Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology

The nature of the muscarinic receptor subtype mediating the endothelium-dependent relaxation of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted arterial segments. Relaxation induced by several agonists (acetylcholine (ACh), acetyl-beta-methylcholine, oxotremorine, carbachol and McN-A-343) was recorded. The ability of selective (pirenzepine, dicyclomine, adiphenine, AF-DX 116, methoctramine, gallamine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD] and non-selective antagonists (atropine, scopolamine and quinuclidinyl benzilate (QNB] to block the relaxation induced by ACh was also estimated. The weak activity of the poorly selective M1 muscarinic receptor as together with the intermediate affinity of pirenzepine and adiphenine tend to exclude the M1 muscarinic receptor as the primary mediator of the cholinergic relaxation. The low affinity of AF-DX 116 and methoctramine further suggested that the cerebrovascular muscarinic receptor does not correspond to the M2 cardiac subtype. In contrast, 4-DAMP and HHSiD potently inhibited the ACh-induced relaxation with affinities similar to those reported at the M3 glandular receptor. We conclude that a similar to the pharmacological M3 muscarinic receptor subtype is responsible for the cholinergic relaxation of the cat middle cerebral artery.     

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.     

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 suppression of the evoked N-wave by oxotremorine-M recorded in the guinea-pig olfactory cortex slice

The effect of the muscarinic agonist oxotremorine-M has been studied on the surface-negative field potential (N-wave) evoked by orthodromic stimulation of the lateral olfactory tract in slices of guinea-pig olfactory cortex. Bath-application of oxotremorine-M (5-80 microM) or carbachol (10-300 microM) produced a reversible depression of the N-wave amplitude without affecting the lateral olfactory tract compound action potential. Oxotremorine-M was approximately 5 times more potent than carbachol in this respect, and the effects of both agonists were competitively blocked by telenzepine (5-100 nM), a selective M1-receptor antagonist. In contrast, methoctramine or AF-DX 116, two 'cardioselective' M2-receptor antagonists, had little or no blocking effect on the agonist responses. It is suggested that oxotremorine-M (like carbachol) inhibits the evoked field potential by activating presynaptic M1-type muscarinic receptors in the olfactory cortex slice.     

Different muscarinic receptor subtypes mediating the phasic activity and basal tone of pig isolated intravesical ureter.

1. We have studied the effects of muscarinic cholinoceptor agonists and specific antagonists on both phasic activity and basal tone of the isolated intravesical ureter of the pig by means of isometric techniques in vitro. 2. Acetylcholine in the presence and absence of physostigmine increased both phasic activity and basal tone of ureteral strips in a concentration-dependent manner. Moreover carbachol, methacholine and oxotremorine-M increased both contractile parameters while bethanechol and McN-A-343 evoked only increases in tone without affecting the frequency of the phasic contractions. 3. The nicotinic receptor blocker, hexamethonium (10(-6)-10(-4) M), failed to modify the contractions evoked by a single dose of carbachol (10(-5) M), whilst the muscarinic antagonist, atropine inhibited both phasic and tonic responses. 4. The muscarinic M1 (pirenzepine), M2 (AF-DX 116 and methoctramine), M3 (4-DAMP, HHSiD and p-F-HHSiD), and putative M4 receptor (tropicamide) antagonists significantly reversed increases in both frequency of phasic activity and baseline tone induced by a submaximal dose of carbachol (10(-5) M). The pIC50 values for inhibition of the induced phasic activity were: atropine (10.16) > 4-DAMP (9.12) > HHSiD (8.22) = methoctramine (7.98) = p-F-HHSiD (7.88 > tropicamide (7.62) = pirenzepine (7.53) = AF-DX 116 (7.45) and for inhibition of basal tone were: atropine (10.73) > 4-DAMP (9.32) > HHSiD (8.65) = pirenzepine (8.43) = p-F-HHSiD (8.38) > methoctramine (7.79) > tropicamide (7.53) > AF-DX 116 (7.04).(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.     

Involvement of M1-muscarinic receptors in the excitation of neocortical neurones by acetylcholine

The technique of microelectrophoresis was used to investigate the cholinoceptor pharmacology of spontaneously active single neurones in the parietal cortex of the rat. Acetylcholine, carbachol and the selective M1-muscarinic receptor agonist, McN-A-343, were each potent excitants (rank order of apparent potency: carbachol greater than acetylcholine greater than McN-A-343). When measured in vitro, the apparent mobilities of carbachol and acetylcholine were similar although significantly less than that of McN-A-343, suggesting that the lower potencies of acetylcholine and McN-A-343 probably reflect a genuine biological phenomenon. In addition to excitation, carbachol also evoked biphasic (excitation/depression) and depressant responses. In contrast to the other cholinoceptor agonists, nicotine produced weak and inconsistent excitations. Excitatory responses to acetylcholine and carbachol were significantly attenuated by the selective M1-muscarinic receptor antagonist, pirenzepine, at a time when the excitatory response to McN-A-343 was also significantly reduced. Responses to phenylephrine were not diminished. On several cells an excitatory response to carbachol was converted to a depression by pirenzepine. These results suggest that the excitatory responses of cortical neurones to cholinoceptor agonists are mediated predominantly by M1-muscarinic receptors. The identity of the receptor mediating the depressant response to carbachol remains uncertain, although nicotinic cholinoceptors do not appear to be involved.     

