Analysis of circadian clock mechanism in birds and mammals.

鸟类和哺乳动物的生物钟机制分析。

基本信息

批准号：
08660367
负责人：
MURAKAMI Noboru
金额：
$ 1.47万
依托单位：
MIYAZAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

在明暗循环下，鸡培养的松果体细胞释放的褪黑激素在黑暗期增加，在光照期减少，并且这种节律性分泌在恒定条件下保持，周期接近24h。昼夜节律振荡器在恒定条件下驱动褪黑激素节律的机制尚未得到充分阐明。我们检查了环AMP依赖性蛋白激酶的可能性。 A参与持续黑暗下培养的小鸡松果体细胞主观夜间褪黑激素释放的增加。在静态细胞培养物中，主观夜间褪黑激素释放量的增加受到 H8（蛋白激酶抑制剂）和 H89（特定蛋白激酶 A 抑制剂）的剂量依赖性抑制，但不受钙磷蛋白 C（特定蛋白激酶 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 9 小时 H8 和 H-89 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，而不会引起相移。细胞内 Ca2+ 螯合剂 BAPTA-AM 和细胞外 Ca2+ 螯合剂 BAPTA 和 EGTA 可以剂量依赖性地抑制主观夜间褪黑激素释放和 cAMP 水平的增加。这些直接证据有力地支持了以下假设：cAMP 依赖性蛋白激酶 A 可能参与了鸡松果体细胞主观夜间褪黑激素释放的增加，并且细胞内 Ca2+ 在松果体腺苷酸环化酶激活中起着重要作用。