Prostaglandin D₂ Induces Ca²⁺ Sensitization of Contraction without Affecting Cytosolic Ca²⁺ Level in Bronchial Smooth Muscle

Abstract

Prostaglandin D₂ (PGD₂) is one of the key lipid mediators of allergic airway inflammation, including bronchial asthma. However, the role of PGD₂ in the pathogenesis of asthma is not fully understood. In the present study, the effect of PGD₂ on smooth muscle contractility of the airways was determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In isolated bronchial smooth muscles (BSMs) of naive mice, application of PGD₂ (10⁻⁹–10⁻⁵ M) had no effect on the baseline tension. However, when the tissues were precontracted partially with 30 mM K⁺ (in the presence of 10⁻⁶ M atropine), PGD₂ markedly augmented the contraction induced by the high K⁺ depolarization. The PGD₂-induced augmentation of contraction was significantly inhibited both by 10⁻⁶ M laropiprant (a selective DP₁ antagonist) and 10⁻⁷ M Y-27632 (a Rho-kinase inhibitor), indicating that a DP₁ receptor-mediated activation of Rho-kinase is involved in the PGD₂-induced BSM hyperresponsiveness. Indeed, the GTP-RhoA pull-down assay revealed an increase in active form of RhoA in the PGD₂-treated mouse BSMs. On the other hand, in the high K⁺-depolarized cultured human BSM cells, PGD₂ caused no further increase in cytosolic Ca²⁺ concentration. These findings suggest that PGD₂ causes RhoA/Rho-kinase-mediated Ca²⁺ sensitization of BSM contraction to augment its contractility. Increased PGD₂ level in the airways might be a cause of the AHR in asthma.

1. Introduction

Augmented airway responsiveness to a wide variety of nonspecific stimuli, called airway hyperresponsiveness (AHR), is a common feature of allergic asthma. A cause of the AHR is hypercontraction of smooth muscle cells of the airways [1,2,3,4,5]. Rapid remission from airway limitation in asthma attack by inhalation of short-acting beta-stimulant also suggests an involvement of increased airway smooth muscle contraction in the airway obstruction. It is thus important for development of asthma therapy to understand the disease-associated alterations of the contractile signaling of airway smooth muscle cells.

Prostaglandin D₂ (PGD₂), one of the cyclooxygenase (COX) metabolites, is the major lipid mediator released from mast cells in allergic reaction, and has been suggested to be involved in the pathogenesis of bronchial asthma. An increase in PGD₂ level in bronchoalveolar lavage (BAL) fluids was demonstrated in experimental asthma models [6,7]. In asthmatic subjects, allergen challenge to the airways caused an increase in PGD₂ in BAL fluids [8,9]. It has been suggested that PGD₂ mediates allergic inflammation, including the airway inflammation in asthma. In mice lacking receptors for PGD₂ (DP₁ receptors), both airway eosinophilia and upregulation of proinflammatory cytokines in BAL fluids induced by allergen challenge were diminished as compared to wild-type animals [10]. PGD₂ also caused cytokine release via an activation of a PGD₂ receptor, CRTH2 (also called as DP₂ receptor), in Th2 lymphocytes [11].

On the other hand, the functional role of PGD₂ on airway smooth muscle remains unclear. Application of PGD₂ to the isolated smooth muscle strips caused contraction in guinea pig trachea [12] and dog bronchus [13]. In contrast, PGD₂ elicited a relaxation in the murine tracheal smooth muscle precontracted with carbachol [10]. In the present study, to elucidate its role in the development of AHR in asthma, the effect of PGD₂ on smooth muscle contractility was determined using bronchial rings isolated from mice.

2. Results

2.1. Effects of Prostaglandin D₂ (PGD₂) on Bronchial Smooth Muscle (BSM) Function

The RT-PCR analyses revealed that both DP₁ and DP₂ receptors were expressed both in hBSMCs and murine BSMs (Figure 1), indicating that PGD₂ could directly act on BSM cells. To determine the role of PGD₂ on the BSM function, its effect on the isometric tension of smooth muscles was examined in BSM tissues isolated from naive control mice. Application of PGD₂ (10⁻⁹–10⁻⁵ M) had no effect on basal tone of the BSM tissues (Figure 2A). However, when the BSMs were precontracted with 30 mM K⁺, application of PGD₂ caused an enhancement of the contraction induced by high K⁺ depolarization, in a PGD₂ concentration-dependent manner (10⁻⁶ and 10⁻⁵ M: Figure 2B,C).

PGD₂ has been known to act on G protein-coupled receptors (GPCRs), mainly the PGD₂ receptor 1 (DP₁) and 2 (DP₂). To elucidate receptor(s) responsible for the enhanced contraction induced by PGD₂, effects of laropiprant (a selective DP₁ receptor antagonist [14]) and fevipiprant (a selective DP₂ receptor antagonist [15]) on the PGD₂-induced augmentation of contraction were tested. As a result, the enhanced contraction induced by PGD₂ was inhibited by laropiprant (10⁻⁶ M: Figure 3A,B), whereas fevipiprant (10⁻⁶ M) had no effect on it (Figure 3C).

2.2. Effects of Prostaglandin D₂ (PGD₂) on Cytosolic Ca²⁺ Level in Human Bronchial Smooth Muscle Cells (hBSMCs)

Due to the difficulty in preparing isolated BSM cells with high purity from the mouse tissues, change in cytosolic Ca²⁺ level was measured using commercially available human BSM cells (hBSMCs) in the present study. The hBSMCs were loaded with a green fluorescent Ca²⁺ indicator, Fluo-8 [16]. As shown in Figure 4A,B, in the hBSMCs incubated with Fluo-8/AM, stimulation of the cells with a Ca²⁺ ionophore A23187 (10⁻⁵ M) caused a marked increase in F/F₀, that is, an increase in cytosolic Ca²⁺ concentration, indicating a successful loading of Fluo-8 into the cells. In the Fluo-8-loaded hBSMCs, stimulation of the cells with 30 mM K⁺ caused a slight but distinct increase in cytosolic Ca²⁺ concentration (Figure 4B,C). Interestingly, PGD₂ had no effect on the K⁺ depolarization-induced increase in cytosolic Ca²⁺ level (Figure 4B,C). PGD₂ also did not alter the basal cytosolic Ca²⁺ level in the Fluo-8-loaded cells (Figure 4A).

2.3. Activation of RhoA/Rho-Kinase Signaling by Prostaglandin D₂ (PGD₂)

The results that PGD₂ caused an augmentation of contraction (Figure 2B,C) under the constant cytosolic Ca²⁺ level (Figure 4) remind us of the Ca²⁺ sensitization of smooth muscle contraction. In smooth muscle cells including airways, activation of a monomeric G-protein, RhoA, causes Ca²⁺ sensitization of the contraction by activating its downstream Rho-kinases [17,18]. To determine whether PGD₂ activates RhoA protein, the GTP-RhoA pull-down assay was performed in mouse BSMs stimulated by PGD₂. As previously reported [19], acetylcholine (ACh: 10⁻³ M) stimulation caused an increase in GTP-bound, active form of RhoA protein in the BSMs of mice (Figure 5A). Similarly, as shown in Figure 5A, an increase in the active form of RhoA protein was observed when the BSM tissues were stimulated with 10⁻⁵ M PGD₂, the concentration where no contractile response from baseline tone was observed (see above). The tension study also revealed an activation of RhoA/Rho-kinase signaling by PGD₂: the PGD₂-induced augmentation of contraction was blocked by Y-27632 (10⁻⁷ M), a selective inhibitor of Rho-kinases (Figure 5B).

3. Discussion

The current study was carried out to determine the role of prostaglandin D₂ (PGD₂) on smooth muscle function of the airways using the bronchial smooth muscles (BSMs) isolated from mice. Although PGD₂ had no effect on their baseline tension, PGD₂ significantly augmented the BSM contraction induced by high K⁺ depolarization (Figure 2B,C). The PGD₂-induced augmentation of contraction was inhibited both by a DP₁ antagonist, laropiprant, and a Rho-kinase inhibitor, Y-27632 (Figure 3 and Figure 5B). Furthermore, PGD₂ could cause an activation of RhoA protein (Figure 5A). In the high K⁺-depolarized cultured human BSM cells, PGD₂ caused no further increase in cytosolic Ca²⁺ concentration (Figure 4). These findings suggest that PGD₂ acts on DP₁ receptors to cause RhoA/Rho-kinase-mediated Ca²⁺ sensitization of contraction in BSMs.

PGD₂ is an acidic lipid mediator derived from the metabolism of arachidonic acid by the action of cyclooxygenases and downstream PGD₂ synthases, and is mainly released from mast cells when activated by antigen stimulation [20]. Allergen challenge to the airways caused an increase in PGD₂ level in the airways of asthmatics [8,9]. However, the functional role of PGD₂ on airway smooth muscle has not yet been unified. In tracheal smooth muscle strips isolated from the guinea pigs, PGD₂ produced a concentration-dependent contraction [12]. Similarly, PGD₂ caused a contraction in bronchial rings isolated from the dogs [13]. In contrast, PGD₂, at a concentration of 3 µM, elicited a relaxation in the murine tracheal smooth muscle precontracted with carbachol [10]. Currently, PGD₂ had no effect on basal tension in BSMs isolated from the mice (see Results section). Differences in the species, region (tracheal versus bronchial smooth muscles), and/or the experimental condition used may be involved in the difference in the PGD₂ response in smooth muscles of the airways. Thus, note that the current study also contains a certain limitation: cultured human BSM cells (hBSMCs) were used for cytosolic Ca²⁺ measurement whereas functional studies were performed using mouse BSM tissues.

The current RT-PCR analyses showed expression of DP₁ and DP₂ receptors in BSM cells (Figure 1), indicating that PGD₂ could directly act on BSM cells. Although PGD₂ did not affect the basal tension, it augmented the submaximal contraction induced by 30 mM K⁺ in BSMs isolated from the mice (Figure 2). The augmented contraction induced by PGD₂ was inhibited by laropiprant (Figure 3), a DP₁ antagonist [14], but not by fevipiprant (see RESULTS), a DP₂ antagonist [15]. An involvement of TP receptor in the PGD₂-mediated contraction has also been suggested [21]. However, PGD₂ did not increase cytosolic Ca²⁺ in the present study (Figure 4), whereas an induction of contraction with Ca²⁺ mobilization by the TP receptor activation has been demonstrated [22]. In addition, our preliminary study revealed that stimulation of TP receptors with a thromboxane A₂ (TXA₂) mimic, U46619, caused a distinct contraction from baseline tension (without K⁺ depolarization) in the mouse BSMs. Pretreatment of BSMs with ozagrel, an inhibitor of TXA₂ synthase, also did not inhibit the augmented contraction induced by PGD₂ (data not shown). It is thus unlikely that the TXA₂/TP receptor is involved in the PGD₂-mediated response in the mouse BSMs. Thus, an activation of DP₁ receptors on the BSM cells might be responsible for the synergistic contraction induced by PGD₂.

Currently, PGD₂ augmented the contraction induced by high K⁺ depolarization in mouse BSM tissues (Figure 2B,C). In the high K⁺-depolarized cultured hBSMCs, PGD₂ caused no further increase in cytosolic Ca²⁺ concentration (Figure 4). Collectively, these findings suggest that PGD₂ augmented the BSM contraction induced by K⁺ depolarization without any increase in cytosolic Ca²⁺ concentration. The observation that PGD₂ caused an augmentation of contraction under the constant cytosolic Ca²⁺ level reminds us of the Ca²⁺ sensitization of smooth muscle contraction. Indeed, the augmented contraction induced by PGD₂ was inhibited by a Rho-kinase inhibitor, Y-27632 (Figure 5). In addition, stimulation of the BSMs with PGD₂ caused an increase in the active form of RhoA, GTP-bound RhoA (Figure 5). The current study for the first time, to our knowledge, demonstrated that PGD₂ activates the RhoA/Rho-kinase signaling to induce Ca²⁺ sensitization of contraction in the BSMs. Previous studies, including ours, demonstrated that muscarinic receptor stimulation of airway smooth muscle caused both an increase in cytosolic Ca²⁺ concentration and an activation of RhoA/Rho-kinase signaling, resulting in the contraction [17,23,24]. On the other hand, the current study revealed that PGD₂ did not have the ability to increase cytosolic Ca²⁺ level in the BSMs (Figure 4). This may be a reason that PGD₂ did not cause any contraction from the baseline tension: the cytosolic Ca²⁺ level at the baseline tension might not have been enough to induce BSM contraction even if the RhoA/Rho-kinase signaling was activated.