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.     

Central pressor effects induced by muscarinic receptor agonists: evidence for a predominant role of the M2 receptor subtype

The cardiovascular effects induced in the rat by several muscarinic receptor agonists were studied. All the agonists produced a clear decrease in heart rate. This decrease appeared to be peripherally mediated, because it was antagonized by methylscopolamine. The effects on blood pressure varied depending on the presence of anaesthesia, previous treatments and the type of agonists tested. When peripheral muscarinic activity was blocked by administration of methylscopolamine, a dose-dependent hypertension was obtained following the injection of oxotremorine, arecoline and aceclidine, by both intraperitoneal and intracerebroventricular routes. The muscarinic receptor agonist RS 86 produced a slight increase in blood pressure but the increase was weaker than those observed with the agonists cited above. On the other hand, the muscarinic receptor agonists pilocarpine, AF-30 and McN-A-343, considered as partially M1-selective compounds, did not produce any effect on blood pressure. Moreover, the hypertension induced by oxotremorine was completely blocked by intracerebroventricular administration of the non-subtype-selective muscarinic receptor antagonist scopolamine but was unaffected by the M1-selective antagonist pirenzepine. We propose that the central hypertensive response induced by muscarinic receptor agonists in the unanaesthetized rat is, at least partially, mediated through the stimulation of the so-called M2 muscarinic receptor subtype.     

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 the muscarinic receptor mediating contraction of the dog prostate

1 We have studied the effects of muscarinic cholinoceptor agonists and subtype-preferring antagonists on the isometric contraction of smooth muscle strips from dog prostate. 2 Acetylcholine and carbachol induced contraction of prostate strips from the peripheral zone, (‘the capsule’). Bethanechol contracted the tissue but not at lower doses. McN-A-343 and oxotremorine-M showed the same effects. 3 Blocking α- and β-adrenoceptors with phentolamine and propranolol, respectively, did not modify carbachol-induced contractions. 4 The nicotinic receptor blocker, hexamethonium (10^??6–10^??4 m ) did not affect the contractile response evoked by a single dose of carbachol (10^??5 m ), whilst the muscarinic receptor antagonist, atropine (10^??11–10^??9 m ), inhibited it in a competitive manner. 5 The muscarinic M₁ (pirenzepine), M₂[AF-DX 116, himbacine (M₂/M₄) and methoctramine], M₃ (HHSID and f-F-HHSID), and putative M₄ (tropicamide) antagonists reduced significantly the carbachol-induced contractions. The pIC₅₀ values were: atropine (10.01) > himbacine (8.3) > methoctramine (7.85) > AF-DX 116 (7.60) > HHSID (7.21) > p-F-HHSID (7.10) > pirenzepine (7.30) > tropicamide (7.00). 6 The antagonist profile indicates that an predominant M₂ receptor subtype could mediate the muscarinic contraction in the canine prostate.     

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)     

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.     

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.     

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.     

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.     

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.     

Muscarinic acetylcholine response in pyramidal neurones of rat cerebral cortex.

1. The effects of acetylcholine (ACh) on pyramidal neurons acutely dissociated from the rat cerebral cortex were studied in the whole-cell mode, by use of the nystatin-perforated patch recording configuration. 2. ACh induced a net inward current (IACh) accompanied by a membrane conductance decrease at a holding potential (VH) of -40 mV. IACh increased in a concentration-dependent manner with a half-maximum concentration (EC50) of 8.7 x 10(-7) M. 3. IACh mainly resulted from the suppression of the voltage- and time-dependent K+ current (M-current). 4. Muscarine and muscarinic agonists such as McN-A-343, oxotremorine and oxotremorine-M mimicked the ACh response. The potency was in the order of oxotremorine-M > McN-A-343 > or = muscarine > oxotremorine. 5. Pirenzepine shifted the concentration-response curve for ACh to the right and the corresponding Schild plot yielded a pA2 value of 7.81. Other muscarinic antagonists also reversibly blocked IACh in a concentration-dependent manner. The inhibitory potency was in the order of atropine > 4-DAMP > pirenzepine > AF-DX-116. 6. IACh could be induced normally even after pre-incubation of dissociated neurones in external solution with 200 ng ml-1 pertussis toxin (PTX) for 8 h, whereas the inhibitory effect of ACh on high-voltage-activated Ca2+ channels was completely abolished by the PTX treatment.     

