| Co-Investigator(Kenkyū-buntansha) |
FUKAO Mitsuhiro Hokkaido Univ., Grad. School of Med., Assis. Prof., 大学院・医学研究科, 助手 (10250432)
HATTORI Yuichi Hokkaido Univ., Grad. School of Med., Asso. Prof., 大学院・医学研究科, 助教授 (50156361)
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| Research Abstract |
We have so far found that ET-1 activated three types of Ca^<+2>-permeable channel in vascular smooth muscle cells derived from rat thoracic aortae : two types of nonselective cation channel (designated NSCC-1 and NSCC-2) and store-onerated Ca^<2+> channel (SOCC). The purpose of the present study is to clarify the structure, function, and functional significance of voltage-independent Ca^<2+>-permeable channels involved in vascular contractions induced by vasoconstricting agents such as endothelin-1 (ET-1) and noradrenaline, and also to develop drugs specifically acting on these channels. To isolate cDNAs encoding these channels, we decided to use expression cloning strategy with Xenopus oocytes. For this purpose, we developed a screening method using ^<45>Ca^<2+> uptake to specifically detect Ca^<2+> entry. First we screened varying tissues and cell lines for high expression levels of the three channels, and found that expression levels of the three channels were highest in CHO cells. For the first step toward expression cloning, we prepared cRNA which was trascribed in vitro using recombinant cDNA for ET type A receptor (ET_AR) as a template : mRNAs were extracted from CHO cells and fractionated according to their sizes with sucrose-gradient ultracentrifugation. For the first screening, we microinjected cRNA for ET_AR and either of the mRNA fractions into Xenopus oocytes, and assayed for ^<45>Ca^<2+> uptake into the oocytes. From thepositive fraction, cDNA library was prepared using ZAP Express Vector and separated into 10 parts. For the second and the following screening, the oocytes were microinjected with cRNA for ET_AR and cRNA prepared from either of the cDNA library parts, and assayed for ^<45>Ca<2+> uptake. By repeating this type of screening, we finally obtained 16 positive clones. We are now determining sequences and function of each clone. After that, we plan to to develop drugs specifically acting on these channels.
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