McN-A-343, a muscarinic agonist, reduces inflammation and oxidative stress in an experimental model of ulcerative colitis.

AIM: The aim of the present study was to investigate the anti-inflammatory response mediated of the M1 muscarinic acetylcholine receptor (mAChR) during experimental colitis. MATERIAL AND METHODS: After the induction of 6% acetic acid colitis, mice were treated with McN-A-343 0.5, 1.0, and 1.5 mg/kg or dexamethasone (DEXA, 2.0 mg/kg) or pirenzepine (PIR, 10 mg/kg; M1 mAChR antagonist). Colonic inflammation was assessed by macroscopic and microscopic lesion scores, colonic wet weight, myeloperoxidase (MPO) activity, interleukin-1 beta (IL1-ß) levels and tumor necrosis factor alpha (TNF-α), glutathione (GSH), malondialdehyde (MDA) and nitrate and nitrite (NO3/NO2), mRNA expression of IKKα, nuclear factor kappa beta (NF-kB) and cyclooxygenase-2 (COX-2), as well protein expression of NF-kB and COX-2. RESULTS: Treatment with McN-A-343 at a concentration of 1.5 mg/kg showed a significant reduction in intestinal damage as well as a decrease in wet weight, MPO activity, pro-inflammatory cytokine concentration, markers of oxidative stress and expression of inflammatory mediators. The action of the M1 agonist by the administration of pirenzepine, which promoted the blocking of the mAChR M1-mediated anti-inflammatory response, has also been proven. CONCLUSION: The results suggest that peripheral colonic M1 mAChR is involved in reversing the pro-inflammatory effect of experimentally induced colitis, which may represent a promising therapeutic alternative for patients with ulcerative colitis.

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

Mutagenesis of nucleophilic residues near the orthosteric binding pocket of M1 and M2 muscarinic receptors: effect on the binding of nitrogen mustard analogs of acetylcholine and McN-A-343.

Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.

Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors.

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Tyr(177), and Thr(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the (172)EDGE(175) sequence had been neutralized and each Tyr(177) and Thr(423) was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr(177) and Thr(423), rather than the (172)EDGE(175) sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

Transducer abstraction: a novel approach to the detection of partial agonist efficacy in radioligand binding studies.

The properties of the ternary complex model (TCM) of drug action at G protein-coupled receptors (GPCRs) were examined, using theoretical computer simulations, with regard to the predicted effects of the presence of a fixed concentration of one agonist on the competition binding profile of another. Subsequently, the binding properties of the full muscarinic acetylcholine receptor (mAChR) agonists acetylcholine (ACh) and carbachol (CCh), and the partial agonists pilocarpine and McN-A-343, were investigated in competition experiments against [(3)H]N-methylscopolamine using homogenate preparations from Chinese hamster ovary cells, stably expressing the human M(1) or M(2) mAChR. At the M(2) mAChR, all agonists displayed biphasic binding curves and were readily modulated by the non-hydrolyzable GTP analogue, Gpp(NH)p, in accordance with previously established experimental observations. In contrast, agonist binding at the M(1) mAChR showed no significant change in the presence of Gpp(NH)p, even in the case of a full agonist. This phenomenon precludes using the "GTP-shift" to assess agonist efficacy at the M(1) mAChR. When the ACh competition curves were reconstructed in the presence of graded concentrations of either a full or a partial agonist, a significant redistribution of the fraction of the high-affinity state recognized by ACh was observed. However, when the procedure was repeated using the antagonist, atropine, no significant effect on the fraction of either the high or low affinity ACh binding components at the mAChR was observed. Taken together, these results indicate that changes in the profile of full agonist binding isotherms, when constructed in the presence of a partial agonist, may be more sensitive indicators of partial agonist efficacy than regular assays that directly measure partial agonist binding.

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca(2+)-mobilization and Ca(2+)-influx pathways in taenia caeci.

1. We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN-A-343 and AHR-602 with those of carbachol in smooth muscle of guinea-pig taenia caeci. 2. McN-A-343 and AHR-602, as well as carbachol, completely displaced the atropine-sensitive binding of [3H]-quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol > McN-A-343 >> AHR-602. 3. In the presence of 2.2 mM extracellular Ca2+, McN-A-343 and AHR-602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4-DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF-DX 116 (M2 selective). These contractions were inhibited by an L-type Ca2+ channel blocker, verapamil. 4. In Ca(2+)-free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN-A-343 and AHR-602. Application of McN-A-343 or AHR-602 inhibited the carbachol-induced contraction in Ca(2+)-free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5. The EC50 value for carbachol-induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6. The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor-mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN-A-343 and AHR-602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.

