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 oxidative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous system (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, we 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 bax (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a high bax/bcl-2 ratio. Control cells expressed 1.3-fold more microcalpain (requiring microM 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 indicated degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibitor of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpain, 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+ influx.
Ray
S K
SK
Department of Neurology, Medical University of South Carolina, Charleston 29425, USA.
Fidan
M
M
Nowak
M W
MW
Wilford
G G
GG
Hogan
E L
EL
Banik
N L
NL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D017931:DNA Primers; D004151:Dipeptides / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007476:Ionophores / Q000494:pharmacology; D016900:Neurofilament Proteins / Q000032:analysis / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D016716:PC12 Cells; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D017931:DNA Primers; D004151:Dipeptides; D007476:Ionophores; D016900:Neurofilament Proteins; D016877:Oxidants; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D000001:Calcimycin; D006861:Hydrogen Peroxide; D002154:Calpain; D002118:Calcium; C058076:calpain inhibitor III