Muscarinic M2 receptor synthesis: study of receptor turnover with propylbenzilylcholine mustard

We have investigated the rate and the functional responsiveness of the newly synthesised M₂ muscarinic receptors in HEL 299 cells following propylbenzilylcholine mustard treatment at 37°C. Propylbenzilylcholine mustard induced a dose-dependent loss of the hydrophilic ligand [³H]N-methylscopolamine binding sites with 80% inactivation at 0.1 μM. The rate of muscarinic receptor synthesis in these cells, estimated from wash-out experiments following propylbenzilylcholine mustard treatment, was very slow and returned to control values after 36 h of propylbenzilylcholine mustard removal. The recovery of muscarinic receptors was blocked by the cycloheximide pre-treatment, indicating the synthetic pathway for the new receptors. In control cells as well as in cells treated with propylbenzilylcholine mustard and allowed to recover for 12 h, carbachol still inhibited forskolin-induced cAMP accumulation. These results show that (i) the rate of M₂ muscarinic receptor synthesis is slow (ii) the recovery of receptors is mainly through increased synthesis and (iii) the newly synthesised receptors retain their full functional activity.