Increased Cytosolic Ca2+ Amplifies Oxygen Radical-Induced Alterations of the Ultrastructure and the Energy Metabolism of Isolated Rat Pancreatic Acinar Cells

Abstract

Background: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca²⁺ concentration and energy metabolism. Methods: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca²⁺ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. Results: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca²⁺ was found, a change which could be inhibited by BAPTA, reducing cellular damage. Conclusion: Cytosolic Ca²⁺ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.