Journal: Frontiers in Physiology

Article Title: Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

doi: 10.3389/fphys.2016.00339

Figure Lengend Snippet: Stimulation with agonists of OR2AG1 and OR1D2 led to an intracellular Ca 2+ increase in HASMCs. (A) Repetitive stimulation with amyl butyrate (300 μM, duration: 30 s) elicited a reproducible transient increase in intracellular Ca 2+ measured with the ratiometric Ca 2+ indicator FURA-2AM. (B) Amyl butyrate, an agonist for OR2AG1, activated HASMCs in a dose-dependent manner with an EC50 of 253.39 μM ( N = 6). (C) The application of the OR1D2 agonist bourgeonal (100 μM, duration: 30 s) led to an increase in intracellular Ca 2+ , and repetitive stimulation exerted a reproducible effect. (D) Bourgeonal was able to activate HASMCs in a dose-dependent manner with an EC50 of 0.5043 μM ( N = 3–5). (E,F) The application of the OR1D2 agonists lilial (300 μM, duration: 30 s; E) and 4-phenylbutyrate (4-PBA; 300 μM, duration: 30 s; F ) led to an intracellular Ca 2+ increase in ratiometric Ca 2+ imaging experiments. Bars indicate the application duration. Error bars represent the ± SEM of three to four independent experiments.

Article Snippet: Antibodies directed against the ORs OR1D2 (Novus Biologicals, Littleton, CO, USA) and OR2AG1 (Novus Biologicals, Littleton, CO, USA) as well as against the olfactory signaling components ACIII (Santa Cruz Biotechnology, Dallas, TX, USA), Gα olf (Santa Cruz Biotechnology, Dallas, TX, USA), CNGA2 (Santa Cruz Biotechnology, Dallas, TX, USA), and CNGA4 (Santa Cruz Biotechnology, Dallas, TX, USA) were used.

Techniques: Imaging

Journal: Frontiers in Physiology

Article Title: Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

doi: 10.3389/fphys.2016.00339

Figure Lengend Snippet: Analysis of the expression of ORs and olfactory signaling components at the transcript and protein level. (A,B) qPCR analysis with cDNA of three different HASMC donors using specific OR primers. PCR products for OR1D2, OR2AG1, and the smooth muscle marker ACTA2 were detected in an agarose gel at ~250 bp (A) . ΔCt-values were calculated and normalized to the ACTA2 Ct-value ( N = 3). The transcript level of OR2AG1 was higher than that of OR1D2 (B) . Error bars represent the ±SEM of at least three independent experiments. (C) The immunocytochemical staining of HASMCs showed that OR1D2 and OR2AG1 are expressed at the protein level. The cells were co-stained with ACTA2 as a marker of HASMCs. Scale bars: 100 μm. (D,E) Western blot experiments using the cytosolic and membrane protein fractions of HASMCs showed specific bands for OR1D2 (~35 kDa; D) and OR2AG1 (~35 kDa; E) in all fractions. (F–H) Immunohistochemical 3,3′-diaminobenzidine (DAB) staining of human lung tissue with OR1D2 (F) and OR2AG1 (G) antibody. A detail of a bronchus can be seen in both images. L, lumen; RE, respiratory epithelium; LP, lamina propria; SM, smooth muscle layer; SU, submucosa. Specific staining was observed in the apical part of the RE and in the SM (F) as well as in the RE, SM, and cells of the SU (G) . (H) Secondary antibody (anti-rabbit) negative control. Arrows indicate staining of the SM. Scaling bar: 200 μm.

Article Snippet: Antibodies directed against the ORs OR1D2 (Novus Biologicals, Littleton, CO, USA) and OR2AG1 (Novus Biologicals, Littleton, CO, USA) as well as against the olfactory signaling components ACIII (Santa Cruz Biotechnology, Dallas, TX, USA), Gα olf (Santa Cruz Biotechnology, Dallas, TX, USA), CNGA2 (Santa Cruz Biotechnology, Dallas, TX, USA), and CNGA4 (Santa Cruz Biotechnology, Dallas, TX, USA) were used.

Techniques: Expressing, Marker, Agarose Gel Electrophoresis, Staining, Western Blot, Membrane, Immunohistochemical staining, Negative Control

Journal: Frontiers in Physiology

Article Title: Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

doi: 10.3389/fphys.2016.00339

Figure Lengend Snippet: Activation of HASMCs by the OR2AG1-specific agonist amyl butyrate led to an influx of Ca 2+ dependent on cAMP production and opening of CNG channels. (A) Amyl butyrate (300 μM) was applied for 30 s in Ringer's solution with 2 mM extracellular Ca 2+ or in a Ca 2+ -free Ringer's solution with 5 mM EGTA. Under Ca 2+ free conditions, the cytosolic Ca 2+ increase was completely abolished ( N = 3). (B) Amyl butyrate (300 μM) was applied for 30 s after a 5 min incubation with the adenylyl cyclase inhibitor SQ22536 (200 μM). Ca 2+ influx was significantly reduced after incubation with SQ22536 ( N = 3). (C) A 2 min incubation with MDL12330A (50 μM), an adenylyl cyclase inhibitor, and a following co-application with amyl butyrate (300 μM, duration: 30 s) significantly inhibited amyl the butyrate-induced Ca 2+ increase ( N = 4). (D) The CNG channel inhibitor L-cis-diltiazem (100 μM) was co-applied with amyl butyrate (300 μM) for 30 s and significantly reduced the Ca 2+ influx induced by amyl butyrate. The inhibitory effect of L-cis-diltiazem was reversible after a wash-out ( N = 5). Bars of all experiments indicate the stimulus duration. All error bars represent the ±SEM of at three to five independent experiments. Significance was tested with an unpaired two-sample Student's t -test or a Mann-Whitney U test. * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies directed against the ORs OR1D2 (Novus Biologicals, Littleton, CO, USA) and OR2AG1 (Novus Biologicals, Littleton, CO, USA) as well as against the olfactory signaling components ACIII (Santa Cruz Biotechnology, Dallas, TX, USA), Gα olf (Santa Cruz Biotechnology, Dallas, TX, USA), CNGA2 (Santa Cruz Biotechnology, Dallas, TX, USA), and CNGA4 (Santa Cruz Biotechnology, Dallas, TX, USA) were used.

Techniques: Activation Assay, Incubation, MANN-WHITNEY