ROLE OF INTRACELLULAR CALCIUM IN SUPEROXIDE-INDUCED HEPATOCYTE INJURY

The mechanisms of hepatocyte injury caused by exogenous superoxide wer e investigated with the use of cultured rat hepatocytes. Cell viabilit y, cytosolic free calcium concentration and cell surface structure wer e observed. Superoxide was produced by adding hypoxanthine and xanthin e oxidase to the buffer. Cytosolic free calcium concentration was calc ulated by means of ratio imaging of fura 2 fluorescence with multipara meter digitized microscopy. In the buffer containing 1.27 mmol/L of ca lcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the add ition of hypoxanthine and xanthine oxidase, small blebs were recognize d on the cell surface with a scanning electron microscope; then a grad ual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentratio n had risen to about 700 nmol/L. Depriving the buffer of calcium (<10 mu mol/L) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the ba sal level (200 nmol/L). When ethylene glycol-bis (beta-aminoethyl ethe r)-N,N,N',N'-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free cal cium concentration decreased. Superoxide dismutase combined with catal ase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpept in, a Ca2+-dependent neutral protease inhibitor, did not affect the ri se in cytosolic free calcium concentration but prevented cell injury. We concluded that cell injury caused by superoxide is initiated by inf lux of calcium and that the resultant activation of Ca2+-dependent pro tease may play an important role in bleb formation and cell death.