Cholinergic receptor-mediated differential cytoskeletal recruitment of actin- and integrin-binding proteins in intact airway smooth muscle

Hak Rim Kim, Muntasir Hoque, and Chi-Ming Hai

Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912

Submitted 19 February 2004 ; accepted in final form 12 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We stimulated bovine tracheal smooth muscle with carbachol and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. In unstimulated tissues, calponin exhibited the highest basal P-to-S ratio (P/S; 2.74 ± 0.47), whereas vinculin exhibited the lowest P/S (0.52 ± 0.09). Cholinergic receptor stimulation increased P/S of the following proteins in descending order of sensitivity: {alpha}-actinin > talin {approx} metavinculin > {alpha}-smooth muscle actin > vinculin {approx} calponin. Carbachol induced ERK1/2 phosphorylation by 300% of basal value. U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly affect the correlation between {alpha}-actinin P/S and carbachol concentration. This observation indicates that cytoskeletal/membrane recruitment of {alpha}-actinin is independent of ERK1/2 mitogen-activated protein kinase activation. Metavinculin and vinculin are splice variants of a single gene, but metavinculin P/S was significantly higher than vinculin P/S. Furthermore, the P/S of metavinculin but not vinculin increased significantly in response to cholinergic receptor stimulation. Calponin and {alpha}-actinin both belong to the family of calponin homology (CH) domain proteins. However, unlike {alpha}-actinin, the calponin P/S did not change significantly in response to cholinergic receptor stimulation. These findings indicate differential cytoskeletal/membrane recruitment of actin- and integrin-binding proteins in response to cholinergic receptor stimulation in intact airway smooth muscle. {alpha}-Actinin, talin, and metavinculin appear to be key cytoskeletal proteins involved in the recruitment process.

actinin; mitogen-activated protein kinase; metavinculin; vinculin


AIRWAY SMOOTH MUSCLE CONTRACTILITY contributes to the increased airway resistance in airway diseases such as asthma (37). Cytoskeletal remodeling has been proposed as an important regulatory mechanism of airway smooth muscle contraction (7, 1315). In smooth muscle cells, the actin cytoskeleton-membrane complex consists of actin filament and integrin- and actin-binding proteins (4, 22, 31, 39). Hirshman and Emala (18) reported that cholinergic and other G protein-coupled receptor agonists induce actin polymerization in cultured airway smooth muscle cells. Saez et al. (29) observed recruitment of focal adhesion kinase, paxillin, vinculin, and talin from the cytoplasm to the membrane during cholinergic receptor stimulation of single airway smooth muscle cells, as revealed by immunofluorescence microscopy. These cultured and single-cell studies suggest the general hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex.

Several studies suggest that the cytoskeletal/membrane recruitment process may also occur in intact airway smooth muscle. Gunst et al. (13) found that muscle stiffness increased while force remained constant during the tonic phase of isometric contraction in canine tracheal smooth muscle. The force-independent increase in stiffness could be explained by cytoskeletal remodeling. Tseng et al. (35) found that F-actin disruption by cytochalasins led to decreases in cholinergic receptor-mediated force, intracellular Ca2+ concentration ([Ca2+]), and myosin light chain phosphorylation in bovine tracheal airway smooth muscle. This observation suggests that actin filaments are dynamic in intact airway smooth muscle and integrity of the actin cytoskeleton is essential for activation-contraction coupling in airway smooth muscle. Mehta and Gunst (24) reported that G-actin content decreased during cholinergic receptor-mediated contraction of canine tracheal smooth muscle. Pavalko et al. (27) reported phosphorylation of talin and paxillin during cholinergic receptor-mediated contraction of canine tracheal smooth muscle. Tang and coworkers (33, 34) showed that depletion of paxillin and focal adhesion kinase with the antisense approach led to loss of force in canine tracheal smooth muscle. These findings together suggest that the actin cytoskeleton and dense plaque/focal adhesions may undergo reorganization in intact airway smooth muscle in response to cholinergic receptor stimulation. This study determines the differential distributions of multiple actin- and integrin-binding proteins between the cytoskeleton-membrane complex and the cytosol.

