Airway-hyperresponsiveness And Alteration Of G-protein Coupled Receptors Of Smooth Muscle In Murine Organ Culture And Smoking Model

Background and AimAirway hyperresponsiveness consists of an increased sensitivity of the airways to constrictor agonists, which is the major feature of asthma. The airway G-proten coupled receptors like bradykinin receptors, thromboxane A2 (TP) receptors and endothelin receptors, which mediate the airway contractions, may play an important role in the development of the airway hyperresponsiveness. Smokinge from cigarette is a strong risk factor for asthma and chronic airway inflammation. The present study was designed to investigate the related intracellular pathways that are involved in the alteration of G-protein coupled receptor in airway hyperresponsivess in vitro and in vivo.MethodsIn vitro study Rat bronchi were isolated and cut into ring segments. The segments were then organ cultured for up to 96 h. Bradykinin (B₂ receptor agonist), des-Arg⁹-bradykinin (B1 receptor agonist), and U46619 (TP receptor agonist)-induced contractions of the segments were monitored by a sensitive myograph system. Transcriptioanal inhibitor (actinomycin D,AcD), translational inhibitor (cycloheximide,CHX), IκB kinase (IKK) inhibitors BMS-345541 and TPCA-1, MAPK inhibitors (JNK inhibitor SP600125, p38 inhibitor SB203580 and MEK inhibitor PD 98059), calcium-free solution and voltage-operated calcium channel (VOCC) inhibitor nifedipine were applied to examine the different intracellular pathways. Receptors and inflammatory mediator genes were examined by a real-time PCR, and protein expression was examined by immunohistochemisty using confocal microscopy and/or Western-blot.In vivo study Mice were repeatedly exposed to smoke from four cigarettes each day for four weeks. After the first week of the smoke exposure, the mice received either dexamethasone intraperitoneally every other day or GW5074 intraperitoneally every day for three weeks. The tone of the tracheal ring segments was recorded with a myograph system and concentration-response curves were obtained by cumulative administration of agonists. Histopathology was examined under light microscopy. Results1. Organ culture of the bronchial segments up to 96 h induced a time-dependent upregulation of bradykinin B1 and B2 receptors in functional, mRNA and protein levels. The IκB kinase (IKK) inhibitors TPCA-1(0.3μmol/L～10μmol/L) and BMS-345541 (1μmol/L～10μmol/L) abolished the organ culture-enhanced bradykinin B₁ and B₂ receptor-mediated contractions in a concentration-dependent manner. This was paralleled with inhibition of IKK activity, reduced mRNA and protein expressions of bradykinin B1 and B2 receptors and decreased mRNA expression of inflammatory mediators IL-6, MMP-9, COX-2 and iNOS.2. Organ culture of rat bronchial segments for up to 48 h induced a time-dependently increase of airway contractile response to U46619. Organ culture for 24 h enhanced U46619-induced JNK phosphorylation in bronchial smooth muscle cells.The enhanced bronchial contraction was attenuated by TP receptor atagonist GR32191 (0.1μmol/L), whereas the TP receptor protein expression was not affected by organ culture. Transcriptioanal inhibitor AcD (5mg/L) and translational inhibitor CHX (10μmol/L) did not affect the enhanced contraction, while c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10μmol/L), chelation of extracellular calcium and calcium channel blocker nifedipine (10μmol/L) inhibited the enhanced contraction.3. Four weeks of exposure to cigarette smoke significantly increased the mouse airway contractile response to carbachol, endothelin-1 and potassium. Intraperitoneal administration of GW5074 (0.5mg/kg and 2 mg/kg) or dexamethasone (0.3mg/kg and 1mg/kg) significantly suppressed the enhanced airway contractile responses, while isoproterenol-induced relaxation was not affected. In addition, the smoke-induced infiltration of inflammatory cells and mucous gland hypertrophy were attenuated by the administration of GW5074 or dexamethasone.Conclusion:1. Organ culture induces the bradykinin receptor-and TP receptor-mediated bronchial hyperresponsiveness. IKK-mediated inflammatory changes in airways may subsequently result in airway hyperresponsiveness to bradykinin via transcriptional upregulation of bradykinin receptors. Airway hyperresponsiveness to TP receptor agonist may occur via an increase in JNK MAPK activity and elevation of extracellular calcium influx.2. Cigarette smoke induces airway contractile hyperresponsiveness. Inhibition of Raf-1 activity and airway inflammation suppresses smoking-associated airway hyper- responsiveness.