Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells

Calpain, a Ca2+-dependent cysteine protease, has previously been implicated in apoptosis or programmed cell death (PCD) in immune cells. Although oxid ative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous syste m (CNS) due to these agents has not yet been defined. To explore a possible role for calpain in neuronal PCD under oxidative stress and Ca2+ influx, w e examined the effects of H2O2 and A23187 on PC12 cells. Treatments caused PCD (light microscopy and TUNEL assay) with altered mRNA expression (RT-PCR ) of bar (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a h igh bax/bcl-2 ratio. Control cells expressed 1.3-fold more mu calpain (requ iring mu M Ca2+) than mcalpain (requiring mM Ca2+). Expression of mcalpain was significantly increased following exposure to oxidative stress and Ca2 influx. The mRNA levels of calpastatin (endogenous calpain inhibitor) and beta-actin (house-keeping) genes were not changed. Western analysis indicat ed degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibit or of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpa in, degradation of 68 kDa NFP, and occurrence of PCD. These results suggest a role for calpain in PCD of PC12 cells due to oxidative stress and Ca2+ i nflux. (C) 2000 Published by Elsevier Science B.V. All rights reserved.