Journal articles on the topic 'Apha Smooth Muscle Actin'
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1
Puzdrova, V. A., T. V. Kudryashova, D. K. Gaynullina, S. V. Mochalov, C. Aalkjaer, H. Nilsson, A. V. Vorotnikov, R. Schubert, and O. S. Tarasova. "Trophic action of sympathetic nerves reduces arterial smooth muscle Ca2+sensitivity during early post-natal development in rats." Acta Physiologica 212, no. 2 (July 3, 2014): 128–41. http://dx.doi.org/10.1111/apha.12331.
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Gown, A. M., A. M. Vogel, D. Gordon, and P. L. Lu. "A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes." Journal of Cell Biology 100, no. 3 (March 1, 1985): 807–13. http://dx.doi.org/10.1083/jcb.100.3.807.
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Injection of chicken gizzard actin into BALB/c mice resulted in the isolation of a smooth muscle-specific monoclonal antibody designated CGA7. When assayed on methanol-Carnoy's fixed, paraffin-embedded tissue, it bound to smooth muscle cells and myoepithelial cells, but failed to decorate striated muscle, endothelium, connective tissue, epithelium, or nerve. CGA7 recognized microfilament bundles in early passage cultures of rat aortic smooth muscle cells and human leiomyosarcoma cells but did not react with human fibroblasts. In Western blot experiments, CGA7 detected actin from chicken gizzard and monkey ileum, but not skeletal muscle or fibroblast actin. Immunoblots performed on two-dimensional gels demonstrated that CGA7 recognizes gamma-actin from chicken gizzard and alpha- and gamma-actin from rat colon muscularis. This antibody was an excellent tissue-specific smooth muscle marker.
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Chistiakov, D. A., A. N. Orekhov, and Y. V. Bobryshev. "Vascular smooth muscle cell in atherosclerosis." Acta Physiologica 214, no. 1 (February 25, 2015): 33–50. http://dx.doi.org/10.1111/apha.12466.
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Wong, Jean Z., Janet Woodcock-Mitchell, John Mitchell, Patricia Rippetoe, Sheryl White, Marlene Absher, Linda Baldor, John Evans, Kirk M. McHugh, and Robert B. Low. "Smooth muscle actin and myosin expression in cultured airway smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 274, no. 5 (May 1, 1998): L786—L792. http://dx.doi.org/10.1152/ajplung.1998.274.5.l786.
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In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express α- and γ-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50–60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.
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Sulbarán, Guidenn, Lorenzo Alamo, Antonio Pinto, Gustavo Márquez, Franklin Méndez, Raúl Padrón, and Roger Craig. "An invertebrate smooth muscle with striated muscle myosin filaments." Proceedings of the National Academy of Sciences 112, no. 42 (October 6, 2015): E5660—E5668. http://dx.doi.org/10.1073/pnas.1513439112.
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Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components.
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Skalli, O., P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, and G. Gabbiani. "A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation." Journal of Cell Biology 103, no. 6 (December 1, 1986): 2787–96. http://dx.doi.org/10.1083/jcb.103.6.2787.
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A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.
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Conley, Catharine A. "Leiomodin and tropomodulin in smooth muscle." American Journal of Physiology-Cell Physiology 280, no. 6 (June 1, 2001): C1645—C1656. http://dx.doi.org/10.1152/ajpcell.2001.280.6.c1645.
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Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker α-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca2+, both SM-Lmod and Tmod are found near α-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.
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Reneman, Robert S. "Preface to ‘Electrical propagation in smooth muscle organs’." Acta Physiologica 213, no. 2 (December 19, 2014): 346. http://dx.doi.org/10.1111/apha.12433.
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Lammers, W. J., and G. J. van der Vusse. "Introduction to ‘Electrical propagation in smooth muscle organs’." Acta Physiologica 213, no. 2 (December 17, 2014): 347–48. http://dx.doi.org/10.1111/apha.12434.
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Hansson, G. K., M. Hellstrand, L. Rymo, L. Rubbia, and G. Gabbiani. "Interferon gamma inhibits both proliferation and expression of differentiation-specific alpha-smooth muscle actin in arterial smooth muscle cells." Journal of Experimental Medicine 170, no. 5 (November 1, 1989): 1595–608. http://dx.doi.org/10.1084/jem.170.5.1595.
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Differentiation of muscle cells is characterized morphologically by the acquisition of contractile filaments and characteristic shape changes, and on the molecular level by induction of the expression of several genes, including those for the muscle-specific alpha-actin isoforms. IFN-gamma is an inhibitor of proliferation for several cells, including vascular smooth muscle, and is also an inducer of differentiated properties for several hematopoietic cells. We have therefore investigated whether IFN-gamma affects the expression of alpha-smooth muscle actin in cultured arterial smooth muscle cells. Cells exposed to IFN-gamma show a reduction of alpha-smooth muscle actin-containing stress fibers, as detected by immunofluorescence. The effect was observed in all phases of the cell cycle, and was caused by a reduction of the synthesis of alpha-smooth muscle actin protein as revealed by two-dimensional electrophoretic analysis of actin isoforms. RNA hybridization using a cRNA probe that hybridizes to all actin mRNAs showed that IFN-gamma-treated cells have a reduced content of the 1.7-kb mRNA that codes for alpha-smooth muscle actin, and to a lesser extent, also of the 2.1-kb mRNA encoding the beta and gamma-cytoplasmic actins. The reduction of alpha-smooth muscle actin mRNA was confirmed using an alpha-smooth muscle actin-specific cRNA probe. The reduction of alpha-smooth muscle actin mRNA occurs within 12 h, and is dependent on protein synthesis, since cycloheximide treatment reversed the effect. The inhibition of this mRNA species was dose dependent, and detectable by RNA hybridization at a dose of 50 U/ml IFN-gamma. These results suggest that the differentiation of arterial smooth muscle cells is not necessarily coupled to an inhibition of cellular proliferation. Instead, IFN-gamma may regulate the expression of several genes that control both proliferation and expression of differentiation markers.
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Small, J. V., D. O. Fürst, and J. De Mey. "Localization of filamin in smooth muscle." Journal of Cell Biology 102, no. 1 (January 1, 1986): 210–20. http://dx.doi.org/10.1083/jcb.102.1.210.
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The distribution of contractile and cytoskeletal proteins in smooth muscle has been mapped by immunocytochemical methods, with special reference to the localization of the actin-binding protein, filamin. Immunolabeling of ultrathin sections of polyvinylalcohol-embedded smooth muscle distinguished two domains in the smooth muscle cell: (a) actomyosin domains, made up of continuous longitudinal arrays of actin and myosin filaments, and (b) longitudinal, fibrillar, intermediate filament domains, free of myosin but containing actin and alpha-actinin-rich dense bodies. Filamin was found to be localized specifically in the latter intermediate filament-actin domains, but was excluded from the core of the dense bodies. Filamin was also localized close to the cell border at the inner surface of the plasmalemma-associated plaques. In isolated cells the surface filamin label showed a rib-like distribution similar to that displayed by vinculin. It is speculated that the two domains distinguished in these studies may reflect the existence of two functionally distinct systems: an actomyosin system required for contraction and an intermediate filament-actin system, with associated gelation proteins, that is responsible, at least in part, for the slow relaxation and tone peculiar to smooth muscle.
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Gunst, Susan J., and Wenwu Zhang. "Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction." American Journal of Physiology-Cell Physiology 295, no. 3 (September 2008): C576—C587. http://dx.doi.org/10.1152/ajpcell.00253.2008.
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A growing body of data supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues. The increase in the proportion of filamentous actin that occurs in response to the stimulation of smooth muscle cells and the essential role of stimulus-induced actin polymerization and cytoskeletal dynamics in the generation of mechanical tension has been convincingly documented in many smooth muscle tissues and cells using a wide variety of experimental approaches. Most of the evidence suggests that the functional role of actin polymerization during contraction is distinct and separately regulated from the actomyosin cross-bridge cycling process. The molecular basis for the regulation of actin polymerization and its physiological roles may vary in diverse types of smooth muscle cells and tissues. However, current evidence supports a model for smooth muscle contraction in which contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins at the membrane, and proteins within this complex orchestrate the polymerization and organization of a submembranous network of actin filaments. This cytoskeletal network may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. Better understanding of the physiological function of these dynamic cytoskeletal processes in smooth muscle may provide important insights into the physiological regulation of smooth muscle tissues.
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Winder, S. J., C. Sutherland, and M. P. Walsh. "A comparison of the effects of calponin on smooth and skeletal muscle actomyosin systems in the presence and absence of caldesmon." Biochemical Journal 288, no. 3 (December 15, 1992): 733–39. http://dx.doi.org/10.1042/bj2880733.
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Thiosphosphorylated smooth muscle myosin and skeletal muscle myosin, both of which express Ca(2+)-independent actin-activated MgATPase activity, were used to examine the functional effects of calponin and caldesmon separately and together. Separately, calponin and caldesmon inhibited the actin-activated MgATPase activities of thiophosphorylated smooth muscle myosin and skeletal muscle myosin, calponin being significantly more potent in both systems. Calponin-mediated inhibition resulted from the interaction of calponin with actin since it could be reversed by increasing the actin concentration. Caldesmon had no significant influence on the calponin-induced inhibition of the smooth muscle actomyosin ATPase, nor did calponin have a significant effect on caldesmon-induced inhibition. In the skeletal muscle system, however, caldesmon was found to override the inhibitory effect of calponin. This difference probably reflects the lower affinity of skeletal muscle actin for calponin compared with that of smooth muscle actin. Calponin inhibition of skeletal muscle actin-activated myosin MgATPase was not significantly affected by troponin/tropomyosin, suggesting that the thin filament can readily accommodate calponin in addition to the troponin complex, or that calponin may be able to displace troponin. Calponin also inhibited acto-phosphorylated smooth muscle heavy meromyosin and acto-skeletal muscle heavy meromyosin MgATPases. The most appropriate protein preparations for analysis of the regulatory effects of calponin in the actomyosin system therefore would be smooth muscle actin, tropomyosin and thiophosphorylated myosin, and for analysis of the kinetic effects of calponin on the actomyosin ATPase cycle they would be smooth muscle actin, tropomyosin and phosphorylated heavy meromyosin, due to the latter's solubility.
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Lemiaa, Eissa, Mortada M. O. Elhassan, Rasha B. Yaseen, Hassan A. Ali, Haider I. Ismail, and M. C. Madekurozwa. "Immunolocalization of intermediate filaments in the kidney of the dromedary camel (Camelus dromedarius)." European Journal of Anatomy 26, no. 4 (July 2022): 387–97. http://dx.doi.org/10.52083/odpi9847.
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Intermediate filaments belong to a large family of proteins which contribute to the formation of the cytoskeleton. The immunolocalization of cytoskeletal proteins has been used extensively in the diagnosis of various renal pathologies. The present study described the immunolocalization of the cytoskeletal proteins vimentin, desmin, smooth muscle actin, and cytokeratin 19 in the normal kidney of the dromedary camel. Kidney samples from eight adult camels were processed for histology and immunohistochemistry. The kidney was enclosed in a renal capsule composed of vimentin immunoreactive fibroblasts and smooth muscle actin immunoreactive smooth muscle cells. The smooth muscle cells in the renal capsule did not exhibit desmin immunoreactivity. Podocytes forming the visceral layer of the glomerular capsule were immunoreactive for vimentin. Immunoreactivity for vimentin and smooth muscle actin in the parietal layer of the glomerular capsule varied, with both reactive and non-reactive cells observed. Intraglomerular mesangial cells were immunoreactive for smooth muscle actin and desmin, but non-reactive to vimentin. The endothelial lining of blood vessels was vimentin immunoreactive, while smooth muscle actin and desmin were demonstrated in the smooth muscle cells of the vessels. The thin limbs of the loops of Henle in cortical nephrons displayed vimentin immunoreactivity. The proximal and distal convoluted tubules, as well as the collecting ducts were negative to vimentin, smooth muscle actin, desmin and cytokeratin 19 immunostaining. In conclusion, the present study has revealed that similarities and differences exist in the immunolocalization of cytoskeletal proteins in the camel when compared to other mammals. The presence of smooth muscle actin in the parietal cells of the glomerular capsule suggests a contractile function of these cells. The results of the study indicate that vimentin and smooth muscle actin can be used as markers for the identification of podocytes and intraglomerular mesangial cells, respectively, in the camel kidney.
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Vrhovski, Bernadette, Karen McKay, Galina Schevzov, Peter W. Gunning, and Ron P. Weinberger. "Smooth Muscle-specific α Tropomyosin Is a Marker of Fully Differentiated Smooth Muscle in Lung." Journal of Histochemistry & Cytochemistry 53, no. 7 (July 2005): 875–83. http://dx.doi.org/10.1369/jhc.4a6504.2005.
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Tropomyosin (Tm) is one of the major components of smooth muscle. Currently it is impossible to easily distinguish the two major smooth muscle (sm) forms of Tm at a protein level by immunohistochemistry due to lack of specific antibodies. α-sm Tm contains a unique 2a exon not found in any other Tm. We have produced a polyclonal antibody to this exon that specifically detects α-sm Tm. We demonstrate here the utility of this antibody for the study of smooth muscle. The tissue distribution of α-sm Tm was shown to be highly specific to smooth muscle. α-sm Tm showed an identical profile and tissue colocalization with α-sm actin both by Western blotting and immunohistochemistry. Using lung as a model organ system, we examined the developmental appearance of α-sm Tm in comparison to α-sm actin in both the mouse and human. α-sm Tm is a late-onset protein, appearing much later than actin in both species. There were some differences in onset of appearance in vascular and airway smooth muscle with airway appearing earlier. α-sm Tm can therefore be used as a good marker of mature differentiated smooth muscle cells. Along with α-sm actin and sm-myosin antibodies, α-sm Tm is a valuable tool for the study of smooth muscle.
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Goldsmith, Adam M., Marc B. Hershenson, Miguel P. Wolbert, and J. Kelley Bentley. "Regulation of airway smooth muscle α-actin expression by glucocorticoids." American Journal of Physiology-Lung Cellular and Molecular Physiology 292, no. 1 (January 2007): L99—L106. http://dx.doi.org/10.1152/ajplung.00269.2006.
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Airway smooth muscle hypertrophy appears to be present in severe asthma. However, the effect of corticosteroids on airway smooth muscle cell size or contractile protein expression has not been studied. We examined the effects of dexamethasone, fluticasone, and salmeterol on contractile protein expression in transforming growth factor (TGF)-β-treated primary bronchial smooth muscle cells. Dexamethasone and fluticasone, but not salmeterol, each reduced expression of α-smooth muscle actin and the short isoform of myosin light chain kinase. Steady-state α-actin mRNA level and stability were unchanged, consistent with posttranscriptional control. Fluticasone significantly decreased α-actin protein synthesis following treatment with the transcriptional inhibitor actinomycin D, indicative of an inhibitory effect on mRNA translation. Fluticasone also significantly increased α-actin protein turnover. Finally, fluticasone reduced TGF-β-induced incorporation of α-actin into filamentous actin, cell length, and cell shortening in response to ACh and KCl. We conclude that glucocorticoids reduce human airway smooth muscle α-smooth muscle actin expression and incorporation into contractile filaments, as well as contractile function, in part by attenuation of mRNA translation and enhancement of protein degradation.
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Ruzicka, D. L., and R. J. Schwartz. "Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2575–86. http://dx.doi.org/10.1083/jcb.107.6.2575.
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The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger-Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha-actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells.
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Park, Frank, David L. Mattson, Lou A. Roberts, and Allen W. Cowley. "Evidence for the presence of smooth muscle α-actin within pericytes of the renal medulla." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 273, no. 5 (November 1, 1997): R1742—R1748. http://dx.doi.org/10.1152/ajpregu.1997.273.5.r1742.
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This study was designed to determine whether smooth muscle α-actin mRNA and smooth muscle α-actin contractile protein elements were present within the renal medullary pericytes. Extraction of total RNA from microdissected outer medullary descending vasa recta allowed for the detection of smooth muscle α-actin mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR). Expression of smooth muscle α-actin was specific to the descending vasa recta and not a result of tubular contamination because RT-PCR amplification of the vasopressin V2 receptor, which is a specific tubular marker, did not occur. To determine the exact cell type(s) that translate the mRNA into protein, we performed immunohistochemistry on the renal outer and inner medulla using a monoclonal smooth muscle α-actin antibody, whose specificity was determined by immunoblot analysis. Smooth muscle α-actin protein was found selectively within the pericytes surrounding the descending vasa recta from the outer and inner medullary tissue sections. This study demonstrates that the pericytes alone that surround the descending vasa recta within the outer and inner medulla contain smooth muscle α-actin mRNA and protein and are therefore the site of the contractile elements that could play a vasomodulatory role in the control of renal medullary blood flow and its distribution within the renal medulla.
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Hilbert, Lennart, Jenna L. Blumenthal, Genevieve Bates, Horia N. Roman, Nedjma B. Zitouni, Michael C. Mackey, and Anne-Marie Lauzon. "Molecular Mechanical differences between Skeletal Muscle α-Actin and Smooth Muscle γ-Actin in the Presence of Smooth Muscle Tropomyosin." Biophysical Journal 104, no. 2 (January 2013): 480a. http://dx.doi.org/10.1016/j.bpj.2012.11.2653.
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Chen, Xuesong, Kristin Pavlish, and Joseph N. Benoit. "Myosin phosphorylation triggers actin polymerization in vascular smooth muscle." American Journal of Physiology-Heart and Circulatory Physiology 295, no. 5 (November 2008): H2172—H2177. http://dx.doi.org/10.1152/ajpheart.91437.2007.
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A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or β-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in β-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.
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Tang, Dale D., and Susan J. Gunst. "The Small GTPase Cdc42 Regulates Actin Polymerization and Tension Development during Contractile Stimulation of Smooth Muscle." Journal of Biological Chemistry 279, no. 50 (September 27, 2004): 51722–28. http://dx.doi.org/10.1074/jbc.m408351200.
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Contractile stimulation induces actin polymerization in smooth muscle tissues and cells, and the inhibition of actin polymerization depresses smooth muscle force development. In the present study, the role of Cdc42 in the regulation of actin polymerization and tension development in smooth muscle was evaluated. Acetylcholine stimulation of tracheal smooth muscle tissues increased the activation of Cdc42. Plasmids encoding wild type Cdc42 or a dominant negative Cdc42 mutant, Asn-17 Cdc42, were introduced into tracheal smooth muscle strips by reversible permeabilization, and tissues were incubated for 2 days to allow for protein expression. Expression of recombinant proteins was confirmed by immunoblot analysis. The expression of the dominant negative Cdc42 mutant inhibited contractile force and the increase in actin polymerization in response to acetylcholine stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type Cdc42 had no significant effect on force, actin polymerization, or myosin light chain phosphorylation. Contractile stimulation increased the association of neuronal Wiskott-Aldrich syndrome protein with Cdc42 and the Arp2/3 (actin-related protein) complex in smooth muscle tissues expressing wild type Cdc42. The agonist-induced increase in these protein interactions was inhibited in tissues expressing the inactive Cdc42 mutant. We conclude that Cdc42 activation regulates active tension development and actin polymerization during contractile stimulation. Cdc42 may regulate the activation of neuronal Wiskott-Aldrich syndrome protein and the actin related protein complex, which in turn regulate actin filament polymerization initiated by the contractile stimulation of smooth muscle.
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Jansen, Sepp R., Anna M. Van Ziel, Hoeke A. Baarsma, and Reinoud Gosens. "β-Catenin regulates airway smooth muscle contraction." American Journal of Physiology-Lung Cellular and Molecular Physiology 299, no. 2 (August 2010): L204—L214. http://dx.doi.org/10.1152/ajplung.00020.2010.
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β-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for β-catenin in the regulation of smooth muscle force production. β-Catenin colocalized with smooth muscle α-actin (sm-α-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-α-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-α-actin and β-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological β-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced β-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-α-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of β-catenin in BTSM strips reduced β-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-α-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of β-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that β-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.
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Kim, Hak Rim, Cynthia Gallant, Paul C. Leavis, Susan J. Gunst, and Kathleen G. Morgan. "Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent." American Journal of Physiology-Cell Physiology 295, no. 3 (September 2008): C768—C778. http://dx.doi.org/10.1152/ajpcell.00174.2008.
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Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an α-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses α-smooth muscle actin, β-actin, nonmuscle γ-actin, and smooth muscle γ-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during α-agonist contractions involves the remodeling of primarily γ-actin and, to a lesser extent, β-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.
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Blank, R. S., M. M. Thompson, and G. K. Owens. "Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells." Journal of Cell Biology 107, no. 1 (July 1, 1988): 299–306. http://dx.doi.org/10.1083/jcb.107.1.299.
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Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.
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Sawtell, N. M., and J. L. Lessard. "Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma-enteric isoform in differentiating striated myocytes." Journal of Cell Biology 109, no. 6 (December 1, 1989): 2929–37. http://dx.doi.org/10.1083/jcb.109.6.2929.
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The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha-vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.
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LaRock, R. G., and P. E. Ginn. "Immunohistochemical Staining Characteristics of Canine Gastrointestinal Stromal Tumors." Veterinary Pathology 34, no. 4 (July 1997): 303–11. http://dx.doi.org/10.1177/030098589703400406.
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Sections from 35 formalin-fixed, paraffin-embedded, canine gastrointestinal stromal tumors consisting of 14 leiomyomas (five stomach, three small intestine, two colon, four rectum), 18 leiomyosarcomas (one stomach, five small intestine, nine cecum, three rectum), two undifferentiated sarcomas (two stomach), and one neurofibrosarcoma (small intestine) were examined for the expression of vimentin, S-100 protein, α-smooth muscle actin, and desmin via immunoperoxidase methodology using an avidin-biotin complex technique. The leiomyomas were 4/14 (29%) vimentin-positive, 3/14 (21%) S-100 protein-positive, 10/14 (71%) α-smooth muscle actin-positive and 13/14 (93%) desmin-positive. Leiomyosarcomas were 18/18 (100%) vimentin-positive, 11/18 (61%) S-100 protein-positive, 9/18 (50%) α-smooth muscle actin-positive, and 15/18 (83%) desmin-positive. The undifferentiated sarcomas were 2/2 (100%) vimentin-positive, 2/2 (100%) S-100 protein-positive, 1/2 (50%) α-smooth muscle actin-positive, and 0/2 (0%) desmin-positive. The neurofibrosarcoma was vimentin and S-100 protein-positive and α-smooth muscle actin- and desmin-negative. Thirty-one of thirty-five (89%) of all neoplasms demonstrated reactivity for either desmin and/or α-smooth muscle actin. S-100 protein reactivity occurred in 17/35 (49%) of all specimens. Lack of desmin and α-smooth muscle actin reactivity occurred in 4/35 (11%) of all specimens, all of which were vimentin-positive. The immunohistochemical results indicate that the majority of canine gastrointestinal stromal tumors (GIST) with light microscopic features of smooth muscle cells have immunohistochemical staining patterns supporting smooth muscle differentiation. Vimentin reactivity correlated with a light microscopic diagnosis of malignancy. The lack of smooth muscle cell markers in some tumors and the high percentage of cases positive for S-100 protein may suggest a more complex histogenesis or differentiation for subgroups of these tumors.
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Gerthoffer, William T. "Actin cytoskeletal dynamics in smooth muscle contraction." Canadian Journal of Physiology and Pharmacology 83, no. 10 (October 1, 2005): 851–56. http://dx.doi.org/10.1139/y05-088.
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Smooth muscles develop isometric force over a very wide range of cell lengths. The molecular mechanisms of this phenomenon are undefined, but are described as reflecting "mechanical plasticity" of smooth muscle cells. Plasticity is defined here as a persistent change in cell structure or function in response to a change in the environment. Important environmental stimuli that trigger muscle plasticity include chemical (e.g., neurotransmitters, autacoids, and cytokines) and external mechanical signals (e.g., applied stress and strain). Both kinds of signals are probably transduced by ionic and protein kinase signaling cascades to alter gene expression patterns and changes in the cytoskeleton and contractile system. Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plasticity of smooth muscles is a major goal in muscle cell biology. Some of the signaling cascades likely to be important include calcium-dependent protein kinases, small GTPases (Rho, Rac, cdc42), Rho kinase, protein kinase C (PKC), Src family tyrosine kinases, mitogen-activated protein (MAP) kinases, and p21 activated protein kinases (PAK). There are many potential targets for these signaling cascades including nuclear processes, metabolic pathways, and structural components of the cytoskeleton. There is growing appreciation of the dynamic nature of the actin cytoskeleton in smooth muscles and the necessity for actin remodeling to occur during contraction. The actin cytoskeleton serves many functions that are probably critical for muscle plasticity including generation and transmission of force vectors, determination of cell shape, and assembly of signal transduction machinery. Evidence is presented showing that actin filaments are dynamic and that actin-associated proteins comprising the contractile element and actin attachment sites are necessary for smooth muscle contraction.Key words: integrin, muscle mechanics, paxillin, Rho, HSP27.
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Tazzeo, Tracy, Genevieve Bates, Horia Nicolae Roman, Anne-Marie Lauzon, Mukta D. Khasnis, Masumi Eto, and Luke J. Janssen. "Caffeine relaxes smooth muscle through actin depolymerization." American Journal of Physiology-Lung Cellular and Molecular Physiology 303, no. 4 (August 15, 2012): L334—L342. http://dx.doi.org/10.1152/ajplung.00103.2012.
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Caffeine is sometimes used in cell physiological studies to release internally stored Ca2+. We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca2+ store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca2+, suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.
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Soares, A., R. Cunha, F. Rodrigues, and H. Ribeiro. "Smooth muscle autoantibodies with F-actin specificity." Autoimmunity Reviews 8, no. 8 (July 2009): 713–16. http://dx.doi.org/10.1016/j.autrev.2009.02.023.
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Carmichael, Jeffrey D., Steven J. Winder, Michael P. Walsh, and Gary J. Kargacin. "Calponin and smooth muscle regulation." Canadian Journal of Physiology and Pharmacology 72, no. 11 (November 1, 1994): 1415–19. http://dx.doi.org/10.1139/y94-204.
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Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation–dephosphorylation plays a role in regulating smooth muscle contraction.Key words: calponin, smooth muscle, confocal microscopy, phosphorylation–dephosphorylation.
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Damon, Deborah H. "Sympathetic innervation promotes vascular smooth muscle differentiation." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 6 (June 2005): H2785—H2791. http://dx.doi.org/10.1152/ajpheart.00354.2004.
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The sympathetic nervous system (SNS) is an important modulator of vascular smooth muscle (VSM) growth and function. Several lines of evidence suggest that the SNS also promotes VSM differentiation. The present study tests this hypothesis. Expression of smooth muscle myosin (SM2) and α-actin were assessed by Western analysis as indexes of VSM differentiation. SM2 expression (normalized to α-actin) in adult innervated rat femoral and tail arteries was 479 ± 115% of that in noninnervated carotid arteries. Expression of α-actin (normalized to GAPDH or total protein) in 30-day-innervated rat femoral arteries was greater than in corresponding noninnervated femoral arteries from guanethidine-sympathectomized rats. SM2 expression (normalized to α-actin) in neonatal femoral arteries grown in vitro for 7 days in the presence of sympathetic ganglia was greater than SM2 expression in corresponding arteries grown in the absence of sympathetic ganglia. In VSM-endothelial cell cultures grown in the presence of dissociated sympathetic neurons, α-actin (normalized to GAPDH) was 300 ± 66% of that in corresponding cultures grown in the absence of neurons. This effect was inhibited by an antibody that neutralized the activity of transforming growth factor-β2. All of these data indicate that sympathetic innervation increased VSM contractile protein expression and thereby suggest that the SNS promotes and/or maintains VSM differentiation.
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An, Steven S., Rachel E. Laudadio, Jean Lai, Rick A. Rogers, and Jeffrey J. Fredberg. "Stiffness changes in cultured airway smooth muscle cells." American Journal of Physiology-Cell Physiology 283, no. 3 (September 1, 2002): C792—C801. http://dx.doi.org/10.1152/ajpcell.00425.2001.
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Airway smooth muscle (ASM) cells in culture stiffen when exposed to contractile agonists. Such cell stiffening may reflect activation of the contractile apparatus as well as polymerization of cytoskeletal biopolymers. Here we have assessed the relative contribution of these mechanisms in cultured ASM cells stimulated with serotonin (5-hydroxytryptamine; 5-HT) in the presence or absence of drugs that inhibit either myosin-based contraction or polymerization of filamentous (F) actin. Magnetic twisting cytometry was used to measure cell stiffness, and associated changes in structural organization of actin cytoskeleton were evaluated by confocal microscopy. We found that 5-HT increased cell stiffness in a dose-dependent fashion and also elicited rapid formation of F-actin as marked by increased intensity of FITC-phalloidin staining in these cells. A calmodulin antagonist (W-7), a myosin light chain kinase inhibitor (ML-7) and a myosin ATPase inhibitor (BDM) each ablated the stiffening response but not the F-actin polymerization induced by 5-HT. Agents that inhibited the formation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, and Y-27632) attenuated both baseline stiffness and the extent of cell stiffening in response to 5-HT. Together, these data suggest that agonist-evoked stiffening of cultured ASM cells requires actin polymerization as well as myosin activation and that neither actin polymerization nor myosin activation by itself is sufficient to account for the cell stiffening response.
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Gao, Hong, Marlene C. Steffen, and Kenneth S. Ramos. "Osteopontin regulates α-smooth muscle actin and calponin in vascular smooth muscle cells." Cell Biology International 36, no. 2 (December 21, 2011): 155–61. http://dx.doi.org/10.1042/cbi20100240.
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Jones, Rosemary, Margaretha Jacobson, and Wolfgang Steudel. "α -Smooth-Muscle Actin and Microvascular Precursor Smooth-Muscle Cells in Pulmonary Hypertension." American Journal of Respiratory Cell and Molecular Biology 20, no. 4 (April 1999): 582–94. http://dx.doi.org/10.1165/ajrcmb.20.4.3357.
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Saga, Hiroshi, Kazuhiro Kimura, Ken'ichiro Hayashi, Takahiro Gotow, Yasuo Uchiyama, Takuya Momiyama, Satoko Tadokoro, Nozomu Kawashima, Akie Jimbou, and Kenji Sobue. "Phenotype-Dependent Expression of α-Smooth Muscle Actin in Visceral Smooth Muscle Cells." Experimental Cell Research 247, no. 1 (February 1999): 279–92. http://dx.doi.org/10.1006/excr.1998.4339.
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Ngai, P. K., and M. P. Walsh. "The effects of phosphorylation of smooth-muscle caldesmon." Biochemical Journal 244, no. 2 (June 1, 1987): 417–25. http://dx.doi.org/10.1042/bj2440417.
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Caldesmon is a major calmodulin- and actin-binding protein of smooth muscle which interacts with calmodulin in a Ca2+-dependent manner or with actin in a Ca2+-independent manner. Isolated caldesmon is capable of inhibiting the actin-activated Mg2+-ATPase of smooth-muscle myosin, suggesting a possible physiological role for caldesmon in regulating the contractile state of smooth-muscle. Caldesmon can be phosphorylated in vitro by a co-purifying Ca2+/calmodulin-dependent protein kinase and dephosphorylated by a protein phosphatase, both of which are present in smooth muscle. We investigated further the phosphorylation of caldesmon and the effects which phosphorylation has on the functional properties of the protein. The kinetics of caldesmon phosphorylation were similar whether the caldesmon substrate was free or bound to actin, actin/tropomyosin or thin filaments. Caldesmon containing endogenous kinase activity was rapidly phosphorylated (to approx. 1 mol of Pi/mol of caldesmon in 5 min) when reconstituted with actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase in the presence of Ca2+ and MgATP2-. Under conditions in which unphosphorylated caldesmon showed substantial inhibition of the actin-activated myosin Mg2+-ATPase, no inhibition was observed with phosphorylated caldesmon. This was the case whether caldesmon was phosphorylated before addition to the actomyosin Mg2+-ATPase system, or phosphorylation was allowed to take place during the ATPase reaction. Binding studies revealed maximal binding of 1 mol of unphosphorylated caldesmon/9.5 mol of actin and 1 mol of phosphorylated caldesmon/11.7 mol of actin. All the bound phosphorylated caldesmon could be released by Ca2+/calmodulin, with half-maximal release at 0.11 microM-Ca2+, whereas only 62% of the bound unphosphorylated caldesmon could be removed, with half-maximal release at 0.16 microM-Ca2+. However, under conditions in which inhibition of actomyosin Mg2+-ATPase activity by non-phosphorylated but not by phosphorylated caldesmon was observed, both forms of caldesmon would remain bound to the thin filament. These observations suggest a possible mechanism whereby caldesmon phosphorylation may prevent its inhibitory action on the actomyosin Mg2+-ATPase.
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Tang, Dale D., and Jian Tan. "Downregulation of profilin with antisense oligodeoxynucleotides inhibits force development during stimulation of smooth muscle." American Journal of Physiology-Heart and Circulatory Physiology 285, no. 4 (October 2003): H1528—H1536. http://dx.doi.org/10.1152/ajpheart.00188.2003.
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The actin-regulatory protein profilin has been shown to regulate the actin cytoskeleton and the motility of nonmuscle cells. To test the hypothesis that profilin plays a role in regulating smooth muscle contraction, profilin antisense or sense oligodeoxynucleotides were introduced into the canine carotid smooth muscle by a method of reversible permeabilization, and these strips were incubated for 2 days for protein downregulation. The treatment of smooth muscle strips with profilin antisense oligodeoxynucleotides inhibited the expression of profilin; it did not influence the expression of actin, myosin heavy chain, and metavinculin/vinculin. Profilin sense did not affect the expression of these proteins in smooth muscle tissues. Force generation in response to stimulation with norepinephrine or KCl was significantly lower in profilin antisense-treated muscle strips than in profilin sense-treated strips or in muscle strips not treated with oligodeoxynucleotides. The depletion of profilin did not attenuate increases in phosphorylation of the 20-kDa regulatory light chain of myosin (MLC20) in response to stimulation with norepinephrine or KCl. The increase in F-actin/G-actin ratio during contractile stimulation was significantly inhibited in profilin-deficient smooth muscle strips. These results suggest that profilin is a necessary molecule of signaling cascades that regulate carotid smooth muscle contraction, but that it does not modulate MLC20 phosphorylation during contractile stimulation. Profilin may play a role in the regulation of actin polymerization or organization in response to contractile stimulation of smooth muscle.
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Sasaki, Y., Y. Sasaki, K. Kanno, and H. Hidaka. "Disorganization by calcium antagonists of actin microfilament in aortic smooth muscle cells." American Journal of Physiology-Cell Physiology 253, no. 1 (July 1, 1987): C71—C78. http://dx.doi.org/10.1152/ajpcell.1987.253.1.c71.
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To assess the physiological role of intracellular Ca2+ in the organization of actin microfilaments in smooth muscle cells, we employed several types of Ca2+ antagonists. The rabbit aortic smooth muscle cells treated with the putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB 8) at 5-100 microM showed a loss or a decrease in size and length of the actin-containing microfilament structure in a dose-dependent manner. Similar disorganization of actin structure was observed in the smooth muscle cells treated with 1-(5-isoquinolinesulfonyl)-homopiperazine (HA 1077) at 5-100 microM, which is a new type of Ca2+ antagonist different from Ca2+ entry blocker. In contrast, 100 microM verapamil and diltiazem induced no reorganization of the actin microfilament structure. Antimycin A decreased the ATP levels in smooth muscle cells and disorganized the actin-containing structure. Unlike antimycin A, TMB 8 and HA 1077 did not lower the ATP level below the threshold needed to maintain the actin filament structure. Both TMB 8 and HA 1077 directly interacted with neither the actin monomer nor F-actin in a viscometrical assay system. Thus these reagents may induce the disorganization of actin microfilament structure in smooth muscle cells through the indirect reaction(s) with the actin, suggesting that an appropriate level of ATP and Ca2+ and/or its involving reactions may be essential for maintenance of the actin structure.
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North, A. J., M. Gimona, Z. Lando, and J. V. Small. "Actin isoform compartments in chicken gizzard smooth muscle cells." Journal of Cell Science 107, no. 3 (March 1, 1994): 445–55. http://dx.doi.org/10.1242/jcs.107.3.445.
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Differentiated smooth muscle cells typically contain a mixture of muscle (alpha and gamma) and cytoplasmic (beta and gamma) actin isoforms. Of the cytoplasmic actins the beta-isoform is the more dominant, making up from 10% to 30% of the total actin complement. Employing an antibody raised against the N-terminal peptide specific to beta-actin, which labels only the beta-isoform on two-dimensional gel immunoblots, we have shown that this isoform has a restricted localisation in smooth muscle. Using double-label immunofluorescence and immunoelectron microscopy of ultrathin sections of chicken gizzard, beta-actin was localised in the dense bodies and in longitudinal channels linking consecutive dense bodies that were also occupied by desmin. It was additionally found in the membrane-associated dense plaques, but was excluded from the actomyosin-containing regions of the contractile apparatus. Taken together with earlier results these findings identify a cytoskeletal compartment containing intermediate filaments, cytoplasmic actin and the actin cross-linking protein filamin. Using an antibody specific only for muscle actin, labelling was found generally around the myosin filaments of the contractile apparatus, but was absent from the core of the dense bodies that contained beta-actin. Thus, if dense bodies act as dual-purpose anchorage sites, for the cytoskeletal actin and the contractile actin, the thin filaments of the contractile apparatus must be anchored at the periphery of the dense bodies. A model of the structural organisation of the cell is presented and the possible roles of the cytoskeleton are discussed.
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Pritchard, K., and S. B. Marston. "Ca2+-dependent regulation of vascular smooth-muscle caldesmon by S.100 and related smooth-muscle proteins." Biochemical Journal 277, no. 3 (August 1, 1991): 819–24. http://dx.doi.org/10.1042/bj2770819.
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1. We have investigated the ability of bovine brain S.100, and of three related proteins from sheep aorta smooth muscle, to confer Ca(2+)-sensitivity on thin filaments reconstituted from smooth-muscle actin, tropomyosin and caldesmon. 2. At 37 degrees C in pH 7.0 buffer containing 120 mM-KCl, approximately stoichiometric amounts of S.100 reversed caldesmon's inhibition of the activation of myosin MgATPase by smooth-muscle actin-tropomyosin. The [S.100] which reversed by 50% the inhibition by caldesmon (the E.C.50) was 2.5 microM when [caldesmon] = 2-3 microM in the assay mixture. When [KCl] was decreased to 70 mM, E.C.50 = 11.5 microM; at 25 degrees C in 70 mM-KCl, up to 20 microM-S.100 had no effect. When skeletal-muscle actin rather than smooth-muscle actin was used to reconstitute thin filaments, 20 microM-S.100 did reverse inhibition by caldesmon, at 25 degrees C in buffer containing 70 mM-KCl. This dependence on conditions is also characteristic of the calmodulin-caldesmon interaction. 3. These results suggested that S.100 or a related protein might interact with caldesmon in smooth muscle. We therefore attempted to prepare such a protein from sheep aorta. Three proteins were purified: an Mr-17,000 protein (yield 16 mg/kg), an abundant Mr-11,000 protein (yield 48 mg/kg), and an Mr-9000 protein (yield 4 mg/kg). Neither of the last two low-Mr proteins had any effect on activation of myosin MgATPase by reconstituted thin filaments. The protein of Mr 17,000 had Ca(2+)-sensitizing activity, and behaved exactly like brain calmodulin in the assay system.
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Dogan, Murat, Young-Soo Han, Philippe Delmotte, and Gary C. Sieck. "TNFα enhances force generation in airway smooth muscle." American Journal of Physiology-Lung Cellular and Molecular Physiology 312, no. 6 (June 1, 2017): L994—L1002. http://dx.doi.org/10.1152/ajplung.00550.2016.
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Airway inflammation is a hallmark of asthma, triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration ([Ca2+]cyt) and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling, and/or changes in contractile protein content. We hypothesized that 24 h of exposure to TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to 4.0) in control and TNFα-treated groups. Relative phosphorylation of the regulatory myosin light chain (p-MLC) and total actin, MLC, and MyHC concentrations were quantified at pCa 9.0, 6.1, and 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and 4.0. For determination of total cross-bridge formation, isometric ATP hydrolysis rate at pCa 4.0 was measured using an enzyme-coupled NADH-linked fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC, and MyHC content, as well as an increase in actin polymerization and an increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation.
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Shimizu, Richard T., Randal S. Blank, Ramiro Jervis, Susan C. Lawrenz-Smith, and Gary K. Owens. "The Smooth Muscle α-Actin Gene Promoter Is Differentially Regulated in Smooth MuscleversusNon-smooth Muscle Cells." Journal of Biological Chemistry 270, no. 13 (March 31, 1995): 7631–43. http://dx.doi.org/10.1074/jbc.270.13.7631.
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Carroll, S. L., D. J. Bergsma, and R. J. Schwartz. "A 29-nucleotide DNA segment containing an evolutionarily conserved motif is required in cis for cell-type-restricted repression of the chicken alpha-smooth muscle actin gene core promoter." Molecular and Cellular Biology 8, no. 1 (January 1988): 241–50. http://dx.doi.org/10.1128/mcb.8.1.241-250.1988.
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A series of 5' deletion mutations of the upstream flanking sequences of the chicken alpha-smooth muscle (aortic) actin gene was prepared and inserted into the chloramphenicol acetyltransferase expression vector pSV0CAT. Deletion recombinants were transfected into fibroblasts, which actively express the alpha-smooth muscle actin gene, and primary myoblast cultures, which accumulate much lower quantities of alpha-smooth muscle actin mRNAs. The first 122 nucleotides of 5'-flanking DNA were found to contain a "core" promoter capable of accurately directing high levels of transcription in both fibroblasts and myotubes. The activity of this core promoter is modulated in fibroblasts by a "governor" element(s) located at least in part between nucleotides -257 and -123. This region contains sequences potentially conserved between mammalian and avian alpha-smooth muscle actin genes as well as one of a pair of 16-base-pair inverted CCAAT box-associated repeats which are conserved among all chordate muscle actin genes examined to date. A smaller DNA segment (-151 to -123) containing these upstream CCAAT box-associated repeats was sufficient to suppress expression of the core promoter in muscle cultures, suggesting that the upstream CCAAT box-associated repeats play a negative role in the alpha-smooth muscle actin gene promoter.
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Carroll, S. L., D. J. Bergsma, and R. J. Schwartz. "A 29-nucleotide DNA segment containing an evolutionarily conserved motif is required in cis for cell-type-restricted repression of the chicken alpha-smooth muscle actin gene core promoter." Molecular and Cellular Biology 8, no. 1 (January 1988): 241–50. http://dx.doi.org/10.1128/mcb.8.1.241.
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A series of 5' deletion mutations of the upstream flanking sequences of the chicken alpha-smooth muscle (aortic) actin gene was prepared and inserted into the chloramphenicol acetyltransferase expression vector pSV0CAT. Deletion recombinants were transfected into fibroblasts, which actively express the alpha-smooth muscle actin gene, and primary myoblast cultures, which accumulate much lower quantities of alpha-smooth muscle actin mRNAs. The first 122 nucleotides of 5'-flanking DNA were found to contain a "core" promoter capable of accurately directing high levels of transcription in both fibroblasts and myotubes. The activity of this core promoter is modulated in fibroblasts by a "governor" element(s) located at least in part between nucleotides -257 and -123. This region contains sequences potentially conserved between mammalian and avian alpha-smooth muscle actin genes as well as one of a pair of 16-base-pair inverted CCAAT box-associated repeats which are conserved among all chordate muscle actin genes examined to date. A smaller DNA segment (-151 to -123) containing these upstream CCAAT box-associated repeats was sufficient to suppress expression of the core promoter in muscle cultures, suggesting that the upstream CCAAT box-associated repeats play a negative role in the alpha-smooth muscle actin gene promoter.
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Dugina, Vera, Lionel Fontao, Christine Chaponnier, Jury Vasiliev, and Giulio Gabbiani. "Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors." Journal of Cell Science 114, no. 18 (September 15, 2001): 3285–96. http://dx.doi.org/10.1242/jcs.114.18.3285.
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Transforming growth factor β (TGFβ), the most established promoter of myofibroblast differentiation, induces ED-A cellular fibronectin and α-smooth muscle actin expression in fibroblastic cells in vivo and in vitro. ED-A fibronectin exerts a permissive action for α-smooth muscle actin expression. A morphological continuity (called fibronexus), a specialized form of focal adhesion, has been described between actin stress fibers that contain α-smooth muscle actin, and extracellular fibronectin, which contains the ED-A portion, in both cultured fibroblasts and granulation tissue myofibroblasts. We have studied the development of these focal adhesions in TGFβ-treated fibroblasts using confocal laser scanning microscopy, three-dimensional image reconstruction and western blots using antibodies against focal adhesion proteins. The increase in ED-A fibronectin expression induced by TGFβ was accompanied by bundling of ED-A fibronectin fibers and their association with the terminal portion of α-smooth muscle actin-positive stress fibers. In parallel, the focal adhesion size was importantly increased, and tensin and FAK were neoexpressed in focal adhesions; moreover, vinculin and paxillin were recruited from the cytoplasmic pool into focal adhesions. We have evaluated morphometrically the length and area of focal adhesions. In addition, we have evaluated biochemically their content of associated proteins and of α-smooth muscle actin after TGFβ stimulation and on this basis suggest a new focal adhesion classification, that is, immature, mature and supermature.When TGFβ-induced α-smooth muscle actin expression was blocked by soluble recombinant ED-A fibronectin, we observed that the fragment was localised into the fibronectin network at the level of focal adhesions and that focal adhesion supermaturation was inhibited. The same effect was also exerted by the ED-A fibronectin antibody IST-9. In addition, the antagonists of actin-myosin contractility BDM and ML-7 provoked the dispersion of focal adhesions and the decrease of α-smooth muscle actin content in stress fibers of pulmonary fibroblasts, which constitutively show large focal adhesions and numerous stress fibers that contain α-smooth muscle actin. These inhibitors also decreased the incorporation of recombinant ED-A into fibronectin network. Our data indicate that a three-dimensional transcellular structure containing both ED-A fibronectin and α-smooth muscle actin plays an important role in the establishment and modulation of the myofibroblastic phenotype. The organisation of this structure is regulated by intracellularly and extracellularly originated forces.
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Lai, M., D. B. Thomason, and N. W. Weisbrodt. "Effect of intestinal bypass on the expression of actin mRNA in ileal smooth muscle." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 258, no. 1 (January 1, 1990): R39—R43. http://dx.doi.org/10.1152/ajpregu.1990.258.1.r39.
Full textAbstract:
In this study, messenger RNAs (mRNAs) for actin isoforms were assessed in longitudinal smooth muscle from the ileum of unoperated rats and from rats that had undergone bypass of the middle 70% of the small intestine. The plasmid clone pGEM 10C, which contains a DNA insert complementary to the 3' untranslated region and the region of mRNA that codes for the synthesis of alpha-smooth muscle actin protein, was used to synthesize two riboprobes. One probe, complementary to the coding region of the insert, hybridizes to most, if not all, actin isoform mRNAs. The second probe, complementary to the 3' untranslated region of the insert, hybridizes only to alpha-smooth muscle actin mRNA. RNA was isolated from animals 4 to 5 days after operation, size fractionated by denaturing gel electrophoresis, transferred to nylon membranes, and exposed to the two 32P-labeled riboprobes. Both probes hybridized to RNA of about 1.3 kilobases long. Longitudinal muscle from both groups of animals contained alpha-smooth muscle actin mRNA as well as mRNA for other actin isoforms. Dot blots of varying amounts of RNA were hybridized to the riboprobes to determine the proportions of actin mRNAs. The content and concentration of mRNAs for all actins, and of mRNA for alpha-smooth muscle actin, were significantly greater in muscle from the functioning ileum of bypassed animals 4-5 days after the operation. Thus the operation induces a rapid, specific activation of these contractile protein genes.
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Walsh, M. P., J. D. Carmichael, and G. J. Kargacin. "Characterization and confocal imaging of calponin in gastrointestinal smooth muscle." American Journal of Physiology-Cell Physiology 265, no. 5 (November 1, 1993): C1371—C1378. http://dx.doi.org/10.1152/ajpcell.1993.265.5.c1371.
Full textAbstract:
Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.
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Kumar, A., K. Crawford, L. Close, M. Madison, J. Lorenz, T. Doetschman, S. Pawlowski, et al. "Rescue of cardiac -actin-deficient mice by enteric smooth muscle -actin." Proceedings of the National Academy of Sciences 94, no. 9 (April 29, 1997): 4406–11. http://dx.doi.org/10.1073/pnas.94.9.4406.
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Skalli, O., M. F. Pelte, M. C. Peclet, G. Gabbiani, P. Gugliotta, G. Bussolati, M. Ravazzola, and L. Orci. "Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes." Journal of Histochemistry & Cytochemistry 37, no. 3 (March 1989): 315–21. http://dx.doi.org/10.1177/37.3.2918221.
Full textAbstract:
alpha-Smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle cells (SMC) and present in high amounts in vascular SMC, was demonstrated in the cytoplasm of pericytes of various rat and human organs by means of immunocytochemistry at the electron microscopic level. In SMC and pericytes, alpha-sm actin was localized in microfilament bundles, strengthening the assumption that it is the functional isoform in these cell types and supporting the assumption that pericytes exert contractile functions.
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Mounier, N., J. C. Perriard, G. Gabbiani, and C. Chaponnier. "Transfected muscle and non-muscle actins are differentially sorted by cultured smooth muscle and non-muscle cells." Journal of Cell Science 110, no. 7 (April 1, 1997): 839–46. http://dx.doi.org/10.1242/jcs.110.7.839.
Full textAbstract:
We have analyzed by immunolabeling the fate of exogenous epitope-tagged actin isoforms introduced into cultured smooth muscle and non-muscle (i.e. endothelial and epithelial) cells by transfecting the corresponding cDNAs in transient expression assays. Exogenous muscle actins did not produce obvious shape changes in transfected cells. In smooth muscle cells, transfected striated and smooth muscle actins were preferentially recruited into stress fibers. In non-muscle cells, exogenous striated muscle actins were rarely incorporated into stress fibers but remained scattered within the cytoplasm and frequently appeared organized in long crystal-like inclusions. Transfected smooth muscle actins were incorporated into stress fibers of epithelial cells but not of endothelial cells. Exogenous non-muscle actins induced alterations of cell architecture and shape. All cell types transfected by non-muscle actin cDNAs showed an irregular shape and a poorly developed network of stress fibers. beta- and gamma-cytoplasmic actins transfected into muscle and non-muscle cells were dispersed throughout the cytoplasm, often accumulated at the cell periphery and rarely incorporated into stress fibers. These results show that isoactins are differently sorted: not only muscle and non-muscle actins are differentially distributed within the cell but also, according to the cell type, striated and smooth muscle actins can be discriminated for. Our observations support the assumption of isoactin functional diversity.