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.

在黑暗时期，鸡培养的松果细胞释放的褪黑激素会增加，并在光漆循环下的光周期减少，并且这种节奏的分泌在恒定条件下保持在恒定的条件下，其周期接近24小时。这种机制是在恒定条件下如何驱动褪黑激素节奏的机制尚未得到足够的阐明。我们检查了环状AMP依赖性蛋白激酶的可能性。 A参与了在恒定黑暗下培养的小鸡松果细胞释放褪黑激素释放的主观夜间增加。 H8（蛋白激酶抑制剂）和H89（特异性蛋白激酶A抑制剂）抑制了褪黑激素释放的主观夜间释放的主观增加，而不是calphostin c（特异性蛋白激酶C抑制剂）在静态细胞中抑制剂量。在一个细胞灌注实验中，从ZT 9（CT 11.2）h开始的9小时H8和H-89脉冲以剂量依赖性的方式抑制了褪黑激素节奏的主观夜间增加，而不会引起相位移位。细胞内Ca2+螯合剂，BAPTA-AM和细胞外Ca2+螯合剂BAPTA和EGTA抑制了褪黑激素释放的主观夜毒的增加和cAMP水平依赖性剂量。这些直接证据强烈支持以下假设：依赖CAMP的蛋白激酶A可能与小鸡松果细胞释放褪黑激素释放的主观夜间增加有关，而细胞内Ca2+在松氨酰腺苷酸环化酶激活中起重要作用。