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Articles in PresS, published online ahead of print August 22, 2002
Am J Physiol Heart Circ Physiol, 10.1152/ajpheart.00428.2002
Submitted on May 28, 2002
Accepted on August 19, 2002
1 Department of Pharmacology and Toxicology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
2 The iCAPTUR4E Center, St. Paul's Hospital, University of British Columbia, Vancouver, British Columbia, Canada
* To whom correspondence should be addressed. E-mail: pli{at}mcw.edu.
We developed an in situ assay system to simultaneously monitor intracellular Ca 2+ concentration ([Ca 2+] i, fura 2 as indicator) and NO levels (DAF-2 as probe) in the intact endothelium of small bovine coronary arteries by using fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK, 1 µM) 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 µM) induced a gradual and 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 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 eNOS, which provides the first direct evidence for a permissive role of Ca 2+ in tyrosine phosphorylation-induced NO production.
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