Different effects of muscarinic agonists in rat superior cervical ganglion and hippocampal slices.

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.     

Muscarinic activity of McN-A-343 and its value in muscarinic receptor classification

The affinity and potency of McN-A-343 (4-(m-chlorophenyl-carbamoyloxy) -2-butynyltrimethylammonium chloride) has been assessed at a range of M1 and M2 muscarinic receptors. McN-A-343 was shown to act as a full agonist at M2 receptors present in the guinea-pig isolated taenia caeci (-log EC50 = 5.14). McN-A-343 exhibited no agonist action in the guinea-pig ileum, atria, bladder or trachea. McN-A-343 was not selective in terms of affinity since its dissociation constants at M1 and M2 binding sites in the rat cerebral cortex and myocardium respectively, were very similar (cortical pPKi = 5.05; myocardial pKi = 5.22). The selectivity previously reported for the compound may be due to differences in intrinsic efficacy and/or tissue receptor reserve. Based on differential antagonist affinities, the muscarinic receptor profile of the taenia caeci, trachea and bladder was similar to that observed in the ileum, but dissimiliar to that observed in the atria.     

Investigation of the mechanism for the relaxation of rat duodenum mediated via M1 muscarinic receptors

1 Relaxation responses of the rat isolated duodenum to the putative M1 muscarinic receptor agonist, McN-A-343, were examined to determine whether the response was due to the release of known non-adrenergic, non-cholinergic relaxant neurotransmitters and to establish the involvement of M1 muscarinic receptors. 2 The role of ATP was examined with the P2 receptor antagonist, suramin, which at 30 mum antagonized the relaxant responses to alpha,beta-methylene ATP. The same dose, however, failed to inhibit the relaxation by McN-A-343. 3 The role of nitric oxide (NO) was examined with the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 100 microm), which failed to inhibit the responses to McN-A-343. As NO mediates relaxation of the duodenum via cGMP generation through guanylyl cyclase, whether the relaxation by McN-A-343 was also via cGMP was examined with the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxation responses to the NO donor, S-nitroso-N-acetyl penicillamine, were inhibited in the presence of ODQ (3 microm), but not those by McN-A-343. 4 Release of gamma-aminobutyric acid (GABA) was examined with the GABAA receptor antagonist, bicuculline (10 microm), which shifted the concentration-response curves for the relaxation of the duodenum by GABA to the right. There was a similar degree of shift in the concentration-response curve for McN-A-343 by bicuculline indicating that release of GABA from enteric neurones of the duodenum could explain the relaxation response to McN-A-343. 5 To test whether the muscarinic receptors mediating the relaxation of the duodenum were of the M1 subtype, the susceptibility to the selective competitive antagonist, pirenzepine and the selective muscarinic toxin from green mamba, MT7, was examined. Pirenzepine (1 microm) shifted the concentration-response for McN-A-343 to the right in a parallel fashion with a dose ratio of 33.3 +/- 20.2. This yielded a pA2 value of 7.5, which concords with those for other responses reputed to be mediated via M1 muscarinic receptors. The toxin MT7 was used as an irreversible antagonist and following incubation with the duodenum was washed from the bath. An incubation time of 30 min with 100 nm of MT7 caused a significant parallel shift in the concentration-response to McN-A-343 confirming the involvement of M1 muscarinic receptors. 6 This study has confirmed that McN-A-343 relaxes the rat duodenum via muscarinic receptors of the M1 subtype and that these receptors are probably located on enteric neurones from which their stimulation releases GABA.     

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.     

Pharmacological characterization of muscarinic receptors involved in McN-A-343-induced effects on intestinal motility and heart rate in conscious dogs.

