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. 1983 Oct 1;97(4):1062–1071. doi: 10.1083/jcb.97.4.1062

Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells

PMCID: PMC2112589  PMID: 6688623

Abstract

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++- independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++- dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament- intermediate filament organization.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adelstein R. S., Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem. 1980;49:921–956. doi: 10.1146/annurev.bi.49.070180.004421. [DOI] [PubMed] [Google Scholar]
  2. Adelstein R. S., Klee C. B. Purification and characterization of smooth muscle myosin light chain kinase. J Biol Chem. 1981 Jul 25;256(14):7501–7509. [PubMed] [Google Scholar]
  3. Adelstein R. S., Pato M. D., Sellers J. R., de Lanerolle P., Conti M. A. Regulation of actin-myosin interaction by reversible phosphorylation of myosin and myosin kinase. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):921–928. doi: 10.1101/sqb.1982.046.01.086. [DOI] [PubMed] [Google Scholar]
  4. Bailey K. Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem J. 1948;43(2):271–279. doi: 10.1042/bj0430271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barron J. T., Bárány M., Bárány K., Storti R. V. Reversible phosphorylation and dephosphorylation of the 20,000-dalton light chain of myosin during the contraction-relaxation-contraction cycle of arterial smooth muscle. J Biol Chem. 1980 Jul 10;255(13):6238–6244. [PubMed] [Google Scholar]
  6. Cande W. Z. A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells. J Cell Biol. 1980 Nov;87(2 Pt 1):326–335. doi: 10.1083/jcb.87.2.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassidy P., Hoar P. E., Kerrick W. G. Irreversible thiophosphorylation and activation of tension in functionally skinned rabbit ileum strips by [35S]ATP gamma S. J Biol Chem. 1979 Nov 10;254(21):11148–11153. [PubMed] [Google Scholar]
  8. Cooke P. A reversible change in the functional organization of thin filaments in smooth muscle fibers. Eur J Cell Biol. 1982 Apr;27(1):55–61. [PubMed] [Google Scholar]
  9. Dabrowska R., Sherry J. M., Aromatorio D. K., Hartshorne D. J. Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry. 1978 Jan 24;17(2):253–258. doi: 10.1021/bi00595a010. [DOI] [PubMed] [Google Scholar]
  10. Dillon P. F., Aksoy M. O., Driska S. P., Murphy R. A. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science. 1981 Jan 30;211(4481):495–497. doi: 10.1126/science.6893872. [DOI] [PubMed] [Google Scholar]
  11. Fay F. S., Cooke P. H. Reversible disaggregation of myofilaments in vertebrate smooth muscle. J Cell Biol. 1973 Feb;56(2):399–411. doi: 10.1083/jcb.56.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fay F. S., Delise C. M. Contraction of isolated smooth-muscle cells--structural changes. Proc Natl Acad Sci U S A. 1973 Mar;70(3):641–645. doi: 10.1073/pnas.70.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greaser M. L., Gergely J. Reconstitution of troponin activity from three protein components. J Biol Chem. 1971 Jul 10;246(13):4226–4233. [PubMed] [Google Scholar]
  14. Hoar P. E., Kerrick W. G., Cassidy P. S. Chicken gizzard: relation between calcium-activated phosphorylation and contraction. Science. 1979 May 4;204(4392):503–506. doi: 10.1126/science.432654. [DOI] [PubMed] [Google Scholar]
  15. Kelly R. E., Rice R. V. Localization of myosin filaments in smooth muscle. J Cell Biol. 1968 Apr;37(1):105–116. doi: 10.1083/jcb.37.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelly R. E., Rice R. V. Ultrastructural studies on the contractile mechanism of smooth muscle. J Cell Biol. 1969 Sep;42(3):683–694. doi: 10.1083/jcb.42.3.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kendrick-Jones J., Cande W. Z., Tooth P. J., Smith R. C., Scholey J. M. Studies on the effect of phosphorylation of the 20,000 Mr light chain of vertebrate smooth muscle myosin. J Mol Biol. 1983 Mar 25;165(1):139–162. doi: 10.1016/s0022-2836(83)80247-2. [DOI] [PubMed] [Google Scholar]
  18. Marston S. B., Trevett R. M., Walters M. Calcium ion-regulated thin filaments from vascular smooth muscle. Biochem J. 1980 Feb 1;185(2):355–365. doi: 10.1042/bj1850355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mikawa T., Toyo-oka T., Nonomura Y., Ebashi S. Essential factor of gizzard "troponin" fraction. A new type of regulatory protein. J Biochem. 1977 Jan;81(1):273–275. doi: 10.1093/oxfordjournals.jbchem.a131447. [DOI] [PubMed] [Google Scholar]
  20. Perrie W. T., Perry S. V. An electrophoretic study of the low-molecular-weight components of myosin. Biochem J. 1970 Aug;119(1):31–38. doi: 10.1042/bj1190031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Scholey J. M., Taylor K. A., Kendrick-Jones J. Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase. Nature. 1980 Sep 18;287(5779):233–235. doi: 10.1038/287233a0. [DOI] [PubMed] [Google Scholar]
  22. Scholey J. M., Taylor K. A., Kendrick-Jones J. The role of myosin light chains in regulating actin-myosin interaction. Biochimie. 1981 Apr;63(4):255–271. doi: 10.1016/s0300-9084(81)80115-0. [DOI] [PubMed] [Google Scholar]
  23. Sherry J. M., Górecka A., Aksoy M. O., Dabrowska R., Hartshorne D. J. Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. Biochemistry. 1978 Oct 17;17(21):4411–4418. doi: 10.1021/bi00614a009. [DOI] [PubMed] [Google Scholar]
  24. Shoenberg C. F., Needham D. M. A study of the mechanism of contraction in vertebrate smooth muscle. Biol Rev Camb Philos Soc. 1976 Feb;51(1):53–104. doi: 10.1111/j.1469-185x.1976.tb01120.x. [DOI] [PubMed] [Google Scholar]
  25. Shoenberg C. F. The influence of temperature on the thick filaments of vertebrate smooth muscle. Philos Trans R Soc Lond B Biol Sci. 1973 Mar 15;265(867):197–202. doi: 10.1098/rstb.1973.0023. [DOI] [PubMed] [Google Scholar]
  26. Small J. V. Contractile units in vertebrate smooth muscle cells. Nature. 1974 May 24;249(455):324–327. doi: 10.1038/249324a0. [DOI] [PubMed] [Google Scholar]
  27. Small J. V. Studies on isolated smooth muscle cells: The contractile apparatus. J Cell Sci. 1977 Apr;24:327–349. doi: 10.1242/jcs.24.1.327. [DOI] [PubMed] [Google Scholar]
  28. Somlyo A. V., Butler T. M., Bond M., Somlyo A. P. Myosin filaments have non-phosphorylated light chains in relaxed smooth muscle. Nature. 1981 Dec 10;294(5841):567–569. doi: 10.1038/294567a0. [DOI] [PubMed] [Google Scholar]
  29. Suzuki H., Onishi H., Takahashi K., Watanabe S. Structure and function of chicken gizzard myosin. J Biochem. 1978 Dec;84(6):1529–1542. doi: 10.1093/oxfordjournals.jbchem.a132278. [DOI] [PubMed] [Google Scholar]
  30. Walsh M. P., Bridenbaugh R., Hartshorne D. J., Kerrick W. G. Phosphorylation-dependent activated tension in skinned gizzard muscle fibers in the absence of Ca2+. J Biol Chem. 1982 Jun 10;257(11):5987–5990. [PubMed] [Google Scholar]
  31. Walsh M. P., Dabrowska R., Hinkins S., Hartshorne D. J. Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification, and characterization. Biochemistry. 1982 Apr 13;21(8):1919–1925. doi: 10.1021/bi00537a034. [DOI] [PubMed] [Google Scholar]

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