All chemicals were analytical grade or higher. Plasticware was from Costar (High Wycombe, UK). All chemicals and reagents were purchased from Sigma-Aldrich (Poole, UK) unless otherwise stated. The [³H]-myo-inositol was purchased from Perkin Elmer (Bucks, UK). Inositol free DMEM was made to order by Invitrogen (Paisley, UK).

Human airway smooth muscle cells (ASM) were isolated from explants of trachealis muscle obtained from individuals with no history of respiratory disease as described 2430. Briefly, a section of trachealis muscle was isolated by dissection just above the carina and washed in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin (200 U/ml), streptomycin (200 μg/l) and amphotericin B (0.5 μg/l). Explants (0.2 × 0.2 cm) were placed in 6 well plates and allowed to adhere. Cells were allowed to grow with fresh complete medium (10% foetal calf serum and 2 mM glutamine) added regularly, following 7–10 days growth cells approached confluency. As cells reached confluency, explants were removed and the cells were harvested using 5 mg/ml trypsin/2 mg/ml EDTA. Primary ASM cells exhibited >95% cell staining for α-actin. Airway smooth muscle cells were maintained in complete medium and passaged using trypsin/EDTA. Ethical approval for the use of primary cells was obtained from the local ethics committee. Airway smooth muscle cells from four individuals were used.

[³H]-inositol phosphate formation in primary human ASM cells was quantified essentially as described 2430. Briefly, cells were plated in 24 wells at a density of 0.5 × 10⁵ cells/well. Cells were allowed to grow for 2–3 days until ~80% confluent, then medium was replaced with 300 μl of inositol free DMEM, supplemented with 2 mM glutamine and containing [³H]-myo-inositol at a concentration of 8.1 MBq/ml. Cells were then incubated for 24 hours followed by the addition of IL-1β, TNFα, IFNγ (10 ng/ml), IL-13 (50 ng/ml) (PeproTech EC Ltd, London, UK), dexamethasone (1 μM) or salmeterol (1 μM) and incubated for a further 24 hours. The medium was then removed and cells were washed three times with 1 ml Hanks/20 mM HEPES and 300 μl Hanks/20 mM HEPES containing 20 mM LiCl was added. Cells were incubated for 30 min at 37°C, then agonists were added in a volume of 10 μl (100 μM Histamine, 0.1 μM Bradykinin or 10 mM Sodium Fluoride). Cells were incubated for a further 30 min and then the reaction was stopped by removing the medium and addition of 1 ml ice cold 50% methanol/60 mM HCl. Samples were stored at -20°C for at least 24 h. An 800 μl aliquot of each sample was neutralised using ~4.6 ml 5.5 mM Tris-HCl/11 mM NaOH. Total [³H]-inositol phosphates were separated from unincorporated [³H]-inositol using anion-exchange chromatography on Dowex-Cl columns and measured using scintillation counting 2430.

Human ASM cells were transferred to 100 mm Petri dishes at a density of 10⁵ cells/ml. Cells were allowed to grow for 2–3 days until ~80% confluent. Complete medium was replaced with serum free medium and the cells were grown for a further 24 hours followed by the addition of cytokines or drugs (as described above). Isolation of cells for RNA extraction (at 4 and 24 hours post cytokine or drug addition) involved removing medium, a phosphate buffered saline (PBS) wash and collection in 1 ml PBS using a cell scraper. Cells were collected by centrifugation (300 × g 5 min), supernatant discarded and cell pellets resuspended in 50 μl PBS. 450 μl RNAlater was added to each sample followed by mixing and storage at -80°C. Total RNA was extracted from samples using the RNAeasy kit (Qiagen) as directed by the manufacturer. cDNA was generated from 1 μg RNA template using random hexamers and the Superscript kit (Invitrogen, Paisley, UK) as directed by the manufacturer. TaqMan assays specific for the Histamine H1 Receptor (HRH1), Bradykinin B2 Receptor (BDKRB2), Gαq/11 protein (GNA11) and phosholipase C-β1 (PLCB1) transcripts were designed using Primer Express Software (Applied Biosystems, Warrington, UK). See Table 1. Real Time PCR (Applied Biosystems) was carried out as directed by the manufacturer in a reaction volume of 20 μl containing ~100 ng of cDNA, 300 nM of each Primer, 200 nM Probe (Eurogentec Ltd, Southampton, UK) and 10 μl universal mastermix (Applied Biosystems). Differences in the quantity of cDNA template were normalised using a VIC labelled 18s ribosomal RNA endogenous control (Applied Biosystems). PCR was performed using the ABI-7700 and data collected using Sequence Detection Software (Applied Biosystems). Data was analysed as directed by the manufacturer and Ct values were normalised using the 18s ribosomal RNA signal. Relative differences in gene expression were calculated using the 2^dCt value as continuous variable (Sequence Detection System Compendium 7700 Version 4.0, Applied Biosystems).

