INVOLVEMENT OF PROTEIN-KINASE-A IN THE SUBJECTIVE NOCTURNAL RISE OF MELATONIN RELEASE BY CHICK PINEAL CELLS IN CONSTANT DARKNESS

Melatonin release by chick cultured pineal cells increases during the dark periods and decreases during the light periods under light-dark c ycles, and this rhythmic secretion is maintained under constant condit ions with a period of almost 24 hr. The mechanisms by which the circad ian oscillator drives the melatonin rhythm under constant conditions h ave not been elucidated enough. We examined the possibility that cycli c AMP-dependent protein kinase A is involved in the subjective nocturn al increase in melatonin release by chick pineal cells cultured under constant darkness. The subjective nocturnal increase of melatonin rele ase was suppressed dose dependently by H8 (protein kinase inhibitor) a nd H89 (specific protein kinase A inhibitor), but not by calphostin C (specific protein kinase C inhibitor) in static cell cultures. In a ce ll perfusion experiment, 9 hr pulses of H8 and H89 starting at ZT 9 (C T 11.2) hr suppressed the subjective nocturnal increase in melatonin r hythm in dose-dependent manner without causing a phase shift. An intra cellular Ca2+ chelator, -aminophenoxy)ethyleneglycol-N,N,N',N'-tetraac etic acid tetraacetoxymethyl ester (BAPTA-AM), and extracellular Ca2chelators, -aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tet rapotassium salt hydrate (BAPTA) and ethyleneglycol-bis(beta-aminoethy l ether)-N,N,N',N'-tetraacetic acid (EGTA), suppressed both the subjec tive nocturnal increases in melatonin release and cAMP levels dose dep endently. This direct evidence strongly supports the hypothesis that c AMP-dependent protein kinase A may be involved in the subjective noctu rnal increase in melatonin release by chick pineal cells and that intr acellular Ca2+ plays an important role in pineal adenylate cyclase act ivation.