3H]propylbenzilylcholine mustard-labeling of muscarinic cholinergic receptors that selectively couple to phospholipase C or adenylate cyclase in two cultured cell lines

Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma X glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PBCM), and the mobilities of the [3H]PBCM-labeled species of both cells were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1321N1 and NG108-15 cells each primarily expressed a single [3H]PBCM-labeled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. [3H]PBCM labeling was completely inhibited by 1 microM atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the [3H]PBCM-labeled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Since muscarinic receptors are glycoproteins, the contribution of carbohydrate groups to the difference in apparent size of the [3H]PBCM-labeled proteins was determined by treatment of [3H]PBCM-labeled membranes with endoglycosidase F, an enzyme that removes both complex and high mannose type N-linked carbohydrate chains. Endoglycosidase F treatment reduced the apparent size of the [3H]PBCM-labeled species in 1321N1 cells from 92,000 to approximately 77,000 Da and in NG108-15 cells from 66,000 to 45,000 Da. Neuraminidase produced no further reduction of the apparent size of the [3H]PBCM-labeled species from either cell after endoglycosidase F treatment, suggesting the absence of sialic acid containing O-linked carbohydrate chains on the muscarinic receptors of the two cell lines. The results suggest that different muscarinic receptor proteins may be responsible for the two different biochemical responses to muscarinic receptor activation.     

Agonist binding to M1 muscarinic receptors is sensitive to guanine nucleotides

Putative M1 (high-affinity pirenzepine) muscarinic receptors in rabbit hippocampal membranes, treated with 0.1 mM N-ethylmaleimide (NEM), were selectively labeled with [3H]pirenzepine. A single class of binding sites was labeled with a Kd of 3.4 nM, consistent with the pharmacologically-defined M1 subtype of muscarinic receptors. While full muscarinic agonists bound to high- and low-affinity states of [3H]pirenzepine-labeled M1 sites with a KL/KH ratio of approximately 100, the ratio for partial muscarinic agonists was approximately 10. The high-affinity binding of all agonists tested required divalent cations, and was interconverted to low-affinity binding in the presence of the non-hydrolyzable GTP analogue, guanylyl imidodiphosphate (GppNHp). Direct labeling of the high-affinity agonist state of M1 receptors was achieved with 5 nM [3H]oxotremorine-M by selectively uncoupling the high-affinity agonist state of M2 (low-affinity pirenzepine) receptors with NEM. The rate of dissociation of [3H]Pxotremorine-M from M1 receptors was accelerated 6-fold by GppNHp. These results provide further evidence which suggests that putative M1 muscarinic receptors activate second messenger systems by coupling to NEM-insensitive guanine nucleotide-binding proteins.     

Ketamine inhibition of ligand binding to cholinergic receptors and ion channels

Ketamine inhibited the binding of [3H]perhydrohistrionicotoxin and [3H]phencyclidine to the ion channel associated with Torpedo acetylcholine receptors with ID50 values between 11 and 84 micro M, but had not effect on [3H]acetylcholine or [3H]d-tubocurarine binding to the receptor itself. Ketamine's affinity for the ion channel was increased 3-5 fold by receptor agonists. Ketamine also inhibited muscarinic cholinergic receptors with ID50 between 28 and 38 micro M. These results are consistent with biophysical evidence that ketamine interferes with neuromuscular transmission through blockade of the ion channel.     

Dopamine D2 receptor binding subunits of Mr congruent to 140,000 and 94,000 in brain: deglycosylation yields a common unit of Mr congruent to 44,000

The ligand-binding subunit of the porcine striatal dopamine D2 receptor was identified by photoaffinity labeling with [125I]N-azidophenethylspiperone ([125I]NAPS). Upon photolysis, [125I]NAPS covalently incorporated into a broad band of apparent Mr congruent 140,000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Smaller subunits of apparent Mr congruent 94,000 and 34,000 were specifically labeled by [125I]NAPS with an appropriate D2 receptor profile and were similar to the major ligand-binding subunits of photoaffinity-labeled canine striatal D2 receptors. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of the Mr congruent to 140,000/94,000 subunits upon denaturing electrophoresis in either the absence or presence of thiol-reducing/alkylating reagents. In order to investigate the possible basis for the existence of these high molecular weight forms of the D2 receptor, we assessed the carbohydrate nature of photolabeled D2 ligand-binding subunits by the use of lectin affinity chromatography and specific exo- and endoglycosidase treatments. Both photoaffinity-labeled D2 receptor proteins from porcine striatum (Mr congruent to 140,000 and 94,000) were glycoproteins as indexed by their absorption and specific elution from wheat germ agglutinin lectin resins. The exoglycosidase neuraminidase altered the electrophoretic mobility of both the Mr congruent to 140,000 and 94,000 labeled subunits to a single band of apparent Mr congruent to 51,000. Prior removal of sialic acid residues did not alter the reversible binding characteristics of [3H]spiperone to D2 receptors. Complete removal of receptor-associated N-linked carbohydrate by the endoglycosidase glycopeptidase F (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) produced a further increase in the mobility of the Mr congruent to 51,000 subunit to apparent Mr congruent to 44,000. The porcine Mr congruent to 34,000 photolabeled peptide is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase F to a peptide of apparent Mr congruent to 23,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Mediation of separate responses by high affinity and low affinity agonist-receptor conformations

Murine neuroblastoma cells (clone N1E-115) possess muscarinic receptors that mediate multiple responses, including the elevation of cyclic GMP levels and the inhibition of receptor-mediated increases in cyclic AMP. Evidence is presented showing that two muscarinic agonist-receptor conformations in N1E-115 cells each separately mediate a cyclic nucleotide response. Pirenzepine inhibited the [3H]cyclic GMP response to carbachol with a KD value of approximately 6 nM, whereas it inhibited the ability of carbachol to reduce prostaglandin E1-mediated elevations in [3H]cyclic AMP levels with a KD value of 93 nM, thus differentiating between two classes of receptors involved in these responses. Ten muscarinic agonists were studied for their ability to mediate the two cyclic nucleotide responses. Six were as effective as acetylcholine in the reduction of [3H]cyclic AMP levels, but only two were as effective as acetylcholine in elevating [3H]cyclic GMP levels. Four agonists (arecoline, pilocarpine, oxotremorine, and McN-A343) were ineffective in increasing [3H]cyclic GMP levels. These four agonists and bethanecol, which could increase [3H]cyclic GMP levels only 18% as well as acetylcholine, behaved as competitive antagonists in this response to carbachol. These partial agonists, in contrast to carbachol, bound to only one class of muscarinic sites in N1E-115 cells with equilibrium dissociation constants determined by competition binding assays which agreed well with their respective EC50 values for their effect on [3H]cyclic AMP levels. The equilibrium dissociation constants for the partial agonists determined by their inhibition of carbachol in the [3H] cyclic GMP response also agreed well with their respective EC50 values for mediating the [3H]cyclic AMP response. Thus, the partial agonists bound to the same receptors at which carbachol mediated [3H]cyclic GMP formation, but with KD values about the same as their respective EC50 values for inhibition of prostaglandin E1-mediated [3H]cyclic AMP increases. The full agonists acetylcholine and methacholine, like carbachol, bound to two sites in N1E-115 cells. For the six agonists able to stimulate both responses at least to some degree, the ratio of their potencies at each response correlated with their respective efficacies at each response but with much more dependence in the [3H]cyclic GMP response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of phospholipase Cbeta activity by muscarinic acetylcholine and 5-HT(2) receptors in crude and synaptosomal membranes from human cerebral cortex

Stimulation of phospholipase Cbeta by receptor agonists and G proteins has been characterized in crude cerebral membrane preparations, but little is known about their presynaptic localizations and little information is currently available for human brain tissue. The characteristics of phosphoplipase C transmembrane signaling were studied in crude and synaptosomal plasma membranes from postmortem human prefrontal cortex by measuring the hydrolysis of exogenous [(3)H]phosphatidylinositol4,5bisphosphate(PIP(2)) and the immunoreactive levels of phospholipase C (PLC) and G(alphaq/11) proteins. Regulation of PLC activity by Ca(2+) and the 5-HT(2) receptor agonist 5-methyltryptamine, but not by guanosine 5'-O-[3-thiotriphosphate] and the muscarinic acetylcholine receptor agonist carbachol were different between crude and synaptosomal membranes. KCl (20 mM) stimulation was absent in both preparations. Levels of G(alphaq/11)-protein subunits differed between preparations. The functional inhibition carried out with pirenzepine in crude membranes in order to reverse the carbachol-induced PLC stimulation indicates the existence of a component (53%) of the response that is activated by the M(1) muscarinic acetylcholine receptor subtype, and another component (47%) probably mediated by the M(3) muscarinic acetylcholine receptor subtype. In synaptosomal plasma membranes an increased inhibition of carbachol-induced PLC activation through M(1) was found. The PLC activation by 5-methyltryptamine (ketanserin-sensitive in crude membranes) was absent in synaptosomal plasma membranes suggesting the lack of activity mediated by 5-HT(2)-serotonin receptors.     

