Departments of 1 Anesthesiology and 2 Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205
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ABSTRACT |
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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
Gi-2,
G
i-3, or
Gq
. 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
-subunit expression. Oligonucleotide antisense treatment decreased
protein expression of the respective G protein
-subunit. Antisense
depletion of the G
i-2 protein
but not of G
i-3 or
Gq
protein blocked the
carbachol-induced increase in the F- to G-actin ratio. These results
show that the G
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
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INTRODUCTION |
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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 -subunit and two smaller, tightly coupled subunits,
and
. The
-subunits are unique to each G protein, conferring functional specificity. The
chains 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
, transducin 1 and 2, and
gustducin; 3)
Gq
,
G11
,
G14
, and
G15
; and
4)
G12
and
G13
. Additional subfamily
members of
-subunits, as well as splice variants of
Gs
,
G
i-2, and
Go
, have recently been identified (10). In addition to this substantial variability in
-subunits, 5
-subunits and at least 10
-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
-subunits. Because M2
muscarinic receptors are known to couple to members of the
Gi family of heterotrimeric G
proteins and because both G
i-2
and G
i-3 are expressed in ASM
(5) and inactivated by pertussis toxin, antisense oligonucleotides were
designed to specifically bind to mRNA encoding
G
i-2 or
G
i-3. A randomly scrambled
oligonucleotide and an antisense oligonucleotide designed to bind to
mRNA encoding Gq
served as
controls. We found that antisense depletion of
G
i-2 protein but not of
G
i-3 or
Gq
protein inhibited
carbachol-induced actin reorganization in human ASM cells. This study
provides the first evidence that
G
i-2 protein couples muscarinic
receptors to cytoskeletal reorganization in human ASM cells.
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MATERIALS AND METHODS |
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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 -subunits by antisense
oligonucleotides. To determine which G protein
-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
G
i-2,
G
i-3 and
Gq
(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
G
i-2, 5'-AAG TTG CGG TCG ATC
AT-3' for G
i-3, 5'-GCT TGA GCT
CCC GGC GGG CG-3' for Gq
(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
-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
-subunit expression. Successful depletion of the G protein
-subunits was confirmed by Western blot analysis as previously
described (5) with the use of polyclonal antisera specific for
G
i-2,
G
i-3, and
Gq
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%
-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
-subunit. These experiments were conducted to ensure that the
oligonucleotide antisense treatment resulted in specific reduction of
only one G protein
-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
-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.
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RESULTS |
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Carbachol induced actin reorganization in the presence
of antisense for G protein -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 G
i-2 blocked the
carbachol-induced stress fiber formation (Fig. 1C). In contrast, 6 days of
treatment with antisense oligonucleotides directed against
G
i-3 or
Gq
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 G
i-2
antisense treatment but was unaffected by treatment with
G
i-3 or
Gq
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 G
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
G
i-3 or
Gq
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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Immunoblot analysis of G protein
-subunit depletion by antisense
oligonucleotide. To confirm that antisense
oligonucleotide treatment resulted in a decrease in the respective G
protein
-subunit protein, we performed immunoblot analysis on
cultured human ASM cells treated for 6 days with specific G protein
-subunit antisense oligonucleotides. Six days of treatment with
phosphorothioate-modified antisense oligonucleotides directed against
the mRNA encoding G
i-2,
G
i-3, or
Gq
resulted in a significant
reduction in the expression of the respective proteins as determined by
immunoblotting (Fig. 3).
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The band intensities of the
Gi-2 protein decreased in cells
treated for 6 days with G
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 Gq
protein decreased in
cells treated for 6 days with Gq
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 G
i-3 protein were greatly
reduced compared with band intensities for
G
i-2 or
Gq
and required the use of
cells from eight wells (each well, 9 × 9 mm) of the Nunc slide
for G
i-3 immunoblots compared
with cells from only one well for the detection of
G
i-2 or
Gq
. Even with this eightfold
increase in cell numbers, immunoblot detection of
G
i-3 was faint, preventing
quantitative measurement of band intensities despite an obvious visual
decrease in band intensities in lanes treated with
G
i-3 antisense
(n = 4 experiments).
To ensure that the antisense oligonucleotide treatment was specific for
the G protein -subunit it targeted and that equivalent amounts of
protein were loaded into each lane, after the detection of a decrease
in G
i-2 protein after
G
i-2 antisense oligonucleotide treatment, we stripped immunoblots and reprobed them with primary antibody specific for the Gq
protein. The same samples that had shown a decrease in
G
i-2 protein after
G
i-2 antisense oligonucleotide treatment showed no significant difference in the amount of
Gq
protein (0.33 ± 0.02 × 106 counts in control vs.
0.32 ± 0.04 × 106 counts in
G
i-2 antisense treated). These
studies confirm the specificity of the
G
i-2 antisense treatment.
Similarly, the same samples that had shown a decrease in
Gq
protein after
Gq
antisense oligonucleotide
treatment showed no significant difference in the amount of
G
i-2 protein (1.3 ± 0.8 × 106 counts in control vs.
1.2 ± 0.8 × 106 counts
in G
i-2 antisense treated;
n = 3 experiments;
P = 0.93), confirming the specificity
of the Gq
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;
Gi-2, 8,900 ± 229 cells/well (n = 4 experiments;
P = 0.56 vs. control); G
i-3, 7,800 ± 770 cells/well (n = 4 experiments;
P = 0.23 vs. control); and
Gq
, 8,400 ± 550 cells/well
(n = 5 experiments; P = 0.46 vs. control).
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DISCUSSION |
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This study demonstrates that
Gi-2 protein couples muscarinic
receptors to stress fiber formation in human ASM cells. Antisense depletion of the G
i-2 protein
but not G
i-3 or
Gq
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 -subunits of
G
i-2,
G
i-3, and
Gq
resulted in specific
decreases in
-subunit protein expression. This indicated that the
-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
G
i-2, G
i-3, and
Gq
used in our study, as used
by Standifer et al. (25), are complementary to translated regions of
G
i-2,
G
i-3, and
Gq
mRNAs and have very limited
homology between
-subunits. The 30-40% decrease in levels of
-subunit membrane protein observed after 6 days of exposure to
antisense oligonucleotides is consistent with the slow turnover of
-subunit proteins demonstrated in the membranes of other cell types
(3, 16, 24, 26).
It is unlikely that the decreased levels of -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
-subunit protein level was seen only after treatment
with each specific G protein
-subunit antisense oligonucleotide. G
protein
-subunit expression did not change when immunoblots, which
had previously shown decreased
-subunit expression after specific
antisense oligonucleotide treatment, were stripped and reprobed with
primary antibody directed against other G protein
-subunits, indicating that the treatment was indeed
specific for the G protein
-subunit that it targeted and that
equivalent amounts of protein had been loaded into each lane.
In the immunoblot analysis of G protein -subunits, band intensities
for the G
i-3 protein were so
greatly reduced compared with the band intensities of the
G
i-2 and
Gq
protein in human ASM cells
that detection required the use of eight times more cells. This likely
represents a lower level of expression of
G
i-3 than of
G
i-2 or
Gq
in these cells or a lower
affinity of G
i-3 for the
antibody used to detect the protein.
Pretreatment of human ASM cells with oligonucleotide antisense to
Gi-2 produced a partial
inhibition of carbachol-induced stress fiber formation, consistent with
an incomplete loss of G
i-2
membrane protein.
Application of antisense techniques to other cell types has been used
to investigate the relationship of heterotrimeric G protein
-subunits to receptors and second messengers. For example, de
Mazancourt et al. (3) showed that
G
i-3 couples galanin receptors
to inhibition of adenylyl cyclase activity in rat insulinoma cells, and
Tang et al. (26) showed that
G
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
Gi-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
Gi-2 protein but not of
G
i-3 or
Gq
protein significantly
inhibited carbachol-induced stress fiber formation in human ASM cells.
This study provides the first evidence that
G
i-2 couples
M2 muscarinic receptors to Rho
proteins in human ASM cells.
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ACKNOWLEDGEMENTS |
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We thank Ian Hall for kindly providing the human cultured airway smooth muscle cells used in this study.
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bennett, C. F.
Antisense oligonucleotides: is the glass half full or half empty?
Biochem. Pharmacol.
55:
9-19,
1998[Medline].
2.
Chardin, P.,
P. Boquet,
P. Madaule,
M. R. Popoff,
E. J. Rubin,
and
D. M. Gill.
The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells.
EMBO J.
8:
1087-1092,
1989[Abstract].
3.
De Mazancourt, P.,
P. K. Goldsmith,
and
L. S. Weinstein.
Inhibition of adenylate cyclase activity by galanin in rat insulinoma cells is mediated by the G-protein Gi3.
Biochem. J.
303:
369-375,
1994[Medline].
4.
Emala, C. W.,
A. Aryana,
M. A. Levine,
R. P. Yasuda,
S. A. Satkus,
B. B. Wolfe,
and
C. A. Hirshman.
Expression of muscarinic receptor subtypes and M2-muscarinic inhibition of adenylyl cyclase in lung.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L101-L107,
1995
5.
Emala, C. W.,
J. Yang,
C. A. Hirshman,
and
M. A. Levine.
G protein subunits in lung cells.
Life Sci.
55:
593-602,
1994[Medline].
6.
Fernandes, L. B.,
A. D. Fryer,
and
C. A. Hirshman.
M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle.
