Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

The properties of [3H]quinuclidinylbenzilate ( [3H]QNB) binding and [3H]pirenzepine ( [3H]PZ) binding to various regions of rat brain were compared. [3H]PZ appeared to bind with high affinity to a single site, with a Kd value of approximately 15 nM in the cerebral cortex. The rank order of potencies of muscarinic drugs to inhibit binding of either [3H]QNB or [3H]PZ was QNB greater than atropine = scopolamine greater than pirenzepine greater than oxotremorine greater than bethanechol. Muscarinic antagonists (except PZ) inhibited both [3H]PZ and [3H]QNB binding with Hill coefficients of approximately 1. PZ inhibited [3H]QNB binding in cortex with a Hill coefficient of 0.7, but inhibited [3H]PZ binding with a Hill coefficient of 1.0. Hill coefficients for agonists were less than 1. The density of [3H]PZ binding sites was approximately half the density of [3H]QNB binding sites in cortex, striatum and hippocampus. In pons-medulla and cerebellum, the densities of [3H]PZ binding sites were 20 and 0%, respectively, relative to the densities of [3H]QNB binding sites. When unlabeled PZ was used to compete for [3H]QNB binding, the relative number of high-affinity PZ binding sites in cortex, pons and cerebellum agreed with the relative number of [3H]PZ binding sites in those regions. The binding of [3H]PZ and [3H]QNB was nonadditive in cortex. GTP inhibited high-affinity oxotremorine binding, but not PZ binding. Together, these data suggest that [3H]PZ binds to a subset of [3H]QNB binding sites. Whether this subset reflects the existence of subtypes of muscarinic receptors or is a consequence of coupling to another membrane protein remains to be seen.     

Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells

The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 microM atropine sulfate. The Kd and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/10(6) cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 [11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one]. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine greater than pirenzepine greater than AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4 beta-phorbol, 12-beta-myristate, 13-alpha-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.     

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.     

Heterogeneity of high-affinity nicotinic [3H]acetylcholine binding sites

Studies were conducted on high-affinity, nicotinic binding of 3H-labeled acetylcholine ([3H]ACh) to membrane preparations derived from the TE671 human clonal line and the PC12 rat pheochromocytoma. For comparative purposes and to extend results obtained by others in previous studies, [3H]ACh binding to membrane preparations derived from rat brain and from the electric tissue of Torpedo californica also was characterized. In each case, specific [3H]ACh binding (KD values of about 10 nM) could be fit by linear Scatchard and logit-log curves (slope of the latter of about 1.0) indicating that binding occurred to a single class of noninteracting sites, except that a better fit to PC12 cell membrane binding data was obtained using a two-site model. Quantitation of high-affinity binding sites for [3H]ACh and radiolabeled alpha-bungarotoxin and studies of unlabeled toxin competition for [3H]ACh binding indicated that toxin and agonist sites on TE671 cell or Torpedo membranes were closely related, but that toxin and agonist sites had limited or no physical overlap on PC12 cell or rat brain membranes. Generally, drugs of the bisonium and bis-choline series were more effective inhibitors of [3H]ACh binding to TE671 cell or Torpedo membranes, but nicotine and cytisine interacted with PC12 cell or rat brain sites with highest affinity. These results suggest that nicotinic agonists bind with high affinity to specific, membrane-bound sites on presumptive nicotinic ACh receptors expressed on each of these preparations. Moreover, the data are consistent with structural heterogeneity of these nicotinic receptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Napamezole, an alpha-2 adrenergic receptor antagonist and monoamine uptake inhibitor in vitro

