Ischemia is characterized by anoxia and a large decrease of tissue pH. After a critical period of ischemia, reperfusion precipitates irreversible injury. Previous work showed that reperfusion injury to cultured neonatal myocytes was precipitated by a rapid return to physiological pH, a ''pH paradox'' (Bond, J., B. Herman, and J. Lemasters. Biochem. Biophys. Res. Commun. 179: 798-803, 1991). The aim of this study was to measure intracellular pH (pH(i)) and cytosolic free Ca2+ during the pH paradox of reperfusion injury to cultured neonatal rat cardiac myocytes. pH(i) and free Ca2+ were measured by ratio imaging of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura 2 fluorescence. To simulate ATP depletion and acidosis of ischemia, myocytes were incubated with 20 mM 2-deoxyglucose plus 2.5 mM NaCN at pH 6.2. During simulated-ischemia, pH(i) dropped to <6.5 and subsequently remained constant. During this time, some blebbing but little hypercontraction occurred. After 3 or 4 h of simulated ischemia, inhibitors were removed and cells were incubated at pH 7.4 to simulate reperfusion. pH(i) began to increase, blebbing accelerated, and myocytes hypercontracted. As pH(i) increased, viability was lost. The same occurred if pH was increased but metabolic inhibitors were not removed. Monensin, a Na+-H+ ionophore, accelerated the increase of pH after reperfusion and hastened cell killing. Hypercontraction, blebbing, and loss of viability did not occur when inhibitors were removed at pH 6.2 or in the presence of dimethylamiloride, an inhibitor of Na+-H+ exchange. Protection was associated with maintenance of an acidotic pH(i). Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ increased further. When Ca2+ was excluded from the reperfusate, free Ca2+ increased transiently and then began to fall, but lethal reperfusion injury still occurred. If inhibitors were removed at pH 6.2, free Ca2+ remained elevated, but cells did not die. We conclude that return of pH(i) to physiological levels during reperfusion precipitates lethal cell injury. Reperfusion injury does not appear to be triggered by Ca2+ overload.
