Departments of Environmental Health Sciences and Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205
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ABSTRACT |
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Increased sensitivity to intracellular
Ca2+ concentration
([Ca2+]) is an
important mechanism for agonist-induced contraction of airway smooth
muscle, but the signal transduction pathways involved are uncertain. We
studied Ca2+ sensitization with
acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth
muscle by measuring contractions at a constant
[Ca2+] in strips
permeabilized with -toxin or
-escin. The peptide inhibitor G
protein antagonist 2A (GP Ant-2A), which has selectivity for
Gq over
Gi, inhibited contractile
responses to ET-1, ACh, and guanosine
5'-O-(3-thiotriphosphate) (GTP
S), but the
proportional inhibition of ACh responses was less than that of
ET-1. Pretreatment with pertussis toxin reduced ACh
contractions but had no effect on those of ET-1 or GTP
S.
Clostridium
botulinum C3 exoenzyme, which
inactivates Rho family monomeric G proteins, caused similar reductions
in contractile responses to ACh, ET-1, and GTP
S.
Farnesyltransferase inhibition, which inhibits Ras G proteins, reduced
responses to ET-1. We conclude that the heterotrimeric G proteins
Gq and
Gi both contribute to
Ca2+ sensitization by ACh, whereas
ET-1 responses involve Gq but not Gi. Both
Gq and
Gi pathways likely involve Rho
family small G proteins. A Ras-mediated pathway also contributes to
Ca2+ sensitization by ET-1 in
airway smooth muscle.
acetylcholine; airway smooth muscle contraction; endothelin; -escin; heterotrimeric G proteins
Gq and
Gi; monomeric G proteins Ras and
Rho; staphylococcal
-toxin
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INTRODUCTION |
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A RISE IN INTRACELLULAR free Ca2+ concentration ([Ca2+]i) has long been recognized as the normal trigger for smooth muscle contraction. However, it is now widely appreciated that many contractile agonists act both to increase [Ca2+]i and to enhance the effectiveness of Ca2+ for inducing contraction (1, 13, 16, 24, 30, 31, 38). The latter phenomenon can be readily demonstrated in membrane-permeabilized smooth muscle strips as a leftward shift of the force-[Ca2+] curve or as an agonist-induced contraction at a constant [Ca2+]. The signal tranduction pathways involved in the regulation of Ca2+ sensitivity appear to be quite complex and smooth muscle type specific. In most smooth muscle preparations, agonist-induced enhancement of Ca2+ sensitivity or potentiation of Ca2+-induced contractions involves a G protein-mediated cascade that results in inhibition of myosin light chain phosphatase (22, 24, 28). This leads to an increase in the level of myosin light chain phosphorylation, with increased numbers of attached cross bridges and increased force. Tyrosine kinases, protein kinase C, and regulatory thin filaments such as calponin are also involved (5, 19, 37).
Many lines of evidence support the involvement of members of the Ras
superfamily of monomeric G proteins (e.g., Rho A and H-Ras) in the
signaling pathways mediating agonist-induced potentiation of
Ca2+-induced contraction. First,
agonist effects can be mimicked by the poorly hydrolyzable GTP analog
guanosine 5'-O-(3-thiotriphosphate) (GTPS) (12, 18)
and inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP
S) (12, 33). Second, potentiation of
Ca2+-induced contraction by
agonists is associated with increases in myosin phosphorylation and
inhibition of myosin light chain phosphatases (22, 24). Third,
agonist-induced potentiation of
Ca2+-induced contraction is
inhibited by Clostridium botulinum C3 exoenzyme or epidermal cell differentiation inhibitor (12, 16, 18, 20),
specific inhibitors of Rho proteins. Fourth, GTP
S inhibition of
myosin light chain phosphatase is attenuated by C3 exoenzyme (28).
