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Cloning and functional analysis of the genes related to endothelium derived hyperpolarizing factor

内皮源性超极化因子相关基因的克隆及功能分析

基本信息

批准号：
14570074
负责人：
FUAKO Mitsuhiro
金额：
$ 2.69万
依托单位：
SAPPORO MEDICAL UNIVERSITY (2003)Hokkaido University (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570074/
关键词：
EDHF endothelial cell smooth muscle cell artery relaxation ion channel gap junction connexin カリウムイオンチャンネル

项目摘要

The purpose of this project was to clarify the molecular mechanisms of endothelium-derived hyperpolarizing factor(EDHF)-mediated arterial relaxation and membrane hyperpolarization in the rat mesenteric arteries. We tried to identify the ion channels and gap junctional channels involved in the EDHF action. RT-PCR experiment showed that mRNA of small-conductance Ca^<2+>-activated K^+(SK)3, intermediate-conductance Ca^<2+>-activated K^+(IK), large-conductance Ca^<2+>-activated K^+(BK) channels and connexin-37,-40,-43,-45 but not SK1 and SK2 were exist in the rat mesenteric artery. Genes of these ion channels and connexins were cloned by RT-PCR and cDNA library screening. New gene similar to SK3 channel was cloned. Functional analysis using patch clamp techniques showed that the new SK3-related channel has almost the same ion channel functions such as voltage sensitivity and toxin sensitivity. However the Ca^<2+> sensitivity of the channel was less sensitive compared with the reported SK3 channel. The new channel may relate to the arterial function. Immunohistochemical analysis showed that the SK3 channel was expressed in the endothelium. The CX37 and 43 were expressed in both the endothelial and smooth muscle cells, however CX40 was expressed in only the endothelial cells. To clarify the functional role of these genes in the native artery, adenoviruses expressing these genes were constructed. The functional analysis of these genes using adenovirus in the native artery was under estimation.

本研究旨在探讨内皮源性超极化因子（endothelium-derived hyperpolarization factor，EDHF）介导的大鼠肠系膜动脉舒张和膜超极化的分子机制。我们试图确定参与EDHF作用的离子通道和缝隙连接通道。RT-PCR结果显示，大鼠肠系膜动脉中存在小电导Ca^2+激活的K^+（SK）3、中电导Ca^2+激活的K^+（IK）、大电导Ca^2+激活的K^+（BK）通道和连接蛋白37、40、43、45的mRNA，而不存在SK 1和SK 2的mRNA。通过RT-PCR和cDNA文库筛选，克隆了这些离子通道和连接蛋白的基因。克隆了与SK 3通道相似的新基因。利用膜片钳技术进行的功能分析表明，新的SK 3相关通道具有几乎相同的离子通道功能，如电压敏感性和毒素敏感性。然而，该通道的Ca^<2+>敏感性不如已报道的SK 3通道。新通道可能与动脉功能有关。免疫组化结果显示SK 3通道在内皮细胞中表达。CX 37和CX43在内皮细胞和平滑肌细胞中均有表达，而CX40仅在内皮细胞中表达。为了阐明这些基因在天然动脉中的功能作用，构建了表达这些基因的腺病毒。在天然动脉中使用腺病毒对这些基因进行功能分析的估计不足。