title: Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells.
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.
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Ellen E
authors.initials: EE
authors.lastname: Quillen
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Gale C
authors.initials: GC
authors.lastname: Haslam
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Hardeep S
authors.initials: HS
authors.lastname: Samra
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Darius
authors.initials: D
authors.lastname: Amani-Taleshi
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Knight
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Wyatt
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Stephanie C
authors.initials: SC
authors.lastname: Bishop
authors.affiliation: Department of Physical Sciences, Ferris State University, Big Rapids, Michigan 49307.
authors.forename: Kim K
authors.initials: KK
authors.lastname: Colvert
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534. Electronic address: richter@ku.edu.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Richter
authors.affiliation: Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Kitos
mesh_terms: D000263:Adenylate Kinase / Q000502:physiology*; D000818:Animals; D001313:Aurovertins / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009840:Oligomycins / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000502:physiology*; D011817:Rabbits; D013267:Stilbenes / Q000494:pharmacology
chemical_list: D001313:Aurovertins; D004791:Enzyme Inhibitors; D009840:Oligomycins; D013267:Stilbenes; C041525:3,3',4,5'-tetrahydroxystilbene; D000263:Adenylate Kinase; D006180:Proton-Translocating ATPases