1. Intravenous injection of the muscarinic agonist, McN-A-343, in conscious dogs equipped with an ileal Thiry fistula produced a dose-related inhibition of intestinal phasic contractile activity, and an increase in heart rate. 2. The inhibitory action of McN-A-343 on motility was antagonized with different potencies by antimuscarinic drugs. The non-selective drug, N-methylatropine, blocked the McN-A-343 effect as well as the reflex phasic activity. The M1-selective compound, pirenzepine (1-30 micrograms kg-1), was a potent antagonist of the McN-A-343 effect, whereas the cardioselective M2-antagonist, AF-DX 116, and the smooth muscle selective compound, 4-diphenylacetoxy-N-methyl piperidine (4-DAMP), were completely ineffective at the doses tested. 3. The McN-A-343-induced inhibition of intestinal motility was blocked by locally applied lignocaine, suggesting the involvement of a neural inhibitory pathway. The resistance to hexamethonium and (alpha 1-, alpha 2- and beta-) adrenoceptor blocking drugs excluded transmission through a nicotinic synapse or release of catecholamines. 4. McN-A-343-induced tachycardia was also the result of muscarinic receptor activation. It was very sensitive to antagonism by 4-DAMP, while being completely unaffected by AF-DX 116. Pirenzepine displayed an intermediate profile, reducing tachycardia at doses fully active in reversing the agonist-mediated effect on intestinal motility. Propranolol partially reduced McN-A-343 tachycardia, suggesting catecholamine release. 5. The two McN-A-343 effects investigated in the present study appear to be mediated by different muscarinic receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of muscarinic receptor agonists and antagonists on swim-stress-induced 'behavioural despair' in rats

The effects of some selective agonists and antagonists of cholinergic muscarinic receptor subtypes on swim-stress-induced immobility (SI) was investigated in rats. This paradigm has been proposed to assess 'behavioural despair' in rodents as a laboratory model for clinical depression. All the rats were pre-treated with atropine ethoiodide i.p. to obviate any peripheral cholinergic influence. SI was only marginally less in intracerebroventricular (i.c.v.) cannulated rats administered artificial cerebrospinal fluid than in uncan nulated rats administered normal saline i.p. The drugs which do not have access across the blood-brain barrier were administered i.c.v. The muscarinic M(1) receptor agonists, arecholine and McN-A-343, and the non-specific muscarinic receptor agonist, oxotremorine, induced dose-related increases in SI, whereas the muscarinic M(1) receptor antagonists, scopolamine and pirenzepine, and the non-specific antagonist, atropine, produced significant decreases in SI. Carbachol, an M(2) receptor agonist, and physostigmine induced a dose- dependent dual effect, with lower doses attenuating and higher doses augmenting SI. The M(2) receptor antagonists, gallamine and AF-DX 116, increased SI. The data may be interpreted to suggest that muscarinic M(1) receptors may function to accentuate depression whereas muscarinic M(2) receptors may exert an inhibitory modulatory effect.     

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.     

Estimation of relative microscopic affinity constants of agonists for the active state of the receptor in functional studies on M2 and M3 muscarinic receptors

In prior work, we have shown that it is possible to estimate the product of observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist simply through the analysis of their respective concentration-response curves. In this report, we show analytically and through mathematical modeling that this product, termed intrinsic relative activity (RA(i)), is equivalent to the ratio of microscopic affinity constants of the agonists for the active state of the receptor. We also compared the RA(i) estimates of selected muscarinic agonists with a relative estimate of the product of observed affinity and intrinsic efficacy determined independently through the method of partial receptor inactivation. There was good agreement between these two estimates when agonist-mediated inhibition of forskolin-stimulated cAMP accumulation was measured in Chinese hamster ovary cells stably expressing the human M(2) muscarinic receptor. Likewise, there was good agreement between the two estimates when agonist activity was measured on the ileum from M(2) muscarinic receptor knockout mice, a convenient assay for M(3) receptor activity. The RA(i) estimates of agonists in the mouse ileum were similar to those estimated at the human M(3) receptor with the exception of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), which is known to be an M(1)- and M(4)-selective muscarinic agonist. Additional experiments showed that the response to McN-A-343 in the mouse ileum included a non-M(3) muscarinic receptor component. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity and ligand-directed signaling.     

