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IDENTIFICATION OF NON-VOLTAGE DEPENDENT CA^<2+> INFLUX PATHWAY IN CARDIAC MUSCLE AND ITS REGULATORY MECHANISM -Analysis by observation of subcellular Ca^<2+> dynamics using evanescent-field fluorescence microscopy-

心肌中非电压依赖性 CA^<2> 流入途径及其调节机制的鉴定 -使用倏逝场荧光显微镜观察亚细胞 Ca^<2> 动态进行分析-

基本信息

批准号：
13670096
负责人：
KUREBAYASHI Nagomi
金额：
$ 2.24万
依托单位：
Juntendo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Presence of store-operated and/or ligand-operated Ca^<2+> influx pathways have been indicated in many kinds of cells and their important roles in Ca^<2+> homeostasis were reported. On the other hands, in many excitable cells including cardiac and skeletal muscles, voltage-dependent Ca^<2+> channel (VDCC) have been thought to be the major Ca^<2+> influx pathway. However, we recently showed the presence of SOC pathway in skeletal muscle, where the SOC activity was much higher than that of VDCC. We also found that non-VDCC pathway was also present in rat and guinea pig ventricular muscle. We examined properties of the pathway by observing Ca^<2+> dynamics using laser-scanning confocal microscope or evanescent-field microscope and determining SR Ca^<2+> content with Rapid Cooling Contracture (RCC) method. The influx showed marked temperature-dependence. Because the influx was resistant to nifidipine or KB-R7943, the possibility of DHPR or Na^+/Ca^<2+> exchanger was excluded. Among putative inhibitors of various Ca^<2+> influxes, neither 2-aminoethoxydiphenyl borate (2-APB) nor SKF96365 was significantly effective. Interestingly, it was suppressed to a half by 20 μM econazole, a putative inhibitor to TRPV5. With RT-PCR analysis, mRNA for TRPV6, but not for TRPV5, was detected in ventricular muscles, suggesting that TRPV6 may be a candidate for the Ca^<2+> influx pathway. On the contrary, the SOC Ca^<2+> influx in skeletal muscle was very sensitive to 2-APB but not to econazole. These results suggest that molecules for the non-VDCC pathways should be different in cardiac and skeletal muscles. Further studies are required for the localization and identification of the pathways.

许多细胞中都存在由钙离子和/或配体调控的钙离子内流途径，并报道了它们在钙离子稳态中的重要作用。另一方面，在包括心肌和骨骼肌在内的许多兴奋性细胞中，电压依赖性钙通道(VDCC)被认为是主要的钙内流途径。然而，我们最近发现在骨骼肌中存在SOC通路，其SOC活性远高于VDCC。我们还发现非VDCC通路也存在于大鼠和豚鼠的心室肌中。用激光扫描共聚焦显微镜或消失场显微镜观察钙离子动力学变化，用快速冷却收缩(RCC)法测定肌浆网钙离子含量，观察信号转导途径的特性。这种流入表现出明显的温度依赖性。由于内流对硝苯地平或KB-R7943耐药，排除了DHPR或Na~+/Ca~(2+)&gt；交换体的可能性。在各种钙离子内流的假定抑制剂中，2-氨基乙氧基二苯硼酸酯(2-APB)和SKF96365都没有明显的效果。有趣的是，它被20μ甲康唑抑制了一半，这是一种可能的TRPV5的抑制剂。RT-PCR分析显示，在心室肌中检测到TRPV6的mRNA，而不是TRPV5的mRNA，提示TRPV6可能是钙离子内流途径的候选者。骨骼肌SOC钙离子内流对2-APB非常敏感，但对益康唑不敏感。这些结果表明，非VDCC途径的分子在心肌和骨骼肌中应该是不同的。这些途径的定位和鉴定还需要进一步研究。