Characterization of muscarinic receptors of the rabbit ear artery smooth muscle and endothelium

Muscarinic receptors of the rabbit ear artery were characterized by observing the effect of the subtype selective antagonist pirenzepine on functional responses and radioligand binding. Pirenzepine has been shown to bind with high affinity to muscarinic receptors of certain brain regions and peripheral ganglia (M1 subtype) and with low affinity to receptors of the heart and upper gastrointestinal tract (M2 subtype). The affinity (pKB) of pirenzepine for the muscarinic sites of the endothelium was determined by the competitive antagonism of the relaxation response to methacholine. Schild analysis gave a pKB of 6.5 (320 nM) which is consistent with the low affinity, M2, subtype of muscarinic receptor. Removal of the endothelium eliminates any response to muscarinic agonists but does not decrease the density of muscarinic binding sites determined by binding of the specific ligand (-)-[3H]quinuclidinyl benzilate. This indicates a second group of muscarinic receptors most probably located on vascular smooth muscle cells for which there is no known function. The pKi for pirenzepine at these sites, as determined by the inhibition of (-)-[3H]quinuclidinyl benzilate binding, was 6.26 (550 nM) which is also consistent with a low affinity subtype. Thus, both types of vascular muscarinic binding sites, those on the endothelium which mediate relaxation and those on the vascular smooth muscle cells, are of the low affinity, M2, subtype.     

Multiple binding affinities of N-methylscopolamine to brain muscarinic acetylcholine receptors: differentiation from M1 and M2 receptor subtypes

The properties of the specific binding of the muscarinic receptor ligands [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine in rat brain were compared. The specific binding of both ligands was affected equally by heat, phospholipase A2 and trypsin. N-[3H]methylscopolamine labeled only a fraction of the total muscarinic receptors recognized by [3H]quinuclidinyl benzilate in different brain areas and in the heart. Evidence is presented that N-[3H]methylscopolamine, in fact, binds to a subpopulation of [3H]quinuclidinyl benzilate binding sites. The distribution of the high-affinity binding sites of N-[3H]methylscopolamine did not show a different tissue dependence as compared to the total receptor population, and did not parallel the distribution of the pirenzepine-sensitive M1 receptor subtype. Similarly, the affinity of both [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine varied from one tissue to another by a maximum of 2-fold. Although (-)-quinuclidinyl benzilate competed for the specific binding of [3H]quinuclidinyl benzilate in different tissues according to the law of mass-action, N-methylscopolamine showed an anomalous interaction with two binding sites. The low-affinity binding sites of N-methylscopolamine showed saturability of [3H]quinuclidinyl benzilate binding and stereoselectivity. When the binding characteristics of these N-methylscopolamine-inaccessible binding sites of [3H]quinuclidinyl benzilate in the brain were investigated further, it was found that N-methylscopolamine bound exclusively with a single low affinity, whereas pirenzepine still interacted with two receptor populations incorporated in these sites. It is concluded from several lines of evidence that the heterogeneity of binding of N-methylscopolamine to muscarinic receptors does not represent an interaction with the muscarinic M1 and M2 receptor subtypes defined by pirenzepine. Thus, the unique binding profile of pirenzepine to muscarinic receptors cannot be explained merely on the basis of its hydrophilic nature.     

Characterization of high affinity [3H]pirenzepine and (-)-[3H] quinuclidinyl benzilate binding to muscarinic cholinergic receptors in rabbit peripheral lung

We have characterized the binding of the selective muscarinic antagonist [3H]pirenzepine ([3H])PZ) and the classical muscarinic antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) to muscarinic cholinergic sites in rabbit peripheral lung membranes. For both radioligands, high affinity binding with pharmacologic specificity was demonstrated. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 4.5 nM and the Kd for (-)-[3H]QNB binding was 6.2 pM. Comparison of the total binding capacity values determined by saturation experiments with [3H] PZ and (-)-[3H]QNB demonstrates that approximately 78% of the total muscarinic binding sites in rabbit peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (-)-[3H]QNB binding by the muscarinic agonist carbachol in peripheral lung membranes. If the pulmonary muscarinic receptor with high affinity for PZ proves to have an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [3H]quinuclidinyl benzilate or [3H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [3H]quinuclidinyl benzilate in a biphasic manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

The binding properties of (-)-[3H]quinuclidinyl benzilate and [3H] N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-[3H]quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of [3H]N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when [3H]N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-[3H] quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.     

