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

Carol A. Hirshman and Charles W. Emala

Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Galpha i-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Galpha i-2 and Gqalpha were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Galpha i-2 pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Galpha i-2 or a Gqalpha pathway.

G protein; endothelin-1; carbachol; lysophosphatidic acid; antisense oligonucleotide; Galpha i-2; cell culture


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CYTOSKELETON of smooth muscle cells is a filamentous network consisting largely of filamentous actin (F-actin), which provides a scaffold on which motor proteins such as myosin translocate to generate internal stress and alter the mechanical properties of the cells. Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells (4, 12, 19, 25, 34) and Ca2+ sensitization (increased muscle tension under constant-calcium conditions) in intact smooth muscle preparations (4, 5, 9, 17, 27) by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. A downstream effector pathway linking Rho proteins to the actin cytoskeleton and Ca2+ sensitization has recently been elucidated (20). Rho-associated protein kinase, a product of activated Rho (Rho-GTP), phosphorylates and inactivates the myosin-binding subunit of myosin light chain phosphatase, thereby inhibiting myosin light chain dephosphorylation (20). As a result, the phosphorylated form of myosin light chain accumulates, leading to contraction of the actomyosin-based cytoskeleton and Ca2+ sensitization of smooth muscle preparations.

The signaling pathways upstream from Rho are cell-type specific and poorly understood, particularly in airway smooth muscle. Cholinergic agonists (1, 5, 8), endothelin-1 (5), and histamine (8) all sensitize intact airway smooth muscle preparations to Ca2+ via a Rho-mediated pathway (5). Lysophosphatidic acid (LPA), a lipid mediator which induces actin reorganization in Swiss 3T3 cells via a pathway involving Rho proteins (12) and stimulates mitogenesis in airway smooth muscle cells (3), enhances contractility of airway smooth muscle preparations in response to cholinergic stimulation (33). Airway smooth muscle preparations express M2 and M3 muscarinic cholinergic receptors (6, 30), endothelin type A (ETA) and type B (ETB) receptors (14, 39), and presumably at least one type of LPA receptor (3).

Heterotrimeric G proteins are the major upstream entity involved in Rho activation induced by agonists. These G proteins consist of an alpha -subunit and two smaller, tightly coupled subunits, beta  and gamma . The alpha -subunits are unique to each G protein, conferring functional specificity. The alpha -subunits are subdivided into four major families on the basis of their amino acid sequence homology: 1) Gsalpha and Golf; 2) Galpha i-1, Galpha i-2, Galpha i-3, Goalpha , Gzalpha , transducins 1 and 2, and gustducin; 3) Gqalpha , G11alpha , G14alpha , and G15alpha ; and 4) G12alpha and G13. Splice variants of Gsalpha , Galpha i-2, and Goalpha as well as additional subfamily members of alpha -subunits have recently been identified (16). In addition, five beta -subunits and at least 10 gamma -subunits have so far been described (16).

M3 muscarinic receptors couple to phospholipase C to produce increases in inositol trisphosphate and diacylglycerol via the heterotrimeric G protein Gq, whereas activation of M2 muscarinic receptors inhibits adenylyl cyclase via interaction with members of the pertussis toxin-sensitive G protein family Gi. ETA receptors are coupled to the inhibition of adenylyl cyclase via Gi (28) and to the production of inositol trisphosphate via Gq (36). LPA receptors couple to at least three G proteins, including Gq, which links the receptor to phospholipase C; Gi, which triggers Ras activation and adenylyl cyclase inhibition; and G12 /13, which mediates Rho activation (24).

Togashi et al. (34) recently demonstrated that carbachol exposure led to actin reorganization in human airway smooth muscle cells that express mainly M2 muscarinic receptors (37). Moreover, this actin reorganization was blocked by pretreatment with atropine, Clostridium botulinum C3 exoenzyme, or pertussis toxin (34), implicating muscarinic receptors, monomeric G proteins of the Rho family, and pertussis-sensitive heterotrimeric G proteins, respectively, in this pathway. In a subsequent study using antisense oligonucleotides designed to specifically bind to the mRNA encoding Galpha i-2, Galpha i-3, or Gqalpha , Hirshman et al. (18) showed that antisense oligonucleotide depletion of Galpha i-2 protein but not of Galpha i-3 or Gqalpha protein blocked carbachol-induced increases in actin reorganization in the same cells. These data indicate that Galpha i-2 proteins couple M2 muscarinic receptors to Rho proteins and actin reorganization in human airway smooth muscle cells.

