Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120
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ABSTRACT |
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We investigated whether Rho activation is required for Ca2+-insensitive paxillin phosphorylation, myosin light chain (MLC) phosphorylation, and contraction in tracheal muscle. Tyrosine-phosphorylated proteins have been implicated in the Ca2+-insensitive contractile activation of smooth muscle tissues. The contractile activation of tracheal smooth muscle increases tyrosine phosphorylation of the cytoskeletal proteins paxillin and focal adhesion kinase. Paxillin is implicated in integrin-mediated signal transduction pathways that regulate cytoskeletal organization and cell motility. In fibroblasts and other nonmuscle cells, paxillin tyrosine phosphorylation depends on the activation of Rho and is inhibited by cytochalasin, an inhibitor of actin polymerization. In permeabilized muscle strips, we found that ACh induced Ca2+-insensitive contraction, MLC phosphorylation, and paxillin tyrosine phosphorylation. Ca2+-insensitive contraction and MLC phosphorylation induced by ACh were inhibited by C3 transferase, an inhibitor of Rho activation; however, C3 transferase did not inhibit paxillin tyrosine phosphorylation. Ca2+-insensitive paxillin tyrosine phosphorylation was also not inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D, or by the inhibition of MLC phosphorylation. We conclude that, in tracheal smooth muscle, Rho mediates Ca2+-insensitive contraction and MLC phosphorylation but that Rho is not required for Ca2+-insensitive paxillin tyrosine phosphorylation. Paxillin phosphorylation also does not require actomyosin activation, nor is it inhibited by the actin filament capping agent cytochalasin D.
actin cytoskeleton; cytochalasin D; focal adhesion proteins; myosin light chain phosphorylation; Rho guanosine 5'-triphosphatase; Rho kinase
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INTRODUCTION |
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THE CONTRACTILE
ACTIVATION of tracheal smooth muscle tissues by muscarinic and
other agonists increases the tyrosine phosphorylation of the
cytoskeletal proteins paxillin and focal adhesion kinase (FAK; see
Refs. 31, 49, 60). In cultured cell lines, paxillin and FAK localize to
focal adhesion sites where transmembrane integrins bind to
extracellular matrix proteins (5, 57). These
sites serve as a locus for the connection of actin filaments to the cytosolic domains of integrins via cytoskeletal linker proteins such as
vinculin and -actinin and talin, enabling the transmission of
tension between the actin cytoskeleton and the extracellular matrix
(5, 59). The membrane-associated dense plaque
sites of smooth muscle cells have a similar molecular structure to the focal adhesion sites of cultured cells, and these sites play an analogous functional role in force transmission (5,
11, 45, 56).
Focal adhesion sites also serve as a center for the coordination of signaling pathways that mediate many cellular processes (5). Both FAK and paxillin are implicated in integrin-mediated signaling pathways that regulate cytoskeletal organization (7, 29). In fibroblasts and other cell lines, adhesion-induced integrin clustering initiates the tyrosine phosphorylation of paxillin and FAK and the formation of focal adhesions and stress fibers (5, 29, 40). These responses are also induced by the activation of G protein-coupled receptors for neuropeptides and growth factors such as bombesin, bradykinin, endothelin, and vasopressin (37). There is extensive literature documenting the dependence of both G protein-mediated and adhesion-stimulated tyrosine phosphorylation of paxillin and FAK on the small-molecular-weight GTPase Rho (4, 6, 7, 14, 35, 37, 39, 44). The formation of actin stress fibers and focal adhesions can be induced when Rho proteins are introduced into fibroblasts by micropipette (6, 30, 35), and these responses can be inhibited by tyrosine kinase inhibitors (36). Rho protein has also been found to be necessary for the tyrosine phosphorylation of paxillin and FAK in cell types other than fibroblasts (12, 16, 50, 61).
