Gialpha but not Gqalpha is linked to activation of p21ras in human airway smooth muscle cells

Charles W. Emala, Feng Liu, and Carol A. Hirshman

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airway smooth muscle hypertrophy contributes to the narrowing of asthmatic airways. Activation of the mitogen-activated protein kinases is an important event in mediating cell proliferation. Because the monomeric G protein p21ras is an important intermediate leading to activation of mitogen-activated protein kinases, we questioned which heterotrimeric G protein-coupled receptors were linked to the activation of p21ras in cultured human airway smooth muscle and which of the heterotrimeric G protein subunits (alpha  or beta gamma ) transmitted the activation signal. Carbachol and endothelin-1 increased GTP-bound p21ras in a pertussis toxin-sensitive manner [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): control, 30 ± 1.7; 3 min of 1 µM carbachol, 39 ± 1.1; 3 min of 1 µM endothelin-1, 40 ± 1.2], whereas histamine, bradykinin, and KCl were without effect. Transfection of an inhibitor of the G protein beta gamma -subunit [the carboxy terminus (Gly495-Leu689) of the beta -adrenoceptor kinase 1] failed to inhibit the carbachol-induced activation of p21ras. These data suggest that Gi- but not Gq-coupled receptors activate p21ras in human airway smooth muscle cells, and this effect most likely involves the alpha -subunit.

G proteins; carbachol; endothelin; epidermal growth factor; bradykinin; histamine; pertussis toxin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INCREASED MASS of the smooth muscle lining the airway (5, 11) and airway hyperresponsiveness are characteristic features of human asthma, and increased airway smooth muscle DNA synthesis is seen in a number of animal models of airway hyperresponsiveness (12, 14, 21). The association between increased airway smooth muscle mass and airway responsiveness suggests that enhanced airway smooth muscle proliferation may be involved in the pathogenesis of the disease. However, the signaling pathways that regulate the growth and development of airway smooth muscle cells to various stimuli have not been well characterized.

A number of signal transduction pathways leading to the activation of mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs) have been implicated in the control of cell proliferation in airway smooth muscle cells. These include growth factors that stimulate receptor tyrosine kinases (15) and contractile agonists that signal through G protein-coupled receptors (13, 19). The major pathway involved in MAP kinase or ERK activation by growth factors requires the activation of p21ras (4), a membrane-bound 21-kDa monomeric G protein. Heterogeneity exists in the mechanisms by which G protein-coupled receptors activate MAP kinases. Depending on the receptor being activated and on the cell type, MAP kinase activation may be mediated by pertussis toxin-sensitive (1, 27) or pertussis toxin-insensitive G proteins (10) and be either protein kinase C (PKC) (10, 13) or p21ras dependent (1, 25, 27). In airway smooth muscle cells, a major pathway by which agonists activate MAP kinases is pertussis toxin insensitive and is mediated by PKC without the involvement of p21ras (15).

In native airway smooth muscle cells, carbachol activates M2 and M3 muscarinic receptors to activate Gi and Gq pathways, respectively. However, the cultured airway smooth muscle cells used in this study predominantly express M2 muscarinic receptors (26). These M2 muscarinic receptors and endothelin receptors are coupled to the pertussis toxin-sensitive G protein Gi, whereas histamine H1, bradykinin, and endothelin receptors are coupled to Gq (9). Agonists that activate these receptors are present in the airway of humans in both the presence and absence of disease. Acetylcholine is released from parasympathetic postganglionic nerves, histamine and bradykinin are potent inflammatory mediators thought to be important in asthma and allergy, and endothelin-1 is found in increased concentrations in the airways of humans with asthma (22).

