Abstract
Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.
Highlights
IntroductionTo explore the source of extracellular AK in cultures of endothelial cells, a sensitive and versatile method for evaluating AK activity was devised
To explore the source of extracellular AK in cultures of endothelial cells, a sensitive and versatile method for evaluating AK activity was devised. It is described and its suitability for this study confirmed at the beginning “Results.” Its use in studies of human umbilical vein endothelial cells (HUVECs) and a line of human embryonic lung fibroblasts (WI38) in culture suggests that enough AK does leak from cells to account for the observed extracellular ATP formation from ADP
The inoculum for an experiment was prepared by incubating the cell layer at 37 °C in trypsin/EDTA solution (0.01% crystalline porcine trypsin and 0.1% EDTA in DPBSA) until the cells had detached from the dish, terminating the tryptic action by adding a small volume of medium containing 5% FBS
Summary
To explore the source of extracellular AK in cultures of endothelial cells, a sensitive and versatile method for evaluating AK activity was devised. Most of the experiments reported here involved HUVECs from GlycoTech Corp., and the results using them were substantiated with HUVECs from Cascade Biologics and American Type Culture Collection. They were cultivated in HUVEC medium (1:1 Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium supplemented with 100 g/ml heparin, 15 g/ml endothelial cell growth supplement, 10% fetal bovine serum (FBS), 100 units/ml penicillin G, and 100 g/ml streptomycin sulfate) in 100-mm tissue culture plates or T-75 tissue culture flasks and, for experimental purposes, in collagen-coated multiwell plates. The plates were again vortexed, and the A492 values were determined on a multiwell plate reader (AutoReader II, Ortho-Clinical Diagnostics, Inc., Raritan, NJ)
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