Depolarization of endothelial cells enhances platelet aggregation through oxidative inactivation of endothelial NTPDase

Abstract

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 (02-) 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 a, α,β-methylene-5′-diphosphate (100 μmol/L). AMP was decreased by chronic depolarization from 0.54±0.16 to 0.39±L-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.