Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

Abstract

Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca(2+) increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases.

Keywords: contraction; cytokines; olfactory receptor; signaling; smooth muscle cells.

Figure 1

Representative traces of ratiometric Ca²⁺ imaging experiments showing an increase in intracellular Ca²⁺ evoked by amyl butyrate (300 μM; A) and bourgeonal (300 μM; B). Bars indicate the application duration.

Figure 2

Stimulation with agonists of OR2AG1 and OR1D2 led to an intracellular Ca²⁺ increase in HASMCs. (A) Repetitive stimulation with amyl butyrate (300 μM, duration: 30 s) elicited a reproducible transient increase in intracellular Ca²⁺ measured with the ratiometric Ca²⁺ 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 Ca²⁺, 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 Ca²⁺ increase in ratiometric Ca²⁺ imaging experiments. Bars indicate the application duration. Error bars represent the ± SEM of three to four independent experiments.

Figure 3

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.

Figure 4

(A) The immunocytochemical staining of HASMCs showed that the olfactory signaling proteins ACIII, G_olf, CNGA2, and CNGA4 are expressed at the protein level. The cells were co-stained with ACTA2 as a marker for HASMCs. Scale bars: 100 μm. (B–E) Specific bands for ACIII (~130 kDa; B), Gα_olf (~40 kDa; C), CNGA2 (~80 kDa; D), and CNGA4 (~60 kDa; E) were detected in the membrane fraction of HASMCs via western blot analysis of the membrane and cytosolic fractions.

Figure 5

Bourgeonal-induced Ca²⁺ increase in HASMCs is dependent on the extracellular Ca²⁺ concentration, production of cAMP, and opening of CNG channels. (A) The bourgeonal-specific antagonist undecanal significantly inhibited the activation of OR1D2 and, thereby, Ca²⁺ increases in HASMCs. Bourgeonal (100 μM) was first applied for 30 s. After a 3 min wash in Ringer's solution, undecanal (200 μM) and bourgeonal (100 μM) were co-applied for 30 s. The cells were again washed with Ringer's solution, followed by an application of bourgeonal (100 μM) for 30 s. The undecanal-induced inhibition was reversible (N = 5). (B) Bourgeonal (100 μM) was applied for 30 s in Ringer's solution containing 2 mM extracellular Ca²⁺ or in a Ca²⁺-free condition with 5 mM EGTA. Under Ca²⁺ free conditions, the cytosolic Ca²⁺ increase was completely abolished (N = 6). (C) Bourgeonal (100 μM) was applied for 30 s after a 5 min incubation with the adenylyl cyclase inhibitor SQ22536 (200 μM). Ca²⁺ influx was significantly reduced after incubation with SQ22536 (N = 3). (D) MDL12330A (50 μM), an adenylyl cyclase inhibitor, was applied for 2 min, followed by co-application with bourgeonal (100 μM, duration: 30 s), which significantly inhibited bourgeonal-induced Ca²⁺ increase. This effect was reversible after a 4 min washing step (N = 4). (E) The CNG channel inhibitor L-cis-diltiazem (100 μM) was co-applied with bourgeonal (100 μM) for 30 s and significantly reduced the Ca²⁺ influx induced by bourgeonal. The inhibitory effect of L-cis-diltiazem was reversible after a wash-out (N = 6). The bars of all experiments indicate the stimulus duration. All error bars represent the ±SEM of at three to six 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, and ^***p < 0.001.

Figure 6

Activation of HASMCs by the OR2AG1-specific agonist amyl butyrate led to an influx of Ca²⁺ 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 Ca²⁺ or in a Ca²⁺-free Ringer's solution with 5 mM EGTA. Under Ca²⁺ free conditions, the cytosolic Ca²⁺ 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). Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.

Figure 7

Cell contraction assays demonstrating the physiological relevance of the activation of ORs in HASMCs. (A,B,E) Contraction analysis after the incubation of collagen gel-embedded HASMCs with histamine (HIS; 100 μM), amyl butyrate (AB; 100 μM), and co-incubation with histamine (100 μM) and amyl butyrate (100 μM). The gel diameter decrease is exemplarily shown after 30 min of incubation with drug (A). As a control, cells were incubated with DMEM + 0.1% DMSO (DMSO control, Ctrl). Histamine induced a significant decrease in the gel diameter when incubated with HASMC collagen gels for 34 min (B,E). Amyl butyrate itself did not alter the gel diameter under these conditions and produced no significant changes in relation to the medium control. The co-incubation of amyl butyrate and histamine resulted in a non-significant decrease in relation to the medium control but a significant inhibition of the contraction induced by histamine alone (N = 3). The AC inhibitor SQ22536 (SQ; 200 μM) inhibited the amyl butyrate-induced reduction of histamine-mediated contraction (N = 3). (C,D,F) Analysis of the effects of bourgeonal on HASMC contraction. The gel diameter decrease is exemplarily shown after 30 min of incubation with either bourgeonal (BG; 100 μM), undecanal (UD; 200 μM) or bourgeonal (100 μM), and undecanal (BG + UD; 200 μM;) (D). Bourgeonal (100 μM) induced a noticeable decrease in the gel diameter (D,F). Undecanal itself induced a weaker contraction of HASMCs. The bourgeonal-induced effect was significantly inhibited by the co-incubation of the bourgeonal-specific antagonist undecanal (200 μM; N = 4; F). The effect of bourgeonal was also inhibited by SQ22536 (SQ; 200 μM) and histamine (100 μM), but not L-cis-diltiazem (LCD) or amyl butyrate (N = 3). Blue and red dotted lines indicate the gel area. All error bars represent the ±SEM of either three or four experiments. Significance was tested with an unpaired two-sample Student's t-test. In (F) significance is tested compared to bourgeonal-induced decrease of gel diameter. ^*p < 0.05, ^**p < 0.01.

Figure 8

Bourgeonal increases the production of inflammatory cytokines in HASMCs. Serum-deprived HASMCs were stimulated with bourgeonal or amyl butyrate in the presence or absence of undecanal, the ERK inhibitor PD98059, the p38MAPK inhibitor SB203580, or the Jnk inhibitor SP600125 at the indicated concentrations and for the indicated times (A,C), for 72 h (B,D) or for 6 days (E). (A–D) The cytokines in the cell culture supernatants were measured by ELISA. (E) Viable cells were counted by Trypan blue staining. Stimulation with EGF was used as a positive control. Data (A–D, N = 4; e, N = 3) are presented as mean ±SEM. Significance was tested with an unpaired two-sample Student's t-test. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. non-stimulated controls if placed on top of the bars or to values as indicated. n.s., differences to non-stimulated controls are not significant.