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Some rocks contain multiple remanence “components”, each of which preserves a record of a different magnetic field. The temperature ranges over which these remanence components demagnetize can overlap, making it difficult to determine their directions. We present a data analysis tool called Thermal Resolution Of Unblocking Temperatures (TROUT) that treats the process of thermal demagnetization as a function of temperature (or alternating field demagnetization as a function of coercivity). TROUT models the unblocking temperature distributions of components in a demagnetization experiment, allowing these distributions to overlap. TROUT can be used to find the temperatures over which paleomagnetic directions change and when two directional components overlap resulting in curved demagnetization trajectories. When applied to specimens given multi-component Thermoremanent Magnetizations (TRMs) in the laboratory, the TROUT method estimates the temperature at which the partial TRMs were acquired to within one temperature step, even for specimens with significant overlap. TROUT has numerous applications: knowing the temperature at which the direction changes is useful for experiments in which the thermal history of a specimen is of interest (e.g. emplacement temperature of pyroclastic deposits, re-heating of archaeological artifacts, reconstruction of cooling rates of igneous bodies). The ability to determine whether a single component or multiple components are demagnetizing at a given temperature is useful for choosing appropriate ranges of temperatures to use in paleointensity experiments. Finally, the width of the range of temperature overlap may be useful for inferring the domain state of magnetic mineral assemblages.

Earth's magnetic field is recorded as oceanic crust cools, generating lineated magnetic anomalies that provide the pattern of polarity reversals for the past 160 million years

Chronic cough is a burden of many respiratory diseases with no effective & safe treatment. The prostaglandin PGE2 acts on EP receptors (EP1-4) with beneficial & adverse effects in the lungs. We have shown EP4 agonism inhibits activation of airway sensory nerves, blocking cough1. We aimed to determine the signalling downstream of EP4 in airway sensory nerves, to better understand its anti-tussive effect. We isolated guinea pig (GP) vagal ganglia neurons, using retrograde tracer dye to identify airway neurons in vitro. We assessed intracellular calcium & cAMP in these neurons using chemical & virally transfected fluorophores. We also used a grease-gap recording technique to pharmacologically examine EP4 signalling in GP & human vagus nerves in vitro. We have shown that EP4 agonist inhibits capsaicin-induced activation of GP airway neurons. EP4 is known to be a Gαs-coupled GPCR, so we imaged cAMP, observing that EP4 agonism causes cAMP elevation. Dual-fluorophore imaging showed cAMP elevation in neurons where EP4 agonism had inhibitory effects. From previous work we know EP4 agonist inhibits capsaicin-induced GP & human vagus nerve activation. We looked whether the main cAMP effectors (PKA & EPAC) were involved. EPAC (but not PKA) blockers (ESI-09 & KT5720) abolished the inhibitory effect of EP4 agonism. EPAC activator (8-CPT-2Me-cAMP) mimicked the effect of EP4 agonism; this was blocked by EPAC inhibitor. These data reveal a role for cAMP & EPAC in the inhibitory & anti-tussive effects of EP4 agonism on airway sensory nerves. Understanding how EP4 exerts its beneficial effects on airway sensory nerves is important to better inform drug discovery efforts for anti-tussive therapies. 1.Wortley(2019)AJRCCM;199:A2862

Background Individuals with inflammatory bowel disease (IBD) are at increased risk of developing anxiety or depression (A&D). Crohn's disease (CD) and ulcerative colitis (UC) with comorbid A&D are both more challenging to manage. IBD providers need to better understand the causes and impact of A&D in order to improve care for IBD patients. We sought to identify clinical factors that influence development of A&D and healthcare utilization in IBD. Methods This is a retrospective analysis using an IBD natural history registry from a single tertiary care referral center. Presence of A&D was determined based upon responses to the Hospital Anxiety and Depression Scale. Demographic and clinical factors were abstracted to evaluate for significant associations. Results Four hundred thirty-two IBD patients (132 UC, 256 CD, and 44 indeterminate colitis) were included in this study. One hundred ninety-two (44.4%) had anxiety or depression or both, and most were female (59.4%, P < 0.05). History of surgery (P < 0.05), female gender (P < 0.05), smoking (P < 0.05), and extra-intestinal manifestations (P < 0.01) were each independently predictive of A&D. Inflammatory bowel disease patients with A&D more often underwent imaging studies (53.6% vs 36.7%, P < 0.05), visited the ED (30.7% vs 20.8%, P < 0.05), or were hospitalized (31.7% vs 21.7%, P < 0.05). They were also more frequently prescribed corticosteroids (50.5% vs 36.7%, P < 0.01) and biologic medications (62.5% vs 51.3%, P < 0.05). Finally, they were more likely to have had at least 1 "no-show" (29.2% vs 16.7%, P < 0.01) and had a higher mean number of "no-shows" (0.69 +/- 0.1 vs 0.30 +/- 0.1, P < 0.01) over the study period. Discussion Anxiety and depression are common in the setting of IBD and are strongly associated with surgical history, disease complications (including extra-intestinal manifestations), smoking, and female gender. Inflammatory bowel disease patients with A&D are also more likely to require therapy and to utilize healthcare resources. This study refines our understanding of A&D development and its impact in IBD and provides additional considerations for management in this setting.

Commercial plantations are primarily managed for timber production, and are frequently considered poor for biodiversity, particularly for mammalian species. Bats, which constitute one fifth of mammal species worldwide, have undergone large declines throughout Europe, most likely due to widespread habitat loss and degradation. Bat use of modified landscapes such as urban or agricultural environments has been relatively well studied, however, intensively managed plantations have received less attention, particularly in Europe. We assessed three of the largest, most intensively managed plantations in the UK for the occurrence of bats, activity levels and relative abundance in response to environmental characteristics at multiple spatial scales, using an information theoretic approach. We recorded or captured nine species; Pipistrellus pipistrellus and P . pygmaeus were the most commonly recorded species on acoustic detectors and female P . pygmaeus were the most commonly captured. The influence of environmental characteristics on bat activity varied by species or genus, although all bat species avoided dense stands. Occurrence and activity of clutter and edge adapted species were associated with lower stand densities and more heterogeneous landscapes whereas open adapted bats were more likely to be recorded at felled stands and less likely in areas that were predominantly mature conifer woodland. In addition, despite morphological similarities, P . pipstrellus and P . pygmaeus were found foraging in different parts of the plantation. This study demonstrates that with sympathetic management, non-native conifer plantations may have an important role in maintaining and supporting bat populations, particularly for Pipistrellus spp.

Most airway diseases, including chronic obstructive pulmonary disease (COPD), are associated with excessive coughing. The extent to which this may be a consequence of increased activation of vagal afferents by pathology in the airways (e.g., inflammatory mediators, excessive mucus) or an altered neuronal phenotype is unknown. Understanding whether respiratory diseases are associated with dysfunction of airway sensory nerves has the potential to identify novel therapeutic targets.To assess the changes in cough responses to a range of inhaled irritants in COPD and model these in animals to investigate the underlying mechanisms.Cough responses to inhaled stimuli in patients with COPD, healthy smokers, refractory chronic cough, asthma, and healthy volunteers were assessed and compared with vagus/airway nerve and cough responses in a cigarette smoke (CS) exposure guinea pig model.Patients with COPD had heightened cough responses to capsaicin but reduced responses to prostaglandin E2 compared with healthy volunteers. Furthermore, the different patient groups all exhibited different patterns of modulation of cough responses. Consistent with these findings, capsaicin caused a greater number of coughs in CS-exposed guinea pigs than in control animals; similar increased responses were observed in ex vivo vagus nerve and neuron cell bodies in the vagal ganglia. However, responses to prostaglandin E2 were decreased by CS exposure.CS exposure is capable of inducing responses consistent with phenotypic switching in airway sensory nerves comparable with the cough responses observed in patients with COPD. Moreover, the differing profiles of cough responses support the concept of disease-specific neurophenotypes in airway disease. Clinical trial registered with www.clinicaltrials.gov (NCT 01297790).

Background and Purpose Airway microvascular leak (MVL) involves the extravasation of proteins from post-capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. Prostaglandin E2 (PGE2), a product of cyclooxygenase mediated metabolism of arachidonic acid, binds to 4 receptors, termed EP1-4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Experimental Approach Evans Blue dye was used as a marker of airway MVL and selective EP receptor agonists and antagonists were used alongside EP receptor deficient mice to define the receptor subtype involved. Key Results PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2-induced MVL was demonstrated in Ptger2-/- and Ptger4-/- mice and in wild type mice pre-treated simultaneously with EP2 (PF-04418948) and the EP4 (ER-819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2-/- and Ptger4-/- mice. Conclusions and Implications PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2. This article is protected by copyright. All rights reserved.

Acute liver failure (ALF) is a rare life-threatening condition characterized by rapid progression and death. Causes vary according to geographic region, with acetaminophen and drug-induced ALF being the most common causes in the United States. Determining the cause aids in predicting the prognosis and the presentation of manifestations and guides providers to perform cause-specific management. At initial presentation, nonspecific symptoms are present but may progress to complications, including cerebral edema, infection, coagulopathy, renal failure, cardiopulmonary failure, and acid-base and/or metabolic disturbances. Although some cases of ALF resolve with conservative measures, liver transplantation is the ultimate treatment in many cases.

ATP, an endogenous danger signal acting on purinergic receptors including P2X2 & P2X3 on sensory nerves, is elevated in airway disease. A P2X3 antagonist was highly effective in reducing cough counts in chronic cough patients (Abdulqawi Lancet [2015] 385:9974). We have shown that activation of transient receptor potential (TRP) V4 causes cough in guinea pigs (GP) via ATP release, which act on P2X3 to fire A∂-(but not C-) fibres. Activation of the TRPM3 ion channel similarly activates A∂ fibres via P2X3, but also C-fibres independently of P2X3. We examined the distribution of TRPM3, P2X2&P2X3 in single airway neurons. Airway terminating neurons isolated from GPs were individually harvested & single-cell gene expression evaluated. P2X3 was expressed in 23/32 Jugular (J) & 22/37 Nodose (N) neurons, P2X2 was expressed in 1/32 J & 16/37 N, & P2X2 was co-expressed in 15/47 P2X3 neurons. Most A∂-fibres originate from the N ganglion, where P2X2/3 co-expression was predominant, and most C-fibres originate from the J ganglion where P2X3 was predominant. TRPM3 was expressed on 22/32 J and 14/37 N neurons, and co-expressed in 34/49 P2X expressing neurons. These results suggest P2X2/3 expression is A∂-fibre predominant, and required for firing caused by endogenous ATP release. This may explain why TRPV4, which we found to only activate nodose neurons, only fires A∂-fibres. The frequent expression of TRPM3 on J neurons explains P2X2/3-independent firing of C-fibres by TRPM3. This work shows how single cell expression can determine ion channel profiles of different nerve populations, which may aid drug targeting and differentiation for patients with altered sensory reflex responses and enhanced cough in chronic lung diseases.

Heightened cough responses to inhaled capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, are characteristic of patients with chronic cough. However, previously, a TRPV1 antagonist (SB-705498) failed to improve spontaneous cough frequency in these patients, despite small reductions in capsaicin-evoked cough.XEN-D0501 (a potent TRPV1 antagonist) was compared with SB-705498 in preclinical studies to establish whether an improved efficacy profile would support a further clinical trial of XEN-D0501 in refractory chronic cough.XEN-D0501 and SB-705498 were profiled against capsaicin in a sensory nerve activation assay and in vivo potency established against capsaicin-induced cough in the guinea pig. Twenty patients with refractory chronic cough participated in a double-blind, randomized, placebo-controlled crossover study evaluating the effect of 14 days of XEN-D0501 (oral, 4 mg twice daily) versus placebo on awake cough frequency (primary outcome), capsaicin-evoked cough, and patient-reported outcomes.XEN-D0501 was more efficacious and 1,000-fold more potent than SB-705498 at inhibiting capsaicin-induced depolarization of guinea pig and human isolated vagus nerve. In vivo XEN-D0501 completely inhibited capsaicin-induced cough, whereas 100 times more SB-705498 was required to achieve the same effect. In patients, XEN-D0501 substantially reduced maximal cough responses to capsaicin (mean change from baseline, XEN-D0501, -19.3 ± 16.4) coughs; placebo, -1.8 ± 5.8 coughs; P 0.0001), but not spontaneous awake cough frequency (mean change from baseline, XEN-D0501, 6.7 ± 16.9 coughs/h; placebo, 0.4 ± 13.7 coughs/h; P = 0.41).XEN-D0501 demonstrated superior efficacy and potency in preclinical and clinical capsaicin challenge studies; despite this improved pharmacodynamic profile, spontaneous cough frequency did not improve, ruling out TRPV1 as an effective therapeutic target for refractory cough. Clinical trial registered with www.clinicaltrialsregister.eu (2014-000306-36).

Cough is the most common reason to visit a primary care physician, yet it remains an unmet medical need. Fatty acid amide hydrolase (FAAH) is an enzyme that breaks down endocannabinoids, and inhibition of FAAH produces analgesic and anti-inflammatory effects. Cannabinoids inhibit vagal sensory nerve activation and the cough reflex, so it was hypothesised that FAAH inhibition would produce antitussive activity via elevation of endocannabinoids. Primary vagal ganglia neurons, tissue bioassay, in vivo electrophysiology and a conscious guinea pig cough model were utilised to investigate a role for fatty acid amides in modulating sensory nerve activation in vagal afferents. FAAH inhibition produced antitussive activity in guinea pigs with concomitant plasma elevation of the fatty acid amides N-arachidonoylethanolamide (anandamide), palmitoylethanolamide, N-oleoylethanolamide and linoleoylethanolamide. Palmitoylethanolamide inhibited tussive stimulus-induced activation of guinea pig airway innervating vagal ganglia neurons, depolarisation of guinea pig and human vagus, and firing of C-fibre afferents. These effects were mediated via a cannabinoid CB2/Gi/o-coupled pathway and activation of protein phosphatase 2A, resulting in increased calcium sensitivity of calcium-activated potassium channels. These findings identify FAAH inhibition as a target for the development of novel, antitussive agents without the undesirable side-effects of direct cannabinoid receptor agonists.

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.

Prostaglandin D2 (PGD2) causes cough and levels are increased in asthma suggesting that it may contribute to symptoms. Although the prostaglandin D2 receptor 2 (DP2) is a target for numerous drug discovery programmes little is known about the actions of PGD2 on sensory nerves and cough.We used human and guinea pig bioassays, in vivo electrophysiology and a guinea pig conscious cough model to assess the effect of prostaglandin D2 receptor (DP1), DP2 and thromboxane receptor antagonism on PGD2 responses.PGD2 caused cough in a conscious guinea pig model and an increase in calcium in airway jugular ganglia. Using pharmacology and receptor-deficient mice we showed that the DP1 receptor mediates sensory nerve activation in mouse, guinea pig and human vagal afferents. In vivo, PGD2 and a DP1 receptor agonist, but not a DP2 receptor agonist, activated single airway C-fibres. Interestingly, activation of DP2 inhibited sensory nerve firing to capsaicin in vitro and in vivo.The DP1 receptor could be a therapeutic target for symptoms associated with asthma. Where endogenous PGD2 levels are elevated, loss of DP2 receptor-mediated inhibition of sensory nerves may lead to an increase in vagally associated symptoms and the potential for such adverse effects should be investigated in clinical studies with DP2 antagonists.

Background COPD is an inflammatory disease usually associated with cigarette smoking (CS) with an increasing global prevalence and no effective medication. Extracellular ATP is increased in the COPD affected lung and may play a key role in driving CS-induced airway inflammation, but the mechanism involved in ATP release has eluded researchers. Recently, the transient receptor potential (TRP) and pannexin-1 channels have been suggested to play a role in other experimental paradigms. Thus, the aim of this work is to investigate if these channels are involved in CS-induced ATP release in the lung. Methods Primary human cells were exposed to CS and extracellular ATP levels measured. Mice were exposed to mainstream CS and airway inflammation assessed. TRPV1/4 mRNA expression was assessed in human lung parenchyma. Results CS exposure caused a dose-related increase in ATP from primary airway bronchial epithelial cells. This was attenuated by blockers of TRPV1, TRPV4 and pannexin-1 channels. Parallel data was obtained using murine acute CS-driven model systems. Finally, TRPV1/4 mRNA expression was increased in lung tissue samples from patients with COPD. Conclusions Extracellular ATP is increased in the COPD affected lung and may play a key role in driving disease pathophysiology. These experiments uncover a novel mechanism which may be responsible for CS-induced ATP release. These findings highlight novel targets that could lead to the development of medicine to treat this devastating disease.

In chronic lung diseases, activation of airway sensory nerves initiate respiratory reflexes including cough for which there is currently no safe and effective treatment. Ion channels on sensory afferents can activate these reflexes and as such are attractive therapeutic targets. The ion channel TRPM3 is a sensor of noxious heat (Vriens J. et al. Neuron.2011:70.482-494), is activated by hypoosmolarity and is expressed in somatosensory neurons from dorsal root and trigeminal ganglia. We aimed to characterise the role of TRPM3 in airway sensory nerve activation and the cough reflex. Single cell PCR demonstrated that TRPM3 was expressed in airway specific neurons from guinea pig (GP) nodose and jugular ganglia. Functional experiments using selective pharmacological tools indicated that the TRPM3 agonist CIM0216 evoked [Ca 2+ ] i flux in isolated airway neurons, and also depolarisation of mouse, GP and human vagus nerves in an in vitro preparation. Depolarisation by CIM0216 and hypoosmolar solution was inhibited by two selective TRPM3 antagonists and in Trpm3 -/- mice. Preliminary results demonstrate action potential firing by CIM0216 of C-fibres in the anaesthetised GP. These data suggest that TRPM3 is expressed in airway sensory neurons and that activators of TRPM3 cause functionally relevant activation of airway nerves. Recent data using preclinical models and clinical challenge studies has demonstrated differing profiles of cough responses across diseases, supporting the concept of disease-specific neurophenotypes and underlining the need to understand airway neurobiology (Belvisi MG. et al .AJRCCM.2016). The discovery of TRPM3 as a regulator of airway reflexes suggests it may be a novel target for antitussive therapy.

Urban air pollution, specifically particulate matter (PM), is a global health issue. Diesel exhaust particles (DEP) are a key component of PM and epidemiological evidence links exposure with respiratory symptoms. Activation of airway sensory afferents drives respiratory reflexes. Transient receptor potential (TRP) channels expressed on airway afferents can be activated by various stimuli to initiate reflexes, such as cough or bronchoconstriction (Birrell et al. AJRCCM:2009 , 1042–1047). We aimed to determine whether DEP activated airway sensory nerves and whether TRP channels were involved. DEP subunits were ∼50nm in diameter with aggregates up to 2μm. DEP was ∼2% metallic, ∼15% organic and ∼83% carbon. In anaesthetised guinea pigs, intratracheal administration of DEP activated chemosensitive airway single nerve fibres (8.3±0.3 action potential impulses.sec -1 ). Only the organic extract (DEP-OE) activated sensory nerves (measured as depolarisation) of the isolated guinea pig and human vagus and this was inhibited by 83.4±10.2% by a TRPA1 antagonist. DEP generates oxidative stress, a known TRPA1 activator. The antioxidant N-acetyl cysteine (NAC) abolished responses to DEP-OE and polycyclic aromatic hydrocarbons (PAH) found in DEP activate the vagus nerve. The aryl hydrocarbon receptor (AhR) and subsequent mitochondrial ROS, which can activate TRPA1 on nociceptive C-fibres (Nesuashvili et al. MolPharm : 2013,83,1007-1019), are central to the response. To conclude, DEP can activate airway sensory nerves via activation of chemosensitive afferents. These findings provide the first mechanistic insights into how exposure to urban air pollution leads to respiratory symptoms.

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.

Cough is a protective mechanism but can occur excessively in disease. Cough can be modulated by a range of GPCRs which can be either inhibitory or excitatory. Prostaglandin E2 and bradykinin can activate airway sensory nerves via EP3 and B2 receptors receptively and have both been shown to mediate their effects though TRPV1 and TRPA1 receptors. Activation of the β2-adrenoceptor and cannabinoid CB2 receptors can inhibit sensory nerves and prevent cough. It is currently thought that activation of the β2-adrenoceptor causes c-AMP dependent activation of PKA; however, recent research has suggested that the pathway involves PKG-mediated opening of the BKCa channel leading to hyperpolarization.

The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.

Per-oral endoscopic myotomy (POEM) is a relatively novel endoscopic technique for the treatment of achalasia. POEM has been shown to have outcomes comparable to those with Heller myotomy, but it is less invasive and has fewer complications. A 72-year-old man with progressive solid and liquid dysphagia underwent POEM, but soon after the procedure went into cardiac arrest; spontaneous circulation returned after 10 minutes of CPR. He was subsequently found to have tension pneumopericardium as a result of the inadvertent use of air instead of carbon dioxide during the procedure. He had a prolonged hospitalization that required an extended stay in the medical intensive care unit. Although rare, POEM can lead to critical, life-threatening complications.

Cough is an essential reflex, but chronic cough can be a problematic symptom of respiratory diseases, for which no effective and safe medications are available. We have shown that inhibition of fatty acid amide hydrolase (FAAH) elevates endocannabinoids (inc. palmitoylethanolamide, anandamide, oleoylethanolamide & linoleoylethanolamide: PEA, AEA, OEA, LEA) and inhibits citric-acid evoked cough in guinea pigs, and PEA inhibits sensory nerve depolarisation1. We investigated the pathway of endocannabinoid inhibition of sensory nerve/neuron activity. PEA, AEA, OEA & LEA inhibited capsaicin-induced depolarisation of isolated guinea pig vagus nerve via the CB2 (but not CB1) receptor, and capsaicin-induced calcium influx in airway-terminating jugular neurons – i.e. cough reflex relevant vagal neurons. Using vagus nerve from PTX dosed guinea pigs we showed PEA inhibition required the Gαi protein. PEA inhibition also required small conductance K+ channel (SKCa) activation, and concurrent with SKCa mediated suppression of nerve activation, PEA inhibited depolarisation by TRPA1 & TRPV4 agonists. Finally, PEA increases the calcium sensitivity of SKCa via protein phosphatase 2A activation. Key experiments paralleled in human vagus nerve indicate this pathway translates to man. In summary, endocannabinoids inhibit sensory nerve activation in guinea pig and human vagus nerve via CB2–Gαi–PP2A–SKCa. This data implies that inhibiting FAAH/elevating endocannabinoids may be beneficial in conditions driven by excessive peripheral sensory nerve activation including chronic cough. 1. Adcock et al. (2011) Proc Br Pharmacol Soc.9: A025p.

DiSodium Cromoglycate (DSCG) has been used as an effective asthma therapy for over 50 years with a good clinical safety history but with poor pharmacokinetics. An analogue of DSCG with a more preferable profile could aid the development of novel asthma drugs, however, the mechanism of action is currently unknown. Previous pre-clinical data has linked the late asthmatic response (LAR) with activation of the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel on airway sensory nerves 1 and DSCG has been shown to inhibit this response in clinical studies 2 . Thus we hypothesised that the effectiveness of DSCG is via the modulation of sensory nerves. In a rat allergic model of asthma, DSCG inhibited the LAR. Using rat, guinea pig and human isolated vagal nerves and rat airway vagal ganglia cells it was shown that DSCG modulates TRPA1-driven nerve activation and calcium influx. DSCG is an agonist at the orphan G protein coupled receptor 35 (GPR35). TRPA1 and GPR35 are expressed in vagal ganglia cells therefore it was hypothesised that GPR35 is involved in the mechanism by which DSCG inhibits TRPA1 mediated sensory nerve activity. Utilising a range of GPR35 agonists and channel blockers we identified that this effect of DSCG was dependent on GPR35 via signalling through Gi and N-type calcium channels. Finally, using in vivo models we show that DSCG inhibition of airway nerve firing and LAR was attenuated by the GPR35 antagonist, ML 145. DSCG attenuates the TRPA1-mediated nerve-driven asthma symptoms via a direct action on sensory nerves through GPR35. Thus, GPR35 could be a novel, safe and effective target for the development of future asthma therapy. Raemdonck et al (2012) Thorax 67: 19 Howell & Altounyan (1967) Lancet 2: 539.

Asthma is a debilitating disease of the airways characterised by symptoms such as bronchoconstriction, hyperresponsiveness and cough. Current therapies are ineffective in severe disease highlighting the need for novel treatments. Menthol, a cooling compound found in medicinal products and several OTC medications, is commonly thought to activate the Transient Receptor Potential Melastatin 8 (TRPM8) channel and has been reported to possess beneficial effects such as bronchodilation and suppression of cough, however the mechanism of action is unknown. The aim was to study the beneficial properties of menthol in the airways and investigate the role of TRPM8. TRPM8 was expressed in mouse and guinea pig (GP) vagal ganglia. Using a TRPM8 antagonist (JNJ41876666) and Trpm8 -/- mice in an isolated vagal nerve preparation we showed that both WS3 (TRPM8 agonist) and (-)-menthol caused a small activation of human, GP and mouse vagal nerves which was TRPM8 dependent. Interestingly, pre-incubation of (-)-menthol inhibited vagal nerve activation induced by the tussive stimulus, capsaicin, an effect that was not inhibited by JNJ41876666. WS3 and (-)-menthol caused relaxation of human, mouse and GP trachea, which was not abolished by JNJ41876666 nor in the Trpm8 -/- mouse airway. No expression of TRPM8 was detected in human, GP or mouse airway smooth muscle. (-)-menthol caused a small TRPM8-dependent activation but a robust TRPM8-independent inhibition of vagal sensory nerve activity and relaxation of airway smooth muscle. Elucidating the mechanism behind the beneficial effects of (-)-menthol could lead to the development of promising new therapeutic targets for airway diseases such as asthma.

Activation of TRPV4 causes vagal nerve depolarisation, single airway fibre firing and reflex cough in the guinea pig (GP) through activating nodose derived Aδ fibres(Belvisi M. et al. AJRCCM:2013.A5265). Ca 2+ signal and fibre firing demonstrated a short delay prior to activation, suggesting an indirect mechanism. Activation of TRPV4 possibly on the urothelium has been linked to ATP release and activation of P2X3 receptors on bladder afferents (Aizawa N. et al . Neuro Urod,2010 : 148-55), and so the aim of this study was to investigate the role of ATP and P2X3 in TRPV4 induced activation of airway afferents. Ca 2+ imaging of GP airway neurons indicated that αβ-MeATP, an agonist of P2X1 and P2X3 containing receptors, induced Ca 2+ flux in the nodose ganglia and caused depolarisation of GP and human vagal tissue in an in vitro vagal nerve preparation. Depolarisation of human and GP nerves by the TRPV4 agonists GSK1016790a (GSK101) and hypoosmolarity was inhibited following the use of 2 selective P2X3 antagonists; AF-353 and TNP-ATP. A mechanism for ATP release was indicated as GSK101 induced depolarisation was abolished in Pannexin 1 KO mice, indicating that TRPV4 initiates ATP release through the Pannexin 1 pore. In vivo , GSK101 induced firing of airway Aδ fibres was significantly inhibited following AF-353 administration, and cough induced by GSK101 was inhibited by AF-353 to a similar extent as by the TRPV4 antagonist GSK2193874. The data suggests that TRPV4 agonists activate airway sensory nerves and cause cough in the conscious GP through release of ATP and activation of P2X3 on nodose derived Aδ fibres. TRPV4 is therefore a novel modulator of airway sensory nerves and as such is a good candidate for anti-tussive therapies.

Background and Purpose Airway microvascular leak (MVL) involves the extravasation of proteins from post‐capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. PGE2, a product of COX‐mediated metabolism of arachidonic acid, binds to four receptors, termed EP1–4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Experimental Approach Evans Blue dye was used as a marker of airway MVL, and selective EP receptor agonists and antagonists were used alongside EP receptor‐deficient mice to define the receptor subtype involved. Key Results PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2‐induced MVL was demonstrated in Ptger2 −/− and Ptger4 −/− mice and in wild‐type mice pretreated simultaneously with EP2 (PF‐04418948) and EP4 (ER‐819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2 −/− and Ptger4 −/− mice. Conclusions and Implications PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2.

BackgroundAsthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the airway. Current treatment options (long acting β-adrenoceptor agonists and glucocorticosteroids) are not optimal as they are only effective in certain patient groups and safety concerns exist regarding both compound classes. Therefore, novel bronchodilator and anti-inflammatory strategies are being pursued. Prostaglandin E2 (PGE2) is an arachidonic acid-derived eicosanoid produced by the lung which acts on four different G-protein coupled receptors (EP1–4) to cause an array of beneficial and deleterious effects. The aim of this study was to identify the EP receptor mediating the anti-inflammatory actions of PGE2 in the lung using a range of cell-based assays and in vivo models.Methods and resultsIt was demonstrated in three distinct model systems (innate stimulus, lipopolysaccharide (LPS); allergic response, ovalbumin (OVA); inhaled pollutant, cigarette smoke) that mice missing functional EP4 (Ptger4−/−) receptors had higher levels of airway inflammation, suggesting that endogenous PGE2 was suppressing inflammation via EP4 receptor activation. Cell-based assay systems (murine and human monocytes/alveolar macrophages) demonstrated that PGE2 inhibited cytokine release from LPS-stimulated cells and that this was mimicked by an EP4 (but not EP1–3) receptor agonist and inhibited by an EP4 receptor antagonist. The anti-inflammatory effect occurred at the transcriptional level and was via the adenylyl cyclase/cAMP/ cAMP-dependent protein kinase (PKA) axis.ConclusionThis study demonstrates that EP4 receptor activation is responsible for the anti-inflammatory activity of PGE2 in a range of disease relevant models and, as such, could represent a novel therapeutic target for chronic airway inflammatory conditions.

The objective of this study was to examine the relationship of allergen sensitivity to asthma symptoms among inner-city asthmatics seen at our Brooklyn, NY, asthma center. We hypothesized that asthma severity would increase for adults and children with increased cockroach and dust mite allergen sensitivity. Data were gathered from retrospective chart review for all patients who were treated at the center with a diagnosis of asthma and had undergone skin-prick testing (SPT) for allergen sensitivity during 1998 (pediatric, n = 79; adult, n = 29). Asthma severity (determined by National Heart, Lung and Blood Institute [NHLBI] asthma severity class) was examined in relation to allergen sensitivity. Allergen sensitivity was measured by percent positive to skin-prick testing as well as by relative mean diameter of skin prick test wheals. For adults, mite sensitivity prevalence was 61% and cockroach sensitivity prevalence was 41%. For children, mite sensitivity prevalence was 49%; cockroach sensitivity prevalence was 42%. For adults, asthma severity correlated significantly with sensitivity to Cladosporium, tree, and grass as measured by percent positive skin tests and by increasing mean diameter of skin test wheals. There was a significant correlation with severity for adult dust mite sensitivity only as measured by increasing mean wheal diameter. Ragweed sensitivity showed a significant correlation with severity only as measured by percent positive skin tests. There was a significant positive association for adults between increasing asthma severity and total number of allergen sensitivities per subject. There was no significant correlation for children between asthma severity and total number of allergen sensitivities per subject. Among children, no specific allergen sensitization showed a significant positive association with asthma severity. By both measures of allergen sensitization, there was a significant negative association for children between Cladosporium and asthma severity. Among our inner-city asthmatic population significant correlation between mite sensitivity and asthma severity was found only in adults. No significant association was seen with cockroach. However, outdoor allergen sensitivity (Cladosporidium, tree, ragweed, and grass) significantly correlated with asthma for adults in this inner city population.

Background The IL-1 family of cytokines is known to play an important role in inflammation therefore understanding the mechanism by which they are produced is paramount. Despite the recent plethora of publications dedicated to the study of these cytokines, the mechanism by which they are produced in the airway following endotoxin, Lipopolysaccharide (LPS), exposure is currently unclear. The aim was to determine the mechanism by which the IL-1 cytokines are produced after LPS inhaled challenge. Methods Mice were challenged with aerosolised LPS, and lung tissue and bronchiolar lavage fluid (BALF) collected. Targets were measured at the mRNA and protein level; caspase activity was determined using specific assays. Results BALF IL-1b/IL-18, but not IL-1a, was dependent on Ice Protease-Activating Factor (IPAF), and to a lesser extent Apoptosis-associated Speck-like protein containing a CARD (ASC). Interestingly, although we measured an increase in mRNA expression for caspase 1 and 11, we could not detect an increase in lung enzyme activity or a role for them in IL-1a/b production. Further investigations showed that whilst we could detect an increase in caspase 8 activity at later points in the time course (during resolution of inflammation), it appeared to play no role in the production of IL-1 cytokines in this model system. Conclusions TLR4 activation increases levels of BALF IL-1b/IL-18 via an IPAF dependent and caspase 1/11/8 independent pathway. Furthermore, it would appear that the presence of IL-1a in the BALF is independent of these pathways. This novel data sheds light on innate signalling pathways in the lung that control the production of these key inflammatory cytokines.

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.

Background Sensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored. Objective We hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung. Methods We used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues. Results Here we show TRPV4-induced activation of guinea pig airway–specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough. Conclusion This study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP–mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.

Background Asthma and chronic obstructive pulmonary disease are airway inflammatory diseases characterised by airflow obstruction. Currently approved bronchodilators such as long-acting β 2 adrenoceptor agonists are the mainstay treatments but often fail to relieve symptoms of chronic obstructive pulmonary disease and severe asthma and safety concerns have been raised over long-term use. The aim of the study was to identify the receptor involved in prostaglandin E 2 (PGE 2 )-induced relaxation in guinea pig, murine, monkey, rat and human airways in vitro . Methods Using an extensive range of pharmacological tools, the relaxant potential of PGE 2 and selective agonists for the EP 1–4 receptors in the presence and absence of selective antagonists in guinea pig, murine, monkey, rat and human isolated airways was investigated. Results In agreement with previous studies, it was found that the EP 2 receptor mediates PGE 2 -induced relaxation of guinea pig, murine and monkey trachea and that the EP 4 receptor mediates PGE 2 -induced relaxation of the rat trachea. These data have been confirmed in murine airways from EP 2 receptor-deficient mice ( Ptger2 ). In contrast to previous publications, a role for the EP 4 receptor in relaxant responses in human airways in vitro was found. Relaxant activity of AH13205 (EP 2 agonist) was also demonstrated in guinea pig but not human airway tissue, which may explain its failure in clinical studies. Conclusion Identification of the receptor mediating PGE 2 -induced relaxation represents a key step in developing a novel bronchodilator therapy. These data explain the lack of bronchodilator activity observed with selective EP 2 receptor agonists in clinical studies.

Background In allergic asthma, exposure to relevant antigens leads to an early asthmatic response (EAR) followed, in certain subjects, by a late asthmatic response (LAR). Although many subjects with asthma consider LAR to be one of the defining symptoms of their disease, and despite its widespread use in the clinical assessment of new therapeutic entities, the mechanism underlying the LAR remains unclear. Method A study was undertaken using ovalbuminsensitised and challenged Brown Norway rat and C57BL/ 6J mouse models which recapitulate phenotypic features of allergic asthma including the LAR and its susceptibility to clinically effective agents. Results In conscious animals an EAR was followed by a LAR. The LAR was subjectively evidenced by audible (wheeze) and visual signs of respiratory distress associated with quantifiable changes in non-invasive lung function assessment. Treatments that attenuated the EAR failed to impact on the LAR and, while anaesthesia did not impact on EAR, it abolished LAR. A key role for airway sensory neuronal reflexes in the LAR was therefore hypothesised, which was confirmed by the blockade observed after administration of ruthenium red (non-selective cation channel blocker), HC-030031 (TRPA1 inhibitor) and tiotropium bromide (anticholinergic) but not JNJ-17203212 (TRPV1 inhibitor). Conclusion These results suggest that LAR involves the following processes: allergen challenge triggering airway sensory nerves via the activation of TRPA1 channels which initiates a central reflex event leading to a parasympathetic cholinergic constrictor response. These data are supported by recent clinical trials suggesting that an anticholinergic agent improved symptoms and lung function in patients with asthma.