The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety

A review of the literature suggests that the dorsal hippocampal serotonergic system, and, in particular, the postsynaptic 5-HT(1A) receptor, mediates an anxiogenic response, whereas endogenous dorsal hippocampal cholinergic tone mediates an anxiolytic response. Accordingly, it has been shown that direct dorsal hippocampal administration of the 5-HT(1A) receptor agonist, 8-OH-DPAT, the nicotinic receptor antagonist, mecamylamine, and the M(1) muscarinic receptor antagonist, pirenzepine, all have anxiogenic effects in rats tested in the social interaction test. It is therefore surprising that nicotine also has an anxiogenic effect in this test following dorsal hippocampal administration. However, the anxiogenic effects of mecamylamine and nicotine in the dorsal hippocampus are blocked by coadministration of the 5-HT(1A) receptor antagonist, WAY 100635, suggesting that both of these compounds act by enhancing hippocampal serotonergic transmission, thereby stimulating postsynaptic 5-HT(1A) receptors. This conclusion is supported by the observation that both nicotine and mecamylamine stimulate basal [3H]-5-HT release from dorsal hippocampal slices. A possible mechanism by which nicotinic receptor ligands modulate hippocampal 5-HT release is discussed, and it is proposed that the dorsal hippocampal serotonergic and cholinergic systems are tightly coupled and function antagonistically in the modulation of anxiety, as measured in the social interaction test. These systems are relatively unimportant in controlling behaviour on trial 1 in the plus-maze. On trial 2 in the elevated plus-maze, a model of specific phobia, the endogenous cholinergic system, nicotine, and the M(1) receptor agonist, McN-A-343, all mediate an anxiolytic effect, whereas stimulation of 5-HT(1A) receptors mediates an anxiogenic effect. It is proposed that the hippocampus may predominantly control the avoidance components of phobic anxiety, with other regions, such as the dorsomedial hypothalamus, controlling the escape components.     

Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea-pig taenia caeci.

1. The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR-602, and McN-A-343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea-pig caecum. 2. Compared to CCh, ACh was a full agonist for contraction but AHR-602 and McN-A-343 were partial agonists producing 80-85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR-602 and McN-A-343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh. 3. CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mM) was inhibited by atropine (IC50 8.5 nM) and pirenzepine (IC50 450 nM). The accumulation of inositol phosphates in the presence of AHR-602 or McN-A-343 was not significantly different (P greater than 0.05) from basal levels. 4. A concentration of 0.2 mM AHR-602 produced a parallel shift of the concentration-response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mM) was greater than 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN-A-343 (20 microM) produced a flattening of the concentration-response curve to CCh for inositol phosphate accumulation. 5. The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2-muscarinic receptor. AHR-602 and McN-A-343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.     

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.     

Inhibition of field stimulation-induced contractions of rabbit vas deferens by muscarinic receptor agonists: selectivity of McN-A-343 for M1 receptors

