Hypoxia enhances Ecto-5'-Nucleotidase activity and cell surface expression in endothelial cells: role of membrane lipids.
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.
Ledoux
S
S
INSERM U426, Faculté de Médecine Xavier Bichat, Université Paris 7, BP416, 16 rue Henri Huchard, 75870 Paris, Cedex 18, France. ledoux@bichat.inserm.fr
Runembert
I
I
Koumanov
K
K
Michel
J B
JB
Trugnan
G
G
Friedlander
G
G
D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D000860:Hypoxia / Q000503:physiopathology*; D008563:Membrane Lipids / Q000737:chemistry; D010100:Oxygen / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051381:Rats
D008563:Membrane Lipids; D012333:RNA, Messenger; D019308:Palmitic Acid; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase; D010100:Oxygen