SULFHYDRYL-DISULFIDE MODULATION AND THE ROLE OF DISULFIDE OXIDOREDUCTASES IN REGULATION OF THE CATALYTIC ACTIVITY OF NITRIC-OXIDE SYNTHASE IN PULMONARY-ARTERY ENDOTHELIAL-CELLS

The role of sulfhydryl groups (SH) and disulfide bonds as well as disulfide oxidoreductases in regulation of the catalytic activity of the membrane-bound constitutive isoform of nitric oxide (NO) synthase from porcine pulmonary artery endothelial cells (PAEC) was examined. Treatment of intact PAEC or a total membrane preparation isolated from PAEC with the SH alkylating agent N-ethylmaleimide (NEM) (10 to 50 mu M) or with the intramolecular disulfide-forming agent diamide (20 to 100 mu M) resulted in the reduction of NO synthase activity in a dose-dependent fashion, Similar loss of enzyme activity was observed when purified NO synthase from the membrane fraction of PAEC was incubated in the presence of NEM. The loss of membrane protein SH content from NEM- and diamide-treated preparations was associated with loss of NO synthase activity, In contrast, when intact PAEC or isolated total membranes derived from PAEC were treated with increasing concentrations (1 to 5 mM) of the disulfide-reducing agent dithiothreitol (DTT), but not oxidized DTT, NO synthase activity was increased by 20 to 85 %. DTT reduction of native disulfides from NEM-treated preparations or of disulfides formed after diamide treatment of membranes reversed the inhibition of NO synthase activity. Similarly, enzymatic reduction by thioredoxin/thioredoxin reductase, but not by glutaredoxin, reversed the inhibition of membrane fraction and purified NO synthase isolated from diamide-treated cells. This enzyme-catalyzed disulfide reduction was > 1,000-fold more efficient than the DTT-induced reduction. These results demonstrate that modulation of the SH and/or the disulfide bonds of NO synthase is associated with alterations in the catalytic activity of the membrane-bound constitutive isoform of NO synthase in PAEC. This modulation appears to be part of a regulatory mechanism that involves the oxidation-reduction of at least two conformationally vicinal reactive SH located in or near the catalytic domain of NO synthase and indicates that thioredoxin/thioredoxin reductase may play a physiologically important role in the regulation of NO synthase activity through a redox mechanism.