Inhibition of the field stimulation-induced twitch responses of the rabbit vas deferens by the muscarinic receptor agonist, McN-A-343, has been attributed to presynaptic muscarinic receptors of the M1 subtype located on noradrenergic nerve terminals. Stimulation of these receptors causes inhibition of transmitter release and inhibition of the contractile response. However, the selectivity of McN-A-343 for M1 receptors has been questioned and this throws doubt on whether the prejunctional receptors of the rabbit vas deferens are of the M1 subtype. In this study we have undertaken a comprehensive re-evaluation of the inhibition of prostatic and epididymal portions of the rabbit isolated field-stimulated vas deferens by several agonists, including McN-A-343, and quantified the antagonism by M1-selective antagonists, pirenzepine and telenzepine. Prostatic and epididymal portions of vasa deferentia from New Zealand White rabbits were immersed in a low Ca2+ Krebs solution at 32+/-0.5 degrees C gassed with 5% CO2 in oxygen. Yohimbine (1.0mM) was present throughout to block prejunctional alpha2-adrenoceptors. Field stimulation was applied by repeated application of single pulses (30 V, 0.05 Hz, 0.5 ms) and isometric contractions recorded. Carbachol and oxotremorine initially potentiated the epididymal contractions but at higher concentrations there was inhibition. In the prostatic portion, oxotremorine only inhibited. McN-A-343 produced inhibitory responses only in both epididymal and prostatic portions. Pirenzepine shifted the concentration-response curves forthe inhibitory responses to oxotremorine to the right. However, the potentiation of the twitches also became more apparent with the lower concentrations of oxotremorine. Schild plots for the antagonism by pirenzepine yielded pA2 values of 7.96+/-0.004 and 7.7+/-0.02 for the epididymal and prostatic portions, respectively. The concentration-response curves for the inhibition of twitches by McN-A-343 were displaced to the right in a parallel manner by pirenzepine in both prostatic and epididymal portions with no potentiation of the twitches. The Schild plot for this antagonism generated pA2 values of 7.68+/-0.01 and 8.07+/-0.01, respectively. Telenzepine caused parallel shifts of the McN-A-343 concentration-response curves to the right in prostatic portions, the pA2 value being 8.70+/-0.13. Telenzepine (10(-7) M) abolished the inhibitory effect of carbachol to reveal only concentration-dependent potentiation of the contractions. The Schild plot for antagonism of this contractile effect yielded a pA2 value (7.07+/-0.09) that was significantly less by almost two orders of magnitude (1.70) than the value for the antagonism by telenzepine of the McN-A-343-induced inhibitory response. The pA2 values of pirenzepine and telenzepine against the inhibitory responses of the rabbit vas deferens are consistent with the involvement of M1 receptors. This leads to the conclusion that McN-A-343 causes inhibition through this receptor type. The doubts concerning the selectivity of McN-A-343 for M1 receptors are therefore unfounded. The fact that McN-A-343 does not display a selective binding profile suggests that its selectivity does not arise from affinity differences but probably resides in its intrinsic efficacy.     

Emesis induced by 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343): evidence for a predominant central muscarinic M1 mediation

The emetic action of 4-(m-chlorophenylcarbamoyloxy)-2- butynyltrimethylammonium chloride (McN-A-343) was investigated in the unanaesthetized cat, after it was injected into the cerebral ventricles, through chronically-implanted cannulae. Intracerebroventricular injection of McN-A-343 produced dose-dependent and shortlasting emesis, which was not completely abolished after ablation of the area postrema. The predominantly selective muscarinic M1 antagonist, pirenzepine as well as the mixed muscarinic M1 and M2 antagonist, atropine, injected into the cerebral ventricles, attenuated or abolished the emesis evoked by intracerebroventricular McN-A-343. Both atropine and pirenzepine produced dose-dependent inhibition of the emesis evoked by McN-A-343. However, the ID50 value for atropine was approximately five times greater than that for pirenzepine. Abolition of McN-A-343-induced emesis only occurred with the largest dose of atropine (1 mg). On the other hand, selected ganglionic blocking agents, an alpha- and beta-adrenoceptor blocking agent, a dopamine antagonist, a 5-hydroxytryptamine antagonist and an antihistamine, all injected into the cerebral ventricles, had no significant effect on emesis evoked by McN-A-343, similarly injected. The emetic response to intracerebroventricular injection of McN-A-343 was attenuated or abolished in cats pretreated with hemicholinium-3, triethylcholine, reserpine and 6-hydroxydopamine, intracerebroventricularly. On the contrary, the emetic response to intracerebroventricular injection of McN-A-343 was not altered in cats pretreated with intracerebroventricular injections of bretylium, alpha-methyl-p-tyrosine and 5,6-dihydroxytryptamine. It is postulated that the emesis produced by McN-A-343, injected into the cerebral ventricles, is mediated through muscarinic M1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in behavioral responses to a cholinergic agonist in post-pubertal rats with neonatal ventral hippocampal lesions: relationship to changes in muscarinic receptor levels.

