement of evoked release of norepinephrine from rat hippocampal slices by the M1 selective agonist McN-A-343. In hippocampal slices from rats which were lesioned with 192-saporin, the response to McN-A-343 was reduced compared to sham-operated controls. Pirenzepine binding demonstrated no change in M1 receptor number or affinity. However, the curve for displacement of pirenzepine by the muscarinic agonist oxotremorine-M was shifted to the right in hippocampal tissue from lesioned rats. This shift was identical to that produced by addition of the non-hydrolyzable GTP analogue GppNHp, which uncouples the M1 muscarinic receptor/G-protein complex. These results suggest that lesion of septal-hippocampal cholinergic inputs causes uncoupling of the M1 muscarinic receptor, decreasing responsiveness to stimulation. These findings are similar to reports of decreased M1 muscarinic receptor coupling to G-proteins and loss of function in Alzheimer's disease. The 192 IgG-saporin lesion may provide a viable animal model in which to study uncoupling of G-proteins and M1 muscarinic receptors.     

Nitric oxide-dependent relaxation induced by M1 muscarinic receptor activation in the rat small intestine.

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.     

Direct pharmacological comparison of the muscarinic receptors mediating relaxation and contraction in the rabbit thoracic aorta.

The purpose of the present studies was to directly compare the pharmacology of the muscarinic cholinergic receptors coupled to carbachol-induced relaxation and contraction of the intact and the endothelium-denuded rabbit thoracic aorta, respectively. The order of potencies of agonists for producing relaxation in the intact aorta was similar to that for producing contraction in the denuded aorta. In both preparations, the partial agonists pilocarpine, McN-A-343, and RS86 functioned as antagonists, indicating a lack of receptor reserve in both preparations. Further, the pA2 values for antagonists in both tissues were virtually identical and were consistent with the pharmacology of M3 receptors.     

Rat hippocampal muscarinic autoreceptors are similar to the M2 (cardiac) subtype: comparison with hippocampal M1, atrial M2 and ileal M3 receptors.

1. Affinity constants for 15 non-selective or putatively selective muscarinic antagonists were determined at muscarinic autoreceptors and postsynaptic receptors (linked to phosphatidylinositol (PI) hydrolysis) in rat hippocampal slices, at muscarinic receptors mediating contractility in guinea-pig atria or ileal smooth muscle and at binding sites in rat cerebral cortical membranes labelled with [3H]-1-quinuclidinyl benzilate or [3H]-pirenzepine. 2. Comparison of the affinities of these antagonists at central M1 receptors (inositol-monophosphate formation in rat hippocampal slices) with their affinities at peripheral M1 receptors (inhibition by McN-A-343 of electrically stimulated twitches in rabbit vas deferens) provides support for the suggestion that these receptors may differ pharmacologically. 3. Comparison of affinity constants obtained by displacement of specifically bound [3H]-pirenzepine from rat cerebral cortical membranes with those obtained in functional tests showed poor correlations between affinities for binding sites and for functional atrial receptors or for hippocampal autoreceptors. A significant correlation was found between affinities for [3H]-pirenzepine binding and those determined at muscarinic receptors linked to PI turnover in rat hippocampus. A significant correlation was also obtained between the affinities for specific [3H]-pirenzepine binding sites in cortical membranes and the affinities at ileal receptors. 4. Comparison of the affinity values for muscarinic autoreceptors in rat hippocampus with affinity values obtained from in vitro models of muscarinic receptor subtypes showed no significant correlations between these autoreceptors and either M1 or M3 receptors. A significant correlation was found between antagonist affinities for hippocampal autoreceptors and muscarinic receptors in the heart. Therefore, muscarinic autoreceptors in rat hippocampus are pharmacologically similar to the M2 (cardiac) muscarinic receptor subtype.     

Acetylcholine receptor agonists stimulate [H]taurine release from rat sympathetic ganglia

The endogenous taurine content, and the uptake and release of [³H]taurine were examined using the rat superior cervical ganglion. Taurine was found to be one of the most abundant amino acids in the superior cervical ganglion, and the superior cervical ganglion took up [³H]taurine from the incubation medium. Carbachol stimulated the release of [³H]taurine in a concentration-dependent manner with an EC₅₀ of 26 μM and maximal stimulation at 100 μM. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium stimulated release with the same potency but with greater efficacy than carbachol. The nicotinic receptor antagonist hexamethonium (1 mM) inhibited carbachol-stimulated release by 74% (±)-Muscarine stimulated release with an EC₅₀ of 8 μM but with a maximal effect of only 32% of that produced by 100 μM carbachol. Oxotremorine another muscarinic receptor agonist, was ineffective, even at 1 mM. The muscarinic receptor antagonist atropine inhibited carbachol-stimulated release by 30% at 10μM. These results show that ³H taurine release from rat superior cervical ganglion can be stimulated by cholinergic receptor agonists. Release is mediated predominantly by a nicotinic receptor and partially by a muscarinic receptor.     

