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

Carol A. Hirshman1,2, Hideaki Togashi1, Dan Shao1, and Charles W. Emala1

Departments of 1 Anesthesiology and 2 Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

G protein; fluorescein isothiocyanate-labeled phalloidin; Texas Red-labeled deoxyribonuclease I; antisense oligonucleotide

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MUSCARINIC SIGNALING PATHWAYS are important determinants of airway smooth muscle (ASM) tone. ASM expresses both M2 and M3 muscarinic receptors, with >80% of the receptors of the M2 subtype (4, 6, 7, 23). M3 muscarinic receptors couple to phospholipase C to increase inositol phosphate and diacylglycerol formation via the large G protein Gq. Activation of M2 muscarinic receptors inhibits adenylyl cyclase via interaction with the pertussis toxin-sensitive large G protein Gi. Traditionally, activation of M3 muscarinic receptors was thought to contract the muscle, whereas activation of M2 receptors was thought to inhibit relaxation. It is becoming increasingly clear that these G protein-coupled receptors can induce mitogenic signaling via the Ras superfamily of monomeric low-molecular-weight GTP-binding proteins, which regulate cell growth and differentiation, gene expression, actin cytoskeleton assembly, cell motility, and contractility (22, 27, 28).

Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins, are thought to play a pivotal role in determining cell shape (17) by inducing agonist-activated reorganization of the actin cytoskeleton in cells (2, 9, 12, 13, 19, 27) and potentiation of Ca2+-induced contraction or Ca2+ sensitization in intact smooth muscle (2, 8, 11, 21). A signaling pathway linking Rho proteins to the actin cytoskeleton and to Ca2+ sensitization has been elucidated. Rho activation increases the amount of phosphorylated myosin light chain. This results from a decrease in the dephosphorylation rate rather than from an increase in the phosphorylation reaction (20). Rho kinase, a product of activated Rho, was recently shown to inactivate the myosin-binding subunit of myosin phosphatase (14). Thus the increased amount of activated phosphorylated myosin binds to actin and results in actin reorganization and muscle contraction.

The signaling pathways upstream from Rho are cell-type specific. Large G proteins are the major upstream entity involved in Rho activation induced by agonists. These G proteins share a heterotrimeric structure consisting 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  chains 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 , transducin 1 and 2, and gustducin; 3) Gqalpha , G11alpha , G14alpha , and G15alpha ; and 4) G12alpha and G13alpha . Additional subfamily members of alpha -subunits, as well as splice variants of Gsalpha , Galpha i-2, and Goalpha , have recently been identified (10). In addition to this substantial variability in alpha -subunits, 5 beta -subunits and at least 10 gamma -subunits have so far been identified (10).

Using human ASM cells that express mainly M2 muscarinic receptors, Togashi et al. (27) recently demonstrated that carbachol exposure led to reorganization of the actin cytoskeleton and that this reorganization was blocked by pretreatment with atropine, Clostridium botulinum C3 exoenzyme, or pertussis toxin, implicating muscarinic receptors, monomeric G proteins of the Rho family, and pertussis-sensitive heterotrimeric G proteins, respectively, in this pathway. The present study was designed to determine which heterotrimeric G protein couples muscarinic receptors to cytoskeletal reorganization in human ASM cells. We used an antisense approach capable of selectively downregulating individual G protein alpha -subunits. Because M2 muscarinic receptors are known to couple to members of the Gi family of heterotrimeric G proteins and because both Galpha i-2 and Galpha i-3 are expressed in ASM (5) and inactivated by pertussis toxin, antisense oligonucleotides were designed to specifically bind to mRNA encoding Galpha i-2 or Galpha i-3. A randomly scrambled oligonucleotide and an antisense oligonucleotide designed to bind to mRNA encoding Gqalpha served as controls. We found that antisense depletion of Galpha i-2 protein but not of Galpha i-3 or Gqalpha protein inhibited carbachol-induced actin reorganization in human ASM cells. This study provides the first evidence that Galpha i-2 protein couples muscarinic receptors to cytoskeletal reorganization in human ASM cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture and carbachol stimulation. Primary cultures of previously characterized human tracheal smooth muscle cells (29) were a kind gift from Dr. Ian Hall (Nottingham, UK) and were maintained in M-199 medium 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, unless otherwise stated, at 37°C in an atmosphere of 5% CO2-95% air. They were plated on eight-well microscope slides (each well 9 × 9 mm; Nunc chambers, Naperville, IL) and incubated until the cells achieved confluence. The cells were extensively washed and were maintained in 200 µl/well of serum-free M-199 medium for 48 h. Quiescent, serum-starved cells were left untreated (control) or stimulated with carbachol (100 µM) for 5 min.