J. Pharmacol. Exp. Ther.
262:
119-126,
1992[Abstract].
7.
Fryer, A. D.,
and
E. E. El-Fakahany.
Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists.
Life Sci.
47:
611-618,
1990[Medline].
8.
Gong, M. C.,
K. Iizuka,
G. Nixon,
J. P. Browne,
A. Hall,
J. F. Eccleston,
M. Sugai,
S. Kobayashi,
A. V. Somlyo,
and
A. P. Somlyo.
Role of guanine nucleotide-binding proteinsras-family or trimeric proteins or both
in Ca2+ sensitization of smooth msucle.
Proc. Natl. Acad. Sci. USA
93:
1340-1345,
1996
9.
Hall, A.
Small GTP-binding proteins and the regulation of the actin cytoskeleton.
Annu. Rev. Cell Biol.
10:
31-54,
1994.
10.
Hildebrandt, J. D.
Role of subunit diversity in signaling by heterotrimeric G proteins.
Biochem. Pharmacol.
54:
325-339,
1997[Medline].
11.
Hirata, K.,
A. Kikuchi,
T. Sasaki,
S. Kuroda,
K. Kaibuchi,
Y. Matsuura,
H. Seki,
K. Saida,
and
Y. Takai.
Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.
J. Biol. Chem.
267:
8719-8722,
1992
12.
Hotchin, N. A.,
and
A. Hall.
Regulation of the actin cytoskeleton, integrins and cell growth by the rho family of small GTPases.
Cancer Surv.
27:
311-322,
1996[Medline].
13.
Janmey, P. A.
Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly.
Annu. Rev. Physiol.
56:
169-191,
1994[Medline].
14.
Kimura, K.,
M. Ito,
M. Amano,
K. Chihara,
Y. Fukata,
M. Nakafuku,
B. Yamamori,
J. Feng,
T. Nakano,
K. Okawa,
A. Iwamatsu,
and
K. Kaibuchi.
Regulation of myosin phosphatase by rho and rho-associated kinase (rho-kinase).
Science
273:
245-248,
1996[Abstract].
15.
Knowles, G. C.,
and
C. A. G. McCulloch.
Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.
J. Histochem. Cytochem.
40:
1605-1612,
1992
16.
Levis, M. J.,
and
H. R. Bourne.
Activation of the subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity.
J. Cell Biol.
119:
1297-1307,
1992[Abstract].
17.
Mak, J. C. W.,
and
P. J. Barnes.
Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung.
Am. Rev. Respir. Dis.
141:
1559-1568,
1990[Medline].
18.
Neckers, L.,
and
L. Whitesell.
Antisense technology: biological utility and practical considerations.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L1-L12,
1993
19.
Nobes, C. D.,
and
A. Hall.
Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility.
Biochem. Soc. Trans.
23:
456-459,
1995[Medline].
20.
Noda, M.,
C. Yasuda-Fukazawa,
K. Moriishi,
T. Kato,
T. Okuda,
K. Kurokawa,
and
Y. Takuwa.
Involvement of rho in GTPS-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity.
FEBS Lett.
367:
246-250,
1995[Medline].
21.
Otto, B.,
A. Steusloff,
I. Just,
K. Aktories,
and
G. Pfitzer.
Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle.
J. Physiol. (Lond.)
496:
317-329,
1996[Abstract].
22.
Post, G. R.,
and
J. H. Brown.
G protein-coupled receptors and signaling pathways regulating growth responses.
FASEB J.
10:
741-749,
1996
23.
Roffel, A. F.,
C. R. S. Elzing,
G. M. Van Amsterdam,
R. A. deZeeuw,
and
J. Zaagsma.
Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function.
Eur. J. Pharmacol.
153:
73-82,
1988[Medline].
24.
Silbert, S.,
T. Michel,
R. Lee,
and
E. J. Neer.
Differential degradation rates of the G protein o in cultured cardiac and pituitary cells.
J. Biol. Chem.
265:
3102-3105,
1990
25.
Standifer, K. M.,
G. C. Ross,
and
G. W. Pasternak.
Differential blockade of opioid analgesia by antisense oligodeoxynucleotides directed against various G protein subunits.
Mol. Pharmacol.
50:
293-298,
1996[Abstract].
26.
Tang, T.,
J. G. Kiang,
T. E. Cote,
and
B. M. Cox.
Antisense oligodeoxynucleotide to the Gi2 protein subunit sequence inhibits an opioid-induced increase in the intracellular free calcium concentration in ND8-47 neuroblastoma × dorsal root ganglion hybrid cells.
Mol. Pharmacol.
48:
189-193,
1995[Abstract].
27.
Togashi, H.,
C. W. Emala,
I. P. Hall,
and
C. A. Hirshman.
Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L803-L809,
1998
28.
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].
29.
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].