Excitotoxic neonatal ventral hippocampal (NVH) lesion in rats is considered as a putative animal model of schizophrenia as lesioned animals show characteristic post-pubertal emergence of neurochemical and behavioral abnormalities analogous to some of those seen in this disease. Converging evidence points to the involvement of central cholinergic system in this neuropsychiatric disorder, and our previous studies have suggested that cholinergic neurotransmission may be altered in post-pubertal NVH lesioned rats. We investigated here muscarinic receptor reactivity in NVH lesioned animals by measuring the effects of the muscarinic receptor agonist oxotremorine on physiological responses known to be modulated by these receptors such as body temperature, salivation, tremor, pain, and prepulse inhibition of the acoustic startle (PPI). Quantitative receptor autoradiography revealed that post-pubertal NVH lesioned animals display increased levels of [3H]pirenzepine/M1-like and [3H]AFDX-384/M2-like receptor binding sites in the striatum, nucleus accumbens, and in subareas of the dorsal hippocampus. Moreover, in response to the systemic administration of oxotremorine (0.25 mg/kg), post-pubertal NVH lesioned rats exhibited increases in salivation and tremor, and a greater reduction in body temperature compared to sham control animals. Increases in the hot-plate latency were also observed suggesting enhanced antinociceptive effects of oxotremorine in post-pubertal NVH lesioned animals. Finally, oxotremorine (0.1 and 0.25 mg/kg) disrupted PPI in post-pubertal sham control rats while the muscarinic receptor antagonist biperiden (0.5 and 1.0 mg/kg) normalized this behavior in NVH lesioned rats. Taken together, these findings reveal that post-pubertal NVH lesioned rats display enhanced muscarinic receptor responsiveness, which may relate to some behavioral abnormalities reported in this animal model.     

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)     

Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein

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.     

Intrathecal clonidine inhibits mechanical allodynia via activation of the spinal muscarinic M1 receptor in streptozotocin-induced diabetic mice

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

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.     

Cholinergic regulation of thyrotropin secretion in male rats

The cold-stimulated thyrotropin (TSH) secretion in male rats was suppressed by muscarinic agonists, i.e. Oxa-22, McN-A-343 (an M1 agonist), oxotremorine (an M2 agonist) and methacholine (a quaternary compound). The inhibitory effect of Oxa-22 was antagonized by atropine, butylscopolamine and glycopyrrolate as well as by pirenzepine, an M1 antagonist and AF-DX 116, a new M2 antagonist. Various muscarinic antagonists were not active when given alone. Cytisine, a peripheral nicotinic agonist, was not active but nicotine significantly suppressed the cold-stimulated TSH secretion. Its effect was counteracted by mecamylamine but not by hexamethonium. The thyrotropin releasing hormone (TRH)-induced TSH secretion was not inhibited by Oxa-22, nicotine or methacholine. These results show that irrespective of the receptor subtype (muscarinic1 or muscarinic2, nicotinic), cholinergic activation inhibits the cold-stimulated TSH secretion. The results also suggest that this inhibitory effect is at the hypothalamic rather than the anterior pituitary level. The muscarinic action seems to occur outside the blood-brain barrier but the nicotinic action occurs inside this barrier.     

Anxiogenic effects of nicotine in the dorsal hippocampus are mediated by 5-HT1A and not by muscarinic M1 receptors

After direct administration into the dorsal hippocampus nicotine decreased the time spent in social interaction, without changing locomotor activity, indicating an anxiogenic effect. The possibility that post-synaptic M1 muscarinic receptors mediated this effect was examined by determining whether dorsal hippocampal administration of a specific M1 receptor agonist (McN-A-343) had anxiogenic effects, and whether the anxiogenic effect of nicotine could be reversed by co-administration of the M1 receptor antagonist, pirenzepine. McN-A-343 (0.3, 1.6, 3.2, 15.8 nmol) was without effect on social interaction, and pirenzepine (0.7 and 2.4 nmol) injection into the dorsal hippocampus failed to reverse the decrease in social interaction caused by nicotine (6.3 nmol) injection into this area. However, the decrease in social interaction after nicotine (50 nmol) was completely reversed by the specific 5-HT1A receptor antagonist, WAY 100635 (0.4 nmol) after co-administration of both drugs into the dorsal hippocampus. Thus, the anxiogenic effect of nicotine in this brain region seems to be mediated by 5-HT1A, but not M1, receptors. In contrast to the effect of nicotine in naive animals, those retested after a second injection of 50 nmol did not show a significant anxiogenic effect. The theoretical implications of this are discussed and from a practical point of view this suggests caution in the retesting of animals after central injections.