Regional distribution of M1, M2 and non-M1, non-M2 subtypes of muscarinic binding sites in rat brain

The distribution of subtypes of the muscarinic receptor in homogenates of the rat brain was investigated by measuring the competitive inhibition of the binding [3H]N-methylscopolamine by pirenzepine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one). In most brain regions, the competitive binding curves for AF-DX 116 and pirenzepine were consistent with a two-site model. The dissociation constant of pirenzepine for its high-affinity site (M1 receptor) was approximately 10(-8) M, whereas the dissociation constant of AF-DX 116 for its high affinity site (M2 receptor) was approximately 10(-7) M. In many regions, particularly those in the forebrain, the sum of the densities of the M1 and M2 binding sites was substantially less than 100% of the total sites, indicating the existence of a third population of sites lacking high affinity for both pirenzepine and AF-DX 116. We have designated these latter sites as non-M1, non-M2 muscarinic receptors. In general, the densities of the M1 and non-M1, non-M2 binding sites were highest in cerebral cortex, corpus striatum and hippocampus, intermediate in thalamus and hypothalamus, and lowest in midbrain, medulla-pons and cerebellum, whereas the M2 binding site had a relatively low, uniform density throughout the brain. The binding capacity of [3H]N-methylquinuclidinyl benzilate was estimated to be 20 to 30% lower than that of [3H]quinuclidinyl benzilate in various regions of the forebrain, but not in more caudal regions of the brain where the two radioligands had approximately the same binding capacities. Treatment of homogenates of the cerebral cortex with benzilylcholine mustard caused a selective loss of the majority of the [3H]N-methylscopolamine binding sites but spared 25% of the sites labeled by [3H]quinuclidinyl benzilate The results of pirenzepine/[3H]quinuclinyl benzilate competitive binding experiments on cerebral cortex treated with benzilylcholine mustard showed that the residual binding sites for [3H] quinuclidinyl benzilate were enriched in M1 muscarinic receptors.     

Pharmacologic characterization and functional role of muscarinic autoreceptors in the rabbit striatum

The objectives of the present studies were 1) to pharmacologically characterize the muscarinic autoreceptors in the striatum and 2) to examine their role in the regulation of physiologic acetylcholine (ACh) release. Schild plots were generated for atropine and pirenzepine against oxotremorine-induced inhibition of [3H]ACh release. Atropine, a nonselective antagonist, yielded a pA2 of 8.92. The pA2 for pirenzepine, a purported M1-selective antagonist, was 7.14. Both Schild plots had slopes not significantly different from one. Four agonists [oxotremorine, carbachol (CARB), McN-A-343 and pilocarpine] were tested for their effectiveness in inhibiting [3H]ACh release. McN-A-343 and pilocarpine have been reported to be selective for M1 receptors. Oxotremorine and carbachol were effective and potent inhibitors of [3H] ACh release, whereas McN-A-343 and pilocarpine were weak. Although the existence of muscarinic receptor subtypes remains an open question, these data are consistent with the "low" pirenzepine affinity (M2) subtype. Chronic treatments (14 days) with several agents were carried out (in vivo) to assess the role of muscarinic autoreceptors in the regulation of physiologic ACh release. Scatchard analyses of binding studies with [3H]quinuclidinyl benzilate were also performed to assess changes in the muscarinic receptor population in the striatum. Chronic treatment with scopolamine caused a 100% increase in the Bmax for [3H]quinuclidinyl benzilate binding but had no effect on the sensitivity of [3H]ACh release to inhibition by CARB. Fourteen-day treatment with physostigmine (3 mg/kg) produced a decrease in the sensitivity of [3H]ACh release to CARB plus a 42% decrease in Bmax and a 48% decrease in Kd for [3H]quinuclidinyl benzilate binding. Chronic haloperidol treatment caused an increase in the sensitivity of [3H]ACh release to CARB accompanied by a 46% increase in Bmax for 3H quinuclidinyl benzilate binding. These data suggest that muscarinic autoreceptors in the striatum do not regulate physiologic ACh release in the presence of intact acetylcholinesterase and that the interaction of dopaminergic and cholinergic neurons in the striatum may not be simple trans-synaptic inhibition.     

