BD20

Ca²⁺ Redistribution And Mitochondrial Dysfunction During Chemical Anoxia/Reoxygenation In Renal Cell Cultures: A Laser Scanning Confocal Microscopy Study.

N. Takeyama*. K. Yamagami, and T. Tanaka, Dept of Emergency & Critical Care Medicine, Kansai Medical University, Japan.

The aim of this study is to evaluate changes in mitochondrial function of cultured MDCK cell by laser scanning confocal microscopy and a digitized-fluorescence-imaging during chemical anoxia/reoxygenation (A/R). Chemical anoxia was induced with rotenone and 2-deoxyglucose, inhibitor of a site 1 of the electron transport chain and glycolysis, respectively. Oxidative metabolism was restored by the addition of a short odd-chain fatty acid (heptanoate). Cytosolic free Ca²⁺ measured by Fura-2 increased gradually about 50% after 30 min of anoxia and returned to initial value immediately after reoxygenation. Anoxia up to 60 min or 30 min of anoxia followed by 30 min of reoxygenation had no effect on cell viability and mitochondrial membrane potential (m) assessed by DiOC6 and JC-1. In the presence of thapsigargin, a specific inhibitor of the Ca-ATPase of the endoplasmic reticulum, m was stable during 60 min of anoxia. However, m decreased with reoxygenation after 30 min of anoxia. The sensitization to A/R by thapsigargin disappeared in the presence of cyclosporin A, a specific inhibitor of mitochondrial inner membrane pore opening. We conclude that reoxygenation-induced Ca²⁺ redistribution and the mitochondrial membrane permeability transition are involved in A/R injury of MDCK cell.