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J Appl Physiol 91: 1452-1459, 2001;
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Vol. 91, Issue 3, 1452-1459, September 2001

HIGHLIGHTED TOPICS
Signal Transduction in Smooth Muscle
Selected Contribution: Roles of focal adhesion kinase and paxillin in the mechanosensitive regulation of myosin phosphorylation in smooth muscle

Dale D. Tang and Susan J. Gunst

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The increase in intracellular Ca2+ and myosin light chain (MLC) phosphorylation in response to the contractile activation of tracheal smooth muscle is greater at longer muscle lengths (21). However, MLC phosphorylation can also be stimulated by Ca2+-insensitive signaling pathways (19). The cytoskeletal proteins paxillin and focal adhesion kinase (FAK) mediate a Ca2+-independent length-sensitive signaling pathway in tracheal smooth muscle (30). We used alpha -toxin-permeabilized tracheal smooth muscle strips to determine whether the length sensitivity of MLC phosphorylation can be regulated by a Ca2+-insensitive signaling pathway and whether the length sensitivity of active tension depends on the length sensitivity of myosin activation. Although active tension remained length sensitive, ACh-induced MLC phosphorylation was the same at optimal muscle length (Lo) and 0.5 Lo when intracellular Ca2+ was maintained at pCa 7. MLC phosphorylation was also the same at Lo and 0.5 Lo in strips stimulated with 10 µM Ca2+. In contrast, the Ca2+-insensitive tyrosine phosphorylation of FAK and paxillin stimulated by ACh was higher at Lo than at 0.5 Lo. We conclude that the length-sensitivity of MLC phosphorylation depends on length-dependent changes in intracellular Ca2+ but that length-dependent changes in MLC phosphorylation are not the primary mechanism for the length sensitivity of active tension.

myosin light chain phosphorylation; intracellular calcium; cytoskeleton


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY SMOOTH MUSCLE exhibits the property of "length adaptation," also referred to as "mechanical plasticity" (9, 10, 13, 24). The contractile responses of the muscle are not fixed for any particular set of stimulation conditions; instead, the muscle adapts its contractile behavior in response to its mechanical environment, thereby optimizing its contractility to the mechanical conditions under which it is stimulated (9, 10, 12, 13, 24). The plastic or length-adaptive properties of airway smooth muscle play an important role in regulating airway muscle responsiveness under dynamic conditions (11, 12, 27, 28). However, the mechanisms by which smooth muscle cells sense and respond to changes in their mechanical environment are not understood.

In airway smooth muscle and in other smooth muscle tissues, the intracellular calcium concentration ([Ca2+]i) is length sensitive when the muscles are stimulated with contractile stimuli (2, 21, 25). Myosin light chain (MLC) phosphorylation is also length sensitive in smooth muscles stimulated with contractile agonists; both MLC phosphorylation and [Ca2+]i are lower when the muscles are stimulated at a short muscle length than at a longer muscle length (21, 25, 35). As MLC phosphorylation is regulated by [Ca2+]i (17), the length-sensitive regulation of MLC phosphorylation may be mediated by the length-sensitive changes in [Ca2+]i. MLC phosphorylation increases actomyosin ATPase activity and smooth muscle contraction (17). Thus it has been suggested that the length sensitivity of smooth muscle active-tension generation may result from length-sensitive changes in MLC phosphorylation (2, 21, 25).

There is accumulating evidence that integrins can act as mechanoreceptors and transmit mechanical signals between the extracellular matrix and the actin cytoskeleton (6, 34). In smooth muscle tissues, integrins localize to dense plaque sites, which are believed to have a molecular structure similar to that of focal adhesion sites (5). At focal adhesion sites, actin filaments link to the cytoplasmic domains of the beta -subunits of integrin proteins via a number of cytoskeletal proteins, including alpha -actinin, talin, and vinculin. These sites also serve as a center for the coordination of signaling pathways that mediate many cellular processes (4). Two important components of focal adhesion sites, focal adhesion kinase (FAK) and paxillin, are implicated in actin filament remodeling, focal adhesion formation, and cell motility in cultured cells (1, 4, 23).

