INTRACELLULAR PH AND CA2+ HOMEOSTASIS IN THE PH PARADOX OF REPERFUSION INJURY TO NEONATAL RAT CARDIAC MYOCYTES

Ischemia is characterized by anoxia and a large decrease of tissue pH. After a critical period of ischemia, reperfusion precipitates irrever sible 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 myo cytes. pH(i) and free Ca2+ were measured by ratio imaging of 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein and fura 2 fluorescence. To simu late ATP depletion and acidosis of ischemia, myocytes were incubated w ith 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. Du ring this time, some blebbing but little hypercontraction occurred. Af ter 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 incre ase, blebbing accelerated, and myocytes hypercontracted. As pH(i) incr eased, viability was lost. The same occurred if pH was increased but m etabolic inhibitors were not removed. Monensin, a Na+-H+ ionophore, ac celerated the increase of pH after reperfusion and hastened cell killi ng. Hypercontraction, blebbing, and loss of viability did not occur wh en inhibitors were removed at pH 6.2 or in the presence of dimethylami loride, an inhibitor of Na+-H+ exchange. Protection was associated wit h maintenance of an acidotic pH(i). Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ incr eased further. When Ca2+ was excluded from the reperfusate, free Ca2increased transiently and then began to fall, but lethal reperfusion i njury still occurred. If inhibitors were removed at pH 6.2, free Ca2remained elevated, but cells did not die. We conclude that return of p H(i) to physiological levels during reperfusion precipitates lethal ce ll injury. Reperfusion injury does not appear to be triggered by Ca2overload.