It is a remarkable event that the PGD₂-induced augmentation of contraction was inhibited by laropiprant, an antagonist of DP₁ receptor that is known as a Gs protein-coupled receptor. In smooth muscle cells including the airways, the Gs protein activation, such as beta-adrenoceptor stimulation by isoprenaline, causes an increase in cAMP level to induce relaxation [25,26,27]. However, the current study indicated that activation of DP₁ receptor by PGD₂ could cause a response to contractile direction. Although the discrepancy is not explainable now, an activation of extracellular signal-regulated kinase (ERK) signaling by DP₁ receptor stimulation has also been reported in nasal epithelial cells [28]. It is thus possible that, in addition to the classical Gs/cAMP pathway, the DP₁ receptor stimulation activates multiple intracellular signaling, including the RhoA/Rho-kinase signaling. Further studies are needed to make clear the mechanism of action of PGD₂ in the BSMs.

In conclusion, the current study revealed that PGD₂ augmented the BSM contraction by activating the RhoA/Rho-kinase-mediated Ca²⁺ sensitization of contraction via an activation of DP₁ receptors on the BSM cells. Increased PGD₂ level in the airways might be one of the causes of the enhanced airway responsiveness to nonspecific stimuli, one of the characteristic features of bronchial asthma.

4. Materials and Methods

4.1. Animals

Male BALB/c mice were purchased from the Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan) and housed in a pathogen-free facility. All animal experiments were approved by the Animal Care Committee of the Hoshi University, Tokyo, Japan (permission code: 30-086, permission date: 21 June 2018).

4.2. Pharmacological Reagents

Prostaglandin D₂ (PGD₂: Cat. No. 12010) and laropiprant (MK-0524: Cat. No. 10009835) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Fevipiprant was purchased from MedChem Express (Monmouth Junction, NJ, USA: Cat. No. HY-16768).

4.3. Determination of Bronchial Smooth Muscle (BSM) Responsiveness

Mice were sacrificed by exsanguination from abdominal aorta under urethane (1.6 g/kg, i.p.) anesthesia and the airway tissues under the larynx to lungs were immediately removed. About 3 mm length of the left main bronchus (about 0.5 mm diameter) was isolated. The resultant tissue ring preparation was then suspended in a 5 mL organ bath by two stainless-steel wires (0.2 mm diameter) passed through the lumen. For all tissues, one end was fixed to the bottom of the organ bath while the other was connected to a force-displacement transducer (TB-612T, Nihon Kohden, Tokyo, Japan) for the measurement of isometric force. A resting tension of 0.5 g was applied. The buffer solution contained modified Krebs–Henseleit solution with the following composition (mM): NaCl 118.0, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, NaHCO₃ 25.0, KH₂PO₄ 1.2, and glucose 10.0. The buffer solution was maintained at 37 °C and oxygenated with 95% O₂/5% CO₂. After the equilibration period, the tension studies were performed. In case of the high K⁺ depolarization studies, experiments were conducted in the presence of atropine (10⁻⁶ M).

