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.

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 Arrestin Specificity for G Protein-coupled Receptors in Human Airway Smooth Muscle

2001 ◽  
Vol 276 (35) ◽  
pp. 32648-32656 ◽  
Author(s):  
Raymond B. Penn ◽  
Rodolfo M. Pascual ◽  
You-Me Kim ◽  
Stuart J. Mundell ◽  
Vera P. Krymskaya ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Airway Smooth Muscle ◽  
G Protein Coupled Receptors ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
G Protein Coupled

scholarly journals IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility

2019 ◽  
Vol 202 (5) ◽  
pp. 1540-1548 ◽  
Author(s):  
Katarzyna Bulek ◽  
Xing Chen ◽  
Vandy Parron ◽  
Aparna Sundaram ◽  
Tomasz Herjan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Stress Fiber Formation

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.

Giα but not Gqα is linked to activation of p21 ras in human airway smooth muscle cells

1999 ◽  
Vol 276 (4) ◽  
pp. L564-L570 ◽  
Author(s):  
Charles W. Emala ◽  
Feng Liu ◽  
Carol A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Protein Kinases ◽  
G Protein ◽  
Airway Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Mitogen Activated Protein Kinases ◽  
Human Airway ◽  
Mitogen Activated Protein

Airway smooth muscle hypertrophy contributes to the narrowing of asthmatic airways. Activation of the mitogen-activated protein kinases is an important event in mediating cell proliferation. Because the monomeric G protein p21 ras is an important intermediate leading to activation of mitogen-activated protein kinases, we questioned which heterotrimeric G protein-coupled receptors were linked to the activation of p21 ras in cultured human airway smooth muscle and which of the heterotrimeric G protein subunits (α or βγ) transmitted the activation signal. Carbachol and endothelin-1 increased GTP-bound p21 ras in a pertussis toxin-sensitive manner [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): control, 30 ± 1.7; 3 min of 1 μM carbachol, 39 ± 1.1; 3 min of 1 μM endothelin-1, 40 ± 1.2], whereas histamine, bradykinin, and KCl were without effect. Transfection of an inhibitor of the G protein βγ-subunit [the carboxy terminus (Gly495–Leu689) of the β-adrenoceptor kinase 1] failed to inhibit the carbachol-induced activation of p21 ras . These data suggest that Gi- but not Gq-coupled receptors activate p21 ras in human airway smooth muscle cells, and this effect most likely involves the α-subunit.

Muscarinic receptors in human airway smooth muscle are coupled to phosphoinositide metabolism

1989 ◽  
Vol 164 (2) ◽  
pp. 369-371 ◽  
Author(s):  
Herman Meurs ◽  
Adiet Timmermans ◽  
Ronald G.M. Van Amsterdam ◽  
Frans Brouwer ◽  
Henk F. Kauffman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Muscarinic Receptors ◽  
Phosphoinositide Metabolism ◽  
Human Airway Smooth Muscle ◽  
Human Airway

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.

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