Purification of muscarinic acetylcholine receptors by affinity chromatography

Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein.     

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.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

Abstract: We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)-generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 µ M sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N -[³H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 µ M . Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [³H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.     

STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [³H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [³H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [³H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.     

Evidence for Multiple Muscarinic Receptor Subtypes in Human Brain

Pirenzepine, a compound with selective antimuscarinic activity, was used to distinguish muscarinic acetylcholine receptor subtypes in normal human brain. Hill coefficients and IC50 values derived from the inhibition of specific [3H]L-quinuclidinyl benzilate receptor binding suggest the presence of two muscarinic binding sites, differing both in affinity for pirenzepine and in tissue distribution.     

Large scale preparation and characterization of membrane-bound and detergent-solubilized muscarinic acetylcholine receptor from pig atria

The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig atrial membranes by new large scale procedures which result in 30-40 fold enrichment of the receptor in the membrane-bound state and a further three fold enrichment during solubilization. The membrane-bound receptor was prepared by differential and sucrose density gradient centrifugation in 25 mM imidazole, 1 mM EDTA, pH 7.4. A double extraction procedure using a mixed digitonin/cholate detergent was used to solubilize the receptor at a 60-70% yield. The membrane and solubilized preparations had specific activities of 3.5-5 and 8-12 pmol [3H]L-quinuclidinyl benzilate (QNB) binding sites per mg of protein, respectively. The presence of imidazole, which behaved as a weak muscarinic ligand, stabilized the receptor during solubilization and storage. Both the membrane-bound and detergent-solubilized mAcChR bound antagonists at a single class of sites and agonists at two subclasses of QNB sites. The proportion of high affinity agonist sites in the solubilized receptor was about 1/3 that in the membrane receptor. [3H]Propylbenzilylcholine mustard covalently labeled a single prominent atropine-sensitive component with an apparent molecular weight of 70-74,000 on SDS-polyacrylamide gels for both the membrane and solubilized receptor.     

Anomalous binding of [3 H] N-methyl-quinuclidinyl benzilate methyl chloride to human lymphocyte muscarinic receptors

Using the muscarinic cholinergic ligand [³ H] N-methyl quinuclidinyl benzilate methyl chloride ([³ H] NM-QNB), we demonstrated that intact, viable human lymphocytes posses specific muscarinic binding sites. Equilibrium binding studies show that muscarinic acethylcholine receptor are devided into two subtype; high affinity (Ms) and low affinity types (Mw) for the ligand.     

Muscarinic Receptors in the Cochlear Nucleus and Auditory Nerve of the Guinea Pig

The specific-binding properties of l-[3H]quinuclidinyl benzilate, a muscarinic acetylcholine-receptor antagonist, were investigated in synaptic and other membrane preparations of the guinea pig cochlear nucleus and auditory nerve. Binding parameters for all experiments were consistent with a single binding site with a Hill coefficient of 1.0. The binding of the ligand was specific and of high affinity, with values of KD in the range of 30-80 pM. Bmax was 0.352 +/- 0.023 pmol/mg protein for the dorsal cochlear nucleus and 0.215 +/- 0.011 pmol/mg protein for the ventral cochlear nucleus. The dorsal cochlear nucleus/ventral cochlear nucleus ratio for density of muscarinic receptors (1.6/1.0) was maintained across two different buffer systems, which varied with respect to the inclusion of proteolysis inhibitors. The results for auditory nerve indicated a level of binding much below that of the cochlear nucleus, with Bmax = 0.052 +/- 0.011 pmol/mg protein. The results of specific-binding experiments for l-[3H]quinuclidinyl benzilate support a role for acetylcholine as a neurotransmitter in the cochlear nucleus. The greater density of muscarinic receptors in the dorsal cochlear nucleus may indicate greater cholinergic activity in the dorsal relative to the ventral cochlear nucleus.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Identification of Muscarinic Receptors in Rat Cerebral Cortical Microvessels

Microvessels isolated from rat cerebral cortex consist mainly of capillaries (greater than 85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (KD = 0.034 nM). The association and dissociation rate constants (0.37 min-1 nM-1 and 0.0067 min-1, respectively) yield an equilibrium KD of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.     

