The initial bronchoconstrictor response of the asthmatic airway depends on airway smooth muscle (ASM) contraction. Intracellular calcium is a key signaling molecule, mediating a number of responses, including proliferation, gene expression, and contraction of ASM. Ca2+ influx through receptor-operated calcium (ROC) or store-operated calcium (SOC) channels is believed to mediate longer term signals. The mechanisms of SOC activation in ASM remain to be elucidated. Recent literature has identified the STIM and ORAI proteins as key signaling players in the activation of the SOC subtype; calcium release–activated channel current (ICRAC) in a number of inflammatory cell types. However, the role for these proteins in activation of SOC in smooth muscle is unclear. We have previously demonstrated a role for STIM1 in SOC channel activation in human ASM. The aim of this study was to investigate the expression and define the potential roles of the ORAI proteins in SOC-associated Ca2+ influx in human ASM cells. Here we show that knockdown of ORAI1 by siRNA resulted in reduced thapsigargin- or cyclopiazonic acid (CPA)–induced Ca2+ influx, without affecting Ca2+ release from stores or basal levels. CPA-induced inward currents were also reduced in the ORAI1 knockdown cells. We propose that ORAI1 together with STIM1 are important contributors to SOC entry in ASM cells. These data extend the major tissue types in which these proteins appear to be major determinants of SOC influx, and suggest that modulation of these pathways may prove useful in the treatment of bronchoconstriction.

Background

β2-adrenoceptor agonists elicit bronchodilator responses by binding to β2-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β2-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.

Methods

Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β2-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.

Results

Temporal analysis revealed that in hASM cells the β2-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.

Conclusions

We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β2-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β2-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo.

Background

Agonist stimulation of airway smooth muscle (ASM) results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells.

Methods

The expression profile of NCX (which encodes the Na+/Ca2+ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.

Results

NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine peripheral airways.

Conclusions

Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca2+ homeostasis and link store depletion via STIM1 directly with NCX activation.

Background

We have previously identified Urokinase Plasminogen Activator Receptor (PLAUR) as an asthma susceptibility gene. In the current study we tested the hypothesis that PLAUR single nucleotide polymorphisms (SNPs) determine baseline lung function and contribute to the development of Chronic Obstructive Pulmonary Disease (COPD) in smokers.

Methods

25 PLAUR SNPs were genotyped in COPD subjects and individuals with smoking history (n = 992). Linear regression was used to determine the effects of polymorphism on baseline lung function (FEV1, FEV1/FVC) in all smokers. Genotype frequencies were compared in spirometry defined smoking controls (n = 176) versus COPD cases (n = 599) and COPD severity (GOLD stratification) using logistic regression.

Results

Five SNPs showed a significant association (p < 0.01) with baseline lung function; rs2302524(Lys220Arg) and rs2283628(intron 3) were associated with lower and higher FEV1 respectively. rs740587(-22346), rs11668247(-20040) and rs344779(-3666) in the 5'region were associated with increased FEV1/FVC ratio. rs740587 was also protective for COPD susceptibility and rs11668247 was protective for COPD severity although no allele dose relationship was apparent. Interestingly, several of these associations were driven by male smokers not females.

Conclusion

This study provides tentative evidence that the asthma associated gene PLAUR also influences baseline lung function in smokers. However the case-control analyses do not support the conclusion that PLAUR is a major COPD susceptibility gene in smokers. PLAUR is a key serine protease receptor involved in the generation of plasmin and has been implicated in airway remodelling.

Airway smooth muscle (ASM) plays a pivotal role in modulating bronchomotor tone but also orchestrates and perpetuates airway inflammation and remodeling. Despite substantial research, there remain important unanswered questions. In 2006, the National Heart, Lung, and Blood Institute sponsored a workshop to define new directions in ASM biology. Important questions concerning the key functions of ASM include the following: Does developmental dysregulation of ASM function promote airway disease, what key signaling pathways in ASM evoke airway hyperresponsiveness in vivo, do alterations in ASM mass affect excitation–contraction coupling, and can ASM modulate airway inflammation and remodeling in a physiologically relevant manner? This workshop identified critical issues in ASM biology to delineate areas for scientific investigation in the identification of new therapeutic and diagnostic approaches in asthma, chronic obstructive pulmonary disease, and cystic fibrosis.

Background

Asthma and allergy are complex multifactorial disorders, with both genetic and environmental components determining disease expression. The use of molecular genetics holds great promise for the identification of novel drug targets for the treatment of asthma and allergy. Genome-wide linkage studies have identified a number of potential disease susceptibility loci but replication remains inconsistent. The aim of the current study was to complete a meta-analysis of data from genome-wide linkage studies of asthma and related phenotypes and provide inferences about the consistency of results and to identify novel regions for future gene discovery.

Methods

The rank based genome-scan meta-analysis (GSMA) method was used to combine linkage data for asthma and related traits; bronchial hyper-responsiveness (BHR), allergen positive skin prick test (SPT) and total serum Immunoglobulin E (IgE) from nine Caucasian asthma populations.

Results

Significant evidence for susceptibility loci was identified for quantitative traits including; BHR (989 pedigrees, n = 4,294) 2p12-q22.1, 6p22.3-p21.1 and 11q24.1-qter, allergen SPT (1,093 pedigrees, n = 4,746) 3p22.1-q22.1, 17p12-q24.3 and total IgE (729 pedigrees, n = 3,224) 5q11.2-q14.3 and 6pter-p22.3. Analysis of the asthma phenotype (1,267 pedigrees, n = 5,832) did not identify any region showing genome-wide significance.

Conclusion

This study represents the first linkage meta-analysis to determine the relative contribution of chromosomal regions to the risk of developing asthma and atopy. Several significant results were obtained for quantitative traits but not for asthma confirming the increased phenotype and genetic heterogeneity in asthma. These analyses support the contribution of regions that contain previously identified asthma susceptibility genes and provide the first evidence for susceptibility loci on 5q11.2-q14.3 and 11q24.1-qter.

Background

Airway hyper-responsiveness (AHR) is a key feature of asthma and a causal relationship between airway inflammation and AHR has been identified. The aim of the current study was to clarify the effect of proinflammatory cytokines and asthma medication on primary human airway smooth muscle (ASM) inositol phosphate (IPx) signalling and define the regulatory loci involved.

Methods

Primary Human ASM cells were isolated from explants of trachealis muscle from individuals with no history of respiratory disease. The effect of cytokine or asthma medication on histamine or bradykinin induced IPx signalling was assessed by [3H] inositol incorporation. Quantitative Real Time PCR was used to measure mRNA levels of receptors and downstream signalling components. Transcriptional mechanisms were explored using a combination of 5'Rapid Amplification of cDNA Ends (5'RACE) and promoter-reporter techniques.

Results

Treatment of Human ASM cells with IL-13, IFNγ or salmeterol for 24 hours lead to a modest augmentation of histamine induced IPx responses (144.3 +/- 9.3, 126.4 +/- 7.5 and 117.7 +/- 5.2%, p < 0.05). Similarly, TNFα, IFNγ or salmeterol treatment augmented bradykinin induced IPx responses (127.4 +/- 8.3, 128.0 +/- 8.4 and 111.7 +/- 5.0%, P < 0.05). No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus. Analyses of mRNA expression of components of the IPx pathway i.e. H1 Histamine Receptor (HRH1), B2 Bradykinin Receptor (BDKRB2), Gαq/11 and PLC-β1 identified that a significant induction of receptor mRNA (>2 fold) was a feature of these responses explaining the cytokine and spasmogen specificity. The HRH1 and BDKRB2 promoter regions were mapped in ASM and promoter-reporter analyses identified that salmeterol can induce HRH1 (>2 fold) and BDKRB2 (2–5 fold) transcription. The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.

Conclusion

Our results indicate that the spasmogen specific receptor locus may be a key site of regulation determining the magnitude of spasmogen mediated ASM IPx responses during airway inflammation or following asthma medication. These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.

Background

Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular Ca2+ stores can modulate contractile responses, modulate proliferation and regulate synthetic activity. Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified.

Methods

To investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay.

Results

Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp.

Conclusion

Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.

The availability of a draft sequence for the human genome will revolutionise research into airway disease. This review deals with two of the most important areas impinging on the treatment of patients: pharmacogenetics and pharmacogenomics. Considerable inter-individual variation exists at the DNA level in targets for medication, and variability in response to treatment may, in part, be determined by this genetic variation. Increased knowledge about the human genome might also permit the identification of novel therapeutic targets by expression profiling at the RNA (genomics) or protein (proteomics) level. This review describes recent advances in pharmacogenetics and pharmacogenomics with regard to airway disease.

The interleukin-4 (IL-4) signalling cascade has been identified as a pathway potentially important in the development of asthma. Genetic variants within this signalling pathway might contribute to the risk of developing asthma in a given individual. A number of polymorphisms have been described within the IL-4 receptor α (IL-4Rα) gene. In addition polymorphism occurs in the promoter for the IL-4 gene itself. This commentary accompanies a paper by C Ober et al describing the contribution of IL-4Rα polymorphism to susceptibility to asthma and atopy in the Hutterite population and other outbred populations collected during the collaborative studies on the genetics of asthma (CSGA) programme.

A sequence variant (rs7216389-T) near the ORMDL3 gene on chromosome 17q21 was recently found to be associated with childhood asthma. We sought to evaluate the effect of rs7216389-T on asthma subphenotypes and its correlation with expression levels of neighboring genes. The association of rs7216389-T with asthma was replicated in six European and one Asian study cohort (N=4917 cases N=34 589 controls). In addition, we found that the association of rs7216389-T was confined to cases with early onset of asthma, particularly in early childhood (age: 0–5 years OR=1.51, P=6.89·10−9) and adolescence (age: 14–17 years OR=1.71, P=5.47·10−9). A weaker association was observed for onset between 6 and 13 years of age (OR=1.17, P=0.035), but none for adult-onset asthma (OR=1.07, P=0.12). Cases were further stratified by sex, asthma severity and atopy status. An association with greater asthma severity was observed among early-onset asthma cases (P=0.0012), but no association with sex or atopy status was observed among the asthma cases. An association between sequence variants and the expression of genes in the 17q21 region was assessed in white blood cell RNA samples collected from Icelandic individuals (n=743). rs7216389 associated with the expression of GSDMB and ORMDL3 genes. However, other sequence variants showing a weaker association with asthma compared with that of rs7216389 were more strongly associated with the expression of both genes. Thus, the contribution of rs7216389-T to the development of asthma is unlikely to operate only through an impact on the expression of ORMDL3 or GSDMB genes.