title: 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.
abstract: 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, calpeptin, as assessed by nuclear staining with Hoechst 33258 and lactate dehydrogenase release. The processing of procaspase-3 was inhibited by z-VAD-FMK, but not by calpeptin. In contrast, z-VAD-FMK failed to block the proteolytic cleavage of fodrin-alpha, a preferential substrate for calpain. On the other hand, degradation of actin and fodrin-alpha was prevented by calpeptin but not by z-VAD-FMK. In addition, not only protein kinase C (PKC)-alpha but also PKC-delta were cleaved to generate approximately 46 kDa fragments. The PKC fragmentation was inhibited by calpeptin but not by z-VAD-FMK. These findings suggest that the extracellular Ca2+ influx induced by hypoxic stress activates calpain, resulting in the degradation of cytoskeletal proteins and generation of PKC fragments almost independently of caspase activation. Therefore, calpain may play an important role in hypoxic PC12 cell death.
authors.affiliation: Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan. haruki-y@cc.gifu-u.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Yamakawa
authors.forename: Y
authors.initials: Y
authors.lastname: Banno
authors.forename: S
authors.initials: S
authors.lastname: Nakashima
authors.forename: S
authors.initials: S
authors.lastname: Yoshimura
authors.forename: M
authors.initials: M
authors.lastname: Sawada
authors.forename: Y
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Y
authors.initials: Y
authors.lastname: Nozawa
authors.forename: N
authors.initials: N
authors.lastname: Sakai
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism* / Q000473:pathology; D004151:Dipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D009410:Nerve Degeneration / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051381:Rats
chemical_list: D002614:Chelating Agents; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007476:Ionophores; D007527:Isoenzymes; C071482:calpeptin; D000001:Calcimycin; D004533:Egtazic Acid; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D010447:Peptide Hydrolases; D002154:Calpain; D020169:Caspases; D002118:Calcium