Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells

Introduction: Hypoxia induces dilatation of the umbilical vein by releasing autocoids from endothelium; prostaglandins (PGs), adenosine and nitric oxide (NO) have been implicated. ATP is vasoactive, thus we tested whether hypoxia releases ATP from primary Human Umbilical Vein Endothelial Cells (HUVEC). Methods: HUVEC were grown on inserts under no-flow conditions. ATP was assayed by luciferin-luciferase and visualised by quinacrine labeling. Intracellular Ca2+ ([Ca2+](i)) was imaged with Fura-2. Results: ATP release occurred constitutively and was increased by hypoxia (PO2: 150-8 mmHg), similar to 10-fold more from apical, than basolateral surface. Constitutive ATP release was decreased, while hypoxia-induced release was abolished by brefeldin or monensin A, inhibitors of vesicular transport, and LY294002 or Y27632, inhibitors of phosphoinositide 3-kinases (PI3K) and Rho-associated protein kinase (ROCK). ATP release was unaffected by NO donor, but increased by calcium ionophore, by >60-fold from apical, but <25% from basolateral surface. Hypoxia induced a small increase in [Ca2+](i) compared with ATP (10 mu M); hypoxia inhibited the ATP response. Quinacrine-ATP fluorescent lad in the perinuclear space, were diminished by hypoxia and monensin, whereas brefeldin A increased fluorescence intensity, consistent with inhibition of anterograde transport. Discussion.: Hypoxia within the physiological range releases ATP from HUVEC, particularly from apical/adluminal surfaces by exocytosis, via an increase in [Ca2+](i), PI3K and ROCK, independently of NO. We propose that hypoxia releases ATP at concentrations sufficient to induce umbilical vein dilation via PGs and NO and improve fetal blood flow, but curbs amplification of ATP release by autocrine actions of ATP, so limiting its pro-inflammatory effects. (C) 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).