Cell and Tissue Research
Cell Tiss. Res. 176, 463-473 (1977)
9 by Springer-Verlag 1977
Protamine Induced Intracellular Uptake of Horseradish Peroxidase and Vacuolation in Mouse Skeletal Muscle in vitro* Isa Jirmanovfi, R. Libelius, I. Lundquist, and S. Thesleff Institute of Physiology, CzechoslovakAcademyof Sciences, Prague and the Department of Pharmacology, University of Lund, Sweden
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 H R P solution for 60 rain at + 3 7 ~ HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition H R P was found within the transverse tubules. Exposure to H R P for 30 rain at + 3 7 ~ followed by H R P together with a polycationic protein (protamine) for 30 rain at + 3 7 ~ caused an intracellular vesicular uptake of HRP. Intracellular H R P was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular H R P 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 H R P uptake was in general agreement with the morphological results. The uptake of H R P in the presence of protamine was stimulated at + 3 7 ~ and blocked at + 4~ The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis. Key words: Skeletal muscle - Protamine vacuolation - Electron microscopy.
Endocytosis -
Autophagic
Introduction Earlier studies on the binding of a cobra (Naja naja siamensis) c~-neurotoxin, m.w. 7820, to the mouse extensor digitorum longus (EDL) muscle in vitro have Send offprint requests to: Dr. Roll Libelius, Department of Pharmacology, University of Lund, S-22362 Lund, Sweden Acknowledgements. The study was carried out under the auspicies of the Czechoslovak Academy of Sciences and the Royal Academy of Sciences in Sweden. The work was supported by grants from the Medical Faculty, Universityof Lund, Sweden, from the Swedish Medical Research Council, Stockholm, Sweden (14X-3112, 14X-4286, O4P-4289) and from Muscular Dystrophy Association of America, Inc.
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s h o w n a specific, saturable a n d irreversible b i n d i n g to cholinergic receptors a n d also an unspecific, u n s a t u r a b l e a n d irreversible b i n d i n g of the toxin to the muscle tissue (Libelius, 1974; Libelius, 1975; Libelius e t a l . , 1975). The unspecific b i n d i n g of toxin was stimulated at + 3 7 ~ by polycationic proteins ( p r o t a m i n e , histone a n d polylysine) a n d blocked by low t e m p e r a t u r e ( + 4 ~ (Libelius, 1975). Since other investigators have shown that such proteins stimulate endocytosis in isolated t u m o r cells in culture (Ryser a n d H a n c o c k , 1965) it was suggested that the unspecific b i n d i n g o f toxin in muscle tissue could be due to a n intracellular vesicular u p t a k e of this m a c r o m o l e c u l e (Libelius, 1975). P o l y c a t i o n i c proteins n o t only cause a n increase in endocytosis b u t m a y also induce cell d a m a g e a n d v a c u o l a t i o n at high c o n c e n t r a t i o n s a n d / o r long time of exposure ( K o r n g u t h et al., 1961; M a y h e w et al., 1973; Ryser, 1967). W h e t h e r these vacuoles are related to endocytosis is u n k n o w n , however, they are larger in size t h a n m i c r o p i n o c y t o t i c vesicles a n d appear to occur in response to a variety of n o x i o u s cell stimuli (Bessis, 1964; Oudea, 1963; Ryser et al., 1962; Y a n g et al., 1965). I n the present study we have e x a m i n e d the E D L muscle of the m o u s e for m o r p h o l o g i c a l correlates to the increased u p t a k e of m a c r o m o l e c u l e s i n d u c e d by p r o t a m i n e in vitro. As a m a c r o m o l e c u l a r m a r k e r of the extra-cellular fluid we used H R P because it is untoxic, a n d essentially restricted to vesicular transport m e c h a n i s m s (cf. Beck a n d Lloyd, 1969; Straus, 1967) a n d is suitable for b o t h histochemical ( G r a h a m a n d K a r n o v s k y , 1966) a n d chemical ( L u n d q u i s t a n d Josefsson, 1971) detection.
