title: Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences.
abstract: OBJECTIVE
  Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET(B) ) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.
  
  
  METHODS
  ET(B) receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca(2+) , F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A(2) and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively.
  
  
  RESULTS
  Cigarette smoke extract dose-dependently increased ET(B) receptor expression in HPAECs after 24h incubation. CSE-induced ET(B) expression was attenuated by bosentan, the ET(B) receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET(B) receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET(B) receptor expression, mimicking the effect of CSE. CSE-induced ET(B) receptor overexpression caused greater cell contraction; increased intracellular Ca(2+) ; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA(2) and ROS production; and decreased NO after acute ET-1 (10nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine.
  
  
  CONCLUSIONS
  Cigarette smoke extract induced ET(B) receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET(B) receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.
authors.affiliation: Research Unit, University General Hospital Consortium, Valencia, Spain. xmilara@hotmail.com
authors.forename: J
authors.initials: J
authors.lastname: Milara
authors.forename: J L
authors.initials: JL
authors.lastname: Ortiz
authors.forename: G
authors.initials: G
authors.lastname: Juan
authors.forename: R
authors.initials: R
authors.lastname: Guijarro
authors.forename: P
authors.initials: P
authors.lastname: Almudever
authors.forename: M
authors.initials: M
authors.lastname: Martorell
authors.forename: E J
authors.initials: EJ
authors.lastname: Morcillo
authors.forename: J
authors.initials: J
authors.lastname: Cortijo
mesh_terms: D000368:Aged; D000077300:Bosentan; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D019332:Endothelin-1 / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000502:physiology; D005260:Female; D006801:Humans; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology; D009569:Nitric Oxide / Q000032:analysis; D011651:Pulmonary Artery / Q000166:cytology*; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000378:metabolism*; D012906:Smoke; D013449:Sulfonamides / Q000494:pharmacology; D013929:Thromboxane B2 / Q000032:analysis; D014026:Nicotiana; D014162:Transfection; D015854:Up-Regulation; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D019332:Endothelin-1; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D044023:Receptor, Endothelin B; D012906:Smoke; D013449:Sulfonamides; D009569:Nitric Oxide; D013929:Thromboxane B2; D054460:rho-Associated Kinases; D000077300:Bosentan