Crucial role of calpain in hypoxic PC12 cell death: Calpain, but not caspases, mediates degradation of cytoskeletal proteins and protein kinase C-alpha and -delta

Ca2+ influx is one of the main causative events in hypoxic PC12 cell death, because an extracellular Ca2+ chelator:, ethylene glycol bis (2-aminoethyl ether)-N,N,N ' ,N ' -tetraacetic acid (EGTA) inhibited and Ca2+ ionophore A23187 mimicked the hypoxic cell death. The hypoxic cell death was markedly prevented by a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala -Asp-fluoromethylketone (z-VAD-FMK) as well as a calpain inhibitor, calpept in, as assessed by nuclear staining with Hoechst 33258 and lactate dehydrog enase release. The processing of procaspase-3 was inhibited by z-VAD-FMK, b ot not by calpeptin. in contrast, z-VAD-FMK failed to block the proteolytic cleavage of fodrin-alpha a preferential substrate for calpain. On the othe r hand, degradation of actin and fodrin-alpha was prevented by calpeptin bu t nor by z-VAD-FMK. In addition, not only protein kinase C (PKC)-alpha but also PKC-delta were cleaved to generate similar to 46 kDa fragments The PKC fragmentation was inhibited by calpeptin but not by z-VAD-FMK. These findi ngs suggest that the extracellular Ca2+ influx induced by hypoxic stress ac tivates calpain, resulting in the degradation of cytoskeletal proteins and generation of PKC fragments almost independently of caspase activation. The refore, calpain may play an important role in hypoxic PC12 cell death.