Napamezole (2-[3,4-dihydro-2-naphthalenyl)methyl]-4,5-dihydro-1H- imidazole-monohydrochloride) is a selective alpha-2 adrenergic receptor antagonist and a monoamine re-uptake inhibitor in vitro. The alpha adrenergic antagonist activity of napamezole was determined in vitro in rat brain receptor binding assay using [3H]clonidine and [3H]prazosin for alpha-2 and alpha-1 receptors, respectively. The Ki values for napamezole were 28 nM (alpha-2) and 93 nM (alpha-1). The relative potencies for inhibiting [3H]clonidine binding were: phentolamine greater than idazoxan greater than napamezole greater than mianserin greater than yohimbine greater than piperoxan greater than rauwolscine greater than tolazoline much greater than prazosin; and for inhibition [3H]prazosin binding they were: prazosin greater than phentolamine greater than mianserin greater than napamezole greater than yohimbine greater than idazoxan greater than tolazoline. Alpha adrenoceptor antagonism was also assessed in the isolated rat vas deferens. Napamezole reversed clonidine-induced decreased in twitch height in the electrically stimulated rat vas deferens (alpha-2 antagonism with a Kb of 17 nM). The rank order of potency as an alpha-2 antagonist relative to other compounds was phentolamine greater than idazoxan greater than yohimbine greater than piperoxan = napamezole greater than mianserin much greater than prazosin. Napamezole also antagonized methoxamine-induced contractions (alpha-1) of the rat vas deferens with a Kb of 135 nM. The rank order of potency of these compounds as alpha-1 antagonists was prazosin greater than phentolamine greater than mianserin greater than yohimbine greater than napamezole greater than idazoxan.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Stabilization of antagonist binding to cardiac muscarinic acetylcholine receptors by gallamine and other neuromuscular blocking drugs

The effects of neuromuscular blocking drugs and muscarinic agonists and antagonists on the dissociation of [3H]quinuclidinylbenzilate ([3H]QNB) from muscarinic receptors was studied on rat atrial homogenates. In typical experiments the investigated drug was added to the homogenate equilibrated with [3H]QNB and the amount of undissociated [3H]QNB receptor complexes was measured 40 min later. The antagonists atropine and pirenzepine, agonists carbamoylcholine and methylfurmethide and neuromuscular blockers pancuronium, d-tubocurarine and decamethonium caused a concentration-dependent dissociation of [3H]QNB from the receptors, which may be explained by their competition with [3H]QNB for the same (primary) binding sites. In accordance with this, these drugs did not affect the dissociation of [3H]QNB elicited by an excess of atropine, which indicates that the kinetics of dissociation of the [3H]QNB receptor complex remained unchanged in their presence. Neuromuscular blockers alcuronium, gallamine and to a lesser degree tercuronium differed from the other drugs in that 1) their effect on [3H]QNB dissociation was biphasic, being higher at their low (10(-6) to 10(-5) M) than at their high concentrations (10(-4) to 10(-3) and that 2) at high concentrations they strongly inhibited the dissociation of [3H]QNB receptor complexes elicited by the excess of atropine. Their behavior may be rationalized by assuming that at low concentrations they bind to the primary binding site making rebinding of once dissociated [3H]QNB molecules improbable (competitive mechanism), whereas at high concentrations they also act on a secondary (allosteric) binding site stabilizing the [3H]QNB receptor complexes by slowing their off-kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Muscarinic cholinergic regulation of electrogenic chloride secretion in porcine proximal jejunum

Acetylcholine is present in a majority of submucosal neurons which project to the intestinal epithelium. In this study, we examined the role of acetylcholine and the actions of cholinomimetic drugs, such as carbachol (CCH), on ion transport across muscle-stripped sheets of mucosa-submucosa from the proximal jejunum of weaned piglets. Serosal administration of CCH (10 nM-100 microM) produced rapid increases in short-circuit current (Isc) which were attributed to net Cl secretion. Acetylcholine, bethanechol and (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride were partially effective in increasing Isc. Atropine and selective muscarinic cholinergic antagonists produced dextral shifts in the CCH concentration-effect relationship with an order of relative potency of 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) greater than atropine much greater than pirenzepine greater than 11-[[[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one(AF-DX116). The muscarinic receptor blocker [3H]quinuclidinyl benzilate (QNB) bound specifically and saturably to two sites in the mucosa-submucosa having equilibrium dissociation constants of approximately 10 +/- 3 and 890 +/- 120 pM and Bmax = 7 +/- 3 and 47 +/- 9 fmol/mg protein, respectively. Selective cholinergic antagonists competed for [3H]QNB binding with a rank order of affinity of 4-DAMP greater than hexahydrosiladifenidol much greater than AF-DX 116 greater than or equal to pirenzepine. Specific [3H]QNB binding sites were autoradiographically localized in the jejunal wall to the epithelium, submucosa, and muscularis propria. Electrical transmural stimulation (10-300 pulses/10 sec, 0.5 msec duration, 60 V stimulus strength) delivered to mucosal sheets produced tetrodotoxin-sensitive Isc elevations which were proportional to the number of impulses delivered. Mucosal Isc responses to electrical stimulation were attenuated by 10 microM hexamethonium, 1 microM atropine or autotachyphylaxis to CCH. Tetrodotoxin, at 0.1 microM, produced a 20-fold increase in the secretory potency of CCH. These results suggest that acetylcholine released from intramural neurons in porcine proximal jejunum produces transepithelial Cl secretion. Its effects may be mediated through interactions with two populations of muscarinic cholinergic receptors, located on neuronal and non-neuronal cells within the intestinal mucosa and submucosa, which serve to inhibit and promote Cl secretion respectively.     

