Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
Invest Ophthalmol Vis Sci. 2010 May;51(5):2575-86. doi: 10.1167/iovs.09-4303. Epub 2009 Dec 3.
To determine whether retinal neurons become more susceptible to injury by nitric oxide (NO) under hypoxic conditions.
Cells from the RGC-5 line were exposed to different concentrations (0.1-100 microM) of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, under normoxic and hypoxic (1.0% O(2)) conditions with 5.5 mM glucose or with no glucose. In some experiments, carboxy-PTIO, a scavenger of NO, was added with SNAP. The SNAP-induced cell injury was determined by the WST-8 assay and by the assessment of phosphatidylserine externalization and changes in hypodiploid DNAs. Alterations of mitochondrial membrane potential, superoxide anion formation, cellular adenosine triphosphate (ATP) contents, and caspase activity were also determined after exposure to SNAP.
Exposure of RGC-5 cells to SNAP (100 microM) significantly decreased the number of living cells cultured under hypoxic conditions with or without glucose. Coadministration of carboxy-PTIO (1.0 microM) suppressed SNAP-induced cell death. SNAP-induced cell death of cells cultured under hypoxia with glucose was accompanied by increased expression of phosphatidylserine and hypodiploid DNAs. These findings indicated that death was mediated in part by apoptosis. In addition, loss of mitochondrial membrane potential, increase of superoxide formation, and activation of caspase was observed. Cyclosporine A, TEMPOL, and Z-VAD-FMK suppressed cell death. On the other hand, SNAP depleted the ATP contents of cells cultured under hypoxia without glucose, causing mainly necrotic cell death.
These results indicate that RGC-5 cells become susceptible to SNAP under hypoxic conditions in which NO may have greater impact on mitochondrial function.
确定在缺氧条件下，视网膜神经元是否更容易受到一氧化氮 (NO) 的损伤。
将 RGC-5 细胞系暴露于不同浓度（0.1-100 μM）的 S-亚硝基-N-乙酰青霉胺 (SNAP) 下，这是一种 NO 供体，在正常氧和缺氧（1.0% O2）条件下，使用 5.5 mM 葡萄糖或无葡萄糖。在一些实验中，用 SNAP 加入了 NO 清除剂羧基-PTIO。通过 WST-8 测定和磷脂酰丝氨酸外翻以及低二倍体 DNA 的变化来确定 SNAP 诱导的细胞损伤。还在暴露于 SNAP 后测定了线粒体膜电位、超氧阴离子形成、细胞三磷酸腺苷 (ATP) 含量和半胱天冬酶活性的变化。
将 RGC-5 细胞暴露于 SNAP（100 μM）显著降低了在有或无糖的缺氧条件下培养的活细胞数量。共给予羧基-PTIO（1.0 μM）抑制了 SNAP 诱导的细胞死亡。在有葡萄糖的缺氧条件下培养的细胞中，SNAP 诱导的细胞死亡伴随着磷脂酰丝氨酸和低二倍体 DNA 的表达增加。这些发现表明死亡部分是由细胞凋亡介导的。此外，观察到线粒体膜电位丧失、超氧形成增加和半胱天冬酶激活。环孢素 A、TEMPOL 和 Z-VAD-FMK 抑制细胞死亡。另一方面，SNAP 耗尽了在无糖缺氧条件下培养的细胞的 ATP 含量，主要导致坏死性细胞死亡。
这些结果表明，在缺氧条件下，RGC-5 细胞对 SNAP 变得敏感，NO 可能对线粒体功能产生更大的影响。
