Purpose: To identify the presence of endogenous superoxide anion-generating system in rabbit lens epithelial cells.
Methods: The generation of superoxide anion was quantified by using lucigenin-amplified chemiluminescence (LUCL) in intact rabbit lens epithelial cells (N/N 1003A). The LUCL readings were recorded with a luminometer (LumiStar BMG) immediately upon arachidonic acid (AA) addition. To test the effects of superoxide dismutase (SOD) and DPI (non-specific inhibitor to NADPH oxidase), cells were preincubated for 30 minutes with indicated concentrations of inhibitors before stimulation with AA. The expression of NOX family, including NOX1, NOX2 or gp91(phox), NOX3, NOX4 and NOX5, was detected by RT-PCR.
Results: AA at dosage of 30-90 microM proportionally induced luminescence in N/N 1003A cells. Production of superoxide occurred quickly within 30 seconds of AA addition, reached the highest level after 200-260 seconds and dissipated after 600 seconds. The amount of superoxide anions, expressed as relative light unit of luminescence (RLU), was proportional to the concentration of AA used. Dose-dependent effect could be seen. Superoxide generation was inhibited in N/N1003A cells preloaded with SOD. DPI eliminated AA-induced superoxide anion generation. RT-PCR using primers specific for mRNAs of the five isoforms of the NOX proteins documented that mRNA encoding NOX1 through NOX5 were constitutively present in N/N1003A cells. The expression of NOX1 was much weaker than that of the other four NOX isoforms.
Conclusions: NADPH oxidase complex is involved in superoxide anion generation in rabbit lens epithelial cells. N/N 1003A cells constitutively produce mRNA encoding five isoforms of NOX proteins.
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