Muscarinic receptors: biochemical studies.

The characterization of pharmacological receptors in subcellular preparations has, to date, relied mainly on observations of the binding properties of radioactively labelled ligands. Minimal criteria for receptor-specific binding are that a component of binding should saturate with increasing concentrations of the ligand and that only drugs with the appropriate pharmacological specificity, and at concentrations at which they are pharmacologically active, should displace this binding component. These criteria have provided the basis for the biochemical identification of numerous classes of receptors (see, for example, Cuatrecasas, 1974; Snyder & Bennett, 1976; Fewtrell, 1976), but in relatively few cases have quantitative correlations between the directly measured binding parameters and pharmacologically derived values been demonstrated. Difficulties arise in the determination of receptor-binding properties of drugs from dose-response curves in whole tissue, because of the intervention of unknown and probably non-linearly coupled steps between the binding of a drug to a receptor and production of the measured response. The pharmacologically determined parameters have been derived, in general, on the assumption that a drug interacts with a uniform population of binding sites and also, in the case of null methods (Arunlakshana & Schild, 1959; Furchgott, 1966) that equal occupancy of a receptor results in an equal response. These assumptions may not be correct, and thus the pharmacologically determined binding constants could be in error. It is also possible that the binding properties of receptors in tissue homogenates do not reflect the binding properties in the whole tissue. These uncertainties underline the requirement for both determination of the detailed binding properties of the receptor and for the characterization of the processes linking occupancy of the receptor by a drug to the production of the physiological response. The binding properties of muscarinic receptors in subcellular fractions have been investigated in considerable detail by using a variety of radioactively labelled antagonists (see, for example, Paton & Rang, 1965; Farrow & O’Brien, 1973; Fewtrell & Rang, 1973; Burgen et al., 1974a,b; Beld & Ariens, 1974; Yamamura & Snyder, 1974a,b; Birdsall et al., 1976; Hulme et al., 1976; Wells et al., 1977) and agonists (Birdsall et al., 1976; Hulme et al., 1976). In solutions of a physiological ionic composition, muscarinic antagonists bind to a uniform population of receptor sites on the membrane (Birdsall et al., 1976; Hulme et a/., 1976; Wells et al., 1977), whereas muscarinic agonists exhibit a dispersion in their affinity constants for the same receptor sites. The binding properties of muscarinic receptors in membrane fractions from rat and guinea-pig cortex (Burgen et al., 1974b), guinea-pig ileum (Burgen et al., 1974~ ; Yamamura & Snyder, 1974b), neuroblastoma cells (Strange et al., 1977; P. G. Strange, N. J. M. Birdsall & A. S. V. Burgen, unpublished work) and in intact smooth muscle (Young, 1974; Taylor ef al., 1975) are the same, suggesting that the heterogeneity in the agonist-binding sites is not an artifact of homogenization. Experiments have shown that the heterogeneity does not result from receptor desensitization or from negatively co-operative interactions between agonist-binding sites (Birdsall et al., 1976; Hulme et al., 1976). This pattern, in which the binding of antagonists to a receptor is simple, whereas that of agonists is complex and not described by a single affinity constant, also seems to be characteristic of opiate receptors (Birdsall et al., 1976; Hulme, 1977), nicotinic receptors (Michaelson et a/ . , 1974) and glycine receptors (Young & Snyder, 1973). To a first approximation, all the results from binding studies of muscarinic receptors can be fitted to a model in which there are two classes of agonist-binding sites, one having a high affinity (KH) and the other a low affinity (KL) for agonists. The binding of agonist and antagonists is competitive and mutually exclusive, and the antagonist-binding Sites, linked to the two classes of agonist-binding site, have the same affinity for a given