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J. Biol. Chem., Vol. 281, Issue 30, 20728-20737, July 28, 2006

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Ectoadenylate Kinase and Plasma Membrane ATP Synthase Activities of Human Vascular Endothelial Cells^*

Ellen E. Quillen, Gale C. Haslam, Hardeep S. Samra, Darius Amani-Taleshi, Jeffrey A. Knight, Diane E. Wyatt, Stephanie C. Bishop, Kim K. Colvert, Mark L. Richter¹, and Paul A. Kitos

From the Department of Molecular Biosciences, Kansas University, Lawrence, Kansas 66045-7534 and the Department of Physical Sciences, Ferris State University, Big Rapids, Michigan 49307

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 P¹,P⁵-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F₁F₀-ATP synthase and F₁-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.

Received for publication, December 7, 2005 , and in revised form, April 24, 2006.

^* This work was supported by the Higuchi Biosciences Center, Kansas University; the Camille and Henry Dreyfus Foundation (New York); and American Heart Foundation Grant 0151395Z. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045-7534. Tel.: 785-864-3334; Fax: 785-864-5321; E-mail: richter{at}ku.edu.

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