MELATONIN BIOSYNTHESIS IN PHOTORECEPTOR-ENRICHED CHICK RETINAL CELL-CULTURES - ROLE OF CYCLIC-AMP IN THE K-EVOKED, CA2+-DEPENDENT INDUCTIONOF SEROTONIN N-ACETYLTRANSFERASE ACTIVITY()

The roles of cyclic AMP and calcium in the regulation of serotonin N-a cetyltransferase (NAT) activity were studied in low density monolayer cultures of chick retinal photoreceptors and neurons. Photoreceptor-en riched retinal cell cultures were prepared from embryonic day 6 retina s and cultured for 6 days. NAT activity in these cultures could be ind uced by treatment with cyclic AMP protagonists, 8Br-cyclic AMP, forsko lin, and 3-isobutyl-1-methylxanthine (IBMX), or by treatment with depo larizing concentrations of extracellular K+. The stimulatory effect of K+, which involves Ca2+ influx through dihydropyridine-sensitive chan nels, was mediated at least in part by cyclic AMP, as indicated by the following observations. Depolarizing concentrations of K+ stimulated the formation of cyclic AMP, and the stimulatory effects of K+ on both cyclic AMP formation and on NAT activity were synergistically potenti ated by the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1 -methylxanthine (IBMX). MDL 12,330A, a putative adenylate cyclase inhi bitor, inhibited K+-evoked cyclic AMP accumulation and induction of NA T activity over the identical concentration range. In contrast, MDL 12 ,300A failed to inhibit the induction of NAT elicited by 8Br-cyclic AM P. H-89, an inhibitor of cyclic AMP-dependent protein kinase, antagoni zed the induction of NAT activity by either forskolin or K+ with equal potency for both stimuli. These results suggest that cyclic AMP plays an essential role in the induction of NAT activity that occurs as a c onsequence of membrane depolarization. Cyclic AMP and Ca2+ may also in teract at a step distal to adenylate cyclase. In cells treated with a low concentration of 8Br-cyclic AMP, increasing intracellular Ca2+ wit h K+ (20 or 45 mM) or the calcium ionophore A23187 elicited a stimulat ion of NAT activity that was slightly, but consistently greater than t hat expected from the additive effects of each treatment alone. The Ca 2+ channel agonist Bay K 8644, however, did not potentiate the effect of 8Br-cyclic AMP. In contrast to the interaction of calcium elevating agents and 8Br-cyclic AMP, Bay K 8644 or depolarizing concentrations of K+ elicited a large, synergistic potentiation of NAT activity induc ed by treatments that raise the levels of endogenous cyclic AMP, forsk olin and IBMX. These results indicate that the primary role of Ca2+ in flux in the induction of NAT activity in photoreceptor cells is to sti mulate cyclic AMP formation, but a secondary role may be to potentiate the effect of cyclic AMP at a step distal to adenylate cyclase.