MELATONIN BIOSYNTHESIS IN CULTURED CHICK RETINAL PHOTORECEPTOR CELLS - CALCIUM AND CYCLIC-AMP PROTECT SEROTONIN N-ACETYLTRANSFERASE FROM INACTIVATION IN CYCLOHEXIMIDE-TREATED CELLS

The aim of the present study was to examine the roles of membrane depo larization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N-acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 mu M forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivat ion) was determined. After induction, in the presence of cycloheximide , NAT activity declined with a half-life of similar to 30 min. The rat e of inactivation was greatly reduced when depolarizing concentrations of K+, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-i sobutyl-1-methylxanthine were added together with cycloheximide. The a pparent increase in NAT stability caused by K+ was abolished by additi on of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca2+ influx through dihydropyridine-sensitive channels . MDL-12330A, an inhibitor of K+-stimulated cyclic AMP formation, bloc ked the effect of depolarizing concentrations of K+. This result sugge sts that the effect of Ca2+ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolariz ation-evoked Ca2+ influx and cyclic AMP formation have two roles in th e regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein requir ed for NAT activity. Second, they increase the half-life of the enzyme , presumably by regulating the turnover of existing enzyme molecules.