title: 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.
authors.affiliation: Department of Physiology, University of Pittsburgh School of Medicine, PA 15261.
authors.forename: A
authors.initials: A
authors.lastname: Gasbarrini
authors.forename: P
authors.initials: P
authors.lastname: Caraceni
authors.forename: H
authors.initials: H
authors.lastname: Farghali
authors.forename: D H
authors.initials: DH
authors.lastname: Van Thiel
authors.forename: A B
authors.initials: AB
authors.lastname: Borle
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D005632:Fructose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D005632:Fructose; D004533:Egtazic Acid; D007770:L-Lactate Dehydrogenase; D005947:Glucose; D002118:Calcium