Skip to main content
Log in

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells

A freeze-fracture cytochemical investigation

  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Summary

The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3-β-hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.

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

Access this article

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

  • Bittman R (1978) Sterol-polyene antibiotic complexation: probe of membrane structure. Lipids 13:686–691

    Google Scholar 

  • Branton D, Bullivant S, Gilula NB, Karnovsky MJ, Moor H, Mühlethaler K, Northcote DH, Packer L, Satir B, Satir P, Speth V, Staehelin LA, Steere RL, Weinstein RS (1975) Freeze-etching nomenclature. Science 190:54–56

    Google Scholar 

  • Bray DF, Rayns DG, Wagenaar EB (1978) Intramembrane particle densities in freeze-fractured sarcoplasmic reticulum. Canad J Zool 56:140–145

    Google Scholar 

  • Coleman R (1968) Some features of the lipid composition of rat liver surface and cytoplasmic membranes. Chem Phys Lipids 2:144–146

    Google Scholar 

  • Deamer DW, Baskin RJ (1969) Ultrastructure of sarcoplasmic reticulum preparations. J Cell Biol 42:296–307

    Google Scholar 

  • De Kruijff B, Gerritsen WJ, Oerlemans A, Van Dijk PWM, Demel RA, Van Deenen LLM (1974) Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecthin liposomes. II. Temperature dependence of the polyene antibiotic-sterol complex formation. Biochim Biophys Acta 339:44–56

    Google Scholar 

  • Demel RA, De Kruyff B (1976) The function of sterols in membranes. Biochim Biophys Acta 457:109–132

    Google Scholar 

  • Ebashi S, Endo M (1968) Calcium ion and muscle contraction. Prog Biophys Mol Biol 18:123–183

    Google Scholar 

  • Elias PM, Friend DS, Goerke J (1979) Membrane sterol heterogeneity. Freeze-fracture detection with saponins and filipin. J Histochem Cytochem 27:1247–1260

    Google Scholar 

  • Fiehn W, Peter JB, Mead JF, Gan-Elapano M (1971) Lipids and fatty acids of sarcolemma, sarcoplasmic reticulum and mitochondria from rat skeletal muscle. J Biol Chem 246:5617–5620

    Google Scholar 

  • Franzini-Armstrong C (1975) Membrane particles and transmission at the triad. Fed Proc 34:1382–1389

    Google Scholar 

  • Friend DS, Havel CM, Silberklang M, Watson JA (1980) Distribution and effects of cholesterol on 3-β-hydroxysterol-free eukaryotic cells. J Cell Biol 87:196a

    Google Scholar 

  • Headon DR, Barrett EJ, Joyce NM, O'Flaherty J (1977) Cholesterol in muscle membranes. Mol Cell Biochem 17:117–123

    Google Scholar 

  • Heusson-Stiennon J-A, Wanson JC, Drochmans P (1972) Isolation and characterization of the sarcoplasmic reticulum of skeletal muscle. J Cell Biol 55:471–488

    Google Scholar 

  • Jain MH (1975) Role of cholesterol in biomembranes and related systems. Curr Top Membr Transp 6:1–57

    Google Scholar 

  • Kitajima Y, Sekiya T, Nozawa Y (1976) Freeze-fracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasma membranes of Epidermophyton, Saccharomyces and red blood cells. A proposal for polyene-ergosterol complex-induced membrane lesions. Biochim Biophys Acta 445:452–465

    Google Scholar 

  • Klein I, Moore L, Pastan I (1978) Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts. Biochim Biophys Acta 506:42–53

    Google Scholar 

  • Lau YH, Caswell AH, Brunschwig J-P, Baerwald RJ, Garcia M (1979) Lipid analysis and freeze-fracture studies on isolated transverse tubules and sarcoplasmic reticulum subfractions of skeletal muscle. J Biol Chem 254:540–546

    Google Scholar 

  • MacLennan DH (1975) Resolution of the calcium transport system of sarcoplasmic reticulum. Canad J Biochem 53:251–261

    Google Scholar 

  • Madden TD, Chapman D, Quinn PJ (1979) Cholesterol modulates activity of calcium-dependent ATPase of the sarcoplasmic reticulum. Nature 279:538–541

    Google Scholar 

  • Mas-Oliva J (1980) The calcium transporting properties of the cardiac muscle cell membrane. PhD Thesis, University of London

  • Mathias RT, Levis RA, Eisenberg RS (1980) Electrical models of excitation-contraction coupling and charge movement in skeletal muscle. J Gen Physiol 76:1–31

    Google Scholar 

  • Meissner G (1975) Isolation and characterization of two types of sarcoplasmic reticulum vesicles. Biochim Biophys Acta 389:51–68

    Google Scholar 

  • Montesano R, Perrelet A, Vassalli P, Orci L (1979) Absence of filipin-sterol complexes from large coated pits on the surface of culture cells. Proc Natl Acad Sci USA 76:6391–6395

    Google Scholar 

  • Montesano R, Vassalli P, Perrelet A, Orci L (1980) Distribution of filipin-cholesterol complexes at sites of exocytosis — a freeze-fracture study of degranulating mast cells. Cell Biol Int Rep 4:975–984

