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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.

鸡松果体细胞褪黑激素的释放在暗周期中增加，在明暗周期的光周期中减少，这种有节律性的分泌在恒定的条件下保持，周期接近24小时。昼夜节律振荡器如何在恒定的条件下驱动褪黑素节律的机制还不够清楚。我们检测了cAMP依赖的蛋白激酶的可能性。A参与了在持续黑暗条件下培养的雏鸡松果体细胞夜间褪黑激素释放的主观增加。在静态细胞培养中，褪黑激素释放的主观增加可被蛋白激酶抑制剂H8和特异性蛋白激酶A抑制剂H89抑制，但不能被特异性蛋白激酶C抑制剂Calphostin C抑制。在细胞灌流实验中，从ZT9(CT11.2)h开始的9小时H8和H-89脉冲以剂量依赖的方式抑制主观夜间褪黑激素节律的增加，而不会引起相移。细胞内钙离子螯合剂BAPTA-AM和细胞外钙离子螯合剂BAPTA和EGTA均呈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依赖的蛋白激酶A可能参与了鸡松果体细胞夜间褪黑激素释放的主观增加，细胞内钙离子在松果体腺苷酸环化酶激活中起重要作用。