Abstract 776: Protein Disulfide Isomerase Is Involved In Redox Regulation Of Nitric Oxide Output During Laminar Shear Stress In Endothelial Cells
While anti-atherogenic effects of sustained laminar shear (LS) involve NO release from eNOS, increases in LS trigger transient superoxide production via NADPH oxidase. Recently, we showed that NADPH oxidase undergoes thiol-dependent regulation by the thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to promote NO internalization via trans-nitrosation reactions. We hypothesized that PDI-dependent support of NADPH oxidase activity affects NO output during sustained LS. Cultured rabbit aortic endothelial cells (RAEC) submitted to LS (15 dynes/cm2) in a cone-plate system for 18h exhibited (vs. static controls):
Decreased (~50%) superoxide production (HPLC analysis of DHE oxidation);
Decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay);
Decreased mRNA expression of Nox1 (67%) and Nox4 (45%) (real-time QPCR);
Increased eNOS expression (~50%, western blot) and nitrite levels in culture medium (Δ = 7.1±2.5[SD] μM, NO Analyzer and Griess reaction);
Decrease in total and membrane fraction PDI protein expression (~20%) without changes in membrane fraction/total ratio of PDI. RAEC were transfected with c-myc-tagged plasmid coding for wild-type (WT) PDI or PDI mutated in 4 thioredoxin-motif cysteine residues.
Forced expression (2-fold) of mutated but not WT PDI led to increase in nitrite output after LS (18h) (Δmutated = 17.2±3.3 μM vs. ΔWT = 7.0±1.9 μM, n=3, p<0.02). Confocal microscopy indicated similar subcellular localization between WT and mutated PDI. PDI co-imunoprecipitated with p22phox NADPH oxidase subunit, but not with eNOS or caveolin-1, either in static condition or after LS. Fractionation studies in sucrose gradients showed that PDI is distributed throughout several fractions in static conditions, including caveolin-1-enriched fractions, but migrates to higher-density fractions, not containing caveolin-1, during sustained LS. These results suggest that PDI is involved in regulation of NO output during LS via its effects on NADPH oxidase activity.