Fluorescence microscopy. Fluorescence microscopy was performed as previously described (27). In brief, cells were fixed for 15 min by the addition of fresh paraformaldehyde in phosphate-buffered saline (PBS) to the cells in M-199 medium to achieve a final paraformaldehyde concentration of 3.7%. After two washes with PBS, excess aldehyde was quenched with 50 mM NH4Cl for 15 min. After two 500-µl washes with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. Wells were rinsed with 0.1% Triton X-100 in PBS and then blocked (1% BSA 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 filamentous actin (F-actin) and Texas Red-labeled DNase I (Texas Red-DNase I; 10 µg/ml) to localize monomeric actin (G-actin) (15). Wells were washed twice for 5 min in 0.1% Triton X-100 in PBS and once for 5 min in PBS. 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 PC 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 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, we optimally adjusted, at the outset, the time of image capturing, the image intensity gain, the image enhancement, and the image black level in both channels and kept them 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 with Image-Pro Plus software. An increase in the F- to G-actin ratio indicated an increase in stress fiber formation.

Downregulation of G protein alpha -subunits by antisense oligonucleotides. To determine which G protein alpha -subunits were important in carbachol-induced increases in stress fiber formation in human ASM cells, we treated cells with specific antisense oligonucleotides at a concentration of 10 µM for a total of 6 days on the basis of a study by Tang et al. (26). Cells were incubated in M-199 medium in the presence of 1% fetal bovine serum for 4 days and no serum for an additional 2 days, with specific antisense oligonucleotides (10 µM) or no treatment for 6 days (redosed every 2 days), after which the cells were left untreated or treated with carbachol (100 µM) for 5 min in five separate experiments. Specific phosphorothioated antisense oligonucleotides were synthesized to bind to human mRNAs encoding the protein subunits of Galpha i-2, Galpha i-3 and Gqalpha (25). Oligonucleotide sequences were phosphorothioated at the first and last four bases to impair intracellular degradation and resistance to exo- and endonucleases. The oligonucleotides were 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, 5'-GCT TGA GCT CCC GGC GGG CG-3' for Gqalpha (25), and 5'-GGG GGA AGT AGG TCT TGG-3' as a nonspecific oligonucleotide (25). Six days of treatment with these antisense oligonucleotides has been shown to result in reduced protein expression of the respective G protein alpha -subunit (25). These sequences were originally designed against mouse or rat cDNA but have complete homology with the human sequences (18).

To ensure that antisense treatment did not result in nonspecific cell toxicity that would result in a generalized loss of cellular proteins, we performed cell counts after antisense treatments. Cells were trypsinized from wells on Nunc slides after 6 days of no treatment or treatment with each antisense oligonucleotide and were counted in a hemocytometer after Trypan blue staining.

Western blot analysis. Cells in parallel wells were harvested for immunoblot analysis of G protein alpha -subunit expression. Successful depletion of the G protein alpha -subunits was confirmed by Western blot analysis as previously described (5) with the use of polyclonal antisera specific for Galpha i-2, Galpha i-3, and Gqalpha obtained from NEN (Boston, MA). In brief, plasma membranes harvested from one Nunc well, prepared from untreated and antisense-treated human ASM cells, were solubilized in gel loading buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 5% beta -mercaptoethanol) and separated on 10% polyacrylamide gels. Proteins were electrophoretically transferred to polyvinylidene difluoride filters (Millipore, Bedford, MA), blocked for 1 h at 37°C in Tris-buffered saline (50 mM Tris, pH 7.5, 2 mM MgCl2, and 140 mM NaCl) containing 3% BSA, 0.1% Tween 20, and 0.02% NaN3, and then incubated overnight rocking at room temperature in diluted primary antisera in Tris-buffered saline containing 1% BSA, 0.05% Tween 20, 2% Nonidet P-40, and 0.02% NaN3. Primary antibody was detected with a goat anti-rabbit secondary antibody coupled to alkaline phosphatase that was reacted with a chemiluminescent substrate and exposed to film according to the manufacturer's protocol (Bio-Rad, Hercules, CA). Exposed film was scanned with a 1,200 dots/inch Vista Scan Scanner (Umax, Fremont, CA) coupled to a Power Computing (Round Rock, TX) personal computer. Band intensities were quantitated with Mac BAS software (version 2.2) from Fuji Photo.

In some experiments after the immunoblots were exposed to film, they were stripped of the original primary-secondary antibody complexes and reprobed with the primary antibody specific for a different G protein alpha -subunit. These experiments were conducted to ensure that the oligonucleotide antisense treatment resulted in specific reduction of only one G protein alpha -subunit and also to ensure that equal quantities of sample were loaded in each lane. To strip blots, the polyvinylidene difluoride membranes were washed (62.5 mM Tris, pH 6.8, 2% SDS, and 100 mM beta -mercaptoethanol) for 30 min at 65°C, rinsed (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20) and reblocked before incubation with primary antibody.

Materials. Carbamylcholine chloride (carbachol) 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 (Gaithersburg, MD).

Statistical analysis of data. All data are presented as means ± SE. Arbitrary band intensities were log transformed. Analysis of significance of changes was by two-tailed paired t-test. F- to G-actin ratios were compared with control ratios by two-way ANOVA with Bonferroni posttest comparisons with Instat software (Graph Pad, San Diego, CA). P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Carbachol induced actin reorganization in the presence of antisense for G protein alpha -subunits. Exposure of serum-deprived cultured human ASM cells to 100 µM carbachol for 5 min resulted in an increase in the FITC-phalloidin staining intensity of F-actin (Fig. 1, A and B) and a decrease in the Texas Red-DNase I staining intensity of G-actin. Prior treatment of cells for 6 days with phosphorothioate-modified antisense oligonucleotides directed against the mRNA encoding Galpha i-2 blocked the carbachol-induced stress fiber formation (Fig. 1C). In contrast, 6 days of treatment with antisense oligonucleotides directed against Galpha i-3 or Gqalpha or a nonspecific antisense oligonucleotide had no effect on the carbachol-induced stress fiber formation (Fig. 1, D-F, respectively). Carbachol induced an increase in the F- to G-actin ratio that was blocked with Galpha i-2 antisense treatment but was unaffected by treatment with Galpha i-3 or Gqalpha antisense oligonucleotides or a randomly scrambled antisense oligonucleotide (Fig. 2). Carbachol increased the F- to G-actin ratio from a control level of 1.43 ± 0.34 to 2.83 ± 0.64 (n = 5 experiments; P < 0.001). Six days of Galpha i-2 antisense treatment blocked this increase, with an F- to G-actin ratio of 1.80 ± 0.57 (n = 5 experiments; P = 0.34 compared with control cells). Six days of antisense treatment with Galpha i-3 or Gqalpha oligonucleotides or a randomly scrambled antisense oligonucleotide had no effect on the carbachol-induced increase in the F- to G-actin ratio, with ratios of 3.0 ± 0.61, 2.9 ± 0.61, and 2.4 ± 0.53, respectively, in five experiments (P < 0.01 compared with control cells for all three antisense oligonucleotides).


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Fig. 1.   Increased F-actin staining of carbachol-treated human airway smooth muscle (ASM) cells and effect of 6 days of pretreatment with antisense oligonucleotides directed against different G protein alpha -subunits (represents 5 experiments). A: untreated. B: treated with 100 µM carbachol for 5 min. C-F: pretreated for 6 days with 10 µM antisense oligonucleotide directed against Galpha i-2 (C), Galpha i-3 (D), or Gqalpha (E) or a random sequence of oligonucleotide (F) before treatment with 100 µM carbachol for 5 min. Carbachol significantly increased intensity of F-actin staining (B), and this was only attenuated by pretreatment with antisense oligonucleotide directed against Galpha i-2 (C).


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Fig. 2.   F- to G (F/G)-actin fluorescence intensity ratio. Fluorescence intensities of FITC-labeled phalloidin and Texas Red-labeled DNase I in the same cells were calculated from a view containing >15 cells/group for each condition, and F/G actin ratio was calculated. An increase in F/G actin ratio indicates stress fiber formation. Values are means ± SE from 5 experiments. Carbachol (Carb; 100 µM) induced a significant increase in F/G actin ratio that was attenuated by 6 days of pretreatment with 10 µM antisense oligonucleotide (AS) directed against Galpha i-2. * Significantly different from control, P < 0.01.

Immunoblot analysis of G protein alpha -subunit depletion by antisense oligonucleotide. To confirm that antisense oligonucleotide treatment resulted in a decrease in the respective G protein alpha -subunit protein, we performed immunoblot analysis on cultured human ASM cells treated for 6 days with specific G protein alpha -subunit antisense oligonucleotides. Six days of treatment with phosphorothioate-modified antisense oligonucleotides directed against the mRNA encoding Galpha i-2, Galpha i-3, or Gqalpha resulted in a significant reduction in the expression of the respective proteins as determined by immunoblotting (Fig. 3).


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Fig. 3.   Representative immunoblots of Galpha protein expression after 6 days of antisense oligonucleotide treatment in cultured human ASM cells. Confluent cells were treated for 6 days with 10 µM antisense oligonucleotide specifically directed against Galpha i-2, Galpha i-3, or Gqalpha . Cell lysates were subjected to immunoblot analysis with primary antisera specific for each G protein alpha -subunit. Protein expression of all 3 G protein alpha -subunits was decreased by specific antisense oligonucleotide treatment.

The band intensities of the Galpha i-2 protein decreased in cells treated for 6 days with Galpha i-2 antisense oligonucleotide from a control level of 3.2 ± 1.3 × 106 to 2.0 ± 0.85 × 106 counts (n = 5 experiments; P = 0.0067). The band intensities of the Gqalpha protein decreased in cells treated for 6 days with Gqalpha antisense oligonucleotide, from a control level of 1.2 ± 0.14 × 106 to 0.83 ± 0.13 × 106 counts (n = 4 experiments; P = 0.01). Band intensities for the Galpha i-3 protein were greatly reduced compared with band intensities for Galpha i-2 or Gqalpha and required the use of cells from eight wells (each well, 9 × 9 mm) of the Nunc slide for Galpha i-3 immunoblots compared with cells from only one well for the detection of Galpha i-2 or Gqalpha . Even with this eightfold increase in cell numbers, immunoblot detection of Galpha i-3 was faint, preventing quantitative measurement of band intensities despite an obvious visual decrease in band intensities in lanes treated with Galpha i-3 antisense (n = 4 experiments).

To ensure that the antisense oligonucleotide treatment was specific for the G protein alpha -subunit it targeted and that equivalent amounts of protein were loaded into each lane, after the detection of a decrease in Galpha i-2 protein after Galpha i-2 antisense oligonucleotide treatment, we stripped immunoblots and reprobed them with primary antibody specific for the Gqalpha protein. The same samples that had shown a decrease in Galpha i-2 protein after Galpha i-2 antisense oligonucleotide treatment showed no significant difference in the amount of Gqalpha protein (0.33 ± 0.02 × 106 counts in control vs. 0.32 ± 0.04 × 106 counts in Galpha i-2 antisense treated). These studies confirm the specificity of the Galpha i-2 antisense treatment. Similarly, the same samples that had shown a decrease in Gqalpha protein after Gqalpha antisense oligonucleotide treatment showed no significant difference in the amount of Galpha i-2 protein (1.3 ± 0.8 × 106 counts in control vs. 1.2 ± 0.8 × 106 counts in Galpha i-2 antisense treated; n = 3 experiments; P = 0.93), confirming the specificity of the Gqalpha antisense treatment.

Cell viability assays to detect cytotoxic effect of antisense oligonucleotide treatment. No visible change in cell density was apparent by F- and G-actin staining in cells pretreated with antisense oligonucleotides. To confirm that antisense oligonucleotide treatment did not result in cell death, which could account for a reduced amount of immunoreactive protein, we performed trypan blue exclusion assays on cells treated with each antisense oligonucleotide and compared them with untreated cells.

