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.

1 Chronic inflammation is a central feature of asthma. The inflammatory cytokine tumour necrosis factor a (TNFa) has been implicated in this disease, and is known to alter airway smooth muscle functionally. 2 The aim of this study was to investigate the influence of TNFa on tachykinin-induced airway relaxation. Mouse tracheae were cultured in the absence and presence of TNFa for 1 or 4 days. 3 In the absence of TNFa, substance P (SP) and neurokinin A (NKA) induced comparable levels of relaxation in fresh and cultured segments. Functional studies with selective antagonists/inhibitors indicated that the relaxation was mediated by the NK 1 receptor coupled to cyclooxygenase (COX)-2 activation and subsequent release of prostaglandin E 2 (PGE 2 ). TNFa attenuated SP-and NKAinduced relaxation in a time-and concentration-dependent manner, decreasing the ability of PGE 2 to relax tissues. 4 Further studies indicated that TNFa elevated COX-2 activity and that concomitant inhibition of COX-2 reversed TNFa-attenuated PGE 2 relaxation. Culture with PGE 2 decreased SP-and PGE 2 -mediated relaxation, further implicating the activity of COX-2 in the attenuation of tachykinin signalling. 5 Gene expression analysis demonstrated that TNFa increased the expression of smooth muscle COX-2, PGE 2 synthase and EP 2 receptor mRNA, and decreased the expression of the EP 4 receptor. 6 Overall, these results show that NK 1 receptor-mediated relaxation induced by PGE 2 is attenuated by prolonged TNFa stimulation. Increased COX-2 activity induced by TNFa appears to be central to this process.