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 b icarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured w ith 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 P-31 NMR spec troscopy in real time, and intracellular free Mg2+ (Mg2+i) from the ch emical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Ano xia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, wh ile the P(i)/ATP ratio increased 10-fold from 2.75 to 28.3. Under cont rol conditions, the resting cytosolic free calcium was 127 +/- 6 nM. A noxia increased Ca2+i in two distinct phases: a first rise occurred wi thin 15 min and reached a mean value of 389 +/- 35 nM (p < 0.001). A s econd peak reached a maximum value of 1.45 +/- 0.12-mu-M (p < 0.001) a fter 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 firs t hour and increased 6-fold during the second hour of anoxia. Upon reo xygenation, 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, beta-ATP again fell 90%, but Ca2+i, after the first initial p eak, fell below control levels, and LDH release increased only 2.7-fol d. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the c ontrol levels within 45 min. These results suggest that the rise in Ca 2+i induced by anoxia is caused by an influx of Ca2+ from the extracel lular fluid, and that LDH release and cell injury may be related to th e resulting rise in Ca2+i.