Molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium.

Nitric oxide (NO) plays an important role in airway function, and endothelial NO synthase (eNOS) is expressed in airway epithelium. To determine the basis of cell-specific eNOS expression in airway epithelium, studies were performed in NCI-H441 human bronchiolar epithelial cells transfected with the human eNOS promoter fused to luciferase. Transfection with 1624 base pairs of sequence 5' to the initiation ATG (position -1624) yielded a 19-fold increase in promoter activity versus vector alone. No activity was found in lung fibroblasts, which do not express eNOS. 5' deletions from -1624 to -279 had modest effects on promoter activity in H441 cells. Further deletion to -248 reduced activity by 65%, and activity was lost with deletion to -79. Point mutations revealed that the GATA binding motif at -254 is mandatory for promoter activity and that the positive regulatory element between -248 and -79 is the Sp1 binding motif at -125. Electrophoretic mobility shift assays yielded two complexes with the GATA site and three with the Sp1 site. Immunodepletion with antiserum to GATA-2 prevented formation of the slowest migrating GATA complex, and antiserum to Sp1 supershifted the slowest migrating Sp1 complex. An electrophoretic mobility shift assay with H441 versus fibroblast nuclei revealed that the slowest migrating GATA complex is unique to airway epithelium. Thus, cell-specific eNOS expression in airway epithelium is dependent on the interaction of GATA-2 with the core eNOS promoter, and the proximal Sp1 binding site is also an important positive regulatory element.

Endothelial dysfunction in the aorta of transgenic rats harboring the mouse Ren-2 gene.

The renin-angiotensin system plays an important role in the pathophysiology of hypertension. We studied vascular function in the aorta of mouse Ren-2 transgenic rats (TGR(mRen2)27). Changes in isometric tension of isolated aorta of TGR(mRen2)27 and Sprague-Dawley rats (SD) were recorded in organ chambers. Contractions to angiotensin II (AII), big-endothelin and endothelin-1 (ET-1), but not KCl were decreased in TGR. Blockade of nitric oxide (NO)-synthase by L-NAME or removal of the endothelium did not alter these decreased contractions to ET-1 and AII in TGR, suggesting that receptors or signaling pathways of these two agonists are downregulated during hypertension. Contractions to norepinephrine (NE) were also lower in TGR, however blockade of NO-synthase by L-NAME or removal of the endothelium evoked similar contractions to NE in both strains, suggesting that basal release of NO reduces contractions to NE to a greater extent in transgenic than control rats. In the presence of L-NAME, acetylcholine evoked endothelium-dependent contractions (EDCF) in TGR, which were blocked by the thromboxane/prostaglandin H2 receptor antagonists SQ 30741, and partially by the thromboxane synthase inhibitor CGS 13080, suggesting that prostaglandin H2 is the mediator. Endothelium-dependent relaxation to acetylcholine was decreased in TGR, while endothelium-independent relaxations to sodium nitroprusside were similar in both strains. SQ 30741 did not improve relaxations to acetylcholine in TGR indicating that impaired relaxations to acetylcholine are due to a decreased acetylcholine-receptor mediated release of NO rather than increased release of EDCF. Thus, Ren-2 hypertension leads to marked alterations of vascular functions in the aorta. These changes could contribute to hypertension and its vascular complications in TGR(mRen2)27 rats.

Regulation of endothelial nitric-oxide synthase during hypoxia.

The mechanism by which nitric-oxide (NO) production increases during hypoxia is unknown. To explore the effect of hypoxia upon endothelial nitric-oxide synthase (ecNOS) activity and expression, we exposed bovine aortic endothelial cells (BAEC) to hypoxia (1% O2) for 0-24 h and measured levels of ecNOS mRNA, protein, and activity. The amount of ecNOS mRNA increases to more than twice the basal level after 6 h of hypoxia. Incubation of BAEC with actinomycin D during hypoxia prevents this increase, demonstrating that higher levels of mRNA observed during hypoxia are due to increased synthesis, not to increased stability of ecNOS mRNA. Levels of ecNOS protein increase throughout 24 h of hypoxia to more than twice normoxic levels. Although ecNOS expression increases within 2 h of hypoxia, total activity remains unchanged. To explore the transcriptional regulation of ecNOS, we constructed a reporter plasmid containing the ecNOS promoter region upstream of the luc gene and transfected this reporter plasmid into BAEC. In this system, hypoxia induces a linear increase over time in the expression of luciferase driven by the ecNOS promoter. It is concluded that hypoxia induces an increase in transcription of ecNOS in endothelial cells, activating the regulatory region of ecNOS by undefined transcription factors.