Vinburnine decelerates [3H]N-methylscopolamine binding to recombinant human muscarinic M1-M4 acetylcholine receptors

The kinetics of [3H]N-methylscopolamine binding to membranes of Chinese hamster ovary (CHO) cells expressing muscarinic M(1)-M(4) acetylcholine receptors was studied. [3H]N-methylscopolamine dissociation was used for the "single-point" analysis of allosteric modulation by vinburnine (L-eburnamonine). [3H]N-methylscopolamine dissociation was decelerated by vinburnine with EC(50) values of 29.5, 4.1, 9.5 and 15.0 microM for muscarinic M(1)-M(4) receptors, respectively. Acetylcholine doubled the EC(50) of vinburnine for muscarinic M(3) receptors. These kinetic EC(50) values correlated with equilibrium binding constants, supporting the ternary allosteric model. Vinburnine also decelerated the association of [3H]N-methylscopolamine binding, resulting in opposite cooperativity for muscarinic M(1) and M(2) receptors.     

On the involvement of multiple muscarinic receptor subtypes in the activation of phosphoinositide metabolism in rat cerebral cortex

We have examined the activation of phosphoinositide metabolism by muscarinic agonists in rat cerebral cortex, in an attempt to delineate the mechanisms by means of which some selective antagonists inhibit this response in a manner that deviates from simple mass action law. The accumulation of [3H]inositol phosphates induced by the full agonist carbamylcholine in cell aggregates preparations was inhibited by muscarinic antagonists with the following order of potency: telenzepine greater than atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than pirenzepine greater than hexahydro-sila-difenidol greater than AF-DX 116. The same order of potency was found for the competition of these antagonists with [3H]telenzepine binding to M1 muscarinic receptors. The inhibition of the formation of [3H]inositol phosphates activated by acetylcholine, carbamylcholine, and oxotremorine-M by pirenzepine and telenzepine showed biphasic curves, with 62-73% of the response being inhibited with high affinity. Atropine, AF-DX 116, and pirenzepine shifted the concentration-response curves of oxotremorine-M to the right in a parallel manner. However, pirenzepine at micromolar concentrations showed deviation from linearity of the Schild regression. The blockade by high concentrations of pirenzepine and telenzepine showed less than additive dose ratios when assayed in the presence of atropine, suggesting deviation of their antagonism from simple competition. However, after alkylation with propylbenzilylcholine mustard in the presence of low concentrations of pirenzepine, the response to carbamylcholine and oxotremorine-M showed monophasic inhibition curves by pirenzepine and linear Schild regression for this antagonist. These results support the interpretation that the formation of [3H]inositol phosphates is activated by multiple muscarinic receptor subtypes in rat cerebral cortex. The profile of affinities of muscarinic antagonists indicates that a major component of the response is activated by an M1 receptor subtype and a minor component is probably mediated by M3 muscarinic receptors when acetylcholine, carbamylcholine, or oxotremorine-M are used to stimulate the response. Conversely, pirenzepine inhibited the response induced by methacholine and bethanechol in a monophasic manner with high affinity (Ki = 13 nM), suggesting that these agonists can selectively stimulate phosphoinositide metabolism through activation of M1 muscarinic receptors in rat cerebral cortex.     

A novel series of non-quaternary oxadiazoles acting as full agonists at muscarinic receptors

1 A novel series of non-quaternary oxadiazole-based muscarinic agonists demonstrated high affinity for muscarinic receptors. 2. These agonists possessed high efficacy in the nanomolar range at muscarinic receptors in the superior cervical ganglion, atrium and ileum but did not show selectivity across the tissue preparations. 3. Two amino oxadiazoles, one from a quinuclidine series (L-660,863) and one from a 1-azanorbornane series (L-670,207) possessed a high ratio of potency for displacing the binding of [3H]-N-methyl-scopolamine ([3H]-NMS) to potency for displacing the agonist [3H]-oxotremorine-M cortex. 4. The two azanorbornane derivatives L-670,548 and L-670,207 stimulated the turnover of phosphatidylinositol in the cortex with a potency higher than that obtained with any other known muscarinic agonist (ED50 0.26 and 0.18 microM respectively). 5. The maximum response obtained with L-670,207 was greater than that observed for carbachol but was comparable to that of the natural ligand acetylcholine. 6. These oxadiazole muscarinic agonists are among the most potent and efficacious non-quaternary muscarinic agonists ever described.     