Inhibition of 3H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

The specific binding of [3H]5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ([3H]LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. [3H]LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound [3H]LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2 less than 5 min) and full dissociation. Equilibrium saturation analysis of [3H]LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of [3H]LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.     

Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover

(-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems.     

Differential ontogeny of putative M1 and M2 muscarinic receptor binding sites in the murine cerebral cortex and heart

Studies with [3H]pirenzepine [( 3H]PZ) suggest that this nonclassical muscarinic antagonist selectively identifies putative M1 muscarinic receptors. We now compare the ontogeny of these putative M1 sites, identified by high-affinity [3H]PZ binding, with sites identified by the classical antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) in murine cerebral cortical and cardiac homogenates. Dissociation constants (Kd) for [3H]PZ (2.1-6 nM in the cortex and 2.0-21 nM in the heart) and for (-)-[3H]QNB (10-28 pM in the cortex and 10-39 pM in the heart) are similar in adult and neonatal tissues, whereas receptor density (maximum binding, femtomoles per milligram of protein) varies significantly. Cerebral cortical [3H]PZ binding rises from 14% at birth, to 88% of adult levels by day 14, peaks at 128% at day 28 and falls to the mean adult level of 606 fmol/mg of protein. Cerebral cortical (-)-[3H]QNB binding parallels [3H]PZ binding. Conversely, parallel studies show cardiac (-)-[3H]QNB density is 3- to 17-fold greater than the comparable density of high-affinity [3H]PZ binding sites throughout ontogeny. We conclude that: 1) the high ratio of [3H]PZ binding to (-)-[3H]QNB binding identifies the murine cerebral cortex as a tissue which contains predominantly putative M1 muscarinic binding sites; 2) the relatively low ratio of [3H]PZ binding to (-)-[3H]QNB binding throughout ontogeny identifies the murine heart as a tissue which contains primarily the putative M2 muscarinic binding site; and 3) M1 and M2 receptor binding sites show distinct developmental curves in the cerebral cortex and heart.(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)     

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)     

Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes

The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.     

Antagonists and agonists interactions with the muscarinic receptor of the rat large airways

The muscarinic receptor in the rat large airway was characterized by radioligand binding experiments. Using I-quinuclidinyl (phenyl-4-[3H])benzilate ([3H]QNB) as the radioligand, the receptor appears to be homogenous. The receptor density was 23 fmol/mg of protein and the Kd value for [3H]QNB binding was 16 pM. Competition of the [3H]QNB binding for the receptor with selective antagonists and agonists was used to characterize the muscarinic receptor. The K0.5 values for the (M1)-selective antagonists pirenzepine and telenzepine were 210 and 20 nM, respectively. The M2a-selective antagonist AF-DX 116 and the M2b-selective antagonist hexahydrosila-difenidol had K0.5 values of 130 and 120 nM, respectively. By comparing the apparent affinities of these antagonists in the large airways to their affinities in rat heart, the large airway muscarinic receptor appears to be of the M2a type. Agonists competition curves of [3H]QNB binding to the receptor were shallow. The agonist curves were modeled to one- and two-site binding models. All agonists, including M1-selective agonists, gave preferred fits to two-site models. Guanine nucleotide in the assay caused right shifts of the competition curves and decreased the apparent proportion of the receptor population that was in the higher affinity state for the agonists. Thus, it is concluded that: 1) the rat large airway muscarinic receptor interacts with antagonists in a manner which support the hypothesis that the receptors are of the M2a subtype and 2) both the high and low agonist affinity states of the M2a receptor of the rat large airways are capable of interacting with M1 agonists.     

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.     

