Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

Muscarinic binding to mouse brain receptor sites

In mouse brain the binding of [³H]-Atropine to the muscarinic receptor seems to be a simple mass-action determined process as gauged both by approach to equilibrium kinetics and binding at equilibrium. In contrast, using isotopic dilution technique, dissociation measurements indicate the existence of two receptor-ligand complexes. It would appear that association and dissociation rates of binding of the muscarinic antagonists atropine, scopolamine, N-methyl-4-piperidyl benzilate (4NMPB) and 3-quinuclidinyl benzilate (QNB) decrease with increasing affinity based on comparisons of kinetic binding data. The differences between the association rate constants are small whereas those between the dissociation rate constants differ markedly. This kinetic behavior is similar to the well-known time profile of antimuscarinic activity in isolated tissues. These phenomena are discussed in terms of possible isomerization of the receptor-ligand complex, as has been proposed recently for [³H]-scopolamine and [³H]-4NMPB binding.     

A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes

We recently demonstrated that the non-classical muscarinic receptor antagonist [³H]pirenzepine ([³H]PZ) identifies a high affinity population of muscarinic sites in the rat cerebral cortex. We now report that cortical muscarinic sites to which [³H]PZ binds with high affinity are modulated by ions but not guanine nucleotides. We also have examined equilibrium [³H]PZ binding in homogenates of various rat tissues using a new rapid filtration assay. All regional saturation isotherms yielded a similar high affinity dissociation constant (K_d = 2 ? 8 nM) in 10 mM sodium-potassium phosphate buffer. Receptor density (B_max in fmol/mg tissue) varied as follows: corpus striatum = 154.5, cerebral cortex = 94.6, hippocampus = 94.3, ileum = 1.3, cerebellum = 1.0, and heart = 0.45. The cerebral cortex and hippocampus possess 61 percent of striatal binding sites, while the ileum, cerebellum and heart contain only 0.84 percent, 0.65 percent and 0.29 percent of striatal sites respectively. The [³H]PZ sites in heart, ileum, and cerebellum represent 3.1 percent, 9.6 percent, and 10.4 percent of the sites obtained by using [³H](?)quinuclidinyl benzilate. Thus, [³H]PZ labels high affinity muscarinic receptor binding sites with a tissue distribution compatible with the concept of distinct M₁ and M₂ receptor subtypes. Accordingly, regions such as heart, cerebellum, and ileum would be termed M₂, though each have an extremely small population of the M₁ high affinity [³H]PZ site. [³H]PZ therefore appears to be a useful ligand for M₁ receptor identification. Furthermore, the inability to demonstrate a significant effect of guanine nucleotides upon high affinity [³H]PZ binding to putative M₁ receptors suggests that M₁ sites may be independent of a guanine regulatory protein.     

Characterization of Muscarinic Acetylcholine Receptors in Cultured Bovine Aortic Endothelial Cells

Abstract

The binding characteristics of [³H]quinuclidinyl benzilate ([³H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [³H]QNB bound to endothelial cell membranes with high affinity (k_D = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (K_i) were determined by displacement of bound [³H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M₃ antagonist) > pirenzepine (M₁ antagonist) > AFDX-116 (M₂ antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M₁ agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M₃ and M₁ subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [³H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [³H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.     

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.     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

Regulation of ligand binding to cardiac muscarinic receptors by ammonium ion and guanine nucleotides

Guanine nucleotides and Na⁺ are known to regulate ligand binding to cardiac muscarinic receptors, which are netagively couple to the adenylate cyclase system. In the present study, we found that NH₄⁺ was more potent than Na⁺ or other monovalent cations in regulating the affinity of the muscarinic receptor for agonists and antagonists. The effect of NH₄⁺ (or Na⁺) on the binding of the antagonist [³H]quinuclidinyl benzilate (QNB) to muscarinic receptors in homogenates of embryonic chick hearts depended on the assay buffer used. NH₄⁺ increased K_d in phosphate buffer or histidine and increased B_max in Tris. NH_f4⁺ (0.1 M) increased the IC₅₀ value for actylcholine inhibition of [³H]QNB binding 20-fold compared to 3–4-fold with 0.1 M Na⁺ or K⁺. Furthermore, NH₄⁺ could substitute for and was more potent than Na⁺ in producing synergistic effects with Gpp[NH]p to reduce the affinity of the receptor of acetylcholine. Tris depressed these effects. Gpp[NH]p plus 0.4 M NH₄Cl totally converted the receptor population to a low affinity agonist state and increased the IC₅₀ for acetylcholine by more than 2000-fold. Two conclusions can be made from the present results. First, NH₄⁺ appears to be the most potent effector yet studied of the monovalent cation site of the muscarinic receptor system. Second, the use of Tris in muscarinic receptor ligand binding assays will produce anomalous results concerning the properties of both agonist antagonist binding to the receptor.     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Striatal muscarinic receptors: Regulation by dopaminergic agonists

