1 Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of in¯ammatory mediators that a ect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1 ± 32 days, and investigated contractile responses mediated by muscarinic and 5-hydroxytryptamine (5-HT) receptors following long-term exposure to tumour necrosis factor-alpha (TNFa). 2 Following culture, in the absence of TNFa, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not a ected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 3 5-HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFa produced a time-and concentration-dependent increase in the maximal contraction to 5-HT, evidently mediated by 5-HT 2A receptors, whereas, the potency for carbachol was reduced. 4 In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the ®rst days of culture. The timedependent e ect of TNFa indicates the importance of studying the long-term e ect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.

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Hyperresponsiveness to bronchoconstrictor stimuli is a major pathophysiologic feature of asthma, but the molecular mechanisms behind this are not fully understood. The release of TNF-alpha and IL-1beta during the inflammatory process is believed to play an important role in airway hyperresponsiveness. We have previously demonstrated, using a murine in vitro model of chronic airway inflammation, that TNF-alpha up-regulated bradykinin B(1) and B(2) receptors in the airway smooth muscle. By using the same model, the present study was designed to investigate the effects of IL-1beta and its interaction with TNF-alpha on the expression of bradykinin B(1) and B(2) receptors in mouse tracheal smooth muscle. IL-1beta up-regulated bradykinin B(1) and B(2) receptor expression and increased contractile response to bradykinin B(1) and B(2) receptor agonists (des-Arg(9)-bradykinin and bradykinin, respectively) in the tracheal smooth muscle. Transcriptional inhibitor actinomycin D, c-Jun N-terminal kinase (JNK) inhibitors SP600125 and TAT-TI-JIP(153-163), but not extracellular signal-regulated kinase 1 and 2 (ERK 1/2) inhibitor PD98059, significantly attenuated this up-regulation, indicating that a transcriptional mechanism and intracellular JNK signal transduction pathway were involved. In addition, IL-1beta did not affect bradykinin B(1) and B(2) receptor mRNA stability. Remicade, an anti-TNF-alpha antibody, markedly suppressed IL-1beta-induced up-regulation of bradykinin B(1) and B(2) receptors, suggesting that TNF-alpha was involved in the up-regulation, which is further supported by the fact that IL-1beta enhanced TNF-alpha mRNA expression in the tracheae. Intracellular JNK pathway and TNF-alpha might provide key links between inflammatory mediators like IL-1beta and airway hyperresponsiveness to bradykinin.

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The pineal gland hormone melatonin exerts its regulatory roles in a variety of physiological and pathological responses through two G protein-coupled receptors, melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2), which have been recognized as promising targets in the treatment of a number of human diseases and disorders. The MT1 receptor was identified nearly 20 years ago; however, the molecular mechanisms by which MT1-mediated signaling affects physiology remain to be further elucidated. In this study, using HEK293 cells stably expressing the human MT1 receptor, melatonin induced a concentration-dependent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). The melatonin-mediated phosphorylation of ERK1/2 at later time points (≥5 min) was strongly suppressed by pretreatment with pertussis toxin, but only a slight, if any, inhibition of ERK1/2 activation at early time points (≤2 min) was detected. Further experiments demonstrated that the Gβγ subunit, phosphoinositide 3-kinase, and calcium-insensitive protein kinase C were involved in the MT1-mediated activation of ERK1/2 at later time points (≥5 min). Moreover, results derived from cAMP assays combined with a MT1 mutant indicated that the human MT1 receptor could also couple to Gs protein, stimulating intracellular cAMP formation, and that the MT1-induced activation of ERK1/2 at early time points (≤2 min) was mediated by the Gs/cAMP/PKA cascade. Our findings may provide new insights into the pharmacological effects and physiological functions modulated by the MT1-mediated activation of ERK1/2.