A pharmacological study of the responses induced by muscarinic agonists on the isolated superior cervical ganglion of the guinea-pig

We have studied the muscarinic agonist induced responses on the guinea-pig superior cervical ganglion in vitro, as recorded from the internal carotid nerve using a grease-gap. The principal response was a depolarization, but a small hyperpolarizing response could be revealed under certain conditions. We determined the pA2 of a number of muscarinic antagonists against the muscarine induced depolarization. Four selective antagonists and atropine appeared to act competitively. The rank order of their pA2s was 4-DAMP (8.5), atropine (8.4), pirenzepine (8.0), methoctramine (7.2) and AF-DX 116 (6.3). In addition to muscarine, we assessed the potency and relative maximum response of nine other muscarinic compounds to depolarize this preparation: carbachol, 5-methylfurmethide, oxotremorine, oxotremorine-M, pilocarpine, RS 86, AF102B and two novel compounds L-670548 and L-679512. L-670548 was the most potent and AF102B was the least potent agonist tested. Only AF102B evoked a maximum depolarization that was significantly smaller than muscarine. A hyperpolarizing response to carbachol (1 microM) could be recorded when the superfusing medium contained 0.3 microM pirenzepine and only 0.1 mM CaCl2 (cf. usual 2.5 mM). This response was relatively small compared to that evoked on the superior cervical ganglion of the rat. It was blocked by the cardioselective antagonists methoctramine (0.1-0.3 microM) and AF-DX 116 (0.3-1.0 microM). Of the 10 agonists tested, only carbachol, oxotremorine and oxotremorine-M reproducibly evoked a hyperpolarizing response. It was concluded that muscarinic agonists can induce a depolarization of the guinea-pig superior cervical ganglion mediated by M1 receptors. The activation of cardiac-like M2 receptors resulted in a hyperpolarizing response that was relatively small.     

Muscarinic receptor agonist-mediated modulation of neuronal activity in rat cerebral cortex.

Multiple cortical neuronal responses were elicited by the iontophoretic application of muscarinic receptor agonists and antagonists in the rat cerebral sensorimotor cortex in vivo. (1) The muscarinic receptor agonist, oxotremorine-M induced a biphasic effect on spontaneous firing. This was evident as an early brief increase in the firing rate over the spontaneous discharge followed by secondary inhibition of spontaneous activity. The excitation could be blocked by the muscarinic receptor non-selective antagonist atropine and by both the M1 receptor antagonist pirenzepine and the M2 receptor antagonists gallamine or methoctramine. Oxotremorine-M inhibition of spontaneous activity was not affected by the M1 receptor antagonist pirenzepine, while evaluation of its sensitivity to gallamine and methoctramine was not possible since these two M2 receptor antagonists also depressed spontaneous activity, unlike pirenzepine. Of the other two muscarinic receptor agonists, oxotremorine had inconsistent and weak excitatory effects whilst McN-A-343 had only weak excitatory or inhibitory effects on spontaneous activity. (2) Oxotremorine-M, oxotremorine and McN-A-343 had a depressant action on neuronal discharges evoked by glutamate or acetylcholine. A depressant effect of oxotremorine-M was also demonstrated on the early excitation evoked by subsequent applications of oxotremorine-M itself. Of the three muscarinic receptor agonists tested, oxotremorine-M was the most potent in evoking a long-term depression of evoked discharges, lasting from several minutes (greater than 5 min) to as long as 40 min. Oxotremorine-M-induced depression of evoked responses was most sensitive to the M2 receptor antagonists, whereas oxotremorine-induced depression was more sensitive to the M1 receptor antagonist pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential distribution of muscarinic receptor subtypes and their regulation by G-protein in rat brain

1. [3H]quinuclidinyl benzilate ([3H]QNB) binding in rat cerebral and cerebellar synaptosomes had different Bmax values, but similar Kd values. 2. These bindings could be displaced by classic muscarinic agents: pilocarpine (partial agonist), and atropine (antagonist), which both had similar binding affinities in rat cerebral and cerebellar synaptosomes. 3. The new muscarinic M1 selective agents: McN-A-343 (agonist), pirenzepine and trihexyphenidyl (antagonists) and higher affinities for receptor sites in the cerebrum than in the cerebellum. 4. The muscarinic M2 selective agents: carbachol, oxotremorine (agonists), and AF-DX-116 (antagonist) had higher affinities for receptor sites in the cerebellum than in the cerebrum. 5. GPP(NH)p (40 microM) decreased the binding affinities of carbachol and oxotremorine in the cerebellum, but not in the cerebrum. However, it did not decrease the binding affinities of all the antagonists studied in both brain regions. 6. These results reveal that more muscarinic M1 sites are present in the cerebrum than in the cerebellum, while the opposite is true for M2 sites. Furthermore, the regulatory role of G-protein on these muscarinic receptor subtypes in the brain is different.     

Interaction of some muscarinic agonists and antagonists at the prejunctional muscarinic receptor in the rabbit ear artery preparation.

The inhibitory effect of several muscarinic agonists on responses to sympathetic nerve stimulation of the isolated perfused ear artery of the rabbit was compared to that of acetylcholine in preparations pretreated with dyflos, cocaine and yohimbine. In general the potency of the agonists was similar to that observed at peripheral muscarinic sites except for arecaidine propargyl ester and 4-(m-chlorophenylcarbamoyloxy)-2-butynyl trimethylammonium chloride (McN-A-343). The inhibitory effect observed with N-benzyl-3-pyrrolidyl acetate methobromide (AHR-602) was not exerted via muscarinic receptors. With carbachol (CCh) as an agonist, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was found to have a pKB value of 7.74 and thus was 19 fold less active as an antagonist of responses to the agonist, than previously reported for guinea-pig ileum. When McN-A-343 was used as the agonist, the slope of the Schild plot with the antagonist was significantly less than unity. It is suggested that an allosteric interaction of 4-DAMP may be involved with this agonist. By use of an allosteric model, a pKB of 8.56 for 4-DAMP was obtained. Secoverine produced similar pKB values with either CCh (8.19) or McN-A-343 (8.13) as the agonist.