Subclassification of muscarinic receptors in the heart,urinary bladder and sympathetic ganglia in the pithed rat

Summary In pithed normotensive rats muscarinic receptors were characterized heart, urinary bladder and sympathetic ganglia; the selectivity of some classical muscarinic agents for these subtypes was investigated. The potencies in decreasing heart rate, increasing bladder pressure and increasing diastolic blood pressure were measured for the following, intraarterially administered cholinergic agonists: McN-A-343 ([4-m-chlorophenylcarbamoyloxy]-2-butynyltrimethylammonium), pilocarpine, carbachol, oxotremorine, arecoline, acetyl--methylcholine and acetylcholine. The selective M₁-antagonist pirenzepine, the mixed M₁/M₂-antagonist dexetimide and the cardioselective M₂-antagonist gallamine were used as tools for identification of the receptors. All data were obtained after intravenous pretreatment with a high dose of atenolol to eliminate tachycardia induced by stimulating sympathetic ganglionic muscarinic receptors.Dexctimide strongly antagonized the bradycardia as well as the increase in bladder pressure induced by pilocarpine, carbachol, oxotremorine, arecoline, acetyl--methylcholine and acetylcholine, whereas pirenzepine was much less effective. Gallamine antagonized the bradycardia, whereas no influence was found on the bladder contraction. Pilocarpine acted as a partial agonist in reducing heart rate as well as in increasing bladder pressure, whereas McN-A-343 was almost ineffective in doses up to 1 mg/kg.The hypertensive response to pilocarpine and carbachol was less pronounced than that produced by McN-A-343. Pirenzepine and dexetimide significantly antagonized the hypertensive response to McN-A-343 and pilocarpine, whereas gallamine was much less effective. The hypertensive response induced by carbachol was totally blocked by hexamethonium. The other agonists used in this study did not produce a significant increase in diastolic blood pressure in doses that produced a maximal effect on heart rate and urinary bladder pressure.Simultaneously, intraarterially infused acetylcholine dose-dependently and reversibly decreased the pressor response to intravenously administered McN-A-343.These data suggest that muscarinic receptors in rat sympathetic ganglia belong to the M₁-subtype, whereas the muscarinic receptors in rat heart and urinary bladder represent heterogenous populations of M₂-receptors. The agonists used in this study, though, could not discriminate between these heterogenous M₂-receptors.Like McN-A-343, pilocarpine appears to be a rather selective M₁-agonist. In this study the M₁/M₂ selectivity of muscarinic agents with pronounced M₂-agonist activity could not be evaluated since M₂-receptor stimulation interferes with the hypertensive response to M₁-receptor stimulation.     

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.     

Aging and cholinergic responses in bovine trachealis muscle.

1. The relative potencies of muscarinic agonists on bovine tracheal smooth muscle were unchanged as a consequence of aging and were carbachol greater than oxotremorine greater than muscarine greater than pilocarpine greater than McNeil A-343. 2. During aging, the potencies of carbachol, oxotremorine, McNeil A-343 and pilocarpine, but not muscarine, were reduced. 3. Maximal induced tensions to all the agents studied were reduced as a consequence of age. 4. Irreversible antagonism with benzilylcholine mustard showed that agonist efficacy was significantly reduced during aging. 5. Estimated receptor occupancy at the EC50 was significantly greater in tracheal tissues from the mature versus immature cows for every agonist studied. 6. The dissociation constants for full agonists (carbachol, oxotremorine and methacholine) were decreased with maturation while the converse was observed with partial agonists (McNeil A-343, pilocarpine). 7. We conclude that there are significant changes in the properties and coupling of muscarinic receptors during aging. These changes may contribute to the reduced airway reactivity seen in vivo.     

Characterization of muscarinic receptors on the isolated guinea pig ileum at pharmacologically low concentrations

• 1.1. Several selective and non-selective muscarinic agonists (McN-A-343, RS-86, arecoline, oxotremorine-M, pilocarpine, cis-dioxolane, and acetylcholine) were examined for relaxant activity in the isolated guinea-pig ileum at pharmacologically low concentrations.
• 2.2. The concentrations studied include: 1 × 10⁻¹²M, 3 × 10⁻¹²M, 1 × 10⁻¹¹M, 3 × 10⁻¹¹M and 1 × 10⁻¹⁰M.
• 3.3. None of the compounds exhibited relaxant activity in both the field and non-field stimulated ileum.
• 4.4. All of the above compounds exert muscarinic agonist activity in a concentration range of 1 × 10⁻⁹M to 1 × 10⁻⁶M (Williams et al., 1992).
• 5.5. Thus, in the isolated guinea-pig ileum, muscarinic agonists do not exert relaxant activity of the gastrointestinal tract at low concentrations.

     

Muscarinic suppression of the M-current in the rat sympathetic ganglion is mediated by receptors of the M1-subtype.

1. Under voltage-clamp dissociated adult and foetal rat superior cervical ganglion (s.c.g.) cells exhibited a non-inactivating voltage- and time-dependent component of K+ current termed the M-current (IM). IM was detected and measured from the current decay during hyperpolarizing voltage steps applied from potentials where IM was pre-activated. 2. Neither the resting membrane current nor the amplitude of these current decay relaxations were reduced by omitting Ca from the bathing fluid, showing that the M-current was not a 'Ca-activated' K-current dependent on a primary Ca-influx. Concentrations of (+)-tubocurarine sufficient to block the slow Ca-activated K-current IAHP did not inhibit IM or antagonize the effect of muscarinic agonists on IM, showing that IM was not contaminated by IAHP. Tetraethylammonium (1 mM), which blocks the fast Ca-activated K-current IC, produced a small inhibition of IM. This was not due to contamination of IM by IC since muscarinic agonists did not consistently block IC. 3. The muscarinic agonists muscarine, oxotremorine, McN-A-343 and methacholine reversibly suppressed IM, resulting in an inward (depolarizing) current. The rank order of potency was: oxotremorine greater than or equal to muscarine greater than McN-A-343 greater than methacholine. 4. The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5. IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 +/- 0.13 (n = 3) and 6.02 +/- 0.13 (n = 4) respectively. 6. The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 microM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nM. 7. Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 +/- 0.03 (n = 8). 8. These results suggest that the receptors mediating suppression of the M-current accord with those designated pharmacologically as M1 and that these receptors reach maturity at a very early stage in the development of the rat s.c.g.     

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.     

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.     

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)     

Inhibitory cholinergic effects on the autonomously contractile bulbus cordis branchialis of the cephalopod Sepia officinalis L.

In vitro experiments were performed on a standardized preparation of the autonomously contractile bulbus cordis branchialis of the branchial heart of Sepia officinalis to investigate its cholinergic neuroregulation. Apart from acetylcholine, nicotine and carbachol (nicotinic agonists), the muscarinic agonists muscarine, arecoline, pilocarpine, and oxotremorine also exerted concentration-dependent negative inotropic effects on the preparations. As both the muscarinic antagonist quinuclidinylbenzilate and the nicotinic antagonist alpha-bungarotoxin blocked the ACh action there might be a special, possibly mixed muscarinic/nicotinic ACh-receptor system in the myocytes of the bulbus cordis branchialis, which is different from the cholinergic receptor in the central part of the branchial heart.     

Muscarinic receptors in rat sympathetic neurones

1 Potential changes in isolated superior cervical ganglia of the rat produced by muscarinic-receptor agonists were recorded by an extracellular ;air-gap' method.2 Muscarinic agonists produced a delayed low-amplitude ganglion depolarization, frequently preceded by a hyperpolarization. Potentials were enhanced by reducing [K(+)](o) or [Ca(2+)](o).3 Mean ED(50) values (muM) for depolarization at 25 degrees C were: oxotremorine 0.004, methylfurmethide 0.11, (+/-)-muscarine 0.24, furmethide 1.56, pilocarpine 4.81 and AHR-602 (N-benzylpyrrolidylacetate methobromide) 10.8. Responses produced by oxotremorine, pilocarpine and AHR-602 showed some characteristics of ;partial agonism'. ED(50) values (muM) for choline esters (measured in the presence of 2.5 mM hexamethonium) were: acetylcholine 3.2, methacholine 59 and bethanechol 78.4 Responses to muscarine were antagonized by hyoscine (K(I) 0.49 nM) atropine (K(I) 0.24 nM) methylscopolamine (K(I) 0.09 nM) lachesine (K(I) 0.15 nM) and (weakly) by hexamethonium (K(I) 0.2 mM). Propylbenzilylcholine mustard produced irreversible antagonism with an apparent onset rate constant of 2 x 10(5) M(-1)S(-1).5 Depolarization was accompanied by facilitation of submaximal ganglionic transmission.6 Muscarine (1 to 100 muM) initially reduced, then increased, the rate of (86)Rb(+)-efflux from isolated ganglia at both 6 and 120 mM [K(+)](o). These effects were reduced by 1 muM hyoscine.7 No consistent change in the amounts of cyclic 3',5'-guanosine monophosphate in isolated ganglia accompanying muscarinic depolarization could be detected.8 Mean against ED(50) values (muM) for contracting the rat isolated ileum were: oxotremorine 0.012, methylfurmethide 0.29, (+/-)-muscarine 0.48, pilocarpine 7.8 and AHR-602 9.9. Mean antagonist K(I) values (nM) were: hyoscine 0.17, atropine 0.34 and lachesine 0.27.9 It is concluded that ganglionic muscarinic receptors are quite similar to ileal receptors in terms of agonist ED(50) and antagonist K(I) values, and that the major difference between them lies in the greater ;efficacy' of certain agonists (pilocarpine, AHR-602 and McN-A-343) on the ganglion.     