Muscarinic receptor subtypes in bovine adrenal medulla

Muscarinic receptors in bovine adrenal medullary microsomes were characterized by radioligand binding assay, using l-[3H]quinuclidinyl benzilate (QNB), a muscarinic antagonist. Specific [3H]QNB binding to microsomes was rapid, reversible, saturable and of high affinity. Saturation experiments revealed a single class of binding sites for the radioligand with a maximum number of binding sites and an apparent dissociation constant of 162.6 fmoles/mg protein and 40.3 pM respectively. According to computer-assisted nonlinear regression analysis, however, drug/[3H]QNB competition curves indicated the presence of at least two affinity sites for muscarinic agonists (acetylcholine, carbamylcholine, oxotremorine), with a high (K1) and a low (K2) affinity (e.g. K1 = 664.8 nM and K2 = 36.5 microM for acetylcholine). The two affinity sites for acetylcholine showed only minimal regulation by magnesium and guanosine 5'-triphosphate. Furthermore, the presence of two affinity sites was suggested for the antagonists pirenzepine and gallamine, but not for atropine and pilocarpine. The K1 and K2 values for pirenzepine were 23.7 and 429 nM, respectively, with 54.5% of total sites having a high affinity. These results indicate that at least two distinct subtypes of muscarinic receptors exist in the bovine adrenal medulla and that they are distinguished by their relative binding affinity for muscarinic agonists and antagonists. The receptors are predominantly composed of the affinity state termed M1, as described for the receptors of sympathetic ganglia.     

Pharmacological differences between muscarinic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase inhibition

Pharmacological differences between muscarinic cholinergic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase were studied. Stimulation of muscarinic receptors from SK-N-SH human neuroblastoma cells resulted in phosphoinositide hydrolysis, but not in inhibition of cAMP formation. As has been shown previously, stimulation of muscarinic receptors from NG108-15 neuroblastoma x glioma cells, on the other hand, resulted in inhibition of cAMP formation without any observable phosphoinositide hydrolysis. These two cell lines provide a useful model system in which to study differential coupling of muscarinic cholinergic receptors. Inhibition of [3H]N-methyl scopolamine [( 3H]NMS) binding and inhibition of carbachol-stimulated function by the antagonists pirenzepine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were studied in this system. Pirenzepine inhibited [3H]NMS binding in both cell lines with low affinity (Ki of 130 and 160 nM in NG108-15 and SK-N-SH cells respectively), indicating that both cell lines express M2 receptors. None of the three antagonists studied exhibited any clear selectivity for the receptors in one cell line over those of the other. In contrast, several agonists including acetylcholine, bethanechol and carbachol exhibited pronounced selectivity. These agonists inhibited [3H]NMS binding to membranes from SK-N-SH cells with IC50 values that were 17-, 3- and 38-fold higher, respectively, than those of NG108-15 cells. This selectivity was still observed when whole cells rather than membranes were studied. These findings indicate that pharmacological differences between receptors coupled to phosphoinositide turnover and those coupled to cAMP inhibition can be detected with certain agonists, but not with the antagonists pirenzepine, AF-DX 116 or 4-DAMP.     

Muscarinic M1, M2 receptor binding. Relationship with functional efficacy

A comparison has been made between [3H]pirenzepine binding to the M1 receptor population of rat cerebral cortex and [3H]N-methylscopolamine binding to M2 receptors in rat cardiac membranes. Several standard muscarinic antagonists including trihexyphenidyl HCl, benztropine, biperidin and 4-DAMP (4-diphenylacetoxy-N-methyl piperidine methiodide) showed some selectivity for the M1 binding assay. Dicyclomine and hexahydrosiladifenidol were the only antagonists with a selectivity approaching that of pirenzepine. Gallamine and AFDX-116 were the only M2 (cardiac) selective antagonists. Muscarinic agonists displayed profiles which could be classified into two groups, apparently related to their intrinsic activity. One group displayed apparent selectivity for the heart, with low Hill coefficients and contained full agonists such as acetylcholine. The second group displayed less selectivity, intermediate Hill coefficients and contained partial agonists such as pilocarpine. Thus muscarinic agents can distinguish between different tissues not only on the basis of receptor selectivity, but also by recognition of high and low agonist affinity states. Thus the intrinsic activity of a muscarinic agonist may reflect an apparent but not true receptor-mediated selectivity.     