Fifth, Rho-associated kinase, which is activated by GTP-Rho A,
inactivates myosin light chain phosphatase by phosphorylating its
myosin binding unit (21). Sixth, constitutively active mutants of Rho A
(16, 30), of H-Ras (33), and of the mitogen-activated protein kinase
extracellular signal-regulated kinase 2, which is downstream to Ras
(14), all potentiate Ca2+-induced
contraction in smooth muscle preparations. However, the relative importance of particular G proteins in the
Ca2+-sensitizing effects of
specific agonists on specific smooth muscle types is not well
characterized.
Airway smooth muscle expresses M2 and M3 muscarinic receptors, with greater than 80% being of the M2 subtype (7, 9, 11, 32). Both endothelin (ET)A and ETB receptors are present in airway smooth muscle (15). Muscarinic M3 and ET receptors couple to phospholipase C to produce increases in inositol trisphosphate and diacylglycerol via the heterotrimeric G protein Gq. Activation of the M2 muscarinic receptor inhibits adenylyl cyclase via interaction with members of the pertussis toxin-sensitive G protein family Gi. Activation of M3 muscarinic or ET receptors contracts the muscle, whereas activation of M2 receptors inhibits cAMP-induced relaxation. A recent study (36) has shown that agonists that bind to G protein-coupled receptors can also stimulate mitogenic signaling via the Ras superfamily of monomeric G proteins. We therefore investigated the contributions of Gq, Gi, Ras, and Rho to acetylcholine (ACh)- and ET-induced contractions of permeabilized porcine tracheal smooth muscle strips at a constant [Ca2+].
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METHODS |
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Animals and tissue preparation. Young adult swine (50-70 lb) were sedated with ketamine (40 mg/kg im), anesthetized with pentobarbital sodium (30 mg/kg iv), and killed by exsanguination through the femoral arteries. The tracheae were removed and placed in cold physiological salt solution for transport to the laboratory. Tissues were stored for up to 48 h at 4°C in similar solutions bubbled with 95% O2-5% CO2. Pairs of tracheal smooth muscle strips 0.2-0.3 mm in width and ~12 mm in length were dissected from the posterior aspect of the upper half of the trachea with a binocular microscope and tied at either end with 6-0 silk suture for later attachment to a chamber and force transducer (Grass FT-03).
Experimental chambers and solutions.
Pairs of smooth muscle strips were mounted vertically and studied
simultaneously in identical 800-µl chambers made from 1/4-dram
glass shell vials. The chamber solution was stirred continuously with a
magnetic bar. [Ca2+]
was changed by flushing the chambers with ~4 ml of solution added
through polyethylene tubing to the bottom of the chamber. Excess
solution was removed by suction at the top. Solutions contained (in mM)
130 potassium propionate, 20 mono[tris(hydroxymethyl)aminomethane]maleate, 7.0 MgCl2, 4.6 Na2ATP, 2.0 EGTA, 2.0 phosphocreatine, and 1.0 dithiothreitol plus 8 U/ml of creatine
phosphokinase, 106 M
leupeptin, and sufficient CaCl2 to
provide the desired concentration of free
Ca2+, assuming a
Ca2+-EGTA dissociation constant of
3 × 10
7 M. Calmodulin
was not added. Solutions of
10
9 and 3 × 10
4 M free
Ca2+ were prepared and carefully
adjusted to pH 7.10. Solutions of intermediate
[Ca2+] were then made
by mixing these solutions in appropriate proportions. All experiments
were performed at room temperature.
Smooth muscle strip permeabilization.
Permeabilization with staphylococcal -toxin was performed before the
strips were mounted in the chamber by 50-60 min of incubation with
830 U/ml of
-toxin in 300 µl of relaxing solution
([Ca2+] = 10
9 M, composition as
described in Experimental
chambers
and
solutions). Permeabilization with
-escin (60 µM) was performed in the
experimental chambers while isometric force in the presence of
10
5 M
Ca2+ was measured. The
-escin
concentration was increased to 80 µM in some experiments as needed to
increase the rate of increase in force. The
-escin was washed out
with relaxing solution when force had nearly plateaued (after ~45
min).
