Ca2+ removal mechanisms in freshly isolated rabbit aortic endothelial cells

Calcium removal from the cytoplasm was investigated in freshly isolated aortic endothelial cells by monitoring changes in intracellular calcium ([Ca2+](i)) using ratiometric fura-2 fluorimetry. Blockade of the Na+/Ca2+ exchanger (NCX) by replacement of external sodium with equi-molar N-methyl-d-glutamine (0Na PSS) decreased the removal rate by 52%. Blockade of the sarco/encloplasmic reticulum Ca2+ ATPase (SERCA) by cyclopiazonic acid (CPA) decreased the removal rate by 50%. Simultaneous application of CPA and 0Na PSS did not reduce the removal rate any further (53%). The lack of additivity of these two procedures, suggests that SERCA and the NCX function in series to lower [Ca2+](i). In addition, in the absence of extracellular Ca2+, removal of external Na+ markedly reduced the rate of loss of Ca2+ from the ER further supporting the hypothesis that NCX is functionally linked to ER calcium release channels, and thus, plays an important role in ER calcium unloading. To investigate the mechanism for the coupling of NCX and SERCA, the same protocols as described above were repeated after treating the cells with cytochalasin D, which disrupts the cytoskeleton. This treatment uncoupled the NCX from SERCA, as evidenced by the resulting additive inhibitory effects of application of CPA and removal of extracellular Na+ on the rate of Ca2+ removal from the cytoplasm. These data suggest that in endothelial cells NCX and SERCA function in series to remove about half of the free Ca2+ from the cytosol, while PMCA contributes to the other half of the Ca2+ removal process. (C) 2002 Elsevier Science Ltd. All rights reserved.