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


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.
Krötz
Florian
F
Institute of Physiology, München, Germany. fkroetz@lmu.de
Sohn
Hae Young
HY
Keller
Matthias
M
Gloe
Torsten
T
Bolz
Steffen Sebastian
SS
Becker
Bernhard F
BF
Pohl
Ulrich
U
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD; D001081:Apyrase / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D001160:Arterioles / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D004794:Enzyme Repression / Q000502:physiology; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology*; D008647:Mesocricetus; D010084:Oxidation-Reduction; D010710:Phosphates / Q000378:metabolism; D010974:Platelet Aggregation / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology
D015703:Antigens, CD; D017077:Culture Media, Conditioned; D010710:Phosphates; D013481:Superoxides; D000244:Adenosine Diphosphate; D001081:Apyrase; C067869:CD39 antigen