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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:2003.)
© 2002 American Heart Association, Inc.

Vascular Biology

Depolarization of Endothelial Cells Enhances Platelet Aggregation Through Oxidative Inactivation of Endothelial NTPDase

Florian Krötz^*; Hae Young Sohn^*; Matthias Keller; Torsten Gloe; Steffen Sebastian Bolz; Bernhard F. Becker; Ulrich Pohl

From the Institute of Physiology (F.K., M.K., T.G., S.S.B., B.F.B., U.P.), München, Germany; and the Department of Internal Medicine (H.S.S., M.K.), Ludwig-Maximilians-University, München, Germany.

Correspondence to Florian Krötz, MD, Institute of Physiology, Schillerstr. 44, 80336 München, Germany. E-mail fkroetz{at}lmu.de

Objective— The objective of this study was to investigate whether depolarization of cultured endothelial cells (human umbilical vein endothelial cells, HUVECs) affects their ectonucleotidase activity through superoxide (O₂^-) production.

Methods and Results— Depolarization by the cation channel gramicidin (100 nmol/L) or tetrabutylammonium chloride (1 mmol/L) induced O₂^- release from HUVECs (n=4), which was decreased by superoxide dismutase (SOD, 500 U/mL). The activity of endothelial ectonucleotidases was assessed by the production of inorganic phosphate from ADP, which was decreased by chronic depolarization by 25% (n=6, P<0.05) and the amount of AMP derived from ADP in the presence of the 5'-nucleotidase inhibitor ,ß-methylene-5'-diphosphate (100 µmol/L). AMP was decreased by chronic depolarization from 0.54±0.16 to 0.39±0.11 µmol/min/mg protein (n=6, P<0.05). This was abolished in the continuous presence of SOD (n=6). NTPDase protein was predominantly expressed in HUVECs (n=4). Protein abundance, viability of cells, and apoptosis rates were not altered by depolarization (n=10). Only in the presence of depolarized HUVECs, but not with control cells or with HUVECs depolarized in the presence of SOD, did 5 µmol/L of ADP cause irreversible platelet aggregation. Increases in transmural pressure induced endothelial depolarization in intact hamster small arterioles.

Conclusions— Depolarization causes the endothelial production of O₂^-, which inhibits the activity of endothelial ectonucleotidases. Increases in transmural pressure induce endothelial depolarization. In chronically hypertensive diseases, depolarization might favor platelet aggregation.

Key Words: endothelium • chronic depolarization • superoxide • ectonucleotidases • platelet aggregation

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