Development of the optical system for continuous and multi-factorial monitoring of the intracellular signaling network in endothelial and smooth muscle cells in vascular strips.

本研究的目的是开发用于连续和多因素监测血管条内皮细胞和平滑肌细胞内信号网络的光学系统。利用新开发的系统，我们研究了血管张力调节的机制。本研究获得了以下结果：(1)我们开发了一种同时监测[Ca^<2+>]i变化和血管条张力的系统。该系统在David G.Lambert编辑的《钙信号协议》一书的第114卷《分子生物学方法》（Humana Press Inc.1999）中作为“使用前表面荧光法测量平滑肌条中的[Ca^<2+>]i”一章报道。(2)采用fura-2的前表面荧光法，研究了在苯肾上腺素诱导的猪肾小动脉收缩过程中，thapsigargin诱导的三个阶段内皮依赖反应的机制。研究发现，最初的短暂松弛伴随着平滑肌的减少[Ca^<2+>]，这是由于内皮衍生的超极化因子（EDHF）和一氧化氮（NO）的释放。随后的短暂性收缩伴随着[Ca^<2+>]i的增加，这是由于血栓素A_2从内皮细胞释放所致。最后的持续松弛，不伴有[Ca^<2+>]的变化，是由于内皮细胞释放NO。(3)研制了血管平滑肌细胞[Ca^<2+>]i和（pH）i同时监测系统。(4)利用该系统，我们研究了平滑肌细胞碱化诱导张力发展增加的机制。NH_4Cl对原代培养的大鼠主动脉平滑肌细胞产生碱化反应，并伴有[Ca^<2+>]i的升高。发现碱化增加了Ca^<2+>通过skf96365敏感的Ca^<2+>通道进入。Ca^<2+>通过细胞膜的容性进入可能增加[Ca^<2+>]i，从而增加碱化过程中的张力发展。少

The purpose of this study is to develop optical systems for continuous and multi-factorial monitoring of the intracellular signaling network in endothelial and smooth muscle cells in vascular strips. Using newly developed systems, we investigated the mechanisms underlying the regulation of the vascular tonus. Following results were obtained in this study : (1) We have developed a system to monitor changes in [Ca^<2+>]i and tension of the vascular strips simultaneously. This system was reported as a chapter "Measurement of [Ca^<2+>]i in smooth muscle strips using front-surface fluorimetry" 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.1999). (2) Using front-surface fluorimetry of fura-2, we investigated the mechanism underlying the three phasic endothelium-dependent responses induced by thapsigargin during the phenirephrine-induced contraction in porcine renal small arterial strips. … More It was found that the initial transient relaxation accompanied by a reduction of smooth muscle [Ca^<2+>]i was due to both the release of endothelium-derived hyperpolarizing factor(EDHF) and nitric oxide(NO).. The subsequent transient contraction accompanied by an increase in [Ca^<2+>]i was due a release of thromboxane A_2 from the endothelium. The final sustained relaxation, which was not accompanied by the changes in[Ca^<2+>]i was due to release of NO from the endothelium.(3)We have developed a new system to monitor [Ca^<2+>]i and (pH)i of vascular smooth muscle cells simultaneously. (4) Using this system, we investigated the mechanism underlying an increase in the tension development induced by the alkalization of smooth muscle cells. The application of NH_4Cl induced an alkalization which is accompanied by an increase in[Ca^<2+>]i in rat aortic smooth muscle cells in primary culture. It was found that alkalization increases Ca^<2+> entry through the SKF96365-sensitive Ca^<2+> channels. The capacitative entry of Ca^<2+> through cell membrane might increase [Ca^<2+>]i, and thus, increases tension development during alkalization. Less