Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.     

Isolated rat gastric parietal cells: Cholinergic response and pharmacology

Isolated, partially purified or enriched rat gastric muscosal parietal cells were shown to respond to carbamycholine (EC₅₀ = 2 μM) and other muscarinic cholinergic agonists as measured by an increased accumulation of ¹⁴C-aminopyrine, an indirect measure of acid secretion. The secretory response to carbamylcholine was shown to be inhibited stereoselectively and reversibly by nanomolar concentrations of muscarinic cholinergic antagonists. Non-muscarinic antagonists, including cimetidine, were either ineffective or very weak inhibitors. The affinity constants calculated for cholinergic antagonist inhibition of ¹⁴C-aminopyrine accumulation induced by carbamylcholine were similar to those previously calculated from direct binding studies on purified parietal cell particulate fractions using ³H-QNB (1). These studies support the existence of specific parietal cell muscarinic cholinergic receptors with which the natural secretagogue acetylcholine interacts to regulate gastric acid secretion.     

Agonist-specific reverse regulation of muscarinic receptors by transition metal ions and guanine nucleotides

Transition metal ions, e.g. Mn²⁺, Ni²⁺ and Co²⁺ enhance $\text{in vitro}$ agonist binding to muscarinic receptors in mouse cortex or hippocampus. This effect arises mainly from the conversion of low to high affinity binding sites. Binding properties of antagonists in these brain areas, as well as those of both agonists and antagonists of medulla-pons muscarinic receptors, are insensitive to these ions. The induced interconversion can be reversed by either of the following procedures: (i) removal of the ions; (ii) thermal exposure; (iii) addition of micromolar concentrations of guanine nucleotides.     

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.     

Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N-Ethylmaleimide

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.     

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.     

Interactions between the muscarinic receptors, sodium channels, and guanine nucleotide-binding protein(s) in rat atria

The effects of the voltage-sensitive sodium channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors were studied in homogenates of rat atria. Also studied were the effects of muscarinic ligands on the binding of tritium-labeled batrachotoxin ([3H]BTX) to the same preparation. BTX (1 microM), which induces an open state in sodium channels, enhanced the affinity of binding of several agonists to the muscarinic receptors. Analysis of the data indicated that the effect of BTX was to increase the affinity of the agonists toward the high-affinity sites. Binding of antagonists was not affected by BTX. At higher concentrations of toxin, the density of the high affinity muscarinic sites was also affected. The binding of agonists (but not of antagonists) to muscarinic receptors in turn enhanced the specific binding of [3H]BTX to sodium channels. These effects on the muscarinic receptors and on the sodium channels were inhibited in the presence of Gpp(NH)p at concentrations lower than those bringing about conversion of binding sites from the high affinity to the low affinity conformation. On the basis of these findings we suggest that the opening of sodium channels and the binding of agonists to muscarinic receptors in rat atrial membranes are coupled events which are mediated by guanine nucleotide-binding protein(s). Such a hypothesis is consistent with previously proposed models for signal transduction in the membrane.     

Binding of agonists and antagonists to muscarinic receptors

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [³H] acetylcholine. Mechanisms that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.     

Muscarinic receptor binding in rat brain using the agonist, [3H]cis methyldioxolane

Muscarinic receptor binding was measured in rat forebrain preparations using the muscarinic agonist, [³H]cis methyldioxolane ([³H]CD). The results of equilibrium binding studies using [³H]CD concentrations between 0.5–64 nM showed that [³H]CD binding did not saturate in this concentration range, although the binding isotherm was concave downward. Nonlinear regression analysis of the binding data revealed the presence of two populations of muscarinic receptors having dissociation constants of 1.83 and 123 nM and binding capacities of 85 and 1320 fmol/mg protein, respectively. Competitive inhibition experiments showed that [³H]CD binding was readily displaced by several muscarinic agonists and antagonists. The stereospecificity of [³H]CD binding was demonstrated in competitive inhibition experiments using the stereoisomers of benzetimide and acetyl-β-methylcholine. Dexetimide was 10,000 times more potent than levetimide and l-acetyl-β-methylcholine was 520 times more potent than d-acetyl-β-methylcholine. A variety of nonmuscarinic cholinergic drugs were not effective at inhibiting [³H]CD binding at a concentration of 10 μM.     

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase

The binding of [³H](?)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (?)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10^?9 – 10^?8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10^?6M).     

Muscarinic acetylcholine receptor in chick limb bud during morphogenesis

Summary In the chick embryo a cholinesterase activity appears in various organ anlagen which has been correlated with morphogenetic movements (Drews 1975). The cholinesterase activity is present in the mesenchyme of the limb bud during aggregation of the central chondrogenic core. In the present study binding of tritium labelled quinuclidinyl benzilate ((³H)QNB), a muscarinic antagonist, to homogenates of chick limb buds was investigated by a filtration assay. In the homogenate of limb buds at Stage 24 specific binding of (³H)QNB was demonstrated. Determination of binding constants and inhibition of binding by agonists and antagonists was studied at Stage 25/26. Specific binding was defined by the difference in binding in the absence and presence of atropine (1 M). Specific binding of (³H)QNB reflected a muscarinic receptor. The K_d in two experiments was 0.11 nM and 0.16 nM, the binding capacity was 15.7 fmol (³H)QNB/mg protein and 12.0 fmol (³H)QNB/mg protein, respectively. Data on displacement of specific bound (³H)QNB by various nicotinic and muscarinic ligands confirmed the muscarinic nature of the receptor. Muscarinic ligands inhibited the (³H) QNB binding, whereas nicotinic ligands caused no inhibition at pharmacological concentrations. I conclude that a specific muscarinic acetylcholine receptor is part of the cholinergic system whose presence is indicated by cholinesterase activity in the chondrogenic core of the limb bud during morphogenesis.     

