Analysis of circadian clock mechanism in birds and mammals.

鸡松果体细胞在光-暗周期中，暗期褪黑素分泌增加，光期褪黑素分泌减少，这种节律性分泌在恒定条件下维持，周期接近24 h。昼夜节律振荡器如何在恒定条件下驱动褪黑素节律的机制尚未得到充分阐明。我们研究了环腺苷酸依赖性蛋白激酶。A是参与主观夜间增加褪黑激素释放的鸡松果体细胞培养下恒定的黑暗。在静态细胞培养中，H8（蛋白激酶抑制剂）和H89（特异性蛋白激酶A抑制剂）抑制褪黑激素释放的主观夜间增加呈剂量依赖性，但不受calphostin C（特异性蛋白激酶C抑制剂）的抑制。在细胞灌注实验中，从ZT 9（CT 11.2）h开始的H8和H-89的9小时脉冲以剂量依赖性方式抑制褪黑激素节律的主观夜间增加，而不引起相移。细胞内钙离子螯合剂BAPTA-AM和细胞外钙离子螯合剂BAPTA和EGTA抑制褪黑激素释放和cAMP水平的主观性直肠增加，呈剂量依赖性。这些直接的证据有力地支持了这样的假设，即cAMP依赖性蛋白激酶A可能参与主观夜间增加鸡松果体细胞的褪黑激素释放，细胞内Ca 2+在松果体腺苷酸环化酶激活中起着重要作用。

Melatonin release by chick cultured pineal cells increases during the dark periods and decreases during the light periods under light-dark cycles, and this rhythmic secretion is maintained under constant conditions, with a period close to 24h. The mechanisms how circadian oscillator drives the melatonin rhythm under constant conditions have not been elucidated enough. We examined the possibility that cyclic AMP-dependent protein kinase. A is involved in the subjective nocturnal increase in melatonin release by chick pineal cells cultured under constant darkness. The subjective nocturnal increase of melatonin release was suppressed dose-dependently by H8 (protein kinase inhibitor) and H89 (specific protein kinase A inhibitor), but not by calphostin C (specific protein kinase C inhibitor) in static cell cultures. In a cell perfusion experiment, 9 hour pulses of H8 and H-89 starting at ZT 9 (CT 11.2) h suppressed the subjective nocturnal increase in melatonin rhythm in dose-dependent manner without causing a phase shift. An intracellular Ca2+ chelator, BAPTA-AM,and extracellular Ca2+ chelators, BAPTA and EGTA,suppressed both the subjective nocturanal increases in melatonin release and cAMP levels dose-dependently. These direct evidences strongly support the hypothesis that cAMP-dependent protein kinase A may be involved in the subjective nocturnal increase in melatonin release by chick pineal cells, and that intracellular Ca2+ plays an important role in pineal adenylate cyclase activation.