Thioredoxin restores nitric oxide-induced inhibition of protein kinase C activity in lung endothelial cells.
We previously reported that exposure to exogenous nitric oxide (NO) causes diminished expression of thioredoxin/thioredoxin reductase, a critical component of the redox system that regulates the functions of redox-sensitive enzymes, receptors, and transcription factors. Here we examined the role of thioredoxin in NO-induced inhibition of protein kinase C (PKC) isoform(s) and potential interaction of PKC and thioredoxin in pulmonary artery endothelial cells (PAEC) in culture. Exposure to NO gas (8 ppm) significantly diminished the catalytic activity of the representative isoforms of the conventional, novel, and atypical PKCs alpha, epsilon, and zeta, respectively, in PAEC. Further examination of NO's effect on PKC-zeta revealed that NO-induced inhibition of the catalytic activity of PKC-zeta was time-dependent and regulated by a posttranscriptional mechanism. NO-induced loss of the catalytic activity of PKC-zeta was restored by incubation with the disulfide reducing agent dithiothreitol (DTT) as well as by purified thioredoxin or thioredoxin reductase. Confocal imaging studies revealed co-localization of PKC and thioredoxin in PAEC. These results indicate that: (1) NO-induced inhibition of PKC isoforms is associated with S-nitrosylation-mediated disulfide formation of active site thiols in PKC-zeta as the disulfide reducing agent DTT and/or the thioredoxin enzyme system restore PKC-zeta catalytic activity and (2) NO causes oxidation of endogenous thioredoxin as exogenous reduced thioredoxin or thioredoxin reductase are required to reduce thioredoxin and to restore the catalytic activity of PKC-zeta in PAEC.
Kahlos
Katriina
K
Department of Medicine, University of Florida College of Medicine, USA.
Zhang
Jianliang
J
Block
Edward R
ER
Patel
Jawaharlal M
JM
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D042783:Endothelial Cells / Q000201:enzymology*; D008168:Lung / Q000166:cytology*; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D019163:Reducing Agents / Q000494:pharmacology; D013552:Swine; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*; D013997:Time Factors; D000099221:Protein Kinase C zeta
D004220:Disulfides; D004229:Dithiothreitol; D009569:Nitric Oxide; D020033:Protein Isoforms; D011493:Protein Kinase C; D019163:Reducing Agents; D013880:Thioredoxin-Disulfide Reductase; D013879:Thioredoxins; D000099221:Protein Kinase C zeta