Material and Methods All experiments were performed on innervated extensor digitorum longus (EDL) muscles from adult male NMRI mice, weighing about 30 g. Immediately after sacrificing the animal the EDL muscles from both legs were removed and kept in an oxygenated salt solution (Liley, 1956). The muscles were then mounted at about normal resting tension on a perspex holder for subsequent incubation as previously described (Libelius, 1975). Incubation Solutions. Oxygenated salt solution containing HRP (1 mg/ml) with and without prota-
mine (60 ~/ml) was used. In control experiments oxygenated salt solution containing HRP (inactivated by boiling for 3 h) with or without protamine (60 gg/ml), or oxygenated salt solution with only protamine (60 gg/ml) were used. Incubation Procedure. For morphological experiments the following incubation procedures were
used: HRP for 60 min at +37~ HRP for 30 min followed by HRP+protamine for 30 min at +37 ~ C or HRP+protamine for 60 rain at +4 ~ C. Controls were incubated in inactivated HRP +protamine for 60 min at +37~ or in protamine solution without HRP for 60 min at +37~ Muscles used for quantitative chemical assay of HRP uptake were incubated identically to those of the morphological study except that inactivated HRP was not used. Controls were untreated or incubated in protamine for 60 min at +37~ to estimate the endogenous peroxidase activity (Lundquist and Iosefsson, 1971). After incubation the muscles were washed in HRP-free oxygenated salt solution with 4 changes for 60 rain each at +4~ (4x 1 h). Histochemical and Embedding Procedures. Following incubation the muscles were, when still mounted
on the holder, transferred into a fixative (1% paraformaldehyde and 1% glutaraldehyde solution in phosphate buffer, pH 7.3) and fixed for 2 h at +4~ After fixation the muscles were sliced with a razor blade and the slices (about 1-2 mm thick) rinsed overnight in a buffered dextrose
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solution (pH 7.3) at +4~ The tissue blocks were incubated in a saturated solution of 3,3'diaminobenzidine tetrahydrochloride in 0.l M phosphate buffer (pH 7.3) for 30 rain at room temperature (22-24~ further 30 min after the addition of H202 to a final concentration of 0.01%. Following the histochemical incubation the tissue blocks were briefly washed in a buffered dextrose solution and postfixed in a buffered 2% solution of osmium tetroxide for 2.5 h. The material was thereafter dehydrated and embedded in Epon: After inspection of unstained and stained (a mixture of 1% azure, 1% methylene blue solution in 1% sodium borate) semithin sections by light microscopy, ultrathin sections of selected areas were cut. Ultrathin sections, unstained or stained with uranyl acetate, lead citrate or a combination of these stains were examined in a Philips 300 electron microscope.
Chemical Assay of HRP. After HRP incubation and washing for 4• 1 h at +4~
muscles were transferred to ice-cold 0.3 M mannitol solution and homogenized in an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) while chilled with crushed ice. Determination of HRP activity was performed according to the method of Lundquist and Josefsson (1971), which made it possible to analyze peroxidase at concentrations as low as 1 ng/rnl. Protein determination was performed with the method of Lowry et al. (1951).
Chemicals. 3,3'-diaminobenzidine tetrahydrochloride (DAB) and horseradish peroxidase Type VI (Sigma Chemical Co., St. Louis, Mo.). Protamine sulphate (clupein-type, m.w. about 8100, Vitrurn AB). Statistical evaluation was made using Student's t-test. P-values < 0.001 were considered statistically significant.
Results Light Microscopy Semithin transversal sections o f muscles treated with H R P + p r o t a m i n e or prota m i n e alone at + 3 7 ~ displayed a mosaic p a t t e r n o f changes. Some of the fibres, preferentially in the periphery of the muscle, were v a c u o l a t e d (Fig. 1) b u t most of the fibres were intact. A few fibres showed areas of rarefaction located either close to the fibre surface or in the depth of the fibre. L o n g i t u d i n a l sections showed focal v a c u o l a t i o n in otherwise u n a l t e r e d fibres (Fig. 2). The size of vacuoles varied between fibres with the largest sizes in fibres showing signs of rarefaction i n d i c a t i n g a progressive change from v a c u o l a t i o n to fibre d e g e n e r a t i o n a n d necrosis. V a c u o l a t i o n was n o t seen after i n c u b a t i o n in H R P alone or in H R P + p r o t a m i n e at + 4 ~ C. Thus i n c u b a t i o n at low t e m p e r a t u r e ( + 4 ~ C) protected the muscle fibres against the v a c u o l a t i n g effect of p r o t a m i n e . I n muscles i n c u b a t e d in H R P + p r o t a m i n e the extracellular space a n d preferentially the m e m b r a n e of superficial fibres were labelled with H R P as indicated by the d a r k - b r o w n o x i d a t i o n p r o d u c t of DAB. In vacuolated fibres the b r o w n reaction p r o d u c t was present at the i n n e r circumference of some vacuoles. Fibres with regions of rarefaction showed diffuse staining, p r e s u m a b l y due to p l a s m a m e m b r a n e d i s r u p t i o n , a n d they were excluded from further e x a m i n a t i o n . Muscles i n c u b a t e d in H R P w i t h o u t p r o t a m i n e showed labelling o f the extracellular space. N o H R P c o u l d be detected inside the fibres. Similar results were o b t a i n e d for muscles i n c u b a t e d in H R P + p r o t a m i n e at + 4 ~ C. C o n t r o l muscles i n c u b a t e d in h e a t - i n a c t i v a t e d H R P + p r o t a m i n e or in a solution with p r o t a m i n e only showed n o H R P labelling of muscle fibre m e m b r a n e s
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Fig. 1. Cross section of protamine-treated ( + 37~ C) muscle fibres. N u m e r o u s vacuoles in superficial cells. Stained section, x 560 Fig. 2. Longitudinal section of protamine and H R P treated ( + 3 7 ~ C) muscle fibres. Segmental vacuolation of a superficial fibre. Dark-brown reaction product of D A B between fibres. Stained section, x 560
Fig. 3. Cross section of a protamine-treated fibre. Many H R P positive vesicles and bodies (some of which are denoted by arrows) are seen inside the muscle fibre. G Golgi region; n nucleus; m mitochondria. • 37,600 Fig, 4. Cross section of a protamine-treated fibre. N u m e r o u s vacuoles (V) are seen inside the muscle fibre cytoplasm. Note the vacuole lined by H R P (denoted by arrow), m mitochondria; t transverse tubule, x 23,500
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or extracellular space, whereas erythrocytes in capillaries showed a positive reaction most likely due to the peroxidatic activity of hemoglobin.
Electron Microscopy The exposure to protamine at + 37~ caused uptake of H R P in muscle fibres (Fig. 3) and intense cytoplasmic vacuolation (Fig. 4). Besides infiltrating the basal lamina and adsorbed on the sarcolemma H R P was found inside the fibres not only in transverse tubules but also in numerous vesicles, membrane limited bodies and vacuoles (Figs. 3-7). H R P containing structures were of different sizes and were scattered throughout the muscle cell cytoplasm. H R P positive vesicles and bodies seemed to be most numerous at the periphery of muscle fibres, immediately beneath the sarcolemma and in close vicinity to the nucleus and to Golgi cisternae (Figs. 5, 6). They were either completely filled with the reaction product or contained rounded less dense areas; in many of the H R P containing bodies the reaction product occupied only a narrow peripheral rim (Figs. 5-7). The structures shown in Figure 7 probably represent secondary lysosomes containing ingested HRP. in vacuoles which were either clear or contained a moderate amount of membranous or granular material, the reaction product was most frequently seen in the periphery (Fig. 4). Most of the larger clear vacuoles, outlined by a single membrane, did not contain H R P label. Muscle fibres incubated in H R P or H R P + p r o t a m i n e at + 4 ~ showed H R P infiltration of the basal lamina and H R P labelling of the sarcolemma and the transverse tubules but intracellular H R P positive structures as those shown in Figures 3-7 could not be detected. As with light microscopy, muscles incubated in medium with heat-inactivated HRP+_protamine or in protamine only showed no H R P labelling. No attempt was made to grade the density of the sarcolemma or the intracristal space of mitrochondria in DAB incubated control muscles compared to non-incubated specimens. Such a comparison would be difficult because of the small variation in contrast intensity usually present between different grids. Thus, the occasionally observed increased density of the sarcolemma and mitochondria in DAB incubated specimens as reported by other workers (Williams and Wissig, 1975) cannot be proved or disproved. However, no obvious reaction product was seen within these structures in uncontrasted sections.
Fig. 5. Cross section of a protamine-treated muscle fibre. Beneath the sarcolemma H R P is seen in vesicles and membrane limited bodies (denoted by arrows). The vacuole (V) also contains the reaction product. G Golgi region; m mitochondria, x 63,500 Fig. 6. Cross section of a protamine-treated muscle fibre. N u m e r o u s HRP positive vesicles and bodies (some of which are denoted by arrows) are seen in the region close to the nucleus (n). t transverse tubule; in mitochondria, x 63,500 Fig. 7. Cross section of a protamine-treated muscle fibre. Membrane limited bodies containing H R P (denoted by arrows) display clear areas and reaction product of a typical density is seen at their periphery, x 98,700
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~
Protomine 6 0 rnin or untrecJted muscles HRP 6 0 rain
HRP 3 0 rain * HRP and Protamine 3 0 rain
15s
HRP 9 Protc]mine 6 0 min
HRP + Protarnine 6 0 rNn { 4 ~
I.
12~ =
o -~ IO(
E
ii~i!ii!~i
E 5(
Control
37~
l
t-,-
40C -4
Fig. 8. Uptake of ttRP expressed as nanogram HRP/milligram muscle tissue protein (Mean +S.E.M.) (n= 5-6 muscles). Incubation conditions as indicated in the figure with special symbols. After incubation all muscles were washed in HRP-free solution 4 x 1 h at + 4~ C. Control denotes endogenous peroxidase activity of the muscle. Differences between all groups were statistically significant at p < 0.001
Chemical Determination F o l l o w i n g i n c u b a t i o n in H R P s o l u t i o n muscles were washed for 4 x 1 h at + 4 ~ to clear the e x t r a c e l l u l a r space f r o m H R P a n d thus to o b t a i n an a p p r o x i m a t e e s t i m a t i o n o f i n t r a c e l l u l a r H R P u p t a k e . As shown in F i g u r e 8, the u p t a k e o f H R P was s t i m u l a t e d by p r o t a m i n e when the i n c u b a t i o n t e m p e r a t u r e was + 37~ T h e s t i m u l a t i o n was m o r e p r o n o u n c e d when p r o t a m i n e was present d u r i n g 60 m i n o f i n c u b a t i o n t h a n when present d u r i n g 30 min. L o w e r i n g the t e m p e r a ture to + 4 ~ r e d u c e d H R P u p t a k e to values lower t h a n those in muscles w i t h o u t p r o t a m i n e at + 37 ~ Muscles i n c u b a t e d in p r o t a m i n e w i t h o u t H R P a n d w a s h e d for 4 x 1 h at + 4 ~ d i s p l a y e d a very low p e r o x i d a s e activity o f the same m a g n i t u d e as the e n d o g e n o u s p e r o x i d a s e activity f o u n d in u n t r e a t e d muscles (controls). Discussion T h e p r e s e n t study has s h o w n t h a t i n c u b a t i o n o f skeletal muscle with p r o t a m i n e at + 37~ causes i n t r a c e l l u l a r u p t a k e o f H R P . The o x i d a t i o n p r o d u c t o f D A B ,
Induced Uptake of HRP in Skeletal Muscle
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indicating peroxidase activity, was present in vesicles and membrane limited bodies and vacuoles inside the muscle fibres already after 30 rain exposure to protamine. Muscles without protamine did not show any detectable vesicular bound HRP uptake, HRP being present only in the membranes facing the extracellular fluid, i.e. in the basal lamina and the sarcolemma and in the transverse tubular system. Similarly, low temperature (+ 4 ~C) prevented vesicular uptake of HRP in response to protamine. The mechanisms related to the transport of HRP into muscle cells and subsequent accumulation of the protein tracer into organelles some of which probably represent secondary lysosomes requires further investigation. Previous electron microscopic studies have shown that coated vesicles are involved in protein uptake in many types of cells (Bowers, 1964; Rosenbluth and Wissig, 1964). Thus such vesicles are considered to be involved in the uptake of extracellular proteins at i.e. rat neuromuscular junctions (Heuser and Reese, 1973; Zacks and Saito, 1969) and neurosecretory terminals (Douglas et al., 1971; Nagasawa et al., 1971). Moreover, phagocytosis of latex particles was recently reported to occur in cultured muscle cells (Garfield et al., 1975). Further analysis is required, however, to verify a possible role of coated vesicles in the uptake of HRP into muscle fibres following protamine treatment. In support of the morphological observations chemical determinations of HRP showed about 90% increase of intracellular bound HRP in muscles after 30 min exposure to protamine at +37 ~ C, as compared to HRP without protamine. Low temperature (+ 4 ~C) blocked the increase in HRP, giving an approximate estimation of extracellularly bound HRP. It is notable, however, that without any apparent morphological correlate there was a significant increase of HRP in muscles incubated at +37~ even in the absence of protamine. This increase might be due to an increased amount of HRP being bound extracellularly at this higher temperature but also to an intracellular uptake process giving rise to local HRP-concentrations too small to be detected with the present morphological technique. Although much further evidence is needed to establish the presence of such a process in muscle tissue the present results might suggest the possibility of vesicular transport being a physiological uptake mechanism for macromolecules contained in the extracellular fluid. This is supported by a recent observation that 3H-inulin probably is taken up in skeletal muscle by a bulk uptake process (unpublished observation). Exposure to protamine induced vacuolation of muscle fibres. This effect was limited mostly to fibres in the periphery of the muscle. The fibres were frequently focally vacuolated with large unaffected areas between groups of vacuoles. The vacuoles were membrane limited, sometimes containing HRP label and in other instances containing remnants of myofibrous structures. The largest vacuoles were clear without content. When present in large numbers the vacuoles seemed to be part of a general degenerative process in the fibres resulting in sarcolemma disruption and final cell necrosis. The vacuolation process had many similarities with autophagic vacuoles in denervated muscle as described by Schiaffino and Hanzlikova (1972) and in other tissues (cf. Ericsson, 1969). Autophagic activity is generally ascribed to activation of lysosomal enzymes in response to either a direct chemical
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influence or as a result of enhanced endocytosis. Since we have demonstrated that protamine induces vesicular uptake of HRP it seems possible that this endocytosis results in lysosomal activation and subsequently in the formation of autophagic vacuoles, and at a later stage, in cell necrosis. The muscle fibres were affected in a mosaic pattern by protamine and when this led to large scale vacuolation, focal sarcolemma lesions occurred allowing HRP to enter diffusely into the sarcoplasm. It is of interest that in Duchenne dystrophy muscle fibres exhibit a similar pattern of focal degenerative changes with the formation of autophagic vacuoles and sarcolemma lesions (Morki and Engel, 1975). The possibility that processes, similar to those induced by protamine in normal muscle, occur in that type of disease should be considered. 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)
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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., Hanzfikova, 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. lfi, 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)
Accepted August 28, 1976
Cell Tiss. Res. 176, 463-473 (1977)
9 by Springer-Verlag 1977
Protamine Induced Intracellular Uptake of Horseradish Peroxidase and Vacuolation in Mouse Skeletal Muscle in vitro* Isa Jirmanovfi, R. Libelius, I. Lundquist, and S. Thesleff Institute of Physiology, CzechoslovakAcademyof Sciences, Prague and the Department of Pharmacology, University of Lund, Sweden
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 H R P solution for 60 rain at + 3 7 ~ HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition H R P was found within the transverse tubules. Exposure to H R P for 30 rain at + 3 7 ~ followed by H R P together with a polycationic protein (protamine) for 30 rain at + 3 7 ~ caused an intracellular vesicular uptake of HRP. Intracellular H R P was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular H R P 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 H R P uptake was in general agreement with the morphological results. The uptake of H R P in the presence of protamine was stimulated at + 3 7 ~ and blocked at + 4~ The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis. Key words: Skeletal muscle - Protamine vacuolation - Electron microscopy.
Endocytosis -
Autophagic
Introduction Earlier studies on the binding of a cobra (Naja naja siamensis) c~-neurotoxin, m.w. 7820, to the mouse extensor digitorum longus (EDL) muscle in vitro have Send offprint requests to: Dr. Roll Libelius, Department of Pharmacology, University of Lund, S-22362 Lund, Sweden Acknowledgements. The study was carried out under the auspicies of the Czechoslovak Academy of Sciences and the Royal Academy of Sciences in Sweden. The work was supported by grants from the Medical Faculty, Universityof Lund, Sweden, from the Swedish Medical Research Council, Stockholm, Sweden (14X-3112, 14X-4286, O4P-4289) and from Muscular Dystrophy Association of America, Inc.
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s h o w n a specific, saturable a n d irreversible b i n d i n g to cholinergic receptors a n d also an unspecific, u n s a t u r a b l e a n d irreversible b i n d i n g of the toxin to the muscle tissue (Libelius, 1974; Libelius, 1975; Libelius e t a l . , 1975). The unspecific b i n d i n g of toxin was stimulated at + 3 7 ~ by polycationic proteins ( p r o t a m i n e , histone a n d polylysine) a n d blocked by low t e m p e r a t u r e ( + 4 ~ (Libelius, 1975). Since other investigators have shown that such proteins stimulate endocytosis in isolated t u m o r cells in culture (Ryser a n d H a n c o c k , 1965) it was suggested that the unspecific b i n d i n g o f toxin in muscle tissue could be due to a n intracellular vesicular u p t a k e of this m a c r o m o l e c u l e (Libelius, 1975). P o l y c a t i o n i c proteins n o t only cause a n increase in endocytosis b u t m a y also induce cell d a m a g e a n d v a c u o l a t i o n at high c o n c e n t r a t i o n s a n d / o r long time of exposure ( K o r n g u t h et al., 1961; M a y h e w et al., 1973; Ryser, 1967). W h e t h e r these vacuoles are related to endocytosis is u n k n o w n , however, they are larger in size t h a n m i c r o p i n o c y t o t i c vesicles a n d appear to occur in response to a variety of n o x i o u s cell stimuli (Bessis, 1964; Oudea, 1963; Ryser et al., 1962; Y a n g et al., 1965). I n the present study we have e x a m i n e d the E D L muscle of the m o u s e for m o r p h o l o g i c a l correlates to the increased u p t a k e of m a c r o m o l e c u l e s i n d u c e d by p r o t a m i n e in vitro. As a m a c r o m o l e c u l a r m a r k e r of the extra-cellular fluid we used H R P because it is untoxic, a n d essentially restricted to vesicular transport m e c h a n i s m s (cf. Beck a n d Lloyd, 1969; Straus, 1967) a n d is suitable for b o t h histochemical ( G r a h a m a n d K a r n o v s k y , 1966) a n d chemical ( L u n d q u i s t a n d Josefsson, 1971) detection.
Material and Methods All experiments were performed on innervated extensor digitorum longus (EDL) muscles from adult male NMRI mice, weighing about 30 g. Immediately after sacrificing the animal the EDL muscles from both legs were removed and kept in an oxygenated salt solution (Liley, 1956). The muscles were then mounted at about normal resting tension on a perspex holder for subsequent incubation as previously described (Libelius, 1975). Incubation Solutions. Oxygenated salt solution containing HRP (1 mg/ml) with and without prota-
mine (60 ~/ml) was used. In control experiments oxygenated salt solution containing HRP (inactivated by boiling for 3 h) with or without protamine (60 gg/ml), or oxygenated salt solution with only protamine (60 gg/ml) were used. Incubation Procedure. For morphological experiments the following incubation procedures were
used: HRP for 60 min at +37~ HRP for 30 min followed by HRP+protamine for 30 min at +37 ~ C or HRP+protamine for 60 rain at +4 ~ C. Controls were incubated in inactivated HRP +protamine for 60 min at +37~ or in protamine solution without HRP for 60 min at +37~ Muscles used for quantitative chemical assay of HRP uptake were incubated identically to those of the morphological study except that inactivated HRP was not used. Controls were untreated or incubated in protamine for 60 min at +37~ to estimate the endogenous peroxidase activity (Lundquist and Iosefsson, 1971). After incubation the muscles were washed in HRP-free oxygenated salt solution with 4 changes for 60 rain each at +4~ (4x 1 h). Histochemical and Embedding Procedures. Following incubation the muscles were, when still mounted
on the holder, transferred into a fixative (1% paraformaldehyde and 1% glutaraldehyde solution in phosphate buffer, pH 7.3) and fixed for 2 h at +4~ After fixation the muscles were sliced with a razor blade and the slices (about 1-2 mm thick) rinsed overnight in a buffered dextrose
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solution (pH 7.3) at +4~ The tissue blocks were incubated in a saturated solution of 3,3'diaminobenzidine tetrahydrochloride in 0.l M phosphate buffer (pH 7.3) for 30 rain at room temperature (22-24~ further 30 min after the addition of H202 to a final concentration of 0.01%. Following the histochemical incubation the tissue blocks were briefly washed in a buffered dextrose solution and postfixed in a buffered 2% solution of osmium tetroxide for 2.5 h. The material was thereafter dehydrated and embedded in Epon: After inspection of unstained and stained (a mixture of 1% azure, 1% methylene blue solution in 1% sodium borate) semithin sections by light microscopy, ultrathin sections of selected areas were cut. Ultrathin sections, unstained or stained with uranyl acetate, lead citrate or a combination of these stains were examined in a Philips 300 electron microscope.
Chemical Assay of HRP. After HRP incubation and washing for 4• 1 h at +4~
muscles were transferred to ice-cold 0.3 M mannitol solution and homogenized in an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) while chilled with crushed ice. Determination of HRP activity was performed according to the method of Lundquist and Josefsson (1971), which made it possible to analyze peroxidase at concentrations as low as 1 ng/rnl. Protein determination was performed with the method of Lowry et al. (1951).
Chemicals. 3,3'-diaminobenzidine tetrahydrochloride (DAB) and horseradish peroxidase Type VI (Sigma Chemical Co., St. Louis, Mo.). Protamine sulphate (clupein-type, m.w. about 8100, Vitrurn AB). Statistical evaluation was made using Student's t-test. P-values < 0.001 were considered statistically significant.
Results Light Microscopy Semithin transversal sections o f muscles treated with H R P + p r o t a m i n e or prota m i n e alone at + 3 7 ~ displayed a mosaic p a t t e r n o f changes. Some of the fibres, preferentially in the periphery of the muscle, were v a c u o l a t e d (Fig. 1) b u t most of the fibres were intact. A few fibres showed areas of rarefaction located either close to the fibre surface or in the depth of the fibre. L o n g i t u d i n a l sections showed focal v a c u o l a t i o n in otherwise u n a l t e r e d fibres (Fig. 2). The size of vacuoles varied between fibres with the largest sizes in fibres showing signs of rarefaction i n d i c a t i n g a progressive change from v a c u o l a t i o n to fibre d e g e n e r a t i o n a n d necrosis. V a c u o l a t i o n was n o t seen after i n c u b a t i o n in H R P alone or in H R P + p r o t a m i n e at + 4 ~ C. Thus i n c u b a t i o n at low t e m p e r a t u r e ( + 4 ~ C) protected the muscle fibres against the v a c u o l a t i n g effect of p r o t a m i n e . I n muscles i n c u b a t e d in H R P + p r o t a m i n e the extracellular space a n d preferentially the m e m b r a n e of superficial fibres were labelled with H R P as indicated by the d a r k - b r o w n o x i d a t i o n p r o d u c t of DAB. In vacuolated fibres the b r o w n reaction p r o d u c t was present at the i n n e r circumference of some vacuoles. Fibres with regions of rarefaction showed diffuse staining, p r e s u m a b l y due to p l a s m a m e m b r a n e d i s r u p t i o n , a n d they were excluded from further e x a m i n a t i o n . Muscles i n c u b a t e d in H R P w i t h o u t p r o t a m i n e showed labelling o f the extracellular space. N o H R P c o u l d be detected inside the fibres. Similar results were o b t a i n e d for muscles i n c u b a t e d in H R P + p r o t a m i n e at + 4 ~ C. C o n t r o l muscles i n c u b a t e d in h e a t - i n a c t i v a t e d H R P + p r o t a m i n e or in a solution with p r o t a m i n e only showed n o H R P labelling of muscle fibre m e m b r a n e s
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Fig. 1. Cross section of protamine-treated ( + 37~ C) muscle fibres. N u m e r o u s vacuoles in superficial cells. Stained section, x 560 Fig. 2. Longitudinal section of protamine and H R P treated ( + 3 7 ~ C) muscle fibres. Segmental vacuolation of a superficial fibre. Dark-brown reaction product of D A B between fibres. Stained section, x 560
Fig. 3. Cross section of a protamine-treated fibre. Many H R P positive vesicles and bodies (some of which are denoted by arrows) are seen inside the muscle fibre. G Golgi region; n nucleus; m mitochondria. • 37,600 Fig, 4. Cross section of a protamine-treated fibre. N u m e r o u s vacuoles (V) are seen inside the muscle fibre cytoplasm. Note the vacuole lined by H R P (denoted by arrow), m mitochondria; t transverse tubule, x 23,500
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or extracellular space, whereas erythrocytes in capillaries showed a positive reaction most likely due to the peroxidatic activity of hemoglobin.
