Dedkova Elena N, Ji Xiang, Lipsius Stephen L, Blatter Lothar A
Department of Physiology, Loyola University Chicago, 2160 S. First Ave., Maywood, IL 60153, USA.
Am J Physiol Cell Physiol. 2004 Feb;286(2):C406-15. doi: 10.1152/ajpcell.00155.2003. Epub 2003 Sep 24.
Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]mt). Stimulation of mitochondrial Ca2+ uptake by exposure to different cytoplasmic Ca2+ concentrations (1, 2, and 5 microM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]mt was sensitive to the NOS antagonist l-N5-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca2+ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl-beta-cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca2+ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis.
尽管一氧化氮（NO）是血管内皮细胞中已知的细胞呼吸调节剂，但这些细胞中线粒体特异性一氧化氮合酶（mtNOS）的存在仍是一个有争议的问题。我们使用激光扫描共聚焦显微镜结合对NO敏感的荧光染料DAF-2来监测小牛血管内皮（CPAE）细胞线粒体中NO产生的变化。细胞用膜通透性的对NO敏感的染料4,5-二氨基荧光素（DAF-2）二乙酸酯加载，随后用洋地黄皂苷通透以去除胞质DAF-2，从而能够测量线粒体中的NO产生（[NO]mt）。通过暴露于不同的细胞质Ca2+浓度（1、2和5 microM）刺激线粒体Ca2+摄取，导致线粒体NO产生呈剂量依赖性增加。[NO]mt的这种增加对NOS拮抗剂L-N5-（1-亚氨基乙基）鸟氨酸和钙调蛋白拮抗剂氯米达唑（R-24571）敏感，表明NO合成的内源性起源及其对钙调蛋白的依赖性。用质子载体FCCP使线粒体膜电位崩溃，或用钌红阻断线粒体Ca2+单向转运体，以及用抗霉素A联合寡霉素阻断呼吸链，均抑制线粒体NO产生。添加NO供体精胺NONOate导致DAF-2荧光显著增加，而这些处理均未对其产生影响。DAF-2信号的线粒体起源通过与线粒体标记物MitoTracker Red共定位以及观察到用甲基-β-环糊精破坏小窝（细胞质NOS所在位置）形成并不能阻止DAF-2荧光增加而得到证实。线粒体钙摄取的激活刺激mtNOS磷酸化（在Ser-1177处），而FCCP可阻止这种磷酸化。数据表明，刺激线粒体Ca2+摄取可激活CPAE细胞线粒体中的NO产生。这表明存在一种线粒体特异性NOS，其可对NO对细胞呼吸、膜电位和细胞凋亡提供快速的局部调节作用。
