title: Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation.
abstract: Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA-NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K gamma gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kgamma. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.
authors.affiliation: Laboratory of Biochemistry of the Immune System, University of Sevilla, Spain.
authors.forename: J C
authors.initials: JC
authors.lastname: Bernabé
authors.forename: J R
authors.initials: JR
authors.lastname: Tejedo
authors.forename: P
authors.initials: P
authors.lastname: Rincón
authors.forename: G M
authors.initials: GM
authors.lastname: Cahuana
authors.forename: R
authors.initials: R
authors.lastname: Ramírez
authors.forename: F
authors.initials: F
authors.lastname: Sobrino
authors.forename: F J
authors.initials: FJ
authors.lastname: Bedoya
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals; D007093:Imidazoles / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008928:Mitochondria / Q000473:pathology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D005609:Free Radicals; D007093:Imidazoles; D007202:Indicators and Reagents; D007328:Insulin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; D009599:Nitroprusside; D009569:Nitric Oxide; D002374:Catalase; D013482:Superoxide Dismutase; D020928:Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C093642:SB 203580