Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho

1999 ◽  
Vol 277 (3) ◽  
pp. L653-L661 ◽  
Author(s):  
Carol A. Hirshman ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Muscle Cells ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Actin Reorganization ◽  
Endothelin 1 ◽  
In Cells

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 ± 0.3 in control cells, 6.7 ± 0.8 with carbachol, 7.2 ± 0.8 with LPA, and 7.4 ± 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Gαi-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Gαi-2 and Gqα were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Gαi-2pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Gαi-2 or a Gqα pathway.

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.

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.

Isoproterenol increases peripheral [Ca2+]i and decreases inner [Ca2+]i in single airway smooth muscle cells

1995 ◽  
Vol 268 (3) ◽  
pp. C771-C779 ◽  
Author(s):  
H. Yamaguchi ◽  
J. Kajita ◽  
J. M. Madison
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Acetoxymethyl Ester ◽  
Fura 2 ◽  
Low Concentrations ◽  
In Cells

The effects of isoproterenol (Iso) or photolysis of caged adenosine 3',5'-cyclic monophosphate (cAMP) on intracellular Ca2+ concentration ([Ca2+]i) were studied in single airway smooth muscle cells. Changes in [Ca2+]i were measured ratiometrically. In cells loaded with 10 microM fura 2-acetoxymethyl ester (AM), superfusion of Iso (10 microM) increased [Ca2+]i in 20 of 22 cells from 153.2 +/- 21.3 to 252.8 +/- 38.3 nM, and this increase depended on extracellular Ca2+ and was blocked by ryanodine (50 microM). Photolysis of intracellular caged cAMP increased [Ca2+]i by 104.0 +/- 17.3 nM in 20 cells and decreased [Ca2+]i by 49.3 +/- 9.4 nM in 10 cells. With modified confocal microscopy, peripheral [Ca2+]i was increased and inner cytosolic [Ca2+]i was decreased during stimulation with Iso. Iso-induced decreases in [Ca2+]i were also observed with conventional optics in cells loaded with 0.5 microM fura 2-AM. However, in these same cells, Iso increased [Ca2+]i in the presence of low concentrations of ryanodine (1-20 microM). We concluded that Iso decreased [Ca2+]i in the inner cytosol but increased [Ca2+]i in the peripheral cytosol by mechanisms that depended on both extracellular and sarcoplasmic reticulum Ca2+ release channels. Our study suggests that fluorescence from a peripheral cytosol with high [Ca2+]i can sometimes confound the measurement of “cytosolic” [Ca2+]i with conventional optics.

scholarly journals Actin depolymerization via the β-adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway

2001 ◽  
Vol 281 (5) ◽  
pp. C1468-C1476 ◽  
Author(s):  
Carol A. Hirshman ◽  
Defen Zhu ◽  
Reynold A. Panettieri ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Kinase ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Actin Depolymerization ◽  
Human Airway

Actin is a major functional and structural cytoskeletal protein that mediates such diverse processes as motility, cytokinesis, contraction, and control of cell shape and polarity. While many extracellular signals are known to mediate actin filament polymerization, considerably less is known about signals that mediate depolymerization of the actin cytoskeleton. Human airway smooth muscle cells were briefly exposed to isoproterenol, forskolin, or the cAMP-dependent protein kinase A (PKA) agonist stimulatory diastereoisomer of adenosine 3′,5′-cyclic monophosphate (Sp-cAMPS). Actin polymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, forskolin, or Sp-cAMPS induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. The PKA inhibitor Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) completely inhibited forskolin-stimulated depolymerization, whereas it only partially inhibited isoproterenol-induced depolymerization. The protein tyrosine kinase inhibitors genistein or tyrphostin A23 also partially inhibited isoproterenol-induced actin depolymerization. In contrast, the combination of Rp-cAMPS and either tyrosine kinase inhibitor had an additive effect at inhibiting isoproterenol-induced actin depolymerization. These results suggest that both PKA-dependent and -independent pathways mediate actin depolymerization in human airway smooth muscle cells.

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