Photoaffinity labeling of the solubilized, partially purified muscarinic acetylcholine receptor from porcine atria by p-azidoatropine methyl iodide

The synthesis of a tritiated photoaffinity analogue of the muscarinic antagonist atropine, [3H]-p-azidoatropine methyl iodide is described. The compound appeared to bind to a single class of sites in membrane-bound, solubilized, and partially purified preparations of muscarinic receptor from porcine atria with a dissociation constant (determined by competition vs. [3H]-L-quinuclidinyl benzilate) of about 1.0 X 10(-7) M. This value was in agreement with the apparent dissociation constant (8.5 X 10(-8)M) determined by measuring the concentration dependence of covalent incorporation into a partially purified receptor preparation. Competition experiments indicated that the specific covalent labeling could be blocked by the muscarinic agonist carbamylcholine and the antagonists L-quinuclidinyl benzilate and atropine. An apparent molecular weight of 75 000 +/- 5000 was found for specifically labeled peptide(s) in a solubilized, partially purified receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Agonist-induced changes in the modulation of K+ permeability and beating rate by muscarinic agonists in cultured heart cells

The correlation between number of muscarinic cholinergic receptor sites as measured by binding of the muscarinic antagonist [3H]methylscopolamine ([3H]MS) and the ability of muscarinic agonists to mediate a physiologic response was determined in intact heart cells cultured from chick embryos 10 d in ovo. The increase in K+ permeability and the decrease in beating rate mediated by the muscarinic agonist carbamylcholine were the responses studied. Exposure to 10(-3) M carbamylcholine caused a 15% decrease in beating rate and a 33% increase in the rate of 42K+ efflux from cells labeled to equilibrium. An assay for binding of [3H]MS to intact cells was developed. [3H]MS bound specifically to intact heart cells (185 fmol/mg protein) with a Kd of 0.48 nM. Exposure of cells for various times to 10(-3) M carbamylcholine followed by binding of [3H]MS to intact cells demonstrated that a gradual loss of 70% of [3H]MS binding sites took place over the next 6 h with a T 1/2 of 30 min. A decrease in the ability of carbamylcholine to stimulate K+ efflux and to decrease beating rate was observed after pre-exposure of cells to muscarinic agonists. A close correlation was found between the loss of the subclass of muscarinic receptors subject to agonist control and the loss of physiologic responsiveness after agonist exposure. The data suggest the absence of significant numbers of "spare" receptors within this group.     

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.     

Muscarinic acetylcholine receptors of rat lymphocytes

The muscarinic acetylcholine receptors on rat lymphocytes were determined by [3H]quinuclidinyl benzilate binding studies. Binding of [3H]quinuclidinyl benzilate is rapid (half saturation occurred within 120 s) and highly specific. Muscarinic receptors reveal high lability. The number of receptors on plasma membrane depends on time of incubation as well as on composition of incubation medium. Lymphocytes incubated in nutrient-deficient media lose their surface receptors; enrichment of the medium causes reappearance of the receptors. Appearance of [3H]quinuclidinyl benzilate-binding sites in the incubation medium was under conditions in which binding to lymphocytes was decreased. It is concluded that the number of plasma membrane receptors on rat lymphocytes represents the dynamic steady state in which newly synthesized and degraded receptors are balanced.     

Solubilization of muscarinic acetylcholine receptors of bovine brain

The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [³H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors.     

Specific [3H]-N-methyl scopolamine binding without cholinergic function in cultured adult skin fibroblasts

Cultured adult skin fibroblasts were studied for binding and functional evidence of muscarinic receptors in order to assess their utility as a model of cholinergic function in affective illness. Saturable, specific, high affinity binding could be demonstrated in intact cells from some cell lines with [3H]-NMS, but not [3H]-QNB, presumably because of intracellular trapping of unbound [3H]-QNB. [3H]-NMS specific binding indicated a single site with a KD of approximately 210 pM. [3H]-NMS was displaced by cholinergic agonists and antagonists with relative affinities similar to muscarinic receptors in brain. Many cell lines, however, showed no specific binding. No functional response to carbachol could be demonstrated with respect to inhibition of isoproterenol-stimulated cyclic AMP formation, stimulation of cyclic GMP formation or stimulation of phosphoinositide hydrolysis in any cell line regardless of either high or no specific [3H]-NMS binding.     

A human embryonic lung fibroblast with a high density of muscarinic acetylcholine receptors

Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and N-methylscopolamine showed that the human embryonic lung fibroblast CCL137 possesses approximately 2 X 10(5) muscarinic receptors/cell, i.e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin-stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down-regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N-methylscopolamine.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.