EFFECTS OF HIGH AND LOW PH ON CA-I(2+) AND ON CELL INJURY EVOKED BY ANOXIA IN PERFUSED RAT HEPATOCYTES

The effect of high and low pH on anoxic cell injury was studied in fre shly isolated rat hepatocytes cast in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB) saturated with 95% O-2 and 5% CO2. Cytosolic free calcium (Ca-i(2+)) was measured with aequor in, intracellular pH (pH(i)) with BCECF, and lactic dehydrogenase (LDH ) by the increase in NADH absorbance during lactate oxidation to pyruv ate. A 2 h period of anoxia was induced by perfusing the cells with KH B saturated with 95% N-2 and 5% CO2. The extracellular pH (pH(0)) 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 fructo se. In some experiments, anoxia was performed in Ca2+-free media by pe rfusing the cells with KHB without Ca2+ but with 0.1 mM EGTA. Reducing pH(0) to 6.8 during anoxia did not reduce the increase in Ca-i(2+), b ut but completely abolished LDH release. Under these conditions, pH(i) 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 in jury caused by a low pH(0) is associated with a low pH(i) but not with a reduced elevation in Ca-i(2+) Increasing pH(0) to 8.0 during anoxia increased pH(i) above 8.0 +/- 0.01 and doubled LDH release without si gnificantly altering the rise in Ca-i(2+). When 15 mM fructose was pre sent with a pH(0) of 8.0, pH(i) was still 8.0, but there was practical ly no rise in Ca-i(2+), and LDH release was again completely abolished . On the other hand, a Ca2+-free perfusate with a pH(0) of 8.0 kept th e rise in Ca-i(2+) below 400 nM but did not abolish the massive releas e of LDH caused by high pH. Since cell injury is caused by the activat ion of Ca2+-sensitive hydrolytic enzymes such as phospholipase A(2), t hese experiments suggest that a low pH(< 6.5) prevents their activatio n even in the presence of a high Ca-i(2+). Conversly, a high pH (> 8.0 ) can activate hydrolytic enzymes and cause injury even in the absence of an elevated Ca-i(2+). The precise mechanism by which fructose prot ects hepatocytes against cell injury at pH(i) 8.0 is unclear.