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• W1990181657 abstract "The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that ∼40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation. The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that ∼40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation. Cofilin, a 19-kDa protein, and the closely related protein actin depolymerization factor (ADF) 2The abbreviations used are: ADFactin depolymerization factorAChacetylcholineGAPDHglyceraldehyde-3-phosphate dehydrogenasePSSphysiological saline solutionTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]amino}ethanesulfonic acidWTwild typeMLCmyosin light chainPIPES1,4-piperazinediethanesulfonic acid are members of a family of “actin-dynamizing proteins.” These proteins play a critical role in the rapid adaptation of the actin cytoskeleton to localized cellular functions (1Bamburg J.R. McGough A. Ono S. Trends Cell Biol. 1999; 9: 364-370Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 2Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (837) Google Scholar, 3Condeelis J. Trends Cell Biol. 2001; 11: 288-293Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The activation of ADF/cofilin is essential for cell motility and polarized cell migration. actin depolymerization factor acetylcholine glyceraldehyde-3-phosphate dehydrogenase physiological saline solution 2-{[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]amino}ethanesulfonic acid wild type myosin light chain 1,4-piperazinediethanesulfonic acid The cytoskeletal organization of differentiated smooth muscle cells and tissues is dynamic, and it is regulated during contractile stimulation (4Gunst S.J. Fredberg J.J. J. Appl. Physiol. 2003; 95: 413-425Crossref PubMed Scopus (117) Google Scholar, 5Gunst S.J. Zhang W. Am. J. Physiol. 2008; 295: C576-C587Crossref PubMed Scopus (274) Google Scholar, 6Zhang W. Gunst S.J. Proc. Am. Thorac. Soc. 2008; 5: 32-39Crossref PubMed Scopus (99) Google Scholar). Dynamic changes in cytoskeletal organization may enable smooth muscle cells to modulate their structure and contractility in response to changes in their external environment (6Zhang W. Gunst S.J. Proc. Am. Thorac. Soc. 2008; 5: 32-39Crossref PubMed Scopus (99) Google Scholar, 7Gunst S.J. Tang D.D. Opazo-Saez A. Respir. Physiol. Neurobiol. 2003; 137: 151-168Crossref PubMed Scopus (125) Google Scholar). Actin polymerization can be triggered by contractile stimuli in many smooth muscle tissues, and tension development can be dramatically depressed by short term exposure to inhibitors of actin polymerization (5Gunst S.J. Zhang W. Am. J. Physiol. 2008; 295: C576-C587Crossref PubMed Scopus (274) Google Scholar, 8An S.S. Laudadio R.E. Lai J. Rogers R.A. Fredberg J.J. Am. J. Physiol. 2002; 283: C792-C801Crossref PubMed Scopus (143) Google Scholar, 9Cipolla M.J. Gokina N.I. Osol G. FASEB J. 2002; 16: 72-76Crossref PubMed Scopus (195) Google Scholar, 10Hirshman C.A. Emala C.W. Am. J. Physiol. 1999; 277: L653-L661PubMed Google Scholar, 11Jones K.A. Perkins W.J. Lorenz R.R. Prakash Y.S. Sieck G.C. Warner D.O. J. Physiol. (Lond.). 1999; 519: 527-538Crossref Scopus (60) Google Scholar, 12Mehta D. Gunst S.J. J. Physiol. (Lond.). 1999; 519: 829-840Crossref Scopus (217) Google Scholar, 13Tang D.D. Turner C.E. Gunst S.J. J. Physiol. (Lond.). 2003; 553: 21-35Crossref Scopus (61) Google Scholar, 14Tang D.D. Gunst S.J. J. Biol. Chem. 2004; 279: 51722-51728Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (98) Google Scholar, 16Kim H.R. Gallant C. Leavis P.C. Gunst S.J. Morgan K.G. Am. J. Physiol. 2008; 295: C68-C78Google Scholar). In airway smooth muscle, the inhibition of actin polymerization can inhibit tension development in the absence of an effect on myosin light chain phosphorylation, suggesting that actin polymerization regulates tension development by processes independently of cross-bridge cycling (5Gunst S.J. Zhang W. Am. J. Physiol. 2008; 295: C576-C587Crossref PubMed Scopus (274) Google Scholar, 12Mehta D. Gunst S.J. J. Physiol. (Lond.). 1999; 519: 829-840Crossref Scopus (217) Google Scholar, 13Tang D.D. Turner C.E. Gunst S.J. J. Physiol. (Lond.). 2003; 553: 21-35Crossref Scopus (61) Google Scholar, 15Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (98) Google Scholar). Actin is present in both unassembled (globular, G) and filamentous (F) form in all cells. Actin monomers (G-actin) add preferentially to the fast growing (barbed) ends of the actin filaments; the availability of barbed ends is critical for the addition of G-actin monomers to existing actin filaments (3Condeelis J. Trends Cell Biol. 2001; 11: 288-293Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 17Carlier M.F. Pantaloni D. J. Biol. Chem. 2007; 282: 23005-23009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Cofilin activation enhances F-actin dynamics by increasing the dissociation of actin monomers from the pointed ends of actin filaments, which enhances the pool of available actin monomers (18Kiuchi T. Ohashi K. Kurita S. Mizuno K. J. Cell Biol. 2007; 177: 465-476Crossref PubMed Scopus (145) Google Scholar), and by binding to F-actin and severing it to make new barbed ends available for polymerization and depolymerization. High concentrations of active cofilin can nucleate filament assembly (19Andrianantoandro E. Pollard T.D. Mol. Cell. 2006; 24: 13-23Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 20Chen X.M. Huang B.Q. Splinter P.L. Orth J.D. Billadeau D.D. McNiven M.A. LaRusso N.F. Infect. Immun. 2004; 72: 3011-3021Crossref PubMed Scopus (47) Google Scholar). The activity of ADF/cofilin is regulated by phosphorylation at a single site on the amino terminus, serine 3, which inhibits its activity. Phosphorylation at this site abolishes the ability of ADF/cofilin to bind to F-actin and thus inhibits its severing function (1Bamburg J.R. McGough A. Ono S. Trends Cell Biol. 1999; 9: 364-370Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 21Agnew B.J. Minamide L.S. Bamburg J.R. J. Biol. Chem. 1995; 270: 17582-17587Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 22Moriyama K. Iida K. Yahara I. Genes Cells. 1996; 1: 73-86Crossref PubMed Scopus (318) Google Scholar). We hypothesized that ADF/cofilin might play an important role in the regulation of actin dynamics in smooth muscle during contractile activation. In this study, we analyzed the effect of contractile activation on the phosphorylation of ADF/cofilin at Ser-3. To evaluate the function of ADF/cofilin in regulating actin dynamics and tension generation during contraction of smooth muscle tissues, we expressed an inactive cofilin phosphomimetic (cofilin S3E) in the tissues, which has minimal actin severing activity (23Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar). Our results demonstrate that ADF/cofilin undergoes dephosphorylation in response to contractile stimulation in smooth muscle tissues and that ADF/cofilin dephosphorylation is necessary for both actin polymerization and active tension generation. We conclude that the activation of ADF/cofilin is a necessary step for the dynamic reorganization of actin that occurs during the contraction of smooth muscle tissues. Preparation of Smooth Muscle Tissues and Measurement of Force—Mongrel dogs (20–25 kg) were euthanized with pentobarbital sodium (30 mg/kg intravenously) and quickly exsanguinated. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Indiana University School of Medicine. A segment of the trachea was immediately removed and immersed in physiological saline solution (PSS) at 22 °C containing (mm): 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose. PSS was aerated with 95% O2, 5% CO2 to maintain a pH of 7.4. Smooth muscle was dissected free of connective tissue and epithelium and cut into strips (1 mm wide × 0.2–0.5 mm thick × 15 mm long). Muscle strips were placed in PSS at 37 °C in a 25-ml organ bath and attached to a force transducer for measurement of force. At the beginning of each experiment, the optimal length (Lo) for muscle contraction was determined by progressively increasing the length of the muscle until the active isometric force elicited by ACh reached a maximum. All tissues were then maintained at Lo for 30–60 min without stimulation. For experiments involving the introduction of plasmids encoding cofilin proteins, muscle strips were then subjected to the reversible permeabilization procedure described below. Two days were then allowed for expression of the recombinant proteins, at which time the active isometric force in response to ACh at Lo was determined again. Reagents—The following antibodies were used in these studies: mouse monoclonal anti-cofilin (BIOSOURCE), rabbit polyclonal anti-phospho-ADF/cofilin at serine-3 antibody (23Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar), and rabbit polyclonal anti-ADF/cofilin antibody (reacts with both ADF and cofilin) (24Shaw A.E. Minamide L.S. Bill C.L. Funk J.D. Maiti S. Bamburg J.R. Electrophoresis. 2004; 25: 2611-2620Crossref PubMed Scopus (33) Google Scholar) provided by Dr. James Bamburg, Colorado State University; mouse monoclonal anti-actin (Clone AC-40, Sigma); mouse monoclonal GAPDH (RDI, Concord, MA). Polyclonal myosin light chain antibody was custom-made by BABCO (Richmond, CA). pcDNA3.1 vectors (human cytomegalovirus as promoter) encoding human wild type cofilin and inactive mutant cofilin S3E were provided by Dr. J. R. Bamburg (Colorado State University, Fort Collins). Transfection of Smooth Muscle Tissues with Plasmids—Plasmids were introduced into tracheal smooth muscle strips by the method of reversible permeabilization as described previously (13Tang D.D. Turner C.E. Gunst S.J. J. Physiol. (Lond.). 2003; 553: 21-35Crossref Scopus (61) Google Scholar, 15Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (98) Google Scholar, 25Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopus (111) Google Scholar). After initial equilibration and contraction to 10-5 m ACh to obtain maximal force, muscle strips were attached to metal mounts at Lo. The strips were incubated successively in each of the following solutions: Solution 1 (at 4 °C for 120 min) containing (mm): 10 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, and 20 TES; solution 2 (at 4 °C overnight) containing (mm): 0.1 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, 20 TES, and 10 μg/ml plasmids; Solution 3 (at 4 °C for 30 min) containing (mm): 0.1 EGTA, 5 Na2ATP, 120 KCl, 10 MgCl2, 20 TES; and solution 4 (at 22 °C for 60 min) containing (mm): 110 NaCl, 3.4 KCl, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 dextrose. Solutions 1–3 were maintained at pH 7.1 and aerated with 100% O2. Solution 4 was maintained at pH 7.4 and aerated with 95% O2, 5% CO2. After 30 min in Solution 4, CaCl2 was added gradually to reach a final concentration of 2.4 mm. The strips were then incubated in a CO2 incubator at 37 °C for 2 days in serum-free Dulbecco's modified Eagle's medium containing 5 mm Na2ATP, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml plasmids encoding human wild type cofilin or the inactive mutant cofilin S3E. Analysis of ADF/Cofilin Phosphorylation—ADF/cofilin isoforms and phosphorylation were analyzed by one-dimensional and by two-dimensional electrophoresis. Muscle tissues were rapidly frozen using liquid nitrogen-cooled tongs and pulverized using a mortar and pestle. Pulverized muscle tissues were mixed with extraction buffer containing the following: 20 mm Tris-HCl, pH 7.4, 2% Triton X-100, 0.4% SDS, 2 mm EDTA, 2 mm EGTA, phosphatase inhibitors (2 mm sodium orthovanadate, 2 mm molybdate, and 2 mm sodium pyrophosphate, 50 mm sodium fluoride) and protease inhibitors (2 mm benzamidine, 0.5 mm aprotinin, and 1 mm phenylmethylsulfonyl fluoride). For one-dimensional electrophoresis, each sample was centrifuged for the collection of supernatant, and the supernatant was then boiled in sample buffer (1.5% dithiothreitol, 2% SDS, 80 mm Tris-HCl, pH 6.8, 10% glycerol, and 0.01% bromphenol blue) for 5 min. Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose. To measure ADF/cofilin phosphorylation, the nitrocellulose membrane was simultaneously probed with antibodies to phospho-Ser-3 ADF/cofilin and cofilin, followed by fluorophore-conjugated anti-rabbit and anti-mouse immunoglobulins. Fluorescence signals were detected and analyzed using an Odyssey fluorescence scanner (LI-COR Biosciences, Lincoln, NE). For two-dimensional PAGE, protein was precipitated from smooth muscle tissue protein extracts using a methanol/chloroform/water mixture (26Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar). The precipitated total smooth muscle protein was redissolved in ReadyPrep two-dimensional sample buffer (Bio-Rad). Isoelectric focusing was performed in a PROTEAN IEF cell with 11-cm IPG strips pH 3–10 (Bio-Rad) according to the manufacturer's instructions. The focused proteins were then separated by means of an 18% SDS-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane (Bio-Rad). ADF and cofilin were detected using a polyclonal anti-ADF/cofilin antibody that reacts with both ADF and cofilin (24Shaw A.E. Minamide L.S. Bill C.L. Funk J.D. Maiti S. Bamburg J.R. Electrophoresis. 2004; 25: 2611-2620Crossref PubMed Scopus (33) Google Scholar). The ratios of phosphorylated ADF and phosphorylated cofilin to total ADF and total cofilin and the amount of recombinant cofilin expression were analyzed by scanning densitometry. Measurement of Regulatory Myosin Light Chain Phosphorylation—Frozen muscle strips were immersed in dry ice precooled acetone containing 10% w/v trichloroacetic acid and 10 mm dithiothreitol. Proteins were extracted in 8 m urea, 20 mm Tris base, 22 mm glycine, and 10 mm dithiothreitol. Phosphorylated and unphosphorylated myosin lights chains (MLCs) were separated by urea-glycerol PAGE, transferred to nitrocellulose, and then probed using antibody to the 20-kDa myosin light chain (15Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (98) Google Scholar, 27Zhang W.W. Gunst S.J. J. Physiol. (Lond.). 2006; 572: 659-676Crossref Scopus (62) Google Scholar). Proteins were visualized by enhanced chemiluminescence (ECL). The ratio of phosphorylated to unphosphorylated MLC was determined by scanning densitometry. Analysis of F-actin and G-actin—The relative proportions of F-actin and G-actin in smooth muscle tissues were analyzed using a standard assay kit (Cytoskeleton, Denver, CO) as described previously (15Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (98) Google Scholar, 27Zhang W.W. Gunst S.J. J. Physiol. (Lond.). 2006; 572: 659-676Crossref Scopus (62) Google Scholar). Briefly, each of the tracheal smooth muscle strips was homogenized in 200 μl of F-actin stabilization buffer (50 mm PIPES, pH 6.9, 50 mm NaCl, 5 mm MgCl2, 5 mm EGTA, 5% glycerol, 0.1% Triton X-100, 0.1% Non-idet P-40, 0.1% Tween 20, 0.1% β-mercaptoethanol, 0.001% antifoam, 1 mm ATP, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 10 μg/ml benzamidine, and 500 μg/ml tosyl arginine methyl ester). Supernatants of the protein extracts were collected after centrifugation at 150,000 × g for 60 min at 37 °C. The pellets were resuspended in 200 μl of ice-cold distilled water containing 10 μm cytochalasin D and then incubated on ice for 1 h to depolymerize F-actin. The resuspended pellets were gently mixed every 15 min. Four microliters of supernatant (G-actin) and pellet (F-actin) fractions were subjected to immunoblot analysis. The ratios of F-actin to G-actin were determined using densitometry. Statistical Analysis—Comparisons between the two groups were performed using paired Student's t tests. Comparisons among multiple groups were performed using repeated measures analysis of variance. Values refer to the number of tissues used to obtain mean values. p < 0.05 was considered statistically significant. Contractile Stimulation of Tracheal Smooth Muscle Tissues Induces ADF/Cofilin Dephosphorylation on Ser-3 (Fig. 1)—Canine tracheal smooth muscle strips were maintained for 30 min without contractile stimulation and then contracted isometrically with 10-5 m ACh for 15 or 30 s or 1, 5, or 15 min or left unstimulated. ADF/cofilin phosphorylation on Ser-3 was elevated in unstimulated tissues and decreased significantly in response to ACh stimulation. The decrease in ADF/cofilin phosphorylation was evident 15 s (0.25 min) after stimulation with ACh and persisted for the duration of the contraction (Fig. 1, A and B). Differences in ADF/cofilin phosphorylation in ACh-stimulated and unstimulated tissues were statistically significant at all time points (n = 6, p < 0.05). The time course of the decrease in ADF/cofilin phosphorylation was similar to the time course of the increase in force development in response to ACh stimulation. ADF/cofilin phosphorylation 30 or 60 min after stimulation with ACh was not significantly different from ADF/cofilin phosphorylation at 15 min, suggesting that the activation of ADF/cofilin was sustained for the duration of the contraction (data not shown). We also evaluated whether the decline in ADF/cofilin phosphorylation during contractile stimulation was dependent on a receptor-mediated mechanism. Muscles were stimulated with 60 mm KCl, and ADF/cofilin phosphorylation was analyzed 15 and 30 min after stimulation. ADF/cofilin phosphorylation decreased significantly to 61 ± 5% (p < 0.05, n = 6) in response to KCl stimulation, and the decrease in ADF/cofilin phosphorylation persisted for the 30 min duration of the contraction. The decrease in ADF/cofilin phosphorylation was less than during ACh stimulation, but force development in response to KCl stimulation was only about 60% of that observed with ACh stimulation. The results demonstrate that both receptor-mediated agonists and depolarization with KCl stimulate the dephosphorylation of ADF/cofilin and its activation in tracheal smooth muscle tissues. ACh Stimulation Induces Similar Dephosphorylation at Ser-3 of Both Cofilin and ADF in Tracheal Smooth Muscle Tissues (Fig. 2)—We used two-dimensional gel electrophoresis to determine which isoforms of ADF/cofilin are present in tracheal smooth muscle and to quantitate the effects of stimulation with ACh on their phosphorylation (Fig. 2A). We found that cofilin and ADF were represented in similar proportions (54 ± 2 and 46 ± 2%, respectively) (n = 4). Cofilin2 represented less than 7% of the total ADF/cofilin detected in the muscle tissue. In the unstimulated smooth muscle tissues, the phospho-cofilin was 40 ± 3% of total cofilin, and this proportion decreased to 14 ± 3% with ACh stimulation (Fig. 2B)(n = 4). The proportion of phospho-ADF in unstimulated smooth muscle was 38 ± 4%, and this decreased to 14 ± 1% with ACh stimulation. Expression of the Inactive Cofilin S3E Mutant Inhibits the Dephosphorylation of Endogenous ADF/Cofilin Induced by ACh (Fig. 3)—Plasmids encoding wild type cofilin (cofilin WT) or the inactive phosphomimetic cofilin mutant S3E (cofilin S3E) were introduced into smooth muscle strips by reversible permeabilization. Transfected tissues and control untreated tissues were then maintained in an incubator for 2 days to allow for the expression of recombinant proteins. Muscle tissues were stimulated with 10-5 m ACh for 5 min or left unstimulated and then frozen for the analysis of ADF/cofilin phosphorylation on Ser-3. The amount of total cofilin was 70–80% higher in each group of muscle strips treated with cofilin WT or cofilin S3E compared with tissues without plasmid treatment (n = 5 or 6, p < 0.05), consistent with a robust expression of the recombinant proteins in the transfected tissues (Fig. 3C). There were no significant differences in the amount of cofilin in tissues expressing wild type or mutant cofilin constructs. The amount of P-ADF/cofilin in unstimulated muscle tissues treated with either cofilin WT or cofilin S3E was also significantly higher than in control tissues, reflecting the higher levels of cofilin in these tissues. When the P-ADF/cofilin values were normalized to total cofilin, the amount of phosphorylated ADF/cofilin in the cofilin WT-treated and unstimulated cofilin S3E-treated muscles was not significantly different from untreated muscles. Stimulation with ACh caused a significant decrease in the amount of P-ADF/cofilin in untreated and cofilin WT-treated tissues; when normalized either to GAPDH or to total cofilin. In contrast, ACh did not cause a significant decrease in the amount of phosphorylated ADF/cofilin in muscle tissues treated with cofilin S3E, whether normalized to GAPDH or total cofilin. Thus, expression of the cofilin S3E mutant in the muscle tissues markedly suppressed ADF/cofilin dephosphorylation and activation in response to stimulation with ACh (Fig. 3, A and B). We used two-dimensional gel electrophoresis to evaluate the effects of expression of cofilin S3E on the phosphorylation of both ADF and cofilin in response to ACh, and to quantify the amount of cofilin S3E in the smooth muscle tissue (Fig. 4, A and B). Cofilin S3E represented 34 ± 5% of the total ADF/cofilin (n = 4). The expression of cofilin S3E caused comparable inhibition of the dephosphorylation of both cofilin and ADF in response to ACh. Expression of Cofilin S3E Inhibits Tension Development in Smooth Muscle Tissues (Fig. 5)—We evaluated the effects of expression of cofilin S3E and cofilin WT on contractile tension 5 min after the stimulation of muscle tissues with 10-5 m ACh. The contractile force generated in response to stimulation with ACh was significantly inhibited in tissues expressing cofilin S3E. The mean tension in cofilin S3E-treated tissues was 52 ± 4.0% of force in untreated or WT-treated tissues (n = 27; p < 0.01). In contrast, the contractile force in tissues expressing the cofilin WT was not significantly different from force in untreated tissues (Fig. 5, A and B). Expression of Inactive Mutant Cofilin S3E Inhibits Actin Polymerization in Response to ACh (Fig. 6)—Smooth muscle strips incubated without plasmids or with plasmids encoding the cofilin S3E or cofilin WT were stimulated with 10-5 m ACh for 5 min, and the proportions of F-actin to G-actin in muscle extracts were analyzed by cell fractionation and immunoblot. Whereas ACh stimulation significantly increased the ratio of F-actin to G-actin in untreated and cofilin WT-treated smooth muscle strips (n = 6; p < 0.05), ACh stimulation did not significantly alter the ratio of F-actin to G-actin in smooth muscle tissues expressing inactive cofilin S3E (Fig. 6A). In tissues treated with cofilin S3E, the ratio of F/G-actin was significantly elevated in unstimulated muscles and significantly depressed in ACh-stimulated muscles relative to untreated or cofilin WT-treated muscles. The effect of ADF/cofilin inactivation on the pools of G-actin and F-actin was evaluated in cofilin S3E, cofilin WT, and untreated muscles (Fig. 6B). Unstimulated muscles treated with cofilin S3E had significantly less G-actin and more F-actin than untreated or cofilin WT-treated muscles (n = 6, p < 0.05). Cofilin WT-treated muscles exhibited a small but statistically significant increase in G-actin and a decrease in F-actin compared with untreated muscles (n = 6, p < 0.05). The results suggest that expression of the cofilin S3E protein inhibited actin polymerization in muscle strips in response to ACh stimulation, and that cofilin S3E inhibited actin depolymerization in unstimulated muscle tissues. Expression of Inactive Cofilin S3E Does Not Affect MLC Phosphorylation in Response to ACh in Smooth Muscle Tissues (Fig. 7)—The effects of stimulation with ACh on MLC phosphorylation were compared in muscle tissues transfected with the plasmids encoding inactive cofilin S3E and cofilin WT and in tissues incubated with no plasmids. There were no significant differences in MLC phosphorylation in unstimulated or ACh-stimulated muscles expressing the inactive cofilin S3E, cofilin WT, or muscles not treated with plasmids (n = 4, p > 0.05). Thus, the inhibition of contraction by cofilin S3E did not affect the signaling pathways that regulate MLC phosphorylation. Depletion of Intracellular Ca2+ from Muscle Tissues or Treatment of the Tissues with Calcineurin Inhibitors Inhibits the Dephosphorylation of ADF/Cofilin Induced by ACh (Fig. 8)—Calcineurin, the Ca2+-dependent protein phosphatase 2B (PP2B), has been shown to regulate the activation of ADF/cofilin in several non-muscle cell lines (23Meberg P.J. Ono S. Minamide L.S. Takahashi M. Bamburg J.R. Cell Motil. Cytoskeleton. 1998; 39: 172-190Crossref PubMed Scopus (217) Google Scholar, 28Wang Y. Shibasaki F. Mizuno K. J. Biol. Chem. 2005; 280: 12683-12689Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). We evaluated the Ca2+ dependence of ADF/cofilin activation by depleting intracellular Ca2+ from muscle tissues. Tissues were incubated in Ca2+-free PSS containing 0.1 mm EGTA. The tissues were then repeatedly stimulated for 5-min time periods with ACh until the contractile response decreased to a minimum (29Tang D.D. Mehta D. Gunst S.J. Am. J. Physiol. 1999; 276: C250-C258Crossref PubMed Google Scholar). This generally required ∼60 min and resulted in a reduction of isometric force to less than 10% of the maximal force. ADF/cofilin phosphorylation was then evaluated after ACh stimulation of the Ca2+-depleted tissues for 5 min. The depletion of Ca2+ markedly inhibited the decrease in ADF/cofilin phosphorylation stimulated by ACh (p < 0.05, n = 6). This suggests that the dephosphorylation and activation of ADF/cofilin are mediated by Ca2+-dependent mechanisms in this tissue. Muscle tissues were incubated with the calcineurin inhibitors, cyclosporin A (10 μm) or deltamethrin (10 μm), for 2 h to evaluate the role of calcineurin in ADF/cofilin activation during active contraction with ACh (Fig. 9). ADF/cofilin dephosphorylation was inhibited significantly in ACh-stimulated muscle tissues pretreated with calcineurin inhibitors (p < 0.05, n = 5). This suggests that t" @default.
• W1990181657 created "2016-06-24" @default.
• W1990181657 creator A5017649557 @default.
• W1990181657 creator A5027575817 @default.
• W1990181657 creator A5033535840 @default.
• W1990181657 creator A5034574499 @default.
• W1990181657 creator A5058154553 @default.
• W1990181657 date "2008-12-01" @default.
• W1990181657 modified "2023-10-15" @default.
• W1990181657 title "Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle" @default.
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