Melatonin Biosynthesis in Cultured Chick Retinal Photoreceptor Cells: Calcium and Cyclic AMP Protect Serotonin N‐Acetyltransferase from Inactivation in Cycloheximide‐Treated Cells

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Abstract
The aim of the present study was to examine the roles of membrane depolarization, 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 µM forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half‐life of ∼30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K+, forskolin, 8‐bromoadenosine 3′,5′‐cyclic monophosphate, or 3‐isobutyl‐1‐methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K+ was abolished by addition 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, blocked the effect of depolarizing concentrations of K+. This result suggests that the effect of Ca2+ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization‐evoked Ca2+ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half‐life of the enzyme, presumably by regulating the turnover of existing enzyme molecules.

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