4.4. Determination of Active Form of RhoA in BSM

The active form of RhoA, GTP-bound RhoA, in BSMs was measured by GTP-RhoA pull-down assay as described previously [19]. In brief, the isolated main bronchial tissues were equilibrated in oxygenated Krebs–Henseleit solution at 37 °C for 1 h. After the equilibration period, the tissues were stimulated with PGD₂ (10⁻⁵ M) or ACh (10⁻³ M) for 15 min, and were quickly frozen with liquid nitrogen. The tissues were then lysed in lysis buffer with the following composition (mM): HEPES 25.0 (pH 7.5), NaCl 150, IGEPAL CA-630 1%, MgCl₂ 10.0, EDTA 1.0, glycerol 10%, 1× protease inhibitor cocktail (Nakalai tesque, Kyoto, Japan), and 1× phosphatease inhibitor cocktail (Nakalai tesque). Active RhoA in tissue lysates (200 µg protein) was precipitated with 25 µg GST-tagged Rho binding domain (amino acids residues 7–89 of mouse rhotekin; Upstate, Lake Placid, NY, USA), which was expressed in Escherichia coli and bound to glutathione-agarose beads. The precipitates were washed three times in lysis buffer, and after adding the SDS loading buffer and boiling for 5 min, the bound proteins were resolved in 15% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with rabbit polyclonal anti-RhoA (Abcam, Cambridge, UK) as primary antibodies.

4.5. Cell Culture and [Ca²⁺]_cyto Measurement

Normal human BSM cells (hBSMCs; a male donor: purchased from Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) were maintained in SmBM medium (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) supplemented with 5% fetal bovine serum, 0.5 ng/mL human epidermal growth factor (hEGF), 5 µg/mL insulin, 2 ng/mL human fibroblast growth factor-basic (hFGF-b), 50 µg/mL gentamicin, and 50 ng/mL amphotericin B. Cells were maintained at 37 °C in a humidified atmosphere (5% CO₂), fed every 48–72 h, and passaged when cells reached 90–95% confluence. Then the hBSMCs (passages 5–7) were seeded in 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and were cultured without serum. Twenty-four hours after the starvation period, the cells were loaded with Fluo-8/AM (2.5 M: AAT Bioquest, Inc., Sunnyvale, CA, USA) in serum-free SmBM medium for 90 min at 37 °C. The cells were washed with PBS and maintained in Krebs–Henseleit solution described above. The intracellular Fluo-8 fluorescence was monitored using fluorescence microscope (Keyence, Osaka, Japan) with BZ-X filter GFP (470/40, 535/50 nm). Images were pictured using time-lapse imaging (Keyence), and analyzed with BZ-X analyzer (Keyence). Change in the cytosolic Ca²⁺ level was calculated as ratio to the basal fluorescence intensity.

4.6. RT-PCR Analyses

Total RNAs of hBSMCs and mouse BSM tissues were extracted using NucleoSpin™ miRNA (TaKaRa Bio, Inc., Shiga, Japan) according to the manufacturer’s instruction. cDNAs were prepared from the total RNA by using PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer’s instructions. cDNA samples were subjected to PCR with Quick Taq™ HS DyeMix (TOYOBO Co., Ltd., Osaka, Japan) in a final volume of 10 µL. The PCR primer sets used are shown in

Table 1. Primer sequences for RT-PCR used in the present study (human).

Gene Name	RefSeq Accession		Sequence	Amplicon Size
human PTGDR	NM_000953	Sense	5′-TCTGCGCGCTACCTTTCATG-3′	85 bp
		Antisense	5′-TCCTCGTGGACCATCTGGATA-3′
human PTGDR2	NM_004778	Sense	5′-CCTCTGTGCCCAGAGCCCCACGATGTCGGC-3′	114 bp
		Antisense	5′-ATGTAGCGGATGCTGGTGTTG-3′
human PTGER1	NM_000955	Sense	5′-GATGGTGGGCCAGCTTGTC-3′	72 bp
		Antisense	5′-GCCACCAACACCAGCATTG-3′
human PTGER2	NM_000956	Sense	5′-GTGCTGACAAGGCACTTCATGT-3′	87 bp
		Antisense	5′-TGTTCCTCCAAAGGCCAAGTAC-3′
human PTGER3	NM_198714	Sense	5′-AAGGCCACGGCATCTCAGT-3′	76 bp
		Antisense	5′-TGATCCCCATAAGCTGAATGG-3′
human PTGER4	NM_000958	Sense	5′-CTTGGAGGCAGGAATTTGCTT-3′	77 bp
		Antisense	5′-AAAGTCCTCAGTGAGGTGGTGTCT-3′
human PTGFR	NM_000959	Sense	5′-GCACATTGATGGGCAACTAGAA-3′	91 bp
		Antisense	5′-GCACCTATCATTGGCATGTAGCT-3′
human PTGIR	NM_000960	Sense	5′-GCCGATCAGCTGCTGTTTCT-3′	75 bp
		Antisense	5′-TTTCCTCTGTCCCTCACTCTCTTC-3′
human TBXA2R	NM_001060	Sense	5′-ACGGAGAAGGAGCTGCTCATC-3′	84 bp
		Antisense	5′-GCGGCGGAACAGGATATACA-3′
human GAPDH	NM_002046	Sense	5′-GGAGCCAAAAGGGTCATCATCTC-3′	282 bp
		Antisense	5′-AGGGATGATGTTCTGGAGAGCC-3′

(for human) and

Table 2. Primer sequences for RT-PCR used in the present study (mouse).

Gene Name	RefSeq Accession		Sequence	Amplicon Size
mouse Ptgdr1	NM_008962	Sense	5′-CAACCTGGGTGCCATGTAC-3′	112 bp
		Antisense	5′-GGACCCGTGCCTGTAGTCT-3′
mouse Ptgdr2	NM_009962	Sense	5′-CTGCACCTGGCGCTATC-3′	174 bp
		Antisense	5′-GTCCAGGCTAATGGCACT-3′
mouse Ptger1	NM_013641	Sense	5′-TACATGGGATGCTCGAAACA-3′	223 bp
		Antisense	5′-TTTTAGGCCGTGTGGGTAG-3′
mouse Ptger2	NM_008964	Sense	5′-ATGCACCTGCTGCTTATCGT-3′	196 bp
		Antisense	5′-TAATGGCCAGGAGAATGAGG-3′
mouse Ptger3	NM_001359745	Sense	5′-TGCTGGCTCTGGTGGTGAC-3′	258 bp
		Antisense	5′-ACTCCTTCTCCTTTCCCATCTGTG-3′
mouse Ptger4	NM_001136079	Sense	5′-CCATCGCCACATACATGAAG-3′	209 bp
		Antisense	5′-TGCACAGATGGCGAAGAGTG-3′
mouse Ptgfr	NM_008966	Sense	5′-CTGCTCCGGACACAACCACTC-3′	191 bp
		Antisense	5′-GGTTCTCCGTCTGGCAGGTTG-3′
mouse Ptgir	NM_008967	Sense	5′-GGATGAAGTTTACCACCTGATTCTGC-3′	196 bp
		Antisense	5′-AGCCTTTCGGAAAAGGATGAAGAC-3′
mouse Tbxa2r	NM_009325	Sense	5′-TTTCGCCCGGTGAACATC-3′	255 bp
		Antisense	5′-GGCTCGCCAGTCCAACAA-3′
mouse Gapdh	NM_001289726	Sense	5′-CCTCGTCCCGTAGACAAAATG-3′	100 bp
		Antisense	5′-TCTCCACTTTGCCACTGCAA-3′

(for mouse), which was designed from published database, BLAST. The thermal cycle profile used was (1) denaturing for 30 s at 94 °C, (2) annealing primers for 30 s at 60 °C, (3) extending the primers for 1 min at 68 °C, and the reaction was run for 40 cycles. The PCR products were subjected to electrophoresis on 2% agarose gel and visualized by ethidium bromide staining.

4.7. Statistical Analyses

All the data are expressed as means ± SE. Statistical significance of difference was determined by paired t-test (Figure 2B,C) or one-way analysis of variance (ANOVA) with post hoc Bonferroni’s multiple comparison (Figure 3B and Figure 4C) using Prism 5 for Mac OS X (GraphPad Software, La Jolla, CA, USA). A value of p < 0.05 was considered significant.