Regulation of Endothelial Nitric-oxide Synthase during Hypoxia

Abstract

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% O 2 ) 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 ec-NOS by undefined transcription factors.Systemic arteries vasodilate in response to hypoxia, delivering more blood to peripheral tissues. This dilation occurs within seconds after hypoxia, and is maintained for hours. The molecular basis of hypoxic vasodilation is not completely understood. Possible mechanisms include direct relaxation of smooth muscle cells induced by changes in pH, ion channel conductance changes, and decreases in ATP levels. Endothelial cells may also release lower amounts of vasoconstrictors, such as endothelin or thromboxane, or may release increased amounts of vasodilators such as adenosine, prostacyclin, endothelial hyperpolarizing factor, and nitric oxide (NO) 1 (1-3). NO is made in endothelial cells by endothelial nitric-oxide synthase (ecNOS), which converts arginine and oxygen into citrulline and NO (4 -12). Although ecNOS is expressed constitutively in resting endothelial cells, the level of ecNOS expression can be altered by various stimuli. Shear-stress and estrogen can increase ecNOS expression both in vitro and in vivo (13-23). Tumor necrosis factor-␣ decreases expression of ecNOS (17, 24 -27). Thus, although ecNOS is called a "constitutive" isoform of NOS, its expression can be altered.Several studies have shown that hypoxia increases the synthesis of NO from systemic arteries both ex vivo (28 -30) and in vivo (31, 32). The effect of hypoxia on NO synthesis from endothelial cells in vitro is less clear; some studies report an increase, decrease, or no change (33-35). These studies exposed cells isolated from systemic vessels to hypoxia for different periods of time, which may explain the different effects of hypoxia. An increase in NO synthesis during hypoxia could be due to increased levels of calcium prolonging the ...

Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor.

“…Our finding that VEGF-induced vascular dysfunction is prevented by NOS inhibitors is consistent with evidence that a relative or absolute increase in nitric oxide mediates vascular dysfunction in diabetic rats and in retina and nerve of nondiabetic rats with acute hyperglycemia (27,55). Our hypothesis that vascular dysfunction induced by early diabetes is mediated by increased nitric oxide production as a consequence of "hypoxia-like" cytosolic reductive stress is supported by evidence that true hypoxia increases endothelial NOS mRNA, NOS enzyme activity, and increased NO production (56,57) and that blood flow increases immediately in response to mild hypoxia.…”

Influence of Hypoxia on Nitric Oxide Synthase Activity and Gene Expression in Children With Congenital Heart Disease

“…Hypoxic incubation (zero O 2 for 24 hours) decreased eNOS activity and expression in endothelial cells from human umbilical cords 9 and saphenous veins. 21,22 In contrast, Arnet et al 23 reported that eNOS activity and expression were increased in bovine aortic endothelial cells after 24 hours of incubation at 1% O 2 . This variability probably reflects differences in species and vascular beds, methods used to maintain the cells, and duration and severity of the hypoxic exposures.…”

Rho-Kinase Mediates Hypoxia-Induced Downregulation of Endothelial Nitric Oxide Synthase

“…Some animal studies demonstrate that NO production is increased in hypoxia-induced pulmonary hypertension, 25,26 whereas human studies indicate that eNOS expression and activity are substantially reduced. 3 These discrepancies may be due in part to the different causes and stages in the development of pulmonary hypertension and to differences between in vitro and in vivo models.…”