The oxime HGG-12 as a muscarinic acetylcholine receptor antagonist without intrinsic activity in cardiac membranes

Direct interactions of the bispyridinium oxime HGG-12 with muscarinic acetylcholine receptors were investigated in porcine cardiac atrial membranes. Competition binding experiments using the radiolabeled muscarinic receptor antagonist (³H)QNB revealed specific binding of HGG-12 to muscarinic acetylcholine receptors of porcine atrial membranes with a dissociation constant of 3.8×10^–7 mol/l. Muscarinic acetylcholine receptor-stimulated binding of the radiolabeled GTP analog (³⁵S)GTP[S] to guanine nucleotide binding proteins (G-proteins) was used to study antagonistic and possible agonistic effects of HGG-12 at muscarinic acetylcholine receptors. HGG-12 completely inhibited carbachol- and oxotremorine-stimulated (³⁵S)GTP[S] binding to pertussis toxin sensitive and insensitive G-proteins in a competitive manner. Inhibition constants (K_I) of HGG-12 for blockade of carbachol- and oxotremorine-stimulated GTP[S]-binding (9.7×10^–7 mol/l and 1.7×10^–6 mol/l, respectively) were higher by about a factor of 100 than those of the muscarinic acetylcholine receptor antagonist atropine. In the absence of muscarinic acetylcholine receptor agonists, HGG-12 by itself had no stimulatory effect on (³⁵S)GTP[S] binding in porcine atrial membranes. The results of this study show that the oxime HGG-12 is a competitive antagonist without intrinsic activity at porcine atrial muscarinic acetylcholine receptors. The stimulatory action of HGG-12 on muscarinic acetylcholine receptors which has been described by several authors is, therefore, suggested to be due to partial inhibition of acetylcholinesterase by the oxime rather than to direct agonism at muscarinic acetylcholine receptors.     

Characterization of cholinergic muscarinic receptors in the rabbit iris

The sphincter smooth muscle of the iris is innervated by excitatory parasympathetic nerve fibers, and the activation of these fibers results in the breakdown of phosphatidylinositol 4,5-bisphosphate into its derived second messengers, myosin light chain phosphorylation and muscle contraction. The present study characterizes the muscarinic acetylcholine receptors (mAChRs) of the rabbit iris employing [3H]N-methylscopolamine ([3H]NMS) and L-[3H]quinuclidinyl benzilate ([3H]QNB) as probes. Binding studies indicated that [3H]NMS and [3H]QNB bound to homogeneous populations of mAChRs with apparent Bmax values of 0.67 and 1.09 pmol/mg protein respectively. Binding of radioligands was rapid, saturable, stereospecific, reversible, and inhibited by specific muscarinic agonists and antagonists in a competitive manner. [3H]NMS displayed a lower amount of nonspecific binding and a faster association and dissociation rate than [3H]QNB. The relative potencies for displacement of both radioligands, based on their Ki values, were (-)QNB greater than atropine greater than (+)QNB greater than pirenzepine greater than pilocarpine. Antagonist displacement of the radioligands appeared to obey the law of mass action, indicating interaction with a single binding site. However, displacement of the radioligands by the agonists carbamylcholine and methacholine indicated interaction with both high and low affinity binding sites. Comparison of the displacement of [3H]NMS and [3H]QNB by pirenzepine in microsomal fractions from rabbit iris, ileal muscle and cerebral cortex revealed the presence of a single subtype of mAChR in the iris which had an affinity for PZ that was slightly higher than that of ileal M2 receptors, but lower than that of brain M1 receptors. This suggests that the mAChRs in the iris may represent a subclass of receptors within the M2 subtype, or they may constitute an entirely different subtype of mAChRs.     

Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.     

In vitro down-regulation predicts agonist efficacy at central muscarinic cholinergic receptors

Agonist induced short-term down-regulation of central muscarinic cholinergic receptors in mechanically dissociated cells of the mouse brain has been shown to predict the efficacy of agonists at muscarinic receptors. Pretreatment of cells with full agonists such as carbachol or oxotremorine M resulted in a loss of available muscarinic cholinergic receptors of about 30% using [3H]N-methylscopolamine [( 3H]NMS) as radioligand, whereas a second group of agonists e.g. RS 86 were only weakly active in this regard producing a significantly smaller loss of cell surface muscarinic cholinergic receptors. The magnitude of down-regulation of muscarinic receptors induced by pretreatment with several cholinergic drugs correlates fairly well with their ability to stimulate the phosphatidylinositol turnover. It seems that the agonist induced down-regulation of muscarinic cholinergic receptors on mechanically dissociated neurons of the mouse brain is a simple screening method to test for centrally acting cholinergic agonists.     

Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors

The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H]-oxotremorine-M binding preclude their utilisation for the prevention of acetylcholine catabolism in rat brain membranes, the latter being required to estimate the binding of acetylcholine to [3H]-oxotremorine-M-labelled muscarinic receptors. However, fasciculin-2, a potent peptide inhibitor of acetylcholinesterase (IC50 24 nM), did prevent catabolism of acetylcholine in rat brain membranes with an atypical inhibition isotherm of [3H]-oxotremorine-M binding, thus permitting an estimation of the "global affinity" of acetylcholine (Ki 85 nM) for [3H]-oxotremorine-M-labelled muscarinic receptors in rat brain.     

Muscarinic M1 receptors in the lateral septal area mediate cardiovascular responses to cholinergic agonists and bradykinin: supersensitivity induced by chronic treatment with atropine

The infusion of pilocarpine, acetylcholine, bradykinin and the selective M1 muscarinic agonist McNeil-A-343 into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. The M1 muscarinic agonist carbamylcholine that causes a rise in arterial blood pressure when injected into the anterior lateral ventricles did not produce any cardiovascular effects when infused into the lateral septal area. Chronic treatment with atropine induced supersensitivity to the muscarinic agonists and a significant increase in the number of muscarinic receptors. In this study bradykinin failed to produce any significant change in cardiovascular activity. Pirenzepine, a M1 muscarinic blocking agent, inhibited completely the effect of both muscarinic agonists and bradykinin on cardiovascular activity. In fact, in vitro studies shows that the displacement of the binding of [3H]QNB by pirenzepine is compatible with the presence of the M1 subtype of muscarinic receptor in the lateral septal area, where it may play a major role on cardiovascular regulation.     

G(q/11) and G(i/o) activation profiles in CHO cells expressing human muscarinic acetylcholine receptors: dependence on agonist as well as receptor-subtype

1. Profiles of G protein activation have been assessed using a [35S]-GTPgammaS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3 or M4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1 and M3, or M2 and M4 receptors were approximately equal. 2. Maximal [35S]-GTPgammaS binding to G(q/11)alpha stimulated by M1/M3 receptors, or G(i1-3)alpha stimulated by M2/M4 receptors occurred within approximately 2 min of agonist addition. The increases in G(q/11)alpha-[35S]-GTPgammaS binding after M1 and M3 receptor stimulation differed substantially, with M1 receptors causing a 2-3 fold greater increase in [35S]-GTPgammaS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half-maximal response. 3. Comparison of M2 and M4 receptor-mediated G(i1-3)alpha-[35S]-GTPgammaS binding also revealed differences, with M2 receptors causing a greater increase in G(i1-3)alpha activation and requiring 10 fold lower concentrations of methacholine to stimulate a half-maximal response. 4. Comparison of methacholine- and pilocarpine-mediated effects revealed that the latter partial agonist is more effective in activating G(i3)alpha compared to G(i1/2)alpha for both M2 and M4 receptors. More marked agonist/partial agonist differences were observed with respect to M1/M3-mediated stimulations of G(q/11)alpha- and G(i1-3)alpha-[35S]-GTPgammaS binding. Whereas coupling to these Galpha subclasses decreased proportionately for M1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3 receptors for G(i)alpha versus G(q/11)alpha activation. 5. These data demonstrate that mACh receptor subtype and the nature of the agonist used govern the repertoire of G proteins activated. They also provide insights into how the diversity of coupling can be pharmacologically exploited, and provide a basis for a better understanding of how multiple receptor subtypes can be differentially regulated.     

3H]N-Methylscopolamine binding to muscarinic receptors in intact adult rat brain cell aggregates

Intact brain cell aggregates were dissociated from adult rat brains, by a simple sieving technique, and were used to study the binding characteristics of [3H]N-methylscopolamine to muscarinic acetylcholine receptors. The magnitude of binding of this ligand was related linearly to the amount of cell protein in the binding assay, with a high ratio of total to nonspecific binding. In addition, specific binding showed saturability and high affinity. Muscarinic receptor antagonists displaced specific [3H]N-methylscopolamine binding according to the law of mass-action, while it was possible to resolve displacement curves using receptor agonists into high- and low-affinity components. The results are discussed in terms of the usefulness of dissociated intact rat brain cells in studying muscarinic acetylcholine receptors in the central nervous system.