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

• Published in: Brain research 2000-01, Vol.852 (2), p.326-334
• Main Authors: RAY, S. K, FIDAN, M, NOWAK, M. W, WILFORD, G. G, HOGAN, E. L, BANIK, N. L
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - drug effects; Apoptosis - physiology; bcl-2-Associated X Protein; Biological and medical sciences; Blotting, Western; Calcimycin - pharmacology; Calcium - metabolism; Calpain - metabolism; Dipeptides - pharmacology; DNA Primers; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Hydrogen Peroxide - pharmacology; In Situ Nick-End Labeling; Ionophores - pharmacology; Isolated neuron and nerve. Neuroglia; Neurofilament Proteins - analysis; Neurofilament Proteins - metabolism; Neurons - chemistry; Neurons - cytology; Neurons - enzymology; Oxidants - pharmacology; Oxidative Stress - physiology; PC12 Cells; Protein Synthesis Inhibitors - pharmacology; Proto-Oncogene Proteins - analysis; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins c-bcl-2 - metabolism; Rats; RNA, Messenger - analysis; Vertebrates: nervous system and sense organs
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/s0006-8993(99)02148-4

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.