O'Brien William J, Krema Cinder, Heimann Tom, Zhao Hongtao
Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, USA.
Invest Ophthalmol Vis Sci. 2006 Mar;47(3):853-63. doi: 10.1167/iovs.05-1063.
Reactive oxygen- and nitrogen-containing molecules produced in high concentrations are mediators of tissue damage caused by inflammation. The free radical molecules superoxide (O2-) and nitric oxide (NO), when produced at low concentrations, may function as second messengers or regulators of signal transduction. The purpose of these studies was to determine whether corneal epithelial and stromal cells are capable of producing O2-* via an NADPH oxidase complex.
Rabbit corneal epithelial and stromal cells, grown as primary cultures and low-passage isolates, were used as the sources of RNA for RT-PCR with primers specific for mRNAs encoding the proteins that comprise an NADPH oxidase complex. The RT-PCR products were sequenced to confirm their identities. The production of proteins composing the oxidase complex was confirmed, and the proteins were identified by Western blot analysis. The production of superoxide in cell-free preparations was assessed by measurement of NADPH-dependent superoxide dismutase (SOD)-inhibitable cytochrome c reduction and by electron paramagnetic resonance (EPR) with a superoxide specific spin trap.
Cell-free extracts of corneal epithelial and stromal cells produced superoxide in an NADPH-dependent manner, and this production was inhibited by SOD. EPR confirmed the identity of the reaction product as superoxide anion. Both rabbit corneal epithelial and stromal cells constitutively produced mRNAs encoding five proteins known to comprise a classic neutrophil-like NADPH oxidase complex. Production of NOX4, p22phox, p47phox, p67phox, and p40phox was confirmed by Western blot. Both epithelial and stromal cells expressed isoforms of Rac, a putative regulator of the activity of the complex.
A constitutively expressed NADPH oxidase complex that includes NOX4 is a source of O2-* produced by rabbit corneal epithelial and stromal cells. Superoxide produced by the oxidation of NADPH via the NADPH oxidase complex is a potential contributor to signal transduction pathways as well as a potential participant in processes that occur during inflammation.
高浓度产生的活性氧和含氮分子是炎症所致组织损伤的介质。自由基分子超氧阴离子（O2-）和一氧化氮（NO）在低浓度产生时，可能作为第二信使或信号转导的调节因子。这些研究的目的是确定角膜上皮细胞和基质细胞是否能够通过NADPH氧化酶复合物产生O2-*。
以原代培养和低代分离培养的兔角膜上皮细胞和基质细胞作为RNA来源，用编码构成NADPH氧化酶复合物的蛋白质的mRNA特异性引物进行RT-PCR。对RT-PCR产物进行测序以确认其身份。确认了构成氧化酶复合物的蛋白质的产生，并通过蛋白质印迹分析鉴定了这些蛋白质。通过测量NADPH依赖性超氧化物歧化酶（SOD）抑制的细胞色素c还原以及使用超氧阴离子特异性自旋捕获剂的电子顺磁共振（EPR）来评估无细胞制剂中超氧化物的产生。
角膜上皮细胞和基质细胞的无细胞提取物以NADPH依赖性方式产生超氧化物，并且这种产生被SOD抑制。EPR证实反应产物为超氧阴离子。兔角膜上皮细胞和基质细胞均组成性地产生编码已知构成经典中性粒细胞样NADPH氧化酶复合物的五种蛋白质的mRNA。通过蛋白质印迹证实了NOX4、p22phox、p47phox、p67phox和p40phox的产生。上皮细胞和基质细胞均表达Rac的亚型，Rac是该复合物活性的假定调节因子。
包括NOX4的组成性表达的NADPH氧化酶复合物是兔角膜上皮细胞和基质细胞产生O2-*的来源。通过NADPH氧化酶复合物氧化NADPH产生的超氧化物是信号转导途径的潜在贡献者，也是炎症过程中发生的过程的潜在参与者。
