Published online before print March 27, 2003, doi: 10.1161/01.RES.0000069022.95401.FE
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Submitted on May 28, 2002
Revised on March 17, 2003
Accepted on March 18, 2003
Hypoxia Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells. Role of Membrane Lipids
S. Ledoux *;
From INSERM U426 (S.L., I.R., G.F.), Faculté de Médecine Xavier Bichat, Université Paris 7; INSERM U538 (K.K., G.T.), Faculté de Médecine Saint-Antoine; INSERM U460 (J.B.M.), Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France.
* To whom correspondence should be addressed. E-mail: ledoux{at}bichat.inserm.fr.
Extracellular adenosine production by the glycosyl-phosphatidyl-inositol-anchored Ecto-5'-Nucleotidase plays an important role in the defense against hypoxia, particularly in the intravascular space. The present study was designed in order to elucidate the mechanisms underlying hypoxia-induced stimulation of Ecto-5'-Nucleotidase in endothelial cells. For this purpose, aortic endothelial cells (SVARECs) were submitted to hypoxic gas mixture. Hypoxia (0% O2 for 18 hours) induced a 2-fold increase of Ecto-5'-Nucleotidase activity (Vmax 19.78±0.53 versus 8.82±1.12 nmol/mg protein per min), whereas mRNA abundance and total amount of the protein were unmodified. By contrast, hypoxia enhanced cell surface expression of Ecto-5'-Nucleotidase, as evidenced both by biotinylation and immunostaining. This effect was accompanied by a decrease of Ecto-5'-Nucleotidase endocytosis, without modification of Ecto-5'-Nucleotidase association with detergent-resistant membranes. Finally, whereas cholesterol content was unmodified, hypoxia induced a time-dependent increase of saturated fatty acids in SVARECs, which was reversed by reoxygenation, in parallel to Ecto-5'-Nucleotidase stimulation. Incubation of normoxic cells with palmitic acid enhanced Ecto-5'-Nucleotidase activity and cell surface expression. In conclusion, hypoxia enhances cell surface expression of Ecto-5'-Nucleotidase in endothelial cells. This effect could be supported by a decrease of Ecto-5'-Nucleotidase endocytosis through modification of plasma membrane fatty acid composition.
Key words: hypoxia • Ecto-5'-Nucleotidase • membrane lipids
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