Insulin-stimulated L-arginine transport requires SLC7A1 gene expression and is associated with human umbilical vein relaxation.

J Cell Physiol. 2011 Feb 1. doi: 10.1002/jcp.22635. [Epub ahead of print]

González M, Gallardo V, Rodríguez N, Salomón C, Westermeier F, Guzmán-Gutiérrez E, Abarzúa F, Leiva A, Casanello P, Sobrevia L.

Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile; Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, Concepción, Chile.

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 (-1606 and -650 pb 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 to -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. © 2011 Wiley-Liss, Inc.

Copyright © 2011 Wiley-Liss, Inc.