title: Inhibition of rat PC12 cell calpain activity by glutathione, oxidized glutathione and nitric oxide.
abstract: Calpain, a calcium activated neutral protease, is involved in mediating neurotoxicity resulting from conditions of oxidative stress and free radical formation, such as hypoxia and ischemia. Nitric oxide (NO) may also be involved in modulating the cytotoxic effects of oxidative stress. We investigated the roles of reduced glutathione (GSH), oxidized glutathione (GSSG), and NO in modulating calpain activity in PC12 cells. Cell extracts were treated with GSSG, GSH, or the NO-donor S-nitroso-N-acetylpenicillamine. Calpain activity was determined by means of a fluorescent assay. Non-linear regression analysis was used to determine the type of inhibition (competitive, uncompetitive, or non-competitive). GSH displayed uncompetitive inhibition, with K(i)=7.0+/-2.0 mM (Mean+/-SEM) while GSSG exhibited competitive inhibition with K(i)=2.5+/-0.3 mM. NO was an irreversible inhibitor of calpain activity. These results suggest that both GSH and GSSG may be important physiological modulators of calpain activity.
authors.affiliation: Division of Intramural Clinical and Biochemical Research, National Institute on Alcohol Abuse and Alcoholism, NIH 10/ 3C103, 10 Center Drive, MSC 1256, Bethesda, MD 20892-1256, USA.
authors.forename: J
authors.initials: J
authors.lastname: Rackoff
authors.forename: Q
authors.initials: Q
authors.lastname: Yang
authors.forename: P B
authors.initials: PB
authors.lastname: DePetrillo
mesh_terms: D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology
chemical_list: D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D002154:Calpain; D019803:Glutathione Disulfide