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.     

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

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

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.     

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.     

The effect of gallamine, gallopamil and nifedipine on responses to acetylcholine and carbachol in the taenia of the guinea-pig caecum

The effects of gallamine, gallopamil and nifedipine on isotonic contractions of the isolated taenia of the guinea-pig caecum produced by acetylcholine (ACh) or carbachol (CCh) were investigated. Gallamine (0.1 to 0.3 mM) inhibited contractions produced by CCh more than those produced by ACh. The difference was still present after pretreatment of the tissue with paraoxon (10 microM for 20 min) to inhibit cholinesterases or in experiments carried out in the presence of tetrodotoxin (0.3 microM) to exclude possible ganglionic stimulation by the agonists. Gallopamil or nifedipine selectively inhibited the tonic response to ACh in the absence or presence of paraoxon. The phasic response to ACh or the tonic response to CCh (0.1 or 1 microM) was much less affected. Reduction of the Ca2+ content of the bath medium reduced phasic and tonic responses to ACh more than the tonic response to CCh. These results suggest that there are differences in the interaction of ACh and CCh with muscarinic receptors in this muscle.     

The actions of some cholinomimetic drugs on the isolated taenia of the guinea-pig caecum

1. The actions on the taenia of 4-(m-chlorophenylcarbamoyloxy)-2-butynyl-trimethylammonium chloride (McN-A-343), N-benzyl-3-pyrrolidyl acetate methobromide (AHR-602), tetramethylammonium (TMA) and choline phenyl ether have been examined and compared with the actions of acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP).2. Responses of the taenia to these agonists are quantitatively and often qualitatively dependent on the tone of the preparation. A method is described which makes allowance for the effect of tone on heights of contractions.3. Acetylcholine, McN-A-343 and AHR-602 produced only contractions; TMA produced contractions or biphasic responses; and choline phenyl ether, nicotine and DMPP produced contractions, relaxations or biphasic responses.4. The mode of action of these compounds has been analysed by means of hyoscine, ganglion-blocking drugs, tetrodotoxin and local anaesthetics.5. It is concluded that acetylcholine, McN-A-343, AHR-602, TMA and choline phenyl ether act on muscarinic receptors in the smooth muscle. Choline phenyl ether has an additional action on nicotinic receptors of cholinergic neurones. Nicotine and DMPP act on nicotinic receptors of cholinergic neurones and of inhibitory neurones. An action on the latter is sometimes also seen with TMA and choline phenyl ether.6. With nicotine or DMPP, and TMA or choline phenyl ether in the presence of hyoscine, part of the contraction phase of biphasic responses (in which a contraction follows relaxation) is best explained as being triggered by the initial relaxation-that is, as a "rebound contraction".7. None of the compounds tested appeared to exert an atropine-sensitive action on neurones.8. In the presence of hyoscine high concentrations of agonists can act on sites not involved with lower concentrations.     

Differential development of carbachol-induced desensitization in receptor-mediated Ca2+ influx and Ca2+ release pathways in smooth muscle of guinea-pig taenia caeci

1. We have found that development of carbachol (CCh)-induced desensitization to receptor agonists, but not to receptor by-passed stimulation, is transiently interrupted by a Ca2+-dependent resensitization during the early stage in the smooth muscle of guinea-pig taenia caeci. To further characterize the receptor-mediated signal transduction pathways involved in this peculiar desensitization process, we examined the desensitization processes during Ca2+ influx- and Ca2+ release-mediated contractions in response to activation of muscarinic receptors or histamine H1 receptors. 2. Desensitization treatment with 10(-4) mol/L CCh for 30 min in the presence of extracellular Ca2+ resulted in desensitization to the muscarinic agonists McN-A-343 or AHR-602, which are known to induce contraction only in the presence of extracellular Ca2+ in taenia caeci. The development of desensitization to these agonists was interrupted by a transient resensitization at 1 min. In contrast, the transient resensitization phase was lost following removal of extracellular Ca2+ during the desensitization treatment with CCh; under these conditions, the desensitization developed gradually without an apparent resensitization phase. 3. Contractions to 10(-4) mol/L CCh and 10(-4) mol/L histamine in the absence of extracellular Ca2+ were gradually desensitized without a resensitization phase following the CCh desensitization treatment, irrespective of the presence or absence of extracellular Ca2+ during CCh treatment, although the onset of the desensitization was delayed under Ca2+-free conditions. 4. These results suggest that the receptor-mediated Ca2+ influx and Ca2+ release pathways are differentially desensitized to CCh and that the transient resensitization appears to regulate the desensitization process in response to Ca2+ influx-mediated contraction. Such differential processes of desensitization in receptor-mediated bifurcated signalling pathways may determine cellular responsiveness to certain types of stimuli, depending on the different Ca2+ sources required for contraction.     

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.     

Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells

Catecholamine secretion and cyclic GMP levels were measured in chromaffin cells isolated from bovine adrenal medulla. Acetylcholine (ACh) and nicotine, but not muscarine, induced 8- to 10-fold increases in catecholamine secretion, with respective ED₅₀ values of 10 and 2 M. Cyclic GMP levels were also increased from 3- to 5-fold in the presence of ACh, and this stimulation was mimicked by muscarine but not by nicotine. Half-maximum stimulations of cyclic GMP levels with ACh and muscarine were observed at 0.1 and 0.3 M respectively. The order of potency of various cholinergic drugs for cyclic GMP stimulation was as follows: ACh > oxotremorine > methacholine > muscarine > carbamylcholine > furthretonium > arecholine > bethanechol. Pilocarpine, McN-A-343, and AHR-602 were inactive at concentrations between 10^?8 and 10^?3 M. Isobutylmethylxanthine (1 mM), a specific phosphodiesterase inhibitor, caused a 7-fold increase in cyclic GMP and potentiated 3-fold the stimulation of cyclic GMP by ACh. The nicotine-induced catecholamine secretion was inhibited 19 and 33 per cent by the co-stimulation of the muscarinic receptor with 0.2 and 0.5 M ACh, respectively. Isobutylmethylxanthine (1 mM) also caused a 44 per cent inhibition of nicotine-induced catecholamine secretion, and its effect was additive to that of ACh. Atropine (0.1 M) selectively abolished the inhibition caused by ACh. Similar inhibitions were also obtained in the presence of exogenous dibutyryl cyclic GMP or 8-bromo cyclic GMP. These data indicate that the nicotinic stimulation of catecholamine secretion from bovine adrenal chromaffin cells may be regulated by cyclic GMP via the stimulation of a muscarinic receptor.     

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.     

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.     

Muscarinic receptors controlling the carbachol-activated nonselective cationic current in guinea pig gastric smooth muscle cells

Muscarinic receptor subtypes controlling the nonselective cationic current in response to carbachol (ICCh) were studied in circular smooth muscle cells of the guinea pig gastric antrum using putative muscarinic agonists and antagonists. Both oxotremorine-M (an M2-selective agonist) and CCh dose-dependently activated the cationic current with EC50 values of 0.21 +/- 0.01 microm and 0.97 +/- 0.06 microM, respectively. In contrast, pilocarpine and McN-A 343 (an M1-selective and a putative M4 agonist) were weak partial agonists. In response to 10/microM CCh, 4-DAMP, methoctramine and pirenzepine dose-dependently inhibited ICCh and had IC50 values of 1.91 +/- 0.2 nM, 0.46 +/- 0.07 microM and 8.33 +/- 0.4 microM, respectively. 4-DAMP, methoctramine and pirenzepine shifted the concentration-response curves of ICCh to the right without significantly reducing the maximal current. Values of the apparent dissociation constant pA2 obtained from Schild plot analysis were 9.24, 7.72 and 6.62 for 4-DAMP, methoctramine and pirenzepine, respectively. Also, pertussis toxin completely blocked ICCh generation. These results suggest that the M2-subtype plays a crucial role in the activation of the ICCh, and a block of the M3-subtype reduces the sensitivity of the M2-mediated response with no significant reduction of maximum response.     

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.     

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.     

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.     

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 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.     

Heterogeneity of muscarinic receptors in lamb isolated coronary resistance arteries.

1. In vitro experiments in a microvascular myograph were designed to characterize postjunctional muscarinic receptors producing contraction both in the presence and absence of the endothelium in coronary resistance arteries (normalized diameter of 150-450 microns), isolated from the left ventricle of hearts from 3-6 month old lambs. Preferential muscarinic receptor antagonists were used to determine the receptor subtype: pirenzepine (M1 receptor), AFDX 116 (M2 receptor), 4-DAMP and pFHHSiD (M3 receptor). 2. The rank order of potency for muscarinic agonist-induced increases in tension in endothelium-intact preparations was oxotremorine-M = methacholine = acetylcholine (ACh) > carbachol. Removal of the endothelium increased the potency of ACh, but this procedure did not change either the sensitivity or maximal response to carbachol. 3. The contractile response to ACh was reproducible. Incubation with 3 x 10(-7)-3 x 10(-6) M pirenzepine induced non-parallel rightward shifts and depressed the maximum of the concentration-response curve to ACh in endothelium-intact arteries. The slope by Schild analysis was 2.9 +/- 0.8 (P < 0.05, n = 7). Atropine, AFDX 116, 4-DAMP and pFHHSiD produced parallel rightward shifts of the curves to ACh and the slopes of the Schild plots were not significantly different from unity. The pKB values for the antagonists from plots constrained to unity in endothelium-intact segments were: atropine (9.4), 4-DAMP (9.0), pFHHSiD (7.9) and AFDX 116 (6.2). 4. In endothelium-denuded arteries, pirenzepine, AFDX 116 and pFHHSiD caused concentration-dependent, parallel rightward displacements of the concentration-response curves to ACh and the slopes of the Schild plots were not significantly different from unity. The plots constrained to a slope of unity gave the following pKB values: pFHHSiD (8.7), pirenzepine (7.5) and AFDX 116 (6.2). 5. In the presence of the endothelium, low concentrations of pirenzepine (10(-9)-10(-7) M) produced leftward shifts of the ACh concentration-response curves. This potentiating effect of pirenzepine was reversed by endothelial cell removal. In preparations precontracted with the thromboxane-mimetic, U46619, the putative M1-selective agonist, McN-A-343, induced a biphasic relaxation with log IC50 of 8.53 +/- 0.14 and 5.02 +/- 0.08 for the first and second phase of the relaxation, respectively, and maximal relaxations of 22.8 +/- 4.3% and 41.1 +/- 5.4% (n = 16). McN-A-343 relaxed the vessels in the presence of 10(-7) M pFHHSiD and 3 x 10(-7) M AFDX 116, but not after incubation with 10(-9) M pirenzepine.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.