Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells

1. Changes in [Ca2+](i) across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity 2. In the present study, we investigated the effect of ouabain, a specific inhibitor of Na+/K+-ATPase, on NO release and [Ca2+](i) movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca2+](i) using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t(1/2) (half-time of the decline of [Ca2+](i), to basal levels after stimulation with 10 mumol/L bradykinin) was used as an index of [Ca2+](i) extrusion. 3. A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose-response concentration induced by bradykinin. The same dose of ouabain affected [Ca2+](i) movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t(1/2) was significantly increased. 4. Pretreatment of RAEC with Na+-free solution, an inhibitor of the Na+/Ca2+ exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. 5. These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na+/K+-ATPase activity and an increase in intracellular Na+ concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca2+](i) by forward mode Na+/Ca2+ exchange to prolong the Ca2+ signal time.