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.     

Beta-adrenergic receptor in the brain: comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates.

The properties of specific ³H-dihydroalprenolol binding sites resemble the properties of the beta-receptor regulating hormone-sensitive adenylyl cyclase activity in an homogenate of rabbit cerebellum. The rabbit cerebellum has 5 to 6 pmole per gm (wet weight) of high affinity (K_D=1.3 nM) specific binding sites for ³H-dihydroalprenolol. the interaction of several beta-adrenergic agonists and antagonists with the specific binding sites is rapid, reversible, and demonstrates stereospecificity which parallels the properties of the beta receptor. Beta-adrenergic agonists show a similar potency as agonists upon adenylyl cyclase activity and as inhibitors of ³H-dihydroalprenolol binding: i.e. l-isoproterenol > l-epinephrine > l-norepinephrine (suggesting a beta₂ adrenergic receptor). The binding affinities of several beta-adrenergic agonists and antagonists for the specific binding sites approximate the affinities of these compounds for the stimulation of adenylyl cyclase. Thus, the ³H-dihydroalprenolol binding sites have properties similar to the beta-adrenergic receptor regulating adenylyl cyclase activity in a rabbit cerebellar homogenate.     

Photoaffinity labeling of specific muscarinic antagonist binding sites of brain: I. Preliminary studies using two p-azidophenylacetate esters of tropine

Possible photoaffinity probes for muscarinic acetylcholine receptors have been explored for the first time: Specific [³H]-quinuclidinyl benzylate binding sites of several fractions from rat brain can be irreversibly inactivated by photoaffinity labeling with two p-azidophenylacetate esters of tropine. Inactivation of these sites depends on formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes; it is dependent on ligand concentration and length of photolysis. Atropine and oxotremorine, but not d-tubocurarine, afford protection against photoinactivation.These findings suggest the utility of these and related azido derivatives as potent, selective photoaffinity ligands directed against binding sites for muscarinic antagonists.     

A characterization of alpha-bungarotoxin binding in the brain of the horseshoe crab, Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin to membrane fragments prepared from Limulus brain tissue has been investigated. Toxin binding approaches saturation in the range of 30 to 40 nm, with maximum binding of 2 to 6 pmol/mg of protein. The saturation kinetics and the rate of displacement of bound toxin are consistent with multiple toxin binding sites. Pharmacological studies show that binding is inhibited by both cholinergic agonists and antagonists, I₅₀′s for inhibition by d-tubocurarine, nicotine, decamethonium, carbachol, and atropine are 2 × 10^?6, 7 × 10^?6, 2 × 10^?5, 6 × 10^?4, and 3 × 10^?4m, respectively. Nicotinic ligands inhibited binding much more effectively than muscarinic ligands. Toxin binding activity was solubilized with Triton X-100. Velocity sedimentation analysis of the solubilized activity revealed three separate components. Seventy to eighty percent of the binding activity had a sedimentation coefficient of 8.6 S. The remaining activity was composed of two components with sedimentation coefficients of 15.1 and 17 S.     

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.     

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.     

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M₁ receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [³H]N-methylscopolamine binding to cloned muscarinic M₁ and M₄ receptors, and reduced binding to M₅ subtype with lower affinity, while they reversibly inhibited the binding of [³H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M₁ receptors at a site distinct from the classical one, and to have affinity for some -adrenoceptors.     

M1 muscarinic antagonists interact with sigma recognition sites.

The M1-selective muscarinic antagonists aprophen, caramiphen, carbetapentane, 2-DAEX, dicyclomine, hexahydrosiladifenidol, iodocaramiphen, nitrocaramiphen, oxybutynin and trihexyphenidyl potently inhibited binding to sigma sites in brain. Both basic ester and non-ester structural type compounds which exhibit affinity for the muscarinic receptor also demonstrated affinity for the sigma site, while the classical antimuscarinic agents atropine and QNB, and the tricyclic pirenzepine, were ineffective in binding to this site. We also observed a significant correlation between the Ki values for sigma compounds to inhibit [3H]pirenzepine binding and their IC50 values to inhibit carbachol-stimulated phosphoinositide turnover. These observations may aid in elucidating the relationship of sigma binding to inhibition of phosphoinositide turnover stimulated by cholinergic agonists.     

Muscarinic acetylcholine receptor: thermodynamic analysis of the interaction of agonists and antagonists

The thermodynamic parameters of the interaction of agonists and antagonists with heart and brain muscarinic receptors were determined. The binding of quinuclidinyl [3H]benzilate and the inhibition of quinuclidinyl benzilate (QNB) binding by agonists and antagonists were examined at temperatures between 2 degrees C and 27 degrees C. The density of specific binding sites and the relative proportions of high- and low-affinity binding components of drugs were unaffected by the temperature changes. The binding of atropine was entropy driven in brain and heart membranes. In contrast, net values of these thermodynamic parameters for QNB binding and for the high-affinity binding component of pirenzepine to brain membranes were decreased with the enhancement of the temperature. The low-affinity binding component of the agonists carbachol, oxotremorine and pilocarpine was enthalpy driven. Their high-affinity binding component was entropy driven at 2 degrees C and became enthalpy driven when the incubation temperature was increased. The guanine nucleotide Gpp[NH]p partly prevented the temperature-dependent decrease of net entropy and enthalpy values. Considering that the net changes of thermodynamic parameters are relevant of the interactions between the ligand, the receptor protein and the adjoining membranous molecules, a three-state conformational model is proposed for the muscarinic receptor protein. The receptor selectivity is reappreciated owing to these three states of the receptor protein and the different components of the muscarinic receptor complexes.