Characterization of muscarinic cholinergic receptors in intact myocardial cells in vitro.

Muscarinic acetylcholine receptors (mAChR) were studied on heart cells grown in culture by the radioligand binding technique. We used [3H]n-methyl-scopolamine to monitor the level of receptors on intact cardiocytes. The number of mAChR was very low during the first days in culture (23 fmol/dish). It increased gradually until it reached a plateau on the 4th day (180 fmol/dish), where it remained for 1-2 weeks. To determine whether contractile activity affected the level or affinity of mAChR, the cardiocytes were exposed to agents that stimulate or arrest the heart beat. Treatment with triiodothyronine (T3, 10-90 nM) for 48 hr caused a reduction in the level of the receptors by 20-30% without changing significantly the affinity of the receptors. Similarly, electrical stimulation caused a reduction in the level of the receptors by 30-40%, without a significant influence on creatine kinase activity. When the myocardial cells were treated with Ca-channel blocker such as metoxyverapamil (D600) (10-30 micrograms/mL) or diltiazem (10-25 micrograms/mL) the level of the receptors was also reduced by 30-40%. The reduction in the receptor binding sites was accompanied by an increase in Kd from 0.8 to 3.2 nM in D600-treated cells, whereas there was no significant change in the radioligand affinity after application of diltiazem. Treatment with D600 or T3 together with cycloheximide showed that under these experimental conditions the rate of receptor degradation was accelerated. The half-life of the receptors in the control was 27 hr, whereas the half-lives of T3 and D600 were 15 and 18 hr, respectively. It is concluded that regulation of the amount of cholinergic receptors occurs at the level of receptor breakdown, and simple linkage does not exist between the rate of cardiac contractions and the number of mAChR.