Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts
1 Departments of Pathology, Saint Louis University School of Medicine, St. Louis 63104; 2 Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110; and 3 Department of Physiology, Indiana University School of Medicine, Indianapolis, I...
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| Veröffentlicht in: | American Journal of Physiology: Cell Physiology 2004-01, Vol.286 (1), p.C8-21 |
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| container_title | American Journal of Physiology: Cell Physiology |
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| creator | Emmert, Daniel A Fee, Judy A Goeckeler, Zoe M Grojean, Jeremy M Wakatsuki, Tetsuro Elson, Elliot L Herring, B. Paul Gallagher, Patricia J Wysolmerski, Robert B |
| description | 1 Departments of Pathology, Saint Louis University School of Medicine, St. Louis 63104; 2 Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110; and 3 Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 16 September 2002
; accepted in final form 22 August 2003
Thus far, determining the relative contribution of Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK) and Ca 2+ -independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca 2+ , thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO 4 /mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO 4 /mol RLC, respectively, within 2.5 min. Ca 2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.
myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation
Address for reprint requests and other correspondence: R. B. Wysolmerski, Dept. of Pathology, Saint Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: wysolmer{at}slucare1.sluh.edu ). |
| doi_str_mv | 10.1152/ajpcell.00428.2002 |
| format | Article |
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Submitted 16 September 2002
; accepted in final form 22 August 2003
Thus far, determining the relative contribution of Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK) and Ca 2+ -independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca 2+ , thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO 4 /mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO 4 /mol RLC, respectively, within 2.5 min. Ca 2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.
myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation
Address for reprint requests and other correspondence: R. B. Wysolmerski, Dept. of Pathology, Saint Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: wysolmer{at}slucare1.sluh.edu ).</description><identifier>ISSN: 0363-6143</identifier><identifier>EISSN: 1522-1563</identifier><identifier>DOI: 10.1152/ajpcell.00428.2002</identifier><identifier>PMID: 12967916</identifier><language>eng</language><publisher>United States</publisher><subject>Amides - pharmacology ; Animals ; Calcium - physiology ; Calcium-Calmodulin-Dependent Protein Kinases - metabolism ; Cell Line ; Embryo, Mammalian ; Enzyme Inhibitors - pharmacology ; Fibroblasts - drug effects ; Fibroblasts - physiology ; Intracellular Signaling Peptides and Proteins ; Lysophospholipids - pharmacology ; Myosin Light Chains - metabolism ; Myosin-Light-Chain Kinase - metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases - metabolism ; Protein-Serine-Threonine Kinases - physiology ; Pyridines - pharmacology ; Rats ; rho-Associated Kinases ; rhoA GTP-Binding Protein - metabolism ; Thrombin - pharmacology</subject><ispartof>American Journal of Physiology: Cell Physiology, 2004-01, Vol.286 (1), p.C8-21</ispartof><rights>Copyright © 2004 the American Physiological Society 2004</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,315,782,786,887,27931,27932</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12967916$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Emmert, Daniel A</creatorcontrib><creatorcontrib>Fee, Judy A</creatorcontrib><creatorcontrib>Goeckeler, Zoe M</creatorcontrib><creatorcontrib>Grojean, Jeremy M</creatorcontrib><creatorcontrib>Wakatsuki, Tetsuro</creatorcontrib><creatorcontrib>Elson, Elliot L</creatorcontrib><creatorcontrib>Herring, B. Paul</creatorcontrib><creatorcontrib>Gallagher, Patricia J</creatorcontrib><creatorcontrib>Wysolmerski, Robert B</creatorcontrib><title>Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts</title><title>American Journal of Physiology: Cell Physiology</title><addtitle>Am J Physiol Cell Physiol</addtitle><description>1 Departments of Pathology, Saint Louis University School of Medicine, St. Louis 63104; 2 Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110; and 3 Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 16 September 2002
; accepted in final form 22 August 2003
Thus far, determining the relative contribution of Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK) and Ca 2+ -independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca 2+ , thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO 4 /mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO 4 /mol RLC, respectively, within 2.5 min. Ca 2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.
myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation
Address for reprint requests and other correspondence: R. B. Wysolmerski, Dept. of Pathology, Saint Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: wysolmer{at}slucare1.sluh.edu ).</description><subject>Amides - pharmacology</subject><subject>Animals</subject><subject>Calcium - physiology</subject><subject>Calcium-Calmodulin-Dependent Protein Kinases - metabolism</subject><subject>Cell Line</subject><subject>Embryo, Mammalian</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Fibroblasts - drug effects</subject><subject>Fibroblasts - physiology</subject><subject>Intracellular Signaling Peptides and Proteins</subject><subject>Lysophospholipids - pharmacology</subject><subject>Myosin Light Chains - metabolism</subject><subject>Myosin-Light-Chain Kinase - metabolism</subject><subject>Phosphorylation</subject><subject>Protein-Serine-Threonine Kinases - metabolism</subject><subject>Protein-Serine-Threonine Kinases - physiology</subject><subject>Pyridines - pharmacology</subject><subject>Rats</subject><subject>rho-Associated Kinases</subject><subject>rhoA GTP-Binding Protein - metabolism</subject><subject>Thrombin - pharmacology</subject><issn>0363-6143</issn><issn>1522-1563</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kctOwzAQRS0EouXxAyxQVmyqFD-a2NkgoYryUCUkVNaW49iNSxqH2AH697hqimCBF2Np5tw7oxkALhAcI5Tga7FqpKqqMYQTzMYYQnwAhqGAY5Sk5BAMIUlJnKIJGYAT51ZwC6bZMRggnKU0Q-kQPL2UNn4ztXAqXqvCCK-KaCrwKDZ1oRoVQu0jaWvfCumNrSNTR63wkVrn7cZG2uStzSvhvDsDR1pUTp33_yl4nd0tpg_x_Pn-cXo7j0vCmI-RgBrKPNOyYEImeYJ1KojMJaS6kLTQErOETrDSkhKREyonNCOM4kCFvCKn4Gbn23R5GFmq7WwVb1qzFu2GW2H430ptSr60HxwzTGiWBIOr3qC1751ynq-N225S1Mp2jlOUhIdwAC9_d_ppsV9fAOgOKM2y_DSt4k25ccZWdrnhs66qFurL8_5OmKUc8SnjTaGDcvS_shfwvYJ8A0LNmh8</recordid><startdate>20040101</startdate><enddate>20040101</enddate><creator>Emmert, Daniel A</creator><creator>Fee, Judy A</creator><creator>Goeckeler, Zoe M</creator><creator>Grojean, Jeremy M</creator><creator>Wakatsuki, Tetsuro</creator><creator>Elson, Elliot L</creator><creator>Herring, B. 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Paul ; Gallagher, Patricia J ; Wysolmerski, Robert B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-h388t-1a0f0cb9fcd8ac5b52f6a3cbc07fdc7dfc285742efc73ab37c47938726a3574e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amides - pharmacology</topic><topic>Animals</topic><topic>Calcium - physiology</topic><topic>Calcium-Calmodulin-Dependent Protein Kinases - metabolism</topic><topic>Cell Line</topic><topic>Embryo, Mammalian</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Fibroblasts - drug effects</topic><topic>Fibroblasts - physiology</topic><topic>Intracellular Signaling Peptides and Proteins</topic><topic>Lysophospholipids - pharmacology</topic><topic>Myosin Light Chains - metabolism</topic><topic>Myosin-Light-Chain Kinase - metabolism</topic><topic>Phosphorylation</topic><topic>Protein-Serine-Threonine Kinases - metabolism</topic><topic>Protein-Serine-Threonine Kinases - physiology</topic><topic>Pyridines - pharmacology</topic><topic>Rats</topic><topic>rho-Associated Kinases</topic><topic>rhoA GTP-Binding Protein - metabolism</topic><topic>Thrombin - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Emmert, Daniel A</creatorcontrib><creatorcontrib>Fee, Judy A</creatorcontrib><creatorcontrib>Goeckeler, Zoe M</creatorcontrib><creatorcontrib>Grojean, Jeremy M</creatorcontrib><creatorcontrib>Wakatsuki, Tetsuro</creatorcontrib><creatorcontrib>Elson, Elliot L</creatorcontrib><creatorcontrib>Herring, B. Paul</creatorcontrib><creatorcontrib>Gallagher, Patricia J</creatorcontrib><creatorcontrib>Wysolmerski, Robert B</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>American Journal of Physiology: Cell Physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Emmert, Daniel A</au><au>Fee, Judy A</au><au>Goeckeler, Zoe M</au><au>Grojean, Jeremy M</au><au>Wakatsuki, Tetsuro</au><au>Elson, Elliot L</au><au>Herring, B. Paul</au><au>Gallagher, Patricia J</au><au>Wysolmerski, Robert B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts</atitle><jtitle>American Journal of Physiology: Cell Physiology</jtitle><addtitle>Am J Physiol Cell Physiol</addtitle><date>2004-01-01</date><risdate>2004</risdate><volume>286</volume><issue>1</issue><spage>C8</spage><epage>21</epage><pages>C8-21</pages><issn>0363-6143</issn><eissn>1522-1563</eissn><abstract>1 Departments of Pathology, Saint Louis University School of Medicine, St. Louis 63104; 2 Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110; and 3 Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 16 September 2002
; accepted in final form 22 August 2003
Thus far, determining the relative contribution of Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK) and Ca 2+ -independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca 2+ , thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO 4 /mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO 4 /mol RLC, respectively, within 2.5 min. Ca 2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.
myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation
Address for reprint requests and other correspondence: R. B. Wysolmerski, Dept. of Pathology, Saint Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: wysolmer{at}slucare1.sluh.edu ).</abstract><cop>United States</cop><pmid>12967916</pmid><doi>10.1152/ajpcell.00428.2002</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Amides - pharmacology Animals Calcium - physiology Calcium-Calmodulin-Dependent Protein Kinases - metabolism Cell Line Embryo, Mammalian Enzyme Inhibitors - pharmacology Fibroblasts - drug effects Fibroblasts - physiology Intracellular Signaling Peptides and Proteins Lysophospholipids - pharmacology Myosin Light Chains - metabolism Myosin-Light-Chain Kinase - metabolism Phosphorylation Protein-Serine-Threonine Kinases - metabolism Protein-Serine-Threonine Kinases - physiology Pyridines - pharmacology Rats rho-Associated Kinases rhoA GTP-Binding Protein - metabolism Thrombin - pharmacology |
| title | Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts |
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