Summary
The uptake in vitro of horseradish peroxidase (HRP) in mouse skeletal muscle was examined by electron microscopy and chemical determination.
In muscles exposed to an HRP solution for 60 min at +37°C, HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition HRP was found within the transverse tubules. Exposure to HRP for 30 min at +37°C followed by HRP together with a polycationic protein (protamine) for 30 min at +37°C caused an intracellular vesicular uptake of HRP. Intracellular HRP was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular HRP uptake or muscle cell vacuolation could not be detected in the absence of protamine or when the incubation temperature was + 4°C. Chemical determination of HRP uptake was in general agreement with the morphological results. The uptake of HRP in the presence of protamine was stimulated at +31°C and blocked at +4°C.
The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis.
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References
Beck, F., Lloyd, J.B.: Histochemistry and electron microscopy of lysosomes. In: Lysosomes in biology and pathology, 2. (J.T. Dingle and H.B. Fell, eds.). Amsterdam: North-Holland Publishing Company 1969
Bessis, M.: Studies on cell agony and death: an attempt at classification. Ciba Found. Symp. Cell injury 287–316 (1964)
Bowers, B.: Coated vesicles in the pericardial cells of the aphid. Protoplasma 59, 351–367 (1964)
Douglas, W.W., Nagasawa, J., Schultz, R.A.: Coated microvesicles in neurosecretory terminals of posterior pituitary glands shed their coats to become smooth “synaptic” vesicles. Nature (Lond.) 232, 340–341 (1971)
Ericsson, J.L.E.: Mechanism of cellular autophagy. In: Lysosomes in biology and pathology, 2. (J.T. Dingle and H.B. Fell, eds.). Amsterdam: North-Holland Publishing Company 1969
Garfield, R.E., Chacko, S., Blose, S.: Phagocytosis by muscle cells. Lab. Invest. 33, 418–427 (1975)
Graham, R.C. Jr., Karnovsky, M.J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291–302 (1966)
Heuser, J.E., Reese, T.S.: Evidence for recycling of synapting vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 (1973)
Kornguth, S.E., Stahmann, M.A., Anderson, J.W.: Effect of polylysine on the cytology of Ehrlich ascites tumour cells. Exp. Cell Res. 24, 484–494 (1961)
Libelius, R.: Binding of 3H-labelled cobra neurotoxin to cholinergic receptors in fast and slow mammalian muscles. J. Neural Trans. 35, 137–149 (1974)
Libelius, R.: Evidence for endocytotic uptake of cobra neurotoxin in mouse skeletal muscle. J. Neural Trans. 37, 61–71 (1975)
Libelius, R., Eaker, D., Karlsson, E.: Further studies on the binding properties of cobra neurotoxin to cholinergic receptors in mouse skeletal muscle. J. Neural Trans. 37, 165–174 (1975)
Liley, A.W.: An investigation of spontaneous activity of the neuromuscular junction of the rat. J. Physiol. (Lond.) 132, 650–666 (1956)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Lundquist, I., Josefsson, J.-O.: Sensitive method for determination of peroxidase activity in tissue by means of coupled oxidation reaction. Analyt. Biochem. 41, 567–577 (1971)
Mayhew, E., Harlos, J.P., Juliano, R.L.: The effect of polycations on cell membrane stability and transport processes. J. Membrane Biol. 14, 213–228 (1973)
Morki, B., Engel, A.G.: Duchenne dystrophy: Electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscle fibre. Neurology (Minneap.) 25, 1111–1120 (1975)
Nagasawa, J., Douglas, W.W., Schultz, R.A.: Micropinocytotic origin of coated and smooth microvesicles (“Synaptic vesicles”) in neurosecretory terminals of posterior pituitary glands demonstrated by incorporation of horseradish peroxidase. Nature (Lond.) 232, 341–342 (1971)
Oudea, P.R.: Anoxic changes of liver cells. Lab. Invest. 12, 386–394 (1963)
Rosenbluth, J., Wissig, S.L.: The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons. J. Cell Biol. 23, 307–325 (1964)
Ryser, H.J.-P.: Studies on protein uptake by isolated tumor cells. III. Apparent stimulations due to pH, hypertonicity, polycations, or dehydration and their relation to the enhanced penetration of infectious nucleic acids. J. Cell Biol. 32, 737–750 (1967)
Ryser, H., Caulfield, J.B., Aub, J.C.: Studies on protein uptake by isolated tumor cells. I. Electron microscopic evidence of ferritin uptake by Ehrlich ascites tumor cells. J. Cell Biol. 14, 255–268 (1962)
Ryser, H.J.-P., Hancock, R.: Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 150, 501–503 (1965)
Schiaffino, S., Hanzlikova, V.: Studies on the effect of denervation in developing muscle. II. The lysosomal system. J. Ultrastruct. Res. 39, 1–14 (1972)
Straus, W.: Methods for the study of small phagosomes and their relationship to lysosomes with horseradish peroxidase as a “marker protein”. J. Histochem. Cytochem. 15, 375–380 (1967)
Williams, M.C., Wissig, S.L.: The permeability of muscle capillaries to horseradish peroxidase. J. Cell Biol. 66, 531–555 (1975)
Yang, W.C.T., Strasser, F.F., Pomerat, C.M.: Mechanism of drug-induced vacuolization in tissue culture. Exp. Cell Res. 38, 495–506 (1965)
Zacks, S.I., Saito, A.: Uptake of exogenous horseradish peroxidase by coated vesicles in mouse neuromuscular junctions. J. Histochem. Cytochem. 17, 161–170 (1969)
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Jirmanová, I., Libelius, R., Lundquist, I. et al. Protamine induced intracellular uptake of horseradish peroxidase and vacuolation in mouse skeletal muscle in vitro. Cell Tissue Res. 176, 463–473 (1977). https://doi.org/10.1007/BF00231402
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DOI: https://doi.org/10.1007/BF00231402