Pharmacological basis for functional selectivity of partial muscarinic receptor agonists.

Muscarinic receptor agonists activate phosphoinositide hydrolysis and adenylate cyclase in Chinese hamster ovary cells transfected with cDNAs encoding the human muscarinic ml and m3 receptors. Whereas carbachol activates similarly both receptor subtypes, 4-[3-chlorophenyl-carbamoyloxy]-2-butynyltrimethyl ammonium chloride (McN-A-343) preferentially activates the m1 subtype over m3, in regard to both phosphoinositide hydrolysis and adenylate cyclase activity. On the other hand, oxotremorine activates phosphoinositide hydrolysis to a similar extent in both cell lines, but it activates preferentially adenylate cyclase in m1 versus m3 receptor expressing cells. Relative to carbachol, both McN-A-343 and oxotremorine activate preferentially phosphoinositide hydrolysis over adenylate cyclase in both cell lines. Prolonged incubation of cells with either carbachol, McN-A-343, or oxotremorine down-regulated the m1 receptors. This was accompanied by a parallel decrease in adenylate cyclase activity, whereas phosphoinositide hydrolysis remained relatively high. Inactivation of the receptors by alkylation with acetylethylcholine mustard, or by blocking with atropine, reduced carbachol-stimulated adenylate cyclase activity more effectively than carbachol-induced phosphoinositide hydrolysis in both m1 and m3 receptor expressing cells. These findings imply that the receptor reserve in these cell lines is greater for phosphoinositide hydrolysis response than for adenylate cyclase response. Yet, the receptor reserve for each of these responses is similar in both m1 and m3 receptor expressing cells. Since the binding affinities of McN-A-343 and of oxotremorine to m1 and m3 receptors are very similar, and since both cell lines contain similar amounts of spare receptors, we propose that the preferential activation of muscarinic m1 over m3 receptor by partial agonists is related to differences in the abilities of the two receptor subtypes to undergo conformational changes following agonist binding. This hypothesis is supported by results showing that the muscarinic m1 but not m3 receptor exhibits two affinity states in a competition binding assay.

Cholinergic activation of Cl- secretion in rat colonic epithelia.

Acetylcholine receptor agonists and antagonists were used in a pharmacological analysis to identify which muscarinic receptor(s) may be involved in cholinergic regulation of Cl- secretion across rat colonic mucosa in vitro. A comparative ligand binding analysis for each of the antagonists was carried out in parallel. Both studies elicited identical rank order potencies (atropine > or = 4-diphenyl-acetoxy-N-piperidine methiodide (4-DAMP) > pirenzepine > 11-[[2[(diethylamino)methyl]-1-pipiridinyl]acetyl[5,11- dihydro-6H-pyrido[2,3-b]]1,4]benzodiazepine-6-one (AF-DX 116). Cholinomimetic-induced Cl- secretion was predominantly mediated by activation of muscarinic receptors in rat isolated colonic mucosa, with only a modest contribution from nicotinic receptors. Short circuit current responses evoked by the selective muscarinic M1 receptor agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-a minium chloride (McN-A-343) suggest that this receptor subtype, which is thought to be neuronally sited, also plays a minor role in regulation of intestinal ion transport. The principal epithelial cell receptors responsible for acetylcholine receptor-mediated Cl- secretion appear to belong to the M3 class.

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists.

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.

Antagonistic properties of McNeil-A-343 at 5-HT4 and 5-HT3 receptors.

