Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus

Abstract 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 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.</p

Impaired uptake of serotonin by platelets from patients with irritable bowel syndrome correlates with duodenal immune activation

Background &amp; AimsPatients with irritable bowel syndrome with diarrhea (IBS-D) have increased mucosal serotonin (5-hydroxytryptamine [5-HT]) availability, possibly because immune activation reduces activity of the 5-HT transporter (SERT). We investigated the relationship between mucosal and platelet SERT and immune activation of the duodenal mucosa in patients with IBS-D.MethodsWe quantified mucosal intraepithelial lymphocytes (IELs), mast cells, and enterochromaffin cells in blood samples, measured levels of SERT messenger RNA (mRNA) in mucosal samples, and assessed platelet uptake of 5-HT and platelet membrane binding of 3H-paroxetine in samples from 29 healthy volunteers (HVs), 20 patients with IBS-D, and 20 untreated patients with celiac disease.ResultsPatients with IBS-D or celiac disease had increased numbers of IELs and mast cells compared with HVs (both P &lt; .001). Levels of SERT mRNA were reduced in the mucosa of patients with IBS-D or celiac disease and were inversely correlated with numbers of IELs (r = ?0.72, P &lt; .0001). Uptake of 5-HT by platelets from patients with IBS-D or celiac disease was reduced (mean, 17.1 ± 3.5 and 28.3 ± 4.1 nmol • min?1 • mg?1, respectively) compared with HVs (50.8 ± 8.0 nmol • min?1 • mg?1, P &lt; .01 and P = .05, respectively). Binding of paroxetine to membranes of platelets from patients with IBS-D (median [interquartile range], 226 [92–405] fmol/mg protein) was significantly greater than that from HVs (109 [69–175] fmol/mg protein) and correlated inversely with platelet uptake of 5-HT (r = ?0.62, P = .03). Tryptase release from incubated biopsy samples was significantly increased in patients with IBS-D (2.2 [0.42–3.5] vs 0.50 [0.25–0.86] ng • mL?1 • mg?1 for HVs; P = .03).ConclusionsPlatelet SERT is reduced in IBS-D and associated with reduced levels of SERT mRNA and duodenal immune activation.<br/

Effect of salmeterol or cytokine treatment for 4 hours on promoter activity in Human ASM cells transfected with -Luciferase or control constructs

<p><b>Copyright information:</b></p><p>Taken from "Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus"</p><p>Respiratory Research 2007;8(1):68-68.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2117012.</p><p></p> Complete medium was replaced with serum free medium and 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 or SV40 control inserts equimolar amounts of DNA were used. Cells were allowed to grow for 16 hours prior to the addition of medium alone, 10 ng/ml TNFα, 10 ng/ml IFNγ, 50 ng/ml IL-13 or 1 μM salmeterol. Following 4 hours treatment cells were harvested and firefly luciferase was quantified. pGL4-Luc2 (A), pGL4-SV40-Luc2 (B), pGL4-HRH1-1 kb-Luc2 (C), pGL4-HRH1-2 kb-Luc2 (D), pGL4-HRH1-3 kb-Luc2 (E), pGL4-HRH1-4 kb-Luc2 (F) transfections. Data is normalised to the mean luciferase activity of each construct transfection treated with medium alone +/- S.E.M. (n = 4 independent experiments). Dunnett's Multiple Comparison Test (*p < 0.05)

Effect of salmeterol or cytokine treatment on H1 Histamine Receptor and B2 Bradykinin Receptor mRNA expression in Human ASM

<p><b>Copyright information:</b></p><p>Taken from "Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus"</p><p>Respiratory Research 2007;8(1):68-68.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2117012.</p><p></p> ASM cells were serum starved for 24 hours and then treated with medium alone, 10 ng/ml TNFα, 10 ng/ml IFNγ, 50 ng/ml IL-13 or 1 μM salmeterol for 4 or 24 hours. mRNA levels of the H1 Histamine Receptor and B2 Bradykinin Receptor were quantified using Real Time PCR and normalised using the 18s ribosomal RNA endogenous control. HRH1 mRNA quantification following 4 hours (A) and 24 hours (B) stimulation. BDKRB2 mRNA quantification following 4 hours (C) and 24 hours (D) stimulation. Data is normalised to medium control = 100%, n = 3 independent experiments in triplicate, mean +/- S.E.M. Dunnett's Multiple Comparison Test (*p < 0.05)

Effect of salmeterol or cytokine treatment for 4 hours on promoter activity in Human ASM cells transfected with -Luciferase constructs

<p><b>Copyright information:</b></p><p>Taken from "Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus"</p><p>Respiratory Research 2007;8(1):68-68.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2117012.</p><p></p> Human ASM cells were transfected and stimulated as described in Figure 4 except derivatives of pGL4-Luc2 containing the BDKRB2 inserts were used. Following 4 hours treatment cells were harvested and firefly luciferase was quantified. pGL4-BDKRB2-1kb-Luc2 (A), pGL4-BDKRB2-2kb-Luc2 (B), pGL4-BDKRB2-3kb-Luc2 (C), pGL4-BDKRB2-4kb-Luc2 (D) transfections. Data is normalised to the mean luciferase activity of each construct transfection treated with medium alone +/- S.E.M. (n = 4 independent experiments). Dunnett's Multiple Copmarison Test (**p < 0.01)