Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 May 1;104(5):1343–1352. doi: 10.1083/jcb.104.5.1343

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle

PMCID: PMC2114482  PMID: 3553210

Abstract

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold- labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.

Full Text

The Full Text of this article is available as a PDF (4.6 MB).

Selected References

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

  1. Dolber P. C., Sommer J. R. Corbular sarcoplasmic reticulum of rabbit cardiac muscle. J Ultrastruct Res. 1984 May;87(2):190–196. doi: 10.1016/s0022-5320(84)80078-7. [DOI] [PubMed] [Google Scholar]
  2. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983 Jul;245(1):C1–14. doi: 10.1152/ajpcell.1983.245.1.C1. [DOI] [PubMed] [Google Scholar]
  3. Fawcett D. W., McNutt N. S. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969 Jul;42(1):1–45. doi: 10.1083/jcb.42.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Forbes M. S., Sperelakis N. The membrane systems and cytoskeletal elements of mammalian myocardial cells. Cell Muscle Motil. 1983;3:89–155. doi: 10.1007/978-1-4615-9296-9_5. [DOI] [PubMed] [Google Scholar]
  5. Fowler V. M., Bennett V. Erythrocyte membrane tropomyosin. Purification and properties. J Biol Chem. 1984 May 10;259(9):5978–5989. [PubMed] [Google Scholar]
  6. Franzini-Armstrong C. Structure of sarcoplasmic reticulum. Fed Proc. 1980 May 15;39(7):2403–2409. [PubMed] [Google Scholar]
  7. Huggins J. P., England P. J. Sarcolemmal phospholamban is phosphorylated in isolated rat hearts perfused with isoprenaline. FEBS Lett. 1983 Nov 14;163(2):297–302. doi: 10.1016/0014-5793(83)80839-4. [DOI] [PubMed] [Google Scholar]
  8. Iwasa Y., Hosey M. M. Cholinergic antagonism of beta-adrenergic stimulation of cardiac membrane protein phosphorylation in situ. J Biol Chem. 1983 Apr 10;258(7):4571–4575. [PubMed] [Google Scholar]
  9. Jones L. R., Simmerman H. K., Wilson W. W., Gurd F. R., Wegener A. D. Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. J Biol Chem. 1985 Jun 25;260(12):7721–7730. [PubMed] [Google Scholar]
  10. Jorgensen A. O., Campbell K. P. Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum. J Cell Biol. 1984 Apr;98(4):1597–1602. doi: 10.1083/jcb.98.4.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jorgensen A. O., Jones L. R. Localization of phospholamban in slow but not fast canine skeletal muscle fibers. An immunocytochemical and biochemical study. J Biol Chem. 1986 Mar 15;261(8):3775–3781. [PubMed] [Google Scholar]
  12. Jorgensen A. O., Kalnins V., MacLennan D. H. Localization of sarcoplasmic reticulum proteins in rat skeletal muscle by immunofluorescence. J Cell Biol. 1979 Feb;80(2):372–384. doi: 10.1083/jcb.80.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jorgensen A. O., Shen A. C., Campbell K. P. Ultrastructural localization of calsequestrin in adult rat atrial and ventricular muscle cells. J Cell Biol. 1985 Jul;101(1):257–268. doi: 10.1083/jcb.101.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jorgensen A. O., Shen A. C., Daly P., MacLennan D. H. Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle. J Cell Biol. 1982 Jun;93(3):883–892. doi: 10.1083/jcb.93.3.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keller G. A., Tokuyasu K. T., Dutton A. H., Singer S. J. An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5744–5747. doi: 10.1073/pnas.81.18.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kirchberger M. A., Tada M., Katz A. M. Adenosine 3':5'-monophosphate-dependent protein kinase-catalyzed phosphorylation reaction and its relationship to calcium transport in cardiac sarcoplasmic reticulum. J Biol Chem. 1974 Oct 10;249(19):6166–6173. [PubMed] [Google Scholar]
  17. Lamers J. M., De Jonge-Stinis J. T., Hülsmann W. C., Verdouw P. D. Reduced in vitro 32P incorporation into phospholamban-like protein of sarcolemma due to myocardial ischaemia in anaesthetized pigs. J Mol Cell Cardiol. 1986 Feb;18(2):115–125. doi: 10.1016/s0022-2828(86)80464-3. [DOI] [PubMed] [Google Scholar]
  18. Langer G. A. The role of calcium in the control of myocardial contractility: an update. J Mol Cell Cardiol. 1980 Mar;12(3):231–239. doi: 10.1016/0022-2828(80)90037-1. [DOI] [PubMed] [Google Scholar]
  19. Lindemann J. P., Jones L. R., Hathaway D. R., Henry B. G., Watanabe A. M. beta-Adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles. J Biol Chem. 1983 Jan 10;258(1):464–471. [PubMed] [Google Scholar]
  20. Louis C. F., Hogan M., Turnquist J. Properties of the 23,000-Da phosphoproteins in cardiac sarcolemma and sarcoplasmic reticulum. Arch Biochem Biophys. 1986 Apr;246(1):98–107. doi: 10.1016/0003-9861(86)90453-4. [DOI] [PubMed] [Google Scholar]
  21. Manalan A. S., Jones L. R. Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemmal vesicles. J Biol Chem. 1982 Sep 10;257(17):10052–10062. [PubMed] [Google Scholar]
  22. Porzio M. A., Pearson A. M. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochim Biophys Acta. 1977 Jan 25;490(1):27–34. doi: 10.1016/0005-2795(77)90102-7. [DOI] [PubMed] [Google Scholar]
  23. Presti C. F., Scott B. T., Jones L. R. Identification of an endogenous protein kinase C activity and its intrinsic 15-kilodalton substrate in purified canine cardiac sarcolemmal vesicles. J Biol Chem. 1985 Nov 5;260(25):13879–13889. [PubMed] [Google Scholar]
  24. Raeymaekers L., Jones L. R. Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle. Biochim Biophys Acta. 1986 Jun 19;882(2):258–265. doi: 10.1016/0304-4165(86)90163-7. [DOI] [PubMed] [Google Scholar]
  25. Rinaldi M. L., Capony J. P., Demaille J. G. The cyclic AMP-dependent modulation of cardiac sarcolemmal slow calcium channels. J Mol Cell Cardiol. 1982 May;14(5):279–289. doi: 10.1016/0022-2828(82)90206-1. [DOI] [PubMed] [Google Scholar]
  26. Rinaldi M. L., Le Peuch C. J., Demaille J. G. The epinephrine-induced activation of the cardiac slow Ca2+ channel is mediated by the cAMP-dependent phosphorylation of calciductin, a 23 000 Mr sarcolemmal protein. FEBS Lett. 1981 Jul 6;129(2):277–281. doi: 10.1016/0014-5793(81)80183-4. [DOI] [PubMed] [Google Scholar]
  27. Segretain D., Rambourg A., Clermont Y. Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat. Anat Rec. 1981 Jun;200(2):139–151. doi: 10.1002/ar.1092000204. [DOI] [PubMed] [Google Scholar]
  28. Tada M., Inui M. Regulation of calcium transport by the ATPase-phospholamban system. J Mol Cell Cardiol. 1983 Sep;15(9):565–575. doi: 10.1016/0022-2828(83)90267-5. [DOI] [PubMed] [Google Scholar]
  29. Tada M., Katz A. M. Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol. 1982;44:401–423. doi: 10.1146/annurev.ph.44.030182.002153. [DOI] [PubMed] [Google Scholar]
  30. Tada M., Yamamoto T., Tonomura Y. Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol Rev. 1978 Jan;58(1):1–79. doi: 10.1152/physrev.1978.58.1.1. [DOI] [PubMed] [Google Scholar]
  31. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Winegrad S. Calcium release from cardiac sarcoplasmic reticulum. Annu Rev Physiol. 1982;44:451–462. doi: 10.1146/annurev.ph.44.030182.002315. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES