THIOREDOXIN OVEREXPRESSION PREVENTS NO-INDUCED REDUCTION OF NO SYNTHASE ACTIVITY IN LUNG ENDOTHELIAL-CELLS

We recently reported that nitric oxide (NO) induces posttranscriptiona l modulation of lung endothelial cell NO synthase (ecNOS) that results in loss of activity. The loss of activity can be reversed by the redo x regulatory proteins thioredoxin (Thx)/thioredoxin reductase (Thx-R). The present study was designed to examine whether diminished expressi on of endogenous Thx and Thx-R may account for regulation of ecNOS act ivity in NO-exposed cells and whether overexpression of Thx can preven t NO-induced reduction of ecNOS activity in cultured porcine pulmonary artery endothelial cells (PAEC). Exposure to 8.5 ppm NO gas for 24 h resulted in an 80% decrease of Thx and a 27% decrease of Thx-R mRNA ex pression. Similarly, NO exposure caused 30 and 50% reductions in Thx a nd Thx-R protein mass, respectively. This NO-induced decrease in the e xpression of Thx-R mRNA and protein was accompanied by a significant ( P < 0.05) decrease in the catalytic activity of Thx-R but not of gluta redoxin or the cellular levels of reduced glutathione and oxidized glu tathione. Overexpression of Thx gene in PAEC was achieved by transient transfection of these cells with pcDNA 3.1 vector inserted in sense o r antisense (native) orientation in a human Thx cDNA. Thx mRNA and pro tein contents in transfected cells were four- and threefold higher, re spectively, than those in native PAEC. Exposure of native cells to 10 mu M NO solution for 30 min resulted in a significant (P < 0.01) loss of ecNOS activity, whereas ecNOS activity was comparable in Thx-overex pressed cells with or without NO exposure. These results demonstrate t hat NO exposure results in diminished expression of Thx and Thx-R in P AEC. Endogenous levels of Thx are critical to restoring the NO-induced loss of ecNOS activity because overexpression of Thx prevented the NO -induced loss of ecNOS catalytic activity. These results also demonstr ate that NO modulation of ecNOS and Thx proteins is regulated by a phy siologically relevant redox mechanism.