Effects of cocaine and related drugs in nonhuman primates. I. [3H]cocaine binding sites in caudate-putamen

Specific binding sites for [3H]cocaine were identified in caudate-putamen membranes prepared from nonhuman primate brains (Macaca fascicularis and Saimiri sciureus). Saturation of the sites was determined in competition studies using a fixed concentration of [3H]cocaine (2.7 nM) and increasing concentrations of unlabeled cocaine (1 pM-100 microM). Computer resolution of the shallow displacement curve (nH, 0.58) revealed that a two-component binding model [Kd1, 19.2 nM, maximum binding1 (Bmax1), 28.3 pmol/g of tissue; Kd2, 1120 nM, Bmax2, 431 pmol/g of tissue] was statistically preferred over a one-component model (K.50, 283 nM, Bmax, 471 pmol/g of tissue). Binding of [3H]cocaine was NaCl-dependent, with specific binding reduced by 72% when NaCl (100 mM) was omitted from the incubation medium. [3H]Cocaine was displaced stereoselectively by the enantiomers of cocaine and by the diastereoisomers of cocaine and its phenyltropane analog. Cocaine congeners displaced specifically bound [3H]cocaine with IC50 values ranging from 17 nM to over 100 microM in the following rank order of potency: WIN 35,428 greater than WIN 35,065-2 greater than (-)-cocaine greater than WIN 35,981 greater than (-)-norcocaine greater than WIN 35,140 greater than (+)-cocaine, (+)-pseudococaine greater than 3 alpha-tropanyl-1H-indole-carboxylic acid ester greater than 1 alpha H-3 alpha-5 alpha H-tropan-3-yl-3,5-dichlorobenzoate greater than benzoylecgonine, benzoylnorecgonine and (-)-pseudococaine. Several monoamine uptake inhibitors structurally unrelated to cocaine also displaced [3H]cocaine with IC50 values ranging from 1.6 nM to 50 microM. The rank order of potency was: ( +/- )-trans-3-(3',4'-dichlorophenyl)-N-methyl-1-indanamine greater than mazindol greater than nomifensine greater than methylphenidate 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]- 4-(3-phenylpropyl)piperazine, N-[1-(2- benzo(b)thiophenyl)cyclohexyl]piperidine greater than (-)-cocaine greater than 1-amino-4-phenylbicyclo-[2,2,2]-octane greater than bupropion, nisoxetine greater than desipramine, talsupram greater than citalopram. Other drugs, including the dopamine releasing agent (+)-amphetamine and the dopamine receptor agonists (-)-apomorphine, (+)-4-propyl-9-hydroxy-naphthoxazine, quinpirole and SKF 38393 were weak displacers of [3H]cocaine. Monoamine neurotransmitters also were relatively weak, but dopamine was considerably more potent than either norepinephrine or serotonin.(ABSTRACT TRUNCATED AT 400 WORDS)     

3H]pirenzepine and (-)-[3H]quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes

Studies show [3H]PZ identified selectively a subpopulation of muscarinic binding sites compared to classical antagonists like (-)-[3H]QNB in many central and peripheral tissues. We characterized the binding and regulation of selected antagonists to high-affinity [3H]PZ (putative M1) and low-affinity PZ (putative M2) sites in rat cerebral cortex (predominantly M1) and heart (predominantly M2). Saturation isotherms of [3H]PZ and (-)-[3H]QNB were performed under various conditions. Guanyl-5'-yl-imidodiphosphate (30 microM) showed little effect on Kd (dissociation constant) or total binding capacity (total receptor density) values. Higher ionic strength buffers yielded lower affinity values for [3H]PZ and (-)-[3H]QNB. Kinetic studies confirmed high affinity Kd values seen in steady-state assays. We conducted inhibition studies of selected muscarinic antagonists including the reportedly cardioselective (putative M2) drug, AF-DX 116 (11-[(2-(diethylamino)methyl-1-piperidinyl)-acetyl]-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the reportedly M1 selective compound, PZ, and the classical antagonist (-)QNB, using [3H]PZ and (-)-[3H]QNB-labeled cerebral cortical and cardiac homogenates. Assays were done with and without guanyl-5'-yl-imidophosphate at 25 degrees C in 10 mM Na-K-phosphate, 50 mM Na-K-phosphate and modified Krebs-phosphate buffer. Studies showed antagonists generally had higher affinity in 10 mM Na-K-phosphate buffer, were insensitive to guanyl-5'-yl imidodiphosphate and had Hill values (nH) nearly equal to one. Cardiac PZ/[3H]QNB curves were steep.(ABSTRACT TRUNCATED AT 250 WORDS)