title: Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger.
abstract: In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabolites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitochondrial calcium concentration [Ca2+]m. Both agents caused a biphasic increase in [Ca2+]m which was preceded by a rise in cytosolic free calcium concentration [Ca2+]c (18 and 6 seconds for H2O2 and HX-XO, respectively). The peak and plateau elevations of [Ca2+] were consistently higher in the mitochondrial matrix than in the cytosol. In Ca2+-free/EGTA medium, the plateau phase of elevated [Ca2+] evoked by H2O2 due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In contrast 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 were first stimulated with inositol 1,4, 5-trisphosphate-generating agonists or the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition of 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 permeabilized cells, H2O2 mimicked the effects of CGP37157 causing an increase in the basal level of matrix free Ca2+ and decreased efflux. Dissipation of the electrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+]m increases evoked by H2O2. These results demonstrate that the H2O2-induced elevation in [Ca2+]m results from a transfer of Ca2+ secondary to increased [Ca2+]c, and an inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria.
authors: Respiratory Division and Division of Clinical Biochemistry, Department of Internal Medicine, University Hospital, Switzerland. lan.h.jornot@hcuge.ch
authors: L
authors: L
authors: Jornot
authors: P
authors: P
authors: Maechler
authors: C B
authors: CB
authors: Wollheim
authors: A F
authors: AF
authors: Junod
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000331:Aequorin / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002232:Carbocyanines / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000378:metabolism; D002460:Cell Line; D002998:Clonazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000502:physiology*; D013841:Thiazepines / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D001562:Benzimidazoles; D002232:Carbocyanines; D004791:Enzyme Inhibitors; D007211:Indoles; D017382:Reactive Oxygen Species; D019831:Sodium-Calcium Exchanger; D013841:Thiazepines; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000331:Aequorin; D002998:Clonazepam; C079032:CGP 37157; D006632:Histamine; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D014969:Xanthine Oxidase; D002118:Calcium; C000543:cyclopiazonic acid