Inhibition of Nitrobenzylthioinosine-Sensitive Adenosine Transport by Elevated d -Glucose Involves Activation of P _2Y2 Purinoceptors in Human Umbilical Vein Endothelial Cells

Abstract

Abstract-Chronic incubation with elevated D-glucose reduces adenosine transport in endothelial cells. In this study, exposure of human umbilical vein endothelial cells to 25 mmol/L D-glucose or 100 mol/L ATP, ATP-␥-S, or UTP, but not ADP or ␣,␤-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport by D-glucose, ATP, and ATP-␥-S was associated with reduced maximal binding, with no changes in the apparent dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (Ϸ60Ϯ10%, PϽ0.05; nϭ6) in the number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8Ϯ0.1ϫ10 6 in 5 mmol/L D-glucose) and in hENT1 mRNA levels was observed in cells exposed to D-glucose or ATP-␥-S. Incubation with elevated D-glucose, but not with D-mannitol, increased the ATP release by 3Ϯ0.2-fold . The effects of D-glucose and nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P 2Y purinoceptor antagonist), suramin (G␣ s protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (nonselective P 2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine transport via hENT1 in endothelial cells cultured in 25 mmol/L D-glucose could be due to stimulation of P 2Y2 purinoceptors by ATP, which is released from these cells in response to D-glucose. This could be a mechanism to explain in part the vasodilatation observed in the early stages of diabetes mellitus or in response to D-glucose Key Words: endothelium Ⅲ adenosine Ⅲ nitric oxide Ⅲ glucose Ⅲ purinoceptors R emoval of extracellular adenosine is an essential step in the modulation of several of the biological actions of this endogenous nucleoside. 1-4 Plasma and tissue levels of adenosine are regulated by an efficient membrane transport mediated by the Na ϩ -independent, nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (system es or ENT1) 3,4 in human vascular endothelium 5,6 and smooth muscle. 7,8 Human ENT1 (hENT1) expression in Raji cells (a human B-lymphocyte cell line) is dependent on NO levels and the activity of protein kinase C (PKC). 9 Incubation of human umbilical vein endothelial cells (HUVECs) with 25 mmol/L D-glucose for 24 hours has been reported to reduce the NBMPR-sensitive adenosine transport associated with increased protein levels and the activity of endothelial NO synthase, intracellular Ca 2ϩ , PKC, and mitogen-activated protein kinases p42/p44 mapk . 6,10 Thus, hENT1 adenosine transporters could be expressed and modulated in HUVECs.It has been reported that ATP inhibits dipyridamolesensitive adenosine transport in human pulmonary artery endothelium. 11 ATP also induces activation of PKC in endothelium from human umbilical vein, 12 bovine pulmonary artery, 13 and porcine aorta. 14,15 Activation of P 2Y1 and P 2Y2 purinoceptors with ATP induced the phosphorylation of p42 mapk in the human endothelial cell line EAhy 926 16 and p42...

Purinergic Signaling and Blood Vessels in Health and Disease

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Adenosine: An immune modulator of inflammatory bowel diseases

“…The role of the activation of purinergic receptors on adenosine uptake was also studied in vascular endothelial cells and chromaffin cells. ATP has been shown to up-regulate adenosine transport and protein expression in vascular endothelial cells and chromaffin cells [78][79][80] . However, the effect of ATP on adenosine transport in normal and inflamed intestine remains to be elucidated.…”

ADP mediates inhibition of insulin secretion by activation of P2Y13 receptors in mice

“…High glucose levels stimulate the release of ATP into the extracellular space from several tissues and cell types such as endothelial cells, blood vessels, mesangial cells, macrophages and beta cells [1][2][3][4][5]. The net pharmacological effects of extracellular ATP are difficult to predict since ATP by itself can stimulate P2Y 2 , P2Y 4 (in rodents), P2Y 11 and P2X 1 -P2X 7 receptors present on the cell surface [6].…”