Inhibitory mechanisms of docosahexaenoic acid on carbachol-, angiotensin II-, and bradykinin-induced contractions in guinea pig gastric fundus smooth muscle

Abstract

We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca²⁺ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca²⁺, more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca²⁺ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca²⁺ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca²⁺ increase due to Ca²⁺ addition in CPA-treated 293T cells. These findings indicate that Ca²⁺ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions.

Figure 1

Representative traces (a) and summarized data (b: area under the curve (AUC); c: maximum contraction) of the inhibitory actions of docosahexaenoic acid (DHA, 3 × 10⁻⁵ M) on the contractions induced by carbachol (CCh, 6 × 10⁻⁸ M; A), angiotensin II (Ang II, 10⁻⁷ M; B), and bradykinin (BK, 10⁻⁶ M; C) in guinea pig gastric fundus smooth muscle. Data are expressed as means ± standard error of the mean (n = 6 (A, B), n = 5 (C)). *P < 0.05, **P < 0.01 versus EtOH (paired t-test). EtOH, ethanol (0.1%), w, wash out.

Figure 2

Representative traces (a–c) and summarized data (d: area under the curve (AUC); e: maximum contraction) of the inhibitory effects of extracellular Ca²⁺ removal (A) and verapamil (10⁻⁵ M; B) on the contractions induced by carbachol (CCh, 6 × 10⁻⁸ M; a), angiotensin II (Ang II, 10⁻⁷ M; b), and bradykinin (BK, 10⁻⁶ M; c) in guinea pig gastric fundus smooth muscle. Ca²⁺-free solution contained ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 2 × 10⁻⁴ M). Data are expressed as means ± standard error of the mean (n = 5 (A, CCh in B), n = 10 (Ang II in B), n = 8 (BK in B)). w, wash out.

Figure 3

Representative traces (a) and summarized data (b: area under the curve (AUC); c: maximum contraction) of the effects of LOE-908 (LOE, 3 × 10⁻⁵ M; A) and SKF-96365 (SKF, 3 × 10⁻⁵ M; B, C) on the contractions induced by bradykinin (BK, 10⁻⁶ M) in the presence of verapamil (Ver, 10⁻⁵ M; A, B) or Ver plus LOE (C) in guinea pig gastric fundus smooth muscle. Data are expressed as means ± standard error of the mean (n = 9 (A, B), n = 5 (C)). **P < 0.01 versus Ver/Ver plus LOE (paired t-test). DMSO, dimethyl sulfoxide (0.015%), w, wash out.

Figure 4

Representative traces (a) and summarized data (b: area under the curve (AUC); c: maximum contraction) of the inhibitory actions of docosahexaenoic acid (DHA, 3 × 10⁻⁵ M; A and DHA, 10⁻⁴ M; B) on the contractions induced by bradykinin (BK, 10⁻⁶ M) in the presence of verapamil (Ver, 10⁻⁵ M) plus LOE-908 (LOE, 3 × 10⁻⁵ M) in guinea pig gastric fundus smooth muscle. Data are expressed as means ± standard error of the mean (n = 6 each). *P < 0.05, **P < 0.01 versus EtOH (paired t-test). EtOH, ethanol; w, wash out.

Figure 5

Representative traces (a) and summarized data (b) of the inhibitory actions of docosahexaenoic acid (DHA, 3 × 10⁻⁵ M; A) and SKF-96365 (SKF, 3 × 10⁻⁵ M; B) on the contractions induced by cyclopiazonic acid (CPA, 3 × 10⁻⁵ M) in the presence of verapamil (10⁻⁵ M) plus LOE-908 (3 × 10⁻⁵ M) in guinea pig gastric fundus smooth muscle. Data are expressed as means ± standard error of the mean (n = 6 (A), n = 5 (B)). *P < 0.05, **P < 0.01 versus EtOH/control (paired t-test). EtOH, ethanol (0.1%); PPV, papaverine (10⁻⁴ M); w, wash out.

Figure 6

Inhibitory actions of docosahexaenoic acid (DHA, 3 × 10⁻⁵ M; A) and SKF-96365 (SKF, 3 × 10⁻⁵ M; B) on intracellular Ca²⁺ increase due to Ca²⁺ (1.8 mM) addition in cyclopiazonic acid (CPA, 10⁻⁵ M)-treated 293T cells in Ca²⁺-free medium. a: Mean Fura-2 fluorescence intensity ratio (F340/380) changes in the absence and presence of DHA (Aa) and SKF (Ba). Arrows indicate the administration of each drug. b: Summarized data of the peak ratio (F340/380) within 5 min after Ca²⁺ addition in the absence and presence of DHA (Ab) and SKF (Bb). Data are expressed as means ± standard error of the mean (n = 10 (A), n = 6 (B)). **P < 0.01 versus EtOH/H₂O (Welch’s t-test). EtOH, ethanol.

Figure 7

Effects of bradykinin (BK) receptor antagonists on BK-induced contractions in guinea pig gastric fundus smooth muscle (GFSM) (A), and effects of icatibant (B) and docosahexaenoic acid (DHA; C) on intracellular Ca²⁺ increase in BK B₂ receptor-expressing 293T cells (B₂-293T cells). A: Representative traces (Aa, Ab) and summarized data (Ac: area under the curve (AUC); Ad: maximum contraction) of the effects of Lys-(Des-Arg⁹, Leu⁸)-BK (LDALBK, 3 × 10⁻⁵ M; Aa) and icatibant (3 × 10⁻⁵ M; Ab) on BK (10⁻⁶ M)-induced GFSM contractions. B: Changes in mean Fura-2 fluorescence intensity ratio (F340/380) induced by BK (10⁻⁶ M) in the presence and absence of icatibant (3 × 10⁻⁵ M) in B₂-293T cells in normal medium (Ba), and the summarized data of the peak ratio (F340/380) within 5 min after BK administration (Bb). C: Changes in mean Fura-2 fluorescence intensity ratio (F340/380) induced by BK (10⁻⁶ M) in the absence and presence of DHA (3 × 10⁻⁵ M) in B₂-293T cells in Ca²⁺-free medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 2 × 10⁻⁴ M) and Gd³⁺ (10⁻⁵ M) (Ca), and summarized data of the peak ratio (F340/380) within 5 min after BK administration (Cb). Arrows indicate the administration of each drug. Data are expressed as means ± standard error of the mean (n = 12 each). **P < 0.01 versus H₂O (Welch’s t-test). EtOH, ethanol; w, wash out.

Figure 8

Expression levels of Orai (Orai1, Orai2, and Orai3) and Stim (Stim1 and Stim2) mRNA (A) and Trpc (Trpc1, 3–7) mRNA (B) in guinea pig gastric fundus smooth muscle assessed by RT-qPCR. The expression level of each mRNA is shown relative to that of Gapdh, which is set as 1. Data are expressed as means ± standard error of the mean (n = 5 each).