INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

THE PHYSIOLOGICAL BASIS FOR the length-tension behavior of smooth muscle tissues is not understood (12). Although the length-tension behavior of striated muscles has been attributed to differences in the overlap of contractile filaments (10), it is not clear that analogous mechanisms can explain the length-tension behavior of smooth muscle. Smooth muscle exhibits a plasticity of its mechanical response that is not well accounted for on the basis of the traditional sliding filament paradigm. The physical length of smooth muscle at the time that contractile activation is initiated has long-lasting and persistent effects on its mechanical properties for the duration of the period of contractile activation, even when those properties are subsequently measured under identical mechanical conditions (11, 14, 15). We have hypothesized that the plasticity of the mechanical response of smooth muscle may result from an ability of the muscle cell to remodel the organization of its contractile apparatus in response to changes in external stress or strain (13, 14). Actin filament remodeling at the time of contractile activation might be a mechanism by which smooth muscle cells adjust the organization of their contractile apparatus to accommodate to changes in their mechanical environment (13, 14, 22).

The actin filaments of smooth muscle cells link to the membrane at dense plaque sites that are structurally similar to the focal adhesion plaques of cultured cells (4). Smooth muscle dense plaques and focal adhesion sites contain cytoskeletal proteins, including talin, vinculin, and -actinin, that connect actin filaments to transmembrane integrins (4, 8) and thereby enable the transmission of tension between the actin cytoskeleton and the extracellular matrix (35, 36). The adhesion plaques of cultured cells also form a locus for the interaction of signaling molecules that regulate processes involved in adhesion-induced changes in cell physiology, such as cytoskeletal assembly and actin remodeling (3). In cultured cells, focal adhesion kinase (FAK) and paxillin localize to focal adhesion sites and are thought to play a critical role in mediating these signaling processes (3, 21). Both paxillin and FAK undergo phosphorylation during integrin-mediated cell adhesion and during stimulation by a variety of mitogens and growth factors. The phosphorylation of these proteins has been correlated with the assembly of actin stress fibers and with focal adhesion formation (1, 5, 6, 24, 28, 31).

There is growing evidence 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 (30). In cultured endothelial cells, FAK and paxillin undergo tyrosine phosphorylation in response to periods of repetitive mechanical strain (38). In a number of cultured cell types, including endothelial cells and airway smooth muscle cells, cyclic mechanical strain has been shown to induce the alignment of actin filaments along the axis perpendicular to the force vector (16, 29, 32). Paxillin and FAK have been proposed to play an integral role in these strain-induced morphological changes (38).

The contractile activation of tracheal smooth muscle elicits the tyrosine phosphorylation of paxillin (22, 37), indicating that some of the signaling processes that occur in the dense plaques of smooth muscle cells in response to contractile stimuli are similar to those that occur in the adhesion plaques of cultured cells. We therefore hypothesized that paxillin and FAK may be components of an integrin-mediated mechanotransduction pathway in smooth muscle. Such a pathway might initiate signaling events that lead to the modulation of smooth muscle cell shape and contractility. The mechanosensitive modulation of smooth muscle cell contractility might result from the remodeling of actin filaments or from the modulation of signaling pathways that regulate contractile protein activation.

The objectives of the present study were to determine whether a mechanosensitive signal transduction pathway mediated by integrin-associated dense plaque proteins is present in smooth muscle tissue and to evaluate the sensitivity of this pathway to acute changes in muscle length and tension. Changes in the tyrosine phosphorylation of FAK and paxillin were used as indexes of activation of an integrin-mediated mechanotransduction signaling pathway.

	METHODS

Top 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 (PSS) at 22°C (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 2-3 mm in diameter and 12-15 mm in length were dissected from the trachea after removal of the epithelium and connective tissue layer. 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 (L_o) was determined by increasing muscle length progressively until the active force in response to 10⁵ M ACh (Sigma) reached a maximum for that stimulus (F_max). All subsequent changes in muscle length were calibrated as fractions of L_o.

