Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide

Since thiols can undergo nitrosation and the cell membrane is rich in thiol -containing proteins, we considered the possibility that membrane surface t hiols may regulate cellular entry of NO. Recently, protein disulfide isomer ase (PDI), a protein that catalyzes thio-disulfide exchange reactions, has been found on the cell-surface membrane. We hypothesized that cell-surface PDI reacts with NO, catalyzes S-nitrosation reactions, and facilitates NO t ransfer from the extracellular to intracellular compartment. We observed th at PDI catalyzes the S-nitrosothiol-dependent oxidation of the heme group o f myoglobin (15-fold increase in the rate of oxidation compared with contro l), and that NO reduces the activity of PDI by 73.1 +/- 21.8% (P < 0.005). To assess the role of PDI in the cellular action of NO, we inhibited human erythroleukemia (HEL) cell-surface PDI expression using an antisense phosph orothioate oligodeoxynucleotide directed against PDI mRNA. This oligodeoxyn ucleotide decreased cell-surface PDI content by 74.1 +/- 9.3% and PDI foldi ng activity by 46.6 +/- 3.5% compared with untreated or "scrambled" phospho rothioate oligodeoxynucleotide-treated cells (P < 0.0001). This decrease in cell-surface PDI was associated with a significant decrease in cyclic guan osine monophosphate (cGMP) generation after S-nitrosothiol exposure (65.4 /- 26.7% reduction compared with control; P < 0.05), with no effect on cycl ic adenosine monophosphate (cAMP) generation after prostaglandin E-1 exposu re. These data demonstrate that the cellular entry of NO involves a transni trosation mechanism catalyzed by cell-surface PDI. These observations sugge st a unique mechanism by which extracellular NO gains access to the intrace llular environment.