Expression of nonphagocytic NADPH oxidase system in the ocular lens.

PURPOSE The primary goal of this study was to characterize the Rac GTPase associated, NADPH oxidase-mediated Reactive Oxygen Species (ROS)-generating system in the lens tissue. METHODS NADPH oxidase activity in lens tissue was determined by quantifying superoxide-induced lucigenin photoemission. Immunological and PCR/RT-PCR techniques were utilized to determine expression of different components of the NADPH oxidase system in lens tissue. Growth factor stimulated ROS production was determined quantitatively in human lens epithelial cells using dichlorofluorescein diacetate. RESULTS Lens homogenates from different species showed generation of superoxide in a lucigenin-enhanced chemiluminescence assay in the presence of NADPH. This activity was found to be lens protein concentration dependent, heat sensitive, and inhibitable by superoxide dismutase and the flavoprotein inhibitor, diphenyleneiodonium (DPI). The distribution of superoxide generating activity in lens was confined predominantly to the lens epithelium, with very low levels in cortex and none in the nucleus. Immunological assays have demonstrated the presence of p67phox and p47phox in lens tissue, while PCR and RT-PCR reactions amplified DNA products corresponding to the p67phox, p40phox, p22phox, gp91phox, and Rac1 components of the NADPH oxidase complex from human and mouse lens cDNA libraries. Serum starved human lens epithelial cells stimulated with different growth factors including EGF, b-FGF, PDGF, TGF-beta, and LPA demonstrated increased production of ROS, a response which was blocked by inhibitors of NADPH oxidase, such as DPI and the antioxidant-N-acetyl cysteine (NAC). RT-PCR analysis of human lens RNA confirmed readily detectable levels of expression of low molecular weight protein tyrosine phosphatase (LMW-PTP), which is a well-characterized target of redox signaling pathway(s). CONCLUSIONS These data demonstrate the presence of a functional nonphagocytic NADPH oxidase system in lens that is predominantly localized to the lens epithelium. Several growth factors appear to stimulate the activity of lens NADPH oxidase, resulting in increased production of ROS in lens epithelial cells, indicating that redox signaling may have an important role in growth factor effects on lens growth and development.

目的 本研究的主要目的是描述晶状体组织中与Rac GTP酶相关的、NADPH氧化酶介导的活性氧（ROS）生成系统的特征。 方法 通过定量超氧化物诱导的光泽精光发射来测定晶状体组织中的NADPH氧化酶活性。利用免疫学和PCR/RT - PCR技术来测定NADPH氧化酶系统不同组分在晶状体组织中的表达。使用二氯荧光素二乙酸酯定量测定人晶状体上皮细胞中生长因子刺激的ROS产生。 结果 在不同物种的晶状体匀浆中，在有NADPH存在的情况下，光泽精增强化学发光测定显示有超氧化物生成。发现这种活性依赖于晶状体蛋白浓度，对热敏感，并且可被超氧化物歧化酶和黄素蛋白抑制剂二亚苯基碘鎓（DPI）抑制。晶状体中超氧化物生成活性的分布主要局限于晶状体上皮，在皮质中水平极低，在核中没有。免疫学测定已证明晶状体组织中存在p67phox和p47phox，而PCR和RT - PCR反应从人和小鼠晶状体cDNA文库中扩增出了与NADPH氧化酶复合物的p67phox、p40phox、p22phox、gp91phox和Rac1组分相对应的DNA产物。用包括表皮生长因子（EGF）、碱性成纤维细胞生长因子（b - FGF）、血小板源性生长因子（PDGF）、转化生长因子 - β（TGF - β）和溶血磷脂酸（LPA）在内的不同生长因子刺激血清饥饿的人晶状体上皮细胞，显示ROS产生增加，这种反应可被NADPH氧化酶抑制剂如DPI和抗氧化剂 - N - 乙酰半胱氨酸（NAC）阻断。对人晶状体RNA的RT - PCR分析证实了低分子量蛋白酪氨酸磷酸酶（LMW - PTP）的表达水平很容易检测到，它是氧化还原信号通路的一个特征明确的靶点。 结论 这些数据表明晶状体中存在一个功能性的非吞噬性NADPH氧化酶系统，主要定位于晶状体上皮。几种生长因子似乎可刺激晶状体NADPH氧化酶的活性，导致晶状体上皮细胞中ROS产生增加，这表明氧化还原信号在生长因子对晶状体生长和发育的影响中可能具有重要作用。