Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger

In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabol ites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitoc hondrial calcium concentration [Ca2+](m). Both agents caused a biphasic inc rease in [Ca2+]m which was preceded by a rise in cytosolic free calcium con centration [Ca2+](c) (18 and 6 seconds for H2O2 and HX-XO, respectively), T he peak and plateau elevations of [Ca2+] were consistently higher in the mi tochondrial matrix than in the cytosol, In Ca2+-free/EGTA medium, the plate au phase of elevated [Ca2+] evoked by H2O2. due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In c ontrast to H2O2 and HX-XO, ATP which binds the P2Y purinoceptors induced an increase in [Ca2+](m) that was similar to that of [Ca2+](c). When cells we re first stimulated with inositol 1,4,5-trisphosphate-generating agonists o r the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition o f H2O2 did not affect [Ca2+](c), but still caused an elevation of [Ca2+](m) . Moreover, the specific inhibitor of the mitochondrial Ca2+/Na+ exchanger, 7-chloro-3,5-dihydro-5-phenyl-1H-4.1-benzothiazepine-2-on (CGP37157), did not potentiate the effects of H2O2 and HX-XO on [Ca2+](m), while causing a marked increase in the peak [Ca2+](m) and a significant attenuation of the rate of [Ca2+](m) efflux upon addition of histamine or CPA. In permeabilize d cells, H2O2 mimicked the effects of CGP37157 causing an increase in the b asal level of matrix free Ca2+ and decreased efflux, Dissipation of the ele ctrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+](m) increases evoked by H2O2, These results demonstrate that the H2O 2-induced elevation in [Ca2+](m) results from a transfer of Ca2+ secondary to increased [Ca2+](c), and an inhibition of the Ca2+/Na+ electroneutral ex changer of the mitochondria.