Effects of NP-1815-PX, a P2X4 Receptor Antagonist, on Contractions in Guinea Pig Tracheal and Bronchial Smooth Muscles

Abstract

Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10⁻⁵ M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 × 10⁻⁶ M) and ONO-8130 (a prostanoid EP₁ receptor antagonist, 10⁻⁷ M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10⁻⁷ M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10⁻⁵ M) or L-798,106 (a prostanoid EP₃ receptor antagonist, 10⁻⁸ M). NP-1815-PX (10⁻⁵–10⁻⁴ M) strongly suppressed U46619 (a TP receptor agonist)- and prostaglandin F_2α (PGF_2α)-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10⁻⁵–10⁻⁴ M) strongly suppressed the U46619-induced increase in intracellular Ca²⁺ concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10⁻⁴ M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP₁ receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.

INTRODUCTION

NP-1815-PX (Fig. 1A) is a selective P2X4 receptor antagonist.¹⁾ The P2X4 receptor is an ionotropic ATP receptor that is widely expressed in central and peripheral nerves. The activation of P2X4 receptors is thought to be involved in neuropathic pain.^1,2) In fact, intrathecal administration of NP-1815-PX produces anti-allodynic effects in mice with traumatic nerve damage and suppresses the induction of mechanical allodynia in mice with herpetic pain without affecting acute nociceptive pain and motor function.¹⁾ In addition to neuropathic pain, the activation of P2X4 receptors may be involved in multiple pathologies such as airway inflammation in asthma, post-ischemic inflammation, rheumatoid arthritis, neurodegenerative diseases, and metabolic syndrome.³⁾

Fig. 1. Chemical Structure of NP-1815-PX (A) and Relaxant Effect of NP-1815-PX on ATP-Induced Contractions in Guinea Pig Epithelium-Intact Tracheal Smooth Muscle (B–E)

B, C: Representative traces showing the effects of NP-1815-PX (10⁻⁵ M, C) and time-matched control (B) on the contractions induced by ATP (3 × 10⁻⁵ M). D: Summarized data of the relaxant effects of NP-1815-PX (10⁻⁵ M) and time-matched control. E: Summarized data of ATP-induced contractions before the administration of NP-1815-PX. Data are expressed as the mean ± standard error of the mean (n = 5 each). ** p < 0.01 vs. control (Student’s t-test). PPV: papaverine (10⁻⁴ M).

The activation of P2X4 receptors also enhances the immune system in airway mucus, leading to tracheal inflammation and bronchial hypersensitivity.⁴⁾ For example, 1) the expression levels of P2X4 receptor mRNA in bronchoalveolar lavage fluid (BALF), blood monocytes, and blood neutrophils in human asthma patients were reported to be higher than those in normal patients, and 2) the expression level of P2X4 receptor mRNA in lung tissue was reported to increase in ovalbumin (OVA)-sensitized asthma model mice.⁴⁾ In addition, the ATP concentration in BALF was reported to increase 1) when mild allergic asthma patients were sensitized to allergens, and 2) in asthma model mice sensitized with OVA.⁵⁾ Furthermore, the administration of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) (a selective P2X4 receptor antagonist) was reported to suppress the activation of immune cells and improve asthma symptoms in house dust mite extract-induced bronchial asthma model mice.⁴⁾ Therefore, P2X4 receptor antagonists may improve bronchial asthma through excessive immunosuppressive effects.

ATP is also reported to contract guinea pig trachea and this response is inhibited by cyclooxygenase (COX) inhibitors.⁶⁾ In addition, ATP is reported to increase prostaglandin E₂ (PGE₂) levels in rabbit trachea and removal of the epithelium attenuates this ATP-induced PGE₂ release.⁷⁾ Thus, ATP is expected to contract tracheal smooth muscles (TSMs) via epithelial COX-derived prostanoids such as PGE₂. However, the involvement of P2X4 receptors in the contractile function of TSM has rarely been reported.

In this study, in order to clarify the involvement of P2X4 receptors in the contractile response of TSM, we investigated the effects of NP-1815-PX on ATP-induced TSM contractions and the mechanism of these ATP-induced TSM contractions. In addition, we examined pharmacological effects found in TSM and bronchial smooth muscle (BSM) other than the P2X4 receptor antagonistic action of NP-1815-PX.

MATERIALS AND METHODS

Animals

Male Hartley guinea pigs (age: 5–8 weeks; weight: 320–460 g; Kyudo Co., Ltd., Saga, Japan) were housed under controlled conditions (21–22 °C, relative air humidity: 50 ± 5%) and a fixed 12/12 h light/dark cycle (08 : 00–20:00), with food and water available ad libitum. This study was approved by the Toho University Animal Care and User Committee (Approval Nos. 20-51-444, 21-52-444) and was conducted in accordance with the guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University.

Preparation of TSM and BSM

Guinea pigs were anesthetized with isoflurane inhalation and euthanized by exsanguination from the carotid arteries. Thereafter, the tracheal and bronchial tissues (right and left main bronchi) were quickly removed and immersed in Locke–Ringer solution of the following composition (mM): NaCl, 154; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; glucose, 2.8. The tracheal and bronchial tissues were cleaned of unnecessary adipose and connective tissues. Subsequently, the tracheal/bronchial cartilage containing smooth muscle was cut into pieces approximately 2–3/1–2 mm long. In some experiments, the intimal surface of the tracheal and bronchial tissues was gently rubbed with moistened paper to remove as much tracheal/bronchial epithelium as possible.⁸⁾

Recordings of Isometric Tension Changes

Isometric tension changes of TSM/BSM preparations were recorded using the methods described in a previous report.⁹⁾ The TSM and BSM preparations were suspended with stainless steel hooks (outer diameter 200 µm) in a 5-mL organ bath (UC-5; UFER Medical Instrument, Kyoto, Japan) filled with Locke–Ringer solution aerated with mixed gas (95% O₂+ 5% CO₂) and maintained at 32 ± 1 °C (pH = 7.4). Tension changes were isometrically recorded using force displacement transducers (T7-8-240; Orientec Co., Ltd., Tokyo, Japan), a signal conditioner (MSC-2; Primtech Corp., Tokyo, Japan), PowerLab/4sp (ADInstruments Pty. Ltd., Bella Vista, NSW, Australia), and LabChart 7 for Windows (ADInstruments Pty. Ltd.). The preparations were incubated for 60 min while changing the bath solution every 20 min, so that the final passive tension was 2 g. After these procedures, the tissues were incubated for a further 10 min and then contracted twice with histamine (10⁻⁵ M) for 20 min with an interval of ≥20 min. All experiments using epithelium-denuded TSM and BSM preparations were performed in the presence of indomethacin (a COX inhibitor, 3 × 10⁻⁶ M) to eliminate endogenous prostanoids.¹⁰⁾

Assessment of the Effects of Indomethacin on ATP-Induced Contractions in Epithelium-Intact TSM Preparations

After the procedures shown in “Recordings of isometric tension changes,” indomethacin (3 × 10⁻⁶ M) or ethanol (EtOH, the indomethacin vehicle, 0.03%) was added in the bath medium. After ≥5 min of incubation, the epithelium-intact TSM preparations were contracted with ATP (3 × 10⁻⁵ M) for 20 min. This concentration of ATP was able to elicit contractions that approached maximum values. Then, the preparations were completely relaxed using papaverine (PPV, 10⁻⁴ M). In this study, the TSM preparations in which the ATP (3 × 10⁻⁵ M)-induced contractions were ≥10% of the second histamine (10⁻⁵ M)-induced contractions shown in “Recordings of isometric tension changes” were defined as the epithelium-intact preparations.

Assessment of Effects of NP-1815-PX and Various Prostanoid Receptor Antagonists on ATP-Induced Contractions in Epithelium-Intact TSM Preparations

After the procedures shown in “Recordings of isometric tension changes,” the epithelium-intact TSM preparations were contracted using ATP (3 × 10⁻⁵ M) for ≥10 min. After the contractions stabilized, NP-1815-PX (10⁻⁵ M), SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10⁻⁷ M), AL 8810 (a prostanoid FP receptor antagonist, 10⁻⁵ M), ONO-8130 (a prostanoid EP₁ receptor antagonist, 10⁻⁷ M), L-798,106 (a prostanoid EP₃ receptor antagonist, 10⁻⁸ M), EtOH (vehicle for SQ 29,548, AL 8810, and ONO-8130, 0.05%), or dimethyl sulfoxide (DMSO, vehicle for L-798,106, 5 × 10⁻⁴%) was added to the bath medium and incubated for 30 min. Then, the preparations were completely relaxed using PPV (10⁻⁴ M). We selected concentrations of NP-1815-PX/prostanoid receptor antagonists that could sufficiently inhibit the P2X4 receptor/target prostanoid receptors.^1,10–13)

Assessment of the Effects of NP-1815-PX on Contractions Induced by Various Contractile Agents in Epithelium-Denuded TSM and BSM Preparations

After the procedures shown in “Recordings of isometric tension changes,” the epithelium-denuded TSM and BSM preparations were contracted for ≥20 min with the following contractile agents: U46619 (a prostanoid TP receptor agonist, 10⁻⁸ M, TSM; 5 × 10⁻⁸ M, BSM), prostaglandin F_2α (PGF_2α, 5 × 10⁻⁷ M, TSM), carbachol (CCh, 7 × 10⁻⁸ M, TSM; 3 × 10⁻⁸ M, BSM), histamine (3 × 10⁻⁶ M, TSM and BSM), neurokinin A (NKA, 10⁻⁸ M, TSM; 10⁻⁷ M, BSM), and 50 mM high-K in a solution of the following composition (mM): NaCl, 109.6; KCl, 50; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; glucose, 2.8. The concentrations of contractile agents were selected to elicit TSM/BSM contractions that were ≥50% of the second histamine (10⁻⁵ M)-induced contractions described in the section “Recordings of isometric tension changes.” After the contractions stabilized, NP-1815-PX (10⁻⁵–10⁻⁴ M) was added to the bath medium and incubated for 60 min. Then, the preparations were completely relaxed using PPV (10⁻⁴ M).

Assessment of the Effects of SQ 29,548 on U46619/PGF_2α-Induced Contractions in Epithelium-Denuded TSM and BSM Preparations

After the procedures shown in “Recordings of isometric tension changes,” the epithelium-denuded TSM and BSM preparations were contracted for ≥20 min with U46619 (10⁻⁸ M, TSM; 5 × 10⁻⁸ M, BSM) or PGF_2α (5 × 10⁻⁷ M, TSM). After the contractions stabilized, SQ 29,548 (3 × 10⁻⁷–10⁻⁶ M) was added cumulatively to the bath medium. Then, the preparations were completely relaxed using PPV (10⁻⁴ M).

Measurement of Increases in Intracellular Ca²⁺ Concentrations Induced by U46619 in 293T Cell Lines Stably Expressing Human TP Receptor (TP-293T Cells)

Measuring increases in intracellular Ca²⁺ concentrations in TP-293T cells were performed as previously described.¹⁴⁾ Briefly, the cells¹⁴⁾ were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum in a humidified incubator at 37 °C and 5% CO₂. The day before the measurements, the cells were seeded at approximately 90% confluence in a 96-well plate. The next day, the DMEM was replaced with recording medium [(mM): N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 20; NaCl, 115; KCl, 5.4; MgCl₂, 0.8; CaCl₂, 1.8; glucose, 13.8; pH 7.4] containing Fura-2 AM (5 × 10⁻⁶ M), and the Fura-2 AM was loaded into the cells by incubation for 30–60 min at 37 °C and 5% CO₂. The cells were then rinsed with Fura-2 AM-free medium, and their fluorescence intensities were measured using a microplate reader (Infinite F200 Pro, Tecan Group Ltd., Mӓnnedorf, Switzerland) at 510 nm emission generated by excitation at 340  and 380 nm at 25 °C. After the cells were treated for 10 min in the absence and presence of NP-1815-PX (10⁻⁵–10⁻⁴ M), U46619 (10⁻⁸ M) was applied using the injector module, and the fluorescence intensity was measured for 5 min. Changes in the ratio of fluorescence intensities at 510 nm emission generated by excitation at 340  and 380 nm (F340/380) were considered the relative changes in intracellular Ca²⁺ concentration. After the experiment, ionomycin (5 µM) and Mn²⁺ (50 mM) were added to the plates. The fluorescence intensity obtained in the presence of ionomycin and Mn²⁺ (background fluorescence) was subtracted from the fluorescence intensities of all measurements.

Drugs

NP-1815-PX, NC 2600, and gefapixant were kindly provided by Nippon Chemiphar Co., Ltd. (Tokyo, Japan). ATP disodium salt hydrate, indomethacin, histamine dihydrochloride, and carbamylcholine (CCh) chloride were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). SQ 29,548, AL 8810, ONO-8130, L-798,106, and U46619 were purchased from Cayman Chemical Company (Ann Arbor, MI, U.S.A.). Dinoprost (PGF_2α) was purchased from Fuji Pharma Co., Ltd. (Tokyo, Japan). NKA was purchased from Peptide Institute, Inc. (Osaka, Japan).

ATP was dissolved in 25 mM Tris–HCl (pH 8.0) to prepare 2 × 10⁻² M stock solutions, and diluted with 25 mM Tris–HCl (pH 7.4) to prepare 2 × 10⁻³ M working solutions. Indomethacin was dissolved in EtOH to prepare 10⁻² M stock solutions. SQ 29,548 and ONO-8130 were dissolved in EtOH to prepare 2 × 10⁻² M stock solutions. AL 8810 was dissolved in EtOH to prepare 2 × 10⁻³ M stock solutions. L-798,106 was dissolved in DMSO to prepare 2 × 10⁻³ M stock solutions. NC 2600 was dissolved in DMSO to prepare 5 × 10⁻³ M stock solutions. Gefapixant was dissolved in DMSO to prepare 5 × 10⁻² M stock solutions. All other drugs were prepared as aqueous stock solutions and diluted with distilled water to prepare working solutions.

Data Analysis and Statistics

The extent of relaxation induced by all antagonists was calculated relative to the tension level before the application of contractile agents (100% relaxation) and the steady-state tension level prior to the application of each antagonist (0% relaxation). The extent of contraction induced by all contractile agents is shown as relative contraction (%) when the second histamine contraction was set at 100%.

All values are presented as the mean ± standard error of the mean (S.E.M.) for different numbers (n) of preparations. GraphPad Prism™ (GraphPad Software Inc., San Diego, CA, U.S.A.) was used for statistical analyses. Differences were evaluated using Student’s t-test or one-way ANOVA followed by Dunnett’s test. Statistical significance was set at p < 0.05.

RESULTS

Effects of NP-1815-PX on ATP-Induced Contractions in Epithelium-Intact TSM Preparations

Figures 1B–E show representative traces and summarized data of the relaxant effects of NP-1815-PX (10⁻⁵ M) on contractions induced by ATP (3 × 10⁻⁵ M) in epithelium-intact TSM preparations. NP-1815-PX (10⁻⁵ M) almost completely suppressed the ATP-induced contractions. Another P2X4 receptor antagonist (NC 2600, 10⁻⁵ M) also almost completely suppressed the ATP-induced contractions (Supplementary Figs. 1A, B, E, F).

Although a P2X3 receptor antagonist (gefapixant, 10⁻⁵ M) significantly suppressed the ATP-induced contractions (vs. DMSO treatment), the extent of inhibition by gefapixant was very weak compared to that by the P2X4 receptor antagonists (Supplementary Figs. 1C, D, G, H).

Effects of Indomethacin on ATP-Induced Contractions in Epithelium-Intact TSM Preparations

Figure 2 shows representative traces and summarized data of the pretreatment effects of indomethacin (3 × 10⁻⁶ M) on contractions induced by ATP (3 × 10⁻⁵ M) in epithelium-intact TSM preparations. The ATP-induced contractions were completely abolished by pretreatment with indomethacin (3 × 10⁻⁶ M). In addition, indomethacin (3 × 10⁻⁶ M) decreased the basal tension of epithelium-intact TSM preparations.

Fig. 2. Pretreatment Effect of Indomethacin on ATP-Induced Contractions in Guinea Pig Epithelium-Intact Tracheal Smooth Muscle

A, B: Representative traces showing the pretreatment effects of indomethacin (3 × 10⁻⁶ M, B) and its vehicle (ethanol (EtOH, 0.03%, A)) on the contractions induced by ATP (3 × 10⁻⁵ M). C: Summarized data of the pretreatment effects of indomethacin (3 × 10⁻⁶ M) and its vehicle on the ATP-induced contractions. Data are expressed as the mean ± standard error of the mean (n = 5 each). ** p < 0.01 vs. EtOH (Student’s t-test). PPV: papaverine (10⁻⁴ M).

Effects of Prostanoid Receptor Antagonists on ATP-Induced Contractions in Epithelium-Intact TSM Preparations

Figure 3 shows representative traces and summarized data of the effects of various prostanoid receptor antagonists (SQ 29,548, 3 × 10⁻⁷ M; AL 8810, 10⁻⁵ M; ONO-8130, 10⁻⁷ M; L-798,106, 10⁻⁸ M) on contractions induced by ATP (3 × 10⁻⁵ M) in epithelium-intact TSM preparations. The ATP-induced contractions were strongly suppressed by ONO-8130 (10⁻⁷ M). In addition, ONO-8130 (10⁻⁷ M) decreased the basal tension of epithelium-intact TSM preparations. The ATP-induced contractions were suppressed by SQ 29,548 (3 × 10⁻⁷ M) by approx. 35%, although this reduction was not statistically significant (vs. EtOH treatment). The ATP-induced contractions were not affected by AL 8810 (10⁻⁵ M) and L-798,106 (10⁻⁸ M).

Fig. 3. Effects of Various Prostanoid Receptor Antagonists on ATP-Induced Contractions in Guinea Pig Epithelium-Intact Tracheal Smooth Muscle

A–D: Representative traces showing the effects of SQ 29,548 (a TP receptor antagonist, 3 × 10⁻⁷ M, B), AL 8810 (an FP receptor antagonist, 10⁻⁵ M, C), ONO-8130 (an EP₁ receptor antagonist, 10⁻⁷ M, D), and their vehicle (ethanol (EtOH, 0.05%, A)) on the contractions induced by ATP (3 × 10⁻⁵ M). E, F: Representative traces showing the effects of L-798,106 (an EP₃ receptor antagonist, 10⁻⁸ M, F) and its vehicle (dimethyl sulfoxide (DMSO, 5 × 10⁻⁴%, E)) on the contractions induced by ATP. G: Summarized data of the effects of SQ 29,548, AL 8810, ONO-8130, and their vehicle on the ATP-induced contractions. H: Summarized data of ATP-induced contractions before the administration of SQ 29,548, AL 8810, ONO-8130, and their vehicle. I: Summarized data of the effects of L-798,106 and its vehicle on the ATP-induced contractions. J: Summarized data of ATP-induced contractions before the administration of L-798,106 and its vehicle. Data are expressed as the mean ± standard error of the mean (n = 5 each). ** p < 0.01 vs. EtOH (one-way ANOVA followed by Dunnett’s test). PPV: papaverine (10⁻⁴ M).

Effects of NP-1815-PX on Contractions Induced by Contractile Agents in Epithelium-Denuded TSM Preparations

Figure 4 shows representative traces and summarized data of the effects of NP-1815-PX (10⁻⁵–10⁻⁴ M) on contractions induced by U46619 (10⁻⁸ M) and PGF_2α (5 × 10⁻⁷ M) in epithelium-denuded TSM preparations. NP-1815-PX (10⁻⁵–10⁻⁴ M) inhibited both U46619- and PGF_2α-induced TSM contractions in a concentration-dependent manner. Pretreatment with NP-1815-PX (10⁻⁴ M) also suppressed the concentration-response curves for U46619- and PGF_2α-induced TSM contractions (Supplementary Fig. 2). The apparent pA₂ value of NP-1815-PX vs. U46619 was calculated to be 4.59 (n = 5).

Fig. 4. Effects of NP-1815-PX on U46619/PGF_2α-Induced Contractions in Guinea Pig Epithelium-Denuded Tracheal Smooth Muscle

A–F: Representative traces showing the effects of NP-1815-PX (10⁻⁵ M, A, D; 3 × 10⁻⁵ M, B, E; 10⁻⁴ M, C, F) on the contractions induced by U46619 (10⁻⁸ M, A–C) and PGF_2α (5 × 10⁻⁷ M, D–F). G, I: Summarized data of the effects of NP-1815-PX on the U46619 (G)- and PGF_2α (I)-induced contractions. H, J: Summarized data of the U46619 (H)- and PGF_2α (J)-induced contractions before the administration of NP-1815-PX. Data are expressed as the mean ± standard error of the mean (n = 5 each). PPV: papaverine (10⁻⁴ M).

Figure 5 shows representative traces and summarized data of the effects of NP-1815-PX (10⁻⁴ M) on contractions induced by CCh (7 × 10⁻⁸ M), histamine (3 × 10⁻⁶ M), NKA (10⁻⁸ M), and 50 mM high-K in epithelium-denuded TSM preparations. NP-1815-PX (10⁻⁴ M) did not strongly suppress CCh-, histamine-, NKA-, and 50 mM KCl-induced TSM contractions compared with those induced by U46619 or PGF_2α.

Fig. 5. Effects of NP-1815-PX on Carbachol (CCh)-, Histamine-, Neurokinin A (NKA)-, and 50 mM High-K-Induced Contractions in Guinea Pig Epithelium-Denuded Tracheal Smooth Muscle

A–D: Representative traces showing the effects of NP-1815-PX (10⁻⁴ M) on the contractions induced by CCh (7 × 10⁻⁸ M, A), histamine (3 × 10⁻⁶ M, B), NKA (10⁻⁸ M, C), and 50 mM high-K (D). E: Summarized data of the effects of NP-1815-PX (10⁻⁴ M) on the CCh-, histamine-, NKA-, and 50 mM high-K-induced contractions. F: Summarized data of the CCh-, histamine-, NKA-, and 50 mM high-K-induced contractions before the administration of NP-1815-PX. Data are expressed as the mean ± standard error of the mean (n = 5 each). PPV: papaverine (10⁻⁴ M).

Effects of NP-1815-PX on Contractions Induced by Contractile Agents in Epithelium-Denuded BSM Preparations

Figures 6A–C, H, and I show representative traces and summarized data of the effects of NP-1815-PX (10⁻⁵–10⁻⁴ M) on contractions induced by U46619 (5 × 10⁻⁸ M) in epithelium-denuded BSM preparations. NP-1815-PX (10⁻⁵–10⁻⁴ M) inhibited the U46619-induced BSM contractions in a concentration-dependent manner. Since stable, sustained BSM contractions were not obtained for PGF_2α, the effects of NP-1815-PX on PGF_2α-induced BSM contractions were not investigated.

Fig. 6. Effects of NP-1815-PX on U46619-, CCh-, Histamine-, NKA-, and 50 mM High-K-Induced Contractions in Guinea Pig Epithelium-Denuded Bronchial Smooth Muscle

A–C: Representative traces showing the effects of NP-1815-PX (10⁻⁵ M, A; 3 × 10⁻⁵ M, B; 10⁻⁴ M, C) on the contractions induced by U46619 (5 × 10⁻⁸ M). D–G: Representative traces showing the effects of NP-1815-PX (10⁻⁴ M) on the contractions induced by CCh (3 × 10⁻⁷ M, D), histamine (3 × 10⁻⁶ M, E), NKA (10⁻⁷ M, F), and 50 mM high-K (G). H: Summarized data of the effects of NP-1815-PX on the U46619-induced contractions. I: Summarized data of the U46619-induced contractions before the administration of NP-1815-PX. J: Summarized data of the effects of NP-1815-PX (10⁻⁴ M) on the CCh-, histamine-, NKA-, and 50 mM high-K-induced contractions. K: Summarized data of the CCh-, histamine-, NKA-, and 50 mM high-K-induced contractions before the administration of NP-1815-PX. Data are expressed as the mean ± standard error of the mean (n = 5 each). PPV: papaverine (10⁻⁴ M).

Figures 6D–G, J, and K show representative traces and summarized data of the effects of NP-1815-PX (10⁻⁴ M) on contractions induced by CCh (3 × 10⁻⁷ M), histamine (3 × 10⁻⁶ M), NKA (10⁻⁷ M), and 50 mM high-K in epithelium-denuded BSM preparations. NP-1815-PX (10⁻⁴ M) did not strongly suppress CCh-, histamine-, NKA-, and 50 mM KCl-induced BSM contractions compared with those induced by U46619.

Effects of SQ 29,548 on U46619/PGF_2α-Induced Contractions in Epithelium-Denuded TSM and BSM Preparations

Figure 7 shows representative traces and summarized data of the effects of SQ 29,548 (3 × 10⁻⁷–10⁻⁶ M) on contractions induced by U46619 (10⁻⁸ M, TSM; 5 × 10⁻⁸ M, BSM) and PGF_2α (5 × 10⁻⁷ M, TSM) in epithelium-denuded TSM and BSM preparations. The U46619-induced TSM and BSM contractions were completely suppressed by SQ 29,548. The PGF_2α-induced TSM contractions were strongly (approx. 80%) suppressed by SQ 29,548.

Fig. 7. Effects of SQ 29,548 (SQ) on U46619- and PGF_2α-Induced Contractions in Guinea Pig Epithelium-Denuded Tracheal/Bronchial Smooth Muscle

A, B: Representative traces showing the effects of SQ (3 × 10⁻⁷ and 10⁻⁶ M) on the tracheal contractions induced by U46619 (10⁻⁸ M, A) and PGF_2α (5 × 10⁻⁷ M, B). C: Representative traces showing the effects of SQ (3 × 10⁻⁷ and 10⁻⁶ M) on the bronchial contractions induced by U46619 (5 × 10⁻⁸ M). D, E: Summarized data of the effects of SQ on the U46619 (D)- and PGF_2α (E)-induced tracheal contractions. F: Summarized data of the effects of SQ on the U46619-induced bronchial contractions. Data are expressed as the mean ± standard error of the mean (n = 5 each). PPV: papaverine (10⁻⁴ M).

Effects of NP-1815-PX on U46619-Induced Increases in Intracellular Ca²⁺ Concentrations in TP-293T Cells

Figure 8 shows the effects of NP-1815-PX on the changes in intracellular Ca²⁺ concentrations induced by U46619 (10⁻⁸ M) in TP-293T cells. NP-1815-PX (10⁻⁵–10⁻⁴ M) inhibited the increase in Ca²⁺ concentration induced by U46619 (10⁻⁸ M) in a concentration-dependent manner. Although NP-1815-PX (10⁻⁴ M) significantly inhibited the increase in Ca²⁺ concentration induced by PGF_2α (3 × 10⁻⁶ M) in the 293T cell lines stably expressing human FP receptor (FP-293T cells) (vs. control), the extent of inhibition by NP-1815-PX in the FP-293T cells was very weak compared with that in the TP-293T cells (Supplementary Fig. 3).

Fig. 8. Effects of NP-1815-PX on U46619-Induced Increases in Intracellular Ca²⁺ Concentrations in Human TP Receptor-Expressing Cells

A: Mean Fura-2 fluorescence intensity ratio (F340/380) changes induced by U46619 (10⁻⁸ M) in the absence or presence of NP-1815-PX (10⁻⁵–10⁻⁴ M). ● indicates the administration of U46619 (10⁻⁸ M). B: Summarized data of the peak ratio (F340/380) after administration of U46619 within 5 min of its administration. Data are expressed as the mean ± standard error of the mean (n = 14 each). * p < 0.05, ** p < 0.01 vs. Control (one-way ANOVA followed by Dunnett’s test).

NC 2600 (10⁻⁵ M) and gefapixant (10⁻⁵ M) did not affect the increase in Ca²⁺ concentration induced by U46619 (10⁻⁸ M) in TP-293T cells (Supplementary Fig. 4).

DISCUSSION

In this study, to clarify the involvement of P2X4 receptors in the contraction of TSM and the pharmacological effects of NP-1815-PX on TSM and BSM contractions, we investigated the effects of NP-1815-PX on contractile responses in guinea pig TSM and BSM. NP-1815-PX strongly inhibited TSM and BSM contractions mediated through the TP receptor in addition to the P2X4 receptor, whose stimulation induces EP₁ receptor-related mechanisms, which supports the usefulness of NP-1815-PX as a therapeutic drug for asthma.

First, we will discuss the effects of P2X receptor antagonists on ATP-induced contractions in epithelium-intact TSM preparations. The ATP (3 × 10⁻⁵ M)-induced contractions in epithelium-intact TSM preparations were strongly suppressed by the P2X4 receptor antagonists NP-1815-PX (10⁻⁵ M) and NC 2600 (10⁻⁵ M)¹⁵⁾ but were only slightly suppressed by the P2X3 receptor antagonist gefapixant (10⁻⁵ M). Since the IC₅₀ value of NP-1815-PX for ATP (50 µM)-induced intracellular Ca²⁺ increases in 1321N1 cells expressing the human P2X4 receptor was reported to be 0.26 µM,¹⁾ the concentration of NP-1815-PX used in this study (10⁻⁵ M) was sufficient to antagonize the P2X4 receptor. The concentration of gefapixant used in this study (10⁻⁵ M) was also sufficient to antagonize the P2X3 receptor because the IC₅₀ values of gefapixant for the inward currents induced by α,β-methylene ATP (10 µM) in 1321N1 cells expressing the human P2X3 receptor and human P2X2/3 were reported to be 153  and 220 nM, respectively.¹⁶⁾ Therefore, the ATP-induced contractions are considered to be triggered mainly via the P2X4 receptor rather than the P2X3 receptor. Gefapixant is a selective P2X3 receptor antagonist that has been shown to be effective for the treatment of intractable chronic cough¹⁷⁾ and was approved for this treatment in Japan in 2022.¹⁸⁾ Binding of ATP to the P2X3 receptor in the airway sensory nerves has been reported to activate the C fibers of the afferent vagus nerve, causing the cough reflex.¹⁹⁾ Gefapixant is thought to suppress by antagonizing P2X3 receptors on the airway sensory nerves. This study showed that the inhibitory effect of gefapixant (10⁻⁵ M) on ATP-induced contractions was very weak. Therefore, P2X3 receptor antagonists, such as gefapixant, are unlikely to improve chronic cough by directly suppressing TSM contractions.

Next, we will discuss the mechanism of ATP-induced contractions in the epithelium-intact TSM preparations. P2X4 receptors are reported to be expressed in mouse alveolar epithelial type I cells,²⁰⁾ the base of rabbit ciliated cells (epithelial cells),²¹⁾ and even in human airway epithelial cells.²²⁾ Based on the above reports and our results that the ATP-induced TSM contractions were completely suppressed by indomethacin, ATP is thought to stimulate P2X4 receptors in the trachea (epithelium) to activate COX or increase the concentration of arachidonic acid, which is a substrate of COX. Then, COX-derived prostanoids are believed to contract the TSMs. Among the prostanoid receptors, those involved in ATP-induced contractions are thought to be FP, EP₁, EP₃, and TP receptors, since these prostanoid receptors are G_q/G_i protein-conjugated receptors.²³⁾ However, ATP-induced contractions were not suppressed by the selective FP receptor antagonist AL 8810 and the selective EP₃ receptor antagonist L-798,106. Because the pA₂ values of AL 8810 for fluprostenol (a selective FP receptor agonist) in A7r5 cells and 3T3 cells were reported to be 6.68 and 6.34, respectively,¹¹⁾ the concentration of AL 8810 used in this study (10⁻⁵ M) was sufficient to antagonize the FP receptor. Furthermore, the concentration of L-798,106 used in this study (10⁻⁸ M) was sufficient to antagonize the EP₃ receptor since the K_i value of L-798,106 for the human EP₃ receptor was reported to be 0.3 nM.¹²⁾ Therefore, the contribution of FP and EP₃ receptors to the ATP-induced contractions is considered negligible. In contrast, the ATP-induced contractions were partially suppressed by the selective TP receptor antagonist SQ 29,548. The K_i value of SQ 29,548 for the TP receptor is 4.1 nM, and those for the EP, DP, FP, and IP receptors are >100000 nM. Thus, the concentration of SQ 29,548 used in this study (3 × 10⁻⁷ M) should have selectively and strongly antagonized the TP receptor.¹³⁾ In addition, the ATP-induced contractions were strongly suppressed by the selective EP₁ receptor antagonist ONO-8130. The concentration of ONO-8130 used in this study (10⁻⁸ M) selectively suppresses the EP₁ receptor among the EP_1–4 receptors.¹⁰⁾ Moreover, ONO-8130 strongly suppresses the contractions induced by PGE₂ and the EP₁ receptor agonist 17-phenyl trinor PGE₂ in guinea pig trachea.¹⁰⁾ Although the selectivity of ONO-8130 for other prostanoid receptors is unknown, the ATP-induced contractions (COX-mediated responses) are considered to be triggered mostly by the EP₁ receptor and partially by the TP receptor, considering the effects of other prostanoid receptor antagonists. In fact, 1) ATP increases PGE₂ release in a concentration-dependent manner, and removal of the epithelium significantly attenuates this ATP-induced PGE₂ release in rabbit trachea (vs. epithelium-intact trachea)⁷⁾; and 2) the PGE₂ concentration in BALF increases when an asthma mouse model is sensitized with OVA.²⁴⁾ Considering our results and those of previous reports, ATP is thought to stimulate P2X4 receptors in the trachea (mainly epithelia) to produce prostanoids (mainly PGE₂) via COX, and the produced prostanoids are thought to contract TSMs mainly via the EP₁ receptor. Therefore, P2X4 receptor antagonists such as NP-1815-PX and NC 2600 are likely to suppress prostanoid-induced tracheal hypercontraction during asthma by suppressing ATP-mediated prostanoid production, in addition to previously reported inhibitory effects on immune cell activation.⁴⁾

Finally, we will discuss the effects of NP-1815-PX on the contraction of epithelium-denuded TSM and BSM. In epithelium-denuded TSM and BSM, NP-1815-PX suppressed the contractions induced by U46619 and PGF_2α (TSM only) in a concentration-dependent manner but did not strongly suppress the contractions induced by CCh, histamine, NKA, and 50 mM high-K. The TSM and BSM contractions induced by U46619 were almost completely suppressed by SQ 29,548, and the TSM contractions induced by PGF_2α were strongly (approx. 80%) suppressed by SQ 29,548. Thus, the contractions induced by U46619 and PGF_2α are believed to be mediated mainly through the TP receptor, and NP-1815-PX is considered to selectively and strongly suppress the contractions mediated through the TP receptor. NP-1815-PX also suppressed the U46619-induced increase in intracellular Ca²⁺ in human TP receptor-expressing cells in a concentration-dependent manner (Fig. 8) but did not strongly inhibit the PGF_2α-induced increase in intracellular Ca²⁺ in human FP receptor-expressing cells (Supplementary Fig. 3). Therefore, NP-1815-PX may have an antagonistic effect on the TP receptor by directly binding it, but high concentrations of NP-1815-PX may also affect downstream signaling of the TP receptor or other receptors. However, the other P2X receptor antagonists (NC 2600 and gefapixant) did not exhibit antagonistic effects on the TP receptor (Supplementary Fig. 4). These findings suggest that the antagonistic effect on the TP receptor is not a common property of P2X receptor antagonists but a characteristic action of NP-1815-PX. The apparent pA₂ value of NP-1815-PX for the U46619-induced TSM contractions calculated in this study was 4.59, and the antagonistic effect of NP-1815-PX on the TP receptor would not be as strong as the antagonistic effect of NP-1815-PX on the P2X4 receptor (IC₅₀ value = 0.26 µM).¹⁾ However, NP-1815-PX is expected to exert therapeutic effects on asthma by suppressing the hypercontraction of the trachea and bronchi induced by thromboxane A₂ and PGF_2α in addition to suppression of the hypercontraction induced by ATP.

Acknowledgments

NP-1815 PX, NC 2600, and gefapixant were kindly provided by Nippon Chemiphar Co., Ltd.

Conflict of Interest

KO, KY, and YT received a joint research Grant from Nippon Chemiphar Co., Ltd.; RI, MK, and AM have no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Matsumura Y, Yamashita T, Sasaki A, Nakata E, Kohno K, Masuda T, Tozaki-Saitoh H, Imai T, Kuraishi Y, Tsuda M, Inoue K. A novel P2X4 receptor-selective antagonist produces anti-allodynic effect in a mouse model of herpetic pain. Sci. Rep., 6, 32461 (2016).
• 2) Tsuda M. Microglia-mediated regulation of neuropathic pain: molecular and cellular mechanisms. Biol. Pharm. Bull., 42, 1959–1968 (2019).
• 3) Suurväli J, Boudinot P, Kanellopoulos J, Rüütel Boudinot S. P2X4: A fast and sensitive purinergic receptor. Biomed. J., 40, 245–256 (2017).
• 4) Zech A, Wiesler B, Ayata CK, Schlaich T, Dürk T, Hoßfeld M, Ehrat N, Cicko S, Idzko M. P2rx4 deficiency in mice alleviates allergen-induced airway inflammation. Oncotarget, 7, 80288–80297 (2016).
• 5) Idzko M, Hammad H, van Nimwegen M, Kool M, Willart MA, Muskens F, Hoogsteden HC, Luttmann W, Ferrari D, Di Virgilio F, Virchow JC Jr, Lambrecht BN. Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells. Nat. Med., 13, 913–919 (2007).
• 6) Advenier C, Bidet D, Floch-Saint-Aubin A, Renier A. Contribution of prostaglandins and thromboxanes to the adenosine and ATP-induced contraction of guinea-pig isolated trachea. Br. J. Pharmacol., 77, 39–44 (1982).
• 7) Aksoy MO, Borenstein M, Li XX, Kelsen SG. Eicosanoid production in rabbit tracheal epithelium by adenine nucleotides: mediation by P₂-purinoceptors. Am. J. Respir. Cell Mol. Biol., 13, 410–417 (1995).
• 8) Yamaki F, Koike A, Kono H, Zhang X, Yoshioka K, Obara K, Tanaka Y. Effects of catecholamine metabolites on beta-adrenoceptor-mediated relaxation of smooth muscle: evaluation in mouse and guinea-pig trachea and rat aorta. Biol. Pharm. Bull., 43, 493–502 (2020).
• 9) Obara K, Inaba R, Kawakita M, De Dios Regadera M, Uetake T, Murata A, Nishioka N, Kuroki K, Yoshioka K, Tanaka Y. Docosahexaenoic acid selectively suppresses U46619- and PGF_2α-induced contractions in guinea pig tracheal smooth muscles. Biol. Pharm. Bull., 45, 240–244 (2022).
• 10) Säfholm J, Dahlén SE, Delin I, Maxey K, Stark K, Cardell LO, Adner M. PGE₂ maintains the tone of the guinea pig trachea through a balance between activation of contractile EP₁ receptors and relaxant EP₂ receptors. Br. J. Pharmacol., 168, 794–806 (2013).
• 11) Griffin BW, Klimko P, Crider JY, Sharif NA. AL-8810: a novel prostaglandin F_2α analog with selective antagonist effects at the prostaglandin F_2α (FP) receptor. J. Pharmacol. Exp. Ther., 290, 1278–1284 (1999).
• 12) Juteau H, Gareau Y, Labelle M, Sturino CF, Sawyer N, Tremblay N, Lamontagne S, Carrière MC, Denis D, Metters KM. Structure–activity relationship of cinnamic acylsulfonamide analogues on the human EP₃ prostanoid receptor. Bioorg. Med. Chem., 9, 1977–1984 (2001).
• 13) Abramovitz M, Adam M, Boie Y, Carrière M, Denis D, Godbout C, Lamontagne S, Rochette C, Sawyer N, Tremblay NM, Belley M, Gallant M, Dufresne C, Gareau Y, Ruel R, Juteau H, Labelle M, Ouimet N, Metters KM. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim. Biophys. Acta, 1483, 285–293 (2000).
• 14) Yoshioka K, Obara K, Oikawa S, Uemura K, Yamaguchi A, Fujisawa K, Hanazawa H, Fujiwara M, Endoh T, Suzuki T, De Dios Regadera M, Ito D, Ou G, Xu K, Tanaka Y. Docosahexaenoic acid inhibits U46619- and prostaglandin F_2α-induced pig coronary and basilar artery contractions by inhibiting prostanoid TP receptors. Eur. J. Pharmacol., 908, 174371 (2021).
• 15) Jacobson KA, IJzerman AP, Müller CE. Medicinal chemistry of P2 and adenosine receptors: Common scaffolds adapted for multiple targets. Biochem. Pharmacol., 187, 114311 (2021).
• 16) Richards D, Gever JR, Ford AP, Fountain SJ. Action of MK-7264 (gefapixant) at human P2X3 and P2X2/3 receptors and in vivo efficacy in models of sensitisation. Br. J. Pharmacol., 176, 2279–2291 (2019).
• 17) Smith JA, Kitt MM, Morice AH, et al. Gefapixant, a P2X3 receptor antagonist, for the treatment of refractory or unexplained chronic cough: a randomised, double-blind, controlled, parallel-group, phase 2b trial. Lancet Respir. Med., 8, 775–785 (2020).
• 18) An interview form for Lyfnua^® tablets was provided by MSD K.K. (Tokyo, Japan) and Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan).
• 19) Muccino DR, Morice AH, Birring SS, Dicpinigaitis PV, Pavord ID, Assaid C, Kleijn HJ, Hussain A, La Rosa C, McGarvey L, Smith JA. Design and rationale of two phase 3 randomised controlled trials (COUGH-1 and COUGH-2) of gefapixant, a P2X3 receptor antagonist, in refractory or unexplained chronic cough. ERJ Open Res., 6, 00284-02020 (2020).
• 20) Weinhold K, Krause-Buchholz U, Rödel G, Kasper M, Barth K. Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells. Cell. Mol. Life Sci., 67, 2631–2642 (2010).
• 21) Ma W, Korngreen A, Weil S, Cohen EB, Priel A, Kuzin L, Silberberg SD. Pore properties and pharmacological features of the P2X receptor channel in airway ciliated cells. J. Physiol., 571, 503–517 (2006).
• 22) Taylor AL, Schwiebert LM, Smith JJ, King C, Jones JR, Sorscher EJ, Schwiebert EM. Epithelial P2X purinergic receptor channel expression and function. J. Clin. Invest., 104, 875–884 (1999).
• 23) Jones RL. Prostanoid Receptors. xPharm: The Comprehensive Pharmacology Reference. (Enna SJ, Bylund DB eds.) Elsevier, New York, pp. 1–3 (2007).
• 24) Jones VC, Birrell MA, Maher SA, Griffiths M, Grace M, O’Donnell VB, Clark SR, Belvisi MG. Role of EP₂ and EP₄ receptors in airway microvascular leak induced by prostaglandin E₂. Br. J. Pharmacol., 173, 992–1004 (2016).