Simultaneous Measurements of Ca 2+ and Nitric Oxide in Bradykinin-Stimulated Vascular Endothelial Cells

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