title: Insulin-stimulated L-arginine transport requires SLC7A1 gene expression and is associated with human umbilical vein relaxation.
abstract: Insulin causes endothelium-derived nitric oxide (NO)-dependent vascular relaxation, and increases L-arginine transport via cationic amino acid transporter 1 (hCAT-1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC). We studied insulin effect on SLC7A1 gene (hCAT-1) expression and hCAT-transport activity role in insulin-modulated human fetal vascular reactivity. HUVEC were used for L-arginine transport and L-[(3) H]citrulline formation (NOS activity) assays in absence or presence of N-ethylmaleimide (NEM) or L-lysine (L-arginine transport inhibitors). hCAT-1 protein abundance was estimated by Western blot, mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (-1,606 and -650 bp promoter fragments from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites between -177 and -105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein rings. Insulin increased hCATs-L-arginine transport, maximal transport capacity (V(max) /K(m) ), and hCAT-1 expression. NEM and L-lysine blocked L-arginine transport. In addition, it was trans-stimulated (∼7.8-fold) by L-lysine in absence of insulin, but unaltered (~1.4-fold) in presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin increased NO synthesis and caused endothelium-dependent vessel relaxation and reduced U46619-induced contraction, effects blocked by NEM and L-lysine, and dependent on extracellular L-arginine. We suggest that insulin induces human umbilical vein relaxation by increasing HUVEC L-arginine transport via hCATs (likely hCAT-1) most likely requiring Sp1-activated SLC7A1 expression.
authors.affiliation: Cellular and Molecular Physiology Laboratory and Perinatology Research Laboratory, Division of Obstetrics and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors.forename: Marcelo
authors.initials: M
authors.lastname: González
authors.forename: Victoria
authors.initials: V
authors.lastname: Gallardo
authors.forename: Natalia
authors.initials: N
authors.lastname: Rodríguez
authors.forename: Carlos
authors.initials: C
authors.lastname: Salomón
authors.forename: Francisco
authors.initials: F
authors.lastname: Westermeier
authors.forename: Enrique
authors.initials: E
authors.lastname: Guzmán-Gutiérrez
authors.forename: Fernando
authors.initials: F
authors.lastname: Abarzúa
authors.forename: Andrea
authors.initials: A
authors.lastname: Leiva
authors.forename: Paola
authors.initials: P
authors.lastname: Casanello
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008239:Lysine / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000096:biosynthesis; D014471:Umbilical Veins / Q000166:cytology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*
chemical_list: D027201:Cationic Amino Acid Transporter 1; D004791:Enzyme Inhibitors; D007328:Insulin; C420825:SLC7A1 protein, human; D016329:Sp1 Transcription Factor; D014662:Vasoconstrictor Agents; D009569:Nitric Oxide; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D008239:Lysine; D005033:Ethylmaleimide