RhoA also stimulates actomyosin-based contractility by activating Rho kinase, also called ROCK II, a serine-threonine kinase (3, 20). Rho kinase elevates myosin light chain (MLC) phosphorylation either by its ability to phosphorylate and inhibit MLC phosphatase or by directly phosphorylating MLC (3, 20). MLC phosphorylation promotes actin-activated myosin activity and tension generation in both smooth muscle and nonmuscle cells. In fibroblasts, actomyosin activity and tension generation is required for the activation of FAK and for the phosphorylation of paxillin. In these cells, the tyrosine phosphorylation of FAK and paxillin can also be inhibited by cytochalasin, an actin filament capping agent that blocks actin filament polymerization (5, 37).
Rho has also been shown to mediate the Ca2+ sensitization of force and MLC phosphorylation in vascular and visceral smooth muscle tissues (15, 17, 19, 21, 27, 28, 32, 46). There is also evidence that the Ca2+ sensitization of contraction in some smooth muscle tissues depends on protein tyrosine phosphorylation (9, 10, 47). Rho-stimulated tyrosine phosphorylation and actin filament remodeling have been proposed as mechanisms for the Ca2+-insensitive force generation in smooth muscle (9, 10, 47).
We have previously reported that muscarinic stimulation of tracheal smooth muscle increases the tyrosine phosphorylation of paxillin and FAK concurrently with the development of contractile tension, suggesting that FAK and paxillin may play a role in signaling pathways that regulate the contractile activation of smooth muscle (49, 60). We have also found that actin filament polymerization is stimulated during the muscarinic contraction of tracheal smooth muscle (23). Muscarinic activation has also been shown to stimulate actin filament polymerization in cultured tracheal smooth muscle cells (54). Thus FAK and paxillin may be implicated in signaling pathways that regulate cytoskeletal organization during smooth muscle contraction. The tyrosine phosphorylation of both FAK and paxillin can be maximally stimulated in Ca2+-depleted tracheal muscle strips, indicating that signaling to these proteins is not dependent on a rise in intracellular Ca2+ (24, 49). We therefore hypothesized that the Ca2+-insensitive tyrosine phosphorylation of FAK and paxillin in smooth muscle may depend on the activation of Rho.
The objective of the present study was to determine whether the muscarinic receptor-activated signaling pathway for the Ca2+-insensitive tyrosine phosphorylation of paxillin in tracheal smooth muscle depends on the activation of Rho GTPase. We also evaluated the effects of cytochalasin D and actomyosin activation on paxillin phosphorylation.
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METHODS |
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Tissue preparation. Mongrel dogs weighing 20-25 kg were anesthetized with pentobarbital sodium and quickly exsanguinated. A 12- to 15-cm segment of extrathoracic trachea was immediately removed and immersed in physiological saline solution (PSS) at 22°C (composition in mM: 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose). The solution was aerated with 95% O2 and 5% CO2 to maintain a pH of 7.4. Muscle strips (0.1-0.2 mm wide × 7-10 mm long) were dissected from the trachea after removal of the epithelium and connective tissue layer.
Permeabilization of muscle strips and measurement of force.
Muscle strips were pinned in petri plates and permeabilized by a
modification of the method of Kitazawa et al. (21). Strips were incubated at 22°C in a relaxing solution composed of (in mM) 8.5 Na2ATP, 4 K+-EGTA, 1 dithiothreitol (DTT), 10 sodium creatinine phosphate, 20 imidazole, 8.9 MgAc2, 100.5 potassium acetate, and 1 mg/ml creatine phosphokinase (pH 7.1). After
20 min, the strips were then incubated in the same solution with the
addition of 150 µM -escin (Sigma) or 375 U/ml
-toxin
(Calbiochem), 1 µM leupeptin (a protease inhibitor), and 1 µM
carbonyl cyanide m-chlorophenylhydrazone (a mitochondrial
blocker) for another 20-25 min. Intracellular Ca2+
stores were then depleted by incubating the strips in 10 µM
Ca2+ ionophore A-23187 in relaxing solution. An algorithm
of Fabiato and Fabiato (13) was used to calculate the
composition of relaxing or contracting solutions containing free
Ca2+ from pCa 9 to pCa 5. No differences in results were
observed in strips permeabilized with
-toxin or
-escin.
Measurements of the effects of Rho kinase inhibitor and
cytochalasin D in intact and Ca2+-depleted tissues.
Each muscle strip was placed in PSS at 37°C in a 25-ml organ bath and
attached to a Grass force transducer. At the beginning of each
experiment, the optimal length for maximal active force (Lo) was determined by increasing muscle length
progressively until the active force in response to 105 M
ACh (Sigma Chemical) reached a maximum for that stimulus
(Fmax). All subsequent changes in muscle length were
calibrated as fractions of Lo. Up to 14 muscle
strips from a single trachea were contracted isometrically with ACh at
muscle lengths of Lo.
Extraction of muscle proteins. Frozen muscle strips were pulverized in liquid N2 and were transferred to dry ice-cooled Eppendorf tubes. While on ice, 70-80 µl of extraction buffer were added to each of the tubes, after which they were quickly vortexed. The extraction buffer contained 20 mM Tris (pH 7.4), 1% Triton X-100, 0.2% SDS, 2 mM EDTA, phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and 2 mM sodium pyrophosphate), and protease inhibitors (2 mM benzamidine, 0.5 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Each sample was then boiled for 5 min to inactivate phosphatases and proteases after which the samples were maintained at 4°C for 1 h. Insoluble material was removed by centrifugation, and the supernatant was transferred to a new tube. For the extraction of FAK, the concentration of SDS in the extraction buffer was increased to 2%. After extraction, the SDS content was readjusted to 0.2% before determination of the protein concentration. The concentration of protein was determined in an aliquot of supernatant using a standard microbicinchoninic acid protein assay kit (Pierce).
Immunoprecipitation of paxillin or FAK. Paxillin and FAK immunoprecipitation were performed at 4°C, as described previously (31, 49, 60). Muscle extracts containing equal amounts of protein were precleared for 30 min with 50 µl of a 10% suspension of protein A-Sepharose beads. The samples were then centrifuged and incubated overnight with anti-mouse monoclonal paxillin antibody (clone no. 349; Transduction Laboratories) or FAK antibody (clone no. 77; Transduction Laboratories) and for an additional 2 h with 100 µl of a 10% suspension of protein A-Sepharose beads coupled to anti-mouse IgG. The beads from each sample were collected by centrifugation and washed four times with ice-cold immunoprecipitation wash buffer [10 mM Tris · HCl (pH 7.4), 150 mM NaCl, and 0.1% Triton X-100]. Paxillin or FAK was eluted from the beads by boiling the samples for 5 min in sample buffer.
Measurement of tyrosine phosphorylation of paxillin or FAK. Immunoprecipitates of paxillin or FAK or whole muscle extracts (20 µg) were electrophoresed in one dimension on 10% SDS-polyacrylamide gels. Proteins were then transferred to nitrocellulose, blocked with 2% gelatin, and probed with antibody to phosphotyrosine (PY-20; ICN Pharmaceuticals) followed by horseradish peroxidase anti-mouse Ig (Amersham) for visualization by chemiluminescence. Nitrocellulose membranes were then stripped of bound antibodies and were reprobed with a monoclonal antibody against paxillin or FAK to confirm the location of paxillin or FAK and to normalize for minor differences in protein loading. Phosphotyrosine-containing proteins and paxillin or FAK were visualized by chemiluminescence and quantitated by scanning densitometry.
Measurement of MLC phosphorylation. MLC phosphorylation was measured as described previously (24). Because of the small size of the permeabilized strips, it was necessary to pool two to three identically treated muscle strips to obtain each measurement of MLC phosphorylation. Frozen muscle strips were immersed in dry ice-cooled acetone containing 10% (wt/vol) TCA and 10 mM DTT (acetone-TCA-DTT). Strips were thawed in acetone-TCA-DTT at room temperature and then were washed with acetone-DTT. MLCs were extracted for 60 min in 8 mM urea, 20 mM Tris, 22 mM glycine, and 10 mM DTT. Proteins were separated by glycerol-urea PAGE and were blotted to nitrocellulose. MLCs were specifically labeled with polyclonal rabbit anti-MLC 20 antibody. The primary antibody was detected with 125I-labeled recombinant protein A. Unphosphorylated and phosphorylated bands of MLC were localized on nitrocellulose membranes by autoradiography. Bands were cut out and counted in a gamma counter. Background counts were subtracted, and fractional phosphorylation was calculated as the ratio of phosphorylated MLC to total MLC.
Purification of C3 transferase. C3 transferase, produced by Clostridium botulinum, specifically ADP ribosylates and inhibits Rho protein (2, 30). C3 transferase was purified from an Escherichia coli pGEX-2r-bcr recombinant vector expression system (25). Briefly, E. coli cells expressing pGEX vector were grown overnight in Luria broth medium after which they were separated by centrifugation. Cells were then sonicated on ice and centrifuged. C3 transferase was then purified as fusion proteins by conjugating the proteins in the supernatant to glutathione-Sepharose beads. After the beads were washed, C3 transferase was released from the beads by thrombin treatment. Thrombin was removed from C3 transferase by incubating it with p-aminobenzamidine-agarose beads (Sigma). The buffer used for C3 purification contained (in mM) 50 Tris (pH 7.5), 50 NaCl, 2 MgCl2, 1 DTT, and 2.5 CaCl2. After purification, C3 transferase was dialyzed and stored in pCa 9 solution.
ADP ribosylation of Rho by C3 transferase.
Rho protein in tracheal muscle strips was inactivated by ribosylation
with C3 transferase. -Escin-permeabilized muscle strips were
incubated in pCa 9 relaxing solution containing 100 µM NADP and 2.5 µg/ml C3 transferase for 30 min. Parallel strips were subjected to
incubation under the same conditions in the absence of C3 transferase.
Strips were then returned to pCa 7 solution and contracted with
10
4 M ACh. Tension, MLC phosphorylation, and paxillin
phosphorylation were then measured in these strips using protocols
described above.
Statistical analysis. The statistical significance of comparisons among different samples was determined by one-way ANOVA or by paired Student's t-test. All statistical analysis was performed using SigmaStat software. A P < 0.05 was considered to be statistically significant.
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RESULTS |
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ACh induces Ca2+-insensitive increases in force, MLC
phosphorylation, and paxillin tyrosine phosphorylation.
Five to six permeabilized muscle strips were attached to force
transducers and maintained at a Ca2+ concentration of pCa
7. Strips were then stimulated with 104 M ACh or with
GTP
S or by increasing Ca2+ concentration to pCa 6. GTP
S directly activates heterotrimeric G proteins. In separate
experiments, muscle strips were stimulated with 10
4 M ACh
or GTP
S for 1-15 min at pCa 7 or were maintained at pCa 7 for
an equivalent time period. They were then frozen for the measurement of
MLC phosphorylation or the tyrosine phosphorylation of paxillin. Figure
1A shows the increase in the
phosphotyrosine content of paxillin when the muscle strips were
stimulated with ACh at a Ca2+ concentration of pCa 7. The
tyrosine phosphorylation of paxillin increased approximately twofold
under these conditions (Fig. 2). Increasing the Ca2+ concentration from pCa 7 to pCa 6 also
stimulated an increase in paxillin tyrosine phosphorylation (Fig.
1B).
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Effect of Rho inactivation with C3 transferase on force, MLC
phosphorylation, and the tyrosine phosphorylation of paxillin.
-Escin-permeabilized muscle strips were contracted isometrically
with Ca2+ at pCa 6.0. After this test contraction, strips
were relaxed with pCa 9 solution. Strips producing equivalent amounts
of force in response to an increase in Ca2+ concentration
to pCa 6 were paired. They were then incubated for 1 h with or
without 2.5 µg/ml of C3 transferase plus 100 µM NAD in pCa 9 solution. The strips were then contracted with 10
4 M ACh
at pCa 7 (Fig. 3). In separate parallel experiments, untreated muscle
strips and strips pretreated with C3 transferase were stimulated with
10
4 M ACh for 5 min at pCa 7 or were maintained at pCa 7 for an equivalent time period. They were then frozen for the
measurement of MLC phosphorylation or the tyrosine phosphorylation of paxillin.
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Effect of Rho kinase inhibition on paxillin and FAK phosphorylation
in Ca2+-depleted muscle strips.
Ca2+-depleted tracheal muscle strips were incubated with or
without 10 µM Y-27632, a Rho kinase inhibitor, for 60 min. They were
then stimulated with 105 M ACh for 5 min and were frozen
for analysis of the tyrosine phosphorylation of paxillin or FAK. The
increase in the tyrosine phosphorylation of paxillin and FAK in
Ca2+-depleted muscle strips in response to stimulation with
ACh was similar in the strips treated with Y-27632 and in untreated
strips (Figs. 7 and
8). MLC phosphorylation did not
increase significantly in response to stimulation with ACh in
Ca2+-depleted muscle strips, so the effects of Y-27632 on
Ca2+-insensitive MLC phosphorylation could not be assessed
in these strips. However, the treatment of undepleted muscle strips
with Y-27632 resulted in a partial inhibition of contractile force (20-25%) in response to 10
4 M ACh
(n = 4). MLC phosphorylation was also partially
inhibited in these strips (data not shown). In addition, Y-27632
inhibited ACh-induced tension generation in permeabilized muscle strips maintained at pCa 7; 10 µM Y-27632 inhibited force by 60%
(n = 2), whereas 100 µM Y-27632 inhibited force by
70-100% (n = 3).
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Effect of cytochalasin D on paxillin tyrosine phosphorylation.
Intact muscle strips were incubated in 1 µM cytochalasin D, an actin
filament-capping agent (8), for 60 min and then were stimulated with 104 M ACh for 5 min. They were then
frozen for the analysis of paxillin tyrosine phosphorylation and MLC
phosphorylation. Treatment with cytochalasin markedly depressed
contractile force in response to ACh, and it also caused a slight
depression of MLC phosphorylation. However, cytochalasin had no
effect on paxillin tyrosine phosphorylation (Fig.
9).
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Relative Ca2+-sensitivity of ACh-induced increases in
the tyrosine phosphorylation of paxillin and MLC phosphorylation.
The effects of 104 M ACh on force, paxillin
phosphorylation, and MLC phosphorylation was compared in muscle strips
with intracellular Ca2+ maintained at pCa 8, pCa 7.5, or
pCa 7 (Fig. 10). At pCa 8, the addition
of 10
4 M ACh caused little or no increase in force or MLC
phosphorylation; however, ACh elicited a significant increase in
paxillin phosphorylation. When intracellular Ca2+ was
increased to pCa 7.5 or pCa 7, ACh increased both force and MLC
phosphorylation significantly in addition to paxillin phosphorylation (Fig. 10). The increases in paxillin phosphorylation in response to ACh
at pCa 7.5 and pCa 7 were similar to those observed in response to ACh
at pCa 8. ACh did not induce a detectable increase in paxillin
phosphorylation at pCa 9 (data not shown).
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DISCUSSION |
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Our results demonstrate that, in tracheal smooth muscle, the Ca2+-insensitive tyrosine phosphorylation of paxillin that is elicited in response to muscarinic stimulation is not inhibited by inactivation of the small GTPase Rho with C3 transferase. In contrast, under the same conditions of stimulation, Ca2+-independent MLC phosphorylation and tension development induced by ACh in this tissue are markedly inhibited by C3 transferase. We also found that neither the tyrosine phosphorylation of paxillin nor FAK was inhibited by Y-27632, an inhibitor of Rho kinase (p160 ROCK). Paxillin tyrosine phosphorylation was not affected when MLC phosphorylation was inhibited by Ca2+ depletion. Paxillin phosphorylation was also unaffected by the actin filament-capping agent cytochalasin D. Thus, in tracheal smooth muscle tissues, signaling to paxillin induced by muscarinic receptor activation is mediated by a pathway that is independent of Rho activation and actomyosin activity.
Role of Rho in paxillin phosphorylation in nonmuscle cells. Rho has been shown to be an upstream mediator of signaling events leading to the tyrosine phosphorylation of both FAK and paxillin in many cell types (4, 5, 7, 12, 14, 16, 33, 35, 37, 38, 39, 41, 44, 50, 61). The introduction of Rho protein into fibroblasts stimulates the tyrosine phosphorylation of FAK, paxillin, and p130Cas (14, 30, 35). In these cells, C3 transferase inhibits the tyrosine phosphorylation of FAK and paxillin that is stimulated by adhesion, growth factors, or neuropeptide-induced G protein receptor activation (5, 22, 33, 37, 38, 41). In fibroblasts, the phosphorylation of FAK and paxillin has been proposed to occur via the Rho-dependent activation of actin-activated myosin ATPase activity (5, 6, 37, 38). Rho activates Rho kinase, which increases MLC phosphorylation by inhibiting MLC phosphatase or by directly phosphorylating MLC (3, 20, 27). The resulting tension development is proposed to cause the alignment of actin filaments and the aggregation of integrins, thereby activating FAK and associated kinases (e.g., src) that catalyze the tyrosine phosphorylation of paxillin. In fibroblasts and other cells, the tyrosine phosphorylation of paxillin and FAK has also been shown to depend on cytoskeletal integrity and can be inhibited by cytochalasin D, which caps actin filaments at the barbed ends (8) and inhibits their polymerization (1, 16, 26, 37, 42, 43, 50, 62).
A similar Rho dependence of paxillin and FAK phosphorylation has been shown in cell types other than fibroblasts. CCK and epidermal growth factor both stimulate the tyrosine phosphorylation of FAK and paxillin in rat pancreatic acini; the phosphorylation of these proteins is dependent on Rho activation and is inhibited by cytochalasin D (16, 50). In brain vascular endothelial cells, the tyrosine phosphorylation of paxillin and FAK induced by intercellular adhesion molecule-1 cross-linking can be inhibited by the inactivation of Rho (12).Effect of inhibition of Rho and Rho kinase on paxillin
phosphorylation in tracheal muscle.
We found that the incubation of tracheal smooth muscle strips with C3
transferase had no effect on Ca2+-insensitive ACh-induced
paxillin tyrosine phosphorylation, whereas C3 markedly inhibited MLC
phosphorylation and tension development. C3 transferase specifically
ADP ribosylates RhoA, thereby inactivating it (25). We
used -escin-permeabilized tracheal muscle strips to study the
effects of RhoA inactivation with C3 transferase on paxillin and MLC
phosphorylation.
-Escin-permeabilized smooth muscle tissues have
previously been used successfully to introduce C3 transferase into
visceral and vascular smooth muscle tissues (15,
17, 27, 28, 32). We
confirmed that 1 h of incubation with C3 transferase was
sufficient to ribosylate Rho (Fig. 6). The fact that ACh-induced MLC
phosphorylation and force development were substantially inhibited in
tissues that had been treated with C3 transferase provides further
substantiation that Rho was inactivated by the C3 treatment. The
increase in paxillin phosphorylation elicited by ACh was somewhat lower
in permeabilized tracheal smooth muscle strips than in intact or
Ca2+-depleted tracheal muscle strips, whereas
Ca2+-induced paxillin phosphorylation reached comparable
levels in permeabilized and intact muscle strips. The lower levels of
paxillin phosphorylation observed in response to muscarinic receptor
activation in permeabilized muscle strips may have resulted from the
partial disruption of receptor-coupled signaling pathways by the
permeabilization procedure.
Effect of cytochalasin on paxillin phosphorylation.
In fibroblasts and other cell types, paxillin phosphorylation depends
on cytoskeletal integrity and can be blocked by cytochalasin D
(1, 16, 26, 37,
42, 43, 50, 62).
