Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).