The effects of apomorphine on the binding properties of striatal muscarinic receptors were investigated using the specific muscarinic antagonist, [³H](?)3-quinuclidinyl benzilate ([³H](?)QNB). When binding measurements were made in 50 mM sodium/HEPES buffer, pH 7.4, containing Mg⁺², the binding of [³H](?)QNB was consistent with the presence of two binding sites; 57% of the sites had a high affinity dissociation constant of 0.030 nM whereas the remaining sites had a low affinity dissociation constant of 0.64 nM. Apomorphine (1.0 μM) enhanced the binding of [³H](?)QNB by an apparent conversion of low to high affinity sites. A variety of other agents were screened for their ability to enhance [³H](?)QNB binding, and a pattern generally consistent with a dopaminergic effect was observed although some evidence for a β-adrenergic effect was demonstrable. The potent neuroleptics haloperidol, spiperone and sulpiride failed to antagonize the apomorphine enhancement of [³H](?)QNB binding as well as some adrenergic antagonists. However, the potent inhibitors of the dopamine-sensitive adenylate cyclase, α-flupenthixol and fluphenazine, specifically blocked the apomorphine enhancement of [³H](?)QNB binding with K_i values of approximately 0.1 μM.     

Prevention of muscarinic acetylcholine receptor down-regulation by chloroquine: Antilysosomal or antimuscarinic mechanisms

The effect of the antimalarial drug chloroquine on the carbachol-induced down-regulation of muscarinic acetylcholine receptors (mAChRs) was studied in the neuroblastoma-glioma hybrid NG108-15 cells. Chloroquine, which is proposed to have both antilysosomal and antimuscarinic effects (4,11), blocked the loss of both cell surface and total mAChRs as monitored by [³H]N-methyl-scopolamine (NMS) and [³H] quinuclidinyl benzilate (QNB) bindings respectively. To the contrary, NH₄Cl, only an antilysosomal agent, had no effect on the loss of surface receptors, but blocked degradation of internalized receptors following the effect of carbachol. These findings demonstrate that chloroquine prevents the agonist-induced mAChR down-regulation in NG 108-15 cells by both its antilysosomal and antimuscarinic effects.     

Chronic administration of an organophosphorus insecticide to rats alters cholinergic muscarinic receptors in the pancreas

Male rats were treated for 10 days with the organophosphorus insecticide, acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate (disulfoton, 2 mg/kg/day by gavage). At the end of the treatment, binding of [³H]quinuclidinyl benzilate ([³H]QNB) to cholinergic muscarinic receptors and cholinesterase (ChE) activity were assayed in the pancreas. Functional activity of pancreatic muscarinic receptor was investigated by determining carbachol-stimulated secretion of α-amylase in vitro. ChE activity and [³H]QNB binding were significantly decreased in the pancreas from disulfoton-treated rats. The alteration of [³H]QNB binding was due to a decrease in muscarinic receptor density with no change in the affinity. Basal secretion of amylase from pancreas in vitro was not altered, but carbachol-stimulated secretion was decreased. The effect appeared to be specific since pancreozymin was able to induce the same amylase release from pancreases of control and treated rats. The results suggest that repeated exposures to sublethal doses of an organophosphorus insecticide lead to a biochemical and functional alteration of cholinergic muscarinic receptors in the pancreas.     

Myocardial muscarinic acetylcholine receptor: Choline and tris unmask heterogeneity of antagonist binding sites

The binding properties of myocardial muscarinic acetylcholine receptors are altered in the presence of choline or Tris. The binding of the antagonist [³H]quinuclidinyl benzilate is reduced in the presence of choline or Tris buffer, when compared to parallel determinations in a physiologic salt solution or phosphate buffer. Scatchard analysis indicates the reduced binding is due to a decrease in the apparent number of receptor sites. Experiments with other organic buffers exclude the possibility that the reduced binding in Tris is due to the absence of sodium ions. In the presence of choline or Tris up to 45% of the receptors are not accessible to [³H]quinuclidinyl benzilate. The remaining sites maintain their high affinity for the antagonist. A heterogeneity of antagonist sites is evident.     

