Novel identification of the free fatty acid receptor FFAR1 that promotes contraction in airway smooth muscle

Abstract

Obesity is one of the major risk factors for asthma. Previous studies have demonstrated that free fatty acid levels are elevated in the plasma of obese individuals. Medium- and long-chain free fatty acids act as endogenous ligands for the free fatty acid receptors FFAR1/GPR40 and FFAR4/GPR120, which couple to Gq proteins. We investigated whether FFAR1 and FFAR4 are expressed on airway smooth muscle and whether they activate Gq-coupled signaling and modulate airway smooth muscle tone. We detected the protein expression of FFAR1 and FFAR4 in freshly dissected native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by immunoblotting and immunohistochemistry. The long-chain free fatty acids (oleic acid and linoleic acid) and GW9508 (FFAR1/FFAR4 dual agonist) dose-dependently stimulated transient intracellular Ca(2+) concentration ([Ca(2+)]i) increases and inositol phosphate synthesis in HASM cells. Downregulation of FFAR1 or FFAR4 in HASM cells by small interfering RNA led to a significant inhibition of the long-chain free fatty acids-induced transient [Ca(2+)]i increases. Oleic acid, linoleic acid, or GW9508 stimulated stress fiber formation in HASM cells, potentiated acetylcholine-contracted guinea pig tracheal rings, and attenuated the relaxant effect of isoproterenol after an acetylcholine-induced contraction. In contrast, TUG-891 (FFAR4 agonist) did not induce the stress fiber formation or potentiate acetylcholine-induced contraction. These results suggest that FFAR1 is the functionally dominant free fatty acid receptor in both human and guinea pig airway smooth muscle. The free fatty acid sensors expressed on airway smooth muscle could be an important modulator of airway smooth muscle tone.

Keywords: FFAR1; FFAR4; Gq-coupled receptor; airway smooth muscle; free fatty acid.

Fig. 1.

A: representative gel images of immunoblot analyses using antibodies against the free fatty acid receptor 1 (FFAR1) and FFAR4 using total protein prepared from human (i) and guinea pig (ii) tissues: human brain cerebral cortex (50 μg), freshly dissected native human tracheal airway smooth muscle (SM; 100 μg), primary cultured human airway smooth muscle (HASM) cells (100 μg), guinea pig whole brain (150 μg), and freshly dissected native guinea pig tracheal SM (100 μg). Reprobing of blots for GAPDH was performed to demonstrate relative lane loading. B: representative gel images of RT-PCR analyses of total RNA using primers specific for human FFAR4 transcript variant 1 (i) and variant 2 (ii). Total RNA extracted from freshly dissected human tracheal SM or cultured HASM cells was analyzed. Lane 1, base pair standards; lane 2, negative control water blank; lane 3, total RNA from freshly dissected native human tracheal SM; lane 4, total RNA from primary cultured HASM cells; lane 5, total RNA from whole human brain (positive control).

Fig. 2.

A and C: representative photomicrographs of immunohistochemical staining of FFAR1 (A) or FFAR4 (C) in paraformaldehyde-glutaraldehyde-fixed human trachea. E and G: representative photomicrographs of immunohistochemical staining of FFAR1 (E) or FFAR4 (G) in paraformaldehyde-fixed guinea pig trachea. B and D: anti-rabbit IgG isotype negative control in serial section of human trachea. F and H: anti-rabbit IgG isotype negative control in serial section of guinea pig trachea. All sections were counterstained with hematoxylin. Calibration bars: A–D, 100 μm; E–H, 50 μm. ASM, airway smooth muscle. Images are representative of at least 3 independent immunohistochemical analyses from both human and guinea pig trachea.

Fig. 3.

Effects of long-chain free fatty acids (oleic acid or linoleic acid) or a synthetic agonist of both FFAR1 and FFAR4 (GW9508) on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in HASM cells. A: representative traces of fluorescence intensity [change in fluorescence (ΔF) from baseline fluorescence (Fo)] illustrating the characteristics of oleic acid (i; 10 μM)-, linoleic acid (ii; 10 μM)-, or GW9508 (iii; 10 μM)-induced [Ca²⁺]_i increases in HASM cells. At time 0, oleic acid, linoleic acid, or GW9508 was injected into the buffer in the wells. B: concentration-dependent effects (0.1–10 μM) of oleic acid, linoleic acid, or GW9508 on transient (peak) fluorescence increases in HASM cells. Data are means ± SE presented as ΔF/Fo; n = 4–11. C: effects of oleic acid (10 μM), linoleic acid (10 μM), GW9508 (10 μM), vehicle (0.1% DMSO), bradykinin (1 μM), acetylcholine (1 μM), or histamine (10 μM) on transient (peak) fluorescence increases in HASM cells. Data are means ± SE presented as ΔF/Fo; n = 5–11 (shown in parentheses).

Fig. 4.

Involvement of FFAR1 and FFAR4 in transient [Ca²⁺]_i increases induced by long-chain free fatty acids (oleic acid or linoleic acid) in HASM cells. A and B: representative gel images of immunoblot analyses of FFAR1 (A) and FFAR4 (B) protein expression in HASM cells that were transfected with either control nontargeting small interfering RNA (siRNA), FFAR1-specific siRNA, or FFAR4-specific siRNA (targeting for both FFAR4 transcript variant 1 and variant 2) 3 days before analysis. GAPDH was used as loading control. These images are representative of at least 3 independent experiments. C and D: representative traces of fluorescence intensity illustrating the oleic acid (10 μM)-induced [Ca²⁺]_i increases in nontransfected HASM cells, HASM cells transfected with nontargeting siRNA control, or HASM cells transfected with either FFAR1 or FFAR4 siRNA. At time 0, oleic acid was injected to the buffer in the wells. E and F: effect of downregulation of FFAR1 (E; n = 5) or FFAR4 (F; n = 8) by siRNA on oleic acid (10 μM)- or linoleic acid (10 μM)-induced transient [Ca²⁺]_i mobilization in HASM cells. *P < 0.05; **P < 0.01 compared with the HASM cells transfected with nontargeting siRNA control.

Fig. 5.

Effects of pretreatment with U-73122 (5 μM), xestospongin C (Xest C; 20 μM), ryanodine (100 μM), or pertussis toxin (PTX; 100 ng/ml) on peak increase in [Ca²⁺]_i stimulated by ligands for FFAR1/FFAR4 (oleic acid, linoleic acid, or GW9508; 10 μM each) in HASM cells. Data are means ± SE presented as a percentage of the ligand (10 μM)-stimulated fluorescence increases in the absence of inhibitors. *P < 0.05; **P <0.01; ***P < 0.001 compared with FFAR1/FFAR4 ligands (10 μM) alone. Numbers of experiments are shown in parentheses.

Fig. 6.

Effects of ligands for FFAR1/FFAR4 (oleic acid, linoleic acid, or GW9508; 10 μM each) on the classical G_q-coupled receptor agonist bradykinin (1 μM) on the synthesis of inositol phosphate (shown as a percentage of basal level) in HASM cells. *P < 0.05; **P < 0.01 compared with basal level. Data are means ± SE. Numbers of experiments are shown in parentheses.

Fig. 7.

Fluorescent staining ratio of filamentous and globular actin (F/G actin) in HASM cells under untreated (cont) conditions or treated with oleic acid (10 μM; n = 6), linoleic acid (10 μM; n = 7), GW9508 (10 μM; n = 5), or TUG-891 (5 μM; n = 7). Data are means ± SE. An increase in F/G actin indicates an increase in filamentous actin, which is related to smooth muscle cell contraction.

Fig. 8.

Ligands for FFAR1/FFAR4 (oleic acid, linoleic acid, or GW9508) potentiated acetylcholine-induced contractions in guinea pig tracheal rings. A: representative tension tracing in guinea pig tracheal ring illustrating potentiation of acetylcholine (EC₅₀) contraction by the natural ligands for FFAR1/FFAR4 (oleic acid or linoleic acid; 20 μM each) or the_FFAR1/FFAR4-selective agonist GW9508 (20 μM). B: oleic acid, linoleic acid, or GW9508 significantly potentiated the acetylcholine (EC₅₀)-induced contraction, whereas the highly selective FFAR4 agonist TUG-891 (20 μM) did not potentiate the contraction. Data are means ± SE. *P < 0.05 compared with vehicle (0.1% DMSO) control. Numbers of experiments are shown in parentheses.

Fig. 9.

A: representative tension tracing in guinea pig tracheal rings after an acetylcholine (EC₅₀) contraction followed by increasing concentrations of the β-adrenoceptor agonist isoproterenol in the presence of oleic acid (20 μM), linoleic acid (20 μM), GW9508 (20 μM), or their vehicle (0.1% DMSO). B: isoproterenol concentration-response relaxation curves in the presence of vehicle (0.1% DMSO) or oleic acid (20 μM; n = 8), linoleic acid (20 μM; n = 7), or GW9508 (20 μM; n = 6). C: EC₅₀ value of isoproterenol-induced relaxation of an acetylcholine (EC₅₀) contraction in the presence of oleic acid (20 μM; n = 8), linoleic acid (20 μM; n = 7), or GW9508 (20 μM; n = 6) compared with vehicle (0.1% DMSO). D: muscle force remaining after isoproterenol (10^−8.5 M)-induced relaxation of acetylcholine (EC₅₀) contraction in the presence of oleic acid (20 μM; n = 8), linoleic acid (20 μM; n = 7), or GW9508 (20 μM; n = 6) compared with vehicle control.