title: Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells.
abstract: Protein disulfide isomerase (PDI) is an abundant protein primarily found in the endoplasmic reticulum and also secreted into the blood by a variety of vascular cells. The evidence obtained here, suggests that PDI could directly participate in the efflux of NO(+) from red blood cells (RBC). PDI was detected both in RBC membranes and in the cytosol. PDI was S-nitrosylated when RBCs were exposed to nitrite under ∼50% oxygen saturation but not under ∼100% oxygen saturation. Furthermore, it was observed that hemoglobin (Hb) could promote PDI S-nitrosylation in the presence of ∼600 nM nitrite. In addition, three lines of evidence were obtained for PDI-Hb interactions: (1) Hb co-immunoprecipitated with PDI; (2) Hb quenched the intrinsic PDI fluorescence in a saturable manner; and (3) Hb-Fe(II)-NO absorption spectrum decreased in a [PDI]-dependent manner. Finally, PDI was detected on the surface RBC under ∼100% oxygen saturation and released as soluble under ∼50% oxygen saturation. The soluble PDI detected under ∼50% oxygen saturation was S-nitrosylated. Based on these data it is proposed that PDI is taken up by RBC and forms a complex with Hb. Hb-Fe(II)-NO that is formed from nitrite reduction under ∼50% O2, then transfers NO(+) to either Hb-Cys β93 or directly to PDI resulting in S-nitroso-PDI which transverses the RBC membrane and attaches to the RBC surface. When RBCs enter tissues the S-nitroso-PDI is released from the RBC-surface into the blood where its NO(+) is transferred into the endothelium thereby inducing vasodilation, suggesting local oxygen-dependent dynamic interplays between nitrite, NO and S-nitrosylation.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4.
authors.forename: Vasantha Madhuri
authors.initials: VM
authors.lastname: Kallakunta
authors.forename: Anny
authors.initials: A
authors.lastname: Slama-Schwok
authors.forename: Bulent
authors.initials: B
authors.lastname: Mutus
mesh_terms: D002478:Cells, Cultured; D004912:Erythrocytes / Q000201:enzymology* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000378:metabolism*
chemical_list: D006454:Hemoglobins; D009573:Nitrites; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D019704:Protein Disulfide-Isomerases; D010100:Oxygen
keywords: BCA, bicinchoninic acid; EDTA, ethylenediaminetetraacetic acid; Hb, hemoglobin; Hypoxic vasodilation; NOx, nitric oxide related species; NP-40, nonyl phenoxypolyethoxylethanol; Nitrite reductase; PDI, protein disulfide isomerase; PMSF, penylmethylsulfenylfluoride; Protein disulfide isomerase; RBC, red blood cells; Red blood cells; S-nitroso-protein disulfide isomerase; S-nitrosohemoglobin; SDS-PAGE, sodium dodecyl sulfate, poly acrylamide gel electrophoresis; SNO, S-nitrosothiol; SNO-Hb, S-nitrosohemoglobin
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