Journal of Muscle Research & Cell Motility

, Volume 8, Issue 2, pp 135–144 | Cite as

Structural and biochemical analysis of skinned smooth muscle preparations

  • T. Kossmann
  • D. Fürst
  • J. V. Small
Papers
Received:
Revised:

Summary

This paper describes a biochemical and immunocytochemical analysis of smooth muscle strips that were chemically skinned and subjected to contraction and relaxation cycles according to procedures commonly employed in current skinned smooth muscle work. The fate of four major proteins, myosin, filamin, caldesmon and actin, was followed with respect to the proportionate loss of these proteins to the bathing medium as well as to their structural redistribution within the cells in the muscle strips. Large losses (of the order of 50%) of both myosin and filamin occurred at the skinning step, using either Triton X-100 or Saponin as the detergent; losses of actin were up to 30% with Triton X-100 and around 15% with Saponin. Losses of caldesmon were difficult to assess due to the rapid degradation of this protein in the bathing medium. Subsequent cycles of contraction and relaxation resulted in accumulated loss, notably of myosin and filamin, so that after the third contraction as little as 20% and 40% respectively of the original complement of these proteins remained in the muscle strips. These changes in protein composition were accompanied by a drastic redistribution of the proteins in the muscle cells. Most marked were the changes seen with myosin, significant amounts of this protein being already found in the connective tissue space after the first relaxation. These findings point to the need for a careful reappraisal of the conditions currently used in skinned smooth muscle research.

