Intracellular pH and Ca2+ homeostasis in the pH paradox of reperfusion injury to neonatal rat cardiac myocytes

Abstract

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 (pHi) and cytosolic free Ca2+ during the pH paradox of reperfusion injury to cultured neonatal rat cardiac myocytes. pHi 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, pHi 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. pHi began to increase, blebbing accelerated, and myocytes hypercontracted. As pHi 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 pHi. Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ increased further.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothermic in situ perfusion of the porcine liver using Celsior or Ringer-lactate solution

“…This leads to metabolic acidosis secondary to the formation of lactate. Whereas acidotic pH can protect against anoxic cell death, reperfusion of ischemic cells at acidotic pH leads to more cell killing (pH paradox) [27,28]. The minor decrease in arterial pH and base loss in the Celsior group as compared to the Ringer-lactate group is most likely caused by the histidine buffer contained in Celsior.…”

A unique pathway of cardiac myocyte death caused by hypoxia–acidosis

“…Increased activity of the Na + /H + exchanger can cause Ca 2+ overload because the elevated intracellular Na + is subsequently exchanged for Ca 2+ via the Na + /Ca 2+ exchanger (Pierce and Czubryt, 1995). Inhibition of Na + /H + exchange has been shown to protect against ischemic injury, possibly by preventing this increase in Ca 2+ (Bond et al, 1993;Shimada et al, 1996). Conversely, inhibition of the vacuolar ATPase promotes apoptosis, in part by shifting the proton load towards the Na + /H + transporter, thereby increasing Ca 2+ uptake, and in part by reducing the myocyte capacity to control intracellular pH (Gottlieb et al, 1996;Karwatowska-Prokopczuk et al, 1998;Long et al, 1998).…”

Activation of Na+/H+ Exchanger-directed Protein Kinases in the Ischemic and Ischemic-reperfused Rat Myocardium

“…This ischemic treatment is referred to as I1. In other experiments, 10 mM 2-deoxy-Dglucose was added to this medium to inhibit glycolytic ATP production and result in a more severe ischemia (referred to as I2) (30). To simulate reperfusion, the metabolic inhibitor was removed by "washout" with KRH buffer at pH 7.4 (without potassium cyanide) and incubation for 30 min at 37°C.…”

“…We utilized methods that "chemically" induce ischemia and ischemia-reperfusion events similar to that observed in perfused hearts. This "chemical" ischemia acts by inhibition of oxidative phosphorylation (I1), or by inhibiting both oxidative phosphorylation and glycolysis (I2) (30). Fig.…”