General procedures. Up to 14 muscle strips from a single trachea were contracted isometrically with ACh or KCl at muscle lengths of L_o, 0.75 L_o, or 0.5 L_o. Tissues were quickly frozen at desired time points after contractile stimulation, using a liquid nitrogen-cooled clamp, for the determination of the tyrosine phosphorylation of paxillin or FAK. Strips stimulated with KCl and uncontracted strips were studied in the presence of 10⁷ M atropine to block the potential effects of neurotransmitters released from intramural nerves in the tissue.

Ca²⁺ depletion of muscle strips. In some protocols, smooth muscle strips were depleted of intracellular Ca²⁺ before stimulation with contractile agonists. After L_o 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 10⁵ M ACh to the Ca²⁺-free PSS. This step was repeated three to four times with 10⁵ M ACh. Between each stimulation, the strips were incubated in Ca²⁺-free PSS containing 0.1 mM EGTA for 10 min. Stimulation with 10⁵ M ACh initially produced a force of 70% F_max, but subsequent stimulations resulted in progressively smaller contractions. At the end of the depletion protocol, force in response to 10⁵ M ACh was <10% of F_max.

Extraction of muscle proteins. Frozen muscle strips were pulverized under liquid nitrogen, and the powder was transferred to dry ice-cooled centrifuge tubes. While the tubes were on dry ice, 180 µl of extraction buffer were added to the tubes; then the tubes were quickly vortexed. The extraction buffer contained 20 mM Tris (pH 7.4), 2% 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 of extract was boiled for 5 min to inactivate phosphatases and proteases, and then it was kept at 4°C for 1 h. The supernatant was collected after centrifugation at 14,000 rpm for 20 min at 4°C. 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 the determination of protein concentration. The concentration of protein in each sample of supernatant was determined using a standard bicinchoninic acid protein assay kit (Pierce).

Immunoprecipitation of paxillin and FAK. Muscle extracts containing equal amounts of protein were precleared for 30 min with 50 µl of 10% protein A-Sepharose. The precleared extracts were collected after centrifugation at 14,000 rpm for 2 min, and monoclonal antibodies against paxillin (clone 349, Transduction Labs) or FAK (clone 77, Transduction Labs) were added to them. The extracts were incubated with antibodies overnight and then incubated with 125 µl of a 10% suspension of protein A-Sepharose beads conjugated to rabbit anti-mouse IgG for 2 h. Immunocomplexes were washed four times in Tris-buffered saline containing 0.1% Triton X-100. All procedures of immunoprecipitation were performed at 4°C.

Analysis of protein phosphorylation. Whole muscle extracts and immunoprecipitates of paxillin or FAK were boiled in sample buffer (1.54% dithiothreitol, 2% SDS, 80 mM Tris, pH 6.8, 10% glycerol, and 0.01% bromphenol blue) for 5 min and separated by 7.5% (for FAK) or 10% (for paxillin) 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 to anti-mouse IgG (Amersham Life Science) for visualization by enhanced chemiluminescence (ECL). Nitrocellulose membranes were then stripped of bound antibodies and reprobed with monoclonal antibodies against paxillin or FAK to confirm the location of each protein and normalize for minor differences in protein loading. Scanning densitometry of phosphotyrosine blots and paxillin or FAK blots was used to quantitate proteins after the visualization by ECL. The tyrosine phosphorylation of paxillin was analyzed from immunoblots of whole muscle extracts. In each protocol, the results were confirmed for selected points from immunoblots of paxillin immunoprecipitates. No differences were observed between results obtained by analysis of immunoblots of whole muscle extracts and by analysis of immunoblots of paxillin immunoprecipitates (Fig. 1). Phosphorylation of FAK was quantitated from immunoblots of immunoprecipitated FAK. Changes in the tyrosine phosphorylation of paxillin or FAK were expressed as multiples of the phosphorylation of resting tissues at L_o. Each measurement of paxillin phosphorylation represents the average from duplicate muscle strips in a single experiment.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Immunoblots illustrating effect of muscle length on paxillin (Pax) tyrosine phosphorylation in tracheal smooth muscle strips. Results obtained from immunoblots of whole muscle extracts (A) and paxillin immunoprecipitates (B) are compared. Canine tracheal smooth muscle strips were frozen at optimal muscle length (Lo) or 0.5 Lo after 5 min of stimulation with 104 ACh or without stimulation (Resting) to determine paxillin tyrosine phosphorylation. Immunoblots from muscle extracts (A) or paxillin immunoprecipitates (B) were probed with antiphosphotyrosine antibody (Ab), stripped, and reprobed with anti-paxillin Ab. Numbers at left are molecular mass (in kDa) of standard molecular mass markers. Paxillin tyrosine phosphorylation is higher in strips stimulated with ACh at Lo than in strips stimulated at 0.5 Lo. Muscle length had no effect on paxillin tyrosine phosphorylation in unstimulated muscle strips. No significant differences were detected in measurements of paxillin phosphorylation determined from immunoblots of paxillin immunoprecipitates and muscle extracts. Mean increases in paxillin phosphorylation induced by ACh (as multiples of control) were 3.18 ± 0.08 at Lo and 2.17 ± 0.09 at 0.5 Lo when measured from immunoblots of whole muscle extracts (n = 5). Mean increases in paxillin phosphorylation measured from immunoblots of paxillin immunoprecipitates (n = 3; as multiples of control) were 3.21 ± 0.06 at Lo and 2.00 ± 0.17 at 0.5 Lo.

Statistical analysis. All statistical analysis was 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

Mechanosensitivity of the tyrosine phosphorylation of paxillin in ACh-stimulated muscle strips. Canine tracheal smooth muscle strips were isometrically contracted with 10⁴ M ACh at muscle lengths of L_o or 0.5 L_o and then frozen for the analysis of paxillin tyrosine phosphorylation 1, 5, or 10 min after contractile stimulation. Uncontracted strips at L_o were also frozen in the presence of 10⁷ M atropine to determine the effect of muscle length on the resting levels of paxillin tyrosine phosphorylation.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Time course of paxillin tyrosine phosphorylation in muscle strips stimulated with ACh at Lo or 0.5 Lo. Active force (A) and paxillin tyrosine phosphorylation (B) were significantly lower when strips were stimulated with ACh at a muscle length of 0.5 Lo than at a length of Lo. All values are means ± SE (n = 4). Active force is quantitated as percent of maximal response to 104 M ACh at Lo. Paxillin phosphorylation is quantitated as multiples of phosphorylation level obtained in unstimulated tissues at Lo (open circle with dot). Paxillin tyrosine phosphorylation remained higher in muscle strips contracted at Lo for entire 10-min duration of contraction. * Statistically significant differences in force or paxillin phosphorylation between Lo and 0.5 Lo (P < 0.05).

Mechanosensitivity of tyrosine phosphorylation of paxillin in unstimulated muscle strips. The length sensitivity of paxillin tyrosine phosphorylation was compared in unstimulated muscle strips and in muscle strips activated with 10⁴ M ACh for 5 min (Fig. 3). The tyrosine phosphorylation of paxillin was significantly lower in muscles contracted at lengths of 0.75 L_o or 0.5 L_o than in muscles contracted at L_o (n = 5). However, paxillin tyrosine phosphorylation was not significantly different in the unstimulated strips at muscle lengths of L_o, 0.75 L_o, and 0.5 L_o (n = 5).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Mean total force (A) and paxillin tyrosine phosphorylation (B) in unstimulated muscle strips and in muscle strips contracted isometrically with 104 M ACh for 5 min at lengths of 0.5 Lo, 0.75 Lo, and Lo (n = 5). Tyrosine phosphorylation of paxillin increased at longer muscle lengths between 0.5 Lo and Lo in ACh-stimulated muscle strips but not in uncontracted muscle strips. Force is quantitated as percent of total force (active and passive) at Lo 5 min after contraction with 104 M ACh. Paxillin phosphorylation is quantitated as multiples of level in unstimulated tissues at Lo (open circle with dot). Values are means ± SE. * Values at each length are significantly different from each other (P < 0.05).

4

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of tension on paxillin tyrosine phosphorylation. Paxillin tyrosine phosphorylation was compared in unstimulated smooth muscle strips that were stretched to 1.3 Lo in presence of 107 M atropine and in muscle strips stimulated with 104 M ACh at Lo. Although tension in passively stretched strips was similar to that in strips activated at Lo with ACh, paxillin tyrosine phosphorylation in unstimulated strips at 1.3 Lo was significantly lower than that in ACh-stimulated strips at Lo (n = 3, P < 0.05). * Significantly different in unstimulated stretched strips compared with actively contracted strips (P < 0.05).

Effects of muscle length on paxillin tyrosine phosphorylation during isometric contraction with KCl. To evaluate whether the mechanosensitive modulation of paxillin phosphorylation requires receptor activation, canine tracheal smooth muscle strips were contracted isometrically for 5 or 15 min with 60 mM KCl at muscle lengths of L_o or 0.5 L_o for the determination of paxillin phosphorylation (Figs. 5 and 6). Uncontracted strips were also quickly frozen at L_o to determine the resting level of paxillin tyrosine phosphorylation. Additional strips were contracted at L_o for 5 min and quickly shortened to 0.5 L_o and allowed to recontract isometrically for 1 or 10 min and then frozen for the measurement of paxillin tyrosine phosphorylation.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Immunoblot illustrating effect of muscle length on paxillin tyrosine phosphorylation in tracheal smooth muscle strips stimulated with KCl. Canine tracheal smooth muscle strips were frozen at Lo or 0.5 Lo after 5 min of stimulation with 60 mM KCl or without stimulation (Resting) to determine paxillin tyrosine phosphorylation. An additional strip was contracted at Lo for 5 min and then quickly shortened to 0.5 Lo and allowed to recontract isometrically for 1 min. Immunoblots of paxillin immunoprecipitates were probed with antiphosphotyrosine Ab, stripped, and reprobed with anti-paxillin Ab. Numbers at left are molecular mass (in kDa) of standard molecular mass markers. Paxillin tyrosine phosphorylation is higher in strips stimulated with KCl at Lo than in strips at 0.5 Lo, whether strips are shortened to 0.5 Lo before or after stimulation with KCl.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of muscle length and length step on paxillin tyrosine phosphorylation in strips contracted isometrically with 60 mM KCl at lengths of Lo or 0.5 Lo (n = 4). Additional strips were contracted at Lo for 5 min and then quickly shortened to 0.5 Lo and allowed to recontract isometrically for an additional 1 or 10 min (n = 4). Active force (A) and paxillin tyrosine phosphorylation (B) were significantly higher in muscle strips contracted at Lo than in strips contracted at 0.5 Lo (P < 0.05). When muscles were shortened from Lo to 0.5 Lo, paxillin tyrosine phosphorylation in contracted muscle strips decreased to level obtained at 0.5 Lo within 1 min of length change. Force is quantitated as percent of maximal response to 60 mM KCl at Lo. Paxillin phosphorylation is quantitated as multiples of level obtained from unstimulated strips at Lo (open circle with dot). * Values at 0.5 Lo that are significantly different from corresponding values at Lo (P < 0.05).

Length sensitivity of paxillin phosphorylation in Ca²⁺-depleted muscle strips stimulated with ACh. The mechanosensitivity of paxillin phosphorylation was studied in Ca²⁺-depleted muscle strips stimulated with ACh to evaluate its dependence on active tension and intracellular Ca²⁺. Ca²⁺-depleted muscle strips were stimulated with 10⁴ M ACh for 5 or 10 min at muscle lengths of L_o or 0.5 L_o. Additional Ca²⁺-depleted muscle strips were activated with 10⁴ M ACh at 0.5 L_o and then stretched to L_o, at which length paxillin phosphorylation was determined 1 or 5 min after the stretch.

