title: Reduced l-arginine transport and nitric oxide synthesis in human umbilical vein endothelial cells from intrauterine growth restriction pregnancies is not further altered by hypoxia.
abstract: Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C alpha (PKCalpha) involvement. Normal or IUGR HUVEC monolayers were exposed (0-24h) to 5% O(2) (normoxia), and 1 or 2% O(2) (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCalpha or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine(1177), effects reversed by calphostin C (PKC inhibitor) and S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor). However, N(G)-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr(638)-phosphorylated PKCalpha was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCalpha, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR.
authors.affiliation: Perinatology Research Laboratory and Cellular and Molecular Physiology Laboratory, Department of Obstetrics and Gynecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile. pcasane@med.puc.cl
authors.forename: P
authors.initials: P
authors.lastname: Casanello
authors.forename: B
authors.initials: B
authors.lastname: Krause
authors.forename: E
authors.initials: E
authors.lastname: Torres
authors.forename: V
authors.initials: V
authors.lastname: Gallardo
authors.forename: M
authors.initials: M
authors.lastname: González
authors.forename: C
authors.initials: C
authors.lastname: Prieto
authors.forename: C
authors.initials: C
authors.lastname: Escudero
authors.forename: M
authors.initials: M
authors.lastname: Farías
authors.forename: L
authors.initials: L
authors.lastname: Sobrevia
mesh_terms: D000328:Adult; D001120:Arginine / Q000378:metabolism*; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D005260:Female; D005317:Fetal Growth Retardation / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D008954:Models, Biological; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011247:Pregnancy; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology*; D014664:Vasodilation / Q000502:physiology; D055815:Young Adult
chemical_list: D027201:Cationic Amino Acid Transporter 1; D012333:RNA, Messenger; D009569:Nitric Oxide; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051571:Protein Kinase C-alpha