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Neurotoxin-induced hydrolase activity in peripheral nerve
Neuroscience Letters, 24 (1981) 261-265
261
Elsevier/North-Holland Scientific Publishers Ltd.
NEUROTOXIN-INDUCED NERVE
H Y D R O L A S E A C T I V I T Y IN P E R I P H E R A L
ROLAND J. BOEGMAN and BRENDA SCARTH Department of Pharmacology, Queen "s University, Kingston, Ontario K7L 3N6 (Canada)
(Received December 22nd, 1980; Revised version received April 6th, 1981; Accepted April 15th, 1981)
The effect of neurotoxins which block axonal transport or impulse transmission was examined on sciatic nerve hydrolase activity. Subepineural injection of batrachotoxin (BTX) or colchicine resulted in a 4-9-fold increase in acid protease and N-acetylglucosaminidase activity with a smaller increase in acid phosphatase activity. The enzyme activity remained elevated for up to 48 days. Similar injections of tetrodotoxin (TTX) led to a relatively small increase in the activity of these enzymes which returned to normal values within two days. The prolonged increase in hydrolase activity produced by BTX and colchicine suggests that blockade of axonal transport may be mediated by nerve necrosis.
Neurotoxins such as b a t r a c h o t o x i n (BTX), colchicine and tetrodotoxin (TTX), w h e n applied to peripheral nerve in vivo and in vitro, has been used to interrupt axonal transport a n d / o r impulse transmission [2, 5, 6, 10, 11, 20, 21, 23]. The physiological action o f these drugs has been t h o u g h t to be r e m a r k a b l y selective and readily reversible [20, 21]. In this c o m m u n i c a t i o n we show that the hydrolase activity present in peripheral nerve increases following subepineural injections o f neurotoxin and with certain neurotoxins (BTX, colchicine) remains elevated for an extended time period. U n d e r pentobarbital anesthesia 1 #1 o f each o f the following in 10% dextrosesaline was injected subepineurally via a thin glass capillary, into the right sciatic nerve at the sciatic n o t c h o f 35 g male Swiss White mice: B T X (9 x 10- ~2 mol), colchicine (1 x 10 -a mol), T T X (6.3 × 10 -9 mol). E a c h animal received a similar injection o f vehicle alone into the left sciatic nerve to serve as internal c o n t r o l [5, 7]. All surgical procedures were carried out as aseptically as possible. L i m b paralysis immediately following injection o f T T X or B T X was present and was used as an index o f successful toxin application. Paralysis following T T X lasted for 2 