Abstract
Purpose: :
: 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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: ganglion cells • oxidation/oxidative or free radical damage • phosphorylation