Modulation of apoptosis by activation of protein kinasse G

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We investigated the protective effect of nitric oxide (NO) at a low concentration on NO- and hydrogen peroxide-induced cell death and its mechanism in mouse macrophage cell line, RAW264.SNP induced cell death in RAW264 cells at a high concentration (4 mM). Pretreatment with 100 μM SNP or 1 mM dibutylyl-cGMP reduced cytochrome c release from mitochondria and prevented the cell death induced by 4 mM SNP in RAW264 cells. The effect of SNP pretreatment was reduced by LY83583. Pretreatment with dibutylyl-cGMP prevented cell death induced by NOC 18, GSNO or SNP, in a concentration- dependent manner. Pretreatment with dibutylyl-cGMP prevented cytochrome c release induced by NO donors. The protective effect of SNP or dibutylyl-cGMP was significantly attenuated by KT5823 (a protein kinase G inhibitor). These results indicate that NO at a low concentration protects RAW264 cells from the cytotoxicity of NO through cGMP production and activation of PKG.Translocation of Bax from cytosol to mitochon … More dria was observed in the cell death. Untreated RAW264 cells displayed no Bax N-20 antibody (pAb raised against amino acids 11-30 of Bax)-associated immunoreactivity. However, some cells displayed a punctate cytosolic pattern of Bax immunostaining after 4 mM SNP treatment. Bax positive cells displayed a diffuse cytosolic pattern of cytochrome c immunostaining. The number of Bax positive cell was increased after 4 mM SNP treatment. Translocation of Bax and the increase of Bax positive cells induced by 4 mM SNP were inhibited by pretreatment with 100 μM SNP or 1 mM dibutylyl-cGMP. Activation of p38 MAP kinase induced by SNP at a high concentration was prevented by pretreatment with SNP at a low concentration or dibutylyl-cGMP. These results indicate that NO/cGMP signaling pathway inhibits NO-induced apoptpsis of macrophages by suppressing p38 MAP kinase activation, which results in N-terminal conformational change and translocation of Bax.Pretreatment with SNP or 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene (NOC18), at a low concentration for 24 h reduced the H_2O_2-induced cell death, caspase-3 activation and nuclear fragmentation. LY83583 and ODQ, soluble guanylate cyclase inhibitors, inhibited the protective effect of both SNP and NOC18 pretreatment. Protein kinase G inhibitor KT5823 also significantly reduced these cytoprotective effects. These results indicate that NO at a low concentration protects RAW264 cells from H_2O_2-induced apoptosis though cGMP production and activation of protein kinase G. Less