Alteration of nitric oxide synthase activity upon oxidative stress in cultured retinal cells

In the present study we examined the effect of oxidative stress-mediated hydroperoxide formation on the activity of nitric oxide synthase (NOS) in retinal cells in culture. Oxidative stress was induced in the presence of Fe2+ and ascorbate or Fe2+ alone and compared to H2O2-induced maximal cellular oxidation, and was measured by following the formation of intracellular hydroperoxides with the probe DCFH2 (2',7'-dichlorodihydrofluorescein). After a 15-min exposure to the oxidants, formation of hydroperoxides was significantly increased in the presence of 100 microM Fe2+ (about twofold), as compared to the control. Coadministration of Fe2+ and ascorbate (Fe-Asc) did not affect DCF fluorescence, but highly reduced the intracellular pH (pHi = 6.32 +/- 0.08), in comparison with control conditions (pHi = 7.05 +/- 0.11), as determined with the probe BCECF (2',7'-bis-(carboxyethyl)-5(and-6) carboxyfluorescein). Nevertheless, preincubation of Fe-Asc at acidic pH also increased the formation of hydroperoxides. Oxidative stress induced in the presence of Fe-Asc (at pH 6.5) significantly decreased the activity of NOS by 20% of control activity, as determined by the formation of [14C]citrulline. Fe-Asc (pH 6.5) also reduced the production of cyclic GMP (cGMP) in retinal cells by 1.5-fold, although a decrement in pH from 7.4 to 6.5 was not sufficient to decrease cGMP production. These data suggest that NO. production may be compromised in the presence of Fe-Asc. Moreover, neither 4 mM dithiotreitol (DTT) nor 4 mM glutathione (GSH) altered the production of cGMP in retinal cells submitted to oxidative stress. A reduction in NO. generation upon oxidative stress may reduce major damaging effects induced by ONOO- in cultured retinal cells.