Role of Rho in Ca2+-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

Dolly Mehta, Dale D. Tang, Ming-Fang Wu, Simon Atkinson, and Susan J. Gunst

Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 alpha -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 beta -escin (Sigma) or 375 U/ml alpha -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 alpha -toxin or beta -escin.

For the measurement of isometric force, permeabilized muscle strips were mounted in tissue baths and attached to Gould GM-2 force transducers. In each experiment, permeabilization of the strips was verified by contracting the muscles with 10 µM Ca2+.

Strips used for the determination of paxillin or MLC phosphorylation were mounted on wires under tension. They were stimulated either with Ca2+ alone (pCa 6) or with ACh (100 µM) or guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; 100 µM) at a constant Ca2+ concentration. After stimulation for a period of 1-20 min, the strips were frozen quickly in liquid N2 for biochemical analysis. Ten to twelve identically treated muscle strips needed to be pooled to immunoprecipitate sufficient paxillin to obtain a single phosphorylation measurement. Therefore, in each protocol, measurements obtained from paxillin immunoprecipitates were supplemented with additional measurements made from immunoblots of proteins from whole muscle extracts. Only four to five muscle strips needed to be pooled to obtain sufficient paxillin to obtain a measurement of paxillin phosphorylation from blots of whole muscle extracts. As we previously reported, similar increases in paxillin phosphorylation were measured in phosphotyrosine blots of paxillin immunoprecipitates and in phosphotyrosine blots of proteins from whole muscle extracts (49).

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 10-5 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.

The effect of cytochalasin D, an actin filament-capping agent (8), on paxillin phosphorylation was assessed. Intact muscle strips were incubated in cytochalasin D for 60 min before contraction with 10-4 M ACh. After 5 min of contraction, muscle strips were frozen for the measurement of paxillin phosphorylation.

The role of Rho kinase (p160 ROCK) in the regulation of paxillin and FAK phosphorylation was investigated by examining the effect of the pyridine derivative Y-27632, a specific inhibitor of Rho kinase (58), on paxillin and FAK tyrosine phosphorylation in Ca2+-depleted muscle strips. Y-27632 was generously provided by Yoshitomi Pharmaceutical Industries. Smooth muscle strips were depleted of intracellular Ca2+ before stimulation with contractile agonists. After Lo was determined, strips were incubated in Ca2+-free PSS containing 0.1 mM EGTA for 10 min for the removal of extracellular Ca2+. No change in resting tension occurred when the bath was changed from PSS to Ca2+-free PSS containing 0.1 mM EGTA. Muscle strips were then stimulated for 5 min by adding 10-5 M ACh to the Ca2+-free PSS. This step was repeated three to four times with 10-5 M ACh. Between each stimulation, the strips were incubated in Ca2+-free PSS containing 0.1 mM EGTA for 10 min. Stimulation with 10-5 M ACh initially produced a force of 70% Fmax, but subsequent stimulations resulted in progressively smaller contractions. At the end of the depletion protocol, force in response to 10-5 M ACh was <10% of Fmax at Lo.

After Ca2+ depletion, muscle strips were treated with or without 10 µM Y-27632 for 60 min and then were stimulated with 10-5 M ACh for 5 min. Strips were frozen for the determination of FAK or paxillin phosphorylation using a liquid nitrogen-cooled clamp. Each measurement of FAK or paxillin phosphorylation was based on measurements made on immunoprecipitates of protein from a single muscle strip. The effect of Y-27632 on MLC phosphorylation in response to ACh was assessed in intact undepleted tracheal muscle strips.

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. beta -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.

The effectiveness of Rho ribosylation by C3 transferase was evaluated by subjecting extracts of the C3-pretreated and untreated muscle strips to in vitro ribosylation by C3 transferase using [32P]NAD as a ribose donor. Strips pretreated with C3 transferase and untreated strips were frozen and then homogenized in buffer containing 250 mM triethanolamine hydrochloride (pH 7.5), 10 mM MgCl2, 5 mM EDTA, and 5 mM DTT (TEM buffer). ADP ribosylation was then performed at 37°C in a total volume of 50 µl of TEM buffer containing 20 µg of muscle lysate protein, 10 µM [32P]NAD (0.3 µCi), and 50 ng of C3 transferase. After 30 min, the reaction was stopped by adding 10 µl of 5× SDS sample buffer to each sample and boiling it for 10 min. Each sample was then electrophoresed in one dimension on 15% SDS-polyacrylamide gels. The ribosylation of Rho was evaluated by assessing the amount of 32P-labeled Rho on autoradiographs of dried gels. The absence of significant additional ribosylation in C3-pretreated strips (as indicated by a low level of incorporation of 32P) relative to that of untreated strips indicates that Rho protein had been effectively ribosylated by pretreatment of muscle strips with C3 transferase (see Fig. 6).

