MECHANISM OF HYDROGEN-PEROXIDE AND HYDROXYL FREE RADICAL-INDUCED INTRACELLULAR ACIDIFICATION IN CULTURED RAT CARDIAC MYOBLASTS

After a transient ischemic attack of the cardiac vascular system, reac tive oxygen-derived free radicals, including the superoxide (O-2(-.)) and hydroxyl ((OH)-O-.) radicals can be easily produced during reperfu sion. These free radicals have been suggested to be responsible for re perfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O2) is often used as an experimental source of o xygen-derived free radicals. Using freshly dissociated single rat card iac myocytes and the rat cardiac myoblast cell line, H9c2, we have sho wn, for the first time, that an intriguing pH(i) acidification (approx imate to 0.24 pH unit) is induced by the addition of 100 mu mol/L H2O2 and that this dose is without effect on the intracellular free Ca2+ l evels or viability of the cells. Using H9c2 as a model cardiac cell, w e have shown that it is the intracellular production of (OH)-O-., and not O-2(-.) or H2O2, that results in this acidification. We have exclu ded any involvement of (1) the three known cardiac pH(i) regulators (t he Na+-H+ exchanger, the Cl--HCO3 exchanger, and the Na+-HCO3 co-trans porter), (2) a rise In intracellular Ca2+ levels, and (3) inhibition o f oxidative phosphorylation. However, we have found that H2O2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolys is of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been sh own that a small intracellular acidification may severely inhibit cont ractility. Therefore, the sustained pH(i) decrease caused by hydroxyl radicals may contribute, in some pert, to the well-documented impairme nt of cardiac mechanical function (ie, reperfusion cardiac stunning) s een during reperfusion ischemia.