SIMULTANEOUS MEASUREMENTS OF CA2+ AND NITRIC-OXIDE IN BRADYKININ-STIMULATED VASCULAR ENDOTHELIAL-CELLS

The production of endothelium-derived relaxing factor (EDRF), known to be nitric oxide (NO), is triggered by a rise in the cytoplasmic calcium concentration ([Ca2+](i)) subsequent to receptor binding of vasoactive agonists. In vascular endothelial cells, NO is synthesized from L-arginine by the Ca2+/calmodulin-dependent NO synthase. In this study, we report the first simultaneous measurements of [Ca-i(2+]) and [NO] at the level of single endothelial cells. In cultured bovine aortic endothelial cells, extracellular application of bradykinin (BK, 10 to 20 mu mol/L) caused transient (sometimes oscillatory) increase in [Ca2+](i), which was measured with the fluorescent Ca2+ indicator fura 2 and fluorescence imaging microscopy. BK caused an increase in [Ca2+](i), primarily through release from intracellular stores. Under identical experimental conditions, BK caused a transient increase in [NO], which was measured by application of a porphyrinic NO microsensor. [NO] peaked at approximate to 0.5 mu mol/L. Simultaneous measurements of [Ca2+](i) and [NO] in BK-stimulated endothelial cells revealed that a transient increase in [Ca2+](i) was rapidly followed by an increase in [NO] that outlasted the [Ca2+](i) transient.