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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ABSTRACT
INTRODUCTION
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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 10-4 M ACh or with GTPgamma S or by increasing Ca2+ concentration to pCa 6. GTPgamma S directly activates heterotrimeric G proteins. In separate experiments, muscle strips were stimulated with 10-4 M ACh or GTPgamma 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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Fig. 1.   Immunoblot showing effects of stimulation with ACh (A) or intracellular Ca2+ (B) on the tyrosine phosphorylation of paxillin in permeabilized tracheal smooth muscle strips. The tyrosine phosphorylation of paxillin was measured in homogenates of permeabilized muscle strips frozen after 15 min of stimulation with 10-4 M ACh or after 20 min of stimulation with solutions containing Ca2+ at pCa 7 or pCa 6. Blots of paxillin immunoprecipitates were probed with anti-phosphotyrosine antibody and were stripped and reprobed with anti-paxillin antibody.



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Fig. 2.   Changes in force, myosin light chain (MLC) phosphorylation, and tyrosine phosphorylation of paxillin in response to stimulation of permeabilized muscle strips with ACh or guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). Groups of permeabilized muscle strips were stimulated with 10-4 M ACh () for 1-15 min or 100 µM GTPgamma S () for 5 min after incubation at pCa 7. Force in response to ACh (n = 8) or GTPgamma S (n = 4) at pCa 7 was normalized to the maximal force obtained in response to pCa 5. MLC phosphorylation was measured in response to ACh or GTPgamma S at pCa 7 (n = 4). Paxillin tyrosine phosphorylation in response to ACh (n = 4) or GTPgamma S (n = 3) is quantitated as multiples of level of increase over phosphorylation at pCa 7. Data shown are means ± SE. open circle , Mean values of MLC phosphorylation in strips frozen at pCa 9 or pCa 7 in the absence of ACh. All values obtained after stimulation with ACh were significantly different from values at pCa 7 without ACh (P < 0.05).

In muscle strips maintained at pCa 7, stimulation with 10-4 M ACh induced a significant increase in force (Figs. 2 and 3). Stimulation of muscle strips with 10-4 M ACh at pCa 7 also induced a significant increase in MLC phosphorylation. The levels of MLC phosphorylation and paxillin phosphorylation did not change significantly during the period of contractile stimulation (Fig. 2). GTPgamma S also induced significant increases in MLC phosphorylation and in the tyrosine phosphorylation of paxillin.


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Fig. 3.   Effect of C3 exoenzyme on force induced by intracellular Ca2+ and ACh in paired permeabilized tracheal smooth muscle strips. Untreated muscle strips and strips treated with C3 transferase for 60 min were activated by 10-4 M ACh at pCa 7 and then subsequently activated by increasing intracellular Ca2+ to pCa 6. C3 significantly inhibited contractile force in response to ACh at pCa 7; however, C3 did not inhibit contractions induced by increasing intracellular Ca2+ concentration alone. Traces are representative of muscle strips from 5 similar experiments.

Effect of Rho inactivation with C3 transferase on force, MLC phosphorylation, and the tyrosine phosphorylation of paxillin. beta -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.

Treatment of muscle strips with C3 markedly reduced force development induced by ACh, but it had no effect on force induced by increasing intracellular Ca2+ alone (Fig. 3). C3 transferase also inhibited the ACh-induced increase in MLC phosphorylation (Fig. 4). In contrast, C3 had no effect on the ACh-induced tyrosine phosphorylation of paxillin; similar levels of tyrosine phosphorylation of paxillin were observed in C3-treated and in untreated strips (Figs. 4 and 5). The immunoblot shown in Fig. 5 shows phosphotyrosine levels of paxillin in unstimulated muscle strips and in strips stimulated with ACh at pCa 7 with or without treatment with C3 transferase.


