Hypoxia Enhances Ecto-5′-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells

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Abstract
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.

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