Effect of hydrogen peroxide on Ca2+ mobilisation in human platelets through sulphydryl oxidation dependent and independent mechanisms.
Using Fura-2-loaded human platelets we studied the nature of the mechanisms involved in Ca2+ signalling mediated by H2O2. In a Ca2+-free medium, H2O2 (10 microM-100 mM) induced a concentration-dependent increase in [Ca2+]i. Depletion of either agonist-sensitive or mitochondrial Ca2+ pools reduced this effect while depletion of both stores abolished it. Xestospongin C, an inositol 1,3,5-trisphosphate (IP3) receptor inhibitor, reduced Ca2+ release evoked by 1 mM H2O2 by 45%, indicating that H2O2-induced Ca2+ release involves interaction with IP3 receptors. Blockade of the IP3 turnover by lithium or treatment with U-73122 did not modify H2O2-induced Ca2+ release from the agonist-sensitive pool, suggesting the involvement of a mechanism independent of IP3 generation. H2O2 inhibited Ca2+ reuptake into the agonist-sensitive stores mediated by the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Thimerosal (5 microM), a sulphydryl reagent, induced Ca2+ release from the agonist-sensitive stores. This event was impaired by treatment with 2 mM DTT, which also inhibited H2O2-induced Ca2+ release from the agonist-sensitive pool but not from mitochondria. H2O2 reduced the ability of the plasma membrane Ca2+ ATPase (PMCA) to extrude Ca2+ by 75%, an effect that was unaffected by DTT. Consistent with this, thimerosal did not modify the PMCA activity. Finally, exposure to H2O2 triggered platelet aggregation, which was slower than that observed after agonist stimulation. We conclude that H2O2 induced Ca2+ release from agonist-sensitive stores by oxidation of sulphydryl groups in SERCA and the IP3 receptors independently of IP3 generation. In addition, H2O2 induced Ca2+ release from mitochondria and inhibited the PMCA activity by different mechanisms in human platelets.
Redondo
Pedro C
PC
Department of Physiology, University of Extremadura, Av Universidad s/n, 10071 Caceres, Spain.
Salido
Ginés M
GM
Rosado
Juan A
JA
Pariente
José A
JA
D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium