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Evidence suggests that probiotics may enhance the effects of exogenous oestrogens such as hormone replacement therapy and isoflavones, and may lead to improvements in menopausal symptoms, and cardiovascular, bone and vaginal health. The present review aimed to examine the effects of probiotics on menopausal health outcomes and explore whether probiotics may influence the effects of exogenous oestrogenic substances. The following databases were searched: Google Scholar, EMBASE, MEDLINE, Cochrane Library, Scopus, and Web of Science. Thirty-nine studies met the inclusion criteria and contributed data from 3,187 menopausal women. All research was narratively synthesised, and 7 studies were examined via meta-analysis which examined the effects of probiotics versus placebo on menopausal symptoms, including vaginal, vasomotor, and psychological symptoms. Probiotics were associated with improvements in menopausal symptoms, urogenital conditions, and bone health. Meta analysis demonstrated large effects of probiotics on menopausal symptoms (total score), vasomotor symptoms, psychological symptoms, vaginal dryness, and vaginal microbiome health. Findings indicated that probiotics could enhance the efficacy and safety of estriol and isoflavone supplementation on urogenital and menopausal symptoms. These findings suggest that probiotics may offer benefits to menopausal and postmenopausal women, by improving menopausal symptoms and conditions related to low oestrogen levels. Moreover, probiotics may enhance the effects of estriol and isoflavones, potentially benefitting women at risk of oestrogen-dependent cancer where high-dose HRT is contraindicated. While findings are limited by the high risks of bias in the included studies, these results are promising and require further investigation through controlled experimental and observational research.

APOE-ε4 is a main genetic risk factor for developing late onset Alzheimer’s disease (LOAD) and is thought to interact adversely with other risk factors on the brain. However, evidence regarding the impact of APOE-ε4 on grey matter structure in asymptomatic individuals remains mixed. Much attention has been devoted to characterising APOE-ε4-related changes in the hippocampus, but LOAD pathology is known to spread through the whole of the Papez circuit including the limbic thalamus. Here, we tested the impact of APOE-ε4 and two other risk factors, a family history of dementia and obesity, on grey matter macro- and microstructure across the whole brain in 165 asymptomatic individuals (38–71 years). Microstructural properties of apparent neurite density and dispersion, free water, myelin and cell metabolism were assessed with Neurite Orientation Density and Dispersion (NODDI) and quantitative magnetization transfer (qMT) imaging. APOE-ε4 carriers relative to non-carriers had a lower macromolecular proton fraction (MPF) in the left thalamus. No risk effects were present for cortical thickness, subcortical volume, or NODDI indices. Reduced thalamic MPF may reflect inflammation-related tissue swelling and/or myelin loss in APOE-ε4. Future prospective studies should investigate the sensitivity and specificity of qMT-based MPF as a non-invasive biomarker for LOAD risk.

Mid-life hypertension and cerebrovascular dysfunction are associated with increased risk of later life dementia, including Alzheimer’s disease (AD). The classical renin–angiotensin system (cRAS), a physiological regulator of blood pressure, functions independently within the brain and is overactive in AD. cRAS-targeting anti-hypertensive drugs are associated with reduced incidence of AD, delayed onset of cognitive decline, and reduced levels of Aβ and tau in both animal models and human pathological studies. cRAS activity is moderated by a downstream regulatory RAS pathway (rRAS), which is underactive in AD and is strongly associated with pathological hallmarks in human AD, and cognitive decline in animal models of CNS disease. We now show that enhancement of brain ACE2 activity, a major effector of rRAS, by intraperitoneal administration of diminazene aceturate (DIZE), an established activator of ACE2, lowered hippocampal Aβ and restored cognition in mid-aged (13–14-month-old) symptomatic Tg2576 mice. We confirmed that the protective effects of DIZE were directly mediated through ACE2 and were associated with reduced hippocampal soluble Aβ42 and IL1-β levels. DIZE restored hippocampal MasR levels in conjunction with increased NMDA NR2B and downstream ERK signalling expression in hippocampal synaptosomes from Tg2576 mice. Chronic (10 weeks) administration of DIZE to pre-symptomatic 9–10-month-old Tg2576 mice, and acute (10 days) treatment in cognitively impaired 12–13-month-old mice, prevented the development of cognitive impairment. Together these data demonstrate that ACE2 enhancement protects against and reverses amyloid-related hippocampal pathology and cognitive impairment in a preclinical model of AD. Electronic supplementary material The online version of this article (10.1007/s00401-019-02098-6) contains supplementary material, which is available to authorized users.

APOE-e4 is a major genetic risk factor for late-onset Alzheimer’s disease that interacts with other risk factors, but the nature of such combined effects remains poorly understood. We quantified the impact of APOE-e4, family history (FH) of dementia, and obesity on white matter (WM) microstructure in 165 asymptomatic adults (38-71 years old) using quantitative magnetization transfer and neurite orientation dispersion and density imaging. Microstructural properties of the fornix, parahippocampal cingulum, and uncinate fasciculus were compared with those in motor and whole-brain WM regions. Widespread interaction effects between APOE, FH, and waist-hip ratio were found in the myelin-sensitive macromolecular proton fraction from quantitative magnetization transfer. Among individuals with the highest genetic risk (FH+ and APOE-e4), obesity was associated with reduced macromolecular proton fraction in the right parahippocampal cingulum, whereas no effects were present for those without FH. Risk effects on apparent myelin were moderated by hypertension and inflammation-related markers. These findings suggest that genetic risk modifies the impact of obesity on WM myelin consistent with neuroglia models of aging and late-onset Alzheimer's disease.

Three experiments examined the ability of mice to forage efficiently for liquid rewards in pots located in an open field arena. Search behaviour was unconstrained other than by the walls of the arena. All mice acquired the task within 4 days of training, with one trial per day. Experiment 1 tested the hypothesis that hippocampal lesions would disrupt foraging behaviour using extramaze cues. Mice with hippocampal lesions showed normal latency to initiate foraging and to complete the task relative to sham-operated mice. However, lesioned mice showed increased perseverative responding (sensitization) to recently rewarded locations, increased total working memory errors and an increased propensity to search near previously rewarded locations. In Experiment 2, the extramaze cues were obscured and each pot was identified by a unique pattern. Under these conditions, mice with hippocampal lesions showed comparable working memory errors to control mice. However, lesioned mice continued to display increased perseverative responding and altered search strategies. Experiment 3 tested the hypothesis that age-related accumulation of amyloid would disrupt foraging behaviour in transgenic PDAPP mice expressing the V717F amyloid precursor protein (APP) mutation. Consistent with previous findings, PDAPP mice showed both age-dependent and age-independent behavioural changes. More specifically, 14-16 month-old PDAPP mice showed a deficit in perseverative responding and working memory errors. In contrast, changes in search behaviour, such as systematic circling, were present throughout development. The latter indicates that APP overexpression contributed to some features of the PDAPP behavioural phenotype, whereas working memory and flexible responding was sensitive to ageing and β-amyloid burden. In conclusion, the present study provided novel insight into the role of the hippocampus and the effects of APP overexpression on memory and search behaviour in an open-field foraging task.

A major risk factor for the development of Alzheimer's disease (AD) is increasing age, but the reason behind this association has not been identified. It is thought that the changes in endocytosis seen in AD patients are causal for this condition. Thus, we hypothesized that the increased risk of developing AD associated with ageing may be because of changes in endocytosis. We investigated using Western blotting whether the expression of endocytic proteins involved in clathrin-mediated and clathrin-independent endocytosis are altered by increasing age in a mouse model of amyloid pathology. We used mice transgenic for human amyloid precursor protein containing the V717I London mutation. We compared the London mutation mice with age-matched wild-type (WT) controls at three ages, 3, 9 and 18 months, representing different stages in the development of pathology in this model. Having verified that the London mutation mice overexpressed amyloid precursor protein and β-amyloid, we found that the expression of the smallest isoform of PICALM, a key protein involved in the regulation of clathrin-coated pit formation, was significantly increased in WT mice, but decreased in the London mutation mice with age. PICALM levels in WT 18-month mice and clathrin levels in WT 9-month mice were significantly higher than those in the London mutation mice of the same ages. The expression of caveolin-1, involved in clathrin-independent endocytosis, was significantly increased with age in all mice. Our results suggest that endocytic processes could be altered by the ageing process and such changes could partly explain the association between ageing and AD.

A family history (FH) of dementia,APOE-ε4 genotype, and obesity are major risk factors for developing Alzheimer’s disease but their combined effects on the brain and cognition remain elusive. We tested the hypothesis that these risk factors affect apparent white matter (WM) myelin and cognition including spatial navigation and processing speed in 166 asymptomatic individuals (38-71 years). Microstructure in temporal [fornix, parahippocampal cingulum, uncinate fasciculus], motor and whole-brain WM was assessed with myelin-sensitive indices from quantitative magnetization transfer [macromolecular proton fraction (MPF)] and axon density from diffusion imaging. Individuals with the highest genetic risk (FH+ andAPOE-ε4) compared to those with FH+ alone showed obesity-related reductions in MPF and axon density in the right parahippocampal cingulum. No effects were present for those without FH. Furthermore, FH modulated obesity-related effects on spatial navigation behaviour. In summary, an individual’s genetic dementia risk influenced the impact of obesity on WM myelin and cognition.

Introduction Exposure to urban particulate matter (UPM) exacerbates the development of asthma and COPD. UPM exposure triggers the release of ‘alarmins’ by the airway epithelium, which causes an inflammatory response such as acceleration of the activation and maturation of dendritic cells (DC). Previously, we have demonstrated that, in surrogate models of allergic asthma, certain environmental stimuli activate the airway calcium-sensing receptor (CaSR), which drives bronchial hyperresponsiveness, inflammation and remodelling. We have also shown that CaSR antagonists, calcilytics, can abrogate these changes. Whether UPM exerts its effects acting at the CaSR is unknown. Aims To test the ability of calcilytics to: Prevent the effects of UPM in recombinant systems expressing the CaSR and in DC; Suppress airways hyperresponsiveness, inflammation and remodelling in a murine model of alarmin–driven (IL–33) asthma. Methods [Ca2+]i responses to UPM were investigated in HEK293 cells stably transfected with human CaSR (HEK-CaSR) or empty vector (HEK-0), ± calcilytic NPS2143. FACS and cytometric bead array were used to evaluate maturation (%CD83) and cytokine release by human monocyte-derived DC following 24h exposure to UPM ± calcilytics. IL-33 was delivered intranasally to naive mice once daily for 6 consecutive days followed by the calcilytic NPSP795 or vehicle control twice daily from day 2. On day 7, airways resistance was measured (Flexivent) under terminal anaesthesia, after which bronchoalveolar lavage fluid (BALF) analysis was performed, and lungs collected for histomorphology and for measurements of cytokine release. Results UPM increased [Ca2+]i in the HEK-CaSR but not the HEK-0 cells, an effect inhibited by calcilytics. Calcilytics attenuated UPM-induced maturation, and release of the cytokines IL-10 and IL-23p40, but not IL-6 by DC. In vivo, inhaled calcilytics significantly reduced (1) bronchial hyperresponsiveness; (2) BALF inflammatory cell infiltration and lung concentrations of IL-5, IL-13 and IL-6; (3) airways collagen deposition. Conclusions UPM activates the CaSR and induces maturation and activation of DC, an effect inhibited by calcilytics. Furthermore, calcilytics show benefit in alarmin-driven airways inflammation and hyperresponsiveness in an animal asthma surrogate, suggesting that they will be effective against exacerbating stimuli such as UPM.

Introduction: We previously implicated the calcium-sensing receptor (CaSR) in the development of airways inflammation and hyperresponsiveness in human asthma, and that topical CaSR antagonists (calcilytics) reduce both in animal surrogates. Aim: Extend these studies to alarmin-driven airways inflammation by comparing the efficacy of topical (intranasal) calcilytic in a head-to-head comparison in Th2/IgE mediated (OVA) and alarmin (interleukin (IL)-33) driven asthma in a murine surrogate. Methods: IL-33 (naive mice) or OVA (mice IgE-sensitised to OVA) was delivered once daily intranasally for 6 consecutive days followed by the calcilytic NPSP795 or vehicle control twice daily. On day 7, airways resistance was measured (Flexivent) under anaesthesia then the animals euthanised, after which bronchoalveolar lavage fluid (BALF) was collected, one lung paraffin-embedded for histomorphology and the other homogenised to measure cytokines. Results: Topical calcilytic significantly reduced (1) airways hyperresponsiveness (both stimuli); (2) BALF total and differential eosinophil, neutrophil and lymphocyte counts and lung homogenate concentrations of IL-5, IL-13 and IL-6 (variable according to stimulus); (3) peribronchial inflammatory cellular infiltration in the OVA, but not the IL-33 driven model; (4) airways collagen deposition in the IL-33, but not the OVA driven model. Conclusions: Calcilytics show therapeutic benefit in both IgE/Th2 and alarmin-driven airways inflammation and hyperresponsiveness in animal asthma surrogates in vivo, suggesting that they will be effective against any stimulus potentially exacerbating human asthma by inducing an alarmin response in the airways.

Midlife obesity is a risk factor of late onset Alzheimer’s disease (LOAD) but why this is the case remains unknown. As systemic inflammation is involved in both conditions, one possibility is that obesity-related neuroinflammation may contribute to the development of LOAD. Neuroinflammation is closely linked to white matter myelin loss, and this can be measured in vivo with quantitative magnetization transfer (qMT) imaging. Here, we investigated whether differences in obesity measures, i.e., in Waist Hip Ratio (WHR), abdominal visceral and subcutaneous fat volume fractions and Body Mass Index (BMI), were associated with reductions in qMT indices of apparent myelin in temporal white matter pathways involved in LOAD (i.e., the fornix, the parahippocampal cingulum and the uncinate fasciculus compared with whole brain and cortico-spinal white matter) in 166 cognitively healthy individuals (38-71 years of age). Obesity-related effects on myelin-sensitive markers were contrasted with differences in apparent axon density from dual-shell diffusion Neurite Orientation Dispersion and Density Imaging (NODDI). Differences in WHR and in visceral fat volume fractions were negatively correlated with differences in qMT estimates of apparent myelin and tissue metabolism in the fornix but not with any other microstructural components. These correlations were not explained by demographic (age, sex, education), health (hypertension, alcohol consumption, sedentary lifestyle) or genetic (APOE genotype, family history of dementia) risk factors of LOAD. Furthermore, differences in the ratio of plasma concentrations of leptin and adiponectin were also positively correlated with differences in C-Reactive Protein concentrations, and contributed significantly to the correlations between central obesity and myelin-sensitive metrics in the fornix. These results are consistent with the view that visceral fat-related chronic inflammation may damage white matter myelin in limbic regions, known to be vulnerable to LOAD pathology.

Background: Inhibitors of the extracellular calcium-sensing receptor (CaSR), termed calcilytics, were developed to evoke pulsatile changes in plasma parathyroid hormone (PTH) levels as a novel anti-osteoporotic treatment. Recently we have shown that they can suppress airway hyperresponsiveness (AHR) and inflammation in asthma models, providing a potential for a novel asthma treatment strategy1. Objective: To develop a new calcilytic specifically for the treatment of airway disease and test its efficacy in allergic asthma. Methods: The efficacy of a new chemical class of calcilytics to suppress CaSR signalling was tested in vitro in human embryonic kidney 293 cells expressing the CaSR. The most active stereoisomer of one compound (B2.1-E1) was tested for its biological activity in vivo by administering the calcilytic solution to mice via gavage, after which plasma PTH levels were measured. Finally, the efficacy of the inhaled calcilytics to suppress AHR and inflammation was tested in the poly-L-arginine-induced model of AHR and the ovalbumin-sensitised acute asthma model in mice1. Results: B2.1-E1 supressed CaSR signalling in vitro and increased PTH levels in vivo. Inhaled B2.1-E1 led to dose-dependent suppression of AHR evoked by poly-L-arginine, and diminished AHR and inflammation in the acute asthma model in vivo. Conclusions: The novel calcilytic B2.1-E1 suppressed CaSR in vitro, and elicited PTH release in vivo. In addition, B2.1-E1 suppressed AHR and inflammation in mice, providing a rationale for further development of this calcilytic and its chemical class for the treatment of inflammatory lung disease (IP GB1719023.2). References:1Yarova PL et al. (2015). Science TM 7: 284ra60.

Viral exacerbations of allergen-induced pulmonary inflammation in pre-clinical models reportedly reduce the efficacy of glucocorticoids to limit pulmonary inflammation and airways hyper-responsiveness to inhaled spasmogens. However, exacerbations of airway obstruction induced by allergen challenge have not yet been studied. hPIV-3 (human parainfluenza type 3 virus) inoculation of guinea-pigs increased inflammatory cell counts in BAL (bronchoalveolar lavage) fluid and caused hyper-responsiveness to inhaled histamine. Both responses were abolished by treatment with either dexamethasone (20 mg/kg of body weight, subcutaneous, once a day) or fluticasone propionate (a 0.5 mg/ml solution aerosolized and inhaled over 15 min, twice a day). In ovalbumin-sensitized guinea-pigs, allergen (ovalbumin) challenge caused two phases of airway obstruction [measured as changes in sGaw (specific airways conductance) using whole body plethysmography]: an immediate phase lasting between 4 and 6 h and a late phase at about 7 h. The late phase, airway hyper-responsiveness to histamine and inflammatory cell counts in BAL were all significantly reduced by either glucocorticoid. Inoculation of guinea-pigs sensitized to ovalbumin with hPIV-3 transformed the allergen-induced airway obstruction from two transient phases into a single sustained response lasting up to 12 h. This exacerbated airway obstruction and airway hyper-responsiveness to histamine were unaffected by treatment with either glucocorticoid whereas inflammatory cell counts in BAL were only partially inhibited. Virus- or allergen-induced pulmonary inflammation, individually, are glucocorticoid-sensitive, but in combination generate a phenotype where glucocorticoid efficacy is impaired. This suggests that during respiratory virus infection, glucocorticoids might be less effective in limiting pulmonary inflammation associated with asthma.

The greatest risk factor for Alzheimer’s disease (AD) is advanced age, but the reason for this association remains unclear. Amyloid-beta (Aβ) is produced from amyloid precursor protein (APP) primarily after APP is internalised by clathrin-mediated or clathrin-independent endocytosis. Changes in endocytosis in AD have been identified. We hypothesised that endocytic protein expression is altered during ageing, thus influencing the likelihood of developing AD by increasing Aβ production. We explored how levels of endocytic proteins, APP, its metabolites, secretase enzymes and tau varied with age in cortical brain samples from men of three age ranges (young (20–30), middle-aged (45–55) and old (70–90)) with no symptoms of dementia. Aβ40 and Aβ42 were significantly increased in old brains, while APP and secretase expression was unaffected by age. Phosphorylated GSK3β increased significantly with age, a possible precursor for neurofibrillary tangle production, although phosphorylated tau was undetectable. Significant increases in clathrin, dynamin-1, AP180, Rab-5, caveolin-2 and flotillin-2 were seen in old brains. Rab-5 also increased in middle-aged brains prior to changes in Aβ levels. This age-related increase in endocytic protein expression, not described previously, suggests an age-related up-regulation of endocytosis which could predispose older individuals to develop AD by increasing APP internalisation and Aβ generation.

Magnesium sulphate is a potential treatment for acute severe asthma. However, the mechanisms and dose-response relationships are poorly understood. The first objective of this study was to examine whether inhaled magnesium sulphate exerts bronchodilator activity measured as bronchoprotection against histamine-induced bronchoconstriction in conscious guinea-pigs alone and combined with salbutamol. Secondly, we examined whether inhaled magnesium sulphate inhibits airways inflammation and function in models of neutrophilic and eosinophilic lung inflammation induced, respectively, by inhaled lipopolysaccharide or the inhaled antigen, ovalbumin (OVA). Airway function was measured in conscious guinea-pigs as specific airway conductance (sGaw) by whole-body plethysmography. Anti-inflammatory activity was measured against lung inflammatory cell influx induced by OVA inhalation in OVA-sensitised animals or by lipopolysaccharide (LPS) exposure of non-sensitised animals. Airway function (sGaw) was measured over 24 h after OVA exposure. Airway hyperresponsiveness to inhaled histamine and inflammatory cells in bronchoalveolar lavage fluid were recorded 24 h after OVA or LPS challenge. Histamine-induced bronchoconstriction was inhibited by inhaled magnesium sulphate or salbutamol alone and in combination, they produced synergistic bronchoprotection. LPS-induced neutrophil influx was inhibited by 6 days pretreatment with magnesium sulphate. Early and late asthmatic responses in OVA sensitised and challenged animals were attenuated by magnesium sulphate. Lung inflammatory cells were increased by OVA, macrophages being significantly reduced by magnesium sulphate. Nebulised magnesium sulphate protects against histamine-induced bronchoconstriction in conscious guinea-pigs and exerts anti-inflammatory activity against pulmonary inflammation induced by allergen (OVA) or LPS. These properties of magnesium sulphate explain its beneficial actions in acute asthma.

Pre-clinical evaluation of asthma therapies requires animal models of chronic airways inflammation, airway hyperresponsiveness (AHR) and lung remodelling that accurately predict drug effectiveness in human asthma. However, most animal models focus on acute allergen challenges where chronic inflammation and airway remodelling are absent. Chronic allergen challenge models have been developed in mice but few studies use guinea-pigs which may be more relevant to humans. We tested the hypothesis that a chronic rather than acute pulmonary inflammation model would best predict clinical outcome for asthma treatments. Guinea-pigs sensitized with ovalbumin (OVA) received single (acute) or nine OVA inhalation challenges at 48 h intervals (chronic). Airways function was recorded as specific airways conductance (sG(aw)) in conscious animals for 12 h after OVA challenge. AHR to inhaled histamine, inflammatory cell influx and lung histology were determined 24 h after the single or 9th OVA exposure. The inhaled corticosteroid, fluticasone propionate (FP), the phosphodiesterase 4 inhibitor, roflumilast, and the inducible nitric oxide synthase (iNOS) inhibitor, GW274150, orally, were administered 24 and 0.5 h before and 6 h after the single or final chronic OVA exposure. Both models displayed early (EAR) and late (LAR) asthmatic responses to OVA challenge, as falls in sG(aw), AHR, as increased histamine-induced bronchoconstriction, and inflammatory cell influx. Tissue remodelling, seen as increased collagen and goblet cell hyperplasia, occurred after multiple OVA challenge. Treatment with FP and roflumilast inhibited the LAR, cell influx and AHR in both models, and the remodelling in the chronic model. GW274150 also inhibited the LAR, AHR and eosinophil influx in the acute model, but not, together with the remodelling, in the chronic model. In the clinical setting, inhaled corticosteroids and phosphodiesterase 4 inhibitors are relatively effective against most features of asthma whereas the iNOS inhibitor GW274150 was ineffective. Thus, while there remain certain differences between our data and clinical effectiveness of these antiasthma drugs, a chronic pulmonary inflammation guinea-pig model does appear to be a better pre-clinical predictor of potential asthma therapeutics than an acute model.

Aim of the study The present study evaluates the Philippine medicinal plant Artemisia vulgaris for antagonistic activity at selected biogenic amine receptors on smooth muscle of the airways and gastrointestinal tract in order to explain its traditional use in asthma and hyperactive gut. Materials and methods The antagonistic activity of chloroform crude extract (AV-CHCl3) and methanol crude extract (AV-MeOH) of Artemisia vulgaris was studied against concentration–response curves for contractions of the guinea pig ileum and trachea to 5-hydroxytrptamine (5-HT2 receptors), methacholine (M3 muscarinic receptors), histamine (H1 receptors) and β-phenylethylamine (trace amine-associated receptors, TAAR1). Results and discussion The Artemisia vulgaris chloroform (AV-CHCl3) and methanol (AV-MeOH) extract showed histamine H1 antagonism in the ileum and trachea. Further analysis of AV-CHCl3 isolated two major components, yomogin and 1,2,3,4-diepoxy-11(13)-eudesmen-12,8-olide. Yomogin, a sesquiterpene lactone, exhibited a novel histamine H1 receptor antagonism in the ileum. Conclusion The presence of a specific, competitive histamine receptor antagonist and smooth muscle relaxant activity in Artemisia vulgaris extracts on the smooth muscle in ileum and trachea explains its traditional use in the treatment of asthma and hyperactive gut.

Background: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. Methods: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. Results: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. Conclusions: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection.

The cleavage of amyloid precursor protein (APP) by β- and -secretases results in the production of amyloid-β (Aβ) in Alzheimer’s disease. We raised two monoclonal antibodies, 2B3 and 2B12, that recognize the β-secretase cleavage site on APP but not Aβ. We hypothesized that these antibodies would reduce Aβ levels via steric hindrance of β-secretase. Both antibodies decreased extracellular Aβ levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPP from the nonamyloidogenic -secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site, whereas 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Aβ levels. After 60 min incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where β-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPP by β-secretase in a cell-free assay where the effects of antibody internalization and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, generated following β-secretase cleavage (βCTF), were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site IgGs can generically reduce Aβ levels via inhibition of β-secretase by steric hindrance and may provide a novel alternative therapy for Alzheimer’s disease.

Inhaled bradykinin causes bronchoconstriction in asthmatic subjects but not nonasthmatics. To date, animal studies with inhaled bradykinin have been performed only in anesthetized guinea pigs and rats, where it causes bronchoconstriction through sensory nerve pathways. In the present study, airway function was recorded in conscious guinea pigs by whole-body plethysmography. Inhaled bradykinin (1 mM, 20 s) caused bronchoconstriction and influx of inflammatory cells to the lungs, but only when the enzymatic breakdown of bradykinin by angiotensin-converting enzyme and neutral endopeptidase was inhibited by captopril (1 mg/kg i.p.) and phosphoramidon (10 mM, 20-min inhalation), respectively. The bronchoconstriction and cell influx were antagonized by the B2 kinin receptor antagonist 4-(S)-amino-5-(4-{4-[2, 4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]- tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5- oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132) when given by inhalation (1 and 10 μM, 20 min) and are therefore mediated via B2 kinin receptors. However, neither intraperitioneal MEN16132 nor the peptide B2 antagonist icatibant, by inhalation, antagonized these bradykinin responses. Sensitization of guinea pigs with ovalbumin was not sufficient to induce airway hyperreactivity (AHR) to the bronchoconstriction by inhaled bradykinin. However, ovalbumin challenge of sensitized guinea pigs caused AHR to bradykinin and histamine. Infection of guinea pigs by nasal instillation of parainfluenza-3 virus produced AHR to inhaled histamine and lung influx of inflammatory cells. These responses were attenuated by the bradykinin B2 receptor antagonist MEN16132 and H-(4-chloro)DPhe-2'(1-naphthylalanine)-( 3-aminopropyl)guanidine (VA999024), an inhibitor of tissue kallikrein, the enzyme responsible for lung synthesis of bradykinin. These results suggest that bradykinin is involved in virus-induced inflammatory cell influx and AHR

Inhaled corticosteroids are regularly co-administered with beta(2)-adrenoceptor agonists. This study evaluates in conscious guinea-pigs the bronchodilator effect, alone or combined with salbutamol, of TPI 1020, a novel anti-inflammatory corticosteroid and nitric oxide (NO) donor derived from budesonide. Guinea-pigs received inhaled histamine (3 mM) and specific airway conductance (sG(aw)) measured. Responses to histamine were measured before and on the next day 15 min after a 15 min inhalation of vehicle, salbutamol, TPI 1020, budesonide, the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP), or combinations of these drugs. Salbutamol and TPI 1020 caused concentration-dependent bronchodilatation measured as inhibition of histamine-induced bronchoconstriction. TPI 1020-induced bronchodilatation was blocked by the guanylyl cyclise inhibitor, ODQ, indicating cGMP-dependence through released NO. While salbutamol at 80 microM did not exert significant bronchodilatation, significant inhibitions were observed when co-administered with TPI 1020, 0.11 and 0.33 mM. The combined effects of TPI 1020 and salbutamol lasted significantly longer than either drug alone. Inhaled budesonide was a weak bronchodilator and when co-administered with salbutamol there was enhanced bronchodilatation. Addition of the NO-donor, SNAP (0.1 mM), to the budesonide/salbutamol combination, also improved the inhibition of histamine-induced bronchoconstriction. This study has shown that TPI 1020 potentiates the bronchodilator activity of salbutamol, and their combination lasted longer than either drug administered individually. Both the corticosteroid and NO-releasing activities of TPI 1020 appear to be required for the potentiation of salbutamol. Combination of TPI 1020 with a beta(2)-adrenoceptor agonist may therefore be useful against acute bronchoconstriction episodes in asthma, and may offer an opportunity for reducing doses of inhaled beta(2)-adrenoceptor agonists.

Trace amines, including tyramine and β-phenylethylamine (β-PEA), are constituents of many foods including chocolate, cheeses and wines and are generated by so-called ‘friendly’ bacteria such as Lactobacillus, Lactococcus and Enterococcus species, which are found in probiotics. We therefore examined whether these dietary amines could exert pharmacological effects on the gut and its vasculature. In the present study we examined the effects of tyramine and β-PEA on the contractile activity of guinea-pig and rat ileum and upon the isolated mesenteric vasculature and other blood vessels. Traditionally, these amines are regarded as sympathomimetic amines, exerting effects through the release of noradrenaline from sympathetic nerve endings, which should relax the gut. A secondary aim was therefore to confirm this mechanism of action. However, contractile effects were observed in the gut and these were independent of noradrenaline, acetylcholine, histamine and serotonin receptors. They were therefore probably due to the recently described trace amine-associated receptors. These amines relaxed the mesenteric vasculature. In contrast, the aorta and coronary arteries were constricted, a response that was also independent of a sympathomimetic action. From these results, we propose that after ingestion, trace amines could stimulate the gut and improve intestinal blood flow. Restriction of blood flow elsewhere diverts blood to the gut to aid digestion. Thus, trace amines in the diet may promote the digestive process through stimulation of the gut and improved gastrointestinal circulation.

Background and purpose: The dietary trace amines tyramine and β-phenylethylamine (β-PEA) can increase blood pressure. However, the mechanisms involved in the vascular effect of trace amines have not been fully established. The purpose of this study was to evaluate whether trace amine-dependent vasoconstriction was brought about by tyramine and β-PEA acting as indirect sympathomimetic agents, as previously assumed, or whether trace amine-dependent vasoconstriction could be mediated by recently discovered trace amine-associated (TAA) receptors. Experimental approach: The responses to p-tyramine and β-PEA were investigated in vitro in rings of the left anterior descending coronary arteries of pigs. Key results: p-Tyramine induced a concentration-dependent (0.1–3 mM) vasoconstriction. The maximum response and pD2 value for p-tyramine was unaffected by endothelium removal or pre-treatment with antagonists for adrenoceptors, histamine, dopamine or 5-HT receptors. β-PEA also produced a concentration-dependent (0.3–10 mM) vasoconstriction which was unaffected by endothelium removal, β-adrenoceptor or 5-HT receptor antagonists. A substantial, but reduced, response to β-PEA was obtained in the presence of prazosin (1-adrenoceptor antagonist), haloperidol (D2/D3 dopamine receptor antagonist) or mepyramine (H1 histamine receptor antagonist). The pD2 value for β-PEA was unaffected by any of the antagonists tested. Conclusions and implications: Vasoconstriction induced by p-tyramine does not involve an indirect sympathomimetic effect, although vasoconstriction caused by β-PEA may occur, in part, by this mechanism. We therefore propose that trace amine-dependent vasoconstriction is mediated by phenylethylamine-specific receptors, which are closely related to or identical to TAA receptors. These receptors could provide a target for new antihypertensive therapies

Allergic asthma is a chronic disease of the airways, with superimposed acute inflammatory episodes which correspond to exacerbations of asthma. Two novel models of allergic asthma have been developed in mice receiving the same allergen sensitisation, but with acute or chronic allergen exposures, the latter to mimic the human situation more closely. Ovalbumin-sensitised mice were challenged by ovalbumin inhalation twice on the same day for the acute model, and 18 times over a period of 6 weeks for the chronic model. Lung function was monitored in conscious, unrestrained mice immediately after the last challenge for up to 12 h. Airway responsiveness to inhaled methacholine and serum antibody levels were determined 24 h after challenge. Bronchoalveolar inflammatory cell recruitment was determined at 2 or 24 h. Acute and chronically treated mice had similar early and late asthmatic responses peaking at 2 h and 7-8 h, respectively. IgE and IgG antibody levels, compared with naïve mice, and eosinophil infiltration, compared with naïve and saline challenge, were elevated. Airway hyperresponsiveness to methacholine was observed 24 h after challenge in both models. The acute model had higher levels of eosinophilia, whereas the chronic model showed hyperresponsiveness to lower doses of methacholine and had higher levels of total IgE and ovalbumin-specific IgG antibodies. Both novel murine models of allergic asthma bear a close resemblance to human asthma, each offering particular advantages for studying the mechanisms underlying asthma and for evaluating existing and novel therapeutic agents.

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyper-reactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

Purpose: Asthma is associated with reversible airway obstruction, leucocyte infiltration, airways hyperresponsiveness (AHR) and airways remodelling. Fluid accumulation causes pulmonary oedema contributing to airways obstruction. We examined the temporal relationship between the late asthmatic response (LAR) following allergen challenge of sensitised guinea-pigs and pulmonary oedema measured by magnetic resonance imaging (MRI). Materials and Methods: Ovalbumin (OVA) sensitised guinea-pigs received either a single OVA inhalation (acute) or nine OVA inhalations at 48 h intervals (chronic). Airways obstruction was measured as specific airways conductance (sGaw) by whole body plethysmography. AHR to inhaled histamine and bronchoalveolar lavage for leucocyte counts were measured 24 h after a single or the final chronic ovalbumin challenges. MRI was performed at intervals after OVA challenge and high intensity oedemic signals quantified. Results: Ovalbumin caused early bronchoconstriction, followed at 7 h by a LAR and at 24 h AHR and leucocyte influx. The bright intensity MRI oedema signal, peaking at 7 h, was significantly (P

Proteolytic cleavage of amyloid-beta-protein precursor (AbetaPP) by beta- and gamma-secretases results in production of the amyloid-beta peptide (Abeta) that accumulates in the brains of sufferers of Alzheimer's disease (AD). We have developed a monoclonal antibody, 2B12, which binds in the vicinity of the beta-secretase cleavage site on AbetaPP but does not bind within the Abeta region. We hypothesised that this antibody, directed against the substrate rather than the enzyme, could inhibit cleavage of AbetaPP by beta-secretase via steric hindrance and thus reduce downstream production of Abeta. The antibody would enter cells by binding to AbetaPP when it is at the cell surface and then be internalised with the protein. We subsequently demonstrated that, after addition of 2B12 to standard growth media, this antibody was indeed capable of inhibiting Abeta40 production in neuroblastoma and astrocytoma cells expressing native AbetaPP, as measured by an ELISA. This inhibition was both concentration- and time-dependent and was specific to 2B12. We were only able to inhibit approximately 50% of Abeta40 production suggesting that not all AbetaPP is trafficked to the cell surface. We propose that this antibody could be used as a novel, putative therapy for the treatment of AD.

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.

The P2X7 cell surface receptor is responsible for adenosine triphosphate-dependent lysis of immune cells following the formation of non-selective membrane pores. This study examined changes in P2X7 receptor distribution following agonist treatment. Human THP-1 monocytes were exposed to the agonist 2′-3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate for 1–40 min after which P2X7 receptors were immunogold labelled. The number and distribution of P2X7 receptors on the cell membrane were quantified using transmission electron microscopy. Increasing the time of agonist exposure resulted in clustering of P2X7 receptors, a decrease in individual P2X7 receptors and no change in the total number of P2X7 receptors. These results suggest that pore formation does not involve further insertion or initial movement of receptors in the membrane but that P2X7 receptors do cluster together after prolonged activation.

Apparent species differences in the responses of recombinant P2X7 receptors to repeated application of 2′- and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) have been investigated. Repeated application of 100 μM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X7 receptors. Current growth was thought to reflect progressive dilation of the P2X7 ion-channel to a pore permeable to large molecules (MW20 min) and have been suggested to be associated with the dilation of the P2X7 channel (Chessell et al., 1997). In the present study we have further examined the responses of rat, human and mouse P2X7 receptors to repeated and sustained agonist application in order to determine the reasons for the inability to observe current growth at the rat P2X7 receptor. Current growth has been studied using whole-cell patch-clamp electrophysiology while P2X7 receptor-mediated pore formation has been studied by measuring the cellular accumulation of quinolinium,4-[(3-methyl-2 (3H) -benzoxazolylidene) methyl]-1[3-(triethylammonio) propyl]-, diiodide (YO-PRO-1) using confocal microscopy or a 96-well plate reader. YO-PRO-1 is a membrane-impermeant cation (629 Daltons for the di-iodide salt; 375 Daltons for free base) whose fluorescence increases upon binding nucleic acids and thus represents an excellent probe for detecting permeability changes in P2X7 receptor channels (Hickman et al., 1994; Surprenant et al, 1996; Michel et al., 1999).

1The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy-termini only. The murine sst2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization.2This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst2(a) receptor.3In CHO-K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC50 values of 8.3 (sst2(a)) and 8.4 (sst2(b)).4SRIF (3 pM–3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst2(a) cells (pIC50 10.5) and CHO-sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation.5Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx.6This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

Current therapies for Alzheimer’s disease only treat the symptoms of the disease. We have previously developed a novel monoclonal antibody, 2B3, which binds to the [beta]-secretase cleavage site in amyloid precursor protein (APP) and reduces the production of amyloid-[beta] (A[beta]) in human cell lines. To determine whether the antibody was likely to be effective in mouse models of amyloid pathology in vivo, we investigated whether 2B3 could also bind to APP in mouse primary cortical neurones. Primary cortical neurones were produced from E15.5–17.5 C57Bl/6 wild-type and transgenic APP/V717I (London mutation) embryos. The percentage of the neuronal population was determined by immunocytochemistry. Cells were treated with 10 µg/ml 2B3 or an irrelevant IgG for 48 h and A[beta]40 levels determined by ELISA. The population of cells was found to contain over 75% neurones and 2B3 bound effectively to these cells. No differences in A[beta]40 were detected between wild-type and transgenic cells. Importantly, 2B3 significantly inhibited the production of A[beta]40 by 75.15±1.37% of the media control, whereas an irrelevant IgG only significantly reduced A[beta]40 levels by 23.35±5.55% of the media control. The reduction in A[beta]40 produced by 2B3 was significantly greater than that caused by the IgG. These data indicate that 2B3 binds to APP in mouse neurones and can inhibit A[beta]40, similar to our previous findings. The antibody is probably therefore acting by steric hindrance of [beta]-secretase and these data suggest that it will be effective in mice in vivo and could be an alternative potential therapy for Alzheimer’s disease.

The kinetic properties, steady state binding characteristics and autoradiographic distribution of the somatostatin (SRIF) sst2 receptor-selective ligand, [125I]-BIM-23027, have been investigated in the rat central nervous system. Analysis of kinetic, saturation and competition binding data in rat hippocampal membranes was consistent with [125I]-BIM-23207 binding to a single population of non-interacting binding sites. Competition studies, using different SRIF ligands, suggested that [125I]-BIM-23027 was binding to sites similar to that of the recombinant sst2 receptor. The rank order of affinity for displacing specific binding was BIM-23027=SRIF>L-362855⪢BIM-23056. There was a widespread distribution of [125I]-BIM-23027 binding sites in the rat central nervous system. The highest density of binding was observed in the dentate gyrus, medial habenular, amygdala, claustrum and lateral septum as well as in the piriform, cingulate and parietal cortex. The cervical and lumbar spinal cord also displayed moderate levels of binding localized to the substantia gelatinosa. The cellular localization of [125I]-BIM-23027 binding was found to be associated with dendritic terminal fields. In contrast, the cellular signal for sst2 receptor mRNA was restricted to cell somata. The widespread distribution of [125I]-BIM-23027 binding sites within the brain suggests that receptors similar to the recombinant sst2 receptor may mediate a variety of different physiological effects within the central nervous system.

The distribution of nitric oxide synthase (NOS), the enzyme which produces nitric oxide, has previously been studied in the rat central nervous system (CNS) using the NADPH-diaphorase technique and anti-NOS antibodies. However, the former method may not always be selective for NOS while the latter is not quantitative. Therefore a selective, quantifiable method would be desirable. L-NG-Nitro-arginine, an inhibitor of NOS, is available in a tritiated form which we have shown to bind to NOS. We have now examined the regional distribution of [3H]L-NG-nitro-arginine binding in the rat CNS using autoradiography. [3H]L-NG-nitro-arginine specific binding was seen in a number of brain regions with the highest levels in the accessory olfactory bulb, the amygdaloid complex, the Islands of Calleja and the cerebellum. This regional distribution of [3H]L-NG-nitro-arginine binding sites in the rat CNS was, in general, similar to that seen with the NADPH-diaphorase method and anti-NOS antibodies, consistent with the view that all three methods identify NOS in the CNS. Thus, [3H]L-NG-nitro-arginine appears to be a useful radioligand for studying the distribution of NOS in the CNS as its binding is quantifiable and apparently selective for NOS.

Trace amines including β-phenylethylamine (β-PEA) and amphetamines classically exert pharmacological actions via indirect sympathomimetic mechanisms. However, there is evidence for other mechanisms and this study explores the receptors mediating vasoconstriction in rat aorta. β-PEA, d-amphetamine, MDMA, cathinone and methylphenidate caused concentration-dependent contractions of rat isolated aortic rings which were unaffected by prazosin (1 μM), ICI-118,551 (1 μM), cocaine (10 μM) and pargyline (10 μM), to inhibit α1- and β2-adrenoceptors, neuronal transport and monoamine oxidase (MAO), respectively. Octopamine concentration-response curves, however, were shifted to the right. In the presence of the inhibitors, the rate of onset of octopamine contractions was slowed. Lineweaver-Burk analysis of the kinetics of the response generated different KM values for octopamine in the absence (2.35 × 10(-6)M) and presence (6.09 × 10(-5)M) of inhibitors, indicating mediation by different receptors. Tryptamine-induced vasoconstriction also resisted blockade by adrenergic inhibitors and the 5-HT1A, 1B, 1D and 5-HT2A receptor antagonists, methiothepin (50 nM) and ketanserin (30 nM), respectively. Trace amines and amphetamines therefore exert vasoconstriction independently of adrenoceptors, neuronal transport and 5-HT receptor activation. There was no evidence of tachyphylaxis or cross-tachyphylaxis of the vasoconstriction to these amines. Tyramine was a partial agonist and in its presence, β-PEA, d-amphetamine and octopamine were antagonised indicating that they all act through a common receptor for which tyramine serves as an antagonist. We conclude that the vasoconstriction is via TAAR-1, because of structural similarities between amines, ability to stimulate recombinant trace amine-associated receptor 1 (TAAR-1) and the presence of TAAR-1 in rat aorta.

BACKGROUND AND PURPOSE Inhaled corticosteroids, anticholinergics and β2-adrenoceptor agonists are frequently combined for treating chronic respiratory diseases. We examine the corticosteroid, budesonide, and novel NO-donating derivative, TPI 1020, against histamine- and methacholine-induced bronchoconstriction and whether they enhance the β2-adrenoceptor agonist formoterol or muscarinic antagonist tiotropium in conscious guinea pigs. EXPERIMENTAL APPROACH Dunkin-Hartley guinea pigs received inhaled histamine (3 mM) or methacholine (1.5 mM) and specific airway conductance (sGaw) was measured before and 15 or 75 min after treatment with budesonide, TPI 1020, tiotropium or formoterol alone or in combinations. KEY RESULTS Formoterol (0.7–10 µM) and budesonide (0.11–0.7 mM) inhibited histamine-induced bronchoconstriction and tiotropium (2–20 µM) inhibited methacholine-induced bronchoconstriction by up to 70.8 ± 16.6%, 34.9 ± 4.4% and 85.1 ± 14.3%, respectively. Formoterol (2.5 µM) or tiotropium (2 µM) alone exerted small non-significant bronchoprotection. However, when co-administered with TPI 1020 0.11 mM, which alone had no significant effect, there was significant inhibition of the bronchoconstriction (45.7 ± 12.2% and 79.7 ± 21.4%, respectively). Co-administering budesonide (0.11 mM) with tiotropium (2 µM), which alone had no effect, also significantly inhibited the methacholine bronchoconstriction (36.5 ± 13.0%), but there was no potentiation of formoterol against histamine. The NO scavenger, CPTIO, prevented the bronchoprotection by SNAPand TPI 1020. CONCLUSIONS AND IMPLICATIONS TPI 1020 potentiated the bronchoprotection by formoterol and tiotropium. Budesonide also enhanced the effects of tiotropium but not formoterol. Combination of TPI 1020 with a long-acting β2-adrenoceptor agonist or muscarinic receptor antagonist may therefore be a more potent therapeutic approach for treatment of chronic respiratory diseases.