Interactions of agonists with M2 and M4 muscarinic receptor subtypes mediating cyclic AMP inhibition.

In this study the similarities and differences between the M2 and M4 subtypes in their recognition of agonists were explored. A CHO-K1 cell line transfected with the human m2 receptor was used as a homogeneous M2 tissue for comparison with two putative M4 systems (rat striatum and the N1E-115 mouse neuroblastoma cell line). The equilibrium binding dissociation constants and intrinsic efficacies for seven muscarinic agonists were determined for their stimulation of cyclic AMP inhibition via the M2 and M4 receptors. Partial receptor occlusion with propylbenzilylcholine mustard was used to determine binding constants for the more efficacious drugs and the reference agonist oxotremorine-M. The binding dissociation constants and relative efficacies for other agonists were then determined in reference to oxotremorine-M by a null method. For the M2 receptor the agonist binding dissociation constants ranged in potency from oxotremorine (1.5 microM) to bethanechol (171 microM), whereas relative efficacies varied from that of muscarine (relative efficacy = 0.9) to the value for McN-A343 (relative efficacy = 0.04). In general, most agonists bound with similar potencies to M2 and M4 receptors (Kd values within a factor of 2-3). However, oxotremorine bound to the N1E-115 and striatal M4 receptors about 3-fold and 10-fold less potently, respectively, than it did to the M2 receptor. Another exception was pilocarpine, which bound to the N1E-115 receptor (1.9 microM) with 8-fold and 12-fold higher potency than to the CHO-K1 M2 receptor and the striatal M4 receptor, respectively. Despite the low affinity of bethanechol for the M2 receptor, it was an efficacious agonist (maximal response equivalent to that of oxotremorine-M; relative efficacy = 0.6) at this subtype, whereas it was a partial agonist (60%) with lesser efficacy in the clonal M4 system. In contrast, McN-A343 and arecoline were significantly more efficacious at the two M4 receptors than they were at the M2 receptor. The M4 system in the rat striatum displayed some similarity to the N1E-115 M4 system, with regard to the efficacy ranking for certain agonists (arecoline greater than bethanechol greater than McN-A343 greater than or equal to pilocarpine). This rank order was different from the ranking of these four agonists in the M2 system, indicating that these two M4 receptors are more similar to each other in efficacy ranking than they are to the M2 receptor. However, the rat striatal and N1E-115 M4 receptor differed in their binding of oxotremorine and pilocarpine, indicating that these two M4 systems were not pharmacologically identical.(ABSTRACT TRUNCATED AT 400 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.     

Effects of acetylcholine agonists and antagonists on yawning and analgesia in the rat

The ability of acetylcholine muscarinic agonists, injected subcutaneously (s.c.) to elicit yawning and analgesia (tail-flick response) in rats was examined. Yawning was elicited by physostigmine, RS86 and pilocarpine with an inverted 'U'-shaped dose-response relationship; maximal effects occurred at 0.1, 0.5 and 2.0 mg/kg respectively. Neostigmine (0.05-0.2 mg/kg); arecoline (0.5-2.0 mg/kg); bethanecol (0.1-10 mg/kg) and McN-A-343 (5-20 mg/kg) had marginal or no activity. In contrast, dose-related analgesia was obtained following oxotremorine (0.01-0.3 mg/kg) and arecoline (0.5-4.0 mg/kg) and physostigmine (0.1-0.4 mg/kg), RS86 (0.25-2.5 mg/kg) and pilocarpine (0.5-8.0 mg/kg). The effects of acetylcholine antagonists on physostigmine-induced yawning and physostigmine-induced analgesia were also investigated. Following their s.c. injection, trihexyphenidyl, atropine, dicyclomine, secoverine and methylatropine but not pirenzepine, inhibited both yawning and analgesia; there were clear differences in their potencies on the two responses. Pirenzepine, intracerebroventricularly (i.c.v.), inhibited yawning (ED50 value 5.7 micrograms/rat) but not analgesia (3-100 micrograms/rat). The results are discussed in terms of a possible functional differentiation of central muscarinic receptors.     

