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Y. Ha, Y. Dun, M. Thangaraju, V. Ganapathy, S. B. Smith; Analysis of Sigma Receptor (R1) Binding to BiP in Oxidative Stress-Induced Death of Retinal Ganglion Cells (RGC). Invest. Ophthalmol. Vis. Sci. 2009;50(13):120.
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: σR1, a non-opiate, non-PCP binding site thought to complex with BiP (GRP78), may act as a chaperone at the ER-mitochondria interface, which may underlie its neuroprotective effects. Recently, we reported that (+)-pentazocine ((+)-PTZ), a σR1-specific ligand, prevented diabetes-induced apoptotic RGC death in vivo (Smith et al, 2008). Oxidative stress is implicated in RGC death in diabetes and glaucoma. In this study, we used Xanthine:Xanthine Oxidase (X:XO) to induce oxidative stress in RGCs to analyze σR1-BiP binding and whether this might be regulated by phosphorylation.
RGC5 cells were treated 18 h with X:XO (25 µM:10 mU/ml) in presence/absence of (+)-PTZ (3 µM). Cells were prepared for immunoprecipitation (IP), incubated with anti-σR1 antibody; IP proteins were denatured and subjected to SDS-PAGE and analyzed by immunoblotting with mouse monoclonal antibodies to BiP, phosphoserine, or phosphotyrosine. RGCs immunopanned from mouse retina were treated with X:XO (10 µM:2 mU/ml) in the presence/absence of (+)-PTZ. RNA and protein were isolated, pro-and anti-apoptotic genes were analyzed by RT-PCR; those showing changes in expression levels (FasL, caspase 3 and 9, survivin) were analyzed by immunoblotting.
X:XO exposure increased σR1-BiP binding in RGCs, which was eliminated by treatment with (+)-PTZ. It led to phosphorylation of σR1 serine, which was abolished by treatment with (+)-PTZ. Levels of pro-apoptotic proteins: cleaved caspase 3 and 9, FasL were increased in X:XO treated cells but were normal in (+)-PTZ treated cells. Survivin levels decreased in X:XO-treated cells but were normal following (+)-PTZ treatment.
In RGCs, oxidative stress increases σR1-BiP association, which occurs concomitant with an increase in several pro-apoptotic proteins; (+)-PTZ treatment dissociates the σR1-BiP complex and decreases levels of pro-apoptotic proteins while increasing the anti-apoptotic protein survivin. σR1 binding activity under oxidative stress appears to be regulated by phosphorylation.
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