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.
Yi
Fu-Xian
FX
Department of Pharmacology, Medical College of Wisconsin, Milwaukee 53226, USA.
Zhang
Andrew Y
AY
Campbell
William B
WB
Zou
Ai-Ping
AP
Van Breemen
Cornelis
C
Li
Pin-Lan
PL
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D003331:Coronary Vessels / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors
D001152:Arsenicals; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; C029341:oxophenylarsine; D009569:Nitric Oxide; D017027:Protein Tyrosine Phosphatases; D001920:Bradykinin; D002118:Calcium