1. This study describes the in vitro interaction of the muscarinic ligand McNeil-A-343 with two 5-hydroxytryptamine (5-HT) receptor subtypes, the 5-HT4 and 5-HT3 receptors, using functional as well as radioligand binding studies. 2. In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil-A-343 was a competitive antagonist (pA2 6.2) of the 5-HT4 receptor which mediates the relaxation induced by 5-HT. The compound per se relaxed the oesophagus at high concentration only (> or = 10 microM), an effect unchanged by desensitization of the 5-HT4 receptor with 10 microM 5-methoxytryptamine. In the same preparation in the absence of tone, McNeil-A-343 displaced the carbachol concentration-response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors. 3. In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil-A-343 caused a concentration-dependent depolarization that was unaffected by 100 nM ondansetron. The concentration-fast depolarization curve to 5-HT, mediated by the 5-HT3 receptor, was displaced to the right by McNeil-A-343, which showed an apparent affinity (pA2) of 4.8 for the 5-HT3 subtype. 4. In binding studies, McNeil-A-343 recognized a single population of 5-HT4 receptors in pig caudate nucleus, with a pKI of 5.9. The binding affinity of McNeil-A-343 for 5-HT3 receptors in NG 108-15 cells was approximately four times lower (pKI 5.3). Binding affinities (pKI) for muscarinic receptor subtypes in rat tissues were 5.3 (M1, cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively. 5. McNeil-A-343 is an antagonist at 5-HT4 and 5-HT3 receptors; the interaction of the compound with these receptor subtypes (notably the 5-HT4) occurs in a range of concentrations which generally overlaps that relevant to the interaction with muscarinic receptors.

Himbacine discriminates between putative muscarinic M1 receptor-mediated responses.

This study describes the antagonistic properties of himbacine, in comparison with those of pirenzepine, at muscarinic receptors mediating the depolarization of rat superior cervical ganglion, the inhibition of electrically-induced twitch contractions of rabbit vas deferens and the contraction of dog saphenous vein, currently classified as putative muscarinic M1 sites. The affinity of himbacine for the vas deferens site (pA2 8.08) was nearly ten times higher than those for the M1 receptors of rat ganglion and dog saphenous vein (pA2 7.14 and 7.16, respectively); affinity estimates for pirenzepine were similar throughout the different preparations. The present data are consistent with the allocation of ganglion and saphenous vein receptors into the M1 subclass; the profile of the vas deferens site, conversely, appears to be different, and possibly more closely related to that reported for the M4/m4 receptor.

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)

Muscarinic receptor subtypes and sexual behavior in female rats.

Cholinergic muscarinic systems are involved in the regulation of female sexual behavior in rats and hamsters. This series of experiments was designed to determine whether sexual behavior in female rats is controlled preferentially by one of the traditional muscarinic receptor subtypes. Intraventricular infusion of the muscarinic antagonist scopolamine (10 micrograms bilaterally) which binds with high affinity to both M1 and M2 subtypes inhibited sexual behavior, as indicated by the incidence of lordosis, in ovariectomized rats treated with estrogen and progesterone. In contrast, the M1-selective antagonist pirenzepine failed to reduce the incidence of lordosis following intraventricular infusion (10 to 80 micrograms bilaterally). Biochemical analyses revealed that intraventricular infusion of scopolamine (10 micrograms bilaterally) inhibited both M1 and M2 binding in brain tissues while intraventricular infusion of pirenzepine (10 micrograms bilaterally) completely inhibited M1 binding without affecting M2 binding. Intraventricular infusions of the acetylcholinesterase inhibitor physostigmine (10 micrograms bilaterally), the cholinergic agonist carbachol (1 microgram bilaterally), and the muscarinic agonist oxotremorine-M (0.1 micrograms bilaterally) activated lordosis in ovariectomized females primed with low doses of estrogen. In contrast, the putative M1 agonist McN-A-343 failed to significantly increase lordosis following intraventricular infusions (1, 10, 20 micrograms bilaterally). According to biochemical results, the ability of these agents to activate lordosis in female rats was related to their affinities for M2 binding sites not M1 binding sites. In a final experiment, estrogen treatment of ovariectomized rats did not alter muscarinic subtype binding in several brain areas as measured by the M1-selective ligand [3H] pirenzepine and the M2-selective ligand [3H] oxotremorine-M. The results of these experiments confirm that muscarinic systems contribute to the regulation of lordosis in female rats and indicate that M2 binding sites rather than M1 binding sites may be a critical component of this regulation.

Functional determination of McN-A-343 affinity for M1 muscarinic receptors.

The affinity (KA) of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343), acting as an agonist of M1 muscarinic receptors, has been estimated by means of fractional receptor inactivation, employing the irreversible muscarinic antagonist propylbenzylcholine mustard. Two M1-mediated responses elicited by McN-A-343 were studied: relaxation of the isolated rat duodenum and inhibition of twitch contractions in rabbit was deferens. A comparison was made with the affinity of McN-A-343 as an antagonist (KB) of acetylcholine-induced contraction in rat duodenum. Results showed that McN-A-343 displayed similar affinities as an agonist and as an antagonist: -log KA were 4.68 and 5.17 in duodenum and vas deferens, respectively, vs. -log KB of 4.96 in duodenum, indicating that the ability of McN-A-343 to selectively stimulate M1 receptors is not based on a greater affinity for this subtype. In both preparations examined, McN-A-343 reached maximum effect through occupation of a fraction of the total available receptors (approximately 30% in duodenum, approximately 80% in vas deferens), implying that occupation of M1 receptors is translated into effect in a highly efficient way.

Muscarinic receptor subtypes and the selectivity of agonists and antagonists.

Receptors may be grouped into superfamilies, according to their mechanism. Muscarinic receptors belong to one such superfamily, that of the G-protein coupled receptors. Subtypes of muscarinic receptors may be defined according to their primary amino acid sequence, their mechanism and their pharmacology. However, there is as yet no consistent scheme to explain the pharmacology and function of each molecular subtype. The pharmacological tools which distinguish between muscarinic receptor subtypes are described and the emerging evidence for the location of the ligand binding site is discussed.

The interaction of McN-A-343 with pirenzepine and other selective muscarine receptor antagonists at a prejunctional muscarine receptor.

The effect of several muscarine receptor antagonists on responses to carbachol (CCh) and McN-A-343 (McN) were compared in the perfused rabbit ear artery preparation stimulated via noradrenergic nerves at 3 Hz in the presence of cocaine (10 microM) and yohimbine (1 microM). The slope of the dose-response curve to McN was significantly less (P less than 0.05) than that for CCh although both agonists produced up to 100% inhibition of responses to nervous stimulation. All the antagonists investigated produced parallel shifts of the dose-response curve to the agonists and atropine, fenipramide or stercuronium gave a similar pA2 value with either agonist. Pirenzepine was a competitive antagonist when CCh was used, as judged by a slope of 0.96 +/- 0.10 for the Arunlakshana-Schild (A-S) plot (pKB 6.2). Displacement of 3H-(-)QNB binding by pirenzepine gave a pKI value of 6.0 which was not significantly different to the pKB value. When McN was used as the agonist, the dose-ratios obtained with pirenzepine (0.5 microM) were significantly different (P less than 0.01) to those with CCh as agonist and the slope of the A-S plot over the concentration range of 0.1 to 3 microM was significantly less than 1.0 (P less than 0.01), indicating that the inhibition was not a simple competitive interaction. It is suggested that the interaction of McN and pirenzepine may involve an allosteric mechanism.

Pharmacological differences between the high-affinity muscarinic agonist binding states of the rat heart and cerebral cortex labeled with (+)-[3H]cismethyldioxolane.

The high-affinity agonist binding state of muscarinic receptors in the rat heart and cerebral cortex has been pharmacologically characterized in parallel studies. Muscarinic sites were labeled and studied with the aid of a highly specific, rapid filtration binding assay using the potent muscarinic agonist (+)-[3H]CD. Homogenates of both tissues were found to contain a saturable high-affinity (Kd = 1-2 nM), low capacity (6-17% of (-)-[3H]QNB sites) (+)-[3H]CD binding state which demonstrated stereoselectivity and drug specificity typical of a muscarinic site. However, comparative studies of drug potency profiles in competition for myocardial and cerebral cortical (+)-[3H]CD-labeled membranes revealed several major pharmacological differences between muscarinic sites in these tissues. Whereas the muscarinic agonists pilocarpine and McN-A-343, the nonclassical antagonist pirenzepine, and the acetylcholinesterase inhibitor physostigmine reduced (+)-[3H]CD binding in both tissues, their inhibitory effects were more potent (4- to 77-fold) in cerebral cortical membranes. Conversely, gallamine, a nicotinic cholinergic antagonist, demonstrated a 36-fold greater potency at the high-affinity (+)-[3H]CD binding state in myocardial membranes. By comparison, other classical muscarinic agonists and antagonists were nearly equipotent as inhibitors of high-affinity (+)-[3H]CD binding in these two tissues. Thus, these studies for the first time demonstrate that muscarinic receptors in the heart and cerebral cortex can be distinguished pharmacologically by certain drugs which interfere with the high-affinity agonist binding state of the muscarinic recognition site and provide support for the subclassification of these receptors.