Muscarinic receptors of rat lymphocytes--differences in young and old animals

Muscarinic receptors were studied on lymphocytes from young and old Wistar rats. Binding studies were performed by the use of [3H]-QNB, a specific muscarinic antagonist. Some differences between these two groups were observed. Maximal binding of [3H]-QNB and half time of the maximal binding is lower for lymphocytes of old rats [3H]-QNB receptor complexes could not be found in the supernatants derived from lymphocytes of old animals. Higher ability to loose or hide the muscarinic receptors was also observed in this group of rats. All these observations could reflect a more effective degradation, as well as a lower level of muscarinic receptors exposed on lymphocytes from old animals.     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions

Summary Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2–3 fold) the specific binding of [³H]-QNB or [³H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30°C. Magnesium, on the other hand, increased (2–3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased K_d (WP) while magnesium increased K_d (MIC) for [³H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on onist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4°C (24 hr), the stimulatory effect of Gpp(NH)p on [³H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [³H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like G₁,G_o) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.     

Huntington's disease: regional alteration in muscarinic cholinergic receptor binding in human brain.

Huntington's Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntington's Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [³H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [³H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntington's Disease.     

An alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of the guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacity for the muscarinic antagonist, [3H]N-methylscopolamine ( [3H]NMS), was noted without a change in affinity. Similar observations were made in [3H]1-3-quinuclidinyl benzilate ( [3H]1-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both [3H]NMS and [3H]1-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of [3H]1-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 microM. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity.     

Functional muscarinic M2 and M3 receptors and beta-adrenoceptor in cultured rat bladder smooth muscle

A highly purified rat urinary bladder smooth muscle cell culture was obtained by a modified enzymic isolation method, and the presence of functional muscarinic as well as beta-adrenergic receptors were subsequently determined. At 7-10 days of culture, cells became elongated and spindle-shaped showing a typical "hills and valleys" form. They were stained with anti-alpha-actin and anti-myosin antibodies. Radiolabeled ligand binding using [3H]N-methylscopolamine and [3H]CGP12177 showed that these cells expressed muscarinic and beta-adrenergic receptors. Stimulation of cultured cells with carbachol inhibited the forskolin-stimulated cyclic AMP formation, caused an elevation of intracellular Ca2+ concentration measured by fura-2 fluorometry. The latter response was almost completely blocked by 4-DAMP, a selective muscarinic M3 antagonist. On the other hand, stimulation of cultured cells with isoproterenol enhanced the basal cyclic AMP formation, which was reversed by carbachol. Therefore, the presence of functional muscarinic (both M2 and M3) as well as beta-adrenergic receptors was confirmed in pure culture of the rat bladder smooth muscle cells obtained by using an enzymic isolation method.     

Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

Regulation of Muscarinic Receptor-Mediated Cyclic GMP Synthesis by Cultured Mouse Neuroblastoma Cells

Mouse neuroblastoma clone N1E-115 has muscarinic acetylcholine receptors that mediate cyclic GMP synthesis. This receptor-mediated response is not significantly higher than background until the cells have been maintained in the stationary phase for at least 1 week. The basis of the influence of time in culture on the cyclic GMP response was investigated. The relative amount of cyclic GMP synthesized by intact cells was measured by radioactively labeling the GTP pool with [³H]guanine, incubating cells with agonists, and then chromatographically isolating [³H]cyclic GMP. Carbamylcholine-, ionophore X-537A-, and sodium azide-induced cyclic GMP formation increased with time in culture to a maximum of 13-, 9-, and 2.5-fold above basal, respectively. There was no change in the number or the apparent affinity of the muscarinic receptors as measured by [³H]quinuclidinyl benzylate ([³H]QNB) binding. In addition, there was no change in the apparent affinity of the receptors for agonist as measured by the ability of carbamylcholine to displace the specific binding of [³H]QNB. Guanylate cyclase activity per milligram protein and per cell in-creased six- and sevenfold, respectively, from day 0 to day 22. However, this increase in guanylate cyclase appeared to precede the marked increase in sensitivity of the cells to agonists. These data suggest that, in addition to guanylate cyclase and muscarinic receptors, there is another factor which is responsible for the development of this muscarinic receptor-mediated response.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.     

Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Long-term regulation of muscarinic acetylcholine receptors on cultured nerve cells.

Incubation of mouse neuroblastoma cells (clone 1LE-115) with the muscarinic agonist, carbachol, resulted in a time-dependent desensitization to muscarinic receptor-mediated cyclic GMP formation and a decrease in the number and affinity of muscarinic receptors as measured by the binding to intact cells of the muscarinic antagonist, [³H]quinuclidinyl benzilate (QNB). The decrease in responsiveness to cyclic GMP formation reached a maximum after a 15-minute exposure to 1 mM carbachol (short-term desensitization) whereas changes in [³H] QNB binding became apparent only after a one hour exposure and reached a maximum after about 12 hrs (long-term desensitization). Recovery of sensitivity after short-term desensitization was rapid, suggesting that this process may involve a conformational change in the muscarinic receptor. In contrast, recovery after long-term desensitization was prolonged and could be slowed by the inhibition of protein synthesis. These results indicate that different cellular mechanisms are involved in the short-term and long-term desensitization of muscarinic receptors.     

Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine

Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.     

Ketamine prevents seizures and reverses changes in muscarinic receptor induced by bicuculline in rats

The cholinergic system has been implicated in several experimental epilepsy models. In a previous study bicuculline (BIC), known to antagonize GABA-A postsynaptic receptor subtype, was administered to rats at subconvulsant (1 mg/kg) and convulsant (7.5 mg/kg) doses and quinuclidinyl benzilate ([³H]-QNB) binding to CNS membranes was determined. It was observed that ligand binding to cerebellum increases while it decreases in the case of hippocampus. Saturation binding curves showed that changes were due to the modification of receptor affinity for the ligand without alteration of binding site number. The purpose of this study was to assay muscarinic receptors employing other BIC dose (5 mg/kg), which induces seizures and allows the analysis of a postseizure stage as well. To study further muscarinic receptor involvement in BIC induced seizures, KET was also employed since it is a well known anticonvulsant in some experimental models. The administration of BIC at 5 mg/kg to rats produced a similar pattern of changes in [³H]-QNB binding to those recorded with 1.0 and 7.5 mg/kg doses. Here again, changes were observed in receptor binding affinity without alteration in binding site number for cerebellum or hippocampus membranes. Pretreatment with KET (40 mg/kg) prevented BIC seizures and reverted [³H]-QNB binding changes induced by BIC administration. The single administration of KET invariably resulted in [³H]-QNB binding decrease to either cerebellar or hippocampal membranes. KET added in vitro decreased ligand binding likewise. Results of combined treatment with KET plus BIC are hardly attributable to the single reversion of BIC effect since KET alone invariably decreased ligand binding. It is suggested that besides alteration of cholinergic muscarinic receptor other(s) neurotransmitter system(s) may well also be involved.     

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.     

Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)