Modulation of intracellular Ca ion concentrations by endothelium-derived hyperpolarizing factor in vascular smooth muscle

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(1) Vascular smooth muscles for the measurement of intracellular Ca ion concentrations and also for mechanical responses were successfully prepared in the guinea-pig aorta. Smooth muscle of this artery was confirmed to be relaxd by EDHF.(2) Endothelial cells isolated from bovine thoracic aorta using enzymatic dispersion were cultured on microcarrier beeds. Preliminary experiments showed that these cells (3x10^7 cells/each experiment) can produce several types of vasoactive substance in response to bradikinine (Bk).(3) Production of EDHF from the cultured endothelial cells was estimated by using isolated thoracic aortic ring of the guinea-pig. Superfusates of the cultured endothelial cells with Bk relaxd the Bk-contracted aortic ring preparations, and the relaxation was resistant to nitroarginine and indomethacin, but sensitive to high-K solution containing nitroaginine. The results suggested that Bk stimulates the cultured endothelial cells to release vesorelaxtant factors which may be … More different from EDRF or PG12. The effects of these unidentified factors reduced in high-K solution, indicated an involvement of membrane hyperpolarization.4. Intracellular Ca ion concentrations modulated by EDHF were estimated from the fluorescent intensities of Ca-sensitive dye, Fura-2 loaded in primary cultured smooth muscle cells of the guinea-pig aorta (Fura-2 was loaded into the cell in a form of Fura-2 AM). Bk elevated the fluorescent transiently to level equivalent to 300-500nM Ca from the resting level of about 50nM, and them reached the steady level of about 200nM.Bk-containing superfusate elluted from the cultured endothelial cells reduced the Fura-2 fluorescent to the resting level, irrespective to the absence and presence of nitroarginine and indonethacin. The reduction of fluorescent intensity by the endothelial effluents was not clearly detected in high-K solution. The results suggested that hyperpolarization by endothelial factor may be responsible to the reduction of fluorescent. Thus, EDHF relax arterial smooth muscles with reduced Ca concentrations. Less