Skip to main content
SpringerLink
Log in

Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle

  • Papers
  • Published:
Journal of Muscle Research & Cell Motility Aims and scope Submit manuscript

Summary

Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP3 receptor labelling was predominatly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP3 receptor. In aorta (tonic muscle) the InsP3 receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP3 and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP3 receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • ALLBRITTON, N. L., MEYER, T. & STRYER, L. (1992) Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258, 1812–15.

    Google Scholar 

  • BLOCK, B. A., IMAGAWA, T., CAMPBELL, K. P. & FRANZINI-ARMSTRONG, C. (1988) Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J. Cell Biol. 107, 2587–600.

    Google Scholar 

  • BOND, M., KITAZAWA, T., SOMLYO, A. P. & SOMLYO, A. V. (1984) Release and recycling of calcium in guinea pig portal vein smooth muscle. J. Physiol. 355, 677–95.

    Google Scholar 

  • BOND, M., VADASZ, G., SOMLYO, A. V. & SOMLYO, A. P. (1987) Subcellular calcium or magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon. J. Biol. Chem. 262, 15630–6.

    Google Scholar 

  • BURKE, J. M. & ROSS, R. (1979) Synthesis of connective tissue macromolecules by smooth muscle. Internat. Rev. Connect. 8, 119–57.

    Google Scholar 

  • CAMPBELL, K. P., MACLENNAN, D. H., JORGENSEN, A. O. & MINTZER, M. C. (1983) Purification and characterization of calsequestrin from canine sarcoplasmic reticulum and identification of the 53,000 dalton glycoprotein. J. Biol. Chem. 258, 1197–204.

    Google Scholar 

  • CHADWICK, C. C., SAITO, A. & FLEISCHER, S. (1990) Isolation and characterization of the inositol trisphosphate receptor from smooth muscle. Proc. Natl. Acad. Sci. USA 87, 2132–6.

    Google Scholar 

  • DALEN, H., SCHEIE, P., MYKLEBUST, R. & SAETERSDAL, T. (1983) An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum. J. Microscopy 131, 35–46.

    Google Scholar 

  • DEVINE, C. E., SOMLYO, A. V. & SOMLYO, A. P. (1972) Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J. Cell Biol. 52, 690–718.

    Google Scholar 

  • FORBES, M. S., PLANTHOLT, B. A. & SPERELAKIS, N. (1977) Cytochemical staining procedures selective for sarcotubular systems of muscle: modifications and applications. J. Ultrastruct. Res. 60, 306–27.

    Google Scholar 

  • FUJIMOTO, T., NAKADE, S., MIYAWAKI, A., MIKOSHIBA, K. & OGAWA, K. (1992) Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J. Cell Biol. 119, 1507–13.

    Google Scholar 

  • FURIUCHI, T., YOSHIKAWA, S., MIYAWAKI, A., WADA, K., MAEDA, N. & MIKOSHIBA, K. (1989) Primary structure and functional expression of inositol 1,4,5-trisphosphate-binding protein P400. Nature 342, 32–8.

    Google Scholar 

  • FURIUCHI, T., SHIOTA, C. & MIKOSHIBA, K. (1990) Distribution of inositol 1,4,5-trisphosphate receptor mRNA in mouse tissues. FEBS Lett. 267, 85–8.

    Google Scholar 

  • HAINFELD, J. F. & FURUYA, F. R. (1992) A 1.4 nm gold cluster covalently attached to antibodies improves immunolabeling. J. Histochem. Cytochem. 40, 177–84.

    Google Scholar 

  • IINO, M. (1989) Calcium-induced calcium release mechanisms in guinea pig taenia coli. J. Gen. Physiol. 94, 363–83.

    Google Scholar 

  • IINO, M., KOBAYASHI, T. & ENDO, M. (1988) Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea pig. Biochem. Biophys. Res. Comm. 152, 417–22.

    Google Scholar 

  • IKEMOTO, N., BHATNAGAR, G. M., NAGY, B. & GERGELY, J. (1972) Interaction of divalent cation protein component of the sarcoplasmic reticulum. J. Biol. Chem. 247, 7835–7.

    Google Scholar 

  • IKEMOTO, N., NAGY, B., BHATNAGAR, G. M. & GERGELY, J. (1974) Studies on a metal-binding protein of the sarcoplasmic reticulum. J. Biol. Chem. 249, 2357–65.

    Google Scholar 

  • INUI, M., SAITO, A. & FLEISCHER, S. (1987) Purification of the ryanodine receptor and identity with the feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. J. Biol. Chem. 262, 1740–7.

    Google Scholar 

  • JORGENSEN, A. O., KALNINS, V. I., ZUBRZYCKA, E. & MACLENNAN, D. H. (1977) Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures. J. Cell Biol. 74, 287–98.

