Effect of anoxia on intracellular ATP, Na+ i, Ca2+ i, Mg2+ i, and cytotoxicity in rat hepatocytes

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca²⁺ _i) was measured with aequorin, intracellular sodium (Na⁺ _i) with SBFI, intracellular pH (pH_i) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by ³¹P NMR spectroscopy in real time, and intracellular free Mg²⁺ (Mg²⁺ _i) from the chemical shift of β- ATP relative to α-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N₂, 5% CO₂. After 1 h of anoxia, β-ATP fell 66%, and 85% after 2 h, while the P_i/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 ± 6 nM. Anoxia increased Ca²⁺ _i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 ± 35 nM (p <0.001). A second peak reached a maximum value of 1.45 ± 0.12 μM (p <0.001) after 1 h. During the first hour of anoxia, Na⁺ _i increased from 15.9 ± 2.4 mM to 32.2 ± 1.2 mM (p <0.001), Mg²⁺ _i doubled from 0.51 ± 0.05 to 1.12 ± 0.01 mM (p <0.001), and pH_i decreased from 7.41 ± 0.03 to 7.06 ± 0.1 (p <0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca²⁺ _i, Mg²⁺ _i, Na⁺ _i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca²⁺ _i, and whether the increased Ca²⁺ _i was caused by Ca²⁺ influx, the cells were perfused with Ca²⁺-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca²⁺-free medium, β-ATP again fell 90%, but Ca²⁺ _i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca²⁺ _i, ATP, Na⁺ _i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca²⁺ _i induced by anoxia is caused by an influx of Ca²⁺ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca²⁺ _i.