Department of Anesthesiology, Columbia University College of Physicians and Surgeons, New York, New York 10032
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ABSTRACT |
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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 ( or
) 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
-subunit [the carboxy terminus
(Gly495-Leu689)
of the
-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
-subunit.
G proteins; carbachol; endothelin; epidermal growth factor; bradykinin; histamine; pertussis toxin
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INTRODUCTION |
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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 -subunit inhibitor.
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METHODS |
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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).
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RESULTS |
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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].
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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 - or
-subunit of heterotrimeric
G proteins, carbachol-induced
p21ras activation was measured in
cells transiently transfected with the
-ARK1 minigene product
[a known intracellular antagonist of liberated G protein
-subunits (17)]. Carbachol-induced p21ras activation was compared
between untransfected cells, cells transfected with the
-ARK1
minigene, and cells sham transfected with the control pRK5 plasmid
lacking an insert. Transfection with the
-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
-subunits in human airway smooth muscle
cells. Transfection with pRK-
-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).
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To confirm that transfection with the -ARK1 minigene results in
expression of the
-ARK1 protein fragment, immunoblot analysis was
performed with an antibody that recognized the transfected shorter-length carboxy-terminal fragment of
-ARK1. In
pRK-
-ARK1-transfected cells, an immunoreactive band was detected at
~24 kDa (Fig. 5B) consistent with
the expected size of the
-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
-galactosidase showed that ~70% of cells were successfully transfected. These results suggest that abundant expression of the
carboxy terminus
(Gly495-Leu689)
of
-ARK1, the G protein
-subunit antagonist, occurred in the
transfected cells.
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DISCUSSION |
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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 -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
-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,
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
-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 Gq
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
- or
-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 - or
-subunit of
Gi transmitted the signal to
downstream proteins. We utilized a strategy of transiently transfecting
a carboxy-terminal fragment of the
-ARK1 enzyme. This protein
fragment functions as an intracellular scavenger of liberated G protein
-subunits and has been used extensively to distinguish between G
protein
- and
-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-
-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
antagonist.
However, in airway smooth muscle cells transfected with this G protein
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
-subunits but rather is likely
transmitted by the G protein
-subunit. This is in contrast to the
findings of two studies (8, 10) in which the
Gi
-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
-subunits in the majority of cells.
However, this is unlikely considering the abundant expression of the
-ARK1 minigene product identified by immunoblotting in transfected
cells and the ~70% transfection efficiency demonstrated by
-galactosidase staining. Confirming that expression
levels of the
-ARK1 minigene product were sufficiently high to block
a G protein
-mediated event is difficult because the G protein
-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
-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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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