    Google Scholar 

  • JORGENSEN, A. O., SHEN, A. C-Y., DALY, P. & MACLENNAN, D. H. (1982) Localization of Ca2++Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle. J. Cell Biol. 93, 883–92.

    Google Scholar 

  • JORGENSEN, A. O., SHEN, A. C-Y. & CAMPBELL, K. P. (1985) Ultrastructural localization of calsequestrin in adult rat atrial and ventricular muscle cells. J. Cell Biol. 101, 257–68.

    Google Scholar 

  • JORGENSEN, A. O., BRODERICK, R., SOMLYO, A. P. and SOMLYO, A. V. (1988) Two structurally distinct calcium storage sites in rat cardiac sarcoplasmic reticulum: an electron microprobe analysis study. Circ. Res. 63, 1060–9.

    Google Scholar 

  • JORGENSEN, A. O., SHEN, A. C-Y., ARNOLD, W., MCPHERSON, P. S. & CAMPBELL, K. P. (1993) The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle. J. Cell Biol. 120, 969–80.

    Google Scholar 

  • JUNKER, J., SOMMER, J. R., SAR, M. & MEISSNER, G. (1994) Extended junctional sarcoplasmic reticulum of avian cardiac muscle contains functional ryanodine receptors. J. Biol. Chem. 269, 1627–34.

    Google Scholar 

  • KITAZAWA, T., KOBAYASHI, S., HORIUTI, K., SOMLYO, A. V. & SOMLYO, A. P. (1989) Receptor coupled, permeabilized smooth muscle: role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+. J. Biol. Chem. 264, 5339–42.

    Google Scholar 

  • KOBAYASHI, S., KITAZAWA, T., SOMLYO, A. V. & SOMLYO, A. P. (1989) Cytosolic heparin inhibits muscarinic and adrenergic Ca2+ release in smooth muscle. J. Biol. Chem. 264, 17997–8004.

    Google Scholar 

  • KOBAYASHI, S., GONG, M-C., SOMLYO, A. V. & SOMLYO, A. P. (1991) Ca2+-channel blockers distinguish between G-protein coupled pharmacomechanical Ca2+ release and Ca2+ sensitization in smooth muscle. Am. J. Physiol. 260, C364–70.

    Google Scholar 

  • KOWARSKI, D., SHUMAN, H., SOMLYO, A. P. & SOMLYO, A. V. (1985) Calcium release by noradrenaline from central sarcoplasmic reticulum in rabbit main pulmonary artery smooth muscle. J. Physiol. 366, 153–75.

    Google Scholar 

  • LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5.

    Google Scholar 

  • MACLENNAN, D. H. & WONG, P. T. S. (1971) Isolation of a calcium-sequestering protein from sarcoplasmic reticulum. Proc. Nat. Acad. Sci. USA. 68, 1231–5.

    Google Scholar 

  • MARKS, A. R., TEMPST, P., CHADWICK, C. C., RIVIERE, L., FLEISCHER, S. & NADAL-GINARD, B. (1990) Smooth muscle and brain inositol 1,4,5-trisphosphate receptors are structurally and functionally similar. J. Biol. Chem. 265, 20719–22.

    Google Scholar 

  • MÉSZÁROS, L. G., BAK, J. & CHU, A. (1993) Cyclic-ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel. Nature 364, 76–9.

    Google Scholar 

  • MIGNERY, G. A., SÜDHOF, T. C., TAKEI, K. & CAMILLI, P.De. (1989) Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor. Nature 342, 192–5.

    Google Scholar 

  • MILNER, R. E., BAKSH, S., SHEMANKO, C., CARPENTER, M. R., SMILLIE, L., VANCE, J. E., OPES, M. & MICHALAK, M. (1991) Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. J. Biol. Chem. 266, 7155–65.

    Google Scholar 

  • MILNER, R. E., FAMULSKI, K. S. & MICHALAK, M. (1992) Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells. Mol. Cell. Biochem. 112, 1–13.

    Google Scholar 

  • MORAVEC, C. S. & BOND, M. (1991) Calcium is released from the junctional sarcoplasmic reticulum during cardiac muscle contraction. Am. J. Physiol. 260, H989–97.

    Google Scholar 

  • MOUREY, R. J., VERMA, A., SUPATTONE, S. & SNYDER, S. (1990) Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens. Biochem. J. 272, 383–9.

    Google Scholar 

  • Nixon, G. F., Mignery, G. A., Südhof, T. C. & Somlyo, A. V. (1994) Immunogold localization of calsequestrin and inositol 1,4,5-trisphosphate receptors in smooth muscle. Biophys. J. 66 A97 (abstract).

    Google Scholar 

  • OSTWALD, T. J. & MACLENNAN, D. H. (1974) Isolation of a high affinity calcium binding protein. J. Biol. Chem. 249, 974–9.

