title: Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation.
abstract: Two protein signaling systems, phosphorylation and S-nitrosylation, influence most aspects of cellular physiology. S-nitrosylation, which generates a nitrosothiol linkage on cysteine residues, is caused by nitric oxide (NO). NO is believed to act as an anti-apoptotic agent by inhibiting caspase activity in cardiomyocytes, but there is little direct evidence for this. We investigated whether apoptosis inhibition by NO involved S-nitrosylation of caspases in doxorubicin (DOX)-induced myocardial apoptosis. Cardiomyocytes were treated with 1 micromol/l of DOX to induce apoptosis. Pretreatment with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) reduced the apoptosis. This effect was attenuated by treatment with 100 micromol/l of mercury dichloride (HgCl2), which is an agent of denitrosylation. After 24 h DOX-treatment, SNAP reduced the increased caspase-3 activity by 63%, and this effect was reversed by treatment with HgCl2. Immunoblot analysis showed that accumulation of the cleaved caspase-3 protein, an active form that induces apoptosis was inhibited significantly by SNAP. To elucidate nitrosothiol formation on caspase-3 by NO, we did several experiments. First, we prepared an immunoprecipitate of caspase-3 and measured the concentration of NO released from the precipitated complex by HgCl2. Second, S-nitrosylated proteins, purified by immunoprecipitation of caspase-3, were biotinylated and the biotin concentration was estimated by immunoblotting. Third, dual immunofluorescent staining was done with antibodies for S-nitrosocysteine and caspase-3. Results showed that formation of nitrosothiol in caspase-3 in DOX-treated cardiomyocytes with SNAP was increased significantly compared with untreated cardiomyocytes. We reported here that exogenous NO produces an anti-apoptotic effect by suppression of caspase activity via S-nitrosylation in cardiomyocytes.
authors.affiliation: Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Maejima
authors.forename: Susumu
authors.initials: S
authors.lastname: Adachi
authors.forename: Kino
authors.initials: K
authors.lastname: Morikawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ito
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Isobe
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D008627:Mercuric Chloride / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D026403:S-Nitrosothiols / Q000378:metabolism*
chemical_list: D061945:Caspase Inhibitors; D026403:S-Nitrosothiols; C110881:S-nitro-N-acetylpenicillamine; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D008627:Mercuric Chloride; D004317:Doxorubicin; D017869:Cyclic GMP-Dependent Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D010396:Penicillamine