Characterization of [3H]pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both [3H]quinuclidinyl benzilate [( 3H]QNB) and [3H]pirenzepine [( 3H]PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound [3H]QNB binding sites. The Kd values for [3H]QNB and [3H]PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, [3H]PZ appeared to label fewer solubilized binding sites than did [3H]QNB. Maximum binding values for [3H]PZ and [3H]QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of [3H]QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for [3H]PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of [3H]QNB and [3H]PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for [3H]QNB and [3H]PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.     

Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist [3H]pirenzepine [( 3H]PZ) and the classical muscarinic antagonist [3H](-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for [3H](-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of [3H](-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

Direct binding and functional studies on muscarinic cholinoceptors in porcine coronary artery

The muscarinic cholinoceptors in porcine coronary artery were identified and characterized by a binding assay using (-)-[3H]quinuclidinyl benzilate (QNB) and also by pharmacological method. Specific (-)-[3H]QNB binding in the coronary artery was saturable and of high affinity (Kd = 0.08 nM), and it showed a pharmacological specificity as well as stereoselectivity which characterized muscarinic receptors. Muscarinic antagonists competed with the (-)-[3H]QNB binding in order: nonlabeled QNB greater than dexetimide greater than atropine greater than pirenzepine greater than AF-DX 116 greater than levetimide greater than gallamine. Dexetimide was approximately 2000 times as potent as levetimide. The potencies (pKi) of these muscarinic antagonists in competing for (-)-[3H]QNB binding sites in porcine coronary artery correlated well with their pharmacological potencies (pA2 for antagonistic effect of acetylcholine-induced contraction of coronary artery). The decrease in the (-)-[3H]QNB binding by atropine and pirenzepine was due to a reduction in the apparent affinity with little change in the number of maximal binding sites, suggesting a competitive antagonism. Specific (-)-[3H]QNB binding (Kd and maximal number of binding sites) in porcine coronary artery was not changed by the removal of endothelium. We conclude: 1) (-)-[3H]QNB selectively labels the physiologically relevant muscarinic receptors in porcine coronary artery and 2) the majority of these receptors is localized on vascular smooth muscles and the receptors mediate the acetylcholine-induced contractile response of coronary artery.     

Modulation of muscarinic cholinergic receptor affinity for antagonists in rat heart

Modulation of the affinity of agonists and antagonists at muscarinic cholinergic receptors in rat heart membranes was investigated using the radiolabeled antagonist, [3H]quinuclidinyl benzilate ([3H]QNB), and the radiolabeled agonist, [methyl-3H]oxotremorine acetate ([3H]OXO). Receptor affinity for oxotremorine measured in competition binding assays with [3H]QNB or by equilibrium binding of [3H]OXO was increased when the incubation temperature was reduced to 4 degrees C. In contrast, the receptor affinity for [3H]QNB was decreased at lower incubation temperatures and a marked effect of guanine nucleotides on the affinity for [3H]QNB was revealed. Guanine nucleotides increased receptor affinity for [3H]QNB without changing the total number of binding sites. The GTP-induced increase in the affinity for [3H]QNB was reflected by an increase in the rate constant for association of [3H]QNB. At subsaturating ligand concentrations, guanine nucleotides increased [3H]QNB binding and decreased [3H]OXO binding with the same order of potency: GppNHp = GTP gamma S greater than GTP greater than guanosine 5'-diphosphate greater than GMP. Free Mg++ ion was required to observe guanine nucleotide effects on antagonist binding. Pretreatment of heart membranes with N-ethyl-maleimide increased [3H]QNB affinity and blocked the effects of guanine nucleotides. N-Ethylmaleimide also decreased [3H]OXO binding and increased [3H]QNB binding with a similar concentration-effect relationship. Thus, antagonist and agonist binding to muscarinic cholinergic receptors is modulated in a reciprocal manner by a number of factors; this modulation appears to reflect interaction of agonist and antagonist-occupied receptors with a guanine nucleotide regulatory protein, Ni.     