Electron Microscopy The exposure to protamine at + 37~ caused uptake of H R P in muscle fibres (Fig. 3) and intense cytoplasmic vacuolation (Fig. 4). Besides infiltrating the basal lamina and adsorbed on the sarcolemma H R P was found inside the fibres not only in transverse tubules but also in numerous vesicles, membrane limited bodies and vacuoles (Figs. 3-7). H R P containing structures were of different sizes and were scattered throughout the muscle cell cytoplasm. H R P positive vesicles and bodies seemed to be most numerous at the periphery of muscle fibres, immediately beneath the sarcolemma and in close vicinity to the nucleus and to Golgi cisternae (Figs. 5, 6). They were either completely filled with the reaction product or contained rounded less dense areas; in many of the H R P containing bodies the reaction product occupied only a narrow peripheral rim (Figs. 5-7). The structures shown in Figure 7 probably represent secondary lysosomes containing ingested HRP. in vacuoles which were either clear or contained a moderate amount of membranous or granular material, the reaction product was most frequently seen in the periphery (Fig. 4). Most of the larger clear vacuoles, outlined by a single membrane, did not contain H R P label. Muscle fibres incubated in H R P or H R P + p r o t a m i n e at + 4 ~ showed H R P infiltration of the basal lamina and H R P labelling of the sarcolemma and the transverse tubules but intracellular H R P positive structures as those shown in Figures 3-7 could not be detected. As with light microscopy, muscles incubated in medium with heat-inactivated HRP+_protamine or in protamine only showed no H R P labelling. No attempt was made to grade the density of the sarcolemma or the intracristal space of mitrochondria in DAB incubated control muscles compared to non-incubated specimens. Such a comparison would be difficult because of the small variation in contrast intensity usually present between different grids. Thus, the occasionally observed increased density of the sarcolemma and mitochondria in DAB incubated specimens as reported by other workers (Williams and Wissig, 1975) cannot be proved or disproved. However, no obvious reaction product was seen within these structures in uncontrasted sections.
Fig. 5. Cross section of a protamine-treated muscle fibre. Beneath the sarcolemma H R P is seen in vesicles and membrane limited bodies (denoted by arrows). The vacuole (V) also contains the reaction product. G Golgi region; m mitochondria, x 63,500 Fig. 6. Cross section of a protamine-treated muscle fibre. N u m e r o u s HRP positive vesicles and bodies (some of which are denoted by arrows) are seen in the region close to the nucleus (n). t transverse tubule; in mitochondria, x 63,500 Fig. 7. Cross section of a protamine-treated muscle fibre. Membrane limited bodies containing H R P (denoted by arrows) display clear areas and reaction product of a typical density is seen at their periphery, x 98,700
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~
Protomine 6 0 rnin or untrecJted muscles HRP 6 0 rain
HRP 3 0 rain * HRP and Protamine 3 0 rain
15s
HRP 9 Protc]mine 6 0 min
HRP + Protarnine 6 0 rNn { 4 ~
I.