5'RACE was performed in order to identify the putative promoter regions of the BDKRB2 gene in Human ASM. Total RNA was extracted from Human ASM cells derived from two different donors. Total RNA extractions were performed using the RNeasy Mini Kit (Qiagen, Crawley, UK) as described above. 5'RACE was performed using the GeneRacer Kit (Invitrogen) according to the manufacturer's protocol. 2.5 μg of total RNA was used as template. The mRNA was reverse-transcribed with SuperScript II RT (Invitrogen) and the GeneRacer OligodT primer (50 μM) to generate RACE ready cDNA. A nested PCR approach was used to identify the 5' structure of the BDKRB2 gene; PCR1 (GeneRacer 5'Primer/BDKRB2 primer 1 specific for the open reading frame of the BDKRB2 gene). Reaction conditions included; 1 μl RACE ready cDNA template, 0.025 U/μl Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 0.6 μM of each primer, 200 μM dNTPs, 2 mM Magnesium Sulphate and a staged PCR cycling approach; 94°C for 5 min, 94°C for 1 min 30 sec/72°C for 3 min × 5, 94°C for 1 min 30 sec/70°C for 1 min 30 sec/72°C for 3 min × 5, 94°C for 1 min 30 sec/68°C for 1 min 30 sec/72°C for 3 min × 20 and a final extension of 72°C for 10 min. The second stage PCR (PCR 2) utilised nested primers (GeneRacer 5' Nested Primer/BDKRB2 primer 2 specific for the open reading frame of the BDKRB2 gene). Reaction conditions were as described for PCR 1 except 1 μl cDNA (from PCR1) was used as template and PCR cycling involved; 94°C for 5 min, 94°C for 1 min 30 sec/68°C for 1 min 30 sec/72°C for 3 min × 25 and a final extension of 72°C for 10 min. 5'RACE products generated in PCR 2 were cloned into the pCR-4-TOPO vector using the TOPO TA Cloning Kit for Sequencing as directed by the manufacturer (Invitrogen). LB agar plates and LB broths (100 μg/mL ampicillin) were used to select and grow single colonies of positive clones. Plasmid DNA was purified using the FastPlasmid Mini Kit (Eppendorf, Cambridge, UK) and the presence of inserts was confirmed by restriction analysis with EcoRI (Promega, Southampton, UK). Positive clones showing an insert after restriction digest were sequenced using M13 primers (Invitrogen), ABI BigDye Terminator v3.1 Cycle Sequencing Kit and ABI Prism 310 DNA sequencer (Applied Biosystems). Sequences were analysed and the 5' structure of the BDKRB2 gene identified using Chromas v2.0 (31) and BLAST (NCBI).

PCR primers were designed to generate 1, 2, 3 and 4 kb amplicons encompassing the predicted BDKRB2 transcription start sites (TSS) in HASM but not crossing the ExonI/IntronI interface (see Table 1). The most 3' TSS was at position 95,741,003 NCBI Build 35 (102,403 bp within AL355102.5)). A repeat polymorphism [GGTGGGGAC]n exists in this region therefore DNA fragments were generated to include the common n = 2 repeat observed in the Caucasian population 32. Primers were designed to engineer a 5' HindIII and a 3' XhoI site into the DNA fragments to facilitate cloning into the pGL4-Luc2 vector (Promega). The BDKRB2 4 kb PCR included; 1 μl genomic DNA template (Caucasian), 0.025 U/μl Platinum Taq DNA Polymerase High Fidelity, 0.2 μM of each primer, 200 μM dNTPs, 2 mM Magnesium Sulphate and cycling parameters; 94°C for 2 min, 94°C for 30 sec/62°C 30 sec/68°C for 4 min × 30 and a final extension of 68°C for 7 min. The PCR product was cloned into the pCR-4-TOPO vector and sequenced (as described above). The sequenced pCR-4 vector containing the BDKRB2 4 kb fragment was used as PCR template for the generation of the BDKRB2 1, 2 and 3 kb fragments as described above. The 1, 2, 3 and 4 kb BDKRB2 fragments were excised from the pCR-4 vectors using HindIII/XhoI and cloned into pGL4-Luc2 using standard molecular biology techniques. An identical series of pGL4-Luc2 vectors containing 1, 2, 3, and 4 kb HRH1 promoter fragments encompassing all of the identified transcription start sites in Human ASM but not crossing the ExonI/IntronI interface (the most 3' TSS was at 52,754 bp within AC083855, 33) were also generated (Table 1).

CHO-K1 cells were cultured in complete DMEM (10% foetal calf serum and 2 mM glutamine) and harvested using 5 mg/ml trypsin/2 mg/ml EDTA. Cells were plated at a density of 25,000 cells/well into 48-well tissue culture plates (Corning, Sunderland, UK) using complete medium (see above) and allowed to grow for 24 h until ~80% confluent. Complete medium was replaced with serum free medium and the cells were transfected with 0.12 μg of pGL4-Luc2 using Fugene 6 (Roche) at a 3:1 ratio (Fugene:DNA). For derivatives of pGL4-Luc2 containing the HRH1, BDKRB2 or SV40 control inserts equimolar amounts of DNA were used and the amount of Fugene corrected accordingly. Cells were allowed to grow for 40 h prior to a PBS wash and harvested using Passive Lysis Buffer (PLB, 300 μl/well, rocked for 15 min and subjected to freeze/thaw at -80°C). Human ASM experiments were completed in an analogous manner except cells were allowed to grow for 24–72 hours until ~80% confluent. Following transfection cells were allowed to grow for 16 hours prior to the addition of cytokines or drug (at concentrations described above). Following 4 or 24 hours, ASM cells were washed with PBS and harvested using Passive Lysis Buffer as described in a volume of 100 μl/well. Firefly luciferase was quantified using 5 μl (CHO-K1) or 20 μl (ASM) of cell lysate and the luciferase assay system as directed by the manufacturer (Promega) and a Model TD-20e luminometer (Turner Biosystems, Sunnyvale, CA). Data was normalised to fold over the empty pGL4-Luc2 vector. Cytokine or drug treated samples were compared to medium alone treated samples by designated medium treated samples 100%.

Specific transcription factor binding sites were identified using; BioInformatics & Molecular Analysis Section 34, Transcription Element Search System 35, TFSEARCH 36 and WWW Signal Scan 37.

Differences between outcome measures for different treatment groups were compared by Analysis of Variance (ANOVA) in conjunction with Dunnett's Multiple Comparison Post Hoc Test or Students' T test as appropriate. Figures represent mean values (+/- S.E.M). Statistical analyses were completed using GraphPad Prism (GraphPad, San Diego, CA), a P value < 0.05 was considered significant.

In order to assess the effect of the micro-environment on subsequent IPx signalling responses in Human ASM we incubated cells with a series of pro-inflammatory cytokines implicated in the pathogenesis of asthma. These cytokines included; IL-1β, TNFα, IFNγ, or IL-13. The capacity of the ASM cells to produce IPx in response to two specific mediators of airway hyper-responsiveness; histamine and bradykinin and the non-specific Gαq/PLC activator sodium fluoride was determined. Similarly, the effect of two drug classes used routinely in the treatment of asthma was evaluated within the same model; i.e. the long acting β₂-adrenoceptor agonist salmeterol and the steroid dexamethasone (Table 2). Significant differences were observed in the capacity of the ASM cells to produce IPx in response to histamine (p < 0.0001) or bradykinin (p = 0.0004) following cytokine or drug pre-treatment. There was not a statistically significant change in the capacity of the ASM cells to produce IPx following sodium fluoride stimulation (although differences were observed and there was a large degree of variation in these data). More specifically, IL-13, IFNγ and salmeterol were shown to significantly augment the capacity of the ASM cells to produce IPx in response to histamine stimulation (p < 0.05), however, these effects were modest; IL-13 (144.4 +/- 9.3%) compared to medium control (100%)), IFNγ (126.4 +/- 7.5%) and salmeterol (117.7 +/- 5.2%). Similarly, TNFα, IFNγ and salmeterol were shown to augment bradykinin mediated IPx production in ASM cells (p < 0.05), TNFα (127.4 +/- 8.3%), IFNγ (128.0 +/- 8.4%) and salmeterol (111.7 +/- 5.0%).

In order to test the hypothesis that the site of regulation determining the alteration in IPx signalling observed following cytokine or drug pre-treatment occurs at the receptor locus we determined the mRNA expression levels of the H1 Histamine and B2 Bradykinin Receptors in our samples using Real Time PCR, i.e. the two key receptors mediating histamine or bradykinin activation of human ASM cells (2938, Figure 1). Quantification of the HRH1 mRNA levels identified a significant induction of gene expression following drug or cytokine treatment for 4 (p < 0.0001) or 24 hours (p < 0.0001) (Figures 1A and 1B). A significantly elevated level of HRH1 mRNA expression was observed following 4 hours treatment of Human ASM with IL-13 (254 +/- 14.5% compared to medium control (100%)) or IFNγ(228.7 +/- 30.9%). At 24 hours the IL-13 treated cells maintained an elevated level of HRH1 mRNA expression (298.5 +/- 17.0%) and salmeterol treated cells demonstrated a significantly elevated level of HRH1 mRNA expression (217.3 +/- 47.1%). Quantification of BDKRB2 mRNA expression in Human ASM cells following cytokine or drug treatment identified a significant effect on receptor mRNA expression at 4 (p < 0.0001) and 24 hours (p < 0.0001) post treatment (Figures 1C and 1D). A significant induction of BDKRB2 mRNA expression was observed at 4 hours following TNFα (461.0 +/- 20.4%) or IFNγ (322.1 +/- 53.3%) treatment. Following 24 hours treatment of ASM cells an elevated level of BDKRB2 mRNA was observed in the TNFα (156.2 +/- 6.5) and salmeterol (147.1 +/- 14.7%) treated cells. Treatment of Human ASM cells with IL-13 did not significantly influence BDKRB2 mRNA expression.

To further define the potential mechanisms responsible for the alterations in the capacity of Human ASM to generate inositol phosphates we quantified the level of Gαq/11 and PLCβ1 mRNA expression in these samples. The H1 histamine and B2 bradykinin receptors are thought to signal via coupling to the Gαq/11 and phospholipase Cβ1 isoforms 394041. Analysis of GNA11 and PLCB1 mRNA expression in Human ASM cells following 4 or 24 hours treatment with Salmeterol, TNFα, IL-13 or IFNγ did not identify any significant differences in mRNA levels compared to medium treated cells (data not shown). The absence of a significant alteration in GNA11 or PLCB1 expression levels does not exclude the potential that these molecules have altered activation states, which was not evaluated in the current analyses.

In order to facilitate a more comprehensive analysis of the potential transcriptional mechanisms involved in HRH1 and BDKRB2 mRNA induction following Salmeterol, TNFα, IL-13 or IFNγ treatment the BDKRB2 transcription start site(s) and promoter regions were mapped in Human ASM cells using 5'RACE (Figure 2). Figure 2A shows a diagrammatic representation of the identified 5'region and transcript of the BDKRB2 gene in Human ASM. The BDKRB2 transcript was found to be composed of three exons separated by 32,109 bp and 3,223 bp introns respectively spanning ~39 kbps on chromosome 14. The coding region was found to be contained within exon 2 and 3 and two close transcription start sites were identified (Figure 2A and 2B). These data are in excellent agreement with previous analyses using a placental cDNA library 42. The number of sequence clones representing the two potential transcription starts site are shown, with the dominant site (TSS1) being 5' to the previously reported TSS in placental cells (42, Figure 2A and 2B). During the course of this study we also characterised the 5' region of the HRH1 gene in Human ASM which has now been reported elsewhere 33. The HRH1 transcript was found to be predominantly (85%) composed of two exons; a 5' untranslated and an exon containing the coding region separated by a 104.4 kb intron 33. The core HRH1 promoter was identified and three potential transcription start sites were identified 33. 4 kb of each core promoter encompassing all of the identified transcription start sites was used as the basis for the generation of a series of promoter-reporter constructs. In order to potentially identify transcription factor binding sites with relevance to Salmeterol, TNFα, IL-13 or IFNγ treatment cell activation (i.e. NF-κB, AP-1, STAT, CREB and CRE-BP 43) within these 4 kb sequences we completed analyses using transcription factor binding site databases (Figure 2C). Analysis of the HRH1 core promoter sequence using the TFSEARCH database identified the presence of AP-1 (6 sites, between 1–4 kb), NF-κB (1, 1–2 kb), CRE-BP (3, 1–4 kb), CREB (1, 3–4 kb), CRE/CRE-BP (1, 3–4 kb) and STATx (1, 3–4 kb) binding sites (data not shown) however, only the AP-1 (4 sites, between 1–4 kb) and NF-κB (1, 1–2 kb) were identified in two or more databases (Figure 2C). Analysis of the BDKRB2 core promoter using TFSEARCH identified NF-κB (6 sites, between 0–3 kb), AP-1 (5, 1–4 kb), STATx (2, 3–4 kb), CREB (3, 0–4 kb), CREB-BP (4, 0–4 kb) and CREB/CREB-BP (2, 0–4 kb) binding sites (data not shown). Following analyses using multiple databases NF-κB (2, 0–1 kb), CREB (2, 0–4 kb) and AP-1 (9, 0–4 kb) transcription binding sites were observed in two or more analyses (Figure 2C).

Preliminary luciferase experiments using CHO-K1 cells transfected with pGL4 constructs for 40 hours provided clear evidence that all of the HRH1 and BDKRB2 promoter-luciferase plasmids were transcriptionally active (all >15 fold over pGL4-Luc2 control, p = 0.0002 and p = 0.0004 respectively, data not shown). Interestingly the greatest activity in CHO-K1 cells was observed for the plasmids containing the longest segment of the two core promoters i.e. the HRH1-4 kb (106.5 +/- 34.3 fold over pGL4-Luc2, p < 0.01) and BDKRB2-4 kb (219.4 +/- 75.3 fold over pGL4-Luc2, p < 0.01) plasmids. Basal activity of HRH1 promoter containing plasmids transfected into Human ASM showed low level activity over pGL4-Luc2 at 20 hours post transfection with maximal activity observed for the pGL4-HRH1-2 kb transfection samples (2.1 +/- 0.2 fold, Figure 3A). Analyses of luciferase activity in Human ASM cells transfected with the series of BDKRB2 or control constructs for 20 hours demonstrated significant differences in activity between constructs (ANOVA, p = 0.0001). The BDKRB2 1 kb (5.6 +/- 0.9 fold) and 4 kb (3.8 +/- 0.8 fold) constructs showed clear activity (Figure 3B). The pGL4-BDKRB2-3 kb transfection samples showed similar to pGL4-Luc2 activity (Figure 3B). Transfection of Human ASM cells with HRH1 promoter containing constructs for 40 hours again demonstrated a modest transcriptional activity over pGL4-Luc2 as observed for the 20 hour transfections (maximum HRH1 2 kb, 2.5 +/- 0.8 fold, Figure 3C). Transfection of Human ASM cells with BDKRB2 promoter constructs again identified the 1 kb and 4 kb promoter constructs as demonstrating the highest level of activity as shown at 20 hours post transfection (8.7 +/- 1.7 fold and 5.5 +/- 1.1 fold respectively, Figure 3D). pGL4-SV40-Luc2 control luciferase levels were; CHO-K1 (649 +/- 99 fold) and Human ASM (20 hours 1229 +/- 173 fold, 40 hours 1255 +/- 157 fold). These data confirmed that the primary ASM cells were being efficiently transfected using this protocol.

In order to investigate the effect of cytokines or salmeterol on HRH1 promoter activity in Human ASM, cells were transfected with the series of pGL4-HRH1 constructs for 16 hours and then stimulated with the appropriate cytokine or salmeterol for 4 (Figure 4) or 24 (Figure 5) hours prior to luciferase quantification. These analyses were not susceptible to transfection efficiency bias as comparisons were made within transfections. Predominantly, for these pGL4-Luc2 control transfections the background luciferase activity was not affected by stimulation with cytokines or salmeterol, however, a significant reduction in pGL4-Luc2 mediated luciferase activity was observed following 24 hours TNFα treatment (37.4 +/- 5.0 compared to medium control (100%), Figure 5A). Stimulation of Human ASM cells transfected with pGL4-SV40-Luc2 control plasmid did not identify any effect of TNFα, IFNγ, IL-13 or salmeterol treatment for 4 and 24 hours on SV40 mediated luciferase production (Figures 4B and 5B). Analyses of Human ASM cells transfected with the HRH1 1, 2, 3 and 4 kb promoter constructs and stimulated for 4 hours with IFNγ, IL-13 or salmeterol provided evidence that the level of transcription was influenced by treatment for the 3 and 4 kb constructs (ANOVA, p = 0.05 and p = 0.05 respectively Figures 4E and 4F). However, following post-hoc analyses only salmeterol significantly augmented transcription of the 4 kb core HRH1 promoter (224.5 +/- 37.4 versus medium control (100%), p < 0.05 Figure 4F). There was suggestive evidence that IL-13 may influence transcription of the HRH1 3 kb promoter (204.1 +/- 50.1) and IFNγ may influence the 4 kb construct (164.4 +/- 36.8, Figures 4E and 4F) although these differences were not statistically significant. Analyses of Human ASM cells transfected with the HRH1 1, 2, 3 and 4 kb promoter constructs and stimulated for 24 hours with IFNγ, IL-13 or salmeterol provided evidence that the level of transcription was affected for the 1 kb constructs (ANOVA, p = 0.03, Figure 5C) and that salmeterol significantly augments the HRH1 1 kb construct transcriptional activity (250.3 +/- 60.3, p < 0.05, Figure 5C). As previously, there was evidence that IL-13 influences transcription of the HRH1 3 kb promoter (205.3 +/- 90.2, Figure 5E).

In an analogous manner to that described for the HRH1 promoter analyses the effect of TNFα, IFNγ or salmeterol on BDKRB2 mediated transcription was evaluated using the pGL4-BDKRB2-luciferase transfected Human ASM cells. Following 4 hours stimulation with TNFα or IFNγ there were no apparent effects on BDKRB2 mediated transcription for the 1, 2, 3 and 4 kb constructs. Salmeterol treatment resulted in an augmentation of BDKRB2 mediated transcription for the 1, 2, 3 and 4 kb constructs (409.4 +/- 65.7, 446.7 +/- 111.9, 245.3 +/- 30.8 and 542.4 +/- 148.5 respectively, p < 0.01, Figure 6). Following 24 hours stimulation of BDKRB2-luciferase transfected Human ASM cells with TNFα, IFNγ or salmeterol a similar pattern to the 4 hour experiments was observed. TNFα and IFNγ stimulation did not significantly influence BDKRB2 mediated transcription (Figure 7). Salmeterol treatment of BDKRB2-luciferase transfected Human ASM cells for 24 hours significantly augmented BDKRB2 mediated transcription for the 1, 2 and 4 kb constructs (190.9 +/- 30.0, 208.8 +/- 42.7 and 377.7 +/- 110.7 respectively, p < 0.05, Figures 7A, 7B, 7D), however, the effect was apparent for all constructs.