MUSCARINIC RECEPTOR-MEDIATED INHIBITION OF MITOGENESIS VIA A PROTEIN-KINASE C-INDEPENDENT MECHANISM IN M1-TRANSFECTED A9 L-CELLS

Previous studies have shown that activation of various PI-coupled rece ptors stimulates DNA synthesis in some cells, and inhibits DNA synthes is in others. In order to study this effect further, we measured carba chol-mediated [H-3]thymidine incorporation in m1-transfected A9 L cell s and in m1-transfected CHO cells, and found that carbachol profoundly inhibited DNA synthesis in both cell lines. This carbachol response w as observed whether the cells were grown in the presence or in the abs ence of serum. The half-maximal value for carbachol-mediated inhibitio n of [H-3]thymidine incorporation was 5.73 +/- 1.15 mu M in m1-transfe cted A9 L cells. Pre-treatment of m1-transfected A9 L cells with the p rotein kinase C inhibitors staurosporine, sphingosine or phorbol 12-my ristate 13-acetate-induced down-regulation did not prevent the carbach ol inhibition of DNA synthesis, thereby demonstrating that this effect is not mediated via protein kinase C. Inhibition of [H-3-]thymidine i ncorporation was significant at 2 h, but not at 1 h of carbachol treat ment. Recovery of [H-3]thymidine incorporation following carbachol act ivation was also studied by blocking the receptor with atropine follow ing carbachol stimulation. Following this treatment, cells needed at l east 3 h to recover normal [H-3]thymidine incorporation. Taken togethe r, the kinetics of this response suggest that it is not mediated direc tly by a second messenger which typically has much more rapid kinetics .