Identification and role of muscarinic receptor subtypes expressed in rat adrenal medullary cells

The muscarinic receptor is known to be involved in the acetylcholine (ACh)-induced secretion of catecholamines in the adrenal medullary (AM) cells of various mammals. The muscarinic receptor subtype involved and its physiological role, however, have not been elucidated yet. Thus, we investigated these issues in acutely isolated rat AM cells and perfused rat adrenal medulla. The RT-PCR analysis revealed the presence of M(2), M(3), M(4), and M(5) mRNAs. Immunocytochemistry with specific antibodies showed that M(5)-like immunoreactivities (IRs) were detected at half the cell membrane area, which was much larger than that with M(3)- or M(4)-like IRs. Muscarine produced inward currents in a dose-dependent manner. Pilocarpine, McN-A-343, and oxotremorine were less efficient than muscarine; and RS-86, which has no action on the M(5) receptor, produced no current. Electrical stimulation of nerve fibers produced a frequency-dependent increase in the Ca(2+) signal in perfused adrenal medullae. Muscarinic receptors were found to be involved in neuronal transmission in AM cells in the presence of a cholinesterase inhibitor, which suppresses ACh degradation. We concluded that the M(5) receptor is the major muscarinic receptor subtype in rat AM cells and may be involved in neuronal transmission under conditions where ACh spills over the synapse.     

Distinct functional muscarinic receptors in guinea-pig gallbladder, ileum and atria.

OBJECTIVE: The contractile responses of guinea-pig gallbladder smooth muscle cells have been suggested to be mediated by M3 and M4 muscarinic receptors by different research groups. Therefore, in the present study, several pharmacological properties of cholinergic functions in guinea-pig gallbladder, guinea-pig ileum (mediated via M3 receptors), and guinea-pig and rat atria (mediated via M2 receptors) were compared. METHODS: The isometric contractions of isolated guinea-pig ileum, guinea-pig gallbladder, guinea-pig and rat atrial strips in in vitro organ bath were recorded on a polygraph and the effects of carbachol, oxotremorine, McN-A-343, and clozapine have been investigated. RESULTS: Three muscarinic receptor agonists, carbachol, oxotremorine and McN-A-343 showed different order of potencies in their negative inotropic effects and contractile actions in guinea-pig gallbladder suggesting that functional muscarinic receptors in the gallbladder are distinct from those in the atria, and similar to M4-subtypes. Clozapine which was shown to have antagonistic affinity for muscarinic M1, M2, M3 and M5, but partial agonistic affinity for muscarinic M4 receptors, contracted gallbladder concentration-dependently. On the other hand, clozapine antagonised carbachol-induced ileal and gallbladder contractions and negative inotropic effects indicating that it acts like a partial agonist in the gallbladder. CONCLUSION: It was concluded that the contractile muscarinic receptors of guinea-pig gallbladder are distinct from those of atria (M2) and ileum (M3), but seem to be of M4 subtype.     

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.     

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.     

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 study of aggressive behaviour after injection of cholinomimetics,anticholinesterases, nicotinic,and muscarinic ganglionic stimulants into the cerebral ventricles of conscious cats: Failure of nicotinic drugs to evoke aggression

Emotional behaviour, especially aggression, evoked by cholinomimetics, anticholinesterases, nicotinic, and muscarinic ganglionic stimulants injected into the cerebral ventricles of unanesthetized cats was studied and compared. Aggressive behaviour was obtained after intraventricular carbachol, muscarine, arecoline, eserine, neostigmine, and 4-m-chlorophenylcarbamoyloxy-2-butynyl-trimethylammonium chloride (McN-A-343). The aggressive behaviour and the autonomic and motor phenomena of various single doses of cholinomimetics and anticholinesterases were dose-dependent and long-lasting. Neostigmine produced aggressive behaviour only in about one-fourth of the experiments, while arecoline and McN-A-343 induced aggression of slightest intensity. The type of aggressive behaviour caused by carbachol, arecoline, eserine, neostigmine, and McN-A-343 was classified as irritable-type aggressive behaviour, while muscarine induced fear-type aggressive behaviour. Intraventricular administration of nicotine, dimethylphenylpiperazinium, tetramethylammonium, and N-benzyl-3-pyrrolidyl acetate methobromide (AHR-602) did not evoke aggressive behaviour, but they induced fleeting autonomic and motor phenomena. The ability of single intraventricular injections of cholinomimetics and anticholinesterases to trigger and to maintain long-lasting aggressive behavioural phenomena cannot be ascribed to a rapid detonator transmission, but rather to an action that differs from the conventional transmitter function. In addition, we advance a hypothesis that the cholinoceptive neurons sensitive to cholinomimetics and anticholinesterases behave as a biologic generator that when activated produces long-lasting aggression with autonomic and motor phenomena.     

Evidence for a direct relationship between phosphoinositide metabolism and airway smooth muscle contraction induced by muscarinic agonists

The relationship between bovine tracheal muscle contraction and phosphoinositide metabolism was studied with the muscarinic agonists, methacholine, oxotremorine, and McN-A-343. Analysis of the dose-response curves for contraction and inositol phosphates accumulation with these agonists demonstrated a direct relationship between the two parameters, with a considerable reserve of inositol phosphate production for the full contractile agonists, methacholine and oxotremorine, and no reserve for the partial agonist, McN-A-343.