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This Article Full Text Full Text (PDF) All Versions of this Article: 293/5/H2894    most recent 01230.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Østergaard, L. Articles by Simonsen, U. Search for Related Content PubMed PubMed Citation Articles by Østergaard, L. Articles by Simonsen, U.

Diminished NO release in chronic hypoxic human endothelial cells

¹Department of Pharmacology, University of Aarhus, Aarhus; ²Research Laboratory for Biochemical Pathology, Aarhus Sygehus, Aarhus; ³Research Laboratory of Obstetrics and Gynecology, Skejby Hospital, University of Aarhus, Aarhus; and ⁴Research Laboratory, Department of Clinical Biochemistry, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark

Submitted 8 November 2006 ; accepted in final form 12 August 2007

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 µM) 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 Ser¹¹⁷⁷ and eNOS Thr⁴⁹⁵ phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser¹¹⁷⁷, was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of - 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.

endothelial nitric oxide synthase; Akt; actin; endothelium; hypoxia