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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 (β 2 ) are commonly prescribed as bronchodilators for respiratory diseases such as asthma and COPD and yet the intracellular signalling mechanisms are still not fully understood. Recently we have shown, using tracheal tissue from WT and GM mice, that, of the 10 different isoforms of AC, AC6 plays a central role in β-agonist, but not PGE 2 , responses (Birrell et al . BJP 2015). The aim of this study was to determine if this mechanism is apparent in translational studies utilising human airway smooth muscle cells (HASMC). We showed that primary HASMCs express AC6 at the mRNA and protein level. We developed a shRNA sequence and vector that targeted AC6 (60+% knockdown of expression). We showed that cAMP production, as measured by FRET, in response to a β 2 agonist was significantly reduced in HASMCs with reduced AC6 expression, whereas responses to forskolin or EP 4 receptor agonist (receptor responsible for PGE 2 -induced relaxation) were not altered. To confirm AC6 functional role of we employed a technique which can detect the contractile status of cultured cells, Ptychography (VL21 Phasefocus UK). Using this system we showed that ACh induced “contraction”, which is reduced by the β 2 agonist in control cells, was not in the cells treated with AC6-shRNA. Responses to a PKA agonist (downstream from AC) however were comparable. We conclude that AC6 plays a critical role in relaxation of HASMCs to β 2 agonists; these results further unravel the signalling pathway of this extensively prescribed class of medicine.

Introduction: Asthma prevalence has increased world-wide especially in children; thus there is a need to develop new therapies that are safe and effective especially for patients with severe/refractory asthma. CD4 + T cells are thought to play a central role in disease pathogenesis and associated symptoms. Recently, TRPV4 has been demonstrated to regulate the activation and inflammatory properties of CD4 + cells. The aim of these experiments was to determine the role of TRPV4 in an asthma model using a TRPV4 inhibitor and genetically modified (GM) animals. Methods: Mice (wild type or TRPV4 -/- ) and rats were sensitised with antigen (HDM or OVA) and subsequently topically challenged with the same antigen. Key features associated with an allergic asthma type phenotype were measured: lung function (Late asthmatic response [LAR] in the rat model and airway hyperreactivity [AHR] in the mouse model), allergic status (IgE levels) and airway inflammation. Results : Targeting TRPV4 using either GM mice or a pharmacological inhibitor (dosed after sensitisation but before challenge) abolished IgE levels in the mouse model (100% inhibition of the HDM response) but had no impact in the rat. Airway inflammation was significantly decreased in both models (Rat BALF eosinophilia: 92.5% inhibition of the OVA response, mouse BALF eosinophilia: 88.5% inhibition of the HDM response). This was associated with suppression of antigen triggered changes in lung function (Rat LAR: 74.9% inhibition of the signal; mouse AHR: 92.4% inhibition of the response to 3 mg/ml of aerosolised 5-HT). Conclusion : These data would support the evaluation of TRPV4 inhibitors in allergen challenge models in proof of concept clinical trials.

The late asthmatic response (LAR) is a defining symptom of asthma and is regularly used as a model for proof of concept clinical trials with new therapeutic agents, yet the mechanism driving LAR is not fully understood. Recently we have shown using a preclinical model that the LAR involves the following processes: allergen challenge triggering airway sensory nerves which initiate a central reflex event leading to a parasympathetic cholinergic constrictor response (Raemdonck K. et al .Thorax.2012:67:19). We have also shown that activation of the TRPV4-ATP-P2X3 axis can trigger a response in airway Aδ afferent fibres and it is known that ATP levels are increased in the asthmatic airway (Bonvini S.J. et al. JACI.2016). We hypothesised that this axis could be the trigger that engages neuronal reflexes and the subsequent LAR. In a rat model of allergic asthma, antigen challenge triggered a LAR response that was inhibited by two structurally distinct Long Acting Muscarinic Antagonists (LAMA, tiotropium and glycopyrrolate). In naive rats, an inhaled TRPV4 agonist (GSK1016790a) caused a “LAR-like” response which was attenuated by a TRPV4 inhibitor (GSK2193874), a P2X3 inhibitor (AF-353) and a LAMA (glycopyrrolate), suggesting that stimulation of the axis could replicate the LAR. To confirm the role we showed that blockade of TRPV4 or P2X3 attenuated the LAR (74.9% and 88.3%, respectively) in the asthma model. These data suggest that airway sensory nerves can be triggered via a TRPV4-ATP-P2X3 axis to cause the parasympathetic reflex to drive the LAR. Targeting this axis could be a novel and effective treatment for this key feature of asthma and in patients with an allergic asthma phenotype.