Because contractile agonists that elicit proliferative responses in airway smooth muscle cells in culture can be coupled to either Gq or Gi, because MAP kinases are important regulators of cell proliferation, and because p21ras is an important intermediate in MAP kinase activation, we questioned whether stimulation of receptors coupled to Gi and/or Gq induced p21ras activation in human airway smooth muscle cells, and if so, which G protein subunits were involved. We therefore tested the effects of contractile agonists coupled to Gi (carbachol and endothelin) and Gq (histamine, endothelin, and bradykinin) on p21ras activation in human airway smooth muscle cells in the presence and absence of pertussis toxin and a G protein beta gamma -subunit inhibitor.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and 32P loading. Primary cultures of previously characterized human tracheal smooth muscle cells (26) 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 (FBS), unless otherwise stated, at 37°C in an atmosphere of 5% O2-95% air. Cells were grown to confluence in six-well culture plates and used between passages 6 and 12. Confluent cells were rinsed once and cultured overnight in serum-free and phosphate-free DMEM (2 ml medium/well). 32P loading of cells was performed the following day for 4 h by adding 30 µl of phosphorus-32 (9,000 Ci/mmol, 10 mCi/ml) to each well. Medium was then removed, and wells were rinsed once with serum-free and phosphate-free DMEM. One milliliter of serum-free and phosphate-free DMEM was then added to each well, and the indicated effectors were added to wells for the indicated times. Cells were untreated (control) or treated with epidermal growth factor (EGF; 100 ng/ml) for 5 min (positive control) or 1 µM carbachol, 1 µM endothelin-1, 1 µM histamine, or 1 µM bradykinin for 3 min. KCl (40 mM) was used in some experiments to determine whether elevation of cellular calcium alone was sufficient to activate Ras. In some experiments, cells were pretreated with pertussis toxin (100 ng/ml for 4 h) before treatment with carbachol or endothelin-1. Reactions were terminated by the removal of medium and the immediate addition of 500 µl of cold lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 16 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and 10 µg/ml of aprotinin) containing 10 µg/ml of p21ras primary antibody [anti-v-H-ras (Ab-1)]. Plates were incubated on ice for 30 min. Cell lysates were scraped from wells into microcentrifuge tubes and centrifuged in a microcentrifuge for 10 min at 16,000 g at 4°C. Supernatants were transferred to a clean microcentrifuge tube, and an additional 5 µg of p21ras primary antibody were added to each tube and allowed to incubate for 1 h at 4°C. Protein G Sepharose beads were diluted 1:1 with lysis buffer without antibody, and 100 µl of these diluted beads were added to each tube and allowed to incubate for 1 h with gentle mixing at 4°C. Tubes were centrifuged at 82 g for 1 min at 4°C to collect the beads, which were then washed three times for 1 min in 1 ml of washing buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 16 mM MgCl2, and 1% Nonidet P-40). Beads were transferred to a clean tube and washed once more, removing the final supernatant completely. Twenty microliters of elution buffer (2 mM EDTA, 0.2% SDS, and 2 mM 1,4-dithiothreitol) were added to each tube, and tubes were then boiled for 3 min. Beads were pelleted by centrifugation at 16,000 g for 10 min at room temperature. Supernatants were transferred to a new microcentrifuge tube and stored at -20°C until immunoprecipitated [32P]GDP and [32P]GTP were separated and quantitated by thin-layer chromatography (TLC).

TLC was performed on 20 × 20-cm cellulose PEI-F Baker-flex sheets that had been prerun in deionized distilled water and allowed to completely air-dry. Two-microliter aliquots of each sample were counted in a scintillation counter so that equal amounts of radioactivity for each sample could be loaded on TLC plates. Typically, 500 counts/min of each sample in a final volume of 10 µl of elution buffer were spotted 3 cm from the bottom of the TLC plates. Plates were placed in 100 ml (~0.5 cm deep) of tank buffer (0.75 M H2KPO4, pH 3.4) in a glass tank (25 × 27 × 7 cm) with a glass lid. Migration of the tank buffer up the plate was allowed to continue until it reached the top of the plate (~2 h). TLC plates were removed from the tanks, allowed to air-dry, wrapped in plastic wrap, and exposed to Fuji phosphorimager plates overnight. Exposed phosphorimager plates were read in a Fujix BAS 1000 phosphorimager (Fuji Medical Systems, Stanford, CT). The relative intensities of separated [32P]GDP and [32P]GTP were quantitated from these phosphorimages with Mac BAS 2.0 software (Fuji Medical Systems). Data are expressed as the ratio of [32P]GTP to ([32P]GTP + [32P]GDP).

Transfection with pRK-beta -adrenoceptor kinase 1 fragment. Transient expression of the carboxy terminus (Gly495-Leu689) of the beta -adrenoceptor kinase (beta -ARK) 1, also known as the G protein-coupled receptor kinase (GRK) 2, functions as a cellular antagonist for liberated G protein beta gamma -subunits and allows one to distinguish between G protein alpha - and beta gamma -mediated processes (16, 27). The day before transfection, human airway smooth muscle cells were seeded at 70% confluency in six-well plates containing M-199 medium and 10% FBS without antibiotics. Two micrograms of plasmid DNA [pRK-beta -ARK1 or the control plasmid pRK5 lacking an insert (16, 17)] were diluted in 50 µl of M-199 medium. In a separate tube, 10 µg (5 µl) of lipofectamine (GIBCO BRL, Gaithersburg, MD) were diluted in 50 µl of M-199 medium. We slowly pipetted the diluted lipofectamine into the diluted DNA, avoiding excessive agitation. We added 1 µl (1.1 × 109 plaque-forming units) of the replication-deficient adenovirus (Ad5CMVntlacZ) to each tube, gently tapping the tube to mix. The DNA-lipofectamine-virus mixture was allowed to incubate at room temperature for 45 min. Just before transfection, the cells were washed twice with serum-free and antibiotic-free M-199 medium. Five milliliters of serum-free and antibiotic-free M-199 medium were added to each well of the six-well plate. The DNA-lipofectamine-virus mixture was added to the well and incubated at 37°C in an atmosphere of 5% CO2-95% air for 4 h. The transfection mixture was removed, and the cells were rinsed once and then incubated in 5 ml of M-199 medium and 10% FBS. The medium was replaced the following day with M-199 medium with 10% FBS and antibiotics. For immunoblot analysis, cells were incubated for 72 h before being harvested. For p21ras assays, the medium was replaced at 48 h with DMEM without phosphate or serum for assays to be performed at 72 h posttransfection.

Immunoblot analysis of transfected cells. To confirm that transfection was successful, immunoblot analysis was performed on lysed cells that had been transfected with the beta -ARK1 minigene or sham transfected with the control plasmid (pRK5) or were untransfected (control). Seventy-two hours posttransfection, cells in representative wells of six-well plates were rinsed with M-199 medium and then lysed in 0.5 ml of Laemmli buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 5% beta -mercaptoethanol). Aliquots (100 µl) were boiled, subjected to electrophoresis through discontinuous SDS-12% polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes, and immunoblotted as previously described (6) with a rabbit polyclonal primary antibody raised against the carboxy terminus (residues 675-689) of beta -ARK1 (or GRK2) and a secondary antibody coupled to alkaline phosphatase. Immunoreactive bands were identified by chemiluminescence according to the manufacturer's protocol (ImmunoLite II, Bio-Rad, Hercules, CA) and exposed to autoradiographic film.

Materials. Cell culture media, serum, and antibiotics were purchased from GIBCO BRL. Phosphorus-32 (9,000 Ci/mmol, 10 mCi/ml) was purchased from NEN (Boston, MA). Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). The primary antibody to p21ras [anti-v-H-ras (Ab-1)] was purchased from Calbiochem (La Jolla, CA), the primary antibody specific for the beta -ARK1 fragment (GRK2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and protein G Sepharose 4 Fast Flow beads were purchased from Pharmacia Biotech (Piscataway, NJ). TLC plates (20 × 20-cm cellulose PEI-F Baker-flex) were purchased from VWR Scientific Products (Baltimore, MD). pRK-beta -ARK1 minigene and the pRK5 control plasmid were generous gifts from Walter J. Koch (Duke University, Durham, NC) (16, 17). The replication-deficient adenovirus Ad5CMVntlacZ was purchased from the Gene Transfer Vector Core at the University of Iowa (Iowa City, IA). Cultured human airway smooth muscle cells were a kind gift from Dr. Ian P. Hall (Queens Medical Centre, Nottingham, UK).