Keywords

Smooth Muscle Saponin Rapid Degradation Muscle Strip Immunocytochemical Analysis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aberg, A. K. G. &Axelsson, J. (1965) Some mechanical aspects of an intestinal smooth muscle.Acta physiol. Scand. 64, 15–27.Google Scholar
  2. Arner, A. (1982) Mechanical characteristics of chemically skinned guinea-pig taenia coli.Pflügers Arch. 395, 277–84.Google Scholar
  3. Bretscher, A. (1984) Smooth muscle caldesmon.J. biol. Chem. 259, 12873–80.Google Scholar
  4. Endo, M., Kitazawa, T., Yagi, S., Iino, M. &Kakuta, Y. (1977) Some properties of chemically skinned smooth muscle fibers. InExcitationcontraction Coupling in Smooth Muscle (edited byCasteels, R., Godfraind, T. andRüegg, J. C.), pp. 199–209. Amsterdam: North-Holland Publishing Co.Google Scholar
  5. Filo, R. S., Bohr, D. F. &Rüegg, J. C. (1965) Glycerinated skeletal and smooth muscle: calcium and magnesium dependence.Science, N.Y. 147, 1581–3.Google Scholar
  6. Fürst, D. O., Cross, R. A., deMey, J. &Small, J. V. (1986) Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle.EMBO J. 5, 251–7.Google Scholar
  7. Gabella, G. (1976) The force generated by a visceral smooth muscle.J. Physiol, Lond. 263, 199–213.Google Scholar
  8. Gordon, A. R. (1978) Contraction of detergent-treated smooth muscle.Proc. natn. Acad. Sci. U.S.A. 75, 3527–30.Google Scholar
  9. Haeberle, J. R., Coolican, S. A., Evan, A. &Hathaway, D. R. (1985a) The effect of a calcium dependent protease on the ultrastructure and contractile mechanics of skinned uterine smooth muscle.J. Musc. Res. Cell Motility 6, 347–63.Google Scholar
  10. Haeberle, J. R., Hathaway, D. R. &DePaoli-Roach, A. (1985b) Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle.J. biol. Chem. 260, 9965–8.Google Scholar
  11. Iino, M. (1981) Tension responses of chemically skinned fibre bundles of the guinea-pig taenia caeci under varied ionic environments.J. Physiol., Lond. 320, 449–67.Google Scholar
  12. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature, Lond. 227, 680–5.Google Scholar
  13. Laszt, L. &Hamoir, G. (1961) Etude par electrophorese et ultracentrifugation de la composition proteinique de la couche musculaire des carotides de bovide.Biochim. biophys. Acta 50, 430–49.Google Scholar
  14. Lowy, J. &Mulvany, M. J. (1973) Mechanical properties of guinea pig taenia coli muscles.Acta physiol. Scand. 88, 123–36.Google Scholar
  15. Marston, S. B. (1982) The regulation of smooth muscle contractile proteins.Progr. Biophys. molec. Biol. 41, 1–41.Google Scholar
  16. Marston, S. B. &Smith, C. W. J. (1985) The thin filaments of smooth muscle.J. Musc. Res. Cell Motility 6, 669–708.Google Scholar
  17. Matsudaira, P. &Burgess, D. R. (1978) SDS microslab linear gradient polyacrylamide gel electrophoresis.Analyt. Biochem. 87, 386–96.Google Scholar
  18. Meisheri, K. D., Rüegg, J. C. &Paul, R. J. (1985) Studies on skinned fiber preparations. InCalcium and Contractility (edited byGrover, A. k. andDaniel, E. E.) pp. 191–224. The humana Press, Clifton, New Jersey, U. S. A.Google Scholar
  19. Moeremans, M., Daneels, G., vanDijck, A., Langanger, G. & deMey, J. (1984) Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immungold/silver staining.J. Immun. Meth. 74, 353–60.Google Scholar
  20. Paul, R. J., Doerman, G., Zeugner, C. &Rüegg, J. C. (1983) Dependence of unloaded shortening velocity (Vus) on Ca2+, calmodulin and contraction duration in “chemically skinned” smooth muscle.Circ. Res. 53, 342–51.Google Scholar
  21. Peterson, J. W. (1980) Vanadate ion inhibits actomyosin interaction in chemically skinned vascular smooth muscle.Biochem. Biophys. Res. Commun. 95, 1846–53.Google Scholar
  22. Peterson, J. W. (1982) Simple model of smooth muscle myosin phosphorylation and dephosphorylation as rate-limiting mechanism.Biophys. J. 37, 453–9.Google Scholar
  23. Portzehl, H., Caldwell, P. C. &Rüegg, J. C. (1964) The dependence of contraction and relaxation of muscle fibers from the crabMaia squinado on the internal concentration of free calcium ions.Biochim. Biophys. Acta 79, 581–91.Google Scholar
  24. Saida, K. &Nonomura, Y. (1978) Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers.J. gen. Physiol. 72, 1–14.Google Scholar
  25. Schneider, M., Sparrow, M. P. &Rüegg, J. C. (1981) Inorganic phosphate promotes relaxation of chemically skinned smooth muscle of guinea-pig taenia coli.Experientia 37, 980–2.Google Scholar
  26. Small, J. V., Fürst, D. F. & deMey, J. (1986) Localization of filamin in smooth muscle.J. Cell Biol. 102, 210–20.Google Scholar
  27. Small, J. V. &Sobieszek, A. (1980) The contractile apparatus of smooth muscle.Int. Rev. Cytol. 64, 241–306.Google Scholar
  28. Sobieszek, A. &Bremel, R. D. (1975) Preparations and properties of vertebrate smooth muscle myofibrils and actomyosin.Eur. J. Biochem. 55, 49–68.Google Scholar
  29. Sobieszek, A. &Small, J. V. (1976) Myosin linked calcium regulation in vertebrate smooth muscle.J. molec. Biol. 102, 75–92.Google Scholar
  30. Sobue, K., Muramoto, Y., Fujita, M. &Kakiuchi, S. (1981) Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin.Proc. natn. Acad. Sci. U. S. A. 78, 5652–5.Google Scholar
  31. Sparrow, M. P., Mrwa, U., Hofmann, F. &Rüegg, J. C. (1981) Calmodulin is essential for smooth muscle contraction.FEBS Lett. 125, 141–5.Google Scholar
  32. Sparrow, M. P., Pfitzer, G., Gagelmann, M. &Rüegg, J. C. (1984) Effect of calmodulin, Ca2+, and cAMP protein kinase on skinned tracheal smooth muscle.Am. J. Physiol. 246, C308-C314.Google Scholar
  33. Spedding, M. (1981) Comparison of Ca++-antagonists and trifluoperazine in skinned smooth muscle fibres.Br. J. Pharmac. 75, 25P.Google Scholar
  34. Towbin, H., Staehlin, T. &Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. natn. Acad. Sci. U. S. A. 76, 4350–4.Google Scholar
  35. Wang, K. (1977) Filamin, a new high molecular weight protein found in smooth muscle and nonmuscle cells. Purification and properties of chicken gizzard filamin.Biochemistry 16, 1857–65.Google Scholar

Copyright information

© Chapman and Hall Ltd 1987

Authors and Affiliations

  • T. Kossmann
    • 1
  • D. Fürst
    • 2
  • J. V. Small
    • 2
  1. 1.II. Physiologisches InstitutUniversitat HeidelbergHeidelbergWest Germany
  2. 2.Institute of Molecular BiologyAustrian Academy of SciencesBillrothstrasse 11Austria

Personalised recommendations