Effects of high and low pH on Ca2+i and on cell injury evoked by anoxia in perfused rat hepatocytes.
Biochim Biophys Acta
; 1220(3): 277-85, 1994 Feb 17.
Article
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| MEDLINE
| ID: mdl-8305500
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Hipoxia de la Célula
/
Calcio
/
Concentración de Iones de Hidrógeno
/
Hígado
Límite:
Animals
Idioma:
En
Revista:
Biochim Biophys Acta
Año:
1994
Tipo del documento:
Article