Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: neuroprotection • antioxidants • ganglion cells