Multiple in vitro interactions with and differential in vivo regulation of muscarinic receptor subtypes by tetrahydroaminoacridine

Tetrahydroaminoacridine (THA) is known to be a potent centrally acting cholinesterase inhibitor. In this report, the effects of THA in vivo and in vitro on the binding of muscarinic agonists and antagonists to putative M1 and M2 receptor subtypes were assessed in rat brain membranes. THA competitively inhibited labeled agonist and antagonist binding to membranes prepared from M1 and M2 enriched brain regions. The dissociation of radiolabeled antagonists from muscarinic receptors was decelerated markedly by THA. The half-time for dissociation of [3H]oxotremorine-M from the high affinity state of M1 and M2 receptors was unaffected by THA. Chronic THA administration resulted in a selective down regulation in the number of M1 receptors assayed directly with the M1-selective antagonist, [3H]pirenzepine. The decrease in the binding capacity of [3H]pirenzepine was correlated positively with the duration of drug treatment. Saturation analysis of [3H]pirenzepine binding confirmed that this loss in binding capacity was due to a reduction in the number of binding sites and not an altered affinity of the receptor for [3H]pirenzepine. Carbachol-[3H]pirenzepine competition revealed no change in the ratio of high and low affinity agonist states of the M1 receptor with chronic THA administration. In vivo studies demonstrate further that the total number of muscarinic receptors was decreased significantly, whereas putative M2 receptors, measured directly with the agonist [3H]oxotremorine-M or estimated by pirenzepine-[3H]quinuclidinyl benzilate competition, were unchanged. Thus, THA exhibits multiple actions at primary and secondary recognition sites on putative M1 and M2 subclasses of muscarinic receptors. The results suggest further that the clinical pharmacology of THA may represent a composite efficacy of THA at multiple sites on cholinergic synapses.     

Comparison of muscarinic receptor properties in hatched chick heart atrium and ventricle

We have reported previously that chick myocardium responds to muscarinic agonists with a decrease in slow inward current in both atrial and ventricular muscle. A second ionic current, the background potassium current, is increased in the atrium but not in the ventricle. A possible explanation for the modulation of potassium current in atrium only is the existence of a unique muscarinic receptor population in atrium responsible for potassium conductance changes. We looked for differences in atrial and ventricular muscarinic receptors by pharmacological and biochemical techniques. The dissociation constants for binding of l-[3H]quinuclidinyl benzilate were 46 pM in both tissues. Estimates for binding of atropine in competitive binding experiments gave dissociation constants of 1.8 nM in atrium and 2.0 nM in ventricle. Pharmacologic evaluation of atropine occupancy of muscarinic receptor by Schild analysis showed no difference in the dissociation constants in atrium (1.7 nM) and ventricle (1.1 nM). Displacement of 0.1 nM [3H]quinuclidinyl benzilate with carbachol showed the atrium to have a higher apparent affinity for agonist than the ventricle (atrium IC50 = 8.2 X 10(-6) M, ventricle IC50 = 2.1 X 10(-5) M). Computerized curve fitting analysis detected three binding states (super high, high and low affinity) for carbachol in the atrium and ventricle in the absence and presence of 5'-guanylylimidodiphosphate (10(-4) M). We did not detect a qualitative difference between atrial and ventricular muscarinic receptors. Muscarinic-induced potassium conductance changes which occur in the atria do not appear to be due to a unique muscarinic receptor in atria.     

Interaction of a radiolabeled agonist with cardiac muscarinic cholinergic receptors

The interaction of a radiolabeled muscarinic cholinergic receptor agonist, [methyl-3H]oxotremorine acetate [( 3H]OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of [3H]OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of [3H]OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist [3H]quinuclidinyl benzilate. Both muscarinic receptor agonists and antagonists inhibited the binding of [3H]OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of [3H]OXO. This effect was on the maximum binding (Bmax) of [3H]OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The [3H]OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with [3H]OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, [3H]OXO-labeled complex resulted in dissociation of [3H]OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited [3H]OXO binding by 85% with no change in the Kd of [3H]OXO, and with no effect on [3H]quinuclidinyl benzilate binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible and quaternary muscarinic antagonists discriminate multiple muscarinic receptor binding sites in rat brain

