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Neurotrophic effects: Axoplasmic transport involvement in the regulation of skeletal muscle soluble proteins
Neuroscience Letters, 2 (1976) 211--216
211
© ElsevierlNorth-Holland, Amsterdam -- Printed in The Netherlands
NEUROTROPHIC EFFECTS: AXOPLASMIC TRANSPORT INVOLVEMENT IN THE REGULATION OF SKELETAL MUSCLE SOLUBLE PROTEINS
HUGO L. FERNANDEZ and BEATRIZ U. RAMIREZ
Laboratory of Neurophysiology, Gabriela G. Gildemeister, Catholic University of Chile, Santiago (Chile) (Received: April 20th, 1976) (Accepted April 21st, 1976)
SUMMARY
Skeletal muscle soluble proteins were assayed by polyacrylamide gel discelectrophoresis at different times (4--36 days) after injection of 10 mM colchicine into the motor nerve and after denervation. Results showed that effects of drug-induced axoplasmic transport blockage differ from those of denervation as to quantitative changes in the protein concentration of certain bands. Results point to the conclusion that axoplasmic transport is involved in the neural control of a number of muscle proteins. This does not invalidate the participation of acetylcholine and muscle activity in the regulation of muscle metabolism.
Certain features of skeletal muscle protein metabolism are known to be under neural regulation [ 15]. It is uncertain, however, ~vhether this regulation is exclusively mediated by muscle activity and/or acetylcholine (ACh) or also by 'trophic' substances released from motor nerves [ 7 ]. Recently, it has been shown that axoplasmic transport blockage induces some denervation-like changes, in the presence of normal muscle activity and transmitter release [ 2,5]. It follows that substances conveyed by axoplasmic transport might be partly responsible for the regulation of muscle chemosensitive and electrogenic properties [1,4]. In the present work we examined whether the influence of axoplasmic transport also extends to muscle biochemical characteristics. Further, we compared the effects of denervation and of colchicine-induced blockage of axoplasmic transport on the electrophoretic patterns of muscle soluble proteins. Adult cats anesthetized with sodium pentobarbital (36 mg/kg, Nembutal, Abbott) were employed. Hypoglossal nerves were exposed and a local injection of 5 #l saline solution containing 10 mM colchicine (Sigma Chemicals) was applied under the epineurium of one nerve (experimental). The contralateral nerve received an injection of saline (control). In similar experiments,
212
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one of the nerves was transected while.the contralateral was left intact. As previously reported, colchicine treatment causes fibrillation and ACh hypersensitivity in the geniohyoid muscle without affecting nerve conduction or effective neuromuscular transmission [4,5]. These results were confirmed by measuring muscle electric activity and contractile characteristics 4, 10, 21, and 36 days after treatment. Subsequently, the muscles were removed, weighed, and homogenized (1:10, w/v) in 0.15 M phosphate buffer (pH 8.0) at 0°C. The homogenate was centrifuged (Sorval, SM24) at 10,000 X g for 15 min; 20 pl of supernatant -- containing 50--70 ~g of non-collagen protein [13] were assayed b y polyacrylamide gel disc-electrophoresis (7.5% running gel, 4% stacking gel). Tris-glycine (pH 8.3) was used as electrode buffer. Four electrophoretic runs were completed, each consi~_t~ing of 12 gels, loaded as follows: six samples from colchicine preparations {four experimental, two controls) and six from denervated muscles (four experimental, two controls). Following electrophoresis, at 3 mA/gel for 1.5--2.0 h, the gels were fixed in 15% TCA, stained with 0.2% Coomassie blue, and scanned at 605 nm in a KII Canalco microdensitometer [16]. Since collagen, mitochondria, and proteins of the muscle contractile system were for the most part sedimented by centrifugation, the proteins analyzed are mainly those in the microsomal and supernatant fractions. Each gel electrophoretogram consistently showed at least 8 bands that were present in all experimental and control tests (Fig. 1). Although the position of the bands referred to the tracking-dye was reproducible, no obvious qualitative or quantitative alterations could be clearly resolved by eye as to electrophoretic patterns. By analyzing the densitometric tracings (Fig. 1), however, several quantitative band changes were detected both in denervated and colchicine-treated preparations. These changes were evaluated by computing the area under each peak, the relative band content (as percentage from total protein in each gel), the average protein concentration for each band in equal sets of experiments, the individual band coefficient of variation, and the experimental/control ratio. Experimental variability -- due to differences between animals, electrophoretic runs, and protein staining properties -- was accounted for by considering the changes significantly greater than the experimental variation coefficient of the corresponding band and the differences between experimental and control muscles by the t-test of significance. The data embodied in Table I indicate that the effect of colchicine is different from that of denervation. In fact, (a) the protein content of 4 bands (B,
Fig. 1. Densitometric tracings and gel electrophoretograms of soluble proteins from the geniohyoid muscle, 10 days after injection of colchicine into the hypoglossal nerve (C~0) and denervation (Dlo). The contralateral muscles were used for paired comparisons (control). Protein bands are lettered (A to J) from the origin to the buffer front (M). Areas under each peak were computed automatically with a Varian Disc-Integrator and Printer System, model A-25. The length of the disc integrator trace (DIT) beneath each peak is proportional to the peak area (a full pen travel was arbitrarily assigned 100 counts).
Denervation
---33 b
--
-79 c
---
--
4 10 21 36
Colchicine
(6.6 ÷ 0 . 4 ) (25.9)
(13 ~ 0 . 5 ) (14.0) 36 c 51 c ---
C
B
--24 a --31 c --34 c --22 a
--38 c --20 a
m
D ( 2 0 . 5 -+ ?).9) (16.5)
R e l a t i v e c h a n g e in p r o t e i n b a n d
36 c 21 c ---
Time (days)
4 10 21 36
Experimental condition
47 a 51 a ---
G ( 9 . 9 -+ 0 . 7 ) (30.0)
---28 a
--
35 c
--33 a
H (14.5 ± 0.6) (16.7}
D a t a o n l y o f b a n d s ( l e t t e r e d as in Fig. 1 ) t h a t s h o w e d s i g n i f i c a n t c h a n g e s in t h e i r p r o t e i n c o n t e n t . R e s u l t s are a v e r a g e s o f % c h a n g e f r o m c o n t r o l , 4 a n i m a l s f o r e a c h t i m e . F i g u r e s in b r a c k e t s i n d i c a t e d a t a o b t a i n e d f r o m s c a n s o f 32 c o n t r o l e x p e r i m e n t s : u p p e r p a r e n t h e s i s , b a n d p r o t e i n c o n t e n t (%" a v e r a g e + S.E. f r o m t o t a l p r o t e i n ) ; l o w e r p a r e n t h e s i s , c o e f f i c i e n t o f v a r i a t i o n (Cv = s - 1 0 0 ~ / x ) . I n c r e a s e a n d d e c r e a s e f r o m c o n t r o l are i n d i c a t e d b y p o s i t i v e a n d n e g a t i v e n u m b e r s r e s p e c t i v e l y . T h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s b e t w e e n e x p e r i m e n t a l a n d c o n t r o l m u s c l e s is s h o w n b y : a p < 0 . 0 5 ; b p < 0 . 0 2 ; c p < 0 . 0 1 . P ~ 0 . 0 5 w a s considered n o t significant.
MUSCLE S OL U B L E P R O T E I N C H A N G E S AS A F U N C T I O N O F T I M E A F T E R C O L C H I C I N E INJECTION AND AFTER DENERVATION
TABLE I
t~
215
C, D and H) was altered by the drug while that of 5 bands (B, C, D, G and H) was changed by denervation; (b) the individual time course of the alterations was different, except for the initial change in band H; (c) colchicine effects were reversible within 36 days, contrariwise those of denervation (except for G) were not; (d) one band was altered only by denervation (G) and two only by colchicine (B and C, up to day 21); and (e) the protein amount in band C increased and that of band H decreased, as a result of drug treatment; conversely, denervation caused opposite changes. As expected, the metabolic derangement induced by denervation was more drastic than that caused by colchicine. In this respect we must consider that the response of the metabolic system to denervation may well reflect the net influences of muscle atrophy and loss of activity, in addition to the loss of neurogenic 'trophic' substances [8]. In this context it is hard to explain why the protein content of some bands was altered by colchicine and not by denervation. However, the changes in non-collagen protein are complex in that they may partly result from the combined effects of increased and decreased protein synthesis as well as increased proteolytic activity [6,9,11]. Further, it must be noted that our electrophoretic resolution may be insufficient to elucidate the complexity of the proteins contained in each band. Nevertheless, the important point to be stressed is the clearcut differences between colchicine and denervation treatments as to soluble proteins. Thus far, the transsynaptic effects of colchicine have been found to closely mimic those of nerve transection [2,4,5]. Inasmuch as colchicine blocks axoplasmic transport [10,14] our findings point to the first instance in which this blockage causes muscle alterations that are essentially different from those of denervation. In addition, we can conclude that axoplasmic transport is involved in the neural regulation of some muscle proteins. This does not exclude acetylcholine and muscle activity as important factors in this regulation. Therefore, as has been argued for the control of ACh sensitivity [3] and acetylcholinesterase activity [ 12], the trophic regulation of muscle metabolism might consist of multiple mechanisms interacting together or in opposition. ACKNOWLEDGEMENTS
This work was supported by grants from PNUD/UNESCO (RLA 75/047 to H.L.F.), FIUC (208/75 to B.U.R.), and N.I.H. (NS 11122, to P.F.D.). We thank Dr. J.V. Luco and Mr. N.C. Inestrosa (Catholic University of Chile) and Dr. P.F. Davison (Boston Biomedical Institute) for valuable discussions. Technical assistance was given by Mrs. M.B. Ptischel. REFERENCES
Albuquerque, E.X., Warnick, J.E., Sansone, F.M. and Onur, R., The effects of vinblastine and colchicine on neural regulation of muscle, Ann. N.Y. Acad. Sci., 228 (1974) 224--243.
216
2 Albuquerque, E.X., Warnick, J.E., Tasse, J.R. and Sansone, F.M., Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat, Exp. Neurol., 37 (1972) 607--634. 3 Drachman, D.B., The role of acetylcholine as a neurotrophic transmitter, Ann. N.Y. Acad. Sci., 228 (1974) 160--175. 4 Fernandez, H.L., Neurotrophic effects in relation to axoplasmic transport, Acta physiol, lat.-amer., 23 (1973) 189--191. 5 Fernandez, H.L. and Ramirez, B.U., Muscle fibrillation induced by blockage of axoplasmic transport in motor nerves, Brain Res., 79 (1974) 385--395. 6 Goldberg, A.L., Jablecki, C. and Li, J.B., Effects of use and disuse on amino acid transport and protein turnover in muscle, Ann. N.Y. Acad. Sci., 228 (1973) 190--201. 7 Guth, L., Trophic effects of vertebrate neurons, Neurosci. Res. Prog. Bull., 7 (1969) 1--73. 8 Guth, L. and Watson, P.K., The influence of innervation on the soluble proteins of slow and fast muscles of the rat, Exp. Neurol., 17 (1967) 107--117. 9 Hajek, I., Gutmann, E. and Syrovy, I., Proteolytic activity in denervated and reinnervated muscle, Physiol. bohemoslov., 13 (1964) 32--38. 10 Hofmann, W.W. and Peacock, J.H., Postjunctional changes induced by partial interruption of axoplasmic flow in motor nerves, Exp. Neurol., 41 (1973) 345--356. 11 Ionasescu, V., Lewis, R. and Schottelius, B., Neurogenic control of muscle ribosomal protein synthesis, Acta neurol, scand., 51 (1975) 253--267. 12 Linkhart, T.A. and Wilson, B.W., Role of muscle contraction in trophic regulation of chick muscle acetylcholinesterase activity, Exp. Neurol., 48 (1975) 557--568. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265--275. 14 Rodriguez-Echandia, E., Ramirez, B.U. and Fernandez, H.L., Studies on the mechanism of inhibition of axoplasmic transport of neuronal organelles, J. Neurocytol., 2 (1973) 149--156. 15 Samaha, F.J., Guth, L. and Albers, R.W., The neural regulation of gene expression in the muscle cell, Exp. Neurol., 27 (1970) 276--282. 16 Serman, D. and Skreb, N., Colinearity of protein patterns in polyacrylamide gel electrophoresis, Int. J. Biochem., 3 (1972) 657--664.
211
© ElsevierlNorth-Holland, Amsterdam -- Printed in The Netherlands
NEUROTROPHIC EFFECTS: AXOPLASMIC TRANSPORT INVOLVEMENT IN THE REGULATION OF SKELETAL MUSCLE SOLUBLE PROTEINS
HUGO L. FERNANDEZ and BEATRIZ U. RAMIREZ
Laboratory of Neurophysiology, Gabriela G. Gildemeister, Catholic University of Chile, Santiago (Chile) (Received: April 20th, 1976) (Accepted April 21st, 1976)
SUMMARY
Skeletal muscle soluble proteins were assayed by polyacrylamide gel discelectrophoresis at different times (4--36 days) after injection of 10 mM colchicine into the motor nerve and after denervation. Results showed that effects of drug-induced axoplasmic transport blockage differ from those of denervation as to quantitative changes in the protein concentration of certain bands. Results point to the conclusion that axoplasmic transport is involved in the neural control of a number of muscle proteins. This does not invalidate the participation of acetylcholine and muscle activity in the regulation of muscle metabolism.
Certain features of skeletal muscle protein metabolism are known to be under neural regulation [ 15]. It is uncertain, however, ~vhether this regulation is exclusively mediated by muscle activity and/or acetylcholine (ACh) or also by 'trophic' substances released from motor nerves [ 7 ]. Recently, it has been shown that axoplasmic transport blockage induces some denervation-like changes, in the presence of normal muscle activity and transmitter release [ 2,5]. It follows that substances conveyed by axoplasmic transport might be partly responsible for the regulation of muscle chemosensitive and electrogenic properties [1,4]. In the present work we examined whether the influence of axoplasmic transport also extends to muscle biochemical characteristics. Further, we compared the effects of denervation and of colchicine-induced blockage of axoplasmic transport on the electrophoretic patterns of muscle soluble proteins. Adult cats anesthetized with sodium pentobarbital (36 mg/kg, Nembutal, Abbott) were employed. Hypoglossal nerves were exposed and a local injection of 5 #l saline solution containing 10 mM colchicine (Sigma Chemicals) was applied under the epineurium of one nerve (experimental). The contralateral nerve received an injection of saline (control). In similar experiments,
212
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COLCHtCINE (Clo)
OENERVA; IOr~
0 w 0.5"
¢$
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® J.O-
¢t~ 0.~ ol
' i¢t11
CONTROL (CIo)
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213
one of the nerves was transected while.the contralateral was left intact. As previously reported, colchicine treatment causes fibrillation and ACh hypersensitivity in the geniohyoid muscle without affecting nerve conduction or effective neuromuscular transmission [4,5]. These results were confirmed by measuring muscle electric activity and contractile characteristics 4, 10, 21, and 36 days after treatment. Subsequently, the muscles were removed, weighed, and homogenized (1:10, w/v) in 0.15 M phosphate buffer (pH 8.0) at 0°C. The homogenate was centrifuged (Sorval, SM24) at 10,000 X g for 15 min; 20 pl of supernatant -- containing 50--70 ~g of non-collagen protein [13] were assayed b y polyacrylamide gel disc-electrophoresis (7.5% running gel, 4% stacking gel). Tris-glycine (pH 8.3) was used as electrode buffer. Four electrophoretic runs were completed, each consi~_t~ing of 12 gels, loaded as follows: six samples from colchicine preparations {four experimental, two controls) and six from denervated muscles (four experimental, two controls). Following electrophoresis, at 3 mA/gel for 1.5--2.0 h, the gels were fixed in 15% TCA, stained with 0.2% Coomassie blue, and scanned at 605 nm in a KII Canalco microdensitometer [16]. Since collagen, mitochondria, and proteins of the muscle contractile system were for the most part sedimented by centrifugation, the proteins analyzed are mainly those in the microsomal and supernatant fractions. Each gel electrophoretogram consistently showed at least 8 bands that were present in all experimental and control tests (Fig. 1). Although the position of the bands referred to the tracking-dye was reproducible, no obvious qualitative or quantitative alterations could be clearly resolved by eye as to electrophoretic patterns. By analyzing the densitometric tracings (Fig. 1), however, several quantitative band changes were detected both in denervated and colchicine-treated preparations. These changes were evaluated by computing the area under each peak, the relative band content (as percentage from total protein in each gel), the average protein concentration for each band in equal sets of experiments, the individual band coefficient of variation, and the experimental/control ratio. Experimental variability -- due to differences between animals, electrophoretic runs, and protein staining properties -- was accounted for by considering the changes significantly greater than the experimental variation coefficient of the corresponding band and the differences between experimental and control muscles by the t-test of significance. The data embodied in Table I indicate that the effect of colchicine is different from that of denervation. In fact, (a) the protein content of 4 bands (B,
Fig. 1. Densitometric tracings and gel electrophoretograms of soluble proteins from the geniohyoid muscle, 10 days after injection of colchicine into the hypoglossal nerve (C~0) and denervation (Dlo). The contralateral muscles were used for paired comparisons (control). Protein bands are lettered (A to J) from the origin to the buffer front (M). Areas under each peak were computed automatically with a Varian Disc-Integrator and Printer System, model A-25. The length of the disc integrator trace (DIT) beneath each peak is proportional to the peak area (a full pen travel was arbitrarily assigned 100 counts).
Denervation
---33 b
--
-79 c
---
--
4 10 21 36
Colchicine
(6.6 ÷ 0 . 4 ) (25.9)
(13 ~ 0 . 5 ) (14.0) 36 c 51 c ---
C
B
--24 a --31 c --34 c --22 a
--38 c --20 a
m
D ( 2 0 . 5 -+ ?).9) (16.5)
R e l a t i v e c h a n g e in p r o t e i n b a n d
36 c 21 c ---
Time (days)
4 10 21 36
Experimental condition
47 a 51 a ---
G ( 9 . 9 -+ 0 . 7 ) (30.0)
---28 a
--
35 c
--33 a
H (14.5 ± 0.6) (16.7}
D a t a o n l y o f b a n d s ( l e t t e r e d as in Fig. 1 ) t h a t s h o w e d s i g n i f i c a n t c h a n g e s in t h e i r p r o t e i n c o n t e n t . R e s u l t s are a v e r a g e s o f % c h a n g e f r o m c o n t r o l , 4 a n i m a l s f o r e a c h t i m e . F i g u r e s in b r a c k e t s i n d i c a t e d a t a o b t a i n e d f r o m s c a n s o f 32 c o n t r o l e x p e r i m e n t s : u p p e r p a r e n t h e s i s , b a n d p r o t e i n c o n t e n t (%" a v e r a g e + S.E. f r o m t o t a l p r o t e i n ) ; l o w e r p a r e n t h e s i s , c o e f f i c i e n t o f v a r i a t i o n (Cv = s - 1 0 0 ~ / x ) . I n c r e a s e a n d d e c r e a s e f r o m c o n t r o l are i n d i c a t e d b y p o s i t i v e a n d n e g a t i v e n u m b e r s r e s p e c t i v e l y . T h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s b e t w e e n e x p e r i m e n t a l a n d c o n t r o l m u s c l e s is s h o w n b y : a p < 0 . 0 5 ; b p < 0 . 0 2 ; c p < 0 . 0 1 . P ~ 0 . 0 5 w a s considered n o t significant.
MUSCLE S OL U B L E P R O T E I N C H A N G E S AS A F U N C T I O N O F T I M E A F T E R C O L C H I C I N E INJECTION AND AFTER DENERVATION
TABLE I
t~
215
C, D and H) was altered by the drug while that of 5 bands (B, C, D, G and H) was changed by denervation; (b) the individual time course of the alterations was different, except for the initial change in band H; (c) colchicine effects were reversible within 36 days, contrariwise those of denervation (except for G) were not; (d) one band was altered only by denervation (G) and two only by colchicine (B and C, up to day 21); and (e) the protein amount in band C increased and that of band H decreased, as a result of drug treatment; conversely, denervation caused opposite changes. As expected, the metabolic derangement induced by denervation was more drastic than that caused by colchicine. In this respect we must consider that the response of the metabolic system to denervation may well reflect the net influences of muscle atrophy and loss of activity, in addition to the loss of neurogenic 'trophic' substances [8]. In this context it is hard to explain why the protein content of some bands was altered by colchicine and not by denervation. However, the changes in non-collagen protein are complex in that they may partly result from the combined effects of increased and decreased protein synthesis as well as increased proteolytic activity [6,9,11]. Further, it must be noted that our electrophoretic resolution may be insufficient to elucidate the complexity of the proteins contained in each band. Nevertheless, the important point to be stressed is the clearcut differences between colchicine and denervation treatments as to soluble proteins. Thus far, the transsynaptic effects of colchicine have been found to closely mimic those of nerve transection [2,4,5]. Inasmuch as colchicine blocks axoplasmic transport [10,14] our findings point to the first instance in which this blockage causes muscle alterations that are essentially different from those of denervation. In addition, we can conclude that axoplasmic transport is involved in the neural regulation of some muscle proteins. This does not exclude acetylcholine and muscle activity as important factors in this regulation. Therefore, as has been argued for the control of ACh sensitivity [3] and acetylcholinesterase activity [ 12], the trophic regulation of muscle metabolism might consist of multiple mechanisms interacting together or in opposition. ACKNOWLEDGEMENTS
This work was supported by grants from PNUD/UNESCO (RLA 75/047 to H.L.F.), FIUC (208/75 to B.U.R.), and N.I.H. (NS 11122, to P.F.D.). We thank Dr. J.V. Luco and Mr. N.C. Inestrosa (Catholic University of Chile) and Dr. P.F. Davison (Boston Biomedical Institute) for valuable discussions. Technical assistance was given by Mrs. M.B. Ptischel. REFERENCES
Albuquerque, E.X., Warnick, J.E., Sansone, F.M. and Onur, R., The effects of vinblastine and colchicine on neural regulation of muscle, Ann. N.Y. Acad. Sci., 228 (1974) 224--243.
216
2 Albuquerque, E.X., Warnick, J.E., Tasse, J.R. and Sansone, F.M., Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat, Exp. Neurol., 37 (1972) 607--634. 3 Drachman, D.B., The role of acetylcholine as a neurotrophic transmitter, Ann. N.Y. Acad. Sci., 228 (1974) 160--175. 4 Fernandez, H.L., Neurotrophic effects in relation to axoplasmic transport, Acta physiol, lat.-amer., 23 (1973) 189--191. 5 Fernandez, H.L. and Ramirez, B.U., Muscle fibrillation induced by blockage of axoplasmic transport in motor nerves, Brain Res., 79 (1974) 385--395. 6 Goldberg, A.L., Jablecki, C. and Li, J.B., Effects of use and disuse on amino acid transport and protein turnover in muscle, Ann. N.Y. Acad. Sci., 228 (1973) 190--201. 7 Guth, L., Trophic effects of vertebrate neurons, Neurosci. Res. Prog. Bull., 7 (1969) 1--73. 8 Guth, L. and Watson, P.K., The influence of innervation on the soluble proteins of slow and fast muscles of the rat, Exp. Neurol., 17 (1967) 107--117. 9 Hajek, I., Gutmann, E. and Syrovy, I., Proteolytic activity in denervated and reinnervated muscle, Physiol. bohemoslov., 13 (1964) 32--38. 10 Hofmann, W.W. and Peacock, J.H., Postjunctional changes induced by partial interruption of axoplasmic flow in motor nerves, Exp. Neurol., 41 (1973) 345--356. 11 Ionasescu, V., Lewis, R. and Schottelius, B., Neurogenic control of muscle ribosomal protein synthesis, Acta neurol, scand., 51 (1975) 253--267. 12 Linkhart, T.A. and Wilson, B.W., Role of muscle contraction in trophic regulation of chick muscle acetylcholinesterase activity, Exp. Neurol., 48 (1975) 557--568. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265--275. 14 Rodriguez-Echandia, E., Ramirez, B.U. and Fernandez, H.L., Studies on the mechanism of inhibition of axoplasmic transport of neuronal organelles, J. Neurocytol., 2 (1973) 149--156. 15 Samaha, F.J., Guth, L. and Albers, R.W., The neural regulation of gene expression in the muscle cell, Exp. Neurol., 27 (1970) 276--282. 16 Serman, D. and Skreb, N., Colinearity of protein patterns in polyacrylamide gel electrophoresis, Int. J. Biochem., 3 (1972) 657--664.