Irreversible binding of acetylethylcholine mustard to cardiac cholinergic muscarinic receptors.

The interaction of acetylethylcholine mustard (Aech-M) with cardiac muscarinic receptors was studied with radioligand binding techniques and with isolated beating atria. Aech-M and acetylcholine competed about equally for (-)-[3H]quinuclidinyl benzilate (QNB)-binding sites and 5'-guanylylimidodiphosphate reduced the ability of both compounds to compete by 30-fold. Pretreatment of cardiac membranes with 0.25 microM Aech-M, followed by washing, reduced the [3H]QNB binding capacity by 60% without changing the ligand affinity of the receptors left. In the same membranes, a similar fraction of receptors was lost as measured by [3H]N-methyoscopolamine, but a greater fraction was lost when measured by [3H]oxotremorine-M. The loss of [3H]QNB binding capacity was dose, time, and temperature dependent, blocked by atropine and carbachol, and modulated by several guanine nucleotides but not affected by membrane pretreatment with several group-selective reagents. Superfusion of spontaneously beating atria with Aech-M (100 microM) initially reduced the beating rate which returned to control values by 17 min. Atropine blocked the initial reduction in beating rate. In contrast, both carbachol and acetylcholine produced sustained decreases in the beating rate. After pretreatment of atria for 30 min with 100 microM Aech-M alone or 10 microM Aech-M plus 10 microM edrophonium followed by washing, the EC50 value for carbachol inhibition of the beating rate was increased 15.8- and 10.3-fold, respectively, with no change in the ability of isoproterenol to increase the spontaneous beating rate. In addition, there was a 46-47% and a 37-41% reduction in the binding capacity of both [3H]QNB and [3H]oxotremorinel-M in the 100 and 10 microM Aech-M-pretreated atria, respectively. The data indicated that Aech-M is a muscarinic agonist which appears to irreversibly bind to the muscarinic receptor.