Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System

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

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.     

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

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

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.     

Identification of M2 muscarinic receptors in membranes from bovine cerebral basal arteries

Muscarinic receptors were identified in membrane preparations from bovine cerebral arteries by the specific binding of [3H]-quinuclidinyl benzilate. The total amount of binding sites is relatively high: 1.5 pmol/mg protein, as compared to 0.91 pmol/mg for bovine cerebral cortex and 0.08 pmol/mg for heart muscle. In this study we show that the majority of these sites correspond to M2-receptors: 84% of the sites display low affinity for pirenzepine. In addition, GTP causes a rightward shift and steepening of the carbachol competition binding curve. In the presence of GTP, the alkylating reagent N-ethylmaleimide causes a 28-fold increase of the affinity for carbachol. This phenomenon is also observed on bovine heart membranes where muscarinic receptors are known to be of the M2 type. In contrast, muscarinic receptors in cerebral cortex, predominantly of the M1-type, show only a 4-fold increase in agonist affinity by N-ethylmaleimide. These findings suggest that the ability of N-ethylmaleimide to modulate the agonist affinity is an additional criterion for the characterization of muscarinic M2-type receptors.     

Agonist Regulation of Muscarinic Acetylcholine Receptors in Rat Spinal Cord

Abstract: In vitro studies with cultured cells originating from nervous tissue have shown that chronic exposure to muscarinic agonists results in a loss of muscarinic receptors. To determine whether this type of regulation of muscarinic receptor number also occurs in vivo , we infused carbachol into the spinal cords of rats. A single carbachol injection into the lumbar spinal cord caused a significant increase in the nociceptive threshold. This effect of carbachol diminished to control levels after 12 h of repeated agonist injections every 4 h and was blocked by atropine. The desensitization to the antinociceptive effects of carbachol was associated with a loss of muscarinic receptors as determined by the binding of the muscarinic antagonist [³H]quinuclidinyl benzilate. After a 24-h exposure to carbachol given every 4 h, there was about a 60% loss of binding sites. The loss of muscarinic receptors was also blocked by atropine and was reversible. These results represent direct evidence that a muscarinic agonist can regulate receptor number in the central nervous system and suggest that this loss of receptors is associated with a desensitization to the antinociceptive effects of carbachol injected into the spinal cord.     

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.     

Effect of urea-treatment on agonist binding affinity of the muscarinic acetylcholine receptor

Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of ³H-quinuclidinyl benzilate (³H-QNB). This effect was similar to that of Gpp(NH)p. The effects of urea-treatment and Gpp(NH)p were not additive. On the other hand, treatment of the microsome fraction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of urea-treatment and Gpp(NH)p, when these treatments were combined with DTNB.     

Muscarinic cholinergic binding sites on intact human lymphocytes

Using the muscarinic chalinergic ligand [³H]-quinuclidinyl benzilate, we have demonstrated that intact, viable human lymphocytes possess specific muscarinic binding sites. The binding is saturable, proportional to cell number, and is displaceable by atropine, benztropine, trihexyphenidyl and scopolamine. The apparent kd is 67 nM and the number of binding sites per cell is on the order of 5 × 10⁴. Not only do these findings provide a pharmacological basis for the observed effects of muscarinic agents on lymphocyte function, they also demonstrate the utility of human peripheral blood lymphocytes for investigation of abnormalities of the muscarinic cholinergic system.     

Effects of trypsin-treatment on agonist binding affinity to the muscarinic acetylcholine receptor

Trypsin-treatment of the microsome fraction of the ileum and the synaptic membrane fraction of the cerebral cortex of guinea-pig caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of ³H-quinuclidinyl benzilate (³H-QNB). This effect was similar to that of Gpp(NH)p. The effects of trypsin and Gpp(NH)p were not additive. On the other hand, treatment of these fractions with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of trypsin and Gpp(NH)p, when the fractions were treated with two reagents simultaneously.     

Aceclidine and pilocarpine interact differently with muscarinic receptor in isolated rabbit iris muscle

The relationship between muscarinic receptor affinity states and the contractile response to the muscarinic agonists carbachol, aceclidine, and pilocarpine, has been examined in the isolated rabbit iris muscle. Contraction of the iris muscle by carbachol and aceclidine was more potent and/or more efficacious than the response to pilocarpine. Analysis of [³H]-Quinuclidinyl benzilate (QNB) binding showed that while both carbachol and aceclidine bound to high- and low-affinity forms of the muscarinic receptor, pilocarpine bound to one affinity state. The efficacy of carbachol and aceclidine to stimulate contraction of the iris muscle was consistent with receptor occupancy theory only when considering the low-affinity state of the muscarinic receptor, and activation of the low-affinity rather than high-affinity binding state of the receptor is likely to mediate the contraction of iris muscle. Therefore, the typical anti-glaucoma muscarinic agonists aceclidine and pilocarpine may interact differently with their target receptors in isolated rabbit iris muscle.     

Molecular mechanism of the effects of guanine nucleotide and sulfhydryl reagent on muscarinic receptors in smooth muscles studied by radiation inactivation

The molecular sizes of the units concerned in 3-quinuclidinyl benzilate (QNB) binding and in the effects of guanine nucleotide and sulfhydryl reagent on the inhibition of QNB binding by carbachol in smooth muscle of guinea pig ileum were determined to be 76,000, 179,000 and 107,000, respectively by the radiation inactivation method. One or more subunits (GTP subunit) other than the receptor subunit in a muscarinic receptor appeared to be involved in the effect of guanine nucleotide. When guanine nucleotide was present, the receptor subunit seemed to be dissociated from the GTP subunit.     