We have recently shown that FAK and paxillin play essential roles in regulating smooth muscle contraction (31, 32). FAK plays a critical role in the agonist-induced regulation of [Ca2+]i in airway smooth muscle (31). FAK is also a catalyst for the tyrosine phosphorylation of the cytoskeletal protein paxillin. Both FAK and paxillin undergo tyrosine phosphorylation during the contractile activation of tracheal smooth muscle, and the agonist-stimulated tyrosine phosphorylation of these proteins is length sensitive (30). Because of its critical role in the regulation of intracellular Ca2+, FAK may be an upstream mediator of the mechanosensitivity of intracellular Ca2+ and MLC phosphorylation in tracheal smooth muscle.

In airway smooth muscle, MLC phosphorylation can also be regulated by receptor-mediated Ca2+-independent signaling processes (8, 19). The small-molecular-weight GTPase RhoA mediates Ca2+-independent MLC phosphorylation and tension generation in canine tracheal smooth muscle (19). It is not known whether Ca2+-insensitive MLC phosphorylation is length sensitive. The tyrosine phosphorylation of both FAK and paxillin can also be regulated by Ca2+-independent signaling pathways, and the mechanosensitivity of their phosphorylation is retained in Ca2+-depleted muscle strips, suggesting that the mechanotransduction processes mediated by these proteins are upstream of length-sensitive changes in intracellular Ca2+ (20, 30). Thus FAK and paxillin may be part of a mechanosensitive signal transduction mechanism that can regulate intracellular events independently of changes in intracellular Ca2+.

In the present study, we used alpha -toxin-permeabilized tracheal smooth muscle strips to evaluate whether the length-sensitive regulation of [Ca2+]i is required for the mechanosensitive regulation of MLC phosphorylation and whether the length sensitivity of active tension depends on length-sensitive changes in myosin activation. We also evaluated the possibility that FAK and paxillin act as upstream regulators of a Ca2+-insensitive MLC phosphorylation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of tissue. Mongrel dogs (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 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% O2-5% CO2 to maintain a pH of 7.4. Rectangular strips of tracheal muscle 0.1-0.2 mm in diameter and 5-7 mm in length 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 incubated at 22°C in a relaxing solution composed of (in mM) 8.5 Na2ATP, potassium EGTA, 1 dithiothreitol (DTT), 10 sodium creatine phosphate, 20 imidazole, 8.9 MgAc2, 100.5 KAc, 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 alpha -toxin (375 U/ml) (Calbiochem), 1 µM leupeptin (a protease inhibitor), and 1 µM carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial blocker) for another 20-25 min. Intracellular Ca2+ stores were then depleted by incubating the strips in 10 µM calcium ionophore A-23187 in relaxing solution. An algorithm of Fabiato and Fabiato (7) was used to calculate the composition of relaxing or contracting solutions containing free Ca2+ from pCa 9 to pCa 5.

For the measurement of isometric force, the permeabilized muscle strips were mounted in tissue baths and attached to Gould GM-2 force transducers. In each experiment, the permeabilization of the strips was verified by contracting the muscles with 10 µM Ca2+. In addition, at the end of the experiment, the permeabilized strips were returned to normal physiological saline solution and stimulated with ACh. No contractile response was observed under these conditions. In separate experiments, the permeabilized strips were placed in ATP-free relaxing solution and stimulated with ACh at pCa 7. No contractile response to ACh was observed under these conditions, which is further evidence that the muscle strips were fully permeabilized.

Strips used for the determination of MLC phosphorylation or cytoskeletal protein phosphorylation were mounted on wires under tension. They were stimulated either with Ca2+ alone (pCa 5) or with ACh (100 µM) at a constant Ca2+ concentration. The strips were then frozen at the desired time points for biochemical analysis. Multiple identically treated muscle strips (10-12 strips for paxillin, 30-35 strips for FAK) needed to be pooled to immunoprecipitate sufficient paxillin or FAK to obtain a single phosphorylation measurement.

Analysis of MLC phosphorylation. Muscle strips were rapidly frozen 5 min after contractile stimulation and then immersed in acetone containing 10% (wt/vol) TCA and 10 mM DTT, which was precooled with dry ice. Strips were thawed in acetone-TCA-DTT at room temperature and then washed four times with acetone-DTT. Proteins were extracted for 60 min in 8 M urea, 20 mM Tris base, 22 mM glycine, and 10 mM DTT. MLCs were separated by glycerol-urea polyacrylamide gel electrophoresis and transferred to nitrocellulose. The membranes were blocked with 5% bovine serum albumin and incubated with polyclonal affinity-purified rabbit MLC20 antibody. The primary antibody was reacted with 125I-labeled recombinant protein A (New England Nuclear). Unphosphorylated and phosphorylated bands of MLCs were detected by autoradiography. Bands were cut out and counted in a gamma counter. Background counts were subtracted, and MLC phosphorylation was calculated as the ratio of phosphorylated MLCs to total MLCs.

Extraction of the cytoskeletal proteins. Muscle strips were freeze clamped and then pulverized in liquid nitrogen. The pulverized tissue was transferred to dry-ice-cooled centrifuge tubes. While on dry ice, extraction buffer was added to each of the tubes, and they were then quickly vortexed. The extraction buffer contained 20 mM Tris · HCl at pH 7.4, 2% Triton X-100, 2% SDS, 2 mM EDTA, phosphatase inhibitors (in mM: 2 sodium orthovanadate, 2 molybdate, and 2 sodium pyrophosphate) and protease inhibitors (in mM: 2 benzamidine, 0.5 aprotinin, and 1 phenylmethylsulfonyl fluoride). Each sample was boiled for 5 min to inactivate phosphatases and proteases after which it was maintained at 4°C for 1 h. The supernatant was collected after centrifugation at 14,000 rpm for 25 min at 4°C. For the extraction of paxillin, the concentration of SDS in the extraction buffer was decreased to 0.2%. The concentration of protein in each sample was determined using a standard bicinchoninic protein assay kit (Pierce).

Immunoprecipitation of paxillin or FAK. Paxillin and FAK immunoprecipitation were performed at 4°C as described previously (30, 31). Muscle extracts containing equal amounts of protein were precleared for 30 min with 50 µl of 10% suspension of protein A-Sepharose beads. The samples were then centrifuged and incubated overnight with antimouse monoclonal paxillin antibody (clone 349, Transduction Labs) or FAK antibody (clone 77, Transduction Labs) and for an additional 2 h with 100 µl of 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 were eluted from the beads by boiling the samples for 5 min in sample buffer.

Analysis of FAK and paxillin phosphorylation. Immunoprecipitates of FAK or paxillin were boiled in sample buffer (see above) for 5 min and separated by SDS-PAGE. Proteins were transferred to nitrocellulose, blocked with 2% gelatin, and probed with antibody to phosphotyrosine (PY20, ICN Pharmaceuticals) followed by horseradish peroxidase-conjugated anti-mouse Ig (Amerham) for visualization by enhanced chemiluminescence (ECL). Blots were then stripped of bound antibodies and reprobed with monoclonal antibodies against FAK or paxillin to confirm the location of the proteins and normalize for minor differences in protein loading. Phosphotyrosine and FAK or paxillin were quantitated by scanning densitometry after visualization by ECL. The tyrosine phosphorylation of FAK or paxillin was analyzed from immunoblots of FAK or paxillin immunoprecipitates. Changes in the tyrosine phosphorylation of FAK or paxillin were expressed as magnitude of increase over FAK or paxillin phosphorylation of resting tissues.

Statistical analysis. All statistical analyses were performed using SigmaStat software. Comparison among multiple groups was performed by one-way ANOVA or Kruskal-Wallis one-way ANOVA. Differences between pairs of groups were analyzed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Length sensitivity of MLC phosphorylation in the alpha -toxin-permeabilized muscle strips stimulated by Ca2+. We assessed the effect of muscle length on MLC phosphorylation and active force in smooth muscle strips stimulated by constant [Ca2+]i (Fig. 1). Tracheal smooth muscle strips permeabilized with alpha -toxin were stimulated with Ca2+ at pCa 5 and a muscle length of either Lo or 0.5 Lo. Active force was measured in some strips, whereas other strips were frozen after 5 min of stimulation for the measurement of MLC phosphorylation.


