RESUMO
The effect of fasting and anoxia on the intracellular concentration of ATP, Na+, Ca2+, Mg2+, and H+ was studied in isolated perfused rat hepatocytes. ATP and intracellular Mg2+ were measured by 31P-NMR spectroscopy, cytosolic free calcium was measured with aequorin, intracellular Na+ with SBFI, intracellular pH with BCECF, lactic dehydrogenase by NADH absorbance. In hepatocytes from fasted rats, intracellular ATP was depressed 52% (P < 0.001), Nai+ was increased 70% from 16.9 to 27.7 mM (P < 0.02), and Cai2+ was increased 79% from 137 to 245 nM (P < 0.05) when compared to fed rats. Mgi2+ and pHi were unchanged. During anoxia, ATP and the cell phosphorylation potential decreased 90% to practically the same low levels in both fed and fasted groups. On the other hand, in hepatocytes from fasted animals, Cai2+ increased faster and to significantly higher levels than in hepatocytes from fed rats: Cai2+ reached 2.19 microM in 10 min compared to 1.45 microM in 1 h, respectively (P < 0.05). Cell injury assessed by LDH release and trypan blue exclusion also occurred earlier and was more severe in hepatocytes from fasted rats. Fructose and Ca(2+)-free perfusion reduced the rise in Cai2+, abolished LDH release and significantly improved the cell viability measured by Trypan blue exclusion. The data demonstrate that fasting decreases the hepatocytes energy potential and increases Nai+ and Cai2+ which are inversely related to the cell energy potential. Consequently, in hepatocytes isolated from fasted rats, the increase in Cai2+ and the resulting cell injury evoked by anoxia occur earlier and are more severe than in fed rats. These results suggest that Ca2+ plays a crucial role in the development of anoxic cell injury.