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• W2089642295 abstract "The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a β-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and α-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and α-parvin form a stable cytosolic complex and that the ILK·PINCH·α-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with β-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK·PINCH·α-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle. The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a β-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and α-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and α-parvin form a stable cytosolic complex and that the ILK·PINCH·α-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with β-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK·PINCH·α-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle. Smooth muscle tissues from hollow organs are subjected to large changes in shape and volume under physiologic conditions in vivo. When subjected to external mechanical forces, the muscle must rapidly adapt its compliance and contractility to accommodate to changes in external mechanical forces. External forces that are imposed on smooth muscle tissues are transmitted to the interior of the cells via adhesion complexes, which link the cytoskeleton to the extracellular matrix. Transmembrane integrins, which are ligands for extracellular matrix proteins, can mediate the transduction of mechanical signals from the extracellular matrix to the cytoskeleton (1Brakebusch C. Fassler R. EMBO J. 2003; 22: 2324-2333Crossref PubMed Scopus (382) Google Scholar, 2Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 3Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (343) Google Scholar). Recent data has suggested that the cytoskeletal organization of smooth muscle cells is dynamic and that it is regulated during contractile stimulation (4Gunst S.J. Tang D.D. Opazo S.A. Respir. Physiol. Neurobiol. 2003; 137: 151-168Crossref PubMed Scopus (122) Google Scholar, 5Hirshman C.A. Emala C.W. Am. J. Physiol. 1999; 277: L653-L661PubMed Google Scholar, 6Jones K.A. Perkins W.J. Lorenz R.R. Prakash Y.S. Sieck G.C. Warner D.O. J. Physiol. 1999; 519: 527-538Crossref PubMed Scopus (58) Google Scholar, 7Mehta D. Gunst S.J. J. Physiol. 1999; 519: 829-840Crossref PubMed Scopus (214) Google Scholar, 8Tang D.D. Gunst S.J. J. Biol. Chem. 2004; 279: 51722-51728Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) 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. The activation of smooth muscle contraction causes an increase in actin polymerization in airway and other smooth muscles (7Mehta D. Gunst S.J. J. Physiol. 1999; 519: 829-840Crossref PubMed Scopus (214) Google Scholar, 9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 11Herrera A.M. Martinez E.C. Seow C.Y. Am. J. Physiol. 2004; 286: L1161-L1168Crossref PubMed Scopus (63) Google Scholar, 12An S.S. Laudadio R.E. Lai J. Rogers R.A. Fredberg J.J. Am. J. Physiol. 2002; 283: C792-C801Crossref PubMed Scopus (135) Google Scholar, 13Tang D.D. Zhang W. Gunst S.J. J. Biol. Chem. 2005; 280: 23380-23389Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 14Barany M. Barron J.T. Gu L. Barany K. J. Biol. Chem. 2001; 276: 48398-48403Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Cipolla M.J. Gokina N.I. Osol G. FASEB J. 2002; 16: 72-76Crossref PubMed Scopus (188) Google Scholar). In tracheal smooth muscle tissues, contractile stimulation also initiates dynamic changes in the localization of cytoskeletal proteins and an increase in their association with integrin-associated protein complexes (10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar, 16Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopus (108) Google Scholar, 17Kim H.R. Hoque M. Hai C.M. Am. J. Physiol. 2004; 287: C1375-C1383Crossref PubMed Scopus (23) Google Scholar). Furthermore, the inhibition of actin polymerization or cytoskeletal reorganization can inhibit tension development in the absence of an effect on myosin light chain phosphorylation (7Mehta D. Gunst S.J. J. Physiol. 1999; 519: 829-840Crossref PubMed Scopus (214) Google Scholar, 9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 18Tang D.D. Turner C.E. Gunst S.J. J. Physiol. 2003; 553: 21-35Crossref PubMed Scopus (59) Google Scholar). The actin nucleating protein, N-WASp, regulates the initiation of actin polymerization in tracheal smooth muscle during contractile activation (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar). In tracheal smooth muscle, N-WASp activation can be regulated by phosphorylation of the adhesion complex protein, paxillin, which couples the SH2/SH3 adaptor protein CrkII to N-WASp to catalyze its activation by the small GTPase cdc42 (8Tang D.D. Gunst S.J. J. Biol. Chem. 2004; 279: 51722-51728Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 13Tang D.D. Zhang W. Gunst S.J. J. Biol. Chem. 2005; 280: 23380-23389Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Paxillin undergoes tyrosine phosphorylation during the contractile activation of tracheal smooth muscle, and the tyrosine phosphorylation of paxillin is required for tension development and actin polymerization (18Tang D.D. Turner C.E. Gunst S.J. J. Physiol. 2003; 553: 21-35Crossref PubMed Scopus (59) Google Scholar, 20Wang Z. Pavalko F.M. Gunst S.J. Am. J. Physiol. 1996; 271: C1594-C1602Crossref PubMed Google Scholar). However, the mechanisms by which external signals mediated by integrin receptors are coupled to the cytoskeletal signaling pathways that regulate actin polymerization and cytoskeletal dynamics are not known. Integrins are linked to the actin cytoskeleton via macromolecular protein complexes that interact with the cytoplasmic domains of integrin proteins and with actin filaments. These complexes are composed of adaptor and structural proteins as well as proteins with enzymatic activity. Integrin-linked kinase (ILK) 2The abbreviations used are: ILK, integrin-linked kinase; PSS, physiological saline solution; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; EGFP, enhanced green fluorescence protein; ACh, acetylcholine; WT, wild type; MLC, myosin light chain; PIPES, 1,4-piperazinediethanesulfonic acid. 2The abbreviations used are: ILK, integrin-linked kinase; PSS, physiological saline solution; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; EGFP, enhanced green fluorescence protein; ACh, acetylcholine; WT, wild type; MLC, myosin light chain; PIPES, 1,4-piperazinediethanesulfonic acid. is a multidomain protein that binds directly to the cytoplasmic domain of β1 integrins and serves as a scaffolding protein for the organization of cytoskeletal signaling proteins at adhesion complexes (21Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (955) Google Scholar). ILK forms a heterotrimeric complex with the adaptor protein PINCH, an adaptor protein that consists of a tandem array of 5 LIM domains, and α-parvin (also known as actopaxin and CH-ILKBP) (22Attwell S. Mills J. Troussard A. Wu C. Dedhar S. Mol. Biol. Cell. 2003; 14: 4813-4825Crossref PubMed Scopus (121) Google Scholar, 23Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (345) Google Scholar, 24Schmeichel K.L. Beckerle M.C. Cell. 1994; 79: 211-219Abstract Full Text PDF PubMed Scopus (408) Google Scholar). The N-terminal ankyrin domain of ILK binds to the N-terminal LIM domain of PINCH, and the C-terminal domain of ILK binds to α-parvin, which binds to actin filaments (25Tu Y. Huang Y. Zhang Y. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (190) Google Scholar, 26Tu Y.Z. Li F.G. Goicoechea S. Wu C.Y. Mol. Cell. Biol. 1999; 19: 2425-2434Crossref PubMed Scopus (243) Google Scholar, 27Zhang Y.J. Chen K. Tu Y.Z. Velyvis A. Yang Y.W. Qin J. Wu C.Y. J. Cell Sci. 2002; 115: 4777-4786Crossref PubMed Scopus (134) Google Scholar). The C-terminal domain of ILK also binds to paxillin (28Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 29Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2002; 277: 1568-1575Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar); thus ILK may transduce signals from integrin receptors to regulate actin polymerization through its interaction with paxillin (13Tang D.D. Zhang W. Gunst S.J. J. Biol. Chem. 2005; 280: 23380-23389Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Tang D.D. Turner C.E. Gunst S.J. J. Physiol. 2003; 553: 21-35Crossref PubMed Scopus (59) Google Scholar, 30Mishima W. Suzuki A. Yamaji S. Yoshimi R. Ueda A. Kaneko T. Tanaka J. Miwa Y. Ohno S. Ishigatsubo Y. Genes Cells. 2004; 9: 193-204Crossref PubMed Scopus (45) Google Scholar). ILK has also been implicated in the regulation of N-WASp through its interactions with PINCH (31Wu C. Trends Cell Biol. 2005; 15: 460-466Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). ILK and its binding partners are thus positioned to coordinate signaling pathways that regulate cytoskeletal functions and the organization of structural links between integrin proteins and the actin cytoskeleton (22Attwell S. Mills J. Troussard A. Wu C. Dedhar S. Mol. Biol. Cell. 2003; 14: 4813-4825Crossref PubMed Scopus (121) Google Scholar, 32Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Crossref PubMed Scopus (232) Google Scholar). We hypothesized that ILK may play a pivotal role in the regulation of actin polymerization and cytoskeletal dynamics during contractile activation and tension generation in tracheal smooth muscle. To evaluate the role of ILK in these processes, we inhibited the recruitment of ILK to integrin complexes by expressing a truncated N-terminal fragment of PINCH (PINCH LIM1-2) in tracheal smooth muscle tissues. PINCH LIM1-2 contains only the first 2 LIM domains and competes with endogenous PINCH for binding to ILK, thereby disrupting the recruitment of ILK to integrin adhesion sites (33Wu C. J. Cell Sci. 1999; 112: 4485-4489Crossref PubMed Google Scholar). Our results demonstrate that the contractile activation of tracheal smooth muscle stimulates the recruitment of the ILK·PINCH complex to membrane adhesion complexes and increases its interaction with β1 integrins and other adhesion complex proteins. We find that the assembly of the ILK protein complex at membrane adhesion sites in tracheal smooth muscle tissues is critical for the regulation of N-WASp-mediated actin polymerization and tension development during contractile activation. Our findings suggest that integrin-linked kinase is an important mediator of signals from integrin receptors to downstream pathways that regulate actin polymerization and cytoskeletal organization in smooth muscle during contractile stimulation. Reagents and Antibodies—Antibodies used in these studies were obtained from the following sources: mouse β1 integrin (clone 18), mouse PINCH-FL (clone 49, reacts at LIM3 domain of PINCH), polyclonal and monoclonal (JL-8) GFP and paxillin (clone 349), BD Biosciences; ILK (polyclonal antibody), Upstate; PINCH-N (monoclonal antibody, clone N173, reacts at amino acids 14-31 of human PINCH-1) and actin (monoclonal antibody, clone AC40), Sigma; polyclonal vinculin and polyclonal myosin light chain antibodies were custom made by BABCO, Richmond, CA; polyclonal N-WASp (H-100) and polyclonal Arp2 (H-84), Santa Cruz Biochemicals; polyclonal paxillin tyrosine phosphorylation antibodies Y118 and Y31, BIOSOURCE. Monoclonal anti-α-parvin used in immunoprecipitation has been previously described (25Tu Y. Huang Y. Zhang Y. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (190) Google Scholar). Polyclonal anti-α-parvin, from Sigma was used in immunoblot analysis. Secondary antibodies used were: Alexa Fluor 488 and Alexa Fluor 546, obtained from Molecular Probes Co. All other reagents were purchased from Sigma. EGFP-C2 vector encoding human full-length ILK (residues 1-452), EGFP-C2 vectors encoding FLAG-tagged human full-length PINCH (residues 1-315), and the PINCH LIM1-2 domains (residues 1-130) (GFP-PINCH LIM1-2) were used in these studies (34Zhang Y.J. Guo L.D. Chen K. Wu C.Y. J. Biol. Chem. 2002; 277: 318-326Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Preparation of Smooth Muscle Tissues and Measurement of Force—Mongrel dogs (20-25 kg) were anesthetized with pentobarbital sodium (30 mg/kg, intravenously) and quickly exsanguinated in accordance with procedures approved by the Institutional Animal Care and Use Committee, Indiana University School of Medicine. A segment of the trachea was immediately removed and immersed in physiological saline solution (PSS) (composition in mm: 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose). Smooth muscle strips (1.0 × 0.2-0.5 × 15 mm) were dissected free of connective and epithelial tissues. For the measurement of contractile force, muscle tissues were attached to force transducers and maintained within a tissue bath in PSS at 37 °C. Transfection of Smooth Muscle Tissues with Plasmids—Plasmids were introduced into tracheal smooth muscle strips by the method of reversible permeabilization (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar, 13Tang D.D. Zhang W. Gunst S.J. J. Biol. Chem. 2005; 280: 23380-23389Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopus (108) Google Scholar). After determination of the length of maximal isometric force (Lo), 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 (in mm): 10 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, and 20 TES; Solution 2 (at 4 °C overnight) containing (in 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 (in mm): 0.1 EGTA, 5 Na2ATP, 120 KCl, 10 MgCl2, 20 TES; and Solution 4 (at 22 °C for 60 min) containing (in 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 units/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml plasmids. Cell Dissociation and Live Cell Imaging—Smooth muscle cells were enzymatically dissociated from tracheal muscle strips as previously described (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar). Muscle strips were minced and transferred to 5 ml of dissociation solution (in mm: 130 NaCl, 5 KCl, 1.0 CaCl2, 1.0 MgCl2, 10 HEPES, 0.25 EDTA, 10 d-glucose, and 10 taurine, pH 7) with collagenase (type I, 400 units/ml), papain (type IV, 30 units/ml), bovine serum albumin (1 mg/ml), and dithiothreitol (1 mm). All enzymes were obtained from Sigma. The strips were then placed in a 37 °C shaking water bath at 80 oscillations/min for 20-30 min, followed by washing three times with a HEPES-buffered saline solution (in mm: 130 NaCl, 5 KCl, 1.0 CaCl2, 1.0 MgCl2, 20 HEPES, and 10 d-glucose, pH 7.4) and trituration with a pipette to liberate individual smooth muscle cells from the tissue. Live freshly dissociated cells were plated onto glass coverslips and allowed to adhere for 30-120 min before visualization. The localization of GFP-labeled ILK and PINCH was monitored in live cells by scanning them once/s for 60 s using a Zeiss LSM 510 laser scanning confocal microscope with an Apo ×40 (NA 1.2) water immersion objective. EGFP was excited with a 488-nm argon laser light, and fluorescence emissions were collected at 500-530 nm. The optical pinhole was set to resolve optical sections of ∼1 μm in cell thickness. Contraction was stimulated by adding acetylcholine (ACh) to the PSS bathing the cell on the coverslip to achieve a concentration of 100 μm. Analysis of Protein Localization by Immunofluorescence—The effects of ACh stimulation on the localization of endogenous and recombinant cytoskeletal proteins was evaluated in freshly dissociated smooth muscle cells (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar, 16Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopus (108) Google Scholar). The dissociated smooth muscle cells were stimulated with 10−4 m ACh or left unstimulated, fixed, and reacted with primary antibodies specific for the proteins of interest (ILK, PINCH, paxillin, and N-WASp) and with secondary antibodies (Alexa Fluor 488 and Alexa Fluor 546). The cellular localization of fluorescently labeled proteins was determined using a Zeiss LSM 510 laser scanning confocal microscope. Image Analysis—Images of smooth muscle cells were analyzed for regional differences in fluorescence intensity of labeled proteins by quantifying the pixel intensity with a series of six cross-sectional line scans along the entire length of each cell as previously described (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar, 16Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopus (108) Google Scholar). The area of the nucleus was excluded from the analysis. The ratio of pixel intensity between the cell periphery and the cell interior was computed for each line scan by calculating the ratio of the average maximum pixel intensity at the cell periphery to the average minimum pixel intensity in the cell interior. The ratios of pixel intensities between the cell periphery and the cell interior for all of the six line scans performed on a given cell were averaged to obtain a single value for the ratio for each cell. The ratio of fluorescence intensity at the cell periphery to that at the cell interior was compared in cells at rest and in cells stimulated with ACh (10−4 m). Immunoprecipitation of Proteins—Pulverized muscle tissues were mixed with extraction buffer containing 1% Nonidet P-40, 20 mm Tris·HCl, pH 7.4, 0.3% NaCl, 10% glycerol, 2 mm EDTA, phosphatase inhibitors (in mm: 2 sodium orthovanadate, 2 molybdate, and 2 sodium pyrophosphate), and protease inhibitors (in mm: 2 benzamidine, 0.5 aprotinin, and 1 phenylmethylsulfonyl fluoride). Each sample was centrifuged (14,000 × g) for the collection of supernatant. Muscle extracts containing equal amounts of protein were precleared for 30 min with 50 μl of 10% protein A-Sepharose and then incubated overnight with primary antibodies. Samples were then incubated for 2 h with 125 μl of a 10% suspension of protein A-Sepharose beads. Immunocomplexes were washed three times in a buffer containing 50 mm Tris·HCl, pH 7.6, 150 mm NaCl, and 0.1% Triton X-100. All procedures of immunoprecipitation were performed at 4 °C. Measurement of Intracellular Ca2+ Concentration—Tracheal smooth muscle strips were pinned in a dish at a slightly stretched length (1.2 times slack length) and incubated in PSS containing 20 μm fura 2-AM, which was dissolved in 0.5% Me2SO premixed with 0.01% Pluronic 127 for 3.5 h at room temperature. They were then washed in PSS for 30 min to remove extracellular fura 2-AM and allowed time for the hydrolytic conversion of intracellular fura 2-AM to fura 2. Tissues were mounted in a cuvette and attached to a force transducer for the simultaneous measurement of force and fura-2 fluorescence using a ratio fluorescence spectrophotometer (system model C-14, Photon Technology International). The muscle was illuminated alternately at excitation wavelengths of 340 and 380 nm at a frequency of 2 Hz. Emitted light was collected through a single long-pass filter (510 nm) and detected with a photomultiplier tube. The ratio of fluorescence at 340 nm to fluorescence at 380 nm was continuously computed by a dedicated computer. Measurement of Myosin Light Chain (MLC) 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 were separated by glycerol-urea polyacrylamide gel electrophoresis, transferred to nitrocellulose, then immunoblotted for proteins of MLC (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar). The ratio of phosphorylated to unphosphorylated MLCs 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 an assay kit from Cytoskeleton (Denver, CO) as previously described (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) 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% Nonidet 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 using antiactin antibody (clone AC-40; Sigma). The ratios of F-actin to G-actin were determined using densitometry. Endogenous ILK and PINCH Are Recruited to the Cell Membrane during Contractile Stimulation of Smooth Muscle with ACh—The effect of stimulation with acetylcholine (ACh) on the cellular localization of endogenous ILK and PINCH was evaluated in smooth muscle cells freshly dissociated from intact tissues. Freshly dissociated cells were plated onto glass coverslips, stimulated with 10−4 m ACh or left unstimulated, then fixed and stained for immunofluorescence analysis of the distribution of ILK and PINCH (Fig. 1). Fig. 1 shows 4 optical sections from an unstimulated smooth muscle cell and from a stimulated smooth muscle cell, each double stained for ILK and PINCH. Optical sections are shown at the top, bottom, and 2 midsections of the cell. The distribution of ILK was evaluated in midsections of the cell, where a striking difference in the distribution of ILK and PINCH can be seen in unstimulated and ACh-stimulated cells. In cells that were stimulated with ACh, the fluorescence intensity of both ILK and PINCH along the cell membrane relative to the interior was more marked than in the unstimulated cell (Fig. 1A). The cellular distribution of ILK and PINCH was quantified using line scans across a mid-level optical section of each cell as previously described (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar, 16Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopus (108) Google Scholar). Examples of a single profile of pixel intensity from one line scan on each cell are shown in Fig. 1A. In unstimulated cells (n = 32), the ratio of the fluorescence intensity between the cell periphery to the cell interior for both ILK and PINCH was ∼1.3, indicating a higher fluorescence of both proteins at the membrane of the cell relative to the interior. In cells stimulated with ACh (n = 32), the fluorescence intensity ratio rose to about 3 for both ILK and PINCH, suggesting that contractile stimulation with ACh leads to the recruitment of endogenous ILK and PINCH to the membrane of smooth muscle cells. The Stimulation of Freshly Dissociated Tracheal Smooth Muscle Cells with ACh Causes the Rapid Recruitment of GFP-ILK and GFP-PINCH to the Cell Periphery—GFP-ILK and GFP-PINCH were expressed in tracheal smooth muscle tissues to monitor the localization of ILK and PINCH during contractile stimulation. Cells were then enzymatically dissociated from the tissues and evaluated by confocal fluorescence microscopy. As observed previously for other proteins, GFP-ILK or GFP-PINCH fluorescence was observed in ∼90% of the dissociated cells (9Zhang W. Wu Y. Du L. Tang D.D. Gunst S.J. Am. J. Physiol. 2005; 288: C1145-C1160Crossref PubMed Scopus (95) Google Scholar, 10Zhang W. Gunst S.J. J. Physiol. 2006; 572: 659-676Crossref PubMed Scopus (70) Google Scholar, 16Opazo-Saez A. Zhang W. Wu Y. Turner C.E. Tang D.D. Gunst S.J. Am. J. Physiol. 2004; 286: C433-C447Crossref Scopu" @default.
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