The goal of the present study was to investigate whether receptors that couple to the G protein Gq also activate Rho and induce actin reorganization in human airway smooth muscle cells. Using cultured human airway smooth muscle cells that express endothelin, LPA, and M2 muscarinic receptors but not M3 muscarinic receptors, we evaluated the ability of carbachol, endothelin-1, and LPA to induce actin reorganization in these cells. Subsequently, we evaluated the ability of pertussis toxin and C3 exoenzyme to block the receptor-mediated effects. Finally, we used an antisense oligonucleotide approach capable of downregulating individual G protein alpha -subunits. Antisense oligonucleotides were designed to specifically bind to mRNA encoding Galpha i-2, Galpha i-3, and Gqalpha . We found that LPA and endothelin-1, like carbachol, induced actin reorganization. Moreover, pretreatment of the cells with C3 exoenzyme but not with pertussis toxin inhibited endothelin-1- and LPA-induced actin reorganization; and antisense oligonucleotide depletion of both Galpha i-2 and Gqalpha proteins but not either one alone inhibited endothelin-1- or LPA-induced actin reorganization in these cells. This study provides the first evidence that both Galpha i-2 and Gqalpha couple endothelin and LPA receptors to Rho proteins in human airway smooth muscle cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Primary cultures of previously characterized human tracheal smooth muscle cells (kindly provided by Dr. Ian Hall, Nottingham, UK) (13, 37) were maintained in medium 199 containing antibiotics (100 U/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. Preliminary immunohistochemical studies performed in our laboratory confirmed that >90% of the cells expressed alpha -actin. Moreover, immunoblot analysis of these cells identified expression of both alpha -actin and desmin, confirming the smooth muscle phenotype of the cells. The cells were plated on eight-well microscope slides (Nunc Chambers, Naperville, IL) and incubated until the cells achieved confluence. The cells were extensively washed and maintained in serum-free medium 199 for 48 h. Quiescent, serum-starved cells were stimulated with either 100 µM carbachol, 1 µM endothelin-1, or 1 µM LPA for 5 min. Because activation of the cytoskeleton by nonspecific stimuli is a major concern, physical manipulation of the slides was kept to a minimum. Termination of agonist activation was achieved by the addition directly to the medium of an equal volume of 7.4% fresh paraformaldehyde in PBS for 15 min.

In some experiments, the cells were pretreated with pertussis toxin to inactivate Gi proteins or with C3 exoenzyme to block Rho activation before exposure to receptor agonists. The cells were exposed to pertussis toxin (final concentration 100 ng/ml) in serum-free medium for 4 h at 37°C in a cell culture incubator, after which the cells were left untreated or treated with agonist for 5 min. Termination of agonist activation was achieved by the addition of an equal volume of 7.4% fresh paraformaldehyde in PBS for 15 min. C3 exoenzyme pretreatment was performed for 72 h. The cells were incubated with C3 exoenzyme (final concentration 10 µg/ml) for 24 h in medium 199 containing 10% serum and subsequently with 10 µg/ml of C3 exoenzyme for 48 h in serum-free medium. This pretreatment with C3 has been previously shown (34) to block carbachol-induced Rho activation in these cells. The cells were either left untreated or treated with agonist for 5 min. Termination of agonist activation was achieved by the addition directly to the medium of an equal volume of 7.4% fresh paraformaldehyde in PBS for 15 min.

In some experiments, the cells were treated with specific antisense oligonucleotides (final concentration 10 µM) directed against the G protein alpha -subunits Galpha i-2, Galpha i-3, or Gqalpha . The concentration and time of treatment (6 days) has been previously shown by us and others (18, 31, 32) to result in decreased expression of the respective G protein alpha -subunit by immunoblot analysis. These high concentrations of relatively small oligonucleotides are apparently taken up by cells in the absence of lipid or viral vectors, resulting in quantitative decreases in the respective targeted proteins (31, 32). In some experiments, the cells were treated with a combination of antisense oligonucelotides (Galpha i-2 and Gqalpha or Galpha i-3 and Gqalpha ). The oligonucleotide sequences were phosphorothioated at the first and last four nucleotides to impair intracellular degradation and enhance resistance to exo- and endonucleases. The oligonucleotides were commercially synthesized as follows: 5'-CTT GTC GAT CAT CTT AGA-3' for Galpha i-2, 5'-AAG TTG CGG TCG ATC AT-3' for Galpha i-3, and 5'-GCT TGA GCT CCC GGC GGG CG-3' for Gqalpha (18, 31). Antisense oligonucleotide pretreatment was performed for a total of 6 days. The medium was changed and the oligonucleotide was redosed every 2 days. The first 4 days of incubation were performed in medium 199 with 1% fetal bovine serum, whereas the last 2 days of incubation were performed in serum-free medium. This pretreatment has been shown to result in decreased protein expression of the respective G protein alpha -subunit in these cells (18).

Fluorescence microscopy. The staining protocol for F-actin and globular actin (G-actin) was a modification of previous methods of Knowles and McCulloch (21). Preliminary studies were performed comparing the methods of fixation (methanol versus paraformaldeyde) and the addition of fluorescent stains [FITC-labeled phalloidin (FITC-phalloidin) and Texas Red-labeled DNase I (Texas Red-DNase I)] sequentially or concurrently. Methanol fixation resulted in high background staining and was considered unsatisfactory as previously reported by Knowles and McCulloch. We obtained similar results when stains were added sequentially or concurrently. Therefore, the stains were added concurrently to ensure that all wells received identical concentrations of both stains. Preliminary studies also suggested that agonist-induced increases in F-actin staining was time dependent, and, therefore, the fixative was added immediately after 5 min of agonist exposure to stop the agonist effects equally in all wells. After fixation in 3.7% (final concentration) fresh paraformaldehyde in PBS for 15 min, the wells were washed twice with PBS, excess aldehyde was quenched with 50 mM NH4Cl for 15 min, and then the cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After treatment with blocking solution (1% BSA and 0.1% Triton X-100 in PBS) for 10 min, the cells were stained with 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-DNase I (10 µg/ml) to localize monomeric (G) actin (21). The slides were washed twice with 0.1% Triton X-100 in PBS and once with PBS alone, 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 Laboratories, Burlingame, CA). Actin was visualized with a fluorescence microscope (Olympus BHT, Tokyo, Japan), and the image was stored with 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. Measurements were taken from three fields for each treatment and averaged for a single data point. The excitation and emission wavelengths for FITC-phalloidin were 490 and 525 nm, respectively, whereas the excitation and emission wavelengths for Texas Red-DNase I were 596 and 615 nm, respectively. To standardize the fluorescence intensity measurements among experiments, the time of image capturing, image intensity gain, image enhancement, and image black level in both channels were optimally adjusted at the outset and kept constant for all experiments. Images at a maximum diameter were digitized (640 × 484 pixels) with an 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 with Image-Pro Plus software. An increase in the F- to G-actin ratio indicated an increase in actin reorganization.

Materials. Carbachol, LPA, endothelin-1, and FITC-phalloidin were obtained from Sigma (St. Louis, MO). Texas Red-DNase I was obtained from Molecular Probes (Eugene, OR). Phosphorothioate-modified oligonucleotides were obtained from GIBCO BRL (Life Technologies, Gaithersburg, MD). Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). C3 exoenzyme was purchased from Calbiochem (La Jolla, CA).

Statistical analysis of data. To control for day-to-day variations in staining intensity, untreated cells were always compared with treated cells on the same microscope slide because cells on the same slide undergo identical culture, fixation, permeabilization, staining, and microscopy conditions, allowing meaningful comparisons between samples. All data are presented as means ± SE. F- to G-actin ratios were compared by two-way ANOVA with Bonferroni posttest comparisons with Instat software (GraphPad, San Diego, CA). P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure of serum-deprived human airway smooth muscle cells to 100 µM carbachol, 1 µM endothelin, or 1 µM LPA for 5 min resulted in an increase in the FITC-phalloidin staining intensity of F-actin and a decrease in the Texas Red-DNase I staining intensity of G-actin (indicative of reorganization of G-actin into F-actin fibers) compared with that in the untreated cells (Fig. 1). The F- to G-actin fluorescent-staining ratio indicative of actin fiber reorganization significantly increased from 2.4 ± 0.3 in the untreated cells to 6.7 ± 0.8, 7.2 ± 0.8, and 7.4 ± 0.9 in the carbachol-, LPA-, and endothelin-1-treated cells, respectively (P < 0.001 for each agonist compared with control group; n = 14 experiments). The decrease in G actin staining intensity in response to carbachol is presented in Fig. 2.


View larger version (127K):
[in this window]
[in a new window]
 
Fig. 1.   Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate filamentous (F) actin fibers. Carbachol (100 µM; B), lysophaphatidic acid (LPA; 1 µM; C), or endothelin-1 (1 µM; D) for 5 min induced increased F-actin staining compared with that in untreated cells (A). Pretreatment with C3 exoenzyme (E) for 72 h blocked carbachol (F)-, LPA (G)-, or endothelin-1 (H)-induced increases in F actin staining.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2.   Representative photomicrographs of cultured human airway smooth muscle cells stained with Texas Red-DNase I to illustrate globular (G) actin fibers. Under unstimulated control conditions (A), heaviest staining occurred in perinuclear region, whereas carbachol (100 µM; B) decreased staining intensity.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Fluorescent-staining ratios of F- to G- (F/G) actin in cultured human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas Red-DNase I were measured in the same field in triplicate for each treatment. Carbachol, LPA, and endothelin-1 (n = 14 experiments) each increased ratio of F/G actin, indicating actin reorganization. P < 0.001 for each effector compared with control. C3 exoenzyme pretreatment (n = 5 experiments) blocked effect of all 3 agonists, indicating that small G protein Rho is an intermediate in the pathway. * P < 0.05 compared with no C3 pretreatment for each effector.



View larger version (119K):
[in this window]
[in a new window]
 
Fig. 4.   Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Carbachol (100 µM; B), LPA (1 µM; C), or endothelin-1 (1 µM; D) for 5 min induced increased F-actin staining compared with that in untreated cells (A). Four hours of pertussis toxin pretreatment (E) blocked carbachol (F)- but not LPA (G)- or endothelin-1 (H)-induced increases in F-actin staining.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Fluorescent-staining ratios of F/G actin in cultured human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas Red-DNase I were measured in the same field in triplicate for each treatment. Carbachol, LPA, or endothelin-1 (n = 14 experiments) each increased ratio of F/G actin, indicating actin reorganization. P < 0.001 for each effector compared with control. Pertussis toxin pretreatment (n = 5 experiments) blocked effect of carbachol but not of LPA or endothelin-1, indicating that Gi proteins are predominant intermediary heterotrimeric G proteins activated by carbachol, but that other or additional G proteins are intermediates for LPA and endothelin-1. * P < 0.05 for pertussis effect on carbachol only.



View larger version (95K):
[in this window]
[in a new window]
 
Fig. 6.   Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Cells were left untreated (control; A-D) or pretreated for 6 days with antisense oligonucleotides directed against Galpha i-2 (E-H). Subsequently, cells were left untreated (A and E) or were treated with carbachol (100 µM; B and F), LPA (1 µM; C and G), or endothelin-1 (1 µM; D and H) for 5 min before fixation and staining. Carbachol-induced increases in F-actin staining were blocked by pretreatment with Galpha i-2 antisense oligonucleotide, but LPA- and endothelin-1-induced increases were unaffected. P < 0.05 for carbachol compared with carbachol plus Galpha i-2 antisense oligonucleotide.



View larger version (102K):
[in this window]
[in a new window]
 
Fig. 7.   Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Cells were left untreated (control; A-D) or pretreated for 6 days with 2 different antisense oligonucleotides directed against Galpha i-2 and Gqalpha (E-H). Subsequently, cells were left untreated (A and E) or were treated with carbachol (100 µM; B and F), LPA (1 µM; C and G), or endothelin-1 (1 µM; D and H) for 5 min before fixation and staining. Carbachol-, LPA-, or endothelin-1-induced increases in F-actin staining were blocked by pretreatment with combined Galpha i-2 and Gqalpha antisense oligonucleotides. P < 0.05 for each effector compared with effector plus combined antisense oligonucleotide.

In separate experiments, pretreatment of the human airway smooth muscle cells with C3 exoenzyme for 72 h totally inhibited actin reorganization by either carbachol, endothelin-1, or LPA, indicating that Rho proteins are intermediates in the signaling pathway leading from cell surface receptors to actin reorganization. The F- to G-actin fluorescent-staining ratio averaged 2.5 ± 0.1 in the control cells, 1.9 ± 0.3 in the control cells pretreated with C3 exoenzyme, 4.6 ± 0.5 in the carbachol-treated cells, 2.6 ± 0.2 in the carbachol-treated cells pretreated with C3 exoenzyme, 5.7 ± 0.7 in LPA-treated cells, 2.8 ± 0.4 in LPA-treated cells pretreated with C3 exoenzyme, 5.9 ± 0.9 in endothelin-1-treated cells, and 2.7 ± 0.6 in endothelin-1-treated cells pretreated with C3 exoenzyme (P < 0.05 for C3 effect on each agonist; n = 5 experiments; Figs. 1 and 3).


View larger version (121K):
[in this window]
[in a new window]
 
Fig. 8.   Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Cells were left untreated (control; A-D) or were pretreated for 6 days with antisense oligonucleotides directed against Gqalpha (E-H). Subsequently, cells were left untreated (A and E) or were treated with carbachol (100 µM; B and F), LPA (1 µM; C and G), or endothelin-1 (1 µM; D and H) for 5 min before fixation and staining. Carbachol-, LPA-, or endothelin-1-induced increases in F-actin staining were unaffected by pretreatment with Gqalpha antisense oligonucleotide alone. P > 0.05 compared with no antisense oligonucleotide for each effector.

Pretreatment of airway smooth muscle cells with pertussis toxin for 4 h to inactivate the heterotrimeric G protein Gi led to the inhibition of carbachol- but not of endothelin-1- or LPA-induced actin reorganization. The F- to G-actin fluorescent-staining ratio averaged 2.9 ± 0.3 in the control cells, 2.9 ± 0.5 in the pertussis toxin-pretreated controls cells, 6.2 ± 1.2 in the carbachol-treated cells, 3.5 ± 0.5 in the carbachol-treated cells pretreated with pertussis toxin, 6.4 ± 1.6 in the LPA-treated cells, 5.7 ± 1.2 in the LPA-treated cells pretreated with pertussis toxin, 5.5 ± 0.9 in the endothelin-1-treated cells, and 5.5 ± 0.9 in the endothelin-1-treated cells pretreated with pertussis toxin (P < 0.05 for pertussis toxin effect on carbachol only; n = 5 experiments; Figs. 4 and 5). These data suggest that endothelin-1 and LPA couple to actin reorganization via a pathway independent of Gi proteins (e.g., Gq).


View larger version (117K):
[in this window]
[in a new window]
 
Fig. 9.   Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Cells were left untreated (control; A-D) or were pretreated for 6 days with 2 different antisense oligonucleotides directed against Gqalpha and Galpha i-3 (E-H). Subsequently, cells were left untreated (A and E) or were treated with carbachol (100 µM; B and F), LPA (1 µM; C and G), or endothelin-1 (1 µM; D and H) for 5 min before fixation and staining. Carbachol-, LPA-, or endothelin-1-induced increases in F-actin staining were unaffected by pretreatment with combined Galpha i-3 and Gqalpha antisense oligonucleotides. P > 0.05 compared with no antisense oligonucleotide for each effector.

To further characterize the heterotrimeric G proteins that are intermediates leading from receptor activation to actin reorganization, depletion of G protein alpha -subunits with antisense oligonucleotides was performed in a separate series of experiments. Pretreatment of the airway smooth muscle cells with 10 µM antisense oligonucleotide for 6 days resulted in a decrease in protein expression of the respective G protein alpha -subunit (18). Galpha i-2 antisense oligonucleotide pretreatment significantly blocked carbachol-induced actin reorganization but had no significant effect on either LPA- or endothelin-1-induced actin reorganization. The F- to G-actin fluorescent-staining ratio averaged 6.7 ± 0.8 in the carbachol-treated cells, 3.8 ± 0.07 in the carbachol-treated cells pretreated with Galpha i-2 antisense oligonucleotide, 7.2 ± 0.8 in the LPA-treated cells, 6.6 ± 2.3 in the LPA-treated cells pretreated with Galpha i-2 antisense oligonucleotide, 7.4 ± 0.9 in the endothelin-1-treated cells, and 7.1 ± 2.7 in the endothelin-1-treated cells pretreated with Galpha i-2 antisense oligonucleotide (P < 0.05 for Galpha i-2 antisense oligonucleotide effect on carbachol only; n = 3 experiments; Figs. 6 and 10). In contrast, pretreatment with both Galpha i-2 and Gqalpha antisense oligonucleotides blocked not only carbachol-induced actin reorganization but also LPA- and endothelin-1-induced actin reorganization. The F- to G-actin fluorescent-staining ratio averaged 6.7 ± 0.8 in the carbachol-treated cells, 3.8 ± 0.9 in the carbachol-treated cells pretreated with both Galpha i-2 and Gqalpha antisense oligonucleotides, 7.2 ± 0.8 in the LPA-treated cells, 4.0 ± 0.7 in the LPA-treated cells pretreated with both Galpha i-2 and Gqalpha antisense oligonucleotides, 7.4 ± 0.9 in the endothelin-1-treated cells, and 3.8 ± 0.4 in the endothelin-1-treated cells pretreated with both Galpha i-2 and Gqalpha antisense oligonucleotides (P < 0.05 for the combined Galpha i-2 and Gqalpha antisense oligonucleotide effect on each agonist; n = 5 experiments; Figs. 7 and 10). Neither pretreatment with Gqalpha antisense oligonucleotide alone (Figs. 8 and 10) nor the combination of Gqalpha and Galpha i-3 antisense oligonucleotides (Figs. 9 and 10) had a significant effect on actin reorganization induced by carbachol, LPA, or endothelin-1. The F- to G-actin fluorescent-staining ratio averaged 6.7 ± 0.8 in the carbachol-treated cells, 6.1 ± 1.0 in the carbachol-treated cells pretreated with Gqalpha antisense oligonucleotide, 8.0 ± 2.0 in the carbachol-treated cells pretreated with both Galpha i-3 and Gqalpha antisense oligonucleotides, 7.2 ± 0.8 in the LPA-treated cells, 6.9 ± 1.6 in the LPA-treated cells pretreated with Gqalpha antisense oligonucleotide, 9.1 ± 2.2 in LPA-treated cells pretreated with both Galpha i-3 and Gqalpha antisense oligonucleotides, 7.4 ± 0.9 in endothelin-1-treated cells, 6.6 ± 1.9 in endothelin-1-treated cells pretreated with Gqalpha antisense oligonucleotide, and 8.6 ± 2.8 in endothelin-1-treated cells pretreated with both Galpha i-3 and Gqalpha antisense oligonucleotides (n = 3 experiments). Antisense oligonucleotide pretreatment had no significant effect on the F- to G-actin ratios in the untreated (control) cells. The F- to G-actin fluorescent-staining ratios averaged 2.4 ± 0.3 in the untreated cells, 3.8 ± 0.7 in the untreated cells pretreated with Galpha i-2 antisense oligonucleotide, 2.6 ± 0.6 in the untreated cells pretreated Gqalpha antisense oligonucleotide, 2.4 ± 0.4 in the untreated cells pretreated with both Gqalpha and Galpha i-2 antisense oligonucleotides, and 3.9 ± 1.0 in the untreated cells pretreated with Gqalpha and Galpha i-3 antisense oligonucleotide (n = 3 experiments). Taken together, these data suggest that carbachol-induced actin reorganization couples predominantly through a Galpha i-2 pathway, whereas LPA- and endothelin-1-induced actin reorganization couples through both Galpha i-2 and Gqalpha pathways.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 10.   Fluorescent-staining ratios of F/G actin in cultured human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas Red-DNase I were measured in the same field in triplicate for each treatment. Effects of antisense oligonucleotides directed against Galpha i-2, Gqalpha , or Galpha i-3 or combined treatment with Gqalpha and Galpha i-2 or Gqalpha and Galpha i-3 were measured in presence of carbachol, LPA, or endothelin-1. Carbachol-induced increases in F/G actin ratio were blocked by pretreatment with antisense oligonucleotides against Galpha i-2 or combined treatment with antisense oligonucleotides against Galpha i-2 and Gqalpha . In contrast, LPA- or endothelin-1-induced increases in F/G actin ratios were only blocked by pretreatment with a combination of antisense oligonucleotides directed against Gqalpha and Galpha i-2. * P < 0.05 compared with no antisense oligonucleotide for each effector.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates for the first time that activation of receptors activated by either LPA or endothelin-1 led to actin reorganization in human airway smooth muscle cells. Pretreatment of these cells with C3 exoenzyme blocked the ability of these agonists to induce actin reorganization, indicating a role for the monomeric G protein Rho in this pathway. Moreover, the combined antisense oligonucleotide depletion of both the Gqalpha and Galpha i-2 proteins blocked LPA- and endothelin-1-induced actin reorganization, whereas antisense oligonucleotide depletion of the Gqalpha , Galpha i-2, or Galpha i-3 protein alone or the combined depletion of Gqalpha and Galpha i-3 was insufficient to block either LPA- or endothelin-1-induced actin reorganization in these cells. These data implicate for the first time that both Gqalpha and Galpha i-2 are linked to Rho in human airway smooth muscle cells.

The present investigation used dual labeling with FITC-phalloidin and Texas Red-DNase I adapted from the method of Knowles and McCulloch (21) to image and quantify actin reorganization in these 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- and G-actin are thought to be spatially separate and distinct. The labeling of F-actin with FITC-phalloidin and of G-actin with Texas Red-DNase I is known to be specific (21), although variations in staining intensity often occur between studies. Factors that contribute to different absolute values between experiments include the affinity (dictated by the stability of the fluorescent dyes in storage) of the phalloidin-FITC and Texas Red-DNase I conjugates, the amount of photobleaching that occurs during analysis and storage of slides, and the sensitivity of the camera settings. To control for these variations in staining intensity, untreated cells were always compared with treated cells on the same microscope slide because cells on the same slide undergo identical culture, fixation, permeabilization, staining, and microscopy conditions, allowing meaningful comparisons between samples. These fluorescent-staining probes for G- and F-actin do not allow us to differentiate between the different isoforms of actin that might be participating in the polymerization process.

The present study is an extension of previously published data from our laboratory demonstrating that in human airway smooth muscle cells, carbachol induces actin reorganization by a signaling pathway that involves Galpha i-2 (18) and Rho proteins (34). Pretreatment with either C3 exoenzyme, pertussis toxin (34), or antisense oligonucleotides directed against Galpha i-2 (18) almost totally blocked carbachol-induced actin reorganization because human airway smooth muscle cells used in this study express mainly M2 muscarinic receptors (37), in contrast to native airway smooth muscle that expresses both M2 and M3 muscarinic receptors.

The present study measuring the F- to G-actin ratios is consistent with previously published functional data from our laboratory demonstrating that in porcine tracheal smooth muscle, endothlin-1 and muscarinic agonists induce Ca2+ sensitization by a pathway involving Rho proteins (5). Pretreatment with C3 exoenzyme inhibited both acetylcholine- and endothelin-1-induced Ca2+ sensitization in porcine tracheal smooth muscle (5). Moreover, pretreatment with pertussis toxin inhibited acetylcholine- but not endothelin-1-induced Ca2+ sensitization (5) in the tracheal muscle tissue similar to that seen in human airway smooth muscle cells. The present results in airway smooth muscle cells agree with a study (22) in astrocytes showing that endothelin-1-induced actin reorganization was inhibited by C3 exoenzyme but not by pertussis toxin.

In the present study, results with antisense oligonucleotides directed against both Galpha i-2 and Gqalpha demonstrate that in human airway smooth muscle cells, endothelin receptors couple to both Galpha i-2 and Gqalpha . ETA and ETB receptors are known to couple to several heterotrimeric G proteins including Gi, Gs, and Gq (10). Endothelin-1 increases inositol trisphosphate via the ETA receptor in cultured rat (15) and canine (38) airway smooth muscle cells, presumably via Gq, and activates p21ras via a pertussis-sensitive G protein, presumably Gi, in human airway smooth muscle cells (7). The present study identifies a novel signaling pathway for endothelin-1 in airway smooth muscle cells: the activation of Rho with pathways mediated by either Gqalpha or Galpha i-2.

The lack of effect of antisense oligonucleotides directed against G protein alpha -subunits in the control cells demonstrates that human airway smooth muscle cells maintain some basal level of actin polymerization independent of heterotrimeric G proteins. This suggests that additional regulatory pathways dictate the state of actin polymerization in these cells and that activation of cell surface receptors coupled to heterotrimeric G proteins is only one of several pathways modulating actin polymerization in these cells.

The present results in airway smooth muscle cells agree with previously published studies in other cell types in which LPA induces actin reorganization by signaling pathways involving Rho proteins. This LPA-induced actin reorganization occurs in Swiss 3T3 (29), JIC9 (a subclone of Chinese hamster embryo fibroblasts) (11), and mouse NIE-115 neuroblastoma cells (23) and in rat-1 and hamster lung fibroblasts (35). The heterotrimeric G protein(s) used by LPA to induce Rho-mediated actin reorganization appear to be cell-type specific. LPA receptors have been shown to couple to Gq, Gi, and G12 /13. In fibroblasts, LPA-induced actin reorganization is inhibited by pertussis toxin (11, 35), suggesting that Gi couples LPA receptors to Rho-induced actin reorganization, whereas in Swiss 3T3 and neuronal cells, G12 /13 couples LPA receptors to Rho-induced actin reorganization (2, 24). The results from the present study differ from those of previously published studies (2, 11, 24, 35) in that either of two heterotrimeric G proteins, Gqalpha or Galpha i-2, is capable of coupling LPA receptors to Rho-induced actin reorganization in human airway smooth muscle cells. Our study agrees with a study by Nogami et al. (26), who showed that LPA-activated receptors coupled to both pertussis-sensitive and pertussis-insensitive pathways in human airway smooth muscle cells.

The ability of LPA and endothelin-1 to induce actin polymerization by both Gi and Gq implies signaling pathway redundancy in these cells. It is likely that activation of Gi and Gq also leads to activation of other signaling intermediates (characteristic of Gi and Gq, respectively) that are not redundant for these receptors. For example, LPA-induced activation of Gi may lead to inhibition of adenylyl cyclase, whereas its coupling to Gq may lead to activation of phospholipase C. These would be independent and nonredundant functions of LPA-receptor activation. It is possible that important cellular signaling pathways are redundant so that dysfunction of one pathway does not lead to cell death. Because cytoskeletal reorganization is a pivotal process in cell motility, division, secretion, and contraction, it is likely that redundant pathways are a cellular protective mechanism.

In conclusion, this study demonstrates that endothelin-1 and LPA induce actin reorganization in human airway smooth muscle cells via a pathway involving Rho proteins and that antisense oligonucleotide depletion of both Gqalpha and Galpha i-2 proteins significantly inhibited endothelin-1- and LPA-induced actin reorganization in these cells. This study provides the first evidence that Gqalpha as well as Galpha i-2 is linked to Rho proteins in human airway smooth muscle cells.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. A. Hirshman, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 West 168th St., P & S Box 46, New York, NY 10032.

Received 31 December 1998; accepted in final form 10 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akao, M., A. Hirasaki, K. A. Jones, G. Y. Wong, D. H. Bremerich, and D. O. Warner. Halothane reduces myofilament Ca2+ sensitivity during muscarinic receptor stimulation of airway smooth muscle. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L719-L725, 1996[Abstract/Free Full Text].

2.   Buhl, A. M., N. L. Johnson, N. Dhanasekaran, and G. L. Johnson. Galpha 12 and Galpha 13 stimulate rho-dependent stress fiber formation and focal adhesion assembly. J. Biol. Chem. 270: 24631-24634, 1995[Abstract/Free Full Text].

3.   Cerutis, D. R., M. Nogami, J. L. Anderson, J. D. Churchill, D. J. Romberger, S. I. Rennard, and M. L. Toews. Lysophosphatidic acid and EGF stimulate mitogenesis in human airway smooth muscle cells. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L10-L15, 1997[Abstract/Free Full Text].

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

5.   Croxton, T. L., B. Lande, and C. A. Hirshman. Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L748-L755, 1998[Abstract/Free Full Text].

6.   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 the relationship between M2 receptors and inhibition of adenylyl cyclase in lung. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L101-L107, 1995[Abstract/Free Full Text].

7.   Emala, C. W., F. Liu, and C. A. Hirshman. Gialpha but not Gqalpha is linked to activation of p21ras in human airway smooth muscle cells. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L564-L570, 1999[Abstract/Free Full Text].

8.   Gerthoffer, W. T. Agonist synergism in airway smooth muscle contraction. J. Pharmacol. Exp. Ther. 278: 800-807, 1996[Abstract].

9.   Gong, M. C., K. Iizuka, G. Nixon, J. P. Brown, A. Hall, J. F. Eccleston, M. Sugai, S. Kobayashi, A. 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].

10.   Goto, K., H. Hama, and Y. Kasuyu. Molecular pharmacology and pathophysiological significance of endothelin. Jpn. J. Pharmacol. 72: 261-290, 1996[Medline].

11.   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].

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

13.   Hall, I. P., and M. Kotlikoff. Use of cultured airway myocytes for study of airway smooth muscle. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L1-L11, 1995[Abstract/Free Full Text].

14.   Hay, D. W. P., M. A. Luttman, M. Pullen, and P. Nambi. Functional and binding characterization of endothelin receptors in human bronchus: evidence for a novel endothelin B receptor subtype? J. Pharmacol. Exp. Ther. 284: 669-677, 1998[Abstract/Free Full Text].

15.   Henry, P. J. Endothelin-1 (ET-1)-induced contraction in rat isolated trachea: involvement of ETA and ETB receptors and multiple signal transduction systems. Br. J. Pharmacol. 110: 435-441, 1993[Abstract].

16.   Hildebrandt, J. D. Role of subunit diversity in signaling by heterotrimeric G proteins. Biochem. Pharmacol. 54: 325-339, 1997[Medline].

17.   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].

18.   Hirshman, C. A., H. Togashi, D. Shao, and C. W. Emala. Galpha i-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L911-L916, 1998[Abstract/Free Full Text].

19.   Janmey, P. A. The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol. Rev. 78: 763-781, 1998[Abstract/Free Full Text].

20.   Kimura, K., M. Ito, M. Amano, K. Chihara, Y. Fukata, M. Nakafuku, B. Yamamori, J. Feng, T. Nakano, K. Okawa, A. Iwamatsu, and K. Kaibuchi. Regulation of myosin phosphatase by rho and rho-activated kinase (rho-kinase). Science 273: 245-248, 1996[Abstract].

21.   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].

22.   Koyama, Y., and A. Baba. Endothelin-induced cytoskeletal actin re-organization in cultured astrocytes: inhibiton by C3 ADP-ribosyltransferase. Glia 16: 342-350, 1996[Medline].

23.   Kranenburg, O., M. Poland, M. Gebbink, L. Ooomen, and W. H. Moolenaar. Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of Rho A. J. Cell Sci. 110: 2417-2427, 1997[Abstract/Free Full Text].

24.   Moolenaar, W. H., O. K. Kranenburg, F. R. Postma, and C. M. Zondag. Lysophosphatidic acid: G-protein signalling and cellular responses. Curr. Opin. Cell Biol. 9: 168-173, 1997[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.   Nogami, M., S. M. Whittle, D. J. Romberger, S. I. Rennard, and M. L. Toews. Lysophosphatidic acid regulation of cyclic AMP accumulation in cultured human airway smooth muscle cells. Mol. Pharmacol. 48: 766-773, 1995[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.   Ponicke, K. M., M. Vogelsang, M. Heinroth, K. Becker, O. Zolk, M. Bohm, H. Zerkowski, and O.-E. Brodde. Endothelin receptors in the failing and nonfailing human heart. Circulation 97: 744-751, 1998[Abstract/Free Full Text].

29.   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].

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

31.   Standifer, K. M., G. C. Ross, and G. W. Pasternak. Differential blockade of opioid analgesia by antisense oligodeoxynucleotides directed against various G protein alpha  subunits. Mol. Pharmacol. 50: 293-298, 1996[Abstract].

32.   Tang, T., J. G. Kiang, T. E. Cote, and B. M. Cox. Antisense oligodeoxynucleotide to the Gi2 protein alpha  subunit sequence inhibits an opioid-induced increase in the intracellular free calcium concentration in ND8-47 neuroblastoma x dorsal root ganglion hybrid cells. Mol. Pharmacol. 48: 189-193, 1995[Abstract].

33.   Toews, M. L., E. E. Ustinova, and H. D. Schultz. Lysophosphatidic acid enhances contractility of isolated airway smooth muscle. J. Appl. Physiol. 83: 1216-1222, 1997[Abstract/Free Full Text].

34.   Togashi, H., C. W. Emala, I. P. Hall, and C. A. Hirshman. Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L803-L809, 1998[Abstract/Free Full Text].

35.   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].

36.   Vogelsang, M., A. Broede-Sitz, E. Schafer, H.-R. Zerkowski, and O.-E. Brodde. Endothelin ET-A receptors couple to inositol phosphate formation and inhibition of adenylate cyclase in human right atrium. J. Cardiovasc. Pharmacol. 23: 344-347, 1994[Medline].

37.   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].

38.   Yang, C. M., Y.-L. Yo, R. Ong, and J.-T. Hsieh. Endothelin- and sarafotoxin-induced hydrolysis in cultured canine tracheal smooth muscle cells. J. Neurochem. 62: 1440-1448, 1994[Medline].

39.   Yoshimura, H., J. Nishimura, C. Sakihara, S. Kobayashi, S. Takahashi, and H. Kanaide. Expression and function of endothelins, endothelin receptors, and endothelin converting enzyme in the porcine trachea. Am. J. Respir. Cell Mol. Biol. 17: 471-480, 1997[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 277(3):L653-L661
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society