A synergistic effect of extracellular hypocalcemic condition for hyperoxic reoxygenation injury in rat hepatocytes.
BACKGROUND
Calcium accumulation of cells and mitochondria during reperfusion or reoxygenation has been implicated as a potential factor in cell injury as the result of mitochondrial damage. The objective of this study was to disclose whether or not low extracellular calcium ion concentration ([Ca2+]ex) in the medium at the time of reoxygenation might prevent calcium accumulation and attenuate hepatocytes injury after severe hypoxia.


METHODS
Isolated rat hepatocytes were incubated under a hyperoxic or hypoxic atmosphere for 60 min. During the ensuing 60-min hyperoxic reoxygenation, medium [Ca2+]ex was varied from 0.6 microM to 2.0 mM by altering total calcium and addition of chelators.


RESULTS
Incubation in low [Ca2+]ex reduced total cellular calcium and mitochondrial calcium in both the hyperoxic and hypoxic group. Under hyperoxic/hyperoxic incubation (control), hepatocytes were able to maintain potassium balance when [Ca2+]ex was >3.0 microM (pCa=5.5) and cellular viability (% lactate dehydrogenase release) at all levels of extracellular calcium. Under hypoxic/hyperoxic incubation (reoxygenation), however, loss of the ability to restore potassium balance as well as apparent increase in lactate dehydrogenase release were observed at severely low [Ca2+]ex (<30 microM; pCa=4.5). This low [Ca2+]ex-induced exacerbation of hepatocytes viability could not be generated under mild reoxygenation such as normoxia.


CONCLUSIONS
In normal isolated hepatocytes, very low [Ca2+]ex levels produce only very subtle changes in membrane permeability of isolated hepatocytes. After hypoxia, however, hypocalcemia acts synergistically with hyperoxic reoxygenation to produce more severe damage. These results suggested that [Ca2+]ex should be maintained on the physiological level to attenuate hepatocytes injury after severe hypoxia.
Division of Pediatric Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA. kobayasi@med.m.chiba-u.ac.jp
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Kobayashi
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Miescher
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Clemens
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007770:L-Lactate Dehydrogenase; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D010100:Oxygen / Q000633:toxicity*; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D004533:Egtazic Acid; D007770:L-Lactate Dehydrogenase; D011188:Potassium; D010100:Oxygen; D002118:Calcium