The maximal number of binding sites (Bmax) of [3H]quinuclidinyl benzilate (QNB) binding was greater than the Bmax of N-[3H]methylscopolamine (NMS) binding to homogenates of rat brain. The competition of NMS for [3H]QNB demonstrated that NMS discriminates multiple muscarinic binding sites. Similarly, pirenzepine competition of [3H]QNB binding also revealed multiple muscarinic binding sites. Pirenzepine competition for [3H]NMS also was shallow and demonstrated the presence of binding sites with similar affinities to those labeled by [3H]QNB. These data were consistent with the presence of at least three populations of muscarinic binding sites with similar affinities for [3H]QNB: the M1 and M2 binding sites having high and low affinity for pirenzepine, respectively, but which cannot be discriminated by [3H]NMS, and a third site with high affinity for [3H]QNB which has low affinity for NMS. The classical muscarinic antagonists, atropine and scopolamine, also appear to have slightly different affinities for the putative M1 and M2 binding sites. The use of the irreversible antagonists, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and propylbenzilylcholine mustard (PBCM), were used to elucidate the distinct properties of these multiple muscarinic binding sites. Both PBCM and EEDQ irreversibly decreased the Bmax of [3H]QNB and [3H]NMS binding in cortex. PBCM did not appear to discriminate putative M1 and M2 binding sites but selectively alkylated the high affinity NMS and QNB binding sites. In contrast, EEDQ modified the low affinity NMS binding sites such that they still bound [3H]QNB but their affinity for other muscarinic antagonists was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic cholinergic receptor subtype on frog esophageal peptic cells: binding and secretion studies

The muscarinic receptors coupled to pepsinogen secretion on isolated frog esophageal peptic cells have been characterized using functional and radioligand binding techniques. N-[3H]methylscopolamine [( 3H]NMS) binding to intact cells was complex and indicative of a high affinity, low capacity site and a high capacity uptake site. Binding to the high capacity site was inhibited by atropine with high affinity (IC50, 3 nM) and by imipramine and propranolol with IC50 values of 70 and 270 nM, respectively. After inhibition of uptake by 30 microM propranolol, [3H]NMS bound to a single population of high affinity sites (KD, 125 +/- 16 pM), which exhibited binding site maximum of 2.1 fmol/10(6) cells, equivalent to 1260 sites/cell. Binding to these sites was reversible, stereoselective and inhibited by muscarinic receptor agonists with an order of potency: oxotremorine greater than acetylcholine greater than carbachol greater than bethanechol and by antagonists with an order of potency:atropine greater than 4-diphenylacetoxy-N-methylpiperidine methobromide greater than pirenzepine greater than AF-DX 116 (11-2[2-[[diethylamino) methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one). Pepsinogen secretion was stimulated by the agonists with an order of potency: acetylcholine greater than or equal to carbachol greater than oxotremorine greater than bethanechol. Atropine, pirenzepine and AF-DX 116 competitively inhibited carbachol-stimulated pepsinogen secretion with pA2 values of 9.58, 7.37 and 6.68, respectively, which correlated with their log (inhibition constants) for receptor binding. By contrast, agonists with significant efficacy exhibited EC50 values which were 20 to 90 times lower than their inhibition constants for binding which suggests the possibility of "spare" muscarinic receptors. Our findings indicate that functional muscarinic receptors on peptic cells exhibit similar characteristics to the high affinity sites labeled by [3H]NMS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the striatal M2 muscarinic receptor mediating inhibition of cyclic AMP using selective antagonists: a comparison with the brainstem M2 receptor