No difference was seen in total cell counts after antisense oligonucleotide treatment: control, 8,300 ± 475 cells/well; Galpha i-2, 8,900 ± 229 cells/well (n = 4 experiments; P = 0.56 vs. control); Galpha i-3, 7,800 ± 770 cells/well (n = 4 experiments; P = 0.23 vs. control); and Gqalpha , 8,400 ± 550 cells/well (n = 5 experiments; P = 0.46 vs. control).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study demonstrates that Galpha i-2 protein couples muscarinic receptors to stress fiber formation in human ASM cells. Antisense depletion of the Galpha i-2 protein but not Galpha i-3 or Gqalpha protein blocked the carbachol-induced increase in the F- to G-actin ratio. The present investigation used dual labeling with FITC-phalloidin and Texas Red-DNase I, adapted by our laboratory (27) from the method of Knowles and McCulloch (15), to image and quantify stress fiber formation. These data extend previously published data from our laboratory (27) demonstrating that M2 muscarinic receptor activation induces stress fiber formation via a pathway involving a pertussis-sensitive G protein and Rho proteins in these cells. Unlike intact ASM cells that express both M2 and M3 muscarinic receptors, the human cultured ASM cells used in this study express mainly M2 muscarinic receptors (29). Thus treatment of these cells with carbachol would be expected to activate mainly M2 muscarinic receptors, which are known to couple to pertussis-sensitive G proteins of the Gi family.

Exposure of human ASM cells to antisense oligonucleotides directed against the alpha -subunits of Galpha i-2, Galpha i-3, and Gqalpha resulted in specific decreases in alpha -subunit protein expression. This indicated that the alpha -subunit antisense oligonucleotides, which had been modified with phosphorothioate groups at the two ends of each nucleotide, were able to enter the cells and were not significantly degraded. The mechanisms by which oligonucleotides are taken up by cells are not well understood. Both passive diffusion and active transport have been described (1). The sequences of Galpha i-2, Galpha i-3, and Gqalpha used in our study, as used by Standifer et al. (25), are complementary to translated regions of Galpha i-2, Galpha i-3, and Gqalpha mRNAs and have very limited homology between alpha -subunits. The 30-40% decrease in levels of alpha -subunit membrane protein observed after 6 days of exposure to antisense oligonucleotides is consistent with the slow turnover of alpha -subunit proteins demonstrated in the membranes of other cell types (3, 16, 24, 26).

It is unlikely that the decreased levels of alpha -subunit protein observed after specific antisense oligonucleotide treatment resulted from a decrease in cell number due to cell death in culture. No visible change in cell density was apparent in cells treated with antisense oligonucleotides nor did total cell counts decrease. Furthermore, the decrease in alpha -subunit protein level was seen only after treatment with each specific G protein alpha -subunit antisense oligonucleotide. G protein alpha -subunit expression did not change when immunoblots, which had previously shown decreased alpha -subunit expression after specific antisense oligonucleotide treatment, were stripped and reprobed with primary antibody directed against other G protein alpha -subunits, indicating that the treatment was indeed specific for the G protein alpha -subunit that it targeted and that equivalent amounts of protein had been loaded into each lane.

In the immunoblot analysis of G protein alpha -subunits, band intensities for the Galpha i-3 protein were so greatly reduced compared with the band intensities of the Galpha i-2 and Gqalpha protein in human ASM cells that detection required the use of eight times more cells. This likely represents a lower level of expression of Galpha i-3 than of Galpha i-2 or Gqalpha in these cells or a lower affinity of Galpha i-3 for the antibody used to detect the protein.

Pretreatment of human ASM cells with oligonucleotide antisense to Galpha i-2 produced a partial inhibition of carbachol-induced stress fiber formation, consistent with an incomplete loss of Galpha i-2 membrane protein.

Application of antisense techniques to other cell types has been used to investigate the relationship of heterotrimeric G protein alpha -subunits to receptors and second messengers. For example, de Mazancourt et al. (3) showed that Galpha i-3 couples galanin receptors to inhibition of adenylyl cyclase activity in rat insulinoma cells, and Tang et al. (26) showed that Galpha i-2 couples opioid receptors to increases in intracellular free Ca2+ in ND8-47 neuroblastoma cells.

The present study does not determine which pertussis-sensitive G proteins couple muscarinic M2 receptors to other signaling pathways in human ASM cells. Moreover, our study does not address the question of whether Galpha i-2 mediates Rho activation and stress fiber formation by agents other than muscarinic agonists in these cells.

In summary, this study shows that antisense depletion of Galpha i-2 protein but not of Galpha i-3 or Gqalpha protein significantly inhibited carbachol-induced stress fiber formation in human ASM cells. This study provides the first evidence that Galpha i-2 couples M2 muscarinic receptors to Rho proteins in human ASM cells.

    ACKNOWLEDGEMENTS

We thank Ian Hall for kindly providing the human cultured airway smooth muscle cells used in this study.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-58519 and HL-62340.

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

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.

Received 27 March 1998; accepted in final form 22 July 1998.

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Abstract
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Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(5):L911-L916
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