    Google Scholar 

  • RAEYMAEKERS, L., VERBIST, J., WUYTACK, F., PLESSERS, L. & CASTEELS, R. (1993) Expression of Ca2+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscle. Cell Calcium 14, 581–9.

    Google Scholar 

  • RENARD-ROONEY, D. C., HAJNOCZKY, G., SEITZ, M. B., SCHNEIDER, T. M. & THOMAS, A. P. (1993) Imaging of inositol 1,4,5-trisphosphate-induced Ca2+ fluxes in single permeabilized hepatocytes. J. Biol. Chem. 268, 23601–10.

    Google Scholar 

  • RIZZUTO, R., BRINI, M., MURGIA, M. & POZZAN, T. (1993) Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 262, 744–7.

    Google Scholar 

  • SLOT, J. W., POSTHUMA, G., CHANG, L-Y., CRAPO, J. D. & GEUZE, H. J. (1989) Quantitative aspects of immunogold labeling in embedded and in nonembedded sections. Am. J. Anatomy 185, 271–81.

    Google Scholar 

  • SOMLYO, A. P. (1984) Cellular site of calcium regulation. Nature 308, 516–17.

    Google Scholar 

  • SOMLYO, A. P. & HIMPENS, B. (1989) Cell calcium and its regulation in smooth muscle. FASEB J. 3, 2266–76.

    Google Scholar 

  • SOMLYO, A. P. & SOMLYO, A. V. (1968) Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Pharmacol. Exp. Ther. 159, 129–45.

    Google Scholar 

  • SOMLYO, A. P. & SOMLYO, A. V. (1990) Flash photolysis studies of excitation contraction coupling, regulation, and contraction in smooth muscle. Ann. Rev. Physiol. 52, 857–74.

    Google Scholar 

  • SOMLYO, A. P., DEVINE, C. E., SOMLYO, A. V. & NORTH, S. R. (1971) Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium free solutions. J. Cell Biol. 51, 722–41.

    Google Scholar 

  • SOMLYO, A. P., SOMLYO, A. V., DEVINE, C. E., PETERS, P. D. & HALL, T. A. (1974) Electron microscopy and electron probe analysis of mitochondrial cation accumulation in smooth muscle. J. Cell Biol. 61, 723–42.

    Google Scholar 

  • SOMLYO, A. P., SOMLYO, A. V. & SHUMAN, H. (1979) Electron probe analysis of vascular smooth muscle: Composition of mitochondria, nuclei and cytoplasm. J. Cell Biol. 81, 316–44.

    Google Scholar 

  • SOMLYO, A. P., URBANICS, R., VADASZ, G., KOVACH, A. G. B. & SOMLYO, A. V. (1985c) Mitochondrial calcium and cellular electrolytes in brain cortex frozen in situ: electron probe analysis. Biochem. Biophys. Res. Commun. 132, 1071–8.

    Google Scholar 

  • SOMLYO, A. P., WALKER, J. W., GOLDMAN, Y. E., TRENTHAM, D. R., KOBAYASHI, S., KITAZAWA, T. & SOMLYO, A. V. (1988) Inositol trisphosphate, calcium and muscle contraction. Phil. Trans. R. Soc. Lond. B 320, 399–414.

    Google Scholar 

  • SOMLYO, A. V. (1979) Bridging structures spanning the junctional gap at the triad of skeletal muscle. J. Cell. Biol. 80, 743–50.

    Google Scholar 

  • SOMLYO, A. V. (1980) Ultrastructure of vascular smooth muscle. In The Handbook of Physiology. The Cardiovascular System: Vol. II Vascular Smooth Muscle (edited by BOHR, D. F., SOMLYO, A. P. & SPARKS, H. V.), pp. 33–67, Bethesda, MD: American Physiological Society.

    Google Scholar 

  • SOMLYO, A. V. & FRANZINI-ARMSTRONG, C. (1985) New views of smooth muscle structure using freezing, deep-etching and rotary shadowing. Experientia 41, 841–56.

    Google Scholar 

  • SOMLYO, A. V., BOND, M., SOMLYO, A. P. & SCARPA, A. (1985a) Inositol trisphosphate calcium release and contraction in vascular smooth muscle. Proc. Natl. Acad. Sci. USA 82, 5231–5.

    Google Scholar 

  • SOMLYO, A. V., MCCLELLAN, G., GONZALEZ-SERRATOS, H. & SOMLYO, A. P. (1985b) Electron probe X-ray microanalysis of post tetanic Ca and Mg movements across the sarcoplasmic reticulum in situ. J. Biol. Chem. 260, 6801–7.

    Google Scholar 

  • SOMLYO, A. V., HORIUTI, K., TRENTHAM, D. R. KITAZAWA, T. & SOMLYO, A. P. (1992) Kinetics of Ca2+ release and contraction induced by photolysis of caged D-myo-inositol 1,4,5-Trisphosphate in smooth muscle. J. Biol. Chem. 267, 22316–22.

    Google Scholar 

  • SOMMER, J. R. & JOHNSON, E. A. (1979) Ultrastructure of cardiac muscle. In Handbook of Physiology. Section 2: The Cardiovascular System. Vol. 1. The Heart (edited by BERNE, R. M., SPERELAKIS, N. & GEIGER, S. R.) pp. 113–87. Bethesda, MD: American Physiological Society.

    Google Scholar 

  • SZOLLOSI, D. & HUNTER, R. H. F. (1973) A special attachment complex between the rough endoplasmic reticulum and mitochondria in porcine oocytes. J. Microscopie 16, 105–10.

    Google Scholar 

  • TAKEI, K., MIGNERY, G. A., MUGNIANI, E., SÜDHOF, T. C. & CAMILLI, P.De (1994) Inositol 1,4,5-trisphosphate receptor causes formation of ER cisternal stacks in transfected fibroblasts and in cerebellar Purkinje Cells. Neuron 12, 327–42.

    Google Scholar 

  • THARIN, S., DZIAK, E., MICHALAK, M. & OPAS, M. (1992) Wide-spread tissue distribution of rabbit calreticulin, a non-muscle functional analogue of calsequestrin. Cell Tissue. Res. 269, 29–37.

    Google Scholar 

  • TOKUYASU, K. T. (1973) A technique for ultracryomicrotomy of cell suspensions and tissues. J. Cell Biol. 57, 551–65.

    Google Scholar 

  • TOKUYASU, K. T. (1989) Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem. J. 21, 163–71.

    Google Scholar 

  • TOKUYASU, K. T. & SINGER, S. J. (1976) Improved procedures for immunoferritin labeling of ultrathin frozen sections. J. Cell Biol. 71, 894.

    Google Scholar 

  • Van BERGEN EN HENEGOUWEN, P. M. P. (1989) Immunogold labeling of ultrathin cryosections. In: Colloidal Gold. Principles, Methods and Applications, Vol. 1. (edited by HAYAT, M. A.) pp. 191–216. San Diego, CA: Academic Press.

    Google Scholar 

  • VILLA, A., PODINI, P., CLEGG, D. O., POZZAN, T. & MELDOLESI, J. (1991) Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of ER lumenal protein, Bip. J. Cell Biol. 113, 779–91.

    Google Scholar 

  • VILLA, A., PODINI, P., PANZERI, M. C., SOLING, H. D., VOLPE, P. & MELDOLESI, J. (1993) The endoplasmic-sarcoplasmic reticulum of smooth muscle: Immunocytochemistry of vas deferens fibers reveals specialized subcompartments differently equipped for control of Ca2+ homeostasis. J. Cell Biol. 121, 1041–51.

    Google Scholar 

  • WAUGH, R. A. & SOMMER, J. R. (1974) Lamellar junctional sarcoplasmic reticulum. A specialization of cardiac sarcoplasmic reticulum. J. Cell Biol. 63, 337–43.

    Google Scholar 

  • WIBO, M. & GODFRAIND, T. (1994) Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study. Biochem. J. 297, 415–23.

    Google Scholar 

  • WUYTACK, F., RAEYMAEKERS, L., VERBIST, J., JONES, L. R. & CASTEELS, R. (1987) Smooth muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin. Biochem. Biophys. Acta 899, 151–8.

    Google Scholar 

  • YAMAMOTO, T., IINO, M., YAMAZAWA, T. & ENDO, M. (1991) Caffeine-sensitive and-insensitive intracellular calcium stores in vascular smooth muscle cells of the guinea pig. J. Vasc. Med. Biol. 3, 154–60.

    Google Scholar 

  • YAMAZAWA, T., IINO, M. & ENDO, M. (1992) Presence of functionally different compartments of the Ca2+ store in single intestinal smooth muscle cells. FEBS Lett. 301, 181–4.

    Google Scholar 

Download references

Author information

Author notes

  1. Gregory A. Mignery

    Present address: Department of Physiology, Loyola University, Chicago, IL, USA

Authors and Affiliations

  1. Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, 22908, Charlottesville, Virginia, USA

    Graeme F. Nixon & Avril V. Somlyo

  2. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Gregory A. Mignery

Authors
  1. Graeme F. Nixon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gregory A. Mignery
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Avril V. Somlyo
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nixon, G.F., Mignery, G.A. & Somlyo, A.V. Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle. J Muscle Res Cell Motil 15, 682–700 (1994). https://doi.org/10.1007/BF00121075

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00121075

Keywords

Search

Navigation