title: Insulin-increased L-arginine transport requires A(2A) adenosine receptors activation in human umbilical vein endothelium.
abstract: Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A(2A) adenosine receptors (A(2A)AR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A(2A)AR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2A)AR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K(m) for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606) or pGL3-hCAT-1(-650) constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606), and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2A)AR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors: Enrique
authors: E
authors: Guzmán-Gutiérrez
authors: Francisco
authors: F
authors: Westermeier
authors: Carlos
authors: C
authors: Salomón
authors: Marcelo
authors: M
authors: González
authors: Fabián
authors: F
authors: Pardo
authors: Andrea
authors: A
authors: Leiva
authors: Luis
authors: L
authors: Sobrevia
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D027182:Amino Acid Transport System y+ / Q000235:genetics; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D055815:Young Adult
chemical_list: D027182:Amino Acid Transport System y+; D027201:Cationic Amino Acid Transporter 1; D007328:Insulin; D043705:Receptor, Adenosine A2A; C420825:SLC7A1 protein, human; D013868:Thioinosine; D001120:Arginine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine