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 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 microM 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 approximately 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, and 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.
Alonso-Gómez
A L
AL
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322-3090, USA.
Iuvone
P M
PM
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D002642:Chick Embryo; D000242:Cyclic AMP / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008550:Melatonin / Q000096:biosynthesis*; D010786:Photoreceptor Cells / Q000201:enzymology*; D012160:Retina / Q000201:enzymology*; D014316:Tritium
D014316:Tritium; D003513:Cycloheximide; D000242:Cyclic AMP; D001191:Arylamine N-Acetyltransferase; D007930:Leucine; D008550:Melatonin; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine