Diminished NO release in chronic hypoxic human endothelial cells.
The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 microM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser(1177) and eNOS Thr(495) phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser(1177), was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of beta- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.
Ostergaard
Louise
L
Department of Pharmacology, University of Aarhus, Aarhus, Denmark.
Stankevicius
Edgaras
E
Andersen
Malene R
MR
Eskildsen-Helmond
Yvonne
Y
Ledet
Thomas
T
Mulvany
Michael J
MJ
Simonsen
Ulf
U
D015687:Cell Hypoxia / Q000502:physiology; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010766:Phosphorylation; D011247:Pregnancy
D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen