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

Abstract

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 Ca(2+) ([Ca(2+)]i) was imaged with Fura-2.

Results: ATP release occurred constitutively and was increased by hypoxia (PO2: 150-8 mmHg), ∼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 [Ca(2+)]i compared with ATP (10 μM); hypoxia inhibited the ATP response. Quinacrine-ATP fluorescent loci 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 [Ca(2+)]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.

Keywords: ATP; Exocytosis; HUVEC; Hypoxia; Vesicles.

Fig. 1

Effect of hypoxia on ATP release from apical and basolateral surfaces of HUVEC. Columns show mean ± SEM; *, ***: hypoxia vs normoxia; P < 0.05, <0.01 respectively (n = 9, N = 3 in each case; n: number of inserts, N: number of donors). Note different scales for ordinates.

Fig. 2

Effect of pharmacological antagonists on constitutive and hypoxia-induced ATP release from HUVEC. Effects of vesicular transport inhibition with brefeldin A or monensin (A), and effects of PI₃K and ROCK inhibition with LY294002 or Y27632 (B). In each case, apical: left, basolateral: right. Hypoxia-induced ATP release in absence and presence of inhibitors shown in black and hatched columns respectively. All data are shown as % of constitutive release in normoxia (mean ± SEM). §, §§§: hypoxia vs normoxia; P < 0.05, P < 0.001 respectively, *, ***: vs normoxic or hypoxic control; P < 0.05, 0.01 respectively (n = 18, N = 6 in each case; n = number of inserts, N: number of donors).

Fig. 3

Effect of Ca²⁺ ionophore A23187 (top) and NO donor (bottom) on ATP release from apical and basolateral surfaces of HUVEC in normoxia. Columns show mean ± SEM. *** vs control; P < 0.01 (n = 9, N = 3; n = number of inserts, N = number of donors). Note different scales for ordinates showing apical (left) and basolateral (right) release of ATP.

Fig. 4

Changes in [Ca²⁺]_i evoked in HUVEC by ATP in normoxia and acute hypoxia, and by hypoxia. Top: original recordings of [Ca²⁺]_i in HUVEC in response to ATP (10 μM) in normoxia (A), ATP (10 μM) in normoxia vs hypoxia (B), hypoxia (C), note different scale for ordinate. Bottom: dose response relationship for ATP-induced increase in [Ca²⁺]_i relative to that evoked by 100 μM ATP (A), mean change in [Ca²⁺]_i evoked by ATP (10 μM) in normoxia vs hypoxia (B), mean change in [Ca²⁺]_i evoked by hypoxia compared with that evoked by ATP (10 μM, C). Columns show mean ± SEM (5 or 6 samples from 6 different donors in each case). ***: P < 0.01, ATP in normoxia vs ATP in hypoxia, or vs hypoxia.

Fig. 5

Images of HUVEC showing quinacrine-staining of ATP in normoxia and hypoxia. A: example of confocal image of HUVEC monolayer taken at 1 μm intervals, dual-labeled with quinacrine and DAPI. Dashed line drawn around cell boundary. Nucleus appears blue; ATP loci green (white arrows) in perinuclear region (Image representative of 4 samples from 2 donors) Below: fluorescent images of HUVEC stained with quinacrine before (B) and after (C) exposure to hypoxia (representative of 3 samples from 2 donors, both in normoxia and hypoxia).

Fig. 6

Effects of brefeldin A and monensin on quinacrine-stained HUVEC. Fluorescent and bright field images of quinacrine-stained HUVEC with vehicle (A), after brefeldin A (B), after monensin (C). Left; epi-illumination showing quinacrine-induced fluorescence, middle; epi-illumination + bright-field trans-illumination, right; overlay of quinacrine fluorescence (pseudo-colored in red) over bright-field image. Brefeldin A led to accumulation of quinacrine fluorescence in perinuclear region, whereas monensin led to loss of quinacrine fluorescence. Representative in each case of 3 samples from 2 donors.