title: Nicotine enhances murine airway contractile responses to kinin receptor agonists via activation of JNK- and PDE4-related intracellular pathways.
abstract: BACKGROUND
  Nicotine plays an important role in cigarette-smoke-associated airway disease. The present study was designed to examine if nicotine could induce airway hyperresponsiveness through kinin receptors, and if so, explore the underlying mechanisms involved.
  
  
  METHODS
  Murine tracheal segments were cultured for 1, 2 or 4 days in serum-free DMEM medium in presence of nicotine (1 and 10 microM) or vehicle (DMSO). Contractile responses induced by kinin B1 receptor agonist, des-Arg9-bradykinin, and B2 receptor agonist, bradykinin, were monitored with myographs. The B1 and B2 receptor mRNA expressions were semi-quantified using real-time PCR and their corresponding protein expressions assessed with confocal-microscopy-based immunohistochemistry. Various pharmacological inhibitors were used for studying intracellular signaling pathways.
  
  
  RESULTS
  Four days of organ culture with nicotine concentration-dependently increased kinin B1 and B2 receptor-mediated airway contractions, without altering the kinin receptor-mediated relaxations. No such increase was seen at day 1 or day 2. The airway contractile responses to 5-HT, acetylcholine and endothelin receptor agonists remained unaffected by nicotine. Two different neuronal nicotinic receptor antagonists MG624 and hexamethonium blocked the nicotine-induced effects. The enhanced contractile responses were accompanied by increased mRNA and protein expression for both kinin receptors, suggesting the involvement of transcriptional mechanisms. Confocal-microscopy-based immunohistochemistry showed that 4 days of nicotine treatment induced activation (phosphorylation) of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. Inhibition of JNK with its specific inhibitor SP600125 abolished the nicotine-induced effects on kinin receptor-mediated contractions and reverted the enhanced receptor mRNA expression. Administration of phosphodiesterase inhibitors (YM976 and theophylline), glucocorticoid (dexamethasone) or adenylcyclase activator (forskolin) suppressed the nicotine-enhanced airway contractile response to des-Arg9-bradykinin and bradykinin.
  
  
  CONCLUSIONS
  Nicotine induces airway hyperresponsiveness via transcriptional up-regulation of airway kinin B1 and B2 receptors, an effect mediated via neuronal nicotinic receptors. The underlying molecular mechanisms involve activation of JNK- and PDE4-mediated intracellular inflammatory signal pathways. Our results might be relevant to active and passive smokers suffering from airway hyperresponsiveness, and suggest new therapeutic targets for the treatment of smoke-associated airway disease.
authors.affiliation: Division of Ear, Nose and Throat Diseases, CLINTEC, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
authors.forename: Yaping
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lars-Olaf
authors.initials: LO
authors.lastname: Cardell
mesh_terms: D000818:Animals; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D009538:Nicotine / Q000008:administration & dosage*; D043783:Receptor, Bradykinin B1 / Q000819:agonists / Q000378:metabolism*; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014132:Trachea / Q000187:drug effects / Q000502:physiology*
chemical_list: D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D009538:Nicotine; D048670:MAP Kinase Kinase 4; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4