Docosahexaenoic acid and eicosapentaenoic acid strongly inhibit prostanoid TP receptor-dependent contractions of guinea pig gastric fundus smooth muscle

Abstract

The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT-qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D₂ and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP₃ >> FP > EP₁ . The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L-798,106 (an EP₃ receptor antagonist). DHA and EPA suppressed high KCl-induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage-dependent Ca²⁺ channel (VDCC) inhibitor, by 40%-85%. Although LA did not suppress high KCl-induced contractions, it suppressed U46619-induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619-induced Ca²⁺ increases in TP receptor-expressing cells. In contrast, LA inhibited U46619-induced contractions in the presence of verapamil, which was also suppressed by SKF-96365 (a store-operated Ca²⁺ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid-induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA-induced inhibition of U46619-induced contractions.

Keywords: docosahexaenoic acid; eicosapentaenoic acid; gastric fundus smooth muscle; n−3 polyunsaturated fatty acids; prostanoid TP receptor; prostanoids.

FIGURE 1

Representative traces showing the effects of docosahexaenoic acid (DHA, a), eicosapentaenoic acid (EPA, b), and linoleic acid (LA, c) (each 3 × 10⁻⁵ M) on guinea pig gastric fundus smooth muscle contractions induced by prostaglandin (PG) A₂ (3 × 10⁻⁶ M, A), PGD₂ (3 × 10⁻⁶ M, B), PGE₂ (10⁻⁷ M, C), PGF_2α (10⁻⁶ M, D), PGI₂ (10⁻⁶ M, E), and U46619 (3 × 10⁻⁶ M, F). w, wash out

FIGURE 2

Quantified data of the effect of ethanol (EtOH, 0.1%), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) (each 3 × 10⁻⁵ M) on the area under the curve for 10 min (AUC, a) and maximum contractions (b) of guinea pig gastric fundus smooth muscle responses induced by prostaglandin (PG) A₂ (3 × 10⁻⁶ M, A), PGD₂ (3 × 10⁻⁶ M, B), PGE₂ (10⁻⁷ M, C), PGF_2α (10⁻⁶ M, D), PGI₂ (10⁻⁶ M, E), and U46619 (3 × 10⁻⁶ M, F) shown in Figure 1. Data are expressed as the means ± SEM. (n = 13 (LA in F), n = 7 (EPA in F), n = 6 (LA in B), and n = 5 (all others)). *p < .05, **p < .01 versus EtOH (post hoc Dunnett's test after one‐way ANOVA)

FIGURE 3

Comparison of prostanoid receptor mRNA expression levels in guinea pig gastric fundus smooth muscle. The mRNA expression levels of TP (Tbxa2r), FP (Ptgfr), EP₁ (Ptger1), EP₂ (Ptger2), EP₃ (Ptger3), EP₄ (Ptger4), DP₁ (Ptgdr), DP₂ (Ptgdr2), and IP (Ptgir) receptors were determined using RT‐qPCR. The expression level of each mRNA is shown relative to the mRNA expression level of glyceraldehyde 3‐phosphate dehydrogenase (Gapdh), which is set as 1. Data are expressed as the means ± SEM (n = 5 each)

FIGURE 4

Representative traces (A) and quantified data (B) showing the effects of SQ 29,548 (3 × 10⁻⁵ M) on the area under the curve (AUC, Ba) and maximum contractions (Bb) of guinea pig gastric fundus smooth muscle responses induced by prostaglandin (PG) A₂ (3 × 10⁻⁶ M, Aa), PGD₂ (3 × 10⁻⁶ M, Ab), PGE₂ (10⁻⁷ M, Ac), PGF_2α (10⁻⁶ M, Ad), PGI₂ (10⁻⁶ M, Ae), and U46619 (3 × 10⁻⁶ M, Af). Data are expressed as the means ± SEM (n = 11 (PGF_2α), n = 10 (PGD₂ and PGI₂), and n = 5 (all others)). EtOH, ethanol (1.5%); w, wash out

FIGURE 5

Representative traces (A) and quantified data (B) showing the effects of L‐798,106 (3 × 10⁻⁷ M) on the area under the curve (AUC, Ba) and maximum contractions (Bb) of guinea pig gastric fundus smooth muscle responses induced by prostaglandin (PG) A₂ (3 × 10⁻⁶ M, Aa), PGD₂ (3 × 10⁻⁶ M, Ab), PGE₂ (10⁻⁷ M, Ac), PGF_2α (10⁻⁶ M, Ad), and PGI₂ (10⁻⁶ M, Ae) in the presence of SQ 29,548 (3 × 10⁻⁵ M). Data are expressed as the means ± SEM (n = 12 (PGD₂ and PGI₂), n = 10 (PGF_2α), and n = 5 (all others)). *p < .05, **p < .01 versus SQ 29,548 + DMSO (multiple t‐tests). EtOH: ethanol (1.5%); DMSO, dimethyl sulfoxide (0.015%); w, wash out

FIGURE 6

Relationships between the inhibitory effects of docosahexaenoic acid (DHA) (A), eicosapentaenoic acid (EPA) (B), and linoleic acid (LA) (C) on the area under the curve (AUC) of the contractions induced by prostanoids and U46619 (shown in Figure 2Aa–Fa) versus the inhibitory effects of SQ 29,548 on those contractions (shown in Figure 4Ba) (a) and versus the additional inhibitory effects of L‐798,106 on those contractions in the presence of SQ 29,548 (shown in Figure 5Ba) (b). Data are expressed as the means ± SEM. r: correlation coefficient; r ²: coefficient of determination

FIGURE 7

Effects of docosahexaenoic acid (DHA) (A) and eicosapentaenoic acid (EPA) (B) on the concentration‐response curve of U46619. (a) Summarized data of the effects of DHA (10⁻⁵ M and 3 × 10⁻⁵ M) on the concentration‐response curves of U46619. Data are presented as means ± SEM (n = 12 for control of Aa, n = 6 for 10⁻⁵ M DHA, n = 6 for 3 × 10⁻⁵ M DHA, n = 23 for control of Ba, n = 11 for 10⁻⁵ M EPA, n = 12 for 3 × 10⁻⁵ M EPA). (b) Schild plot analysis of DHA (A)/EPA (B) versus U46619. The slope and pA ₂ values are presented as means with 95% confidence intervals (n = 12 for Ab and n = 22 for Bb)

FIGURE 8

Representative traces (A) and quantified data (B) showing the effects of docosahexaenoic acid (DHA) (Aa), eicosapentaenoic acid (EPA) (Ab), and linoleic acid (LA) (Ac) (each 3 × 10⁻⁵ M) on the area under the curve (AUC) (Ba) and contractions (Bb) of guinea pig gastric fundus smooth muscle responses induced by 80 mM KCl. The quantified data of the effects of ethanol (EtOH, 0.1%) and verapamil (10⁻⁵ M) on those responses are also shown in Ba and Bb. Data are expressed as the means ± SEM (n = 7 (EPA) and n = 5 (all others)). *p < .05, **p < .01 versus EtOH (post hoc Dunnett's test after one‐way ANOVA). w, wash out

FIGURE 9

Representative traces (A) and quantified data (B) showing the effects of verapamil (10⁻⁵ M) on the area under the curve (AUC) (Ba) and maximum contractions (Bb) of guinea pig gastric fundus smooth muscle contractions induced by prostaglandin (PG) A₂ (3 × 10⁻⁶ M, Aa), PGD₂ (3 × 10⁻⁶ M, Ab), PGE₂ (10⁻⁷ M, Ac), PGF_2α (10⁻⁶ M, Ad), PGI₂ (10⁻⁶ M, Ae), and U46619 (3 × 10⁻⁶ M, Af). Data are expressed as the means ± SEM (each n = 5). w, wash out

FIGURE 10

Representative traces (A, B) and quantified data (C, D) showing the effects of LOE 908 (3 × 10⁻⁵ M, A, C) and SKF‐96365 (3 × 10⁻⁵ M, B, D) on the area under the curve (AUC) (a) and maximum contractions (b) of guinea pig gastric fundus smooth muscle contractions induced by U46619 (3 × 10⁻⁶ M) in the presence of verapamil (10⁻⁵ M). Data are expressed as the means ± SEM (each n = 5). **p < .01 versus verapamil/verapamil + DMSO (paired t‐tests). DMSO, dimethyl sulfoxide (0.015%); w, wash out