Zhang J, Li Y D, Patel J M, Block E R
Department of Medicine, University of Florida, and Medical Research Service, Department of Veterans Affairs Medical Center, Gainesville, Florida 32608-1197, USA.
Am J Physiol. 1998 Aug;275(2):L288-93. doi: 10.1152/ajplung.1998.275.2.L288.
We recently reported that nitric oxide (NO) induces posttranscriptional modulation of lung endothelial cell NO synthase (ecNOS) that results in loss of activity. The loss of activity can be reversed by the redox regulatory proteins thioredoxin (Thx)/thioredoxin reductase (Thx-R). The present study was designed to examine whether diminished expression of endogenous Thx and Thx-R may account for regulation of ecNOS activity in NO-exposed cells and whether overexpression of Thx can prevent 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 expression. Similarly, NO exposure caused 30 and 50% reductions in Thx and Thx-R protein mass, respectively. This NO-induced decrease in the expression 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 glutaredoxin or the cellular levels of reduced glutathione and oxidized glutathione. Overexpression of Thx gene in PAEC was achieved by transient transfection of these cells with pcDNA 3.1 vector inserted in sense or antisense (native) orientation in a human Thx cDNA. Thx mRNA and protein contents in transfected cells were four- and threefold higher, respectively, than those in native PAEC. Exposure of native cells to 10 microM NO solution for 30 min resulted in a significant (P < 0.01) loss of ecNOS activity, whereas ecNOS activity was comparable in Thx-overexpressed cells with or without NO exposure. These results demonstrate that NO exposure results in diminished expression of Thx and Thx-R in PAEC. 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 demonstrate that NO modulation of ecNOS and Thx proteins is regulated by a physiologically relevant redox mechanism.
我们最近报道，一氧化氮（NO）可诱导肺内皮细胞一氧化氮合酶（ecNOS）的转录后调节，导致其活性丧失。氧化还原调节蛋白硫氧还蛋白（Thx）/硫氧还蛋白还原酶（Thx-R）可逆转这种活性丧失。本研究旨在探讨内源性Thx和Thx-R表达降低是否可解释NO暴露细胞中ecNOS活性的调节，以及Thx过表达是否可预防培养的猪肺动脉内皮细胞（PAEC）中NO诱导的ecNOS活性降低。暴露于8.5 ppm NO气体24小时导致Thx mRNA表达降低80%，Thx-R mRNA表达降低27%。同样，NO暴露分别导致Thx和Thx-R蛋白量降低30%和50%。NO诱导的Thx-R mRNA和蛋白表达降低伴随着Thx-R催化活性的显著（P < 0.05）降低，但谷胱甘肽还原酶或细胞内还原型谷胱甘肽和氧化型谷胱甘肽水平未降低。通过用插入人Thx cDNA有义或反义（天然）方向的pcDNA 3.1载体瞬时转染PAEC，实现了Thx基因在PAEC中的过表达。转染细胞中Thx mRNA和蛋白含量分别比天然PAEC高4倍和3倍。天然细胞暴露于10 microM NO溶液30分钟导致ecNOS活性显著（P < 0.01）丧失，而在有或无NO暴露的Thx过表达细胞中，ecNOS活性相当。这些结果表明，NO暴露导致PAEC中Thx和Thx-R表达降低。内源性Thx水平对于恢复NO诱导的ecNOS活性丧失至关重要，因为Thx过表达可预防NO诱导的ecNOS催化活性丧失。这些结果还表明，NO对ecNOS和Thx蛋白的调节受生理相关的氧化还原机制调控。
