Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries

We developed an in situ assay system to simultaneously monitor intracellular Ca(2+) concentration (Ca(2+)](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 microM) stimulated a rapid increase in Ca(2+)](i) followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 microM) induced a gradual, small increase in Ca(2+)](i) and a slow increase in intracellular NO levels. Removal of extracellular Ca(2+) and depletion of Ca(2+) stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of Ca(2+)](i) by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of Ca(2+)](i) and intracellular NO production in the intact endothelium is a powerful tool to study Ca(2+)-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca(2+) in tyrosine phosphorylation-induced NO production.