School of Pharmacy and Pharmaceutical Research and Technology Institute, Kinki University, Higashi-Osaka, Osaka, Japan.
Curr Eye Res. 2012 Oct;37(10):889-97. doi: 10.3109/02713683.2012.689070. Epub 2012 May 25.
We previously found two mechanisms for the dysfunction in Ca(2+) regulation caused by excessive nitric oxide (NO) using the lenses of hereditary cataract model rats: the first is that NO causes a decrease in Adenosine-5'-triphosphate (ATP) level via cytochrome c oxidase (CCO), resulting in a decrease in ATPase function; the second is that NO causes enhanced lipid peroxidation, resulting in the oxidative inhibition of Ca(2+)-ATPase. In this study, we demonstrate the effect of excessive NO on lipid peroxidation and ATP production in human lens using a human lens epithelial cell line, SRA 01/04 (human lens epithelial (HLE) cells).
Excessive NO via inducible NO synthase (iNOS) was induced by stimulating cells with a combination of interferon-gamma (1000 IU IFN-γ) and lipopolysaccharide (100 ng/mL LPS). CCO activity was measured using a Mitochondrial Isolation kit and Cytochrome c Oxidase Assay kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200.
Cytochrome c oxidase activity and ATP levels were decreased in HLE cells stimulated with IFN-γ and LPS, and aminoguanidine (AG) and diethyldithiocarbamate (DDC) added 6 h before cell collection significantly attenuated these decreases in cells stimulated with the IFN-γ and LPS for 24-30 h. However, the lower CCO activity and ATP levels in HLE cells stimulated with the IFN-γ and LPS for 30 h were not changed by treatment with AG or DDC for 6-12 h, while the CCO activity and ATP levels in HLE cells treated with AG or DDC for 18 were recovered.
Excessive NO causes a decrease in CCO activity and ATP levels, and the recovery time for CCO activity is related to exposure time to NO in HLE cells.
我们之前使用遗传性白内障模型大鼠的晶状体发现了两种由过量一氧化氮（NO）引起的钙（Ca 2+）调节功能障碍机制：第一种是 NO 通过细胞色素 c 氧化酶（CCO）导致三磷酸腺苷（ATP）水平降低，从而降低 ATP 酶功能；第二种是 NO 引起脂质过氧化增强，导致 Ca 2+-ATP 酶的氧化抑制。在这项研究中，我们使用人晶状体上皮细胞系 SRA 01/04（人晶状体上皮（HLE）细胞）证明了过量 NO 对人晶状体中脂质过氧化和 ATP 产生的影响。
通过用干扰素-γ（1000 IU IFN-γ）和脂多糖（100 ng/mL LPS）刺激细胞来诱导诱导型一氧化氮合酶（iNOS）产生过量的 NO。使用线粒体分离试剂盒和细胞色素 c 氧化酶测定试剂盒测定 CCO 活性，使用 Sigma ATP 生物发光测定试剂盒和光度计 AB-2200 测定 ATP 水平。
IFN-γ 和 LPS 刺激的 HLE 细胞中 CCO 活性和 ATP 水平降低，在收集细胞前 6 小时添加氨基胍（AG）和二乙基二硫代氨基甲酸盐（DDC）可显著减弱 IFN-γ 和 LPS 刺激 24-30 小时的细胞中的这些降低。然而，IFN-γ 和 LPS 刺激 30 小时的 HLE 细胞中的较低 CCO 活性和 ATP 水平不能通过 6-12 小时的 AG 或 DDC 处理改变，而用 AG 或 DDC 处理 18 小时的 HLE 细胞中的 CCO 活性和 ATP 水平恢复。
过量的 NO 导致 CCO 活性和 ATP 水平降低，CCO 活性的恢复时间与人晶状体上皮细胞中 NO 的暴露时间有关。
