Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells.
Changes in [Ca(2+)](i) across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity. In the present study, we investigated the effect of ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, on NO release and [Ca(2+)](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 [Ca(2+)](i) using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t((1/2)) (half-time of the decline of [Ca(2+)](i) to basal levels after stimulation with 10 micro mol/L bradykinin) was used as an index of [Ca(2+)](i) extrusion. 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 [Ca(2+)](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. Pretreatment of RAEC with Na(+)-free solution, an inhibitor of the Na(+)/Ca(2+) exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. 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 [Ca(2+)](i) by forward mode Na(+)/Ca(2+) exchange to prolong the Ca(2+) signal time. From these results, we suggest that nanomolar levels of ouabain modulate [Ca(2+)](i) movements and NO production in RAEC.
Dong
Xian Hui
XH
Department of Clinical Sciences and Laboratory Medicine, Kansai Medical University, Moriguchi, Osaka, Japan.
Komiyama
Yutaka
Y
Nishimura
Noriko
N
Masuda
Midori
M
Takahashi
Hakuo
H
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D007424:Intracellular Fluid / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis*; D010042:Ouabain / Q000008:administration & dosage*; D051381:Rats; D017207:Rats, Sprague-Dawley
D009569:Nitric Oxide; D010042:Ouabain; D001920:Bradykinin; D002118:Calcium