Abstract
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 (pHi) 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 beta-ATP relative to alpha-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, beta-ATP fell 66%, and 85% after 2 h, while the Pi/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 less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 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 less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 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 Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-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.
Highlights
From the $Department of Physiology and the §Departmentof Surgery, Universityof Pittsburgh School of Medicine, Pittsburgh, Pennsylvania15261
Ca2+i,ATP, Na+i,and Lactic Dehydrogenase (LDH) returned near the control levels within 45 min. These results suggest that the rise in Ca2+iinduced 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
After 1 h of control, 2 h of anoxia, and 1h of recovery, trypan blue uptake increased 27.7 f 2.9% ( p < 0.05). These experimentsprovide the first direct measurement of a massive increase in cytosolic free calcium in isolated hepatocytes induced by simple anoxia and obtained with a specific Ca2+indicator
Summary
Hepatocytes-Freshly isolated hepatocytes were prepared from adult male Sprague-Dawley rats weighing between 150 and 250g. Intracellular Free Magnesium (Mg2'i)-The concentration of Mg"; was calculated according to Malloy et al [17] from the 31PNMR spectra obtained during the control period, the 2 h of anoxia, and during recovery. The cells were washed, cast in agarose gel threads, placed in a quartz cuvette, and perfused with KHB a t a rateof 0.6 ml/min at 37 "C.Fluorescencewas measured in a SPEX dual excitation spectrofluorometer. The aequorin-loaded cells were imbedded in agarose gel threads, placed in the cuvette of an aequorin luminescence photometer, and perfused at a rate of 0.6 ml/min with KHB at37 "C, as previously described [20]. Viability was examined immediately after isolation, in cells cast in agarose gel threads during the control period, at the end of 2 h of anoxia and after reoxygenation. Ap value less than 0.05 was considered to be significant
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