Molecular Pharmacology of Capacitative Ca^<2+> Entry Channel of Human Aortic Endothelial Cell

在血管内皮细胞中，细胞内钙离子浓度([Ca^&lt；2+&gt；]i)随着各种血管活性物质和机械刺激的反应而升高，引起内皮活性分子的合成和释放。在本研究中，1)针对人血管内皮细胞膜上5‘翻译起始点的反义寡核苷酸作用于人血管内皮细胞。交换可能在调节[Ca^&lt；2+&gt；]i中起主要作用。2)为了研究[Ca^&lt；2+&gt；]i升高与氯电流的功能关系，我们观察了米贝地尔对人主动脉内皮细胞[Ca^&lt；2+&gt；]i和组胺增加的氯离子电流的影响。米贝拉地尔以浓度依赖的方式降低组胺引起的[Ca~(2+)]i升高和Cl~+电流。3)利用RT-PCR法从大鼠脑c DNA中分离到3个(α，β，γ)TRPC4变异体，它们被认为是电容钙通道的分子基础。在HEK293细胞中，基础[Ca~(2+)]i升高，但卡巴胆碱或thapsigargin诱导的Ca~(2+)&gt；α的表达不受影响。结果表明，rTRPC4α在HEK293细胞中瞬时表达时，在功能上参与了基础钙离子水平的调节，可能需要细胞内因素作为容量性钙离子通道。

In vascular endothelial cells, intracellular Ca^<2+> concentration ([Ca^<2+>]_i) increases in response to various vasoactive substances and mechanical stimulation, causing synthesis and release of endothelial active molecules.In this study, 1) human aortic endothelial cells were treated with antisense oligodeoxynucleotides targeting the 5' translation start site of the human membrane Ca^<2+> ATPase isoform 1. Results provide the evidence of functional role of plasma membrane Ca^<2+> ATPase, although other mechanisms including Na^+/Ca^<2+> exchange may play the primary role in regulating [Ca^<2+>]i.2) To examine the functional relationship between the increase in [Ca^<2+>]i and the Cl^- current, we investigated the effects of mibefradil on [Ca^<2+>]i and the Cl^- current increased by histamine in human aortic endothelial cells. Mibefradil decrease the histamine-induced [Ca^<2+>]i elevation and Cl^- current in a concentration-dependent manner. Those results indicate, the functional relationship between the capacitative Ca^<2+> entry channel and the Cl^- channel.And, 3) we isolated three (α, β, γ) TRPC4 variants, thought to be candidates for the molecular basis of capacitative Ca^<2+> entry channels, from rat brain cDNA using RT-PCR. In HEK-293 cells, basal [Ca^<2+>]i was increased, but carbachol or thapsigargin-induced Ca^<2+> entry were not affected by expression of rTRPC4α. Results indicate that rTRPC4α is functionally involved in the regulation of basal Ca^<2+> levels when transiently expressed in HEK-293 cells and may need intracellular factors for acting as the capacitative Ca^<2+> entry channel.