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 receptors stimulates DNA synthesis in some cells, and inhibits DNA synthesis in others. in order to study this effect further, we measured carbachol-mediated [ 3H]thymidine incorporation in m1-transfected A9 L cells and in m1-transfected CHO cells, and found that carbachol profoundly inhibited DNA synthesis in both cell lines. This carbachol response was observed whether the cells were grown in the presence or in the absence of serum. The half-maximal value for carbachol-mediated inhibition of [ 3H]thymidine incorporation was 5.73 ± 1.15 gmM in m1-transfected A9 L cells. Pre-treatment of m1-transfected A9 L cells with the protein kinase C inhibitors staurosporine, sphigosine or phorbol 12-myristate 13-acetate-induced down-regulation did not prevent the carbachol inhibition of DNA synthesis, thereby demonstrating that this effect is not mediated via protein kinase C. Inhibition of [ 3H]thymidine incorporation was significant at 2 h, but not at 1 h of carbachol treatment. Recovery of [ 3H]thymidine incorporation following carbachol activation was also studied by blocking the receptor with atropine following carbachol stimulation. Following this treatment, cells needed at least 3 h to recover normal [ 3H]thymidine incorporation. Taken together, the kinetics of this response suggest that it is not medited directly by a second messenger which typically has much more rapid kinetics.