Drug treatments. Smooth muscle strips
used in experiments involving pertussis toxin or farnesyltransferase
inhibitor FPT III were incubated before -toxin permeabilization in
the presence or absence of the drug for 18-20 h in 500 µl of
medium in a 24-well plate at 31°C in a humidified atmosphere of 5%
CO2 in air. Preliminary results
indicated better preservation of contractile responses in tissues
pretreated at this reduced temperature. The culture medium was
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 250 ng/ml of amphotericin B, and 100 U/ml of nystatin. Pertussis toxin was
prepared as a 500 µg/ml stock solution in water and was added at a
final concentration of 20 µg/ml. FPT III was used at a final
concentration of 5 × 10
4 M. Smooth muscle strips
used for studies of G protein antagonist 2A (GP Ant-2A) were
permeabilized with
-escin to allow entry of the peptide antagonist.
GP Ant-2A (5 × 10
5 M)
was added during permeabilization, to the relaxing solution used to
wash out
-escin, and to the 3.6 × 10
7 M
Ca2+ solution used during the
experimental period. Incubation with C3 exoenzyme (2.5 µg/ml) was
similar to that with GP Ant-2A except that 3 × 10
5 M
-NAD was added to
both treated and control tissues during the treatment and C3 exoenzyme
was washed out before the experimental period.
Experimental protocols. In all cases,
drug effects were evaluated in pairs of smooth muscle strips that were
preincubated, permeabilized, and studied in parallel by identical
methods, except for the presence or absence of the stated drug.
Preliminary experiments indicated that baseline forces of ~2 mN were
optimal for active force generation in these tissues. Hence, after
permeabilization with either -toxin or
-escin, tissues were
bathed with relaxing solution
(10
9 M free
Ca2+) and were stretched until a
stable baseline force of ~2 mN was obtained. This and all solutions
used subsequently contained
10
6 M calcium ionophore
A-23187 to deplete intracellular
Ca2+ stores. After a 20-min
exposure to A-23187, the
[Ca2+] was increased
to a value that typically increased force by ~5% of the maximum
Ca2+ response
(10
7 and 3.6 × 10
7 M for experiments
employing
-toxin and
-escin, respectively). In experiments
involving pertussis toxin, GP Ant-2A or FPT III, cumulative additions
of drugs were then made at 10- to 15-min intervals in the following
order: GTP, ACh, atropine, ET-1, and GTP
S. All drugs were then
washed out with 3 × 10
4 M free
Ca2+ solution, and the difference
between the maximum force obtained in this solution and the force
obtained in the relaxing solution before the protocol was used to
normalize the drug-induced changes in force in that smooth muscle
strip. To limit the duration of experiments involving C3 exoenzyme, ACh
and ET-1 responses were studied in separate groups of tissues by
abbreviated protocols.
Drugs and chemicals. -Toxin, C3
exoenzyme, FPT III
{(E,E)-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino]ethyl]phosphonic acid; (2,2-dimethyl-1-oxopropoxy)methyl ester, sodium}, and
GP Ant-2A
(H-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2) were purchased from Calbiochem (La Jolla, CA). Culture reagents were
from Life Technologies (Gaithersburg, MD). All other drugs and
chemicals were obtained from Sigma (St. Louis, MO).
Data analysis. Force vs.
[Ca2+] curves were
fitted by least squares with the Hill equation to obtain estimates of
the [Ca2+] required
for 50% maximum active force
(EC50) in each tissue. Mean
log(EC50) values were compared
by paired t-tests. Unless stated
otherwise, drug-induced changes in isometric force at a constant
[Ca2+] were normalized
by the change in force induced by high
[Ca2+] in that strip.
The GTPS response was measured relative to the value obtained before
addition of GTP. Effects of pretreatments on force responses were
analyzed by paired two-tailed t-tests, with n = number of animals
and P < 0.05 considered significant.
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RESULTS |
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As has been previously shown in tracheal smooth muscle strips from
other species (1, 29), ACh produced a leftward shift of the
[Ca2+]-force curve of
porcine tracheal smooth muscle (Fig. 1).
Log(EC50) values for
Ca2+ were 6.71 ± 0.09 and
6.42 ± 0.10 log(M) in the presence and absence of
10
5 M ACh, respectively
(P = 0.01;
n = 4). A free
[Ca2+] of
10
4 M produced an
essentially maximum force response. We chose to study the pathways that
contribute to this sensitizing effect by measuring agonist-induced
contractions at a constant
[Ca2+] in
permeabilized smooth muscle strips. Figure
2A shows a
typical protocol in which ACh produced contraction of
-toxin-permeabilized strips at
10
7 M free
Ca2+ in the presence of 5 × 10
6 M GTP. Atropine
reversed the ACh response, and subsequent additions of ET-1 and GTP
S
to the same tissues produced similar contractions.
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Figure 2B shows results from an
experiment similar to that of Fig. 2A
but with a strip permeabilized with -escin. In general, active
forces recorded in
-escin-permeabilized strips were smaller than
those obtained with
-toxin-permeabilized strips of similar size
(Fig.
3A). In
21 control strips permeabilized with
-escin, the active force
induced by Ca2+ (3.24 ± 0.25 mN) tended to be less than that recorded in 18
-toxin-permeabilized strips studied in the absence of drugs (5.39 ± 0.75 mN;
P = 0.054). Contractile responses to
10
4 M GTP
S were
significantly less in 13
-escin-permeabilized strips than in 18
-toxin-permeabilized strips (P = 0.0001). Responses to 10
7 M
ET-1 were also significantly different in these same tissues (P = 0.0017). Responses to
10
4 M ACh were less in
-escin- than in
-toxin-permeabilized strips (0.29 ± 0.05 mN,
n = 15 vs. 0.53 ± 0.28 mN,
n = 5), but this difference was not
significant (P = 0.79). Differences
between
-escin and
-toxin remained significant when the responses
to ET-1 and GTP
S were normalized by the
Ca2+-induced force in the same
strips (P = 0.0037 and 0.0001, respectively; Fig. 3B). However,
when normalized by the contractile response to GTP
S in the same
strip, contractile responses to ET-1 were not significantly different
in
-escin- and
-toxin-permeabilized strips (36 ± 4 vs. 48 ± 4%; P = 0.06; Fig.
3C).
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With the use of the protocol illustrated in Fig. 2, we studied the
effects of the Gq-selective
inhibitor GP Ant-2A on agonist-induced contractions at a constant
[Ca2+] in
-escin-permeabilized strips. As shown in Fig.
4, contractions induced by ACh and ET-1
were significantly reduced in the presence of GP Ant-2A
(P = 0.007 and
P = 0.008, respectively;
n = 6). The proportional
decrease in the ACh response (~28%) was approximately one-half that
of the ET-1 response (~46%). GP Ant-2A attenuated the contractile
response to GTP
S by ~47% (P = 0.006). The force response to high
[Ca2+] was slightly
different between treated and control tissues (2.41 ± 0.26 vs. 2.80 ± 0.23 mN; P = 0.025;
n = 6).
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To further investigate the role of large G proteins in the potentiation
of Ca2+-induced contractions by
ET-1 and ACh, we performed similar studies in strips pretreated with
pertussis toxin to inactivate Gi.
Strips were permeabilized with -toxin and studied as shown in Fig.
2. Contractile responses to ACh were diminished in pertussis
toxin-treated strips, whereas responses to ET-1 and to GTP
S were
unaffected. A summary of data from 11 experiments with smooth muscle
strips from 9 animals is given in Fig. 5.
