An investigation of β2-adrenoceptor-mediated signalling in guinea pig trachea

James Buckley1, Mark Birrell1, Deborah Clarke1, Mark Giembycz2, Maria Belvisi1. 1Imperial College London, London, UK, 2University of Calgary, Calgary, Alberta, Canada.

β2-Adrenoceptor agonists (β2-agonists) are the gold standard bronchodilator therapy for asthma and COPD. Whilst it has been established as dogma that the cyclic AMP/cyclic AMP dependent kinase (PKA) cascade is the signalling pathway through which these drugs act, the supporting experimental evidence is often lacking and publications have begun to question this dogma (Spicuzza et al., 2001; Koike et al., 2004; Tanaka et al., 2003). In this investigation, we comprehensively studied the mechanism by which β2-agonists induce relaxation in airway smooth muscle in vitro, using guinea pig (GP) trachea to model human β2-agonist pharmacology.

Using organ baths and a range of pharmacological tools, we investigated how the relaxation of airway tissue induced by activators of selective β-adrenoceptors, adenylyl cyclase (AC) and PKA was affected by the presence of antagonists selective for specific elements of the signalling cascade in isolated GP trachea under 1 µM carbachol induced tone.

β2-Agonist-induced relaxation of guinea pig isolated airways was mediated predominantly via β2-adrenoceptors; the selective β2-antagonist ICI118551 (100 nM) antagonised fenoterol-induced relaxation (pA2 = 9.1 ± 0.2, n = 4). A prominent role for PKA and BKCa channels in β2-agonist-induced relaxation of airway smooth muscle was also identified; fenoterol-induced relaxation was inhibited by the PKA inhibitor Rp-8-CPT-cAMPS (300 µM, 82.6 ± 4.0 vs. 52.5 ± 12.3% maximum relaxation, n = 4) and the selective BKCa blocker paxilline (1 µM, 90.0 ± 2.4 vs. 46.0 + 15.3% maximum relaxation, n = 4). The guanylyl cyclase inhibitor ODQ (1 µM), the PKG inhibitor KT5823 (10 µM) and selective blockers of KATP (glibenclamide, 10 µM), SK (apamin, 1 µM) and IK (clotrimazole, 10 µM) K+ channels did not substantially affect fenoterol-induced relaxation (all n = 4). Similar to previous studies (Koike et al., 2004; Tanaka et al., 2003) we were unable to provide evidence for a role for AC in the signalling cascade, beyond showing that the AC activator forskolin induces relaxation in GP trachea (30 µM forskolin induced maximal relaxation, n = 4). Structurally distinct inhibitors of AC - MDL12330a and SQ22536 (both 10 µM) - consistently failed to inhibit fenoterol- and forskolin-induced relaxation; in fact the compounds often induced relaxation of the tissues.

Elucidation of the pathway through which β2-agonists function may identify new targets and eventually lead to novel pharmacotherapies with improved safety and efficacy profiles. Whilst we have found evidence that PKA and BKCa channels have important roles in β2-adrenoceptor-mediated signalling, it is currently unclear whether the failure to identify evidence that AC mediates the response is due the enzyme not being involved, or due to issues with the AC-selective tools used in this investigation.

Figure 1: Inhibition of fenoterol-induced relaxation by the PKA inhibitor RP-8-CPT-cAMPS and the BKCa blocker paxilline in isolated GP trachea under induced tone (n = 4, data points represent mean values ± s.e.m.)

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