We hypothesize that cholinergic receptor stimulation induces the recruitment of actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We further hypothesize that ERK1/2 mitogen-activated protein kinase (MAPK) is the putative regulatory mechanism based on the calponin-ERK-caldesmon model proposed by Morgan and Gangopadhyay (25). In this model, calponin serves as an adaptor molecule that carries phospho-ERK to the vicinity of caldesmon, leading to caldesmon phosphorylation and contraction. We tested this hypothesis by stimulating bovine tracheal smooth muscle with the cholinergic receptor agonist carbachol over a range of concentrations from 0.01 to 100 µM. We then homogenized the tissue in an extraction buffer and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. The pellet represents the cytoskeletal/membrane fraction, whereas the supernatant represents the cytosolic fraction as described by Goldberg et al. (12) and Harrington et al. (16). Finally, we measured {alpha}-smooth muscle (SM) actin, {alpha}-actinin, calponin, metavinculin, vinculin, and talin in the pellet and supernatant by Western blotting. To determine the role of ERK1/2 MAPK in regulating the cytoskeletal/membrane recruitment process, we measured ERK1/2 phosphorylation in unstimulated and carbachol-stimulated tissues and investigated the effect of the MEK1/2 inhibitor U0126 on ERK1/2 phosphorylation and the P-to-S ratio (P/S) of actin- and integrin-binding proteins in unstimulated and carbachol-stimulated tissues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Bovine tracheas were collected from a local abattoir and transported to the laboratory in cold (4°C) physiological salt solution (PSS) of the following composition (in mM): 140.1 NaCl, 4.7 KCl, 1.2 Na2HPO4, 2.0 MOPS (pH 7.4), 0.02 Na2EDTA, 1.2 MgSO4, 1.6 CaCl2, and 5.6 D-glucose. The smooth muscle layer together with the adventitial and mucosal layers were excised from the trachea by making longitudinal cuts along their attachments to the cartilage. The adventitial and mucosal layers were then carefully dissected away in cold PSS with microdissecting scissors and fine forceps under a dissecting microscope. Smooth muscle strips were prepared by making cuts along the direction of smooth muscle bundles, corresponding to the circumferential direction in vivo.

Isometric contractions. One end of each muscle strip was clamped to a stainless steel clip connected to a force transducer (Grass FT.03), and the other end was clamped to a stainless steel clip connected to a length manipulator (Narishige). Muscle strips were then equilibrated for 2 h in PSS (37°C, pH 7.4) bubbled with air and adjusted to optimal length (Lo) for maximal active force development as described previously (1). The dimensions of each muscle strip were ~15-mm length, 5-mm width, and 0.5-mm thickness. After equilibration, each muscle strip was first stimulated for 10 min by K+ depolarization with K-PSS, a solution similar in composition to PSS except that 104.95 mM NaCl was substituted for by an equimolar concentration of KCl. The force (Fo) developed in this contraction was used as an internal control to normalize force developed by the same muscle strip in subsequent contractions. Muscle strips were then allowed to relax in PSS for 1 h before stimulation by carbachol for 30 min. For U0126 (MEK1/2 inhibitor) experiments, muscle strips were allowed to relax for 1 h in PSS containing 10 µM U0126 (Cell Signaling Technology, Beverly, MA) before stimulation by carbachol, also in the presence of 10 µM U0126.

Tissue homogenization and fractionation. After 30 min of stimulation by carbachol, muscle strips were quickly homogenized in a cold (4°C) extraction buffer of the following composition (in mM): 20 Tris·HCl (pH 7.5), 2 EDTA, 2 EGTA, 6 mercaptoethanol, 0.1 Na3VO4, 1 PMSF, and 50 NaF with 50 µg/ml aprotinin and 50 µg/ml leupeptin. Tissue homogenates were centrifuged at 100,000 g for 1 h to separate the cytoskeletal/membrane and cytoplasmic fractions. The pellet represents the cytoskeletal/membrane fraction, whereas the supernatant represents the cytosolic fraction as described by Goldberg et al. (12) and Harrington et al. (16). Low ionic strength was shown by Katoh et al. (19) to be important in preserving the filamentous state of actin in the isolation of stress fibers from cultured cells. A low-ionic-strength extraction buffer similar to the extraction buffer used in this study was used by Tsutsumi et al. (36) to extract the cytosolic and cytoskeletal fractions from various tissues, including muscle organs such as the heart and stomach, in studying the translocation of proline- and alanine-rich Ste20-related kinase from the cytosol to the cytoskeleton. Therefore, low-ionic-strength extraction buffer has been shown to be effective in the extraction of cytosolic and cytoskeletal fractions in multiple studies. We did not further separate the pellet into cytoskeleton and membrane fractions because membrane-cytoskeletal interactions are important determinants of the binding affinities of many cytoskeletal proteins (9, 23, 26, 30, 32, 38). After ultracentrifugation, pellets were homogenized in a SDS buffer (1% SDS, 10% glycerol, and 20 mM dithiothreitol) at 20 mg/ml as described previously (35). The supernatant volume was measured, and an equal volume of 2x SDS buffer was added. The pellet and supernatant samples were stored at –80°C. The differences in supernatant volumes among samples were relatively small (<5%), which was factored into the calculation of P/S.

