Modulation of apoptosis by activation of protein kinasse G

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15590082
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Setsunan University
• Principal Investigator: MAEDA Sadaaki Setsunan Univ., Faculty of Pharmaceut.Sci., Professor, 薬学部, 教授 (00135732)
• Co-Investigator(Kenkyū-buntansha): MATSUDA Toshio Osaka Univ., Faculty of Pharmaceut.Sci., Professor, 薬学部, 教授 (00107103) ; YOSHIOKA Yasuhiro Setsunan Univ., Faculty of Pharmaceut.Sci., Research associate, 薬学部, 助手 (40330360) ; YAMAMURO Akiko Setsunan Univ., Faculty of Pharmaceut.Sci., Research associate, 薬学部, 助手 (20340862)
• Project Period (FY): 2003 – 2004
• Keywords: Nitric oxide / Apoptosis / cyclic GMP / Protein kinase G / Hydrogen peroxide / RAW264 cell / Bax / p38 MAP kinas

Research Abstract

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 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.