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-

储存操作和/或配体操作的Ca^<2+>内流通路已在多种细胞中发现，它们在Ca^<2+>稳态中起重要作用。另一方面，在包括心肌和骨骼肌在内的许多可兴奋细胞中，电压依赖性Ca^<2+>通道（VDCC）被认为是Ca^<2+>内流的主要途径。然而，我们最近发现骨骼肌中存在SOC通路，其中SOC活性远高于VDCC。我们还发现，在大鼠和豚鼠心室肌中也存在非vdcc通路。利用激光扫描共聚焦显微镜或瞬变场显微镜观察Ca^<2+>的动力学特性，并采用快速冷却收缩法（RCC）测定SR中Ca^<2+>的含量。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+>交换剂的可能性。在各种Ca^<2+>流入的推定抑制剂中，2-氨基乙氧基二苯硼酸盐（2- apb）和SKF96365都没有显著的效果。有趣的是，它被20 μM的econazole（一种假定的TRPV5抑制剂）抑制到一半。通过RT-PCR分析，在心室肌中检测到TRPV6的mRNA，但未检测到TRPV5的mRNA，这表明TRPV6可能是Ca^<2+>内流途径的候选者。相反，骨骼肌SOC Ca^<2+>内流对2- apb非常敏感，而对econazole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。需要进一步的研究来定位和识别这些通路。

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.