SDS-PAGE and Western blotting. Proteins were separated by SDS-PAGE with a separating gel of 7.5% acrylamide. Pellet and supernatant samples from the same tissue were loaded next to each other to minimize variations during Western blotting. Equal volumes of pellet and supernatant samples were loaded onto the same gel. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane electrophoretically (Transblot, Bio-Rad, Hercules, CA). The nitrocellulose membranes for detecting cytoskeletal proteins were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS). The membranes for detecting ERK1/2 and phospho-ERK1/2 were blocked with 0.1% BSA in TBS. After blocking, nitrocellulose membranes were incubated with appropriate primary antibodies with gentle shaking at 4°C overnight. Next, the nitrocellulose membranes were washed with TBS and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected with enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Piscataway, NJ) and XAR autoradiography film (Kodak, Rochester, NY). Film images were captured and analyzed using the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290) and Kodak 1D image analysis software. Because the loading volumes for pellet and supernatant samples were the same for each gel, we calculated P/S for each protein using the following equation: pellet band density x pellet sample volume/supernatant band density x supernatant sample volume.

Reagents. Primary antibodies against {alpha}-SM actin, {alpha}-actinin, calponin, vinculin, and talin and peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Sigma (St. Louis, MO). According to the manufacturer (Sigma), the calponin antibody (Clone hCP) used in this study does not cross-react with nonmuscle calponin. Primary antibodies against phospho-ERK1/2 (Thr202/Tyr204) and ERK1/2 were purchased from Cell Signaling Technology. SDS-PAGE reagents and nitrocellulose membrane were purchased from Bio-Rad.

Statistics. Data are presented as means ± SE; n represents the number of animals. Student's t-test was used for the comparison of two means (P < 0.05 considered significant). Correlation between P/S and carbachol concentration ([carbachol]) was analyzed by performing linear regression analysis (P < 0.05 considered significant).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Carbachol-stimulated isometric contractions. As shown in Fig. 1, unstimulated bovine tracheal smooth muscle developed a basal force of 0.14 ± 0.04 Fo, where Fo represents the maximum force induced by K+ depolarization. Basal force represents the force developed by an unstimulated muscle above the passive force that was reached immediately after a quick release of a muscle strip to optimal length. Cholinergic receptor stimulation by carbachol stimulated contraction of bovine tracheal smooth muscle in a concentration-dependent manner from 0.01 to 100 µM. As shown in Fig. 1, carbachol-stimulated active forces were significantly higher than basal force at all measured concentrations (P < 0.05). Furthermore, the correlation between carbachol-induced active force and [carbachol] was statistically significant (P < 0.05). The maximal force (1.03 ± 0.06 Fo) developed at 10 µM carbachol was near 1, which is similar to the maximum force (Fo) stimulated by K+ depolarization. The same concentrations of carbachol were used to study the effect of cholinergic receptor stimulation on cytoskeletal/membrane recruitment of actin- and integrin-binding proteins.



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Fig. 1. Carbachol-stimulated contractions of bovine tracheal smooth muscle. Open bar represents basal force developed by unstimulated tissues. Filled bars represent active forces stimulated by carbachol. Active force is expressed as fraction of the active force (Fo) stimulated by K+ depolarization at the beginning of all experiments. Data are presented as means ± SE (n = 9). *Significant difference from basal force (P < 0.05). [carbachol], carbachol concentration.

 
Carbachol-stimulated cytoskeletal/membrane recruitment of actin- and integrin-binding proteins. Figure 2 shows the basal P/S of {alpha}-SM actin, {alpha}-actinin, calponin, metavinculin, vinculin, and talin in unstimulated bovine tracheal smooth muscle. Basal P/S of {alpha}-actinin (0.74 ± 0.16), vinculin (0.52 ± 0.09), and metavinculin (0.78 ± 0.12) were <1. In contrast, basal P/S of {alpha}-SM actin (1.67 ± 0.10), calponin (2.74 ± 0.47), and talin (1.79 ± 0.15) were >1. It is noteworthy that, despite the close homology between vinculin and metavinculin, the P/S of vinculin (0.52 ± 0.09) and metavinculin (0.78 ± 0.12) were significantly different.



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Fig. 2. Basal pellet (P)-to-supernatant (S) ratio (P/S) of {alpha}-smooth muscle (SM) actin, {alpha}-actinin, calponin, metavinculin, vinculin, and talin in unstimulated bovine tracheal smooth muscle. Bands of individual proteins in the pellet and supernatant fractions on Western blots are shown. Identical volumes of pellet and supernatant samples were loaded into adjacent lanes of the same gel to minimize variations during Western blotting. Total pellet and supernatant volumes from a given tissue were similar. P/S values were calculated with the following equation: pellet band density x pellet sample volume/supernatant band density x supernatant sample volume. Data are presented as means ± SE (n = 6–11). A P/S value represents the partition of a given protein between the cytoskeleton-membrane complex and the cytoplasm.

 
Figure 3 shows P/S of the two integrin-binding proteins, {alpha}-actinin and talin, in unstimulated and carbachol-stimulated tissues. The {alpha}-actinin P/S increased significantly with [carbachol] in a concentration-dependent manner, from a basal value of 0.74 ± 0.16 in unstimulated tissues to 2.17 ± 0.47 in tissues stimulated by 10 µM carbachol (Fig. 3A). The talin P/S also increased significantly with [carbachol] from 1.79 ± 0.15 in unstimulated tissues to 2.94 ± 0.43 in tissues stimulated by 10 µM carbachol (Fig. 3B).



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Fig. 3. P/S of {alpha}-actinin (A) and talin (B) in unstimulated and carbachol-stimulated tissues. Open bars represent the basal P/S of {alpha}-actinin and talin in unstimulated tissues. Filled bars represent P/S of {alpha}-actinin and talin in carbachol-stimulated tissues. Data are presented as means ± SE (n = 4–6 for {alpha}-actinin data; n = 8 or 9 for talin data). *Significant difference from basal P/S (P < 0.05).

 
Vinculin and metavinculin are homologous proteins derived from a single gene by alternative splicing. As shown in Fig. 4A, the vinculin P/S did not change significantly with [carbachol]. As shown in Fig. 4B, the metavinculin P/S increased significantly with [carbachol] from a basal value of 0.78 ± 0.12 in unstimulated tissues to 1.26 ± 0.10 in tissues stimulated by 100 µM carbachol. Metavinculin P/S at 10 and 100 µM carbachol were significantly higher than the basal P/S in unstimulated tissues. It is noteworthy that, despite the close homology between vinculin and metavinculin, the P/S of vinculin and metavinculin were significantly different in both unstimulated and carbachol-stimulated tissues at all concentrations measured in this study (Fig. 5).



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Fig. 4. P/S of vinculin (A) and metavinculin (B) in unstimulated and carbachol-stimulated tissues. Open bars represent basal P/S of vinculin and metavinculin in unstimulated tissues. Filled bars represent P/S of vinculin and metavinculin in carbachol-stimulated tissues. Data are presented as means ± SE (n = 6–8 for vinculin data; n = 7 or 8 for metavinculin data). *Significant difference from basal P/S (P < 0.05).

 


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Fig. 5. Comparison between vinculin and metavinculin P/S. Open bars represent P/S of vinculin in unstimulated and carbachol-stimulated tissues. Filled bars represent P/S of metavinculin in unstimulated and carbachol-stimulated tissues. Data shown in this figure are the same as those shown in Fig. 4. *Significant difference between vinculin and metavinculin P/S (P < 0.05).

 
Figure 6 shows P/S of {alpha}-SM actin and calponin in unstimulated and carbachol-stimulated tissues. The {alpha}-SM actin P/S increased significantly with [carbachol] from a basal value of 1.67 ± 0.10 in unstimulated tissues to 2.32 ± 0.26 in tissues stimulated by 100 µM carbachol (Fig. 6A). The calponin P/S did not change significantly with [carbachol], with values ranging from 2.74 ± 0.47 in unstimulated tissues to 2.49 ± 0.46 in tissues stimulated by 1 µM carbachol (Fig. 6B).



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Fig. 6. P/S of {alpha}-SM actin (A) and calponin (B) in unstimulated and carbachol-stimulated tissues. Open bars represent basal P/S of {alpha}-SM actin and calponin in unstimulated tissues. Filled bars represent P/S of {alpha}-SM actin and calponin in carbachol-stimulated tissues. Data are presented as means ± SE (n = 8 for actin data; n = 6 for calponin data). *Significant difference vs. basal P/S (P < 0.05).

 
Carbachol-stimulated ERK1/2 phosphorylation in cytoskeletal/membrane fraction. As shown in Fig. 7A, cholinergic receptor stimulation significantly increased values of phospho-ERK1 in the cytoskeletal/membrane (pellet) fractions of carbachol-stimulated tissues in a concentration-dependent manner, up to 395 ± 97% of basal value at 100 µM carbachol. Similarly, cholinergic receptor stimulation also significantly increased the values of phospho-ERK2 in the cytoskeletal/membrane fractions of carbachol-activated tissues, up to 312 ± 92% of basal value at 100 µM carbachol (Fig. 7B). In these experiments, the same pellet weight of unstimulated and carbachol-stimulated tissues was loaded onto each lane of the same gel during SDS-PAGE. Using an anti-ERK1/2 antibody, we confirmed that the total amounts of ERK1 and ERK2 loaded onto individual lanes were indeed not significantly different (data not shown). Therefore, values of phospho-ERK1 and phospho-ERK2 as shown in Fig. 7 reflect levels of ERK1/2 phosphorylation.



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Fig. 7. Phosphorylation of ERK1 (A) and ERK2 (B) in cytoskeletal/membrane fractions of unstimulated and carbachol-stimulated tissues. Open bars represent basal values of phospho-ERK1 and phospho-ERK2 in cytoskeletal/membrane fraction (pellet) of unstimulated tissues, which were used for normalizing values of phospho-ERK1 and phospho-ERK2 in carbachol-stimulated tissues. Filled bars represent values of phospho-ERK1 and ERK2 in the cytoskeletal/membrane fractions of carbachol-activated tissues, expressed as fractions of the basal phosphorylation. Western blotting with an anti-ERK1/2 antibody confirmed that the same amounts of ERK1 and ERK2 were loaded onto individual lanes (data not shown). Therefore, values of phospho-ERK1 and phospho-ERK2 reflect levels of ERK1/2 phosphorylation. Data are presented as means ± SE (n = 5–8 for both phospho-ERK1 and phospho-ERK2 data sets). *Significant difference from basal value of phospho-ERK1 or phospho-ERK2 (P < 0.05).

 
To compare the distributions of ERK1/2 and phospho-ERK1/2 between the cytoplasmic and cytoskeletal fractions, we compared P/S of unphosphorylated and phosphorylated ERK1 and ERK2 in unstimulated and carbachol-stimulated tissues. As shown in Fig. 8A, P/S of phospho-ERK1 ranged from 1.56 ± 0.24 to 2.98 ± 0.48, whereas P/S of ERK1 ranged from 0.31 ± 0.09 to 0.50 ± 0.19. P/S of phospho-ERK1 and ERK1 did not correlate significantly with [carbachol]. Paired statistical comparisons indicated that the P/S of phospho-ERK1 was significantly higher than the P/S of ERK1 in both unstimulated and carbachol-stimulated tissues. These data suggest that phospho-ERK1 was predominantly localized at the cytoskeleton-membrane complex. As shown in Fig. 8B, P/S of phospho-ERK2 ranged from 5.26 ± 1.67 to 7.22 ± 2.91 whereas P/S of ERK2 ranged from 2.72 ± 0.64 to 5.15 ± 2.27. However, paired statistical comparisons of P/S of phospho-ERK2 and ERK2 indicated that the differences were not statistically significant.



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Fig. 8. P/S of ERK1/2 and phospho-ERK1/2 in unstimulated and carbachol-stimulated tissues. A: P/S of ERK1 (open bars) and phospho-ERK1 (filled bars) in unstimulated and carbachol-stimulated tissues. B: P/S of ERK2 (open bars) and phospho-ERK2 (filled bars) in unstimulated and carbachol-stimulated tissues. Data are presented as means ± SE (n = 6–10). *Significant difference between P/S of unphosphorylated and phosphorylated ERK1 (or ERK2) in paired statistical comparisons (P < 0.05).

 
Effect of U0126 on carbachol-stimulated contraction and cytoskeletal/membrane recruitment of {alpha}-actinin, vinculin, metavinculin, {alpha}-SM actin, and calponin. U0126 is a MEK1/2 inhibitor and is therefore expected to inhibit ERK1/2 phosphorylation. Indeed, we confirmed complete inhibition of ERK1/2 phosphorylation in U0126-treated tissues by performing Western blotting using an anti-phospho-ERK1/2 antibody (data not shown). As shown in Fig. 9, 10 µM U0126 inhibited basal force in unstimulated tissues and active force developed by tissues stimulated by 0.01 µM carbachol. However, U0126 did not significantly affect the maximum active force stimulated by carbachol at concentrations >0.01 µM. Furthermore, active force remained significantly correlated with [carbachol] in U0126-treated tissues (P < 0.05).



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Fig. 9. Effect of 10 µM U0126 on basal force in unstimulated tissues and active force developed by carbachol-stimulated tissues. Open bars represent basal and active forces developed by tissues not treated with U0126 (data from Fig. 1). Filled bars represent basal and active forces developed by tissues treated with 10 µM U0126 (n = 6). Basal and active forces are expressed as fractions of Fo stimulated by K+ depolarization measured at the beginning of all experiments. Data are presented as means ± SE. *Significant difference between untreated (control) and U0126-treated tissues.

 
As shown in Fig. 10, 10 µM U0126 significantly increased the {alpha}-actinin P/S in unstimulated and carbachol-stimulated tissues at 0.01, 0.1, and 100 µM carbachol. As shown in Fig. 11, 10 µM U0126 significantly increased the vinculin P/S only at 1 µM carbachol and did not significantly change the metavinculin P/S. As shown in Fig. 12, in the presence of U0126 vinculin and metavinculin P/S were not significantly different. This is noteworthy because P/S of vinculin and metavinculin were significantly different in untreated tissues (Fig. 4). As shown in Fig. 13A, U0126 (10 µM) significantly increased the {alpha}-SM actin P/S in unstimulated and carbachol-activated tissues at 0.01 and 1 µM carbachol. However, U0126 did not significantly alter the calponin P/S in either unstimulated or carbachol-activated tissues (Fig. 13B).



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Fig. 10. Effect of U0126 on {alpha}-actinin P/S in unstimulated and carbachol-stimulated tissues. Open bars represent P/S of {alpha}-actinin in tissues not treated with U0126 (data from Fig. 3A). Filled bars represent P/S of {alpha}-actinin in tissues treated with U0126 (n = 7). Data are presented as means ± SE. *Significant difference between untreated (control) and U0126-treated tissues (P < 0.05)

 


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Fig. 11. Effect of U0126 on P/S of vinculin (A) and metavinculin (B) in unstimulated and carbachol-stimulated tissues. Open bars represent P/S of vinculin and metavinculin in tissues not treated with U0126 (data from Fig. 4). Filled bars (U0126) represent P/S of vinculin and metavinculin in tissues treated with U0126 (n = 4 or 5). Data are presented as means ± SE. *Significant difference between untreated (control) and U0126-treated tissues (P < 0.05).

 


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Fig. 12. Comparison between vinculin and metavinculin P/S in U0126-treated tissues. Open bars represent P/S of vinculin in unstimulated and carbachol-treated tissues. Filled bars represent P/S of metavinculin in unstimulated and carbachol-treated tissues. Data shown in this figure are taken from Fig. 11.

 


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Fig. 13. Effect of U0126 on P/S of {alpha}-SM actin (A) and calponin (B) in unstimulated and carbachol-stimulated tissues. Open bars represent P/S of {alpha}-SM actin and calponin in tissues not treated with U0126 (data taken from Fig. 6). Filled bars represent P/S of {alpha}-SM actin and calponin in tissues treated with U0126 (n = 6). Data are presented as means ± SE. *Significant difference between untreated and U0126-treated tissues (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The first major finding of this study is that cholinergic receptor stimulation differentially modulates the cytoskeletal/membrane recruitment of {alpha}-SM actin, {alpha}-actinin, calponin, vinculin, metavinculin, and talin in intact airway smooth muscle. An intriguing finding of this study was that cholinergic receptor activation caused the translocation of some cytoskeletal proteins such as {alpha}-actinin, talin, {alpha}-SM actin, and metavinculin out of the cytosol. The findings from this study are consistent with the confocal imaging studies of Saez et al. (29) showing that paxillin and vinculin were recruited to the membrane of airway smooth muscle cells during cholinergic receptor stimulation. Thus these two different experimental approaches are consistent in suggesting that airway smooth muscle cells possess mechanisms that regulate the movement of some cytoskeletal proteins in and out of the cytosol. In this study, we separated the tissue homogenate of intact bovine tracheal smooth muscle into the cytoskeletal/membrane fraction (pellet) and cytoplasmic fraction (supernatant) as described by Goldberg et al. (12) and Harrington et al. (16) and took P/S as a measure of cytoskeletal/membrane recruitment. We did not further separate the pellet into cytoskeletal and membrane fractions because membrane-cytoskeleton interactions are important determinants of the binding affinities of many cytoskeletal proteins (9, 23, 26, 30, 32, 38). We found that, in unstimulated tissues, the various actin- and integrin-binding proteins exhibited highly different basal P/S as well as different sensitivities to cholinergic receptor activation. For example, calponin had the highest basal P/S of 2.74 ± 0.47 in unstimulated tissues (Fig. 2). In contrast, vinculin had the lowest basal P/S of 0.52 ± 0.09 in unstimulated tissues (Fig. 2). Talin had a basal P/S of 1.79 ± 0.15. This finding of a more than threefold difference in the basal P/S of talin and vinculin was unexpected because both vinculin and talin are integral components of dense plaque and focal adhesions (4, 22, 31, 39).

{alpha}-Actinin exhibited the greatest increases in P/S in response to cholinergic receptor stimulation (Fig. 3). The P/S of {alpha}-actinin increased by 193% in response to cholinergic receptor stimulation (Fig. 3). In contrast, the P/S of calponin and vinculin did not change significantly in response to cholinergic receptor stimulation (Figs. 4 and 6). The P/S of {alpha}-SM actin, metavinculin, and talin increased modestly by 30%, 62%, and 64%, respectively, in response to cholinergic receptor stimulation (Figs. 3, 4, and 6). Therefore, cholinergic receptor stimulation induces cytoskeletal/membrane recruitment of the following proteins in descending order of sensitivity: {alpha}-actinin > talin {approx} metavinculin > {alpha}-SM actin > vinculin {approx} calponin. Hirshman and Emala (18) and Mehta and Gunst (24) reported cytoskeletal/membrane recruitment of actin during cholinergic receptor stimulation of airway smooth muscle cells and tissue. Recently, Saez et al. (29) reported recruitment of paxillin and vinculin to the membrane of freshly dissociated airway smooth muscle cells during cholinergic receptor stimulation. However, to our knowledge, this is the first report of differential cytoskeletal/membrane recruitment of {alpha}-SM actin, {alpha}-actinin, calponin, metavinculin, vinculin, and talin in intact airway smooth muscle.

Carbachol induced phosphorylation of ERK1/2 MAPK by 300% of basal value in bovine tracheal smooth muscle (Fig. 7). This observation is consistent with the finding of Gerthoffer et al. (8) in canine tracheal smooth muscle. In addition, we found that phospho-ERK1/2 exhibited relatively high P/S (Fig. 8), indicating that phospho-ERK1/2 were localized predominantly at the cytoskeleton-membrane complex. This observation is consistent with the calponin-ERK-caldesmon model proposed by Morgan and Gangopadhyay (25), in which the phospho-ERK1-calponin complex translocates to the actin filament. We found that U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly change the maximum force induced by carbachol. This observation is consistent with the findings of Hedges et al. (17) that ERK1/2 phosphorylation induced by M2 receptor stimulation also did not significantly affect contraction of canine tracheal smooth muscle. It is noteworthy that U0126 inhibited contractile force induced by a lower concentration of carbachol. Therefore, we calculated EC50 values for individual concentration-force relations by interpolation and then performed statistical comparisons of the two sets of EC50 values. We found that the control (0.023 ± 0.009 mM) and U0126 (0.053 ± 0.013 mM) EC50 values were not significantly different. U0126 (10 µM) significantly increased the P/S of {alpha}-actinin and {alpha}-SM actin in both unstimulated and carbachol-stimulated tissues (Figs. 10 and 13A). As a result, U0126 did not significantly affect the correlation between P/S and [carbachol]. This observation indicates that cholinergic receptor-mediated cytoskeletal/membrane recruitment of {alpha}-actinin is independent of ERK1/2 MAPK activation. The apparent augmentation of cytoskeletal/membrane recruitment of {alpha}-actinin by U0126 was unexpected because ERK1/2 phosphorylation increased in parallel with {alpha}-actinin P/S in response to cholinergic receptor stimulation (Figs. 3 and 7). Phospho-ERK has been found to target newly formed focal adhesions in fibroblasts (6). The observed high P/S of phospho-ERK1/2 (Fig. 8) suggests the possibility that phospho-ERK1/2 may target dense plaques in airway smooth muscle cells. It was noteworthy that U0126 inhibited the basal force developed by unstimulated tissues and the force developed by tissues stimulated by lower concentrations of carbachol, and the inhibition of force was accompanied by significant increases in the cytoskeletal/membrane recruitment of both {alpha}-SM actin and {alpha}-actinin. The observed correlation between the inhibition of force and cytoskeletal/membrane recruitment of {alpha}-SM actin and {alpha}-actinin indicates that cytoskeletal recruitment of {alpha}-SM actin and {alpha}-actinin does not necessarily lead to force development. It is noteworthy that significant increases in {alpha}-SM actin, {alpha}-actinin, and metavinculin all occurred at higher [carbachol] after isometric force had already reached maximum. These observations together suggest that cytoskeletal/membrane recruitment of {alpha}-SM actin, {alpha}-actinin, and metavinculin is not directly involved in contractile function but perhaps is important in strengthening the cytoskeletal architecture of airway smooth muscle cells.

