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

✍ Scribed by Ana Cristina Rego; Catarina R. Oliveira

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 136 KB
• Volume: 51
• Category: Article
• ISSN: 0360-4012
• DOI: 10.1002/(sici)1097-4547(19980301)51:5<627::aid-jnr10>3.0.co;2-#

✦ Synopsis

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 Fe 2؉ and ascorbate or Fe 2؉ alone and compared to H 2 O 2 -induced maximal cellular oxidation, and was measured by following the formation of intracellular hydroperoxides with the probe DCFH 2 (2Ј,7Ј-dichlorodihydrofluorescein). After a 15-min exposure to the oxidants, formation of hydroperoxides was significantly increased in the presence of 100 µM Fe 2؉ (about twofold), as compared to the control. Coadministration of Fe 2؉ and ascorbate (Fe-Asc) did not affect DCF fluorescence, but highly reduced the intracellular pH (pH i ‫؍‬ 6.32 ؎ 0.08), in comparison with control conditions (pH i ‫؍‬ 7.05 ؎ 0.11), as determined with the probe BCECF (2Ј,7Ј-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 [ 14 C]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.