Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells

• Published in: American journal of physiology. Lung cellular and molecular physiology 2006-11, Vol.291 (5), p.L1079-L1093
• Main Authors: John, Theresa A, Ibe, Basil O, Usha Raj, J
• Format: Article
• Language: English
• Subjects: Animals; Antibody Specificity; Caveolin 1 - immunology; Caveolin 1 - metabolism; Cells, Cultured; Down-Regulation - physiology; Endocytosis; Endocytosis - physiology; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Female; Genes; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; Hypoxia - drug therapy; Hypoxia - metabolism; Lung - blood supply; Lung - embryology; Lung - growth & development; Microcirculation - drug effects; Microcirculation - enzymology; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - immunology; Nitric Oxide Synthase Type III - metabolism; Oxygen; Oxygen - pharmacology; Pertussis Toxin - pharmacology; Phosphorylation; Pregnancy; Prenatal development; Protein Kinase C - metabolism; Reactive Oxygen Species - metabolism; Receptors, G-Protein-Coupled - metabolism; Serine - metabolism; Sheep; Threonine - metabolism
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00526.2005

Division of Neonatology and Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance; and Geffen School of Medicine at University of California Los Angeles, Los Angeles, California Submitted 13 December 2005 ; accepted in final form 19 June 2006 We determined the effect of oxygen [ 100 Torr (normoxia) and 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 µM 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 µg/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 P O 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. hypoxia; protein kinase C; G protein i -subunit Address for reprint requests and other correspondence: T. A. John, Rm. 207, RB-1, Los Angeles Biomedical Research Institute at Harbor UCLA Medical Center, 1124 West Carson St., Torrance, CA 90502 (e-mail: bjohn{at}labiomed.org )