Studies on molecular cell biology of the inhibition of coronary vasoconstriction and intimal thickening.

为了预防冠状动脉血管收缩和内膜增厚，我们进行了以下研究：在平滑肌和内皮细胞中，(1)细胞内信号转导网络的表征，(2)阐明细胞周期进展与细胞功能之间的关系，(3)阐明信号因子与细胞功能/结构之间的关系。(4)实验研究结果的临床应用。得到以下结果/结论：(1)我们开发了一种监测血管条[Ca]i变化的方法，该方法在David G.Lambert编辑的《钙信号协议》一书第114卷（Humana Press Inc., Totowa， NJ， USA, 1999）中被h.c anaide报道为“使用前表面荧光法测量平滑肌条中的[Ca]i”一章（P269-P277）。利用这一技术和细胞膜剥皮技术，我们对平滑肌和内皮细胞的信号转导系统进行了表征。特别是，我们描述了平滑肌细胞中收缩器官的钙敏感性，以及内皮细胞中[Ca]i与NO生成之间的关系。(2)在平滑肌细胞中，钙通道（T、L）的类型随细胞周期的进展而变化。内皮细胞生长接触抑制期肌球蛋白磷酸酶出现膜下定位。(3)利用DNA突变体，我们确定了钙调蛋白与肌球蛋白磷酸酶130和20 KDa亚基的蛋白结构和功能之间的关系。(4)缺血性心脏病患者β 3肾上腺素受体DNA中存在Trp64Arg突变。然而，本研究结果的临床应用仍是我们面临的下一个问题。

For the prevention of coronary vasoconstriction and intimal thickening, following studies were performed : In smooth muscle and endothelial cells, (1)the characterization of the intracellular signal transduction networks, (2) the elucidation of the relationships between cell cycle progression and the cellular functions, and (3) the elucidation of the relationships between signaling factors and cellular functions/structures. (4) The clinical applicatopn of the results of these experimental studies.Following results/conclusions were obtained : (1) We have developed a method to monitor [Ca]i changes of the vascular strips, which was reported as the chapter "Measurement of [Ca]i in smooth muscle strips using front-suface fluorimetry" (P269-P277) by H.Kanaide in the book "Calcium signaling protocols" edited by David G.Lambert, which is the Vol.114, of series of "Methods in Molecular Biology" (Humana Press Inc., Totowa, NJ, USA, 1999). Using this technique and skinning of cell membrane, we have characterized the signal transduction system of smooth muscle and endothelial cells. In particular, we have characterized the Ca-sensitivity of the contractile apparatus in smooth muscle cells and relationships between [Ca]i and NO production in endothelial cells. (2) We found that types of Ca-channels(T,L) varied during the progression of cell cycles in smooth muscle cells. There appeared submembranous localization of myosin phosphatase at the phase of contact inhibition of the growth in endothelial cells. (3) Using DNA mutants, we have characterized the relationships between protein structure and funtion of calmodulin and 130 and 20 KDa subunits of myosin phosphatase. (4) There is a mutation of Trp64Arg in DNA of beta 3 adrenoceptors among the patients with ischemic heart disease. However, the clinical application of the results of the present study has remained to be the next problem which confronts us.