Cytochalasin binds to the barbed (fast-growing) ends of actin filaments
and acts as an actin-capping protein, preventing polymerization
(8). In many cell types, actin polymerization and
cytoskeletal remodeling depend on Rho. Rho mediates actin
polymerization by modulating phospholipid metabolism through the
regulation of its downstream effector phosphatidylinositol 4,5-kinase
(51). Phosphatidylinositol 4,5-bisphosphate interacts with
many proteins that are linked to the actin cytoskeleton, including
gelsolin, capZ, -actinin, and profilin (48). We
therefore evaluated the effects of cytochalasin D on muscarinic
receptor-induced phosphorylation of paxillin and FAK in smooth muscle.
We found that cytochalasin D inhibited ACh-induced force by 50%, with
only a slight effect on MLC polymerization, indicating that its effects
on force are likely to result from its effects on actin filaments
(23). However, cytochalasin D had no effect on paxillin
tyrosine phosphorylation induced by ACh. At this concentration,
cytochalasin D does not cause detectable effects on the cytoskeletal
organization of tracheal muscle, as assessed by electron microscopy and
by immunofluorescence imaging (23). Thus the fact that
cytochalasin D did not inhibit paxillin tyrosine phosphorylation in
tracheal smooth muscle may be due to the relative stability of actin
filaments in this tissue.
Role of tyrosine phosphorylation in Ca2+ sensitization
of force in smooth muscle.
Rho proteins have been shown to mediate agonist-induced
Ca2+ sensitization of force in intact and
-escin-permeabilized smooth muscle tissues (15,
17, 19, 27, 28,
32). The agonist-induced Ca2+ sensitization of
contraction is also inhibited by tyrosine kinase inhibitors in visceral
and vascular smooth muscles (9, 10, 47). This has led to the suggestion that cross talk
between Rho and tyrosine kinases may occur in smooth muscle as in
nonmuscle cells and that this may play a role in the regulation of
receptor-mediated Ca2+ sensitization of force and MLC
phosphorylation (32).
Role of paxillin in cytoskeletal remodeling. In nonmuscle cells, FAK and paxillin have both been implicated in the integrin-mediated signaling pathways that regulate cytoskeletal organization (5, 29). There is evidence that paxillin plays a central role in the reorganization of the cytoskeleton required for cell motility and cell spreading (34, 53, 55), and paxillin may also be required for Rho-dependent stress fiber formation (55).
We have recently demonstrated that muscarinic activation stimulates actin polymerization in tracheal smooth muscle tissues (23). Muscarinic activation has also been shown to stimulate actin filament polymerization in primary cultures of tracheal muscle cells, and in these cells this occurs by a Rho-dependent mechanism (54). However, the pathway that links muscarinic receptor activation to cytoskeletal remodeling in smooth muscle remains undetermined. Paxillin and/or FAK might be involved in regulating cytoskeletal organization during the contractile activation of smooth muscle by modulating the anchoring or remodeling of actin filaments. We conclude that the tyrosine phosphorylation of paxillin stimulated by the muscarinic activation of tracheal smooth muscle is independent of the activation of Rho or Rho kinase. Paxillin phosphorylation also does not depend on actomyosin activity and is not inhibited by actin filament capping by cytochalasin. In contrast, we found that Rho activation is necessary for Ca2+-insensitive tension generation and MLC phosphorylation in this tissue. Thus, in differentiated smooth muscle, the activation of signaling pathways mediated by paxillin and FAK may occur by pathways that are different from those in cultured cells. ![]() |
ACKNOWLEDGEMENTS |
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We appreciate the generous donation of Y-27632 by Yoshitomi Pharmaceutical Industries.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-29289, the Midwest Affiliate of the American Heart Association, and the Showalter Foundation.
Address for reprint requests and other correspondence: S. J. Gunst, Dept. of Physiology and Biophysics, Indiana Univ. School of Medicine, 635 Barnhill Rd., Indianapolis, IN 46202-5120 (E-mail: sgunst{at}iupui.edu).
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 18 November 1999; accepted in final form 16 February 2000.
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