Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, PC191

Poster Communications

Insulin induces hCATs- activity dependent relaxation in human umbilical vein

N. Rodriguez1, P. Avila1, V. Gallardo1, S. Luis2, M. González1

1. Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, University of Concepcion, Concepcion, Chile. 2. Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Pontificia Universidad Cat?lica de Chile, Santiago, Chile.

Insulin increases nitric oxide (NO) synthesis via the endothelial NO synthase (eNOS), and L-arginine transport via cationic amino acid transporters (hCATs) in human umbilical vein endothelial cells (HUVECs)(González et al. 2004). These effects of insulin could induce changes in vascular reactivity, but there is not information regarding this hormone effect in human foetal vasculature. We investigated vascular reactivity, and the involvement of hCATs, in response to insulin in human umbilical vein. Umbilical vein rings and HUVECs were isolated from normal pregnancies (ethics committee approval and informed patient consent were obtained). Rings were mounted on an isometric force transducer and registered the highest contractile response (90 mM KCl). Vessels were washed and constricted with 100 nM U46619 (thromboxane A2 analogue). Once stable maximum contraction was reached, rings were exposed to insulin (10-10-10-2 nM) in absence or presence of 200 µM N-ethylmaleimide (NEM, CATs inhibitor). Cells were isolated by collagenase digestion (370C) and cultured in medium 199 (M199) supplemented with 20% newborn and fetal calf sera. L-[3H]Arginine transport (100 µM L-arginie, 2 µCi/ml, 37°C, 1 min), protein abundance (western blotting) and mRNA (real time RT-PCR) for hCAT-1 were measured in absence or presence of insulin (0-10 nM, 8 h of incubation) in HUVECs monolayers (passage 2). Significant (upaired Student’s t test, P<0.05, n=8) dilatation of endothelium-intact human umbilical vein rings pre-constricted with U46619 was reached when 10 nM or higher concentrations of insulin, an effect blocked by NEM. U46619 vasoconstriction was reduced by pre-incubation (8 h) with 1 nM insulin. L-Arginine transport was increased by insulin, within 8 h of incubation, blocked by NEM, with a half-maximal stimulatory concentration (SC50) of 0.2 ± 0.02 nM (mean ± S.E.M.) in HUVECs. Insulin also increased the Vmax (SC50= 0.025 ± 0.003 nM), with no significant (P>0.05) changes in the apparent Km of transport. Insulin increased hCAT-1 isoform expression, an effect maintained for 8 h for protein abundance (3.2 ± 0.1 fold) and mRNA number of copies (2.2 ± 0.2 fold). We suggest that hCAT-1 expression and activity are under regulation by insulin in HUVEC. The fact that insulin alters vascular human umbilical vein reactivity depending on L-arginine transport via hCATs, implies a crucial physiological relevance of these type of cationic amino acid transporters, most likely hCAT-1, in human foetal endothelium, particularly in pregnancy diseases associated with altered L-arginine and insulin metabolism such as gestational diabetes. Supported by FONDECYT (1070865 & 1080534), DIUC 210.033.103-1.0 and CONICYT (AT-23070213 & PIA ACT-73).

Where applicable, experiments conform with Society ethical requirements