4

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effect of Ca2+ depletion on paxillin tyrosine phosphorylation in paxillin immunoprecipitates from extracts of muscle strips stimulated with 104 M ACh for 5 min at muscle lengths of Lo or 0.5 Lo. Immunoblots of paxillin immunoprecipitates were probed with anti-phosphotyrosine Ab and then stripped and reprobed with anti-paxillin Ab. Paxillin tyrosine phosphorylation was significantly lower during stimulation of Ca2+-depleted strips with ACh at 0.5 Lo than at Lo. In unstimulated muscle strips, muscle length had no significant effect on paxillin tyrosine phosphorylation.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of Ca2+ depletion on sensitivity of paxillin tyrosine phosphorylation to muscle length. Ca2+-depleted muscle strips were stimulated for 5 or 10 min with 104 M ACh at muscle lengths of Lo or 0.5 Lo. Additional strips were contracted at 0.5 Lo for 5 min and then quickly lengthened to Lo and allowed to contract isometrically for an additional 1 or 5 min. Paxillin tyrosine phosphorylation was lower in strips stimulated with ACh for 5 or 10 min at 0.5 Lo than in strips stimulated at Lo (n = 5). In strips stimulated at 0.5 Lo and stretched to Lo, paxillin tyrosine phosphorylation increased to level obtained at Lo within 1 min of length increase. ACh caused little or no active force development in Ca2+-depleted muscle strips (n = 5). Active force (A) is quantitated as percent of maximal response to 104 M ACh at Lo. Paxillin phosphorylation is quantitated as multiples of level obtained in resting undepleted tissues at Lo (open circle with dot). * Values at Lo that are significantly different from corresponding values at 0.5 Lo (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Effect of muscle length on force and paxillin phosphorylation in Ca2+-depleted and undepleted muscle strips stimulated with ACh for 5 min. Although there was minimal active tension in response to ACh in Ca2+-depleted strips stimulated at Lo (solid bar) or 0.5 Lo (hatched bar), tyrosine phosphorylation of paxillin was similar at Lo under both conditions. In both undepleted and Ca2+-depleted muscle strips, tyrosine phosphorylation of paxillin was lower in strips stimulated with ACh at 0.5 Lo than in strips stimulated at Lo. * Values of paxillin phosphorylation or force that are significantly lower at 0.5 Lo than at Lo.

Length sensitivity of the tyrosine phosphorylation of FAK during stimulation with ACh. The length sensitivity of the tyrosine phosphorylation of FAK was measured in smooth muscle strips stimulated with 10⁴ M ACh for 5 min. The tyrosine phosphorylation of FAK was higher in muscles stimulated at L_o than at 0.5 L_o (Figs. 10 and 11). The tyrosine phosphorylation of FAK did not change significantly over a 5-min period of contraction with ACh. FAK phosphorylation at L_o increased by 5.9 ± 0.3-fold over resting levels by 1 min of stimulation with ACh and remained elevated by 5.1 ± 0.2-fold after 5 min of stimulation (n = 3; data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 10.   Tyrosine phosphorylation of focal adhesion kinase (FAK) immunoprecipitates in extracts from undepleted (A) and Ca2+-depleted (B) muscle strips stimulated with 104 M ACh for 5 min at muscle lengths of Lo or 0.5 Lo. Tyrosine phosphorylation of FAK in both undepleted and Ca2+-depleted muscle strips stimulated with ACh was lower at 0.5 Lo than at Lo. Ca2+ depletion did not affect tyrosine phosphorylation of FAK in unstimulated muscle strips.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Mean tyrosine phosphorylation of FAK immunoprecipitates from undepleted and Ca2+-depleted muscle strips stimulated with 104 M ACh for 5 min at Lo (solid bar) or 0.5 Lo (hatched bar). Tyrosine phosphorylation of FAK was lower at 0.5 Lo than at Lo in both Ca2+-depleted and undepleted muscle strips. * Values of FAK phosphorylation that are significantly lower at 0.5 Lo than at Lo (n = 3, P < 0.05).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Summary. In this study, we demonstrate that tyrosine phosphorylation of the integrin-associated proteins paxillin and FAK is sensitive to muscle length during the contractile stimulation of tracheal smooth muscle. In addition, this is the first demonstration that the contractile stimulation of a smooth muscle tissue elicits the tyrosine phosphorylation of FAK. The phosphorylation of both paxillin and FAK is higher when tracheal muscles are stimulated isometrically at a long muscle length than at a short length. Differences in the tyrosine phosphorylation of paxillin can be distinguished in muscles contracted at lengths of 0.5 L_o, 0.75 L_o, and L_o (Fig. 3). The length sensitivity of paxillin tyrosine phosphorylation is observed for the entire duration of a 10-min period of contractile stimulation (Fig. 2). A length step imposed on the muscle after contractile activation results in a rapid change in paxillin tyrosine phosphorylation, indicating that the mechanosensitive response mediated by paxillin can occur rapidly in response to an acute change in the mechanical environment of the muscle (Figs. 5, 6, and 8). These results suggest that integrin-mediated mechanotransduction may be an important mechanism by which smooth muscle cells can modulate signaling pathways in response to changes in their external mechanical environment.

Ca²⁺ dependence of mechanosensitive protein phosphorylation. We have previously demonstrated that the phosphorylation of paxillin is Ca²⁺ sensitive (19, 37). We and others have also shown that intracellular Ca²⁺ and myosin light chain (MLC) phosphorylation are modulated in response to changes in muscle length in tracheal smooth muscle (20, 39). We therefore evaluated whether the mechanosensitivity of paxillin phosphorylation can be mediated by changes in intracellular Ca²⁺ by assessing paxillin phosphorylation in muscles activated by K⁺ depolarization at different muscle lengths. Our results show that paxillin phosphorylation is mechanosensitive when muscles are activated by K⁺ depolarization, indicating that the length sensitivity of paxillin phosphorylation can be mediated by a Ca²⁺-sensitive pathway (Figs. 5 and 6).

Tension vs. length as the stimulus for mechanosensitive signal transduction. Although paxillin and FAK phosphorylation is mechanosensitive in actively contracted muscle strips, their phosphorylation is unaffected by muscle length in unstimulated smooth muscle strips (Fig. 3). This observation suggested that tension per se might be the stimulus for the mechanosensitive regulation of FAK and paxillin phosphorylation in this tissue. However, we observed that the length sensitivity of paxillin and FAK phosphorylation is similar in Ca²⁺-depleted and in undepleted muscles, despite the absence of active tension in the Ca²⁺-depleted tissues (Figs. 9 and 11). As the differences in tension at muscle lengths of L_o and 0.5 L_o are much smaller in Ca²⁺-depleted tissues than in undepleted tissues, this indicates that mechanosensitive signal transduction does not result from a tension-sensitive mechanism. The contractile stimulation of Ca²⁺-depleted tissues does not increase MLC phosphorylation significantly (19); thus these results also demonstrate that the activation of contractile proteins is not required for mechanosensitive signal transduction. Our observation that paxillin phosphorylation increased only slightly when high levels of passive tension were generated by stretching uncontracted muscles strips (Fig. 4) provides further support for our conclusion that muscle length rather than tension is the primary stimulus for mechanosensitive signal transduction in tracheal smooth muscle.

Molecular mechanism for mechanosensitive signal transduction. The mechanically induced changes in the phosphorylation of paxillin and FAK observed in this study may be mediated by transmembrane integrins. In cultured cells, paxillin and FAK colocalize with integrin molecules in focal adhesion complexes at the membrane termini of actin stress fiber bundles (3, 33). Extracellular matrix proteins bind to the extracellular domain of integrins, whereas the cytosolic domain of integrin molecules binds to cytosolic proteins, including talin, -actinin, and vinculin, that link the integrin molecules to actin filaments (3, 4). These complexes serve as loci for the transmission of tension between the actin cytoskeleton and the extracellular matrix (27, 35, 36). Mechanical strain or tension applied directly to the extracellular domain of integrins results in increased protein tyrosine phosphorylation, cytoskeletal stiffening, and the activation of downstream signaling pathways, suggesting that integrins can function as mechanotransducers (27, 30, 35, 36).

Role of FAK and paxillin in the regulation of smooth muscle contraction. We have hypothesized that the stimulation of smooth muscle cells with contractile agonists initiates active processes that regulate the organization of the actin cytoskeleton and the attachment of actin filaments to the membrane at membrane-associated dense plaque sites (13, 14, 22). According to our hypothesis, these processes occur in parallel to the activation of contractile proteins and enable force development to be optimized to the mechanical environment of the smooth muscle cell at the time of contractile activation.