scholarly journals The short-chain free fatty acid receptor FFAR3 is expressed and potentiates contraction in human airway smooth muscle

2020 ◽  
Vol 318 (6) ◽  
pp. L1248-L1260 ◽  
Author(s):  
Kentaro Mizuta ◽  
Haruka Sasaki ◽  
Yi Zhang ◽  
Atsuko Matoba ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Airway Smooth Muscle ◽  
Fiber Formation ◽  
Short Chain ◽  
Human Airway Smooth Muscle ◽  
Camp Synthesis ◽  
Human Airway ◽  
Chronic Activation

Emerging evidence suggests that gut microbiota-derived short-chain fatty acids (SCFAs; acetate, propionate, and butyrate) are important modulators of the inflammatory state in diseases such as asthma. However, the functional expression of the Gi protein-coupled free fatty acid receptors (FFAR2/GPR43 and FFAR3/GPR41) has not been identified on airway smooth muscle (ASM). Classically, acute activation of Gi-coupled receptors inhibits cyclic AMP (cAMP) synthesis, which impairs ASM relaxation and can also induce crosstalk between Gi- and Gq-signaling pathways, potentiating increases in intracellular Ca2+ concentration ([Ca2+]i), favoring ASM contraction. In contrast, chronic activation of Gi-coupled receptors can sensitize adenylyl cyclase resulting in increased cAMP synthesis favoring relaxation. We questioned whether the Gi-coupled FFAR2 or FFAR3 is expressed in human ASM, whether they modulate cAMP and [Ca2+]i, and whether SCFAs modulate human ASM tone. We detected the protein expression of FFAR3 but not FFAR2 in native human ASM and primary cultured human airway smooth muscle (HASM) cells. In HASM cells, acute activation of FFAR3 with SCFAs inhibited forskolin-stimulated cAMP accumulation, but chronic activation did not sensitize cAMP synthesis. SCFAs induced [Ca2+]i increases that were attenuated by pertussis toxin, gallein, U73122, or xestospongin C. Acute treatment with SCFAs potentiated acetylcholine-stimulated [Ca2+]i increases and stress fiber formation in cells and contraction of ex vivo human airway tissues. In contrast, chronic pretreatment of human ASM with propionate did not potentiate airway relaxation. Together, these findings demonstrate that FFAR3 is expressed in human ASM and contributes to ASM contraction via reduced cAMP and increased [Ca2+]i.

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

2015 ◽  
Vol 309 (9) ◽  
pp. L970-L982 ◽  
Author(s):  
Kentaro Mizuta ◽  
Yi Zhang ◽  
Fumiko Mizuta ◽  
Hiroshi Hoshijima ◽  
Toshiya Shiga ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Smooth Muscle ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Guinea Pig ◽  
Free Fatty Acids ◽  
Airway Smooth Muscle ◽  
Muscle Tone ◽  
Fiber Formation ◽  
Long Chain

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 Ca2+ concentration ([Ca2+]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 [Ca2+]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.

Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells

1998 ◽  
Vol 274 (5) ◽  
pp. L803-L809 ◽  
Author(s):  
Hideaki Togashi ◽  
Charles W. Emala ◽  
Ian P. Hall ◽  
Carol A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Stress Fiber Formation

To determine whether M2 muscarinic receptors are linked to the monomeric G protein Rho, we studied the effect of carbachol on actin reorganization (stress fiber formation) in cultured human airway smooth muscle cells that expressed mainly M2 muscarinic receptors by dual- fluorescence labeling of filamentous (F) and monomeric (G) actin. F-actin was labeled with FITC-labeled phalloidin, and G-actin was labeled with Texas Red-labeled DNase I. Carbachol stimulation induced stress fiber formation (increased F-actin staining) in the cells and increased the F- to G-actin ratio 3.6 ± 0.4-fold (mean ± SE; n = 5 experiments). Preincubation with pertussis toxin, Clostridium C3 exoenzyme, or tyrosine kinase inhibitors reduced the carbachol-induced increase in stress fiber formation and significantly decreased the F- to G-actin ratio, whereas a mitogen-activated protein kinase inhibitor, a phosphatidylinositol 3-kinase inhibitor, and a protein kinase C inhibitor were without effect. This study demonstrates that in cultured human airway smooth muscle cells, muscarinic-receptor activation induces stress fiber formation via a pathway involving a pertussis-sensitive G protein, Rho proteins, and tyrosine phosphorylation.

scholarly journals Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells

2001 ◽  
Vol 281 (4) ◽  
pp. L824-L831 ◽  
Author(s):  
Ryan E. Lesh ◽  
Charles W. Emala ◽  
H. Thomas Lee ◽  
Defen Zhu ◽  
Reynold A. Panettieri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Actin Reorganization ◽  
Human Airway ◽  
Geranylgeranyltransferase I

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric Gi protein (the Gγ subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.

Gαi-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells

1998 ◽  
Vol 275 (5) ◽  
pp. L911-L916 ◽  
Author(s):  
Carol A. Hirshman ◽  
Hideaki Togashi ◽  
Dan Shao ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Airway Smooth Muscle ◽  
Muscarinic Receptors ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Human Airway Smooth Muscle ◽  
Α Subunit ◽  
Human Airway ◽  
Stress Fiber Formation

To determine which heterotrimeric G protein couples muscarinic receptors to stress fiber formation [measured by an increase in the filamentous (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (ASM) cells, cultured human ASM cells expressing the M2 muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 μM antisense oligonucleotides designed to specifically bind to the mRNA encoding Gαi-2, Gαi-3, or Gqα. A randomly scrambled oligonucleotide served as a control. F- to G-actin ratios were measured with dual-fluorescence labeling after 5 min of carbachol exposure, which is known to increase the F- to G-actin ratio. Cells in parallel wells were harvested for immunoblot analysis of G protein α-subunit expression. Oligonucleotide antisense treatment decreased protein expression of the respective G protein α-subunit. Antisense depletion of the Gαi-2 protein but not of Gαi-3 or Gqα protein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the Gαi-2 protein couples muscarinic receptors to stress fiber formation in ASM.

Melatonin MT2 receptor is expressed and potentiates contraction in human airway smooth muscle

2021 ◽  
Author(s):  
Haruka Sasaki ◽  
Yi Zhang ◽  
Charles W Emala ◽  
Kentaro Mizuta
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Airway Smooth Muscle ◽  
Muscle Tone ◽  
Fiber Formation ◽  
Melatonin Receptors ◽  
Human Airway Smooth Muscle ◽  
Nocturnal Asthma ◽  
Smooth Muscle Tone ◽  
Human Airway

Nocturnal asthma is characterized by heightened bronchial reactivity at night, and plasma melatonin concentrations are higher in patients with nocturnal asthma symptoms. Numerous physiological effects of melatonin are mediated via its specific G protein-coupled receptors (GPCRs) named the MT1 receptor which couples to both Gq and Gi proteins, and the MT2 receptor which couples to Gi. We investigated whether melatonin receptors are expressed on airway smooth muscle, whether they regulate intracellular cyclic AMP (cAMP) and calcium concentrations ([Ca2+]i) which modulate airway smooth muscle tone, and whether they promote airway smooth muscle cell proliferation. We detected the mRNA and protein expression of the melatonin MT2 but not the MT1 receptor in native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by RT-PCR, immunoblotting, and immunohistochemistry. Activation of melatonin MT2 receptors with either pharmacological concentrations of melatonin (10 - 100 µM) or the non-selective MT1/MT2 agonist ramelteon (10 µM) significantly inhibited forskolin-stimulated cAMP accumulation in HASM cells, which was reversed by the Gαi protein inhibitor pertussis toxin or knockdown of the MT2 receptor by its specific siRNA. Although melatonin by itself did not induce an initial [Ca2+]i increase and airway contraction, melatonin significantly potentiated acetylcholine-stimulated [Ca2+]i increases, stress fiber formation through the MT2 receptor in HASM cells, and attenuated the relaxant effect of isoproterenol in guinea pig trachea. These findings suggest that the melatonin MT2 receptor is expressed in ASM, and modulates airway smooth muscle tone via reduced cAMP production and increased [Ca2+]i.

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