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Fig. 4.   Effect of C3 transferase on force, MLC phosphorylation, and tyrosine phosphorylation of paxillin during stimulation of tracheal muscle strips with ACh at pCa 7. C3 significantly inhibited force (n = 5) and MLC phosphorylation (n = 4) in response to ACh; however, it had no significant effect on the tyrosine phosphorylation of paxillin (n = 5). Force is normalized to the maximal force obtained in response to 10-4 M ACh in untreated muscle strips. Paxillin tyrosine phosphorylation is quantitated as multiples of level of increase over phosphorylation at pCa 7. Data shown are means ± SE. * Significant decrease in response to ACh in strips treated with C3 (P < 0.05).



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Fig. 5.   Immunoblot showing effect of C3 transferase on ACh-induced tyrosine phosphorylation of paxillin (Pax). The tyrosine phosphorylation of paxillin was measured in C3-treated and untreated permeabilized muscle strips frozen after 5 min of stimulation with 10-4 M ACh at pCa 7. Immunoblots were probed with anti-phosphotyrosine antibody and were stripped and reprobed with anti-paxillin antibody.

Muscle strips treated with C3 transferase and untreated muscle strips were homogenized and assayed for the in vitro ADP ribosylation of Rho to verify the effectiveness of the C3 treatment. Figure 6 shows the autoradiograph of in vitro ADP ribosylation of Rho by C3 transferase. Homogenates of untreated muscle strips underwent much greater ADP ribosylation in vitro, as indicated by the incorporation of 32P, than homogenates of C3-treated strips, indicating that most of the Rho in the C3-treated strips had been ribosylated during the incubation of the permeabilized strips with C3.


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Fig. 6.   Autoradiograph of ADP ribosylation of homogenates from C3-treated and untreated muscle strips. Muscle strips treated with C3 and untreated strips were frozen, homogenized, and assayed for ADP ribosylation of Rho by adding C3 transferase to the homogenates in the presence of [32P]NAD. Proteins were then separated by electrophoresis on 15% SDS-polyacrylamide gels. The ribosylation of Rho was evaluated by autoradiography. C3 induced a marked increase in the ADP ribosylation of Rho in untreated strips; however, little labeled ADP ribosylation of Rho occurred in muscle strips pretreated with C3 transferase. Autoradiograph is representative of results of 5-6 similar experiments.

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 10-5 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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Fig. 7.   Effect of Rho kinase inhibition on tyrosine phosphorylation of paxillin during stimulation of Ca2+-depleted tracheal muscle strips with ACh. Ca2+-depleted and undepleted muscle strips were incubated with and without 10 µM Y-27632 (Y), a Rho kinase inhibitor. Strips were then stimulated with 10-5 M ACh after which they were frozen to measure paxillin phosphorylation. The increase in paxillin phosphorylation induced by ACh was similar in strips that had been treated with Y-27632 and in untreated strips. Paxillin tyrosine phosphorylation is quantitated as multiples of level of increase over phosphorylation of unstimulated muscle strips. Data shown are means ± SE (n = 4).



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Fig. 8.   Effect of Rho kinase inhibition on tyrosine phosphorylation of focal adhesion kinase (FAK) during stimulation of Ca2+-depleted tracheal muscle strips with ACh. Ca2+-depleted tracheal muscle strips were incubated with or without 10 µM Y-27632, an inhibitor of Rho kinase. Strips were then stimulated with 10-5 M ACh after which they were frozen to determine the tyrosine phosphorylation of FAK. The increase in FAK phosphorylation was similar in strips treated with Y-27632 and in untreated strips. A: immunoblot of FAK immunoprecipitates probed with anti-phosphotyrosine antibody and stripped and reprobed with anti-FAK antibody. B: FAK phosphorylation in Y-27632-treated and untreated muscle strips quantitated as multiples of level of increase over phosphorylation in unstimulated muscle strips. Data shown are means ± SE (n = 4).

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 10-4 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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Fig. 9.   Effect of cytochalasin D (CCD) on tyrosine phosphorylation of paxillin during stimulation of tracheal muscle strips with ACh. Tracheal muscle strips were incubated with or without 1 µM cytochalasin D, an inhibitor of actin polymerization. Strips were then stimulated with 10-4 M ACh for 5 min and were frozen to determine the paxillin or MLC phosphorylation. Cytochalasin treatment caused a marked inhibition of force and a small but significant reduction in MLC phosphorylation (n = 6); however, paxillin phosphorylation was not significantly different in untreated strips and in strips treated with cytochalasin D (n = 4). Paxillin tyrosine phosphorylation is quantitated as multiples of level of increase over phosphorylation in unstimulated muscle strips. Data shown are means ± SE. * Significantly different from untreated muscle strips (P < 0.05).

