Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912
Submitted 19 February 2004 ; accepted in final form 12 July 2004
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ABSTRACT |
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actinin; mitogen-activated protein kinase; metavinculin; vinculin
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 -smooth muscle (SM) actin,
-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.
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MATERIALS AND METHODS |
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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 -SM actin,
-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).
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RESULTS |
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DISCUSSION |
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-Actinin exhibited the greatest increases in P/S in response to cholinergic receptor stimulation (Fig. 3). The P/S of
-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
-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:
-actinin > talin
metavinculin >
-SM actin > vinculin
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
-SM actin,
-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 -actinin and
-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
-actinin is independent of ERK1/2 MAPK activation. The apparent augmentation of cytoskeletal/membrane recruitment of
-actinin by U0126 was unexpected because ERK1/2 phosphorylation increased in parallel with
-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
-SM actin and
-actinin. The observed correlation between the inhibition of force and cytoskeletal/membrane recruitment of
-SM actin and
-actinin indicates that cytoskeletal recruitment of
-SM actin and
-actinin does not necessarily lead to force development. It is noteworthy that significant increases in
-SM actin,
-actinin, and metavinculin all occurred at higher [carbachol] after isometric force had already reached maximum. These observations together suggest that cytoskeletal/membrane recruitment of
-SM actin,
-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 -actinin both belong to the family of calponin homology (CH) domain proteins (10, 20). However, unlike the
-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
-actinin contains two CH domains in tandem (19). The possible involvement of CH domain(s) in the cytoskeletal/membrane recruitment of
-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: -actinin > talin
metavinculin >
-SM actin > vinculin
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,
-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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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