    Google Scholar 

  • Morré DJ (1977) The Golgi apparatus and membrane biogenesis. In: Poste G, Nicolson GL (eds) The synthesis, assembly and turnover of cell surface components. Cell Surface Reviews Vol 4. ElsevierNorth-Holland Biomedical Press, Amsterdam, p 1–83

    Google Scholar 

  • Nakajima Y, Bridgman PC (1981) Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions. J Cell Biol 88:453–458

    Google Scholar 

  • Orci L, Miller RG, Montesano R, Perrelet A, Amherdt M (1980 a) Opposite polarity of filipin-induced deformations in the membrane of condensing vacuoles and zymogen granules. Science 210:1019–1021

    Google Scholar 

  • Orci L, Montesano R, Brown D (1980 b) Heterogeneity of toad bladder granular cell luminal membranes. Distribution of filipin-sterol complexes in freeze-fracture. Biochim Biophys Acta 601:443–452

    Google Scholar 

  • Orci L, Montesano R, Meda P, Malaisse-Lagae F, Brown D, Perrelet A, Vassalli P (1981 a) Heterogeneous distribution of filipin-cholesterol complexes across the cisternae of the Golgi apparatus. Proc Natl Acad Sci USA 78:293–297

    Google Scholar 

  • Orci L, Singh A, Amherdt M, Brown D, Perrelet A (1981 b) Microheterogeneity of protein and sterol content in kidney podocyte membrane. Nature 293:646–647

    Google Scholar 

  • Podolsky RJ, Hall T, Hatchett SL (1970) Identification of oxalate precipitates in striated muscle fibres. J Cell Biol 44:699–702

    Google Scholar 

  • Robenek H, Greven H (1981) Freeze-fracture evidence for high cholesterol content in nuclear membranes of a larval urodelan epidermis. Eur J Cell Biol 25:131–135

    Google Scholar 

  • Robenek H, Jung W, Gebhardt R (1982) The topography of filipin-cholesterol complexes in the plasma membrane of cultured hepatocytes and their relation to cell junction formation. J Ultrastruct Res 78:95–106

    Google Scholar 

  • Robinson JM, Karnovsky MJ (1980) Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J Histochem Cytochem 28:161–168

    Google Scholar 

  • Ryan DM, Shafiq SA (1980) A freeze-fracture study of the anterior and posterior latissimus dorsi muscles of the chicken. Anat Rec 198:147–161

    Google Scholar 

  • Sekiya T, Kitajima Y, Nozawa Y (1979) Effects of lipid phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells, as studied by freeze-fracture electron microscopy. Biochim Biophys Acta 550:269–278

    Google Scholar 

  • Severs NJ (1981 a) Freeze-fracture detection of cholesterol in filipin-treated cardiac muscle cell membranes. J Mol Cell Cardiol 13 (Suppl) 1:86

    Google Scholar 

  • Severs NJ (1981 b) Plasma membrane cholesterol in myocardial muscle and capillary endothelial cells. Distribution of filipin-induced deformations in freeze-fracture. Eur J Cell Biol 25:289–299

    Google Scholar 

  • Severs NJ (1981 c) Localization of cholesterol in the Golgi apparatus of cardiac muscle cells. Experientia 37:1195–1198

    Google Scholar 

  • Severs NJ, Warren RC, Barnes SH (1981) Analysis of membrane structure in the transitional epithelium of rat urinary bladder. 3. Localization of cholesterol using filipin and digitonin. J Ultrastruct Res 77:160–188

    Google Scholar 

  • Sommer JR, Johnson EA (1979) Ultrastructure of cardiac muscle. In: Berne RM, Sperelakis N, Geiger SR (eds) Handbook of Physiology Section 2: The cardiovascular system, Vol 1, The heart. American Physiological Society, Bethesda Maryland, p 113–186

    Google Scholar 

  • Sommer JR, Waugh RA (1976) The ultrastructure of the mammalian cardiac muscle cell —with special emphasis on the tubular membrane systems. Am J Pathol 82:192–211

    Google Scholar 

  • Sommer JR, Dolber PC, Taylor I (1980 a) Filipin-cholesterol complexes in the sarcoplasmic reticulum of frog skeletal muscle. J Ultrastruct Res 72:272–285

    Google Scholar 

  • Sommer JR, Wallace NR, Junker J (1980 b) The intermediate cisterna of the sarcoplasmic reticulum of skeletal muscle. J Ultrastruct Res 71:126–142

    Google Scholar 

  • Tillack TW, Kinsky SC (1973) A freeze-etch study of the effects of filipin on liposomes and human erythrocyte membranes. Biochim Biophys Acta 323:43–54

    Google Scholar 

  • Verkleij AJ, De Kruijff B, Gerritsen WF, Demel RA, Van Deenen LLM, Ververgaert PHJ (1973) Freeze-etch electron microscopy of erythrocytes, Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B. Biochim Biophys Acta 291:577–581

    Google Scholar 

  • Winegrad S (1965) Autoradiographic studies of intracellular calcium in frog skeletal muscle. J Gen Physiol 48:455–479

    Google Scholar 

  • Winegrad S (1970) The intracellular site of calcium activation of contraction in frog skeletal muscle. J Gen Physiol 55:77–88

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Severs, N.J. Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells. Cell Tissue Res. 224, 613–624 (1982). https://doi.org/10.1007/BF00213756

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00213756

Key words

Navigation