Decrease or increase in cardiac muscarinic cholinergic receptor number in rats treated with methacholine or atropine

The effects of chronic treatment of the rat with methacholine and atropine on the cardiac muscarinic cholinergic receptors were investigated. [³H]Quinuclidinyl benzilate ([³H]QNB) was used to directly estimate the number and affinity of the receptors in the heart ventricular membrane. Methacholine treatment decreased, in a dose-related and time-dependent manner, the specific binding of [³H]QNB by 34% as compared to the control. Atropine treatment, on the other hand, resulted in a dose-related increase (28 to 66%) in the number of the receptors. The equilibrium dissociation constant (K_D) of the receptors for the ligand was the same (about 200 pM) for the control and the methacholine treated groups of rats, whereas a dose-related increase (39 to 105%) in the K_D was noted for the atropine treated rats. Similarly, the concentration of acetylcholine causing a 50 percent inhibition (IC₅₀) of the [³H]QNB binding was unaltered for the methacholine treated rats (4 μM), but it was increased 43% for the atropine treated rats.     

A cholinergic receptor site on murine lymphocytes with novel binding characteristics

To further analyze functionally important cholinergic receptors on lymphocytes, we studied the binding of the muscarinic antagonist Quinuclidinyl benzilate (QNB) to murine splenic lymphocytes. Studies of displacement of [³H]QNB by unlabelled QNB on lymphocytes revealed at least two binding sites. Scatchard analysis of equilibrium binding isotherms also distinguished two sites with apparent Kds of 480 nM and 16 μM. There was greater specific QNB binding to B cell-enriched lymphocyte fractions than to T cell fractions. Lymphocyte binding demonstrated temperature-dependent dissociability, and specific binding occurred on isolated lymphocyte membranes as well. Both muscarinic and nicotinic ligands competed for QNB binding to lymphocytes with low and nearly equal affinity. Therefore, QNB binding sites on lymphocytes appear to be of low affinity and of mixed muscarinic and nicotinic character.     

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.     

Regulation of putative muscarinic cholinergic receptor subtypes in rat brain

Transection of the fimbria/fornix, producing a 75% reduction in the activity of the cholinergic marker choline-o-acetyltransferase (CAT EC. 2.3.1.6) in rat hippocampus, did not change the binding characteristics of the non-subtype selective, muscarinic cholinergic receptor antagonist ligand [³H](−)quinuclidinyl benzilate {[³H](−)QNB}. Pirenzepine competition for [³H](−)QNB binding in the hippocampus was best described by a computer derived model assuming two binding sites of high affinity (putative M₁ receptors) and low affinity (putative M₂ receptors). There was no change in the proportion of high and low affinity pirenzepine binding sites in the hippocampus following cholinergic deafferentation. Thus, these data provide no evidence for a discrete localization of either putative subtype of muscarinic receptor discriminated by pirenzepine restricted to the terminals of CAT containing neurons innervating the rat hippocampus.Chronic scopolamine treatment produced a 48% increase in the B_max of [³H](−)QNB binding in the hippocampus, but again there was no change in the proportions of the sites discriminated by pirenzepine demonstrating that both putative subtypes were regulated identically. Similarly, carbachol competition for [³H](−)QNB was unaltered following cholinergic deafferentation or chronic scopolamine treatment. Furthermore, similar guanylyl-5′-imidodiphosphate [Gpp(NH)p] modulation of the proportions of high and low affinity carbachol binding sites was found in the hippocampus following transection of the fimbria/fornix or chronic scopolamine treatment. Thus there is no adaptation of receptor-effector coupling following these treatments that is reflected by changes in receptor recognition site characteristics.Carbachol competition for [³H]pirenzepine binding to putative M₁ receptors in the hippocampus was biphasic and was also modulated by Gpp(NH)p. In the brainstem, there was a homogeneous population of putative M₂ [³H](−)QNB binding sites having low affinity for pirenzepine. Carbachol competition for these binding sites was also biphasic and modulated by guanine nucleotides. Thus, both putative M₁ and M₂ muscarinic receptors, as defined by high or low affinity for pirenzepine respectively, may mediate their effects in rat brain via a guanine nucleotide regulatory subunit.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

Muscarinic cholinergic binding in human erythrocyte membranes

Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([³H]QNB). The apparent K_D is 1.3 × 10^?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system.     

Muscarinic receptor binding in the mouse heart: the selective modulatory effect of fluoride ion on agonist binding

The effect of fluoride ion on the binding of the specific muscarinic agonist ligand [³H]c is methyldioxolane ([³H]CD) to the mouse cardiac muscarinic receptor was investigated. Utilizing equilibrium ligand binding experiments, sodium fluoride (10mM) was shown to decrease [³H]CD binding, measured at a concentration of 2 nM, by 52%. Studies with several different ions demonstrated that the reduction in [³H]CD binding was a specific effect of fluoride. This fluoride modulation was selective for agonist binding, as no effect of fluoride on the binding of the muscarinic antagonist [³H](?) quinuclidinyl benzilate (QNB) was observed.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.