Search

Background and Purpose Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC-dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to β-adrenoceptor agonists. Experimental Approach Receptors mediating responses to β-adrenoceptor agonists in airway smooth muscle (ASM) and sensory nerve were identified in isolated tissue systems. Expression of mRNA for the AC isoforms in ASM and neurones was determined by qPCR. Functional responses were assessed in AC isoform KO mice and wild-type controls. Key Results Airway and vagal tissue expressed mRNA for various isoforms of AC. AC6 was the most prominent isoform. Responses to β-adrenoceptor agonists in tissues from AC6 KO mice were virtually abolished. Conclusions and Implications AC6 played a critical role in relaxation of ASM to β1-adrenoceptor agonists and in modulation of sensory nerves by β1-3-adrenoceptor agonists. These results further unravel the signalling pathway of this extensively prescribed class of medicine.

BACKGROUND AND PURPOSE Understanding the role of the EP2 receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP2 receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP2 receptor over other EP receptors using a range of isolated tissue systems. EXPERIMENTAL APPROACH PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP1); ONO-AE1-259 and PGE2- induced relaxation of mouse and guinea pig trachea (EP2); PGE2-induced depolarization of guinea pig isolated vagus (EP3); PGE2-induced relaxation of human and rat trachea (EP4). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays. KEY RESULTS In bioassay systems, where assessment of potency/selectivity is made against the ‘native’ receptor, PF-04418948 only acted as an antagonist of EP2 receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE2 respectively. However, the affinity of PF-04418948 was not equal in the two preparations. CONCLUSIONS AND IMPLICATIONS Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP2-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP2 receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE2 and the role of EP2 receptors in health and disease.

Background The incidence of asthma is increasing at an alarming rate and while the current available therapies are effective in the majority of patients they fail to adequately control symptoms at the more severe end of the disease spectrum. In the search to understand disease pathogenesis and find effective therapies animal models are often employed. As exposure to house dust mite (HDM) has a causative link, it is thought of as the allergen of choice for modelling asthma. The objective was to develop a HDM driven model of asthmatic sensitisation and characterise the role of key allergic effector cells/mediators. Methods Mice were sensitised with low doses of HDM and then subsequently challenged. Cellular inflammation, IgE and airway responsiveness (AHR) was assessed in wild type mice or CD4+/CD8+ T cells, B cells or IgE knock out mice. Results Only those mice sensitised with HDM responded to subsequent low dose topical challenge. Similar to the classical ovalbumin model, there was no requirement for systemic alum sensitisation. Characterisation of the role of effector cells demonstrated that the allergic cellular inflammation and AHR was dependent on CD4+ and CD8+ T cells but not B cells or IgE. Finally, we show that this model, unlike the classic OVA model, appears to be resistant to developing tolerance. Conclusions This CD4+/CD8+ T cell dependent, HDM driven model of allergic asthma exhibits key features of asthma. Furthermore, we suggest that the ability to repeat challenge with HDM means this model is amenable to studies exploring the effect of therapeutic dosing in chronic, established disease. Electronic supplementary material The online version of this article (doi:10.1186/s12931-016-0359-y) contains supplementary material, which is available to authorized users.

Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC-dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to β-adrenoceptor agonists.Receptors mediating responses to β-adrenoceptor agonists in airway smooth muscle (ASM) and sensory nerve were identified in isolated tissue systems. Expression of mRNA for the AC isoforms in ASM and neurones was determined by qPCR. Functional responses were assessed in AC isoform KO mice and wild-type controls.Airway and vagal tissue expressed mRNA for various isoforms of AC. AC6 was the most prominent isoform. Responses to β-adrenoceptor agonists in tissues from AC6 KO mice were virtually abolished.AC6 played a critical role in relaxation of ASM to β1 -adrenoceptor agonists and in modulation of sensory nerves by β1-3 -adrenoceptor agonists. These results further unravel the signalling pathway of this extensively prescribed class of medicine.

776 volume 17 | number 7 | july 2011 nature medicine To the Editor: We read with interest the article by Deshpande et al.1 on bitter taste receptors (TAS2Rs) in the lungs and the expression of TAS2Rs on human airway smooth muscle (ASM). The authors describe experiments in which TAS2R agonists, such as chloroquine, evoked relaxation of mouse tracheal rings that was threefold greater in magnitude than that elicited by b-adrenergic receptor agonists1 (the current gold-standard bronchodilator therapy for asthma and chronic obstructive pulmonary disease2). We sought to investigate the uncharacteristically weak bronchodilator activity of isoproterenol in human tissue. After repeating key experiments from the paper by Deshpande et al.1 in our laboratories in the UK and in the laboratories of our collaborator in the US (B.J.C.), we obtained data that is consistent with a body of literature3 and clinical practice that suggests that isoproterenol is a potent and efficacious relaxant of human airway smooth muscle, eliciting a relaxation 4 log fold (10,000×) more potent than chloroquine, although both agents had similar efficacy (Fig. 1a,b). This difference in potency was apparent when we precontracted the human airway smooth muscle to 60–80% of the maximum attainable contraction with either 10 μM histamine (Fig. 1a) or 1 μM carbachol (Fig. 1b). We obtained comparable results in studies using airway smooth muscle preparations from guinea pigs, a species in which relaxation to b-agonists is elicited through the b2-adrenergic receptor (M.G.B., N.D., M.A.B. and B.J.C., unpublished observations). Several explanations could be proffered as to why there is a discrepancy between our results and those obtained by Deshpande et al.1 When performing in vitro tissue bath experiments to investigate the relaxant properties of agonists on ASM, standard practice is to precontract the tissue to establish a baseline level of bronchoconstrictor tone. The tone induced would normally be submaximal to avoid functional antagonism of the potential relaxant response4. However, the concentration of acetylcholine used by Deshpande et al.1 (0.1 mM) is supramaximal, which could functionally antagonize the relaxant response to isoproterenol, thereby making it appear smaller than expected. When we repeated these experiments under conditions of supramaximal tension (0.1 mM ACh), we also found that the relaxation to isoproterenol was compromised (60% of the maximal relaxation evoked by papaverine instead of 100%; Fig. 1c,d). We should emphasize that we do not question the author’s conclusions that airway smooth muscle from humans and mice express bitter taste receptors. This is potentially a physiologically important observation. We would respectfully suggest, however, that our data and the published literature cast doubt on the claim that TAS2R agonists are more effective bronchodilators than currently prescribed b2-adrenergic receptor agonists. Note: Supplementary information is available on the Nature Medicine website.

Chronic Obstructive Pulmonary Disease (COPD) is a cigarette smoke (CS)-driven inflammatory airway disease with an increasing global prevalence. Currently there is no effective medication to stop the relentless progression of this disease. It has recently been shown that an activator of the P2X7/inflammasome pathway, ATP, and the resultant products (IL-1β/IL-18) are increased in COPD patients. The aim of this study was to determine whether activation of the P2X7/caspase 1 pathway has a functional role in CS-induced airway inflammation. Mice were exposed to CS twice a day to induce COPD-like inflammation and the role of the P2X7 receptor was investigated. We have demonstrated that CS-induced neutrophilia in a pre-clinical model is temporally associated with markers of inflammasome activation, (increased caspase 1 activity and release of IL-1β/IL-18) in the lungs. A selective P2X7 receptor antagonist and mice genetically modified so that the P2X7 receptors were non-functional attenuated caspase 1 activation, IL-1β release and airway neutrophilia. Furthermore, we demonstrated that the role of this pathway was not restricted to early stages of disease development by showing increased caspase 1 activation in lungs from a more chronic exposure to CS and from patients with COPD. This translational data suggests the P2X7/Inflammasome pathway plays an ongoing role in disease pathogenesis. These results advocate the critical role of the P2X7/caspase 1 axis in CS-induced inflammation, highlighting this as a possible therapeutic target in combating COPD.