Abstract

AimsWe studied whether transforming growth factor ?1 (TGF-?1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-?1 in this cell type. TGF-?1 increases NO synthesis via activation of TGF-? receptor type II (T?RII), and NO inhibits hENT1 expression and activity in HUVECs.Methods and resultsHUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-?1, N G-nitro-l-arginine methyl ester (l-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-?1 receptor type II (TT?RII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-?1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-?1 effect was blocked by l-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-?1 was ineffective in cells expressing TT?RII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-?1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385.ConclusionhENT1 is down-regulated by activation of T?RII by TGF-?1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-?1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.