12~ =
o -~ IO(
E
ii~i!ii!~i
E 5(
Control
37~
l
t-,-
40C -4
Fig. 8. Uptake of ttRP expressed as nanogram HRP/milligram muscle tissue protein (Mean +S.E.M.) (n= 5-6 muscles). Incubation conditions as indicated in the figure with special symbols. After incubation all muscles were washed in HRP-free solution 4 x 1 h at + 4~ C. Control denotes endogenous peroxidase activity of the muscle. Differences between all groups were statistically significant at p < 0.001
Chemical Determination F o l l o w i n g i n c u b a t i o n in H R P s o l u t i o n muscles were washed for 4 x 1 h at + 4 ~ to clear the e x t r a c e l l u l a r space f r o m H R P a n d thus to o b t a i n an a p p r o x i m a t e e s t i m a t i o n o f i n t r a c e l l u l a r H R P u p t a k e . As shown in F i g u r e 8, the u p t a k e o f H R P was s t i m u l a t e d by p r o t a m i n e when the i n c u b a t i o n t e m p e r a t u r e was + 37~ T h e s t i m u l a t i o n was m o r e p r o n o u n c e d when p r o t a m i n e was present d u r i n g 60 m i n o f i n c u b a t i o n t h a n when present d u r i n g 30 min. L o w e r i n g the t e m p e r a ture to + 4 ~ r e d u c e d H R P u p t a k e to values lower t h a n those in muscles w i t h o u t p r o t a m i n e at + 37 ~ Muscles i n c u b a t e d in p r o t a m i n e w i t h o u t H R P a n d w a s h e d for 4 x 1 h at + 4 ~ d i s p l a y e d a very low p e r o x i d a s e activity o f the same m a g n i t u d e as the e n d o g e n o u s p e r o x i d a s e activity f o u n d in u n t r e a t e d muscles (controls). Discussion T h e p r e s e n t study has s h o w n t h a t i n c u b a t i o n o f skeletal muscle with p r o t a m i n e at + 37~ causes i n t r a c e l l u l a r u p t a k e o f H R P . The o x i d a t i o n p r o d u c t o f D A B ,
Induced Uptake of HRP in Skeletal Muscle
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indicating peroxidase activity, was present in vesicles and membrane limited bodies and vacuoles inside the muscle fibres already after 30 rain exposure to protamine. Muscles without protamine did not show any detectable vesicular bound HRP uptake, HRP being present only in the membranes facing the extracellular fluid, i.e. in the basal lamina and the sarcolemma and in the transverse tubular system. Similarly, low temperature (+ 4 ~C) prevented vesicular uptake of HRP in response to protamine. The mechanisms related to the transport of HRP into muscle cells and subsequent accumulation of the protein tracer into organelles some of which probably represent secondary lysosomes requires further investigation. Previous electron microscopic studies have shown that coated vesicles are involved in protein uptake in many types of cells (Bowers, 1964; Rosenbluth and Wissig, 1964). Thus such vesicles are considered to be involved in the uptake of extracellular proteins at i.e. rat neuromuscular junctions (Heuser and Reese, 1973; Zacks and Saito, 1969) and neurosecretory terminals (Douglas et al., 1971; Nagasawa et al., 1971). Moreover, phagocytosis of latex particles was recently reported to occur in cultured muscle cells (Garfield et al., 1975). Further analysis is required, however, to verify a possible role of coated vesicles in the uptake of HRP into muscle fibres following protamine treatment. In support of the morphological observations chemical determinations of HRP showed about 90% increase of intracellular bound HRP in muscles after 30 min exposure to protamine at +37 ~ C, as compared to HRP without protamine. Low temperature (+ 4 ~C) blocked the increase in HRP, giving an approximate estimation of extracellularly bound HRP. It is notable, however, that without any apparent morphological correlate there was a significant increase of HRP in muscles incubated at +37~ even in the absence of protamine. This increase might be due to an increased amount of HRP being bound extracellularly at this higher temperature but also to an intracellular uptake process giving rise to local HRP-concentrations too small to be detected with the present morphological technique. Although much further evidence is needed to establish the presence of such a process in muscle tissue the present results might suggest the possibility of vesicular transport being a physiological uptake mechanism for macromolecules contained in the extracellular fluid. This is supported by a recent observation that 3H-inulin probably is taken up in skeletal muscle by a bulk uptake process (unpublished observation). Exposure to protamine induced vacuolation of muscle fibres. This effect was limited mostly to fibres in the periphery of the muscle. The fibres were frequently focally vacuolated with large unaffected areas between groups of vacuoles. The vacuoles were membrane limited, sometimes containing HRP label and in other instances containing remnants of myofibrous structures. The largest vacuoles were clear without content. When present in large numbers the vacuoles seemed to be part of a general degenerative process in the fibres resulting in sarcolemma disruption and final cell necrosis. The vacuolation process had many similarities with autophagic vacuoles in denervated muscle as described by Schiaffino and Hanzlikova (1972) and in other tissues (cf. Ericsson, 1969). Autophagic activity is generally ascribed to activation of lysosomal enzymes in response to either a direct chemical
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influence or as a result of enhanced endocytosis. Since we have demonstrated that protamine induces vesicular uptake of HRP it seems possible that this endocytosis results in lysosomal activation and subsequently in the formation of autophagic vacuoles, and at a later stage, in cell necrosis. The muscle fibres were affected in a mosaic pattern by protamine and when this led to large scale vacuolation, focal sarcolemma lesions occurred allowing HRP to enter diffusely into the sarcoplasm. It is of interest that in Duchenne dystrophy muscle fibres exhibit a similar pattern of focal degenerative changes with the formation of autophagic vacuoles and sarcolemma lesions (Morki and Engel, 1975). The possibility that processes, similar to those induced by protamine in normal muscle, occur in that type of disease should be considered. 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)
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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., Hanzfikova, 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. lfi, 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)
Accepted August 28, 1976
