Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC106

Poster Communications

Characterising nucleotide release from the EA.hy926 human endothelial cell line during metabolic stress.

C. Thompson1, H. Burrell2, A. Simpson1

1. Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom. 2. Pharmacy and Biomolecular Science, Liverpool John Moores University, Liverpool, United Kingdom.

During hypoxia and cellular damage, ATP is released into the extracellular environment (1, 2). Once released, ATP acts as a signalling molecule via P2 purinoceptors. The aim of this study is to confirm ATP release under cellular stress and to investigate the presence of other nucleotides, such as ADP. Nucleotide release was measured from confluent cells grown in multi-well plates after incubation in a Na+ HEPES-buffered physiological saline for 60 minutes. During the incubation period, the cells were poisoned with a cocktail of, sodium cyanide (4mM), 2-deoxy-D-glucose (10mM) and ionomycin (5µM) for 5-40 minutes. A sample of the buffer was added to ATP monitoring reagent (AMR, ViaLight®). The light emitted (RLU) was measured using a Berthold tube luminometer (LB955). ADP was measured after conversion to ATP, using phosphoenolpyruvate and pyruvate kinase. The RLU was re-measured and the corresponding ATP RLU subtracted. Calibration curves for ATP and ADP were constructed and used to convert the RLU to nucleotide concentrations. Released nucleotides are subject to interconversion enzymes including, alkaline phosphatase (AP) and ATPases (which dephosphorylate ATP) or ecto-adenosine kinase, nucleoside diphosphokinase and F1F0 ATP synthase (which generate ATP from ADP) (3, 4). ATP and ADP assays were therefore carried out in the presence and absence of the ATP hydrolysis inhibitors, levamisole (10mM), ebselen (30µM) and ARL 67156 (100µM). ATP and ADP concentrations per 106 cells are presented as means ± S.E.M. The data shows that after 40 minutes, metabolic poisoning elevates the observed extracellular concentration of ATP from 6.5±2nM to 87±12nM and ADP from 893±178nM to 5244±618nM. ATP hydrolysis inhibitors further significantly elevated both extracellular nucleotides. Levamisole increased ATP to 159±21nM and ADP to 7176±893nM. Ebselen increased ATP to 190±6nM and ADP to 7714±330nM. ARL 67156 increased ATP to 165±12nM and ADP to 7858±189nM (p<0.01 for each when compared with the nil control, ANOVA, n>6). Since both ATP and ADP are elevated in the presence of the ATP hydrolysis inhibitors, it is not yet clear whether the extracellular ADP is derived from extracellular hydrolysis of ATP or whether it is ‘released’ directly from the cells.

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