Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells.
We determined the effect of oxygen [approximately 100 Torr (normoxia) and approximately 30-40 Torr (hypoxia)] on functions of endothelial nitric oxide (NO) synthase (NOS-3) and its negative regulator caveolin-1 in ovine fetal and neonatal lung microvascular endothelial cells (MVECs). Fetal NOS-3 activity, measured as NO production with 0.5-0.9 microM 4-amino-5-methylamino-2,7-difluorofluorescein, was decreased in hypoxia by 14.4% (P < 0.01), inhibitable by the NOS inhibitor N-nitro-L-arginine, and dependent on extracellular arginine. Caveolar function, assessed as FITC-BSA (160 microg/ml) endocytosis, was decreased in hypoxia by 13.5% in fetal and 22.8% in neonatal MVECs (P < 0.01). NOS-3 and caveolin-1 were physically associated, as demonstrated by coimmunoprecipitation and colocalization, and functionally associated, as shown by cross-activation of endocytosis, by their specific antibodies and activation of NOS by albumin. Caveolin peptide, containing the sequence for the PKC phosphorylation site of caveolin, and caveolin antiserum against the site increased NO production and endocytosis by 12.3% (P < 0.05) and 16% (P < 0.05), respectively, in normoxia and increased endocytosis by 25% (P < 0.001) in hypoxia. PMA decreased NO production in normoxia and hypoxia by 19.32% (P < 0.001) and 11.8% (P < 0.001) and decreased endocytosis in normoxia by 20.35% (P < 0.001). PKC kinase activity was oxygen sensitive, and threonine phosphorylation was enhanced in hypoxia. Pertussis toxin increased caveolar and NOS functions. These data support our hypothesis that increased Po(2) at birth promotes dissociation of caveolin-1 and NOS-3, with an increase in their activities, and that PKC and an oxygen-sensitive cell surface G protein-coupled receptor regulate caveolin-1 and NOS-3 interactions in fetal and neonatal lung MVECs.
John
Theresa A
TA
Division of Neonatology, Rm. 207, RB-1, Los Angeles Biomedical Research Institute at Harbor UCLA Medical Center, 1124 West Carson St., Torrance, CA 90502, USA. bjohn@labiomed.org
Ibe
Basil O
BO
Usha Raj
J
J
D000818:Animals; D000918:Antibody Specificity; D051242:Caveolin 1 / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D004705:Endocytosis / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D000860:Hypoxia / Q000188:drug therapy / Q000378:metabolism*; D008168:Lung / Q000098:blood supply* / Q000196:embryology / Q000254:growth & development; D008833:Microcirculation / Q000187:drug effects / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000276:immunology / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D012756:Sheep; D013912:Threonine / Q000378:metabolism
D051242:Caveolin 1; D017382:Reactive Oxygen Species; D043562:Receptors, G-Protein-Coupled; D013912:Threonine; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D010100:Oxygen