Properties of the muscarinic cholinergic receptors in rat atrium.

Abstract

1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3′-quinuclidinyl benzilate ([3H]-QNB), a potent muscarinic antagonist. 2. The specific [3H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [3H]-QNB binding sites. However, α- and β-adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [3H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (KD) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [3H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [3H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [3H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.