Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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ABSTRACT |
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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
Gi-2 alone, LPA- and
endothelin-1-induced actin reorganization were only blocked when both
G
i-2 and
Gq
were depleted. These data
indicate that in human airway smooth muscle cells, carbachol induces
actin reorganization via a G
i-2
pathway, whereas LPA or endothelin-1 induce actin reorganization via
either a G
i-2 or a
Gq
pathway.
G protein; endothelin-1; carbachol; lysophosphatidic acid; antisense oligonucleotide; Gi-2; cell culture
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INTRODUCTION |
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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
-subunit and two smaller, tightly coupled subunits,
and
. The
-subunits are unique to each G protein, conferring functional specificity. The
-subunits are subdivided into four major families on the basis of their amino acid sequence homology:
1)
Gs
and Golf;
2)
G
i-1,
G
i-2,
G
i-3,
Go
,
Gz
, transducins 1 and 2, and gustducin; 3)
Gq
,
G11
,
G14
, and
G15
; and
4)
G12
and
G13. Splice variants of
Gs
,
G
i-2, and
Go
as well as additional
subfamily members of
-subunits have recently been identified
(16). In addition, five
-subunits and at least 10
-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 Gi-2,
G
i-3, or
Gq
, Hirshman et al. (18) showed
that antisense oligonucleotide depletion of
G
i-2 protein but not of
G
i-3 or Gq
protein blocked
carbachol-induced increases in actin reorganization in the same cells.
These data indicate that G
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 -subunits. Antisense oligonucleotides were designed to
specifically bind to mRNA encoding G
i-2,
G
i-3, and
Gq
. 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
G
i-2 and
Gq
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
G
i-2 and
Gq
couple endothelin and LPA
receptors to Rho proteins in human airway smooth muscle cells.
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MATERIALS AND METHODS |
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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 -actin. Moreover, immunoblot analysis
of these cells identified expression of both
-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 -subunits G
i-2,
G
i-3, or
Gq
. 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
-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 (G
i-2 and
Gq
or
G
i-3 and
Gq
). 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
G
i-2, 5'-AAG TTG CGG TCG ATC
AT-3' for G
i-3, and 5'-GCT TGA
GCT CCC GGC GGG CG-3' for
Gq
(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
-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.
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RESULTS |
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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.
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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).
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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).
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To further characterize the heterotrimeric G proteins that are
intermediates leading from receptor activation to actin reorganization, depletion of G protein -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
-subunit (18). G
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 G
i-2 antisense oligonucleotide, 7.2 ± 0.8 in the LPA-treated cells, 6.6 ± 2.3 in the LPA-treated cells pretreated with
G
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
G
i-2 antisense oligonucleotide
(P < 0.05 for
G
i-2 antisense oligonucleotide effect on carbachol only; n = 3 experiments; Figs. 6 and
10). In contrast, pretreatment with both
G
i-2 and
Gq
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 G
i-2 and
Gq
antisense oligonucleotides,
7.2 ± 0.8 in the LPA-treated cells, 4.0 ± 0.7 in the
LPA-treated cells pretreated with both
G
i-2 and
Gq
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
G
i-2 and
Gq
antisense oligonucleotides (P < 0.05 for the combined
G
i-2 and
Gq
antisense oligonucleotide effect on each agonist; n = 5 experiments; Figs. 7 and 10). Neither pretreatment with
Gq
antisense oligonucleotide
alone (Figs. 8 and 10) nor the combination of
Gq
and
G
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 Gq
antisense
oligonucleotide, 8.0 ± 2.0 in the carbachol-treated cells
pretreated with both G
i-3 and
Gq
antisense oligonucleotides, 7.2 ± 0.8 in the LPA-treated cells, 6.9 ± 1.6 in the
LPA-treated cells pretreated with
Gq
antisense oligonucleotide,
9.1 ± 2.2 in LPA-treated cells pretreated with both
G
i-3 and
Gq
antisense oligonucleotides,
7.4 ± 0.9 in endothelin-1-treated cells, 6.6 ± 1.9 in
endothelin-1-treated cells pretreated with
Gq
antisense oligonucleotide,
and 8.6 ± 2.8 in endothelin-1-treated cells
pretreated with both G
i-3 and
Gq
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
G
i-2 antisense oligonucleotide,
2.6 ± 0.6 in the untreated cells pretreated
Gq
antisense oligonucleotide,
2.4 ± 0.4 in the untreated cells pretreated with both
Gq
and
G
i-2 antisense
oligonucleotides, and 3.9 ± 1.0 in the untreated cells pretreated
with Gq
and
G
i-3 antisense oligonucleotide
(n = 3 experiments). Taken together, these data suggest that carbachol-induced actin reorganization couples
predominantly through a G
i-2
pathway, whereas LPA- and endothelin-1-induced actin reorganization
couples through both G
i-2 and
Gq
pathways.
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DISCUSSION |
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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 Gq and
G
i-2 proteins blocked LPA- and
endothelin-1-induced actin reorganization, whereas antisense
oligonucleotide depletion of the
Gq
,
G
i-2, or
G
i-3 protein alone or the
combined depletion of Gq
and
G
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
Gq
and
G
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 Gi-2 (18) and Rho
proteins (34). Pretreatment with either C3 exoenzyme, pertussis toxin
(34), or antisense oligonucleotides directed against
G
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 Gi-2 and
Gq
demonstrate that in human airway smooth muscle cells, endothelin receptors couple to both G
i-2 and
Gq
.
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 Gq
or G
i-2.
The lack of effect of antisense oligonucleotides directed against G
protein -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, Gq
or G
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 Gq and
G
i-2 proteins significantly inhibited endothelin-1- and LPA-induced actin reorganization in these
cells. This study provides the first evidence that
Gq
as well as
G
i-2 is linked to Rho proteins
in human airway smooth muscle cells.
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FOOTNOTES |
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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.
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REFERENCES |
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Akao, M.,
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