Pharmacological profiles of the striatal and brainstem M2 receptors were developed with a group of selective muscarinic antagonists. The striatal M2 muscarinic receptor was identified by its inhibition of [3H]cyclic AMP levels, whereas the brainstem M2 receptor was characterized using competition with [3H]quinuclidinyl benzilate binding. The potency of pirenzepine does not differentiate clearly between the striatal M2 receptor (Ki approximately 300 nM) and the brainstem M2 receptor (Ki = 219 nM) or peripheral M2 receptors. In the present study, we used 4-diphenylacetoxy-N-methylpiperidine methbromide, hexahydrosiladifenidol, AF-DX 116 and methoctramine to characterize the striatal and brainstem M2 receptors in more pharmacological detail. For comparison, the potencies of these antagonists were also measured at cortical M2 receptors (using competition with [3H]pirenzepine binding). The potencies of 4-diphenylacetoxy-N-methylpiperidine methbromide (KB = 0.19 nM) and hexahydrosiladifenidol (KB = 14 nM) in blocking the striatal M2 receptor suggested similarity to those M2 receptors localized in certain smooth muscles or in glands. However, AF-DX 116 (KB = 155 nM) and methoctramine (KB = 47 nM) were considerably more potent in blocking the striatal M2 receptor than as reported in functional studies in smooth muscle or glands. Thus, the profile of the striatal M2 receptor obtained with these antagonists did not match in all respects with either glandular (probable M4 gene product) or cardiac (probable M2 gene product) muscarinic receptors. In contrast, our data with the brainstem M2 receptor was highly correlated (r = 0.93) with literature data regarding the cardiac muscarinic system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the properties of muscarinic cholinergic receptors in the developing chick myocardium

The number of muscarinic cholinergic receptors detected with [3H]quinuclidinyl benzilate (QNB) was constant in embryonic chick heart membranes but increased 2.5-fold by 3 days posthatching; the KD for [3H]QNB did not change. The affinities of the muscarinic receptors for agonists, as determined in in vitro [3H]QNB competition experiments, differed during development. The IC50 values were lowest for 10- to 14-day embryonic heart receptors, intermediate for 18- to 20-day embryonic receptors and highest for 3- to 7-day newborn heart receptors. These apparent differences in agonist affinity were not overcome by guanine nucleotides or monovalent cations alone, but were greatly diminished in the presence of a combination of guanylylimidodiphosphate plus NH4+. Modeling of [3H]QNB/oxotremorine competition curves indicated the presence of three agonist affinity states in membranes from embryonic heart and two in newborn hearts. The KD values for oxotremorine increased during development. The proportion of receptors displaying the highest affinity for oxotremorine was constant at all ages tested whereas the proportion displaying the lowest affinity decreased from 16% in membranes from 12-day embryonic hearts to zero in newborn hearts. The physiological significance of the differences in the properties of the receptors was investigated by assessing their ability to attenuate adenylate cyclase. The efficacy of agonists to attenuate basal adenylate cyclase increased 1.5-fold after birth, whereas the potency of agonists to produce this effect was similar at all ages tested.     

Analysis of cardiac muscarinic receptors recognized selectively by nonquaternary but not by quaternary ligands

Three muscarinic receptor antagonists, [3H]quinuclidinyl benzilate ([3H]QNB), N-[3H]methylscopolamine ([3H]NMS and N-[methyl-3H]QNB ([3H]NMeQNB), each bind to an apparently homogeneous population of receptors on intact chick heart cells. [3H]QNB binds to approximately 9500 sites/cells, whereas [3H]NMS and [3H]NMeQNB bind to approximately 5000 sites/cell. Atropine and scopolamine compete with all three radioligands with a single, high affinity. Their quaternary analogs N-methylatropine and NMS and the quaternary agonist carbachol also show a single affinity for [3H]NMS and [3H]NMeQNB binding sites, but have biphasic competition curves for [3H]QNB sites with low "apparent" affinity for a subpopulation of sites. When 10 nM or greater propylbenzilylcholine mustard is used to alkylate receptors virtually all [3H]NMS binding is abolished, whereas [3H]QNB still labels a significant fraction of the binding sites seen in control cells. The sites with low apparent affinity for quaternary ligands are shown to have characteristics of muscarinic receptors, but do not appear necessary for muscarinic receptor-mediated phosphoinositide hydrolysis. We suggest that a subpopulation of nonfunctional muscarinic receptors are sequestered within the membrane or otherwise inaccessible to hydrophilic or charged ligands.     

Distinction between high-affinity [3H]phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle

[3H]Phencyclidine ([3H]PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of [3H]PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the unionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific [3H]PCP binding was displaced effectively by several PCP analogs and Ca++ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of [3H]quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities .