Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC242

Poster Communications

Role of tyrosine kinases activity in vasodilator mechanisms induced by insulin in human fetal vein.

L. Cabrera1, P. Ávila1, C. Palma1, M. Nuñez1, V. Gallardo1, C. Aguayo2, L. Sobrevia3, M. Gonzalez1

1. Vascular Physiology Laboratory, Department of Physiology, Universidad de Concepcion, Concepcion, Chile. 2. Department of Clinical Biochemistry and Immunology, Universidad de Concepcion, Concepcion, Chile. 3. Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Pontificia Universidad Cat

Insulin increases nitric oxide (NO) synthesis via the endothelial NO synthase (eNOS) and L-arginine transport via the cationic amino acid transporters (hCATs) in human umbilical vein endothelial cells (HUVECs)(González et al. 2004). Ex vivo experiments demonstrated that acute (30 min) and chronic (8 h) insulin causes relaxation of human umbilical vein by an endothelium and hCATs dependent mechanism (González et al., 2011). We here investigated the involvement of tyrosine kinases pathways in the acute effect of insulin in vascular reactivity of human chorionic vein, and chronic insulin effect on hCAT-1 expression in human umbilical vein. Chorionic 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 nM) in absence or presence of genistein (tyrosine kinases inhibitor) or wortmannin [phosphatidylinositol 3 kinase (PI3K) inhibitor]. Cells were isolated by collagenase digestion and cultured in medium 199 (M199) supplemented with 20% newborn and fetal calf sera. Protein abundance (western blotting) and mRNA (real time RT-PCR) for hCAT-1 were measured in absence or presence of insulin (1 nM, 8 h), genistein, wortmannin or calphostin C (PKC inhibitor)in HUVECs monolayers (passage 2). Significant (unpaired Student’s t test, P<0.05, n=5-10) dilatation of endothelium-intact human chorionic vein rings pre-constricted with U46619 was induced by insulin (24±1.2 %), genistein (45±5.3 %) and wortmannin (16±1.3 %). Co-incubation of chorionic vein with insulin and genistein potentiates dilatation caused by these molecules itself (69±3.4 % of dilatation). In HUVEC, insulin increases the mRNA expression (2.2 ± 0.2 fold) and protein abundance (3.2 ± 0.1 fold) of hCAT-1, effects blocked by genistein and wortmannin, but unaltered by calphostin C. We suggest that in acute ex vivo experiments insulin induces relaxation of placental vessels in a PI3K or other tyrosine kinases independent manner. Interestingly, the relaxation induced by insulin and genistein are summative, suggesting two different mechanisms in this phenomena. In other hands, insulin effect on hCAT-1 expression is dependent of tyrosine kinases activity, confirming the role of PI3K in the transcriptional effects of insulin in HUVEC.

Where applicable, experiments conform with Society ethical requirements