Effects of high and low pH on Ca2+i and on cell injury evoked by anoxia in perfused rat hepatocytes

Abstract

The effect of high and low pH on anoxic cell injury was studied in freshly isolated rat hepatocytes cast in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB) saturated with 95% O2 and 5% CO2. Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular pH (pHi) with BCECF, and lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate. A 2 h period of anoxia was induced by perfusing the cells with KHB saturated with 95% N2 and 5% CO2. The extracellular pH (pHo) was maintained at 7.4, 6.8 or 8.0 by varying the bicarbonate concentration. The substrate was either 5 mM glucose, 15 mM glucose or 15 mM fructose. In some experiments, anoxia was performed in Ca(2+)-free media by perfusing the cells with KHB without Ca2+ but with 0.1 mM EGTA. Reducing pHo to 6.8 during anoxia did not reduce the increase in Ca2+i, but but completely abolished LDH release. Under these conditions, pHi decreased to 6.56 +/- 0.3 when glucose was the substrate and to 6.18 +/- 0.25 with 15 mM fructose. Apparently, protection against anoxic injury caused by a low pHo is associated with a low pHi but not with a reduced elevation in Ca2+i. Increasing pHo to 8.0 during anoxia increased pHi above 8.0 +/- 0.01 and doubled LDH release without significantly altering the rise in Ca2+i. When 15 mM fructose was present with a pHo of 8.0, pHi was still 8.0, but there was practically no rise in Ca2+i, and LDH release was again completely abolished. On the other hand, a Ca(2+)-free perfusate with a pHo of 8.0 kept the rise in Ca2+i below 400 nM but did not abolish the massive release of LDH caused by high pH. Since cell injury is caused by the activation of Ca(2+)-sensitive hydrolytic enzymes such as phospholipase A2, these experiments suggest that a low pH (< 6.5) prevents their activation even in the presence of a high Ca2+i. Conversely, a high pH (> 8.0) can activate hydrolytic enzymes and cause injury even in the absence of an elevated Ca2+i. The precise mechanism by which fructose protects hepatocytes against cell injury at pHi 8.0 is unclear.