Abstract
Abstract: :
Purpose:Superoxide may be produced by any of several enzyme systems in cells. The goal of these studies was to determine whether corneal cells could produce superoxide via a neutrophil like NADPH oxidase. Methods:Cell cultures of either rabbit corneal epithelial or stromal cells were established and treated with proinflammatory cytokines. RNA was isolated to evaluate transcription of the genes for each of the proteins that compose the oxidase complex by RT–PCR and cDNA sequencing. Proteins were isolated from cultured cells and steady state levels of each of the proteins in the complex assessed by Western blotting. The ability of the complex to produce superoxide was measured by cytochrome C reduction and electron spin resonance. Results:Fractionation of cultures of either corneal epithelial cells or stromal cells produced membrane fractions capable of producing superoxide in a manner that was inhibitable by DPI, superoxide dismutase (SOD), and gp91ds–tat. SOD inhibitable superoxide production was not inhibited by allopurinol, rotenone, L–NAME, 1400W or azide. Superoxide was produced using NADPH but not NADH as the substrate. Epithelial cells produced superoxide constitutively at 0.98 +/– 0.08 nmole/min/mg and stromal cells at 1.26 +/– 0.07 nmole/min/mg (n= 4). Sequencing of RT–PCR products from total RNA documented that the messages for each of the proteins required to produce a functional NADPH complex including the regulatory protein RAC were present in both cell types. The data indicated some interesting differences between the complexes contained in the two cell types. Stromal cells contained NOX4 and not gp91phox and the complex functioned in the absence of p40phox. Corneal epithelial cells contained either NOX1 or NOX4 or both as well as p40phox. Conclusions:Corneal epithelial and stromal cells produce superoxide in a constitutive manner from NADPH using NOX1 or NOX4 type NADPH complexes. The superoxide produced in this manner may function as a second messenger in signal transduction cascades that regulate corneal inflammation and neovascularization.
Keywords: cornea: basic science • oxidation/oxidative or free radical damage • inflammation