An ~28% decrease in ACh-induced force in pertussis toxin-treated
strips was significant (P = 0.027; n = 9), whereas responses to other
stimuli did not differ between treated and control tissues
(P = 0.65 and 0.69, respectively, for
ET-1 and GTP
S). Maximum
Ca2+-induced increases in force
did not differ between treated and control strips (6.02 ± 1.02 vs.
6.37 ± 1.16 mN; P = 0.55;
n = 9).
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To evaluate the role of Rho in agonist enhancement of
Ca2+-induced contractions, we
studied -escin-permeabilized tracheal smooth muscle strips after
pretreatment with C3 exoenzyme, which inactivates that family of small
G proteins by ADP ribosylation (2). C3 exoenzyme decreased the
contractile responses at a constant
[Ca2+] produced by
ACh, ET-1, and GTP
S (P = 0.029, 0.009, and <0.0001, respectively;
n = 8, 6, and 7, respectively; Fig.
6). The proportional decrease
in contraction produced by C3 exoenzyme was similar for all three
stimuli. High
[Ca2+]-induced force
was not significantly different in C3 exoenzyme-treated strips and
control strips (3.17 ± 0.34 vs. 3.46 ± 0.45 mN;
P = 0.21; n = 8 for strips
contracted with ACh and 3.40 ± 0.42 vs. 3.50 ± 0.58 mN;
P = 0.71;
n = 6 for strips contracted with
ET-1).
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A summary of data obtained with smooth muscle strips preincubated in
the presence or absence of the farnesyltransferase inhibitor FPT III is
shown in Fig. 7. Inhibition of Ras with
this drug caused a significant reduction in ET-1 contraction at a
constant Ca2+ (18.6 ± 2.1 vs. 25.7 ± 3.4%; P = 0.02; n = 7) but had no significant effect on contractions induced by ACh
(P = 0.33;
n = 5) or GTPS (P = 0.43;
n = 7). High
[Ca2+]-induced forces
did not differ between FPT III-treated and untreated strips (4.51 ± 0.78 vs. 4.72 ± 0.66 mN; P = 0.66;
n = 7).
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DISCUSSION |
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Permeabilized smooth mucle preparations have been used extensively to
characterize the cellular mechanisms that regulate
Ca2+ sensitivity. One concern with
the use of these methods is that the chemicals used to permeabilize the
cell membrane may alter the function of relevant cellular pathways.
Staphylococcal -toxin is thought to be less injurious to tissues
than detergents such as
-escin, but our use of
large-molecular-weight drugs (e.g., C3 exoenzyme) required that we
perform certain experiments in
-escin-permeabilized strips. To
compare these permeabilization methods, we evaluated contractile
responses in control tissues permeabilized with
-toxin or
-escin.
Although all strips were of similar size, we generally obtained lower
force responses to ACh, ET-1, GTP
S, and high
Ca2+ in those permeabilized with
-escin relative to those permeabilized with
-toxin (Figs. 2 and
3A) The attenuation of high
Ca2+ responses suggests loss of
"distal" cellular components such as calmodulin, myosin light
chain kinase, or actin in
-escin-permeabilized strips. Addition of
calmodulin to the bathing solutions has been shown to reduce the
decline of force over time in saponin-permeabilized smooth muscle
strips (3).
Significant differences in ET-1 and GTPS responses persisted after
normalization by the high
Ca2+-induced contraction (Fig.
3B). These data suggest an
additional attenuation of pathways activated by G proteins, possibly
due to diffusive loss of small G proteins. However, responses to ACh and ET-1 were similar in
-toxin- and
-escin-permeabilized strips when normalized by the GTP
S response. This indicates that the "upstream" portions of pathways activated by these contractile agonists (e.g., receptors and heterotrimeric G proteins) are not differentially affected by the method of permeabilization. Although quantitative differences do exist, we conclude that both methods of
permeabilization are useful for studies of agonist-induced Ca2+ sensitization. However,
results obtained by different methods of permeabilization should
probably not be compared numerically.
Muscarinic cholinergic receptors of types M3 and M2 are known to couple to the heterotrimeric (large) G proteins Gq and Gi, respectively, in airway smooth muscle (25). ETA and ETB receptors are expressed in porcine airway smooth muscle (15), and each of these receptor subtypes can couple to both Gq and Gi (34). Gq activation causes contraction of airway smooth muscle through release of Ca2+ from intracellular stores and enhancement of Ca2+ entry, whereas Gi activation antagonizes relaxation through inhibition of adenylyl cyclase (6, 9, 35). The roles of Gq and Gi in the enhancement of [Ca2+]i sensitivity are not known. We initially addressed this issue in experiments with GP Ant-2A, an analog of substance P that has been shown to inhibit activation of large G proteins with selectivity for Gq over Gi (27). Preliminary experiments indicated that a higher concentration of GP Ant-2A was required to have effects in our smooth muscle strips than was reported to be effective in a vesicular preparation (27). We attribute this relative insensitivity to limitations of drug access but cannot rule out the possibility of nonspecific effects in our experiments. GP Ant-2A inhibited the contractile responses to ACh and to ET-1 in permeabilized smooth muscle strips studied at a constant [Ca2+] (Fig. 4), consistent with the hypothesis that activation of Gq contributes to the potentiation of Ca2+-induced contractions by these agonists. The effect of GP Ant-2A was more pronounced on ET-1-induced contractions than on ACh-induced contractions, suggesting that the Ca2+-sensitizing effects of ET-1 may be mediated more exclusively by the Gq pathway than are the effects of ACh. However, comparisons of drug effects on ACh and ET-1 responses in this study might be misleading because the magnitude of the contractions induced by these agonists was not comparable. We therefore sought more direct evidence for a Gq-independent pathway in the Ca2+-sensitizing effects of ACh.
Resistance of ACh-induced contractions at a constant
[Ca2+] to inhibition
by GP Ant-2A could reflect involvement of a
Gi-mediated pathway. We tested
this possibility with tissues incubated overnight in the absence or
presence of pertussis toxin, which is known to ADP-ribosylate and
irreversibly inactivate Gi (10).
Tracheal smooth muscle strips treated with pertussis toxin showed
smaller ACh-induced contractions at a constant
[Ca2+], whereas the
responses to ET-1 and GTPS were unaltered (Fig. 5). These data
indicate a novel function for Gi
in mediating increases in Ca2+
sensitivity and, hence, indicate that
Gi contributes directly to
contraction. This conclusion contradicts the generally held belief that
the M2
receptor-Gi pathway contributes to
contraction only indirectly via inhibition of cAMP-mediated relaxation
(6). In fact, our results agree with studies of the
M2-selective antagonist methoctramine in guinea pig airways that yielded Schild plots with
slopes significantly less than unity (17), consistent with contraction
through a heterogeneous population of receptors. The lack of effect of
pertussis toxin on ET-1 responses indicates that ET couples primarily
to Gq in porcine tracheal smooth
muscle, consistent with other data from this laboratory showing
stimulation of inositol trisphosphate production by ET-1 but no
inhibition of GTP-stimulated adenylyl cyclase activity (8).
Studies (12, 16, 18, 20, 28) in a variety of nonairway smooth muscles
have indicated that the Rho p21 family of small G proteins mediates, at
least in part, the
Ca2+-sensitizing responses to
activation of large G protein-coupled receptors. Because ACh and ET-1
appeared to differ with regard to the relative importance of
Gq and
Gi in their
Ca2+-sensitizing effects, we
questioned whether inhibition of Rho family G proteins by C3 exoenzyme
would have differential effects on the responses to these agonists. C3
exoenzyme treatment reduced the contractile responses to ACh, ET-1, and
GTPS (Fig. 6), demonstrating that small G proteins of the Rho family
are important mediators of Ca2+
sensitivity in airway smooth muscle. However, the proportional inhibition was similar for all three stimuli. Hence, our data are
consistent with a convergent pathway in which
Gq and
Gi both lead to activation of Rho
family monomeric G proteins. Incomplete inhibition by C3 exoenzyme of
agonist-induced contractions in this study could result either from
incomplete inactivation of Rho by our treatment or from the presence
of a pathway for Ca2+
sensitization that does not involve Rho.