Statistical analysis. All data are presented as means ± SE; n indicates number of separate experiments. Control versus treated groups were analyzed for an increase in the [32P]GTP-to-([32P]GTP + [32P]GDP) ratio by ANOVA, with Bonferroni posttest comparisons between groups. A P value < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cultured human tracheal smooth muscle cells exposed to 1 µM carbachol or endothelin-1 for 3 min had a significant increase in the amount of [32P]GTP immunoprecipitated by v-H-ras-specific antibodies compared with untreated control cells (Fig. 1). In the control cells, the [32P]GTP-to-([32P]GTP + [32P]GDP) ratio averaged 30 ± 1.7, whereas in carbachol-treated cells, the ratio increased to 39 ± 1.1 (P < 0.01 compared with control value; n = 7) and in endothelin-1-treated cells, the ratio increased to 40 ± 1.2 (P < 0.001 compared with control value; n = 11). In cells treated with the positive control EGF, which activates p21ras independent of a heterotrimeric G protein (3, 27), the [32P]GTP-to-([32P]GTP + [32P]GDP) ratio increased to 59 ± 1.8 (P < 0.001 compared with control value; n = 5; Fig. 1). Pretreatment of cells for 4 h with pertussis toxin blocked the ability of carbachol (Fig. 2) or endothelin-1 (Fig. 3) to activate p21ras [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): carbachol alone, 39 ± 1.1; carbachol + pertussis toxin, 31 ± 1.0 (n = 7); endothelin-1 alone, 40 ± 1.2; endothelin-1 + pertussis toxin, 29 ± 1.1 (n = 10)]. Pertussis toxin pretreatment had no significant effect on either basal levels of p21ras activation or on the activation of p21ras by EGF [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): basal, 30 ± 1.7; pertussis alone, 29 ± 1.7 (n = 12); EGF alone, 59 ± 1.8 (n = 9); EGF + pertussis toxin, 56 ± 0.9 (n = 6); Fig. 4].


View larger version (28K):
[in this window]
[in a new window]
 


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 1.   A: Ras activation in human airway smooth muscle cells. Cells were exposed for 3 min to 1 µM carbachol, 1 µM endothelin-1, 1 µM bradykinin, 1 µM histamine, or 40 mM KCl or for 5 min to 100 ng/ml of epidermal growth factor (EGF; positive control). Ras-bound [32P]GTP and [32P]GDP were immunoprecipitated, separated by TLC, and quantitated by phosphorimaging. Activation of Gi-coupled receptors by carbachol and endothelin-1, but not of Gq-coupled receptors by bradykinin or histamine, resulted in a significant increase in amount of immunoprecipitated [32P]GTP. Elevation of cellular calcium alone with KCl was insufficient to activate Ras, whereas positive control EGF, which does not couple to heterotrimeric G proteins, was a potent activator of Ras. GTP/GTP+GDP, ratio of [32P]GTP to ([32P]GTP + [32P]GDP). * P < 0.05 compared with control group. B: representative phosphorimage of TLC separation of immunoprecipitated [32P]GDP and [32P]GTP bound to Ras after indicated agonist exposure.


View larger version (34K):
[in this window]
[in a new window]
 


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 2.   Pertussis toxin inhibition of carbachol-induced Ras activation in human airway smooth muscle cells. Cells in parallel 6-well plates were preincubated with 100 ng/ml of pertussis toxin or medium alone for 4 h during 32P loading. Cells were exposed to 1 µM carbachol for 3 min, and Ras-bound [32P]GTP and [32P]GDP were immunoprecipitated, separated by TLC, and quantitated by phosphorimaging. Pretreatment with pertussis toxin blocked carbachol-induced activation of Ras, indicating that carbachol was activating Ras via a pertussis toxin-sensitive heterotrimeric G protein. * P < 0.05 compared with carbachol alone. B: representative phosphorimage of TLC separation of immunoprecipitated [32P]GTP and [32P]GDP bound to Ras after carbachol exposure with and without pertussis toxin pretreatment.


View larger version (32K):
[in this window]
[in a new window]
 


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 3.   Pertussis toxin inhibition of endothelin-1-induced Ras activation in human airway smooth muscle cells. Cells in parallel 6-well plates were preincubated with 100 ng/ml of pertussis toxin or medium alone for 4 h during 32P loading. Cells were exposed to 1 µM endothelin-1 for 3 min, and Ras-bound [32P]GTP and [32P]GDP were immunoprecipitated, separated by TLC, and quantitated by phosphorimaging. Pretreatment with pertussis toxin blocked endothelin-1-induced activation of Ras, indicating that endothelin-1 was activating Ras via a pertussis toxin-sensitive heterotrimeric G protein. * P < 0.05 compared with endothelin-1 alone. B: representative phosphorimage of TLC separation of immunoprecipitated [32P]GTP and [32P]GDP bound to Ras after endothelin-1 exposure with and without pertussis toxin pretreatment.


View larger version (40K):
[in this window]
[in a new window]
 


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 4.   Pertussis toxin inhibition of basal or EGF-induced Ras activation in human airway smooth muscle cells. Cells in parallel 6-well plates were preincubated with 100 ng/ml of pertussis toxin or medium alone for 4 h during 32P loading. Cells were exposed to medium alone or EGF for 5 min, and Ras-bound [32P]GTP and [32P]GDP were immunoprecipitated, separated by TLC, and quantitated by phosphorimaging. Pretreatment with pertussis toxin had no effect on basal levels or EGF-induced activation of Ras. B: representative phosphorimage of TLC separation of immunoprecipitated [32P]GTP and [32P]GDP bound to Ras after no stimulation (control) or EGF exposure with and without pertussis toxin pretreatment.

In contrast to the activation of p21ras by carbachol or endothelin-1, 1 µM histamine or 1 µM bradykinin for 3 min had no effect on p21ras activation. The [32P]GTP-to-([32P]GTP + [32P]GDP) ratio after histamine was 32 ± 1.8 (n = 5) and after bradykinin was 32 ± 1.3 (n = 7) compared with the control value of 30 ± 1.7 (n = 20). Elevation of cellular calcium alone with 40 mM KCl did not significantly activate p21ras (30 ± 3.3; n = 4) compared with control (Fig. 1).

In an attempt to determine whether the carbachol-mediated activation of p21ras in airway smooth muscle cells was mediated through the alpha - or beta gamma -subunit of heterotrimeric G proteins, carbachol-induced p21ras activation was measured in cells transiently transfected with the beta -ARK1 minigene product [a known intracellular antagonist of liberated G protein beta gamma -subunits (17)]. Carbachol-induced p21ras activation was compared between untransfected cells, cells transfected with the beta -ARK1 minigene, and cells sham transfected with the control pRK5 plasmid lacking an insert. Transfection with the beta -ARK1 minigene did not effect carbachol-induced activation of p21ras (Fig. 5A), suggesting that the activation of p21ras by carbachol is not mediated via G protein beta gamma -subunits in human airway smooth muscle cells. Transfection with pRK-beta -ARK1 also had no effect on basal or EGF-induced p21ras activation (data not shown). Sham transfection with the empty pRK5 plasmid had no effect on basal or carbachol- or EGF-mediated p21ras activation (data not shown).


View larger version (39K):
[in this window]
[in a new window]
 


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   A: carbachol-induced Ras activation in transfected human airway smooth muscle cells. To determine whether carbachol activation of Ras was mediated via G protein alpha - or beta gamma -subunit in human airway smooth muscle cells, cells were either not transfected (None), transiently transfected with an empty control plasmid (pRK5; Sham), or transiently transfected with a plasmid encoding carboxy-terminal fragment of beta -adrenoceptor kinase 1 (pRK-beta -ARK1; beta ARK1), which functions as a cellular G protein beta gamma antagonist before measurement of carbachol-induced Ras activation. Transient transfection with beta -ARK1 fragment had no effect on carbachol-induced Ras activity. B: representative immunoblot analysis of human airway smooth muscle cell lysates after no transfection, sham transfection, or beta -ARK1 transfection. An expected immunoreactive protein of ~24 kDa (arrowhead) was detected with a primary antibody directed against beta -ARK1 only in cells transfected with beta -ARK1 minigene. A larger band of ~80 kDa, likely representing native full-length beta -ARK-related proteins, was seen in all cells.

