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.

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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