Direct binding studies of 125I-α-bungarotoxin and 3H-quinuclidinyl benzilate interaction with axon plasma membrane fragments. Evidence for nicotinic and muscarinic binding sites

The attachment of ¹²⁵I-α-bungarotoxin (BgTx) which is reportedly bound exclusively to “nicotinic” acetylcholine receptors, as well as ³H-atropine and ³H-3-quinuclidinyl benzilate (QNB), which reportedly bind exclusively to “muscarinic” receptors, was measured in isolated lobster axon plasma membrane fragments and in the soluble axonal protein fraction. ¹²⁵I-α-BgTx binding was also measured in lysolecithin-solubilized fragments. Binding assays were adapted for these studies and are described in detail. High affinity, saturable binding of all three ligands to membrane fragments was observed, as well as binding of BgTx to a macromolecule present in both the soluble fraction and the membrane fragments. These experiments provide the first evidence for the very tight binding of both “nicotinic” and “muscarinic” ligands in peripheral nerve.     

Muscarinic regulation of phosphatidylinositol turnover and cyclic nucleotide metabolism in the heart

Stimulation of cardiac muscarinic receptors leads to increases in the synthesis and hydrolysis of the membrane phospholipid phosphatidylinositol (PI). Carbachol stimulates PI hydrolysis in right and left murine atria as well as in murine ventricule and dissociated embryonic chick heart cells. Muscarinic stimulation of PI hydrolysis is markedly attenuated in calcium-free medium, is not antagonized by isoproterenol, occurs after a latency of several minutes, and is half-maximally activated by approximately 10 microM carbachol. In contrast, muscarinic inhibition of cyclic AMP accumulation in the same preparations is calcium independent, is opposed by the effect of isoproterenol, is maximal in minutes, and is half-maximally activated by 0.1 microM carbachol. These differences demonstrate that the two muscarinic receptor-mediated events are probably unrelated and independent responses. The concentration of carbachol that causes half-maximal activation of PI hydrolysis is almost identical to that causing half muscarinic receptor occupancy as assessed by 3H-labeled (-)-quinuclidinyl benzilate binding. Thus activation of the PI response by carbachol appears to be closely linked to receptor occupancy, whereas cyclase inhibition may occur when only a small percentage of receptors are occupied. The possible role of the PI response in generating intracellular signals such as arachidonic acid release, cyclic GMP synthesis, or C-kinase activation is discussed.     

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.     

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.     

Maturation of mammalian myocardial muscarinic cholinergic receptors

Biochemical alterations of the cardiac muscarinic binding sites have not been correlated with physiological observations. The development of responsiveness to acetylcholine in the fetal mouse heart occurs during the third trimester. We tested the hypothesis that the altered physiological response was related to changes in the muscarinic cholinergic binding site assayed by using the potent antagonist [³H]-quinuclidinyl benzilate (QNB). Analysis of saturation isotherms of specific (atropine displaceable) [³H]-QNB binding gave an apparent dissociation constant (K_Dapp) of 27 ± 3 (SEM) pM for adult heart. Receptor density increased significantly during the third trimester of pregnancy to 48% of adult, while the K_Dapp did not change. The increase in receptor density parallels the demonstrated increased responsiveness to acetylcholine.     

Muscarinic acetylcholine receptors in the brain of the zebrafish (Danio rerio) measured by radioligand binding techniques

Muscarinic acetylcholine receptors (mAChRs) play a role in learning, memory and behavior in vertebrate animals. We measured the muscarinic cholinergic receptor levels in extracts from zebrafish (Danio rerio) brain by radioligand binding techniques. Saturation binding experiments with the radioligand [3H]-quinuclidinyl benzilate (QNB) were used to determine receptor number and relative affinity for several agonists and antagonists. Affinity at zebrafish brain receptors was relatively high with a K(d) of 40 +/- 5 pM. The number of receptors, represented by Bmax, was 63 +/- 16 fmol/mg protein. Oxotremorine and carbachol, agonists at muscarinic acetylcholine receptors, bound with displacement curves indicating multiple binding sites. In addition, oxotremorine bound with a higher affinity than did carbachol. The antagonist potency profile at zebrafish receptors in brain was determined to be atropine>pirenzipine>p-fluoro-hexahydro-sila-difenidol>otenzepad. The results obtained with zebrafish brain compare favorably to those found in insect, fish and mammalian species. Taken together, the binding results and favorable comparisons to mammalian systems indicate that zebrafish may provide a useful model organism for evaluating the role of cholinergic systems in learning, memory and behavior.     

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

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

Changes in brain muscarinic acetylcholine receptors and behavioral responses to atropine and apomorphine in chronic atropine-treated rats

Chronic blockade of cholinergic transmission with atropine resulted in a decrease in atropine-induced activity in the rats, whereas apomorphine - induced locomotion was enhanced. Maximal binding of ³H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, to homogenate of cerebral cortex, striatum and hippocampus was significantly higher in chronic atropine-treated rats than in control animals. No difference was observed in K_D value of the specific ³H-QNB binding or in ID₅₀ value of oxotremorine in inhibiting ³H-QNB binding. No change in the specific binding of ³H-spiroperidol, a dopaminergic antagonist, was observed in those three regions of brains of chronic atropine-treated rats when it was compared with that of control animals. The role of brain muscarinic acetylcholine receptors in behavioral responses is discussed relating an effect of dopaminergic neurons on cholinergic activities.