Intrauterine growth restriction (IUGR) is associated with reduced fetal growth (Bird et al. 2003) and inhibition of L-arginine transport (via cationic amino acid transporters, hCATs) and nitric oxide (NO) synthesis in human umbilical vein endothelium (HUVEC)(Casanello & Sobrevia, 2002). L-Arginine/NO pathway activity is increased by insulin and adenosine in HUVEC (Sobrevia et al. 1997, 1998). We studied whether insulin modulates L-arginine/NO pathway and adenosine transport (via human equilibrative nucleoside transporters, hENTs) in IUGR pregnancies. HUVEC from normal or IUGR pregnancies (Ethics Committee approval and informed patient consent were obtained) were cultured in medium 199 (containing 20% bovine sera, 3.2 mM L-glutamine) and exposed (8 h) to human insulin (0.1 nM) or wortmannin (30 nM). Transport of L-[3H]arginine (15-1000 µM, 2 µCi/ml, 37°C, 1 min) or [3H] adenosine (15-500 µM, 4 µCi/ml, 37°C, 20 s) was measured. Adenosine transport was also determined in presence of nitrobenzylthioinosine (NBMPR, 10 µM, 30 min). hCAT-1, hCAT-2B, hENT1 and eNOS mRNAs were amplified by real time reverse transcriptase-polymerase chain reactions. IUGR reduced maximal L-arginine transport (V max 1.2 ± 0.2 vs 5.4 ± 1 pmol/µg protein/min)(P<0.05, unpaired Students t test, means ± S.E.M.), with no significant (P>0.05) changes in apparent K m (194 ± 30 vs 150 ± 34 µM, IUGR vs normal, respectively). Insulin increased the V max for L-arginine transport in normal (6.4-fold) and IUGR (2.7-fold) cells, but reduced the apparent K m in IUGR cells (21 ± 6 µM). IUGR reduced hCAT-1 (94 ± 2%), hCAT-2B (95 ± 2%), eNOS (73 ± 7%) and hENT1 (93 ± 4%) mRNA. Insulin partially reversed the effect of IUGR, and increased hCATs, hENT1 and eNOS mRNA in normal cells. IUGR increased apparent K m for NBMPR-sensitive adenosine transport (26 ± 3 vs 13 ± 2 µM), with not significant changes in V (0.28± 0.04 vs 0.22 ± 0.04 pmol/µg protein/min, normal vs IUGR, respectively). Insulin increased 10 µM adenosine uptake in cells from normal (2.7-fold) or IUGR (4.5-fold) pregnancies. All effects of insulin were blocked by wortmannin. In summary, insulin could protect against the effect of IUGR on L-arginine/NO pathway and adenosine transport in HUVEC.
University of Glasgow (2004) J Physiol 557P, PC47
Communications: Insulin restores intrauterine growth restriction-induced inhibition of L-arginine and adenosine transport, and nitric oxide synthesis in human umbilical vein endothelial cells.
V. Mezzano (a),F.Sanhueza (a),R. Vasquez (a),G.Munoz (a),P.Casanello (a) and L. Sobrevia (a)
(a) Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, PO Box 114-D, Chile and (b) Program of Pathophysiology, ICBM, University of Chile, Santiago, Chile
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Where applicable, experiments conform with Society ethical requirements.