Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, PC159
Transcriptional modulation of the human equilibrative nucleoside transporter 1 in fetal endothelium from gestational diabetes mellitus
San Martin, R ; Farias, M ; Sobrevia, L ;
1. Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre, School of Medicine, Pontificia Universidad Catolica de Chile, P.O. Box 114-D, Santiago, Chile.
Gestational diabetes mellitus (GDM) is associated with foetal endothelial dysfunction, and human umbilical vein endothelial cells (HUVEC) isolated from pregnancies with GDM show increased nitric oxide (NO) synthesis and L-arginine transport. These effects of GDM involve MAP kinases, protein kinase C (PKC) and NO cell signaling cascades and are blocked by the A2a purinoceptor antagonist ZM-241385 (Vázquez et al. 2004). GDM is also associated with reduced adenosine transport in HUVEC (Sobrevia et al. 1994) and increased extracellular adenosine (Vázquez et al. 2004). We studied the transcriptional regulation of the human equilibrative nucleoside transporter 1 (hENT1) by GDM and the involvement of A2a purinoceptors. HUVEC from normal or GDM pregnancies (Ethic committee approval and informed patient consent were obtained) were cultured in medium 199 (M199, containing 20% bovine sera, 3.2 mM L-glutamine) up to passage 2. hENT1 mRNA levels was quantified with real-time PCR using SYBR green in cells cultured in M199 containing nitrobenzil-tyoinosine (NBMPR, 1 mM) or ZM-241385 (100 nM). Genomic DNA was extracted and several fragments of the promoter region of the hENT1 gene were amplified by PCR (−3100, −2056, −1016 and −697 bp). The PCR products were cloned into pGL3 upstream luciferase reporter gene. These constructs were used to transfect HUVEC by electroporation (320 Volt, 30 msec) in M199 (8-10% efficiency) using GFP as transfection control. GDM reduced hENT1 mRNA expression (32 ± 3%, unpaired Student's t test, means ± S.E.M., n=4). The inhibitory effect of GDM on the hENT1 mRNA level was blocked by ZM-241385. To determine elements implicated in the pathological down-regulation of hENT1 transcription, HUVEC were transfected with plasmid constructs containing the different fragment of the promoter region. Endothelial cells from normal pregnancies show repressor elements from −2056 to −3100 bp. In addition, preliminary assays show that in GDM HUVEC the inhibitory effect of these elements are blocked by ZM-241385. These results support the possibility that the reported reduced adenosine transport in HUVEC from GDM could be associated with reduced hENT1 transcription modulated by distal repressor elements in the gene promoter region of hENT1, involving the activation of A2a purinoceptors.
Where applicable, experiments conform with Society ethical requirements