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

Hideaki Togashi1, Charles W. Emala1, Ian P. Hall2, and Carol A. Hirshman1,3

Departments of 1 Anesthesiology and 3 Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; and 2 Department of Medicine, University Hospital, Queen's Medical Center, Nottingham N67 2UH, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

M2 muscarinic receptor; fluoroscein isothiocyanate-labeled phalloidin; Texas Red-labeled deoxyribonuclease; Gi proteins; Rho proteins; stress fiber formation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE PARASYMPATHETIC nervous system plays an important role in regulating airway smooth muscle tone. The effects of acetylcholine released from postganglionic parasympathetic nerves are mediated by muscarinic receptors present on the smooth muscle. Five different subtypes of muscarinic receptors have been cloned and designated as M1-M5. The M1, M3, and M5 receptors couple to phospholipase C, mediating increases in inositol trisphosphate and diacylglycerol via the heterotrimeric G protein Gq, whereas the M2 and M4 muscarinic receptors mediate the inhibition of adenylyl cyclase activity via interaction with members of the pertussis toxin-sensitive Gi protein family (11). Airway smooth muscle expresses both M2 and M3 muscarinic receptors, with >80% of the muscarinic receptors being of the M2 subtype (9, 12, 13, 34). Activation of M3 muscarinic receptors contracts the muscle, whereas activation of the M2 muscarinic receptors inhibits relaxation. More recent studies have shown that agonists that bind to G protein-coupled receptors can, in addition, stimulate mitogenic signaling via the Ras superfamily of monomeric low-molecular-weight GTP-binding proteins, which regulate cell growth and differentiation, gene expression, actin cytoskeletal assembly, and cell motility (29, 40).

Rho proteins are a subfamily of the Ras superfamily that are thought to play a pivotal role in the determination of cell shape (3). They mediate agonist-activated potentiation of Ca2+-induced contraction in intact vascular, intestinal, and vas deferens smooth muscle (4, 14, 18, 27) and are well accepted as regulators of the actin cytoskeleton induced by extracellular signals in isolated cells (16, 19, 21, 25, 30). The mammalian Rho subfamily consists of nine members: RhoA, RhoB, RhoC, RhoG, Rac1, Rac2, Cdc42, G25K, and TC10 (16). RhoA, Rac, and Cdc42 play an essential role in controlling the assembly of the actin cytoskeleton. Cdc42 regulates the formation of filopodia, whereas Rac proteins regulate lamellipoida formation and membrane ruffling (33).

RhoA regulates the formation of stress fibers, which are bundled actin filaments that transverse the cell and terminate in the plasma membrane at focal adhesion complexes (33). Filamentous (F) actin is the major microfilament of stress fibers. Three lines of evidence suggest that RhoA is necessary for stress fiber formation in many cells. First, microinjection of Clostridium botulinum C3 exoenzyme, which inactivates RhoA (2, 4), into an epidermal or neuronal cell line results in complete loss of stress fibers within 5 min (20, 31). Second, microinjection of a constitutively active RhoA mutant into a fibroblast cell line induces stress fiber formation (28). Third, C3 exoenzyme pretreatment inhibits lysophosphatidic acid (LPA)- or thrombin-induced stress fiber formation in a fibroblast cell line (16).

The signaling pathways by which the heterotrimeric G proteins couple to the monomeric G proteins are known to be cell specific, and little information exists on the pathways mediating stress fiber formation in airway smooth muscle cells. Because muscarinic signaling pathways are important in determining airway smooth muscle tone and because the vast majority of the muscarinic receptors in airway smooth muscle are of the M2 muscarinic subtype, we evaluated the signaling pathways by which the M2 muscarinic receptor is linked to stress fiber formation in airway smooth muscle, as actin filaments in freshly dispersed airway smooth muscle cells are dynamically regulated (38). Using cultured human airway smooth muscle cells that mainly express M2 muscarinic receptors (42), we evaluated the ability of this receptor to alter actin filament reorganization. Subsequently, we evaluated the ability of pertussis toxin and C3 exoenzyme to block muscarinic receptor-mediated effects. Finally, we evaluated the role of tyrosine kinases, mitogen-activated protein (MAP) kinases, phosphatidylinositol 3-kinase (PI-3-kinase), and protein kinase C (PKC) enzymes in the pathway linking M2 muscarinic receptors to actin reorganization in airway smooth muscle cells. The present studies provide the first evidence that carbachol induces stress fiber formation in human airway smooth muscle cells by a signaling pathway that involves Gi proteins, Rho proteins, and tyrosine phosphorylation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Primary cultures of previously characterized human tracheal smooth muscle cells (42) were maintained in M-199 medium containing antibiotics (100 units/ml of penicillin G, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B) and 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2-95% air. They were plated on eight-well microscope slides (Nunc Chambers, Naperville, IL) and incubated until the cells achieved confluence. The cells were extensively washed and were maintained in serum-free M-199 medium for 48 h. Quiescent, serum-starved cells were stimulated with carbachol (100 µM) for 5 min.

Fluorescence microscopy. Cells were fixed in 3.7% fresh paraformaldehyde in PBS for 15 min. After two washes with PBS, excess aldehyde was quenched with 50 mM NH4Cl for 15 min. Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After treatment with blocking solution (1% BSA-0.1% Triton X-100 in PBS) for 10 min, cells were stained with FITC-labeled phalloidin (FITC-phalloidin; 1 µg/ml) in blocking solution for 20 min in a dark room at room temperature to localize F-actin and Texas Red-labeled DNase I (Texas Red-DNase I; 10 µg/ml) to localize monomeric (G) actin (22). Slides were washed with 0.1% Triton X-100 in PBS twice and PBS alone once, each for 5 min. Incubation and washing were performed in parallel for all wells on a slide. A coverslip was mounted on the slide with Vectashield H-1000 (Vector, Burlingame, CA). Actin was visualized with a fluorescence microscope (Olympus BHT; Tokyo, Japan), and the image was stored using Image-Pro Plus software (Medica Cybernetics, Silver Spring, MD) on a personal computer.

The fluorescence intensities of FITC-phalloidin and Texas Red-DNase I were simultaneously calculated from a view containing >15 cells. The excitation and emission wavelengths for FITC-phalloidin are 490 and 525 nm, respectively, whereas the excitation and emission wavelengths for Texas Red-DNase I are 596 and 615 nm, respectively. To standardize the fluorescence intensity measurements among experiments, the time of image capturing, the image intensity gain, the image enhancement, and the image black level in both channels were optimally adjusted at the outset and kept constant for all experiments. Images at maximum diameter were digitized (640 × 484 pixels) with eight-bit gray-level resolution of 0 (minimum) to 256 (maximum) intensity. Cumulative fluorescence intensities for FITC-phalloidin and Texas Red-DNase I were recorded using Image-Pro Plus software. An increase in the F- to G-actin ratio indicated an increase in stress fiber formation.

Pertussis toxin and C3 exoenzyme pretreatment. To determine whether the carbachol-induced increase in stress fiber formation involved Gi proteins, cells were incubated in M-199 medium with either pertussis toxin dissolved in H2O (100 ng/ml) or no treatment for 4 h, after which the cells were left untreated or treated with carbachol (100 µM) for 5 min in five separate experiments.

To determine whether the carbachol-induced increase in stress fiber formation involved Rho proteins, cells were incubated with C3 exoenzyme dissolved in H2O (10 µg/ml) or no treatment for 24 h in M-199 medium containing 10% serum and subsequently with 10 µg/ml of C3 exoenzyme for 48 h in serum-free medium for a total of 72 h of C3 exoenzyme treatment. Treatment of cultured cells for 72 h with 10 µg/ml of C3 exoenzyme has been shown to specifically block subsequent ADP ribosylation of Rho in Swiss 3T3 cells (43), in several tumor cell lines (39), and in a human monocytic cell line (1). Cells were either left untreated or were treated with carbachol (100 µM) for 5 min in five separate experiments.

Pretreatment with tyrosine kinase inhibitors, a MAP kinase inhibitor, a PI-3-kinase inhibitor, or a PKC inhibitor. To determine whether tyrosine kinases were necessary in carbachol-induced increases in stress fiber formation in human airway smooth muscle cells, cells were pretreated with either genistein (20 µM) or vehicle (DMSO; 0.05%) for 20 min in five experiments and either tyrphostin A23 (150 µM) or vehicle (DMSO; 0.05%) for 20 min in an additional five experiments before the addition of carbachol (100 µM) for 5 min. To determine whether MAP kinases were involved in the carbachol-induced increase in actin fiber formation, cells were pretreated with either PD-98059 (10 µM) or vehicle (DMSO; 0.05%) for 20 min before the addition of carbachol in five experiments. To determine whether PI-3-kinases were involved in the carbachol-induced increase in stress fiber formation, cells were pretreated with either wortmannin (500 nM) dissolved in H2O or no treatment for 20 min before the addition of carbachol in five experiments. To determine whether PKC was involved in the carbachol-induced increase in stress fiber formation, cells were exposed to either GF-109203X (100 nM) or vehicle (DMSO; 0.05%) for 20 min before stimulation by carbachol for 5 min in five additional experiments.

Statistical analysis of data. All data are presented as means ± SE. Data from individual representative experiments are displayed (see Figs. 1-3). The average values for all experiments are presented in the text (and see Fig. 4). Analysis of significance of changes was by one-way ANOVA followed by Fisher's protected least significant differences multiple comparison test using StatView software (Abacus Concepts, Berkeley, CA). P < 0.05 was considered significant.

Materials. Carbamylcholine chloride (carbachol), wortmannin, and FITC-phalloidin were obtained from Sigma (St. Louis, MO). C3 exoenzyme, genistein, tyrphostin A23, GF-109203X, and PD-98059 were purchased from Calbiochem (La Jolla, CA). Pertussis toxin was purchased from Lists Biological Laboratories (Campbell, CA). Texas Red-DNase I was obtained from Molecular Probes (Eugene, OR).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Stress fiber formation induced by carbachol. There was no autofluorescence seen in unlabeled cells. Figure 1 shows human airway smooth muscle cells labeled with FITC-phalloidin, which stains F-actin. In untreated cells (Fig. 1, a and b), few actin filaments were visible. However, after exposure to carbachol (100 µM) for 5 min, the number of actin filaments greatly increased, appearing as a dense network of parallel fibers distributed evenly throughout the cytoplasm (Fig. 1, c and d; n = 5 experiments). In preliminary experiments, pretreatment of cells with atropine (100 µM) completely blocked this effect of carbachol.


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Fig. 1.   Representative of 5 experiments showing increased filamentous (F) actin staining of carbachol-treated human airway smooth muscle cells labeled with FITC-phalloidin and visualized by fluorescence microscopy. Cells were serum starved for 48 h before carbachol treatment for 5 min. a and b: Untreated cells. c and d: Cells treated with 100 µM carbachol. +, Presence; -, absence. Original magnifications are shown at top. Bars = 25 µm.

Figure 2 is an example of dual-fluorescence labeling of human airway smooth muscle cells using FITC-phalloidin and Texas Red-DNase I to stain F-actin and G-actin, respectively. Untreated cells (control) displayed few F-actin filaments (Fig. 2a). G-actin was distributed throughout the cytoplasm, with some increase in perinuclear intensity (Fig. 2b). In response to carbachol, the intensity of F-actin staining increased throughout the cytoplasm (Fig. 2c), whereas the intensity of G-actin staining decreased throughout the cytoplasm (Fig. 2d; n = 5 experiments).


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Fig. 2.   Representative of 5 experiments showing dual-fluorescence labeling of F-actin and monomeric (G) actin in human airway smooth muscle cells using FITC-phalloidin and Texas Red-DNase I. Configuration of F- and G-actin in same cells was visualized with fluorescence microscopy. a and b: Untreated cells. c and d: Cells treated with 100 µM carbachol for 5 min. Note increased F-actin staining (c) and decreased G-actin staining (d) in cells treated with carbachol. Bars = 25 µm.

Inhibition of carbachol-induced stress fiber formation by pertussis toxin and C3 exoenzyme. To determine whether Gi and/or Rho proteins are intermediates in the signaling pathway leading from muscarinic-receptor activation by carbachol to increases in stress fiber formation in human airway smooth muscle cells, we evaluated the ability of pertussis toxin and C3 exoenzyme to inhibit stress fiber formation induced by carbachol. In the absence of either pertussis toxin or C3 exoenzyme, cells treated with carbachol showed increased F-actin staining (Fig. 3b) compared with cells not treated with carbachol (Fig. 3a). Carbachol, moreover, significantly increased the F- to G-actin ratio 3.6 ± 0.4-fold (n = 5 experiments, P < 0.05; Fig. 4). Pertussis toxin pretreatment alone (Fig. 3c) had no effect on F-actin staining or on the F- to G-actin ratio but greatly attenuated the ability of carbachol to increase F-actin staining (Fig. 3d). Moreover, pertussis toxin pretreatment significantly reduced the increase in the F- to G-actin ratio induced by carbachol from 3.6 ± 0.4- to 1.5 ± 0.2-fold (n = 5 experiments, P < 0.05; Fig. 4).


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Fig. 3.   Representative of 5 experiments demonstrating that pertussis toxin, C3 exoenzyme, and tyrosine kinase inhibitors block carbachol-induced increase in F-actin staining. Cells were stained with both FITC-phalloidin and Texas Red-DNase I. Untreated cells are shown at left (a, c, e, g, i, k, m, and o). Cells treated with 100 µM carbachol for 5 min are shown at right (b, d, f, h, j, l, n, and p). c and d: Pretreatment with pertussis toxin (PTX; 100 ng/ml) for 4 h. e and f: Pretreatment with C3 exoenzyme (C3; 10 µg/ml) for 72 h. g and h: Pretreatment with genistein (20 µM) for 20 min. i and j: Pretreatment with tyrphostin A23 (150 µM) for 20 min. k and l: Pretreatment with PD-98059 (10 µM) for 20 min. m and n: Pretreatment with wortmannin (500 nM) for 20 min. o and p: Pretreatment with GF-109203X (100 nM) for 20 min.


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Fig. 4.   F- to G-actin fluorescence intensity ratio. Fluorescence intensities of FITC-phalloidin and Texas Red-DNase I in same cells were calculated from a view containing >15 cells/group for each condition, and ratio (F- to G-actin intensity) was calculated. An increase in F- to G-actin ratio indicates stress fiber formation. Values are means ± SE from 5 experiments. * Significantly different from carbachol in absence of pretreatment.

C3 exoenzyme pretreatment alone had no effect on F-actin staining (Fig. 3e) or on the F- to G-actin ratio. In cells pretreated with C3 exoenzyme for 72 h and then challenged with carbachol, F-actin staining did not increase (Fig. 3f). Moreover, C3 exoenzyme pretreatment significantly reduced the increase in the F- to G-actin ratio induced by carbachol from 3.6 ± 0.4- to 1.7 ± 0.2-fold (n = 5 experiments, P < 0.05; Fig. 4).

Inhibition of carbachol-induced stress fiber formation by genistein and tyrphostin A23. To investigate whether tyrosine phosphorylation is required for carbachol-induced increases in stress fiber formation in human airway smooth muscle cells, we studied two protein kinase inhibitors with preferential specificity for tyrosine kinases, genistein, and tyrphostin A23. Pretreatment of cells for 20 min with either genistein (20 µM; Fig. 3g) or tyrphostin A23 (150 µM; Fig. 3i) alone had no effect on F-actin staining or on the F- to G-actin ratio. However, both genistein and tyrphostin inhibited carbachol-induced increases in stress fiber formation (Fig. 3, h and j) and significantly attenuated the increase in the F- to G-actin ratio induced by carbachol. Genistein reduced the carbachol-induced increase in the F- to G-actin ratio from 3.6 ± 0.4- to 1.8 ± 0.2-fold (n = 5 experiments, P < 0.05; Fig. 4), whereas tyrphostin pretreatment reduced the carbachol-induced increase in the F- to G-actin ratio from 3.4 ± 0.4- to 1.4 ± 0.1-fold (n = 5 experiments, P < 0.05; Fig. 4).

Effects of PD-98059, wortmannin, and GF-109203X on carbachol-induced increases in stress fiber formation. To investigate whether MAP kinases, PI-3-kinases, or PKC is required for carbachol-induced increases in stress fiber formation, we pretreated human airway smooth muscle cells with PD-98059, wortmannin, or GF-109203X for 20 min before carbachol exposure for 5 min or no carbachol treatment for the control cells. The inhibitors alone had no significant effect on either F-actin staining (Fig. 3, k, m, and o, respectively) or on the F- to G-actin ratio (Fig. 4). Pretreatment with the inhibitors of MAP kinase, PI-3-kinase, or PKC did not significantly inhibit the carbachol-induced increase in F-actin staining (Fig. 3, l, n, and p, respectively) or the F- to G-actin ratio (Fig. 4). In the cells pretreated with PD-98059, carbachol increased the F- to G-actin ratio 3.2 ± 0.5-fold (n = 5 experiments, P > 0.05); in the cells pretreated with wortmannin, carbachol increased the F- to G-actin ratio 3.2 ± 0.6-fold (n = 5 experiments, P > 0.05); and in the cells pretreated with GF-109203X, carbachol increased the F- to G-actin ratio 3.7 ± 0.7-fold (n = 5 experiments, P > 0.05). The increase in the F- to G-actin ratio was not significantly different from that induced by carbachol in the absence of pretreatment (3.6 ± 0.4-fold).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study demonstrated that activation of muscarinic receptors with carbachol led to reorganization of the actin cytoskeleton in airway smooth muscle cells. We identified several intermediate proteins that participated in this signaling pathway using bacterial toxins and exoenzymes specific for G proteins and inhibitors of protein kinases. Pretreatment of airway smooth muscle cells with pertussis toxin or C3 exoenzyme blocked the ability of muscarinic-receptor activation to induce stress fiber formation, indicating a role for pertussis toxin-sensitive heterotrimeric G proteins and monomeric G proteins of the Rho family in this pathway. Moreover, pretreatment with tyrosine kinase inhibitors blocked carbachol-induced stress fiber formation, whereas inhibitors of MAP kinase, PI-3-kinase, and PKC were without effect, indicating that tyrosine phosphorylation is necessary for this effect.

The present investigation used dual labeling with FITC-phalloidin and Texas Red-DNase I adapted from the method of Knowles and McCulloch (22) to image and quantify actin rearrangement in human airway smooth muscle cells. This method allows for simultaneous observation of the relative amounts and configuration of F- and G-actin in the same cell because the staining patterns of F-actin and G-actin are thought to be spatially separate and distinct (22). The labeling of F-actin with FITC-phalloidin and of G-actin with Texas Red-DNase I is known to be specific (22). In response to carbachol, F-actin reorganized into a networklike pattern, accompanied by an increase in F-actin fluorescence, a simultaneous decrease in G-actin fluorescence, and an increase in the F- to G-actin ratio.

Unlike intact airway smooth muscle cells, which express both M2 and M3 muscarinic receptors, the human cultured airway smooth muscle cells used in the present study express mainly M2 muscarinic receptors (42). Thus treatment of these cells with carbachol would be expected to activate mainly M2 muscarinic receptors, which are linked to Gi. Indeed, pertussis toxin pretreatment blocked the ability of carbachol to increase F-actin staining and increase the F- to G-actin ratio, suggesting that the M2 muscarinic receptor is linked to the formation of stress fibers through a Gi protein in human airway smooth muscle cells, since airway smooth muscle does not express Go proteins (10). Both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins have been shown to couple to stress fiber formation in cells activated with LPA, which couples to both Gi and Gq. Pertussis toxin inhibited LPA-induced stress fiber formation in IIC9 cells (15) but failed to suppress LPA-induced stress fiber formation in Swiss 3T3 cells (17, 23, 33) or phenylephrine-induced stress fiber formation in cardiac myocytes (35). Although carbachol did not significantly increase the F- to G-actin ratio in pertussis toxin-treated cells, the inhibition by pertussis toxin may not have been total. It is possible that higher concentrations of pertussis toxin or longer periods of incubation are needed to totally inhibit the Gi protein. It is also possible, but less likely, that carbachol could have activated a small number of residual M3 muscarinic receptors that could also be linked to stress fiber formation via the pertussis toxin-insensitive G protein Gq because carbachol produces only small increases in inositol phosphates in these cells (42). Nevertheless, our results clearly demonstrate that in human airway smooth muscle cells, a pertussis toxin G protein is involved in carbachol-induced stress fiber formation.

In this study, we used C3 exoenzyme to explore the possible role of Rho proteins in the carbachol-induced stress fiber formation in human airway smooth muscle cells. C3 exoenzyme ADP-ribosylates an asparagine residue at the codon 41 position and inactivates the protein (2). It is very specific for Rho proteins and is at least two orders of magnitude less active on the closely related Cdc42 and Rac proteins (33). A signaling role for RhoA in cytoskeletal organization was first suggested by Ridley and Hall (32, 33) and by Hall (16), who showed that in serum-starved Swiss 3T3 fibroblasts, activated RhoA induced stress fiber formation (28, 32), whereas ADP-ribosylated Rho inhibited serum- and LPA-induced stress fiber formation (33). Our results in human airway smooth muscle cells agree with the fibroblast model in that C3 exoenzyme inhibited carbachol-induced stress fiber formation, suggesting a role for Rho proteins in this response. However, carbachol-induced increases in stress fiber formation in the present study were linked to Rho via a pertussis toxin-sensitive G protein, whereas in the fibroblast model, LPA-induced stress fiber formation was linked to Rho via a pertussis toxin-insensitive G protein (23, 33, 41). Our results also differ from those of Schmidt et al. (36), who found that carbachol was linked to Rho by a pertussis toxin-insensitive G protein in human embryonic kidney cells overexpressing M3 muscarinic receptors. It is possible that in human airway smooth muscle cells, both pertussis toxin-sensitive and -insensitive G proteins couple to Rho in the pathway mediating agonist-induced stress fiber formation, but carbachol can only activate pertussis toxin-sensitive G proteins because the M3 muscarinic receptors are absent.

Inhibitors of several classes of protein kinases were evaluated for their ability to block the effect of carbachol on stress fiber formation in human airway smooth muscle cells. Two inhibitors of tyrosine kinases blocked this effect, whereas inhibitors of MAP kinase, PI-3-kinase, and PKC were without effect. Genistein has been shown in vitro to be a potent inhibitor of tyrosine kinases and to have little effect on the activity of other kinases at the concentration used in the present study (20 µM) (33). Tyrphostin is also tyrosine kinase specific and does not inhibit protein kinase A, PKC, or other serine/threonine kinases (24). We cannot determine from the present study whether tyrosine kinases are involved upstream and/or downstream from Rho in human airway smooth muscle cells. In this respect, the human airway smooth muscle cell resembles the fibroblast model (33) in that tyrosine phosphorylation is required for agonist-induced stress fiber formation.

Protein tyrosine phosphorylation is an important mechanism for regulating smooth muscle contraction. Tyrosine kinase inhibitors suppress agonist-induced contraction in many smooth muscle preparations (8, 26) including airway smooth muscle (5). Tyrosine phosphorylation via protein tyrosine kinases has been implicated in mechanisms that couple receptor activation to both increases in intracellular Ca2+ (6) and modulation of Ca2+ sensitivity (7, 37). The data from the present study in human airway smooth muscle cells are consistent with the data obtained by Chopra et al. (5) in that tyrosine kinase inhibitors suppressed the response. The airway smooth muscle of the rat bronchiole most likely contains both M2 and M3 muscarinic receptors, whereas the airway smooth muscle cells used in the present study contain mainly M2 receptors. One may speculate that the M2 receptor in airway smooth muscle couples to actin polymerization and cytoskeletal reorganization via tyrosine kinases, whereas the M3 receptor couples to activation of PKC via its classic coupling to the Gq-phospholipase C pathway.

This study provides the first evidence linking M2 muscarinic receptors to Rho proteins and to cytoskeletal reorganization in airway smooth muscle cells. Although the link between actin reorganization and muscle cell contraction is not fully understood, it is generally appreciated that reorganization of the cell cytoskeleton is necessary for cells to move or contract and that actin filaments in freshly dispersed airway smooth muscle cells are dynamically regulated (38). Thus, in addition to the inhibition of adenylyl cyclase activity and the inhibition of beta -adrenergic receptor-mediated airway smooth muscle relaxation, M2 muscarinic-receptor activation may contribute to Rho-mediated Ca2+-independent smooth muscle contraction.

    FOOTNOTES

Address for reprint requests: C. A. Hirshman, The Johns Hopkins School of Hygiene, Div. of Physiology, Rm. 7006, 615 N. Wolfe St., Baltimore, MD 21205.

Received 4 August 1997; accepted in final form 3 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aepfelbacher, M. ADP-ribosylation of rho enhances adhesion of U937 cells to fibronectin via the alpha 5beta 1 integrin receptor. FEBS Lett. 363: 78-80, 1995[Medline].

2.   Aktories, K. Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins. Mol. Cell. Biochem. 138: 167-176, 1994[Medline].

3.   Bussey, H. Cell shape determination: a pivotal role for Rho. Science 272: 224-225, 1996[Medline].

4.   Chardin, P., P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 8: 1087-1092, 1989[Abstract].

5.   Chopra, L. C., D. Hucks, C. H. C. Twort, and J. P. T. Ward. Effects of protein tyrosine kinase inhibitors on contractility of isolated bronchioles of the rat. Am. J. Respir. Cell Mol. Biol. 16: 372-378, 1997[Abstract].

6.   Di Salvo, J., S. R. Nelson, and N. Kaplan. Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium. Proc. Soc. Exp. Biol. Med. 214: 285-301, 1997[Abstract].

7.   Di Salvo, J., G. Pfitzer, and L. A. Semenchuk. Protein tyrosine phosphorylation, cellular Ca2+, and Ca2+ sensitivity for contraction of smooth muscle. Can. J. Physiol. Pharmacol. 72: 1434-1439, 1994[Medline].

8.   Di Salvo, J., A. Steusloff, L. Semenchuk, S. Satoh, K. Kolquist, and G. Pfitzer. Tyrosine kinase inhibitors suppress agonist-induced contraction in smooth muscle. Biochem. Biophys. Res. Commun. 190: 968-974, 1993[Medline].

9.   Emala, C. W., A. Aryana, M. A. Levine, R. P. Yasuda, S. A. Satkus, B. B. Wolfe, and C. A. Hirshman. Expression of muscarinic receptor subtypes and M2-muscarinic inhibition of adenylyl cyclase in lung. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L101-L107, 1995[Abstract/Free Full Text].

10.   Emala, C. W., J. Yang, C. A. Hirshman, and M. A. Levine. G protein subunits in lung cells. Life Sci. 55: 593-602, 1994[Medline].

11.   Felder, C. C. Muscarinic acetylcholine receptors: signal transduction through multiple effectors. FASEB J. 9: 619-625, 1995[Abstract/Free Full Text].

12.   Fernandes, L. B., A. D. Fryer, and C. A. Hirshman. M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle. J. Pharmacol. Exp. Ther. 262: 119-126, 1992[Abstract].

13.   Fryer, A. D., and E. E. El-Fakahany. Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists. Life Sci. 47: 611-618, 1990[Medline].

14.   Gong, M. C., K. Iizuka, G. Nixon, J. P. Browne, A. Hall, J. F. Eccleston, M. Sugai, S. Kobayashi, A. V. Somlyo, and A. P. Somlyo. Role of guanine nucleotide-binding proteins---ras-family or trimeric proteins or both---in Ca2+ sensitization of smooth muscle. Proc. Natl. Acad. Sci. USA 93: 1340-1345, 1996[Abstract/Free Full Text].

15.   Ha, K.-S., E.-J. Yeo, and J. H. Exton. Lysophosphatidic acid activation of phosphatidylcholine-hydrolysing phospholipase D and actin polymerization by a pertussis toxin-sensitive mechanism. Biochem. J. 303: 55-59, 1994[Medline].

16.   Hall, A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell Biol. 10: 31-54, 1994.

17.   Hill, C. S., J. Wynne, and R. Treisman. The Rho family GTPases rhoA, rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81: 1159-1170, 1995[Medline].

18.   Hirata, K., A. Kikuchi, T. Sasaki, S. Kuroda, K. Kaibuchi, Y. Matsuura, H. Seki, K. Saida, and Y. Takai. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J. Biol. Chem. 267: 8719-8722, 1992[Abstract/Free Full Text].

19.   Hotchin, N. A., and A. Hall. Regulation of the actin cytoskeleton, integrins and cell growth by the Rho family of small GTPases. Cancer Surv. 27: 311-322, 1996[Medline].

20.   Jalink, K., E. J. Van Corven, T. Hengeveld, N. Morii, S. Narumiya, and W. H. Moolenaar. Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J. Cell Biol. 126: 801-810, 1994[Abstract].

21.   Janmey, P. A. Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annu. Rev. Physiol. 56: 169-191, 1994[Medline].

22.   Knowles, G. C., and C. A. G. McCulloch. Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods. J. Histochem. Cytochem. 40: 1605-1612, 1992[Abstract/Free Full Text].

23.   Koch, W. J., B. E. Hawes, L. F. Allen, and R. J. Lefkowitz. Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by Gbeta gamma activation of p21ras. Proc. Natl. Acad. Sci. USA 91: 12706-12710, 1994[Abstract/Free Full Text].

24.   Levitzki, A. Tyrphostins---potential antiproliferative agents and novel molecular tools. Biochem. Pharmacol. 40: 913-918, 1990[Medline].

25.   Nobes, C. D., and A. Hall. Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility. Biochem. Soc. Trans. 23: 456-459, 1995[Medline].

26.   Ohanian, J., V. Ohanian, L. Shaw, C. Bruce, and A. M. Heagerty. Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries. Br. J. Pharmacol. 120: 653-661, 1997[Abstract].

27.   Otto, B., A. Steusloff, I. Just, K. Aktories, and G. Pfitzer. Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle. J. Physiol. (Lond.) 496: 317-329, 1996[Abstract].

28.   Paterson, H. F., A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall. Microinjection of recombinant p21 rho induces rapid changes in cell morphology. J. Cell Biol. 111: 1001-1007, 1990[Abstract].

29.   Post, G. R, and J. H. Brown. G protein-coupled receptors and signaling pathways regulating growth responses. FASEB J. 10: 741-749, 1996[Abstract/Free Full Text].

30.   Ridley, A. J. Signal transduction through the GTP-binding proteins Rac and Rho. J. Cell Sci. 18: 127-131, 1994.

31.   Ridley, A. J., P. M. Comoglio, and A. Hall. Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol. Cell. Biol. 15: 1110-1122, 1995[Abstract].

32.   Ridley, A. J., and A. Hall. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399, 1992[Medline].

33.   Ridley, A. J., and A. Hall. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J. 13: 2600-2610, 1994[Abstract].

34.   Roffel, A. F., C. R. S. Elzing, G. M. Van Amsterdam, R. A. deZeeuw, and J. Zaagsma. Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function. Eur. J. Pharmacol. 153: 73-82, 1988[Medline].

35.   Sah, V. P., M. Hoshijima, K. R. Chien, and J. H. Brown. Rho is required for Galpha q and alpha 1 adrenergic receptor signaling in cardiomyocytes. J. Biol. Chem. 271: 31185-31190, 1996[Abstract/Free Full Text].

36.   Schmidt, M., C. Bienek, U. Rumenapp, C. Zhang, G. Lummen, K. H. Jakobs, I. Just, K. Aktories, M. Moos, and C. Eichel-Streiber. A role for rho in receptor- and G protein-stimulated phospholipase C reduction in phosphatidylinositol 4,5-biphosphate by Clostridium difficile toxin B. Naunyn Schmiedebergs Arch. Pharmacol. 354: 87-94, 1996[Medline].

37.   Steusloff, A., E. Paul, L. A. Semenchuk, J. Di Salvo, and G. Pfitzer. Modulation of Ca2+ sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation. Arch. Biochem. Biophys. 320: 236-242, 1995[Medline].

38.   Tseng, S., R. Kim, T. Kim, K. G. Morgan, and C.-M. Hai. F-actin disruption attenuates agonist-induced [Ca2+], myosin phosphorylation, and force in smooth muscle. Am. J. Physiol. 272 (Cell Physiol. 41): C1960-C1967, 1997[Abstract/Free Full Text].

39.   Udagawa, T., and B. W. McIntyre. ADP-ribosylation of the G protein Rho inhibits integrin regulation of tumor cell growth. J. Biol. Chem. 271: 12542-12548, 1996[Abstract/Free Full Text].

40.   Van Biesen, T., L. M. Luttrell, B. E. Hawes, and R. J. Lefkowitz. Mitogenic signaling via G protein-coupled receptors. Endocr. Rev. 17: 698-714, 1996[Medline].

41.   Van Corven, E. J., P. L. Hordijk, R. H. Medema, J. L. Bos, and W. H. Moolenaar. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc. Natl. Acad. Sci. USA 90: 1257-1261, 1993[Abstract].

42.   Widdop, S., K. Daykin, and I. P. Hall. Expression of muscarinic M2 receptors in cultured human airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 9: 541-546, 1993[Medline].

43.   Yamamoto, M., N. Marui, T. Sakai, N. Morii, S. Kozaki, K. Ikai, S. Imamura, and S. Narumiya. ADP-ribosylation of the rho A gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle. Oncogene 8: 1449-1455, 1993[Medline].


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