Article

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

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.
AJP Lung Cellular and Molecular Physiology (Impact Factor: 3.52). 12/2006; 291(5):L1079-93. DOI: 10.1152/ajplung.00526.2005
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
31 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nitric oxide synthase (NOS) is negatively regulated by protein-protein interactions with caveolin-1 before extracellular activating signals release it for nitric oxide (NO) production. Smooth muscle protein kinase G (PKG) is a down-stream effector of NO signaling for relaxation of vascular smooth muscle cells (SMC). The PKG is also found in endothelial cells and it inhibits activated NOS within endothelial cells. We used confocal fluorescence microscopy to colocalize the inhibitors caveolin-1 and PKG with NOS in freshly isolated neonatal lamb endothelial cells in order to corroborate the speculation of their differential effects on NOS. The roles of caveolin-1 and PKG as regulators of NOS were investigated by examining their respective subcellular sites of colocalization with NOS using qualitative fluorescence immunohistochemistry and confocal microscopy. Caveolin-1 was colocalized with NOS in the plasma membrane and Golgi. The PKG1-beta isoform was colocalized with serine116 phosphorylated NOS in the cytosol and in vesicular structures seen in the endoplasmic reticulum and in the nuclear region. We conclude that unlike caveolin-1, a known pre-activation inhibitor of nascent NOS, PKG may be a post-activation inhibitor of NOS, possibly important for the recycling of the spent enzyme.
    African health sciences 12/2011; 11(4):526-34.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.
    Journal of Neurochemistry 09/2008; 107(2):478-87. · 3.97 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Effective treatment and/or prevention strategies for neonatal persistent pulmonary hypertension of the newborn (PPHN) have been an important topic in neonatal medicine. However, mechanisms of impaired pulmonary vascular structure in hypoxia-induced PPHN are poorly understood and consequently limit the development of effective treatment. In this study, we aimed to explore the molecular signaling cascades in the lungs of a PPHN animal model and used primary cultured rat pulmonary microvascular endothelial cells to analyze the physiological benefits of ghrelin during the pathogenesis of PPHN. Randomly selected newborn rats were exposed to hypoxia (10-12%) or room air and received daily s.c. injections of ghrelin (150 μg/kg) or saline. After 2 weeks, pulmonary hemodynamics and morphometry were assessed in the rats. Compared with the control, hypoxia increased pulmonary arterial pressure, right ventricle (RV) hypertrophy, and arteriolar wall thickness. Ghrelin treatment reduced both the magnitude of PH and the RV/(left ventricle+septum (Sep)) weight ratio. Ghrelin protected neonatal rats from hypoxia-induced PH via the upregulation of phosphorylation of glycogen synthase kinase 3β (p-GSK3β)/β-catenin signaling and associated with β-catenin translocation to the nucleus in the presence of growth hormone secretagogue receptor-1a. Our findings suggest that s.c. administration of ghrelin improved PH and attenuated pulmonary vascular remodeling after PPHN. These beneficial effects may be mediated by the regulation of p-GSK3β/β-catenin expression. We propose ghrelin as a novel potential therapeutic agent for PPHN.
    Journal of Molecular Endocrinology 04/2011; 47(1):33-43. · 3.58 Impact Factor