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 alpha,beta-methylene-5'-diphosphate (100 micromol/L). AMP was decreased by chronic depolarization from 0.54+/-0.16 to 0.39+/-0.11 micromol/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 micromol/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.
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 alpha,beta-methylene-5'-diphosphate (100 micromol/L). AMP was decreased by chronic depolarization from 0.54+/-0.16 to 0.39+/-0.11 micromol/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 micromol/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.
Authors: Charlotte Roy; Julie Tabiasco; Antoine Caillon; Yves Delneste; Jean Merot; Julie Favre; Anne Laure Guihot; Ludovic Martin; Daniele C Nascimento; Bernhard Ryffel; Simon C Robson; Jean Sévigny; Daniel Henrion; Gilles Kauffenstein Journal: Purinergic Signal Date: 2017-12-13 Impact factor: 3.765
Authors: Janahan Dharmarajah; Jane F Arthur; Christopher G Sobey; Grant R Drummond Journal: Naunyn Schmiedebergs Arch Pharmacol Date: 2010-09-01 Impact factor: 3.000
Authors: Jonathan Watt; Marie-Ann Ewart; Fiona H Greig; Keith G Oldroyd; Roger M Wadsworth; Simon Kennedy Journal: Thromb Res Date: 2012-04-19 Impact factor: 3.944