Modulation of apoptosis by activation of protein kinasse G

我们研究了低浓度一氧化氮（NO）对小鼠巨噬细胞RAW264中一氧化氮和过氧化氢诱导的细胞死亡的保护作用及其机制。高浓度（4 mM） SNP诱导RAW264细胞死亡。预处理100 μM SNP或1 mM二丁基- cgmp可减少线粒体中细胞色素c的释放，并可预防4 mM SNP诱导的RAW264细胞死亡。LY83583降低了SNP预处理的效果。二丁基- cgmp预处理对noc18、GSNO或SNP诱导的细胞死亡具有浓度依赖性。二丁基- cgmp预处理可抑制NO供体诱导的细胞色素c释放。KT5823（蛋白激酶G抑制剂）显著减弱SNP或二丁基cgmp的保护作用。这些结果表明，低浓度NO通过产生cGMP和激活pkg来保护RAW264细胞免受NO的细胞毒性，Bax从细胞质转移到线粒体，在细胞死亡中观察到更多的dridria。未经处理的RAW264细胞未显示Bax N-20抗体（pAb针对Bax的氨基酸11-30）相关免疫反应性。然而，一些细胞在4 mM SNP处理后显示Bax免疫染色的点状细胞质模式。细胞色素c免疫染色显示Bax阳性细胞呈弥漫性胞浆型。经4mm SNP处理后，Bax阳性细胞数量增加。100 μM SNP或1 mM二丁基- cgmp预处理均可抑制4 mM SNP诱导的Bax易位和Bax阳性细胞的增加。低浓度SNP或二丁基cgmp预处理可以阻止高浓度SNP诱导的p38 MAP激酶活化。这些结果表明，NO/cGMP信号通路通过抑制p38 MAP激酶的激活，抑制NO诱导的巨噬细胞凋亡，导致n端构象改变和Bax易位。低浓度SNP或1-羟基-2-氧-3,3-二-（2-氨基乙基）-1-三氮杂烯（no18）预处理24 h可降低h_2o_2诱导的细胞死亡、caspase-3活化和核断裂。可溶性鸟苷酸环化酶抑制剂LY83583和ODQ抑制了SNP和no18预处理的保护作用。蛋白激酶G抑制剂KT5823也显著降低了这些细胞保护作用。结果表明，低浓度NO通过产生cGMP和激活蛋白激酶G. Less来保护RAW264细胞免受h_2o_2诱导的凋亡

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