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1 Departments of Pharmacology and Toxicology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 2 The iCAPTUR4E Center, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada V6Z 1Y6
We developed an in situ assay system to simultaneously monitor intracellular Ca2+ concentration ([Ca2+]i, fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 µM) stimulated a rapid increase in [Ca2+]i followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 µM) induced a gradual, small increase in [Ca2+]i and a slow increase in intracellular NO levels. Removal of extracellular Ca2+ and depletion of Ca2+ stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca2+]i by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca2+]i and intracellular NO production in the intact endothelium is a powerful tool to study Ca2+-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca2+ in tyrosine phosphorylation-induced NO production.
endothelium-derived relaxing factor; signal transduction; protein kinase; coronary circulation; heart
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