Dual Effect of Lipid Peroxidation on the Membrane Order of Retinal Cells in Culture

The effect of lipid peroxidation, induced by ascorbic acid and ferrous sulfate (Fe2+) at pH 7.4 or pH 6.5, on the membrane order of retinal cells in culture was examined. Membrane order was measured by fluorescence anisotropy using 1-[4-(trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene as a fluorescent probe. Alterations of cellular membrane order were correlated with the susceptibility to peroxidation and viability of these cells. At pH 7.4, 1.5 mM ascorbate/7.5 μM Fe2+ induced a low production of thiobarbituric acid-reactive substances (3.47 ± 0.26 nmol TBARS/mg protein), while 5 mM ascorbate/100 μM Fe2+ significantly increased TEARS production to 11.17 ± 1.43 nmol/mg protein. At pH 6.5, in the presence of 5 mM ascorbate/100 μM Fe2+, cellular oxidation was mostly increased following 15 min incubation (19.33 ± 1.66 nmol TBARS/mg protein) and decreased thereafter as a result of a prolonged exposure to the oxidizing agents to levels of 11.02 ± 0.66 nmol TBARS/mg protein, after 180 min peroxidation. The membrane order of control retinal cells treated at pH 6.5 was not changed compared to controls at pH 7.4. Moreover, the membrane order of retinal cells peroxidized at pH 7.4 was not significantly different compared to controls, in the absence of ascorbate/Fe2+. However, significant time-dependent alterations were found in the membrane order of cells peroxidized with 5 mM ascorbate/100 μM Fe2+, at pH 6.5: cellular membrane order decreased after 15 min peroxidation, while longer peroxidative incubation periods, from 60 and up to 180 min, induced an increase in the membrane order. The dual effect of lipid peroxidation, under moderately acidic conditions (pH 6.5), on the membrane order of retinal cells was shown to be prevented upon cellular pretreatment with vitamin E, supplemented to the culture medium. Moreover, vitamin E pretreatment increased the viability of control retinal cells and reduced the production of TBARS after 15 min peroxidation with 5 mM ascorbate/100 μM Fe2+, at pH 6.5. Vitamin E was also shown to reduce conjugated dienes formation after 15 or 60 min peroxidation at pH 6.5. These data suggest that when cells are submitted to oxidative stress the changes in the physical properties of the membranes depend upon the antioxidant status of the living cells. The enhancement of peroxidation observed at pH 6.5 may help to explain oxidative cell damage occurring during ischemia/reperfusion.