To confirm that transfection with the beta -ARK1 minigene results in expression of the beta -ARK1 protein fragment, immunoblot analysis was performed with an antibody that recognized the transfected shorter-length carboxy-terminal fragment of beta -ARK1. In pRK-beta -ARK1-transfected cells, an immunoreactive band was detected at ~24 kDa (Fig. 5B) consistent with the expected size of the beta -ARK1 minigene protein product (17). No immunoreactive band within this molecular-mass range was detected in nontransfected control cells or in control cells sham transfected with the empty pRK5 plasmid. Preliminary experiments in which transfection was performed with a plasmid encoding beta -galactosidase showed that ~70% of cells were successfully transfected. These results suggest that abundant expression of the carboxy terminus (Gly495-Leu689) of beta -ARK1, the G protein beta gamma -subunit antagonist, occurred in the transfected cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the current study, we found that carbachol and endothelin-1 but not histamine, bradykinin, or KCl activated the monomeric G protein p21ras in cultured human airway smooth muscle cells. Activation of p21ras by carbachol or endothelin-1 was blocked by pertussis toxin but not by overexpression of a G protein beta gamma -subunit scavenger, suggesting that muscarinic and endothelin receptors couple to a heterotrimeric Gi protein that activates the monomeric G protein p21ras via the G protein alpha -subunit. The current study is the first study in airway smooth muscle cells to directly measure p21ras activation and to link this activation to a G protein-coupled pathway.

In native airway smooth muscle cells, carbachol activates M2 and M3 muscarinic receptors to activate Gi and Gq pathways, respectively. However, the cultured airway smooth muscle cells used in this study predominantly express M2 muscarinic receptors (26). The very low levels of M3 muscarinic receptors are demonstrated by minimal stimulation by carbachol of inositol phosphate synthesis in these cells (9). Endothelin also couples to both the Gi and Gq pathways, and thus pertussis toxin was used with both carbachol and endothelin to implicate a Gi-mediated pathway. Histamine and bradykinin, which couple to the Gq pathway in these airway smooth cells (9), did not activate p21ras. The effect of pertussis toxin in inhibiting the carbachol and endothelin activation of p21ras appeared to be specific for Gi activation because pertussis toxin had no effect on basal or EGF-induced levels of activated p21ras.

Activation of Gq by histamine or bradykinin or elevation of cellular calcium alone with KCl was insufficient to activate p21ras. These data are not in conflict with a previously published study on signaling in airway smooth muscle cells (15) because that study of mitogenic signaling measured the activation or phosphorylation of the distal MAP kinases, not p21ras activation directly as in the current study. Directly measuring p21ras activation is necessary to determine whether p21ras is a signaling intermediate leading to activation of MAP kinases because receptors coupled to Gq can activate MAP kinases by both p21ras-dependent and p21ras-independent pathways (10, 23). Our results are therefore consistent with those of Kelleher et al. (15) in cultured bovine airway smooth muscle cells in which the Gq-coupled receptor 5-hydroxytryptamine type 2 activated MAP kinases via a pathway proposed to be independent of p21ras. Thus the findings of Kelleher et al. together with those of the present study suggest that in airway smooth muscle cells MAP kinase activation via the Gq pathway occurs through PKC and is p21ras independent, whereas MAP kinase activation via the Gi pathway is p21ras dependent.

Although initial studies in a variety of cell types suggested that Gi-coupled receptors (M2 muscarinic, alpha 2-adrenergic, lysophosphatidic acid, and thrombin) activate MAP kinases through p21ras activation (1, 24, 27) and Gq-coupled receptors activate MAP kinases via a PKC-dependent, p21ras-independent pathway (10), exceptions to these suppositions are emerging. In a recent study (18) in transfected COS-7 cells, stimulation of MAP kinase by thyrotropin-releasing hormone was shown to be pertussis toxin insensitive and partially impaired by scavenging of G protein beta gamma -subunits. Additionally, a pertussis toxin-insensitive, p21ras-dependent pathway that activated MAP kinase has been shown for both thrombin and angiotensin receptors in fibroblasts and myocytes, respectively (2, 20), and a constitutively active Gqalpha mutant activates p21ras in NIH/3T3 cells (25). The emerging picture is that the responsible heterotrimeric G protein, the transmission of the signal by G protein alpha - or beta gamma -subunit, and the involvement of PKC and/or p21ras in the activation of MAP kinases are cell-type dependent.

Our results also agree with the findings of Koch and colleagues (16, 17) in fibroblasts and COS-7 cells in which the Gi protein mediated the activation of p21ras. Having incriminated the Gi pathway in the activation of p21ras in these cells, we next sought to determine whether the G protein alpha - or beta gamma -subunit of Gi transmitted the signal to downstream proteins. We utilized a strategy of transiently transfecting a carboxy-terminal fragment of the beta -ARK1 enzyme. This protein fragment functions as an intracellular scavenger of liberated G protein beta gamma -subunits and has been used extensively to distinguish between G protein alpha - and beta gamma -mediated processes (16, 17). Unlike standard liposome-mediated transfection protocols that have been successful with bovine airway smooth muscle (13), successful transient transfection in the human airway smooth muscle cells used in this study was only obtained when a liposome formulation was combined with a replication-deficient adenovirus that together mediated entry of the pRK-beta -ARK1 plasmid construct (7). Immunoblot analysis revealed abundant expression of an immunoreactive protein at an expected molecular mass of ~24 kDa, indicative of successful expression of this G protein beta gamma antagonist.

However, in airway smooth muscle cells transfected with this G protein beta gamma antagonist, carbachol activated p21ras to a similar level to that seen in untransfected cells or cells transfected with a control plasmid without an insert. These results suggest that the Gi-mediated activation of p21ras in these cells is not transmitted via the G protein beta gamma -subunits but rather is likely transmitted by the G protein alpha -subunit. This is in contrast to the findings of two studies (8, 10) in which the Gi beta gamma -subunit was responsible for the activation of MAP kinase pathways in fibroblasts or COS-7 cells. This difference may be explained by unique signaling pathways among different cell types. It is also possible that the efficiency of transfection was too low in the current study to block liberated G protein beta gamma -subunits in the majority of cells. However, this is unlikely considering the abundant expression of the beta -ARK1 minigene product identified by immunoblotting in transfected cells and the ~70% transfection efficiency demonstrated by beta -galactosidase staining. Confirming that expression levels of the beta -ARK1 minigene product were sufficiently high to block a G protein beta gamma -mediated event is difficult because the G protein beta gamma -subunit has not been shown to activate a specific cellular signal in human airway smooth muscle cells. Other cells have pathways known to utilize the G protein beta gamma -subunit; however, successful transfection and signal blockade in another cell type does not confirm successful transfection and sufficient expression in an airway smooth muscle cell.

In summary, this study shows that activation of Gi- but not of Gq-coupled receptors in airway smooth muscle leads to the activation of the monomeric G protein p21ras via liberated G protein alpha -subunits. Activation of p21ras by M2 muscarinic and endothelin receptors by chronic release of acetylcholine by parasympathetic nerves and endothelin by inflammatory cells, respectively, likely contributes to mitogenesis of airway smooth muscle cells and may contribute to the asthmatic process.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Ian P. Hall (Queens Medical Centre, Nottingham, UK) for providing the human airway smooth muscle cells used in this study.


    FOOTNOTES

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

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. W. Emala, Dept. of Anesthesiology, Columbia-Presbyterian Medical Center, 630 W. 168th St., PH 525, New York, NY 10032 (E-mail: cwe5{at}columbia.edu).

Received 27 August 1998; accepted in final form 21 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alblas, J., E. J. Van Corven, P. L. Hordijk, G. Milligan, and W. H. Moolenaar. Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts. J. Biol. Chem. 268: 22235-22238, 1993[Abstract/Free Full Text].

2.   Chen, Y. H., D. Grall, A. E. Salcini, P. G. Pelicci, J. Pouysségur, and E. V. Obberghen-Schilling. Shc adapter proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor. EMBO J. 15: 1037-1044, 1996[Abstract].

3.   Cobb, M. H., T. G. Boulton, and D. J. Robbins. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 2: 965-978, 1991[Medline].

4.   DeVries-Smits, A. M. M., B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos. Involvement of p21ras in activation of extracellular signal-regulated kinase 2. Nature 357: 602-604, 1992[Medline].

5.   Ebina, M., H. Yaegashi, R. Chiba, T. Takahashi, M. Motomiya, and M. Tanemura. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. Am. Rev. Respir. Dis. 141: 1327-1332, 1990[Medline].

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

7.   Fasbender, A., J. Zabner, M. Chillon, T. O. Moninger, A. P. Puga, B. L. Davidson, and M. J. Welsh. Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo. J. Biol. Chem. 272: 6479-6489, 1997[Abstract/Free Full Text].

8.   Garnovskaya, M. N., T. van Biesen, B. E. Hawes, S. C. Ramos, R. J. Lefkowitz, and J. R. Raymond. Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein beta gamma -subunit-initiated pathway. Biochemistry 35: 13716-13722, 1996[Medline].

9.   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].

10.   Hawes, B. E., T. van Biesen, W. J. Koch, L. M. Luttrell, and R. J. Lefkowitz. Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J. Biol. Chem. 270: 17148-17153, 1995[Abstract/Free Full Text].

11.   Hossain, S., and B. E. Heard. Hyperplasia of bronchial muscle in chronic bronchitis. J. Pathol. 101: 171-184, 1970[Medline].

12.   Hershenson, M. B., S. Aghili, N. Punjabi, C. Hernandez, D. W. Ray, A. Garland, S. Glagov, and J. Solway. Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L263-L269, 1992[Abstract/Free Full Text].

13.   Hershenson, M. B., T. O. Chao, M. K. Abe, I. Gomes, M. D. Kelleher, J. Solway, and M. R. Rosner. Histamine antagonizes serotonin and growth factor-induced mitogen-activated protein kinase activation in bovine tracheal smooth muscle cells. J. Biol. Chem. 270: 19908-19913, 1995[Abstract/Free Full Text].

14.   Hershenson, M. B., A. Garland, M. D. Kelleher, A. Zimmerman, C. Hernandez, and J. Solway. Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness. Am. Rev. Respir. Dis. 146: 1294-1300, 1992[Medline].

15.   Kelleher, M. D., M. K. Abe, T. O. Chao, M. Jain, J. M. Green, J. Solway, M. R. Rosner, and M. B. Hershenson. Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L894-L901, 1995[Abstract/Free Full Text].

16.   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 &Ggr; activation of p21ras. Proc. Natl. Acad. Sci. USA 91: 12706-12710, 1994[Abstract/Free Full Text].

17.   Koch, W. J., B. E. Hawes, J. Inglese, L. M. Luttrell, and R. J. Lefkowitz. Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attentuates Gbeta gamma -mediated signaling. J. Biol. Chem. 269: 6193-6197, 1994[Abstract/Free Full Text].

18.   Palomero, T., F. Barros, D. del Camino, C. G. Viloria, and P. Pena. A G protein beta gamma dimer-mediated pathway contributes to mitogen-activated protein kinase activation by thyrotropin-releasing hormone receptors in transfected cos-7 cells. Mol. Pharmacol. 53: 613-622, 1998[Abstract/Free Full Text].

19.   Panettieri, R. A., P. A. Yadvish, A. M. Kelly, N. A. Rubinstein, and M. I. Kotlikoff. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L365-L371, 1990[Abstract/Free Full Text].

20.   Sadoshima, J. I., and S. Izumo. The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. EMBO J. 15: 775-787, 1996[Abstract].

21.   Sapienza, S., T. Du, D. H. Eidelman, N. S. Wang, and J. G. Martin. Structural changes in the airways of sensitized Brown Norway rats after antigen challenge. Am. Rev. Respir. Dis. 144: 423-427, 1991[Medline].

22.   Sofia, M., M. Mormile, S. Faraone, M. Alifano, S. Zofra, L. Romano, and L. Carratu. Increased endothelin-like immunoreactive material on bronchoalveolar lavage fluid from patients with bronchial asthma and patients with interstitial lung disease. Respiration 60: 89-95, 1993[Medline].

23.   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].

24.   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].

25.   Watanabe, T., I. Waga, Z. Honda, K. Kurokawa, and T. Shimizu. Prostaglandin F2alpha stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway. J. Biol. Chem. 270: 8984-8990, 1995[Abstract/Free Full Text].

26.   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].

27.   Winitz, S., M. Russell, N. X. Qian, A. Gardner, L. Dwyer, and G. L. Johnson. Involvement of ras and raf in the Gi-coupled acetylcholine muscarinic M2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase. J. Biol. Chem. 268: 19196-19199, 1993[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 276(4):L564-L570
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society