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

Abstract

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.

Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress

“…Moreover, NOS inhibitors protected against excitotoxic neuronal injury [43]. Although we found previously that nitro G -w-arginine reduced the activity of NOS in retinal cells in culture [29], exposure to nitro G -w-arginine was not effective against retinal cell injury up to 12 h upon chemical ischemia, hypoglycemia or oxidative stress ( Table 1). The failure of nitro G -warginine to increase the viability of retinal cells upon hypoglycemia or ischemia may also be related with a reduced involvement of the NMDA receptors in cell injury (Table 1), because the generation of nitric oxide has been implicated in the excitotoxic neuronal injury mediated by activation of the NMDA receptors [43,44].…”

“…By using nitrendipine [Ca 2ϩ ] i changes, in response to activation of ionotropic glutamate receptors [27] or upon K ϩ -depolarization [28], were previously shown to involve the L-type Ca 2ϩ channels in retinal cells in culture. Furthermore, we showed that nitro G -w-arginine decreased the activity of NOS in retinal cells in culture [29] by decreasing the conversion of citrulline from arginine. Nevertheless, retinal cell injury induced by oxidative stress, chemical ischemia or hypoglycemia was not significantly affected after incubation with MK-801, nitrendipine or nitro G -w-arginine ( Na ϩ medium without Mg 2ϩ , but containing Ca 2ϩ , for 15 min plus 12 h postincubation, did not influence significantly the release of LDH (not shown).…”

“…The failure of nitro G -warginine to increase the viability of retinal cells upon hypoglycemia or ischemia may also be related with a reduced involvement of the NMDA receptors in cell injury (Table 1), because the generation of nitric oxide has been implicated in the excitotoxic neuronal injury mediated by activation of the NMDA receptors [43,44]. Recently, we showed an inhibition of NOS activity after 15 min ascorbate/Fe 2ϩ -induced oxidative stress in retinal cells [29], explaining the inefficacy of the inhibitor of NOS after withdrawal of the oxidant inducers.…”

Stress‐induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide

“…2008), and DCF fluorescence were measured in retinal cells using a spectrometer (e.g. Rego and Oliveira 1998; Cacicedo et al. 2005), but not the combination of time‐lapse in the retina.…”

FK506 prevents mitochondrial-dependent apoptotic cell death induced by 3-nitropropionic acid in rat primary cortical cultures

“…Measurement of intracellular pH (pHi) was assessed by following the changes in fluorescence of the pH-sensitive probe 2V,7V-bis-(carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) similarly as described by Rego and Oliveira (1998). Cortical cells were loaded with 5 AM BCECF-AM for 45 min, at 378C, in culture medium, in an atmosphere of 95% air and 5% CO 2 , to avoid major changes in cellular pH.…”