Cardiac muscarinic cholinergic receptors. Biochemical identification and characterization

The parasympathetic nervous system modulates heart rate and myocardial contractility. We have labeled the parasympathetic muscarinic cholinergic receptors in rabbit, rat, and guinea pig hearts using the specific high affinity ligand, [³H]quinuclidinyl benzilate ([³H]QNB). Specific [³H]QNB binding in rabbit heart homogenates was defined as the difference in binding between the absence and presence of atropine (1 μM). At all [³H]QNB concentrations studied, specific binding increased linearly with tissue concentration in the range of 0.025 to 0.5 mg of protein/ml. Analyses of saturation isotherms at progressively decreasing tissue concentrations gave an apparent dissociation constant, K(D)app, of 27.1 PM which was similar to the K(D)app for rat brain homogenates (20.0 pM). The rate constants at 37°C for formation and dissociation of the QNB/receptor complex in rabbit heart were 1.03 x 10⁹ M^-1 min^-1 and 2.45 x 10^-2 min^-1, respectively. The mean value for the dissociation constant from the pairs of rate constants (K(D) = k_-1/k₊₁ = 26.8 pM) was similar to the value determined from saturation isotherms. Stereospecificity of the receptor was demonstrated in experiments in which [³H]QNB binding was inhibited by dexetimide in the nanomolar range (K(i)= 0.69 nM) and by levetimide, its stereoisomer in the micromolar range (K(i) = 1.45 μM). Values of K(i) (nanomolar) for muscarinic cholinergic drugs that inhibited specific binding were: atropine 1.05, oxotremorine 144, acetylcholine 490, carbamylcholine 528. The nicotinic cholinergic agents α-bungarotoxin and nicotine were ineffective in displacing [³H]QNB binding at concentrations of 5 and 100 μM, respectively. The antiarrhythmic agents quinidine, lidocaine, and procainamide were also inhibitory with K(i) values (micromolar) of 2, 18, and 48, respectively. The tricyclic antidepressant imipramine, also was an inhibitor with a value for K(i) of 0.17 μM. Ouabain, propranolol, phentolamine, dopamine, and norepinephrine were ineffective in inhibiting QNB binding at 100 μM concentrations. In the rabbit heart, receptor densities (picomoles per g of protein) were: whole heart 57.2, left atrium 302, right atrium 200, ventricular septum 58, right ventricle 53, and left ventricle 37. We conclude that: a population of specific, saturable, high affinity, muscarinic cholinergic receptors in the mammalian heart has been identified and characterized; these binding sites appear to be similar to muscarinic cholinergic receptors in the brain; the regional distribution of these receptors is consistent with classical physiological observations and with the relative distribution of acetylcholine and choline acetyltransferase within the heart; cardiotonic agents, tricyclic antidepressants, and antipsychotic neuroleptics can interact with these myocardial cholinergic receptors.