title: 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 (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.
authors.affiliation: Institute of Physiology, München, Germany. fkroetz@lmu.de
authors.forename: Florian
authors.initials: F
authors.lastname: Krötz
authors.forename: Hae Young
authors.initials: HY
authors.lastname: Sohn
authors.forename: Matthias
authors.initials: M
authors.lastname: Keller
authors.forename: Torsten
authors.initials: T
authors.lastname: Gloe
authors.forename: Steffen Sebastian
authors.initials: SS
authors.lastname: Bolz
authors.forename: Bernhard F
authors.initials: BF
authors.lastname: Becker
authors.forename: Ulrich
authors.initials: U
authors.lastname: Pohl
mesh_terms: 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
chemical_list: D015703:Antigens, CD; D017077:Culture Media, Conditioned; D010710:Phosphates; D013481:Superoxides; D000244:Adenosine Diphosphate; D001081:Apyrase; C067869:CD39 antigen