Docosahexaenoic Acid Selectively Suppresses U46619- and PGF_2α-Induced Contractions in Guinea Pig Tracheal Smooth Muscles

Abstract

We investigated the potential inhibitory effects of docosahexaenoic acid (DHA) on the contractions of guinea pig tracheal smooth muscles in response to U46619 (a thromboxane A₂ (TXA₂) mimetic) and prostaglandin F_2α (PGF_2α) to examine whether this n-3 polyunsaturated fatty acid suppresses prostanoid-induced tracheal contractions. DHA (3 × 10⁻⁵ M) significantly suppressed tracheal contractions elicited by lower concentrations of U46619 (10⁻⁸ M) and PGF_2α (5 × 10⁻⁷ M) (vs. control), although it did not suppress the contractions induced by higher concentrations (U46619: 10⁻⁷ M; PGF_2α: 10⁻⁵ M). Supporting these findings, DHA (4 × 10⁻⁵ M/6 × 10⁻⁵ M) shifted the concentration-response curves for U46619 (10⁻⁹–10⁻⁶ M) and PGF_2α (10⁻⁸–10⁻⁵ M) to the right. However, the slope of the regression line in the Schild plot of DHA vs. U46619/PGF_2α was larger than unity. The tracheal contractions induced by U46619 (10⁻⁸ M) and PGF_2α (5 × 10⁻⁷ M) were significantly suppressed by the prostanoid TP receptor antagonist SQ 29,548 (10⁻⁶ M) (vs. ethanol-treated). In contrast, DHA (4 × 10⁻⁵ M) did not show significant inhibitory effects on the contractions induced by acetylcholine (10⁻⁸–10⁻⁴ M), histamine (10⁻⁸–10⁻⁴ M), and leukotriene D₄ (10⁻¹¹–10⁻⁷ M) (vs. ethanol-treated). These findings indicate that DHA selectively suppresses tracheal contractions induced by U46619 and PGF_2α. Therefore, DHA may be a useful therapeutic agent against asthma associated with tracheal/bronchial hyper-constriction caused by prostanoids including TXA₂ and PGF_2α.

INTRODUCTION

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid present in fish oil. A long-term intake of DHA prevents cardiovascular disease,¹⁾ potentially by inhibiting the production of inflammatory prostanoids.²⁾ We reported previously that DHA selectively and immediately inhibits the prostanoid TP receptor-mediated vascular contractions induced by U46619 (a thromboxane A₂ (TXA₂) mimetic) and prostaglandin F_2α (PGF_2α).^3–6) We showed using human prostanoid TP receptor-expressing cells that the TP receptor and/or its signaling can be targeted by DHA to suppress U46619- and PGF_2α-induced vascular contractions.⁶⁾

Prostanoids including TXA₂ are important mediators in the pathophysiology of bronchial asthma.⁷⁾ Clinical applications of a thromboxane synthase inhibitor and TP receptor antagonists for bronchial asthma also indicate a substantial role of TXA₂ as a bronchial asthma exacerbator.⁷⁾ Chronic administration of DHA helps treat bronchial asthma through its anti-inflammatory effect.²⁾ However, the immediate effects of DHA have not been studied in isolated tracheal smooth muscle (TSM) tissues. In this study, we showed that DHA potently and selectively inhibited U46619- and PGF_2α-induced contractions in guinea pig TSM tissues.

MATERIALS AND METHODS

Animals

Male guinea pigs (4–10 weeks old; weighing 265–540 g; total number, 30; Japan SLC, Hamamatsu, Japan, or 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 Use Committee (Approval Nos. 20-51-444, 21-52-444) and was conducted following the guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University.

Isometric Tension Changes in TSMs

Epithelium-removed tracheal tissues were prepared and isometric tension changes of TSM preparations were recorded using the methods described in a previous report.⁸⁾ The preparations were incubated for 70–90 min with the bath solution being changed every 20 min so that the final passive tension was 2.0 g. The bath solution was a Locke–Ringer solution with the following composition (mM): NaCl, 154; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and D-(+)-glucose, 2.8. The solution was aerated with mixed gas (95% O₂ + 5% CO₂) and maintained at 32 ± 1 °C (pH = 7.4). After this procedure, the TSMs were contracted twice with histamine (10⁻⁵ M) for 20 min. The TSMs were then incubated for ≥20 min until the next procedure. All experiments were performed in the presence of indomethacin (3 × 10⁻⁶ M) to prevent the production of endogenous prostanoids.⁹⁾

Effects of DHA and SQ 29,548 on U46619- and PGF_2α-Induced TSM Contractions

After conducting the primary procedures described in the previous section, the TSMs were contracted with U46619 (10⁻⁸ M/10⁻⁷ M) or PGF_2α (5 × 10⁻⁷ M/10⁻⁵ M) for 20 min. After washing and incubation for ≥20 min, 0.3% ethanol (EtOH, DHA vehicle) or DHA (3 × 10⁻⁵ M) was added to the bath medium. After 30 min of incubation, the TSMs were contracted with the same concentration of U46619 or PGF_2α for 20 min.

For the SQ 29,548 experiment, TSM contractions induced by U46619 (10⁻⁸ M) or PGF_2α (5 × 10⁻⁷ M) were observed in the presence of SQ 29,548 (10⁻⁶ M) after observing these contractions in the presence of 0.05% EtOH (SQ 29,548 vehicle). SQ 29,548 (10⁻⁶ M) selectively antagonizes the TP receptor without affecting other prostanoid receptors.¹⁰⁾

Effects of DHA on Concentration–Response Curves (CRCs) for U46619, PGF_2α, Acetylcholine (ACh), Histamine, and Leukotriene D₄ (LTD₄)

After the primary procedures, 0.4/0.6% EtOH (DHA vehicle) was added to the bath medium. After 30 min of incubation, U46619 (10⁻⁹–10⁻⁶ M), PGF_2α (10⁻⁸–10⁻⁵ M), ACh (10⁻⁸–10⁻⁴ M), and histamine (10⁻⁸–10⁻⁴ M) were cumulatively added to the bath medium. After washing and incubation for ≥60 min, DHA (4 × 10⁻⁵ M/6 × 10⁻⁵ M) was added to the bath medium. After 30 min of incubation, the same agonist was cumulatively added to the bath medium.

Since the reactivity of LTD₄ changed with repeated administration, cumulative administration of LTD₄ (10⁻¹¹–10⁻⁷ M) was performed only once in the presence of 0.4% EtOH/DHA (4 × 10⁻⁵ M), and the results of different preparations were compared. LTD₄ experiments were performed in the presence of L-serine borate (45 mM) and L-cysteine (5 mM) to inhibit the metabolism of LTD₄.¹¹⁾

Drugs

The following drugs were used: DHA, U46619, LTD₄, and SQ 29,548 (Cayman Chemical Co., Ann Arbor, MI, U.S.A.), dinoprost (PGF_2α) (Fuji Pharma Co. Ltd., Tokyo, Japan); indomethacin and histamine dihydrochloride (Sigma-Aldrich Co. LLC, MO, U.S.A.), and ACh chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan). All other chemicals were purchased from generic suppliers and were of reagent grade.

DHA, SQ 29,548, and indomethacin were dissolved in pure EtOH to obtain stock solutions of 10⁻² M (DHA and indomethacin) or 2 × 10⁻³ M (SQ 29,548). U46619 was dissolved in 70% ethanol to obtain a 10⁻⁴ M stock solution. L-Serine borate was prepared by mixing L-serine and borate and adjusting the pH to 7.4 with a sodium hydroxide solution to obtain a 0.9–1.0 M stock solution. All other drugs were prepared as aqueous stock solutions and diluted with distilled water.

Statistical Analysis

The contractions induced by a single administration of any agonist were measured 20 min after agonist administration. All contractions are shown as relative values, with the second contractions induced by 10⁻⁵ M histamine during the primary procedures representing 100%.

To construct CRCs for any agonist, the tension level before cumulative application of the agonist was defined as 0% contraction, and the second contractions induced by 10⁻⁵ M histamine were designated as 100%. The data were plotted as a function of agonist concentration and fitted using GraphPad Prism™ (version 6.0; GraphPad Software, Inc., San Diego, CA, U.S.A.). The pA₂ value of DHA vs. U46619/PGF_2α was calculated from a Schild plot analysis of DHA vs. U46619/PGF_2α. All values are expressed as mean ± standard error of the mean (S.E.M.) or mean (95% confidence interval (CI)) of the data obtained from different numbers (n) of preparations. GraphPad Prism software was used for statistical analyses. Differences between values were evaluated using paired t-tests or post hoc Šidák’s test after two-way ANOVA. Statistical significance was set at p < 0.05.

RESULTS

Effects of DHA on U46619- and PGF_2α-Induced TSM Contractions

Figure 1 shows representative traces (a, b) and quantified data (c, d) of the effects of 0.3% EtOH (a, c) and DHA (3 × 10⁻⁵ M, b, d) on TSM contractions induced by U46619 (10⁻⁸ M, A; 10⁻⁷ M, B) and PGF_2α (5 × 10⁻⁷ M, C; 10⁻⁵ M, D). EtOH did not significantly affect the contractions induced by U46619 (10⁻⁸ M, Aa, Ac; 10⁻⁷ M, Ba, Bc) or PGF_2α (5 × 10⁻⁷ M, Ca, Cc; 10⁻⁵ M, Da, Dc) (vs. control).

Fig. 1. Representative Traces (a, b) and Quantified Data (c, d) Showing the Effects of Ethanol (EtOH) (0.3%, a, c) and Docosahexaenoic Acid (DHA) (3 × 10⁻⁵ M, b, d) on Guinea Pig Tracheal Smooth Muscle Contractions Induced by U46619 (10⁻⁸ M, A; 10⁻⁷ M, B) and Prostaglandin F_2α (PGF_2α) (5 × 10⁻⁷ M, C; 10⁻⁵ M, D)

Bar graphs show mean ± standard error of the mean (S.E.M.) (n = 6 (Dc, Dd), and n = 5 (all others)). * p < 0.05, ** p < 0.01 vs. Ctrl (paired t-test). w: wash out; Ctrl: control.

DHA significantly suppressed the TSM contractions elicited with lower concentrations of U46619 (10⁻⁸ M, Ab, Ad) and PGF_2α (5 × 10⁻⁷ M, Cb, Cd) by approx. 90 and approx. 50%, respectively (vs. control). The magnitude of these contractions was approximately half that induced by 10⁻⁵ M histamine. In contrast, DHA did not suppress the contractions induced by higher concentrations of U46619 (10⁻⁷ M, Bb, Bd) and PGF_2α (10⁻⁵ M, Db, Dd) whose magnitudes were 75–100% of those induced by 10⁻⁵ M histamine. Although DHA significantly enhanced 10⁻⁵ M PGF_2α-induced contractions (Db, Dd), we determined that this change was negligible.

Effects of DHA on CRCs of U46619, PGF_2α, ACh, Histamine, and LTD₄

Figure 2 shows the effects of DHA on CRCs of U46619 (A), PGF_2α (B), ACh (C), histamine (D), and LTD₄ (E) in TSMs. DHA (4 × 10⁻⁵ M, a; 6 × 10⁻⁵ M, b) shifted the CRCs for U46619 (Aa, Ab) and PGF_2α (Ba, Bb) to the right. The slope of the regression line in the Schild plot of DHA vs. U46619 (Ac)/PGF_2α (Bc) was 2.58 (95% CI: 0.59–4.58, Ac)/2.89 (95% CI: 0.81–4.97, Bc), which was larger than unity. Therefore, DHA apparently did not competitively antagonize U46619/PGF_2α. The tentative reference pA₂ value of DHA vs. U46619/PGF_2α was calculated to be 4.40 (95% CI: 4.33–4.74, n = 20)/4.32 (95% CI: 4.25–4.43, n = 18). In contrast, DHA (4 × 10⁻⁵ M) did not show significant inhibitory effects on the CRCs of ACh (C), histamine (D), and LTD₄ (E) (vs. EtOH-treated).

Fig. 2. Effects of Docosahexaenoic Acid (DHA) (4 × 10⁻⁵ M, Aa, Ba, C–E; 6 × 10⁻⁵ M, Ab, Bb) on Concentration–Response Curves of U46619 (A), Prostaglandin F_2α (PGF_2α) (B), Acetylcholine (ACh) (C), Histamine (D), and Leukotriene D₄ (LTD₄) (E) in Guinea Pig Tracheal Smooth Muscles

Ac, Bc: Schild plot analyses of DHA vs. U46619 (Ac)/ PGF_2α (Bc) shown in Aa, Ab/Ba, Bb. Data are expressed as the mean ± S.E.M. (n = 10 (Aa, Ab, Ba, Bb), n = 9 (Ba, Bb), n = 6 (D), and n = 5 (C, E)). Schild plot is expressed as each data (Ac, Bc) and slope and pA₂ values are presented as mean with 95% confidence interval (n = 20 (Ac), and n = 18 (Bc)). * p < 0.05, ** p < 0.01 vs. 0.4%/0.6% EtOH (post hoc Šidák’s test after two-way ANOVA).

Effects of SQ 29,548 on U46619- and PGF_2α-Induced TSM Contractions

Figure 3 shows representative traces (a) and quantified data (b) of the effects of SQ 29,548 (10⁻⁶ M) on TSM contractions induced by U46619 (10⁻⁸ M, A) and PGF_2α (5 × 10⁻⁷ M, B). SQ 29,548 significantly suppressed the TSM contractions induced by U46619 (10⁻⁸ M, A) and PGF_2α (5 × 10⁻⁷ M, B) by approx. 100 and approx. 40%, respectively.

Fig. 3. Representative Traces (a) and Quantified Data (b) Showing the Effects of SQ 29,548 (a TP Receptor Antagonist, 10⁻⁶ M) on Guinea Pig Tracheal Smooth Muscle Contractions Induced by U46619 (10⁻⁸ M, A) and Prostaglandin F_2α (PGF_2α, 5 × 10⁻⁷ M, B)

Bar graphs show the mean ± S.E.M. (n = 6 for each). ** p < 0.01 vs. EtOH (paired t-test). EtOH: 0.05% ethanol; w: wash out.

DISCUSSION

This study revealed that DHA selectively and immediately suppressed the contractions induced by U46619 and PGF_2α in guinea pig TSMs without affecting those induced by ACh, histamine, and LTD₄ (Fig. 4). The ability of DHA to immediately suppress the contractions mediated by the TP receptor suggests that DHA may have preventive and ameliorative effects on asthma attacks.

Fig. 4. A Schematic Summary of This Study

DHA: docosahexaenoic acid; PGF_2α: prostaglandin F_2α; ACh: acetylcholine; LTD₄: leukotriene D₄; M-R: muscarinic receptor; H-R: histamine receptor; CysLT-R: cysteinyl leukotriene receptor.

Based on the following results and reports, the potential targets of the immediate inhibitory effect of DHA on the TSM contractions induced by U46619 and PGF_2α include the TP receptor. 1) The inhibition rates of DHA (3 × 10⁻⁵ M) for the contractions induced by U46619 (10⁻⁸ M)/PGF_2α (5 × 10⁻⁷ M) were approx. 90/50%. The inhibition rates of SQ 29,548 (10⁻⁶ M) were similar for these contractions (approx. 100/40%, U46619/PGF_2α). 2) DHA also selectively suppresses U46619/PGF_2α-induced contractions in guinea pig thoracic aorta, rat thoracic aorta/mesenteric arteries, and porcine coronary arteries and basilar arteries.^3–6) 3) DHA strongly suppressed the increase in intracellular Ca²⁺ concentrations induced by U46619 and PGF_2α in human TP receptor-expressing cells but did not affect those induced by PGF_2α in human prostanoid FP receptor-expressing cells.⁶⁾ 4) DHA suppressed the specific binding of [³H]U46619 and [³H]SQ 29,548 in human platelets.^12,13)

Competitive antagonistic action on the TP receptor partly accounts for DHA inhibitory effects against U46619/PGF_2α-induced contractions because 1) DHA inhibits the specific binding to the TP receptor^12,13) and 2) DHA competitively antagonizes U46619-induced human platelet agglutination and pig coronary artery contractions.^6,12) However, in TSM, other mechanisms in addition to TP receptor inhibition seemed to contribute to the inhibitory effects of DHA on U46619/PGF_2α-induced contractions as the slope of the regression line in the Schild plot of DHA vs. U46619/PGF_2α was larger than unity. A target of DHA during its inhibitory effect on U46619/PGF_2α-induced contractions could be the large-conductance calcium-activated potassium channel (BK channel). This assumption is supported by the following reports: 1) DHA and its CYP metabolites activate BK channels in rat coronary arteries.¹⁴⁾ 2) The TP receptor and BK channel physically associate and display a high degree of proximity.¹⁵⁾ 3) The TP receptor’s first intracellular loop and C-terminus interact with the voltage-sensing conduction cassette of the BK channel.¹⁵⁾ 4) U46619 inhibits the BK channel in human coronaries and rat aorta.¹⁵⁾ However, we assume that the BK channel targeted by DHA might be exclusive to the U46619/PGF_2α-inhibitable channel because DHA effects are restricted to these TP receptor agonists. Further studies are required to confirm these speculations.

Active intake of DHA may prevent asthma attacks through inhibition of TP receptors for the following reasons. 1) Prostanoids such as TXA₂ are also known to exacerbate bronchial asthma.⁷⁾ In previous studies, the blood concentration of TXB₂ (a metabolite of TXA₂) was higher in the asthma attack group than in the non-attack group and healthy subjects, and the plasma concentration of PGF_2α was higher in asthma patients than in healthy subjects.^16,17) 2) A thromboxane synthase inhibitor and TP receptor antagonists have been used for the treatment of bronchial asthma.⁷⁾ In addition, chronic administration of DHA has been reported to improve bronchial asthma due to its anti-inflammatory effects, especially its inhibition of leukotriene production.²⁾ The TP receptor antagonism by DHA might be involved in these improvements. Interestingly, on intake of DHA supplements, the DHA concentration in blood could easily reach the concentration used in this study (≤ 6 × 10⁻⁵ M).¹⁸⁾

Acknowledgments

This work was supported in part by the JSPS KAKENHI Grants-in-Aid for Scientific Research (C) (20K11519 to Y.T. and 21K11686 to K.O.) and Grants-in-Aid for Early Career Scientists (18K17981 to K.O. and 21K17666 to K.Y.).

Conflict of Interest

The authors declare no conflict of interest.

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