Expression of NADPH Oxidase in Rabbit Corneal Epithelial and Stromal Cells in Culture

Abstract

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.