Hong Jangja, Nakano Yasuhiro, Yokomakura Aya, Ishihara Kenji, Kim Soonjoo, Kang Young-Sook, Ohuchi Kazuo
Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
J Pharmacol Exp Ther. 2006 Nov;319(2):672-81. doi: 10.1124/jpet.106.109280. Epub 2006 Aug 8.
In the mouse leukemic monocyte cell line RAW 264.7, the vacuolar-type (H(+))-ATPase (V-ATPase) inhibitors bafilomycin A1 and concanamycin A induced nitric oxide (NO) production through the expression of inducible nitric-oxide synthase mRNA and its protein and decreased cell growth and survival as determined by 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bafilomycin A1 and concanamycin A activated nuclear factor (NF)-kappaB and activator protein-1 and decreased the level of IkappaB-alpha and increased that of phosphorylated c-Jun N-terminal kinase (JNK). NO production induced by these V-ATPase inhibitors was suppressed by the NF-kappaB inhibitor Bay 11-7082 [(E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one] in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na(+),K(+)-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce NO production at which concentrations the MTT response was decreased. The NO donor S-nitroso-N-acetyl-dl-penicillamine further lowered the V-ATPase inhibitor-induced decrease in the MTT response, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) alleviated it partially. Mitochondrial depolarization, an index of apoptosis, was induced by bafilomycin A1 and concanamycin A. On treatment with the nitric-oxide synthase inhibitor N(G)-monomethyl-l-arginine acetate, the disruption of mitochondrial membrane potential induced by bafilomycin A1 and concanamycin A was alleviated partially in parallel with the decrease in NO production. Carboxy-PTIO also alleviated it partially. Our findings suggest that the V-ATPase inhibitors bafilomycin A1 and concanamycin A similarly induce NO production and the newly produced NO participates partially in the V-ATPase inhibitor-induced apoptosis in RAW 264.7 cells.
在小鼠白血病单核细胞系RAW 264.7中，液泡型（H(+)）-ATP酶（V-ATP酶）抑制剂巴弗洛霉素A1和 concanamycin A通过诱导型一氧化氮合酶mRNA及其蛋白的表达诱导一氧化氮（NO）生成，并如通过3-（4,5-二甲基（噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）分析所测定的那样降低细胞生长和存活率。巴弗洛霉素A1和concanamycin A激活核因子（NF）-κB和激活蛋白-1，并降低IκB-α水平，提高磷酸化c-Jun氨基末端激酶（JNK）的水平。这些V-ATP酶抑制剂诱导的NO生成被NF-κB抑制剂Bay 11-7082 [（E）-3- [（4-甲基苯基）磺酰基] -2-丙烯腈]和JNK抑制剂SP600125 [蒽[1,9-cd]吡唑-6（2H）-酮]抑制，同时V-ATP酶抑制剂诱导的MTT反应降低得到部分缓解。Na(+)，K(+)-ATP酶抑制剂丁卡因和F-ATP酶抑制剂寡霉素在降低MTT反应的浓度下未诱导NO生成。NO供体S-亚硝基-N-乙酰基-dl-青霉胺进一步降低V-ATP酶抑制剂诱导的MTT反应降低，而NO清除剂2-（4-羧基苯基）-4,4,5,5-四甲基咪唑啉-1-氧基-3-氧化物钠盐（羧基-PTIO）部分缓解了这种降低。线粒体去极化是细胞凋亡的一个指标，可以被巴弗洛霉素A1和concanamycin A诱导。在用一氧化氮合酶抑制剂N(G)-单甲基-L-精氨酸醋酸盐处理后，巴弗洛霉素A1和concanamycin A诱导的线粒体膜电位破坏与NO生成的减少同时得到部分缓解。羧基-PTIO也部分缓解了这种破坏。我们的研究结果表明，V-ATP酶抑制剂巴弗洛霉素A1和concanamycin A同样诱导NO生成，新生成的NO部分参与了RAW 264.7细胞中V-ATP酶抑制剂诱导的细胞凋亡。