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  		Fig. 1.   alpha -Toxin-permeabilized smooth muscle strips were stimulated with Ca2+ at pCa 5 or pCa 6 for 5 min at optimal muscle length (Lo) and 0.5 Lo. A: active force is normalized to the contractile response to pCa 5. B: myosin light chain (MLC) phosphorylation is not length sensitive in permeabilized smooth muscle strips activated with Ca2+ at pCa 5 or pCa 6. All values are means ± SE (n = 4). *Statistically different values at 0.5 Lo compared with Lo values (P < 0.05).

Active force in the permeabilized smooth muscle strips stimulated with pCa 5 at 0.5 Lo was 22.9 ± 4.4% of the active force in the muscle strips stimulated with pCa 5 at Lo (n = 4, P < 0.05). At pCa 9, MLC phosphorylation in the permeabilized smooth muscle strips was similar at Lo and at 0.5 Lo (0.17 ± 0.02 and 0.19 ± 0.03 mol Pi/mol MLC, respectively; n = 4). Stimulation of the permeabilized strips with Ca2+ at pCa 5 increased MLC phosphorylation to 0.41 ± 0.01 mol Pi/mol MLC at Lo and 0.39 ± 0.02 mol Pi/mol MLC at 0.5 Lo (n = 4). The increases in MLC phosphorylation stimulated by Ca2+ at pCa 5 at Lo and at 0.5 Lo were not significantly different (P > 0.05). When permeabilized muscle strips were stimulated with Ca2+ at pCa 6, MLC phosphorylation was also similar at Lo (0.34 ± 0.01 mol Pi/mol MLC) and 0.5 Lo (0.33 ± 0.01 mol Pi/mol MLC) (n = 4), whereas force at 0.5 Lo was 21.1 ± 2.2% of force at Lo.

Length sensitivity of Ca2+-insensitive MLC phosphorylation in the alpha -toxin-permeabilized muscle strips stimulated by ACh. Tracheal smooth muscle strips were stimulated with 10-4 M ACh at constant Ca2+ (pCa 7) at the muscle lengths of Lo or 0.5 Lo. Active force was measured in some strips, whereas other strips were frozen after 5 min of stimulation for the measurement of MLC phosphorylation (Fig. 2). Nonpermeabilized smooth muscle strips at 22°C were also stimulated with 10-4 M ACh at Lo or 0.5 Lo to verify the length sensitivity of ACh-induced MLC phosphorylation under the same temperature and conditions used for the permeabilized muscles.


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  		Fig. 2.   MLC phosphorylation is not length sensitive in permeabilized muscle strips stimulated with ACh at pCa 7. The permeabilized smooth muscle strips were stimulated with ACh at pCa 7 for 5 min at isometric muscle lengths of Lo or 0.5 Lo. Intact (not permeabilized) smooth muscle strips were also stimulated by ACh at Lo and 0.5 Lo. A: active force is normalized to the contractile response to 10-4 M ACh for the nonpermeabilized strips or to 10-4 M ACh at pCa 7 for the permeabilized strips. B: MLC phosphorylation is similar in the permeabilized strips stimulated with ACh at pCa 7 at Lo or 0.5 Lo. All values are means ± SE (n = 5). *Statistically different values at 0.5 Lo compared with values at Lo (P < 0.05).

Active force in the permeabilized smooth muscle strips stimulated with ACh at pCa 7 at 0.5 Lo was 22.6 ± 9.29% of the active force in the muscle strips stimulated with ACh and pCa 7 at Lo (n = 4, P < 0.05). The mean maximal force of the permeabilized strips stimulated with ACh at pCa 7 at Lo was 126.3 ± 15.9 mg (n = 4). At pCa 7, MLC phosphorylation in the permeabilized smooth muscle strips was similar at Lo and 0.5 Lo, 0.21 ± 0.02 (n = 5) and 0.22 ± 0.02 (n = 4) mol Pi/mol MLC, respectively. Stimulation of the permeabilized strips with ACh at pCa 7 at Lo increased MLC phosphorylation to 0.41 ± 0.03 mol Pi/mol MLC. When the permeabilized strips were stimulated with ACh at pCa 7 at a muscle length of 0.5 Lo, MLC phosphorylation was 0.38 ± 0.03 mol Pi/mol MLC (n = 5). The increase in MLC phosphorylation in the permeabilized muscle strips elicited by ACh at pCa 7 was not significantly different at muscle lengths of 0.5 Lo and Lo (Fig. 2, P > 0.05).

In the nonpermeabilized muscle strips stimulated with ACh at 22°C, force generation at 0.5 Lo was 33.3 ± 8.3% of that at Lo (n = 4, P < 0.05). The maximal force of the nonpermeabilized strips stimulated with ACh at Lo was 127.1 ± 9.2 mg (n = 4). MLC phosphorylation in the unstimulated strips was 0.18 ± 0.03 mol Pi/mol MLC (basal; n = 5). MLC phosphorylation increased to 0.40 ± 0.03 mol Pi/mol MLC when the strips were stimulated with ACh at Lo and to 0.30 ± 0.04 mol Pi/mol MLC when strips were stimulated at 0.5 Lo (Fig. 2, P < 0.05).

Length dependence of FAK tyrosine phosphorylation in the permeabilized muscle strips. We evaluated whether the receptor-mediated Ca2+-insensitive tyrosine phosphorylation of FAK is sensitive to muscle length in alpha -toxin-permeabilized muscle strips. Permeabilized muscle strips at pCa 7 were stimulated with ACh at muscle lengths of Lo or 0.5 Lo and frozen after 5 min of stimulation for the determination of FAK tyrosine phosphorylation.

The tyrosine phosphorylation of FAK at 0.5 Lo and at Lo was evaluated in immunoprecipitates from extracts of permeabilized muscles (Fig. 3A). FAK tyrosine phosphorylation increased by 1.8 ± 0.1-fold in response to ACh at 0.5 Lo and by 3.6 ± 0.5-fold in muscle strips at Lo (Fig. 3B; n = 4, P < 0.05).


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  		Fig. 3.   Effect of muscle length on focal adhesion kinase (FAK) tyrosine phosphorylation in FAK immunoprecipitates from extracts of the permeabilized muscle strips stimulated with ACh at pCa 7. alpha -Toxin-permeabilized smooth muscle strips were stimulated with ACh at pCa 7 for 5 min at muscle lengths of Lo or 0.5 Lo . FAK immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with antiphosphotyrosine antibody and then stripped and reprobed with antiFAK antibody. A: immunoblots show the length sensitivity of FAK tyrosine phosphorylation in the permeabilized muscle strips stimulated with ACh at pCa 7. B: FAK phosphorylation is quantitated as multiples of increase in phosphorylation over the level in unstimulated tissues. Values are means ± SE (n = 4). *Significantly different value at 0.5 Lo compared with Lo value (P < 0.05).

Length dependence of paxillin tyrosine phosphorylation in the permeabilized muscle strips. We evaluated whether the receptor-mediated Ca2+-insensitive tyrosine phosphorylation of paxillin is sensitive to muscle length in alpha -toxin-permeabilized muscle strips. Permeabilized muscle strips at pCa 7 were stimulated with ACh at muscle lengths of Lo or 0.5 Lo and frozen after 5 min of stimulation for the determination of paxillin tyrosine phosphorylation.

The tyrosine phosphorylation of paxillin was evaluated in immunoprecipitates from extracts of permeabilized muscles (Fig. 4A). Paxillin tyrosine phosphorylation increased by 1.4 ± 0.1-fold in response to ACh at 0.5 Lo and by 2.6 ± 0.1-fold in muscle strips at Lo (Fig. 4B; n = 4, P < 0.05).


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  		Fig. 4.   Tyrosine phosphorylation of paxillin is length sensitive in the permeabilized muscle strips stimulated with ACh at pCa 7. The blots of paxillin immunoprecipitates from extracts of the permeabilized strips treated with ACh at pCa 7 for 5 min were reacted with antiphosphotyrosine antibody and then stripped and reprobed with antipaxillin antibody. A: immunoblots show the length-dependent regulation of paxillin tyrosine phosphorylation in the strips. B: paxillin phosphorylation is quantitated as multiples of the levels in unstimulated tissues at Lo. Values are means ± SE (n = 4). *Significantly different value at 0.5 Lo compared with Lo value (P < 0.05).

Summary of results. The effects of muscle length on active force, MLC phosphorylation, and paxillin and FAK phosphorylation for each experimental condition are summarized in Table 1.

                              
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  		Table 1.   Effect of muscle length on active force and protein phosphorylation


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate that the length sensitivity of MLC phosphorylation in smooth muscle is secondary to the length sensitivity of intracellular Ca2+. With the use of alpha -toxin-permeabilized smooth muscle tissues, we evaluated whether MLC phosphorylation is sensitive to muscle length when intracellular Ca2+ is controlled at a constant level independently of changes in muscle length. We found that MLC phosphorylation is not length sensitive in permeabilized tracheal tissues when they are stimulated with ACh at constant intracellular Ca2+ or when they are stimulated with 10 µM Ca2+ (pCa 5) alone. This is true even though both FAK and paxillin phosphorylation remain length sensitive under these conditions. These results suggest that the length sensitivity of MLC phosphorylation is caused by the length-dependent regulation of intracellular Ca2+. The results also indicate that the mechanosensitive regulation of Ca2+-independent FAK and paxillin phosphorylation does not modulate receptor-coupled Ca2+-insensitive MLC phosphorylation. In addition, our results suggest that the length sensitivity of MLC phosphorylation is not critical for the length-dependent regulation of active tension in this tissue.

We have previously shown that the depletion of FAK by antisense oligonucleotides markedly depresses agonist-induced increases in intracellular Ca2+ and MLC phosphorylation in tracheal smooth muscle (31). In differentiated colonic smooth muscle, activated pp60c-Src (Src) has been shown to interact with FAK to mediate the regulation of basal Ca2+-channel activity and the platelet-derived growth factor-induced enhancement of L-type Ca2+ currents (16). In the present study, we have found that the tyrosine phosphorylation of FAK is length sensitive in tracheal smooth muscle stimulated with ACh at constant Ca2+. These observations suggest that the mechanical signal (the change in muscle length) regulates the tyrosine phosphorylation of FAK, which then mediates mechanosensitivity of Ca2+ signaling and thereby regulates the length sensitivity of MLC phosphorylation. The length sensitivity of intracellular Ca2+ has been previously demonstrated in canine tracheal smooth muscle as well as in other smooth muscle tissues (2, 21, 25).

The tyrosine phosphorylation of paxillin is catalyzed by FAK in vitro (3, 33). In cultured fibroblasts, the phosphorylation of paxillin requires the subcellular localization and autophosphorylation of FAK and paxillin (26). We have previously reported that FAK is a necessary component of the signaling pathway leading to paxillin tyrosine phosphorylation in tracheal smooth muscle (31). In the present study, we observed that the agonist-induced Ca2+-insensitive contraction of tracheal muscle is associated with the length-sensitive regulation of the tyrosine phosphorylation of both FAK and paxillin, suggesting that the Ca2+-insensitive tyrosine phosphorylation of paxillin may also be mediated by FAK.

In cultured fibroblasts, integrin activation caused by adhesion to extracellular matrix proteins or by antibody-mediated integrin cross-linking leads to increased tyrosine phosphorylation of FAK and paxillin and recruitment of Src to the integrin-associated protein complex (4, 22). We have previously demonstrated that the tyrosine phosphorylation of FAK is length sensitive in tracheal smooth muscle during contractile activation (30). Our present results indicate that the tyrosine phosphorylation of both FAK and paxillin are also length sensitive in smooth muscle stimulated at constant Ca2+. This suggests that they may be upstream mediators for both Ca2+-insensitive as well as Ca2+-sensitive cellular mechanisms that regulate mechanotransduction in smooth muscle. Evidence from many different cell types indicates that transmembrane integrins can function as mechanotransducers and that the regulation of cellular responses to mechanical stimuli is coordinated by the complex of cytoskeletal proteins that associate with the cytoplasmic domains of integrin molecules (29). Thus FAK and paxillin tyrosine phosphorylation may be essential steps for the integrin-mediated signaling pathways that transmit the extracellular mechanical signals into intracellular effectors in smooth muscle cells.

In canine tracheal smooth muscle, RhoA mediates Ca2+-insensitive MLC phosphorylation and tension development but not FAK and paxillin tyrosine phosphorylation (19). However, there is in vitro evidence that FAK may regulate the Rho family GTPases through a GTPase-activating protein for Rho associated with FAK (GRAF) (15). Thus FAK might mediate a Ca2+-insensitive pathway for Rho-activated MLC phosphorylation and tension development in tracheal smooth muscle, thereby functioning as an upstream regulator of Ca2+-insensitive MLC phosphorylation. In our present study, however, the length-sensitive regulation of FAK and paxillin was not associated with a length-sensitive modulation of agonist-induced Ca2+-insensitive MLC phosphorylation. These results suggest that a Ca2+-insensitive signaling pathway mediated by FAK or paxillin does not regulate Ca2+-insensitive MLC phosphorylation.

It has been suggested that length-sensitive activation of MLC phosphorylation may regulate the length sensitivity of active tension development (2, 21, 25, 35). MLC phosphorylation and active force are higher in smooth muscle tissues when they are stimulated at a long muscle length than when they are stimulated at a shorter length (2, 21, 25). In our present study, the contractile activation of smooth muscle at a shorter length resulted in a lower force development in the absence of a decrease in MLC phosphorylation. Thus the length sensitivity of active tension development was present despite the absence of length-sensitive changes in MLC phosphorylation. In intact (not permeabilized) tracheal smooth muscle strips, a converse situation can be observed. Latrunculin, an inhibitor of actin polymerization, suppresses the length sensitivity of agonist-induced tension development, whereas the length sensitivity of MLC phosphorylation is retained (18). Moreover, Ca2+-activated MLC phosphorylation is insensitive to muscle length in Triton X-100-permeabilized vascular smooth muscle (14). Thus, in both permeabilized and intact muscles, the length sensitivity of MLC phosphorylation can be dissociated from the length sensitivity of tension development. This suggests that the length-sensitive regulation of MLC phosphorylation is not the primary mechanism for regulating the length dependence of active tension in smooth muscle.

We conclude that the length sensitivity of MLC phosphorylation in smooth muscle is secondary to length-sensitive changes in intracellular Ca2+. When intracellular Ca2+ is kept constant, the length sensitivity of MLC phosphorylation is abolished, regardless of whether the muscle is activated by raising intracellular Ca2+ or by agonist-induced activation. However, active tension remains length sensitive under these conditions, suggesting that the length-dependent regulation of contractile protein activation is not the primary mechanism for the length dependence of active tension in tracheal smooth muscle.

In contrast, the length sensitivity of FAK and paxillin is retained when intracellular Ca2+ is kept constant. This is consistent with the possibility that FAK tyrosine phosphorylation and paxillin tyrosine phosphorylation are upstream cellular processes that transduce extracellular mechanical signals to intracellular events. Because FAK is a primary regulator of intracellular Ca2+ in tracheal smooth muscle, it may mediate the mechanosensitive regulation of intracellular Ca2+and thereby regulate Ca2+-sensitive MLC phosphorylation. However, we found that agonist-induced Ca2+-insensitive MLC phosphorylation was not length sensitive under conditions in which the length sensitivity of FAK and paxillin tyrosine phosphorylation is present. This suggests that the phosphorylation of these proteins is not an upstream event in the regulation of Ca2+-insensitive MLC phosphorylation.


    ACKNOWLEDGEMENTS

This work was supported by grants from the American Heart Association Midwest Affiliate and the National Heart, Lung, and Blood Institute (HL-29289).


    FOOTNOTES

Address for reprint requests and other correspondence: S. J. Gunst, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 (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. Section 1734 solely to indicate this fact.

Received 1 March 2001; accepted in final form 5 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 91(3):1452-1459
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