Background & aims: The mechanism of plasma membrane blebbing (dissociation of the lipid bilayer from the membrane cytoskeleton) in hepatocyte injury is not known. The aim of this study was to investigate the role of calpain, a calcium-dependent cytosolic protease, in bleb formation induced by oxidative stress.
Methods: Hepatocytes from Wistar rats were injured with tertbutyl hydroperoxide in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a specific calpain inhibitor, calpeptin (Z-Leu-nLeu-H). Bleb 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 hydroperoxide induced a sustained increase in intracellular calciu, bleb formation, and, ultimately, hepatocyte death. Talin and alpha-actinin were degraded in a time-dependent manner, although no apparent changes of actin filament were observed. Before the cytoskeletal protein degradation, the intermediate form of calpain mu appeared as its proactivated form decreased. In addition, calpeptin or EGTA inhibited not only calpain mu activation but also cytoskeletal protein degradation and bleb formation.
Conclusions: In tert-butyl hydroperoxide-treated hepatocytes, the activation of calpain promotes membrane blebbing via degradation of cytoskeletal proteins.