CALPAIN ACTIVATION IN PLASMA-MEMBRANE BLEB FORMATION DURING TERT-BUTYL HYDROPEROXIDE-INDUCED RAT HEPATOCYTE INJURY

Background & Aims: The mechanism of plasma membrane blebbing (dissocia tion of the lipid bilayer from the membrane cytoskeleton) in hepatocyt e injury is not known, The aim of this study was to investigate the ro le of calpain, a calcium-dependent cytosolic protease, in bleb formati on induced by oxidative stress, Methods: Hepatocytes from Wistar rats were injured with tert-butyl hydroperoxide in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic ac id (EGTA) or a specific calpain inhibitor, calpeptin (Z-Leu-nLeu-H), B leb formation was examined by phase-contrast and transmission electron microscopies. Intracellular calcium concentration was measured using Fura-2, Western blot analyses were performed for cytoskeletal proteins (talin, alpha-actinin, and vinculin) and the intermediate (activated) and proactivated forms of calpain mu. Results: tert-Butyl hydroperoxi de induced a sustained increase in intracellular calcium, bleb formati on, and, ultimately, hepatocyte death, Talin and alpha-actinin were de graded in a time-dependent manner, although no apparent changes of act in filament were observed, Before the cytoskeletal protein degradation , the intermediate form of calpain mu appeared as its proactivated for m decreased, In addition, calpeptin or EGTA inhibited not only calpain mu activation but also cytoskeletal protein degradation and bleb form ation, Conclusions: In tert-butyl hydroperoxide-treated hepatocytes, t he activation of calpain promotes membrane blebbing via degradation of cytoskeletal proteins.