Modulation of apoptosis by activation of protein kinasse G

我们研究了低浓度一氧化氮（NO）对NO和过氧化氢诱导的小鼠巨噬细胞系RAW 264细胞死亡的保护作用及其机制。高浓度（4 mM）SNP诱导RAW 264细胞死亡。用100 μM SNP或1 mM dibutyl-cGMP预处理RAW 264细胞可减少线粒体释放细胞色素c，并阻止4 mM SNP诱导的细胞死亡。LY 83583可降低SNP预处理的作用。用二丁基-cGMP预处理以浓度依赖性方式防止由NOC 18、GSNO或SNP诱导的细胞死亡。用二丁基-cGMP预处理阻止NO供体诱导的细胞色素c释放。蛋白激酶G抑制剂KT 5823可明显减弱SNP或dibutyl-cGMP的保护作用。这些结果表明，低浓度的NO通过产生cGMP和激活PKG来保护RAW 264细胞免受NO的细胞毒性。 ...更多信息 在细胞死亡中观察到DRIA。未处理的RAW 264细胞未显示Bax N-20抗体（针对Bax的氨基酸11-30产生的pAb）相关的免疫反应性。然而，一些细胞在4 mM SNP处理后显示出Bax免疫染色的点状胞质模式。Bax阳性细胞呈弥漫性细胞色素c免疫染色。4 mM SNP处理后Bax阳性细胞数增加。用100 μM SNP或1 mM dibutyl-cGMP预处理可抑制4 mM SNP诱导的Bax易位和Bax阳性细胞的增加。高浓度SNP诱导的p38 MAP激酶激活可被低浓度SNP或二丁基cGMP预处理所阻止。这些结果表明，NO/cGMP信号通路通过抑制p38 MAP激酶的激活，从而导致N端构象改变和Bax的移位，抑制NO诱导的巨噬细胞凋亡。（2-氨乙基）-1-三氮烯（NOC 18）在低浓度下作用24 h，可减少H_2O_2诱导的细胞死亡、caspase-3激活和核碎裂。可溶性鸟苷酸环化酶抑制剂LY 83583和ODQ可抑制SNP和NOC 18预处理的保护作用。蛋白激酶G抑制剂KT 5823也显着降低这些细胞保护作用。提示低浓度NO通过产生cGMP和激活蛋白激酶G对H_2O_2诱导的RAW 264细胞凋亡具有保护作用。少

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