Autocrine and paracrine effects of endothelium-derived relaxing factor on intracellular Ca2+ of endothelial cells and vascular smooth muscle cells. Identification by two-dimensional image analysis in coculture. |
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Authors: | W S Shin T Sasaki M Kato K Hara A Seko W D Yang N Shimamoto T Sugimoto T Toyo-oka |
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Institution: | Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan. |
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Abstract: | To elucidate the effects of endothelium-derived relaxing factor (EDRF) released from vascular endothelial cells (ECs) on handling of intracellular calcium ion (Ca2+i) in ECs themselves and vascular smooth muscle cells (VSMCs), we measured the Ca2+i by two-dimensional digital image analysis of fura-2-loaded ECs and VSMCs in tissue culture. In isoculture of one cell type, adenosine triphosphate (ATP, 1 microM) transiently increased the Ca2+i of both ECs and VSMCs. High-K+ depolarization or angiotensin II also elevated the Ca2+i of VSMCs, whereas neither stimulants changed the Ca2+i of ECs. In coculture of ECs with VSMCs, the same dose of ATP rapidly increased the Ca2+i of ECs and then transiently decreased the Ca2+i of VSMCs to below the resting level. The maximal Ca2+i-modulating effects of ATP on both cell types were reproducible after the second application of ATP. Three kinds of EDRF blockers (L-NG-monomethylarginine, methemoglobin, or methylene blue) potentiated the ATP-induced Ca2+i rise in ECs and attenuated the Ca2+i reduction in VSMCs, suggesting the autocrine and paracrine effects of EDRF on ECs and VSMCs, respectively. However, neither indomethacin, superoxide dismutase, nor neutralizing monoclonal antibody to endothelin-1 altered the second responses. Thus, two-dimensional Ca2+i image analysis of ECs and VSMCs in coculture enabled direct visualization of the EDRF actions in ECs and VSMCs and their modifications. |
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