Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent [3H]N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37 degrees C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system.     

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.     

Characterization of muscarinic cholinergic receptors on rat pancreatic acini by N-[3H]methylscopolamine binding. Their relationship with calcium 45 efflux and amylase secretion

N-[3H]Methylscopolamine (NMS) binding, amylase secretion, and 45Ca efflux from dispersed rat pancreatic acini were investigated in parallel, in the presence or absence of 4 muscarinic agonists and 3 muscarinic antagonists. Scatchard analysis of [3H]NMS saturation isotherms gave a KD of 0.9 nM and an average binding capacity of 24,000 sites per cell. Binding competition curves with the antagonists atropine, dexetimide, and NMS gave KD values of 3.5, 3.5, and 0.5 nM, respectively. With the 3 full agonists oxotremorine, muscarine, and carbamylcholine, the receptor population could be divided into two classes of binding sites: a minor one (15%) with high affinity (KD = 20-35 nM) and a major one (85%) with low affinity (KD = 3-65 microM). There was a receptor reserve of about 50% with respect to carbamylcholine-stimulated amylase secretion. Further analysis of dose-effect curves suggests that low affinity binding sites were involved in the secretory response to muscarinic stimulation. Pilocarpine, like muscarinic antagonists, recognized all binding sites with the same affinity but acted as a partial agonist on amylase secretion and 45Ca efflux.     

Regulation of the solubilized bovine cerebral cortex muscarinic receptor by GTP and Na+

The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities.     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells

The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low-affinity sites on intact cells with an affinity identical to that found for the low-affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (greater than 30 s) high-affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.     

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.     

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.     

Dopaminergic and cholinergic muscarinic receptor effects of L-alphacetylmethadol (LAAM) and its metabolites

In isolated rat hearts L-alphacetylmethadol (LAAM) produced a concentration-dependent decrease in the spontaneous beating rate. This effect was completely prevented by 1.0 microM atropine. Chronic treatment of rats with LAAM increased the number of striatal dopamine receptors measured by [3H]spiroperidol binding. The affinity of these binding sites for [3H]spiroperidol was unchanged by LAAM treatment. There were no significant changes in the number or affinity of binding sites for the labeled muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) with chronic LAAM treatment. The ability of LAAM, nor-LAAM, or dinor-LAAM to antagonize the binding of [3H]spiroperidol (40 pM) or [3H]QNB (125 pM) to striatal membrane fragments was tested. The measured affinity constants for LAAM and metabolites were 100-3000 times higher than the affinity constants of unlabeled spiroperidol at [3H]spiroperidol binding sites. The affinity constants of LAAM and metabolites at muscarinic binding sites were 10-20 times higher than pilocarpine and 5000-8000 times higher than atropine. These results suggest that LAAM can produce some of its effects by acting as a weak agonist at muscarinic receptor sites.     

In vitro effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on muscarinic cholinergic receptors in mouse striatum

1. MPTP significantly lowered Kd of the binding of [3H]QNB to muscarine receptor without affecting Bmax values compared with those of control. Hill coefficients (nH) of control and MPTP (250 microM) added group were 1.15 +/- 0.127 and 0.56 +/- 0.202, respectively. 2. Prior addition of pargyline to MPTP did not prevent the decrease of [3H]QNB binding. The patterns of displacement of [3H]QNB by MPTP and MPP+ were similar to those by some muscarinic agonists, such as acetylcholine, carbamyl choline and methacholine. 3. These results suggest that MPTP might be muscarinic agonist and might play a role to produce Parkinsonism through directly affecting the muscarinic cholinergic receptors in vivo.     

Stimulation by glucose and carbamylcholine of phospholipase C in pancreatic islets

Phosphoinositide hydrolysis in intact pancreatic islet cells was investigated in an indirect but dynamic manner by monitoring the efflux of radioactivity from islets prelabelled with [3H]inositol. A rise in glucose concentration provoked a rapid, modest but sustained increase in effluent radioactivity, this phenomenon being abolished in the absence of extracellular Ca2+ or presence of verapamil. The release of [3H]inositol was also stimulated at high extracellular K+ concentration, but not by gliclazide. Whether in the presence or absence of glucose, carbamylcholine provoked a marked increase in effluent radioactivity. The response to the cholinergic agent was decreased in the presence of verapamil or absence of extracellular Ca2+ and abolished in the presence of atropine or LiCl. These results suggest that an increase in cytosolic Ca activity, as caused by glucose or membrane depolarization, may cause activation of phospholipase C. In response to cholinergic agents, however, the enzymic activation, although modulated by Ca2+ availability, may result directly from the occupation of muscarinic receptors.     

Rat brain and heart muscarinic receptors: modification with tetranitromethane

Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

Reconstitution of solubilized atrial cholinergic muscarinic receptors in liposomes

The reconstitution of solubilized bovine atrial cholinergic muscarinic receptor into liposomes made of exogenous lipids has been achieved by polyethyleneglycol precipitation. Of the different lipid mixtures used, soybean lecithins were shown to be the best on the basis of receptor recovery. The receptor reconsituted into soybean lecithins liposomes exhibited ligand binding properties very similar to those of the native receptor. The dissociation constant of [³H]-N-methyl-scopolamine ([³H]NMS) was 0.46 and 0.30 nM as determined by equilibrium and kinetics experiments respectively. The potency of a range of muscarinic ligands in displacing [³H]NMS binding was atropine > methyl-atropine > scopolamine > pirenzepine oxotremorine > gallamine > carbamylcholine > pilocarpine bethanechol. The Hill slopes of the displacement curves were near 1 for the antagonists and smaller than 1 for the agonists and for gallamine. The agonist binding may be modulated by guanine nucleotides. These results indicate that soybean lecithins fulfill the lipid requirements for the reconstitution of the atrial muscarinic receptor.     

Heterologous desensitization of adenylate cyclase with prostaglandin E1 alters sensitivity to inhibitory as well as stimulatory agonists

Adenylate cyclase in cultured human fibroblasts is activated by prostaglandin E1 (PGE1) or beta-adrenergic agonists, e.g., isoproterenol, and inhibited by muscarinic agonists. Incubation with PGE1 reduced adenylate cyclase responsiveness to both PGE1 and isoproterenol; this so-called heterologous desensitization is believed to result from impaired function of the stimulatory guanyl nucleotide-binding protein of the cyclase complex. The effect of heterologous desensitization by PGE1 on inhibition of adenylate cyclase by the muscarinic agonist oxotremorine was examined. Muscarinic inhibition of basal and isoproterenol-stimulated cAMP accumulation was attenuated following exposure to PGE1; the concentration of oxotremorine required for half-maximal inhibition of cAMP accumulation was increased. In both intact cells and membrane preparations the number of binding sites for [3H]scopolamine, a muscarinic antagonist, was unaltered by desensitization. Following exposure to PGE1, receptor affinity for oxotremorine, assessed by competition with [3H] scopolamine, and the guanyl nucleotide sensitivity of agonist binding were reduced. The amount of inhibitory guanyl nucleotide-binding regulatory protein available for [32P]ADP-ribosylation by pertussis toxin was unaltered by desensitization. Thus, heterologous desensitization of adenylate cyclase with the stimulatory agonist PGE1 alters sensitivity to inhibitory as well as stimulatory ligands.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization.

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.     

Evidence that muscarinic receptors in islet cells are not coupled functionally to adenylate cyclase through the inhibitory guanine nucleotide binding protein (Ni)

The effect of muscarinic agonist on adenylate cyclase was investigated in neonatal islet cells and in a clonal pituitary cell line (GH4C1) following labelling of the intracellular ATP pool with [2,8 3H]adenine. In islet cells carbamylcholine was without effect on basal or glucagon-stimulated adenylate cyclase activity, measured as 3H cyclic AMP production, but inhibited 3H cyclic AMP production in the clonal pituitary cells. The involvement of the inhibitory guanine nucleotide binding protein of adenylate cyclase (Ni) was investigated by the use of the Bordetella pertussis exotoxin, islet activating protein (IAP). Pre-treatment of islet cells with IAP was without effect on adenylate cyclase following carbamylcholine but in the clonal pituitary line abolished the inhibition of 3H cyclic AMP production. It is concluded that in the islet cell, in contrast to the clonal pituitary cell, muscarinic receptors are not effectively coupled through Ni to inhibit adenylate cyclase.     

Mixed Competitive and Allosteric Antagonism by Gallamine of Muscarinic Receptor-Mediated Second Messenger Responses in N1E-115 Neuroblastoma Cells

The antagonistic effects of gallamine on muscarinic receptor-linked responses were investigated in N1E-115 neuroblastoma cells. M1 muscarinic receptor-mediated phosphoinositide hydrolysis induced by carbamylcholine was antagonized by gallamine, with a Ki value of 33 microM. By comparison, gallamine was four- to fivefold less potent in blocking noncardiac M2 muscarinic receptor-mediated inhibition of cyclic AMP formation, with a Ki value of 144 microM. The resulting Arunlakshana-Schild plots of the antagonism of both responses by gallamine were linear and exhibited slopes not differing from 1, a result indicative of a competitive mechanism. To elucidate further the nature of gallamine's inhibitory actions, experiments were performed where the effects of gallamine in combination with the known competitive muscarinic antagonist, N-methylscopolamine (NMS), were studied. In the presence of both antagonists, a supraadditive shift in the carbamylcholine dose-response curve was demonstrated for the two responses, a result suggestive of an allosteric mode of interaction between gallamine and NMS binding sites. Confirmation that gallamine allosterically modifies the muscarinic receptor was provided by radioligand binding studies. Gallamine competition curves with either [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) or [N-methyl-3H]quinuclidinyl benzilate methyl chloride ([3H]NMeQNB) were unusually shallow. Furthermore, gallamine decelerated the rate of dissociation of receptor-bound [3H]NMS greater than [3H]NMeQNB in a dose-dependent manner. The present study demonstrates that whereas gallamine antagonizes carbamylcholine-mediated responses in N1E-115 cells in a competitive manner, an allosteric component of its action is revealed in the presence of muscarinic antagonists such as NMS.