A previous study (33) demonstrated that a constitutively active mutant
of H-Ras p21 produced contraction of -escin-permeabilized mesenteric
microarteries at a constant
[Ca2+]. This result,
in combination with recent data that G protein-coupled receptors can
activate Ras (36), suggests that Ras may mediate, in part, the
Ca2+-sensitizing effects of
contractile agonists. To investigate this possibility, we studied
tracheal smooth muscle strips preincubated in the presence or absence
of a farnesyltransferase inhibitor, FPT III. Farnesyltransferase
catalyzes a posttranslational modification that is important for
activation of Ras (26). Our data showed consistently less contraction
by ET-1 in FPT III-treated strips than in control strips (Fig. 7).
Hence, a farnesylated protein, likely Ras, contributes to the acute
potentiation by ET-1 of
Ca2+-induced contraction. Because
ET-1 responses were not affected by pertussis toxin, we suggest that
Gq may mediate the activation of
Ras. Partial mediation of cholinergic effects via a
Gi pathway could lessen the
contribution of Gq to ACh
responses and account for the lack of effect of FPT III on contractile
responses to that agonist in these experiments. Ras may promote
contraction by stimulation of mitogen-activated protein kinase, which
can phosphorylate and activate myosin light chain kinase (23), or by
activation of a Rho G protein (4) that inhibits myosin light chain
phosphatase (28).
Although GTPS is presumed to activate all G proteins and hence all
of the pathways examined in this study, we found that the contractile
responses to GTP
S were inhibited by only some of the drugs used. C3
exoenzyme was effective, indicating that a substantial portion of the
GTP
S-induced response is mediated by Rho. GP Ant-2A also inhibited
contraction by GTP
S at a constant [Ca2+], suggesting
that GTP
S activation of Gq may
be more effective than its activation of small G proteins or that
Gq may activate some
Ca2+-sensitizing pathway that does
not involve small G proteins. However, an alternative explanation is
that GP Ant-2A is not entirely selective for large G proteins at this
concentration. Despite their ability to attenuate agonist-induced
responses, neither pertussis toxin nor farnesyltransferase inhibition
significantly reduced the contractile effects of GTP
S. Thus, whereas
Gi- and Ras-mediated pathways may
be important for receptor-mediated responses, they appear to account
for only a small fraction of the
Ca2+ sensitization that can be
induced by indiscriminate activation of all G proteins by GTP
S.
In summary, the signal transduction mechanisms of airway smooth muscle that lead to enhanced myofilament Ca2+ sensitivity involve both large and small G proteins, as has been demonstrated in other smooth muscle types. In this tissue, the effects of ET-1 are mediated primarily by Gq, whereas both Gq and Gi contribute to the effects of ACh. Studies with pertussis toxin indicate that Gi enhances airway smooth muscle contraction directly, in addition to its known ability to inhibit relaxation. Rho family small G proteins contribute to the Ca2+-sensitizing effects of both ACh and ET-1. Hence, the M3 receptor-Gq pathway and the M2 receptor-Gi pathway appear to operate in parallel to enhance Ca2+ sensitivity of airway smooth muscle via Rho. This functional redundancy may contribute to the durability of some agonist-induced contractions of airway smooth muscle. At least for ET, mediation of Ca2+ sensitization also involves a farnesylated protein, most likely a monomeric G protein of the Ras family.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant P01-HL-10342.
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FOOTNOTES |
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Address for reprint requests: T. Croxton, Dept. of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, 615 North Wolfe St., Baltimore, MD 21205.
Received 31 October 1997; accepted in final form 28 May 1998.
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