INTERACTION OF A RADIOLABELED AGONIST WITH CARDIAC MUSCARINIC CHOLINERGIC RECEPTORS

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  • 1 January 1983
    • journal article
    • research article
    • Vol. 227  (3) , 570-577
Abstract
The interaction of a radiolabeled muscarinic cholinergic receptor agonist, [methyl-3H]oxotremorine acetate ([3H]OXO), with a washed membrane preparation derived from rat heart, was studied. In binding assays at 4.degree. C, the rate constants for association and dissociation of [3H]OXO were 2 .times. 107/M per min and 5 .times. 10-3/min, respectively. Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of .apprx. 300 pM. The density of [3H]OXO binding sites (90-100 fmol/mg of protein) was .apprx. 75% of that determined for the radiolabeled receptor antagoinst [3H]quinuclidinyl benzilate. Both muscarinic receptor agonists and antagonists inhibited the binding of [3H]OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of [3H]OXO. This effect was on the maximum binding (Bmax) of [3H]OXO with no change occurring in the Kd; the order of potency of 5 nucleotides was guanosine 5''-O-(3-thio-triphosphate) > guanylyl-5''-imidodiphosphate > GTP .gtoreq. GDP > GMP. The [3H]OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization, i.e., membrane receptors that were prelabeled with [3H]OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, [3H]OXO-labeled complex resulted in dissociation of [3H]OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited [3H]OXO binding by 85% with no change in the Kd of [3H]OXO, and with no effect on [3H]quinuclidinyl benzilate binding. No Na+-selective effect on [3H]OXO binding was observed, since a series of monovalent cations, as well as N-CH3-D-glucamine, inhibited [3H]OXO binding with similar concentration dependence.