Vinculin and metavinculin are homologous proteins derived from alternative splicing of a single gene (5, 11, 21). Vinculin is ubiquitous, whereas metavinculin is found only in smooth and cardiac muscle cells (3). However, previous studies failed to detect different cellular localizations of metavinculin and vinculin (2). We were intrigued to observe significantly higher P/S of metavinculin than vinculin in both unstimulated and carbachol-stimulated tissues (Fig. 5). This observation suggests that metavinculin has a relatively higher affinity than vinculin for the cytoskeleton-membrane complex in bovine tracheal smooth muscle. This finding is potentially significant for understanding the cytoskeletal architecture in airway smooth muscle cells, because metavinculin and F-actin appear to form filament webs whereas vinculin and F-actin appear to form filament bundles (28). Furthermore, we observed cytoskeletal/membrane recruitment of metavinculin but not vinculin in response to cholinergic receptor stimulation (Fig. 4). Recently, Saez et al. (29) reported that vinculin was recruited to the cell membrane in enzymatically dissociated canine tracheal smooth muscle cells during cholinergic stimulation. The anti-vinculin antibody (Sigma) used in their study was the same as that used in this study. Therefore, metavinculin is likely to be the species that was recruited to the cytoskeleton-membrane complex during cholinergic stimulation. To our knowledge, this is the first report of differential affinities of metavinculin and vinculin for the cytoskeleton-membrane complex in smooth muscle.

Calponin and {alpha}-actinin both belong to the family of calponin homology (CH) domain proteins (10, 20). However, unlike the {alpha}-actinin P/S, the calponin P/S did not change significantly in response to cholinergic receptor stimulation or U0126 in either unstimulated or carbachol-stimulated tissues (Fig. 13). It is noteworthy that calponin contains only one CH domain whereas {alpha}-actinin contains two CH domains in tandem (19). The possible involvement of CH domain(s) in the cytoskeletal/membrane recruitment of {alpha}-actinin is intriguing but remains to be tested experimentally.

In summary, we found that cholinergic receptor activation led to the following hierarchy of cytoskeletal/membrane recruitment, in order of descending sensitivity, in intact airway smooth muscle: {alpha}-actinin > talin {approx} metavinculin > {alpha}-SM actin > vinculin {approx} calponin. Metavinculin and vinculin are splice variants from a single gene, but metavinculin exhibits a higher affinity than vinculin for the cytoskeleton-membrane complex. Furthermore, metavinculin but not vinculin was recruited to the cytoskeleton-membrane complex during cholinergic receptor stimulation. Therefore, {alpha}-actinin, talin, and metavinculin appear to be the key cytoskeletal proteins involved in cholinergic receptor-mediated cytoskeletal remodeling in airway smooth muscle cells. Isometric contraction experiments do not represent the situation in vivo when airway smooth muscle cells undergo cyclic stretching and lengthening during inspiration and expiration. Muscle length is an important determinant of airway smooth muscle activation as measured by phosphatidylinositol turnover, intracellular [Ca2+], and myosin phosphorylation (1, 15). Therefore, isometric contraction experiments simplify the study of receptor-mediated cross-bridge activation in airway smooth muscle without the complication of length-dependent effects. On the basis of the findings from this study, we are presently investigating the interaction between mechanical strain and receptor activation in the regulation of cytoskeletal remodeling in airway smooth muscle cells.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-52714.


    ACKNOWLEDGMENTS
 
We thank Dr. Elizabeth O. Harrington for providing the protocol for cytoskeletal fractionation. We thank Rhode Island Beef for providing bovine tracheas for the experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C.-M. Hai, Dept. of Molecular Pharmacology, Physiology, and Biotechnology, Brown Univ., Box G-B3, Providence, RI 02912 (E-mail: Chi-Ming_Hai{at}brown.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.


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