Melatonin biosynthesis in photoreceptor-enriched chick retinal cell cultures: role of cyclic amp in the K +-evoked, ca 2+ -dependent induction of serotonin n-acetyltransferase activity

Abstract

The roles of cyclic AMP and calcium in the regulation of serotonin N-acetyltransferase (NAT) activity were studied in low density monolayer cultures of chick retinal photoreceptors and neurons. Photoreceptor-enriched recinal cell cultures were prepared from embryonic day 6 retinas and cultured for 6 days. NAT activity in these cultures could be induced by treatment with cyclic AMP protagonists, 8Br-cyclic AMP, forskolin, and 3-isobutyl-l-methylxanthine (IBMX), or by treatment with depolarizing concentrations of extracellular K +. The stimulatory effect of K +, which involves Ca 2+ influx through dihydropyridine-sensitive channels, 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 potentiated by the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX). MDL 12,330A, a putative adenylate cyclase inhibitor, inhibited K +-evoked cyclic AMP accumulation and induction of NAT activity over the identical concentration range. In contrast, MDL 12,300A failed to inhibit the induction of NAT elicited by 8Br-cyclic AMP. H-89, an inhibitor of cyclic AMP-dependent protein kinase, antagonized 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 consequence of membrane depolarization. Cyclic AMP and Ca 2+ may also interact at a step distal to adenylate cyclase. In cells treated with a low concentration of 8Br-cyclic AMP, increasing intracellular Ca 2+ with K + (20 or 45 mM) or the calcium ionophore A23187 elicited a stimulation of NAT activity that was slightly, but consistently greater than that 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 induced by treatments that raise the levels of endogenous cyclic AMP, forskolin and IBMX. These results indicate that the primary role of Ca 2+ influx in the induction of NAT activity in photoreceptor cells is to stimulate cyclic AMP formation, but a secondary role may be to potentiate the effect of cyclic AMP at a step distal to adenylate cyclase.