Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: Molecular and functional consequences

Abstract

BACKGROUND AND PURPOSE 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. EXPERIMENTAL APPROACH 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. KEY RESULTS Cigarette smoke extract dose-dependently increased ET B receptor expression in HPAECs after 24 h 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 (10 nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine. CONCLUSIONS AND IMPLICATION 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. © 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.