Acetylcholinesterase histochemistry and functional characterization of the muscarinic receptor mediating the contraction of the bovine oesophageal groove

1 By using acetylcholinesterase (AChE) histochemistry and invitro isometric techniques, we have studied the presence and distribution of AChE-positive nerves, as well as the effects of muscarinic cholinoceptor agonists and selective antagonists, in the bovine oesophageal groove. 2 AChE-positive nerves and cells were distributed widely on the oesophageal groove floor. These fibres originated from adventitial ganglia containing bodies with high AChE activity and were shown grouped as large adventitial nerve bundles. 3 Both in the presence and absence of physostigmine, acetylcholine (ACh) induced concentration dependent contractions of bovine oesophageal groove strips. The rank order of the pD₂ values for muscarinic agonists was: oxotremorine-M (7.37) = carbachol (7.14) > acetylcholine plus physostigmine (6.46) > bethanechol (5.42) > McN-A-343 (4.45) > acetylcholine (4.06). 4 Hexamethonium (10^?6–10^?4M ), a nicotinic receptor blocker, did not affect the carbachol concentration–response curve, which was significantly inhibited by the muscarinic antagonist, atropine (10^?9–10^?8M ). 5 The preferential muscarinic antagonists pirenzepine (M₁), 11-(2(-(diethyl-amino)methyl)-1-piperidinylacetyl)-5,-11-dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepine-6-one (AF-DX 116) and methoctramine (M₂), 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and p-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) (M₃) and tropicamide (M₄) evoked rightwards displacements in a parallel manner of the carbachol control curve, and there was no decrease of the maximum response with the highest concentration of antagonist utilized. The muscarinic antagonist affinities, expressed in terms of pA₂ values, were: atropine (9.51) = 4-DAMP (9.32) > p-F-HHSiD (7.78) > tropicamide (7.40) > pirenzepine (6.91) = AF-DX 116 (6.88) = methoctramine (6.71). This muscarinic antagonist profile suggests that an M₃ receptor is involved in the carbachol induced contraction. 6 The present results suggest that a rich network of AChE- positive fibres is present in the oesophageal groove floor, where they form a nerve trunk and thinner branches accompanying blood vessels and sometimes around ganglia. The muscarinic cholinergic contraction of the bovine oesohageal groove seems to be mediated via activation of an M₃ postsynaptic muscarinic receptor.     

Muscarinic M3 receptors mediate total inositol phosphates accumulation in murine HSDM1C1 fibrosarcoma cells

Muscarinic receptors in murine fibrosarcoma HSDM1C1 cells were characterized using both radioligand binding and total inositol phosphates accumulation (IPs). Muscarinic agonists elicited a concentration-dependent enhancement of IPs accumulation with a maximum of 14-fold stimulation above basal level. The following potencies ( - log EC₅₀) were observed for the full agonists: ( + )-cis-dioxolane 5.4, oxotremorine-M 5.3, ( + )-muscarine 5.2 and carbachol 5.0. Bethanechol (4.1) and arecoline (5.0) were partial agonists, evoling 43 and 55%, respectively of the maximum level of stimulation to ( + )-cis-dioxolane, whereas pilocarpine and McN-A-343 were inactive as agonists (1 μmol/1-1 mmol/1) The apparent affinities for muscarinic antagonists ( - log K_B) estimated by Schild regression were: 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) 9.2, dicyclomine 7.0, pirenzepine 6.9, (±)-p-F-HHSiD (para-fluoro-hexahydro-siladifenidol) 7.0, AF-DX 116 6.2, methoctramine 5.7. In saturation binding studies using [³H]N-methylscopolamine a homogeneous population of sites was identified, with a density of 145 pmol/mg protein. In competition radioligand binding studies, the following apparent affinities ( - log K_i) were observed: 4-DAMP 9.7, dicyclomine 8.3, (±)-p-F-HHSiD 7.6, AF-DX 116 6.8, methoctramine 6.6 and gallamine 6.8. In binding studies all antagonists studied recognized a single population of sites, as judged by the Hill coefficients from the displacement isotherms. These data are consistent with HSDM1C1 cells expressing an apparent homogeneous muscarinic M₃ population that mediates a large level of total IPs accumulation. This clonal line may provide a useful model to further elucidate relationship between endogenous muscarinic M₃ receptor stimulation and IPs accumulation. Additionally, these studies illustrate the importance of characterizing muscarinic receptor subtypes by null methods when using biochemical responses in isolated cells.