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W. J. O'Brien, T. Heimann, F. Rizvi; Superoxide Production by NADPH Oxidases in Human Corneal Epithelial and Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4634.
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The purpose of these studies was to determine whether human corneal epithelial and stromal cells were capable of producing superoxide (O2.-) via NADPH oxidases, enzyme complexes believed to be responsible for the localized and limited production of O2.- possessing cell signaling activity.
Human 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 NADPH oxidases. The RT PCR products were sequenced to confirm their identity. siRNA pools designed to degrade sequence specific homologous mRNAs encoding NOX proteins were used to knockdown oxidase activity. The production of proteins composing the oxidase complex was confirmed using western blots. The production of O2.- by whole cells and cell-free preparations was assessed by measurement of NADPH-dependent, superoxide dismutase (SOD) inhibitable cytochrome C reduction.
Permeabilized whole cells and cell-free extracts of corneal epithelial and stromal cells produced O2.- in an NADPH-dependent manner. Human corneal epithelial and stromal cells constitutively expressed mRNAs including NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac. Western blots confirmed the existence of all the proteins required for O2.- production. Stromal cells produced 3 isoforms of NOX thus may contain multiple forms of the oxidase complex. Both epithelial and stromal cells expressed isoforms of Rac, a putative regulator of the activity of the complex. siRNAs specific for NOX5 knocked down levels of the specific transcript thereby confirming the expression of the NOX5 isoform of the oxidase.
Human corneal epithelial and stromal cells constitutively expressed NADPH oxidase complexes in at least 3 isoforms. O2.- was produced by the oxidation of NADPH but not NADH. The O2.- produced by NADPH oxidase is a potential modulator of signal transduction pathways.
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