Relative Ca2+-sensitivity of ACh-induced increases in the tyrosine phosphorylation of paxillin and MLC phosphorylation. The effects of 10-4 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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Fig. 10.   Effects of ACh on force, MLC phosphorylation, and tyrosine phosphorylation of paxillin in response to ACh at pCa 8, 7.5, and 7. Permeabilized muscle strips were stimulated with 100 µM ACh for 5 min while intracellular Ca2+ was maintained at pCa 8, pCa 7.5, or pCa 7. Force measured in response to ACh at pCa 8 (n = 4), pCa 7.5 (n = 4), and pCa 7 (n = 8) was normalized to the maximal force obtained in response to increasing intracellular Ca2+ concentration to pCa 5. MLC phosphorylation was measured in response to ACh at pCa 8 (n = 3), pCa 7.5 (n = 3), and pCa 7 (n = 5). Paxillin tyrosine phosphorylation in response to ACh at pCa 8 (n = 3), pCa 7.5 (n = 2), and pCa 7 (n = 4) is quantitated as multiples of level of increase in phosphorylation in response to ACh elicited at the same pCa. Data shown are means ± SE. * Values are significantly different from values obtained in absence of ACh (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -escin-permeabilized tracheal muscle strips to study the effects of RhoA inactivation with C3 transferase on paxillin and MLC phosphorylation. beta -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.

We sought further confirmation that Rho does not mediate muscarinic receptor-coupled signaling for the regulation of paxillin phosphorylation in tracheal smooth muscle by evaluating the effects of the inhibition of Rho kinase, a downstream effector of Rho, on ACh-induced tension and paxillin and FAK phosphorylation. Rho kinase (p160 ROCK) is a serine-threonine kinase that can be specifically inhibited by the pyridine derivative Y-27632 (58). ACh induces a maximal increase in the tyrosine phosphorylation of paxillin and FAK in Ca2+-depleted tracheal muscle strips (24, 49). In the present study, we found that the inhibition of Rho kinase with Y-27632 did not inhibit the ACh-induced increase in paxillin or FAK tyrosine phosphorylation in Ca2+-depleted tracheal muscle strips, although it caused a small but significant reduction in force and MLC phosphorylation in undepleted tracheal muscles in which Ca2+ signaling remained intact. Y-27632 also inhibited ACh-induced tension development in permeabilized tracheal muscle strips, indicating that ACh-induced Ca2+ sensitization of contraction is mediated by the Rho kinase pathway. Paxillin phosphorylation could also be stimulated in permeabilized muscle strips when intracellular Ca2+ was low enough to inhibit ACh-induced increases in MLC phosphorylation, indicating that paxillin phosphorylation does not depend on actomyosin activation or tension development (Fig. 10). These results suggest that Rho kinase is not an effector in the signaling pathway leading to the tyrosine phosphorylation of FAK or paxillin in tracheal muscle.

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, alpha -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 beta -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).

Our results demonstrate that Rho mediates agonist-induced Ca2+ sensitization of force in tracheal smooth muscle by sensitizing MLC phosphorylation to Ca2+. Our results clearly exclude the possibility that the tyrosine-phosphorylated proteins paxillin or FAK act downstream of Rho to induce Ca2+ sensitization of contraction in tracheal muscle. However, our results do not exclude the possibility that paxillin and FAK are involved in receptor-coupled signal transduction upstream of Rho and that they thereby play an essential role in agonist-induced contraction. Tyrosine-phosphorylated proteins may regulate the activity of guanine exchange factors or GTPase-activating factors that regulate Rho activity (52). For example, a GTPase-activating protein for Rho that binds to FAK (GRAF) can mediate cross talk between FAK and the Rho family GTPases in vitro (18), and GRAF can induce Rho-mediated cytoskeletal changes in some cell types (52).

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

We appreciate the generous donation of Y-27632 by Yoshitomi Pharmaceutical Industries.


    FOOTNOTES

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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