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L. D. Carter-Dawson, R. Rojas, X. Zhao, C. K. Mitchell, A. Chuang; Mechanism of Albumin Protection of RGC-5 Cells Against Oxidative Insult. Invest. Ophthalmol. Vis. Sci. 2009;50(13):119.
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We previously reported that albumin can protect RGC-5 cells in vitro against oxidative insult generated by SIN-1 in a concentration dependent manner. The current study investigates the mechanism of this protective effect, specifically whether this effect is due to albumin ligands, the reactive thiol at cysteine-34 (Cys-34), the drug binding site, or to changes in osmolarity.
To investigate whether albumin ligands contribute to albumin’s protective effect, RGC-5 cell viability (MTT assay) was quantified after exposure to 0 - 2.5g/L fraction V bovine serum albumin (BSA) derived from serum or 0 - 2.5g/L human recombinant albumin (rHSA) devoid of ligands, in normoxic or in oxidative conditions generated by treatment with 2mM SIN-1. Recombinant albumin with a methionine substitution at Cys-34 and a recombinant of domain II were evaluated at varied concentrations for their effect on cell viability in normoxic and oxidative conditions. Cell viabilities were compared between treatment conditions using two-way ANOVA. Differences were considered significant at the 5% level (post-hoc multiple comparisons). A paired t-test was used to compare EC50 values. Osmolarity was measured for each form and concentration of albumin and evaluated for changes.
No significant differences in osmolarity were detected between concentrations or between the different forms of albumin tested in the study. RGC-5 cells experiencing oxidative insult showed similar viabilities when treated with fraction V BSA or with rHSA, indicating no contribution from albumin ligands. Substitution of methionine at Cys-34 in albumin also did not produce a significant change in viability of RGC-5 cells exposed to SIN-1 compared to fraction V BSA containing Cys-34. However, domain II was less effective in protecting the cells than the full length protein.
The protection that albumin provides to RGC-5 cells under oxidative insult is not the result of changes in osmolarity, albumin ligands, Cys-34, or the domain II drug binding site alone, but rather directly related to intrinsic properties of the full albumin molecule. Direct inactivation of extracellular reactive oxygen species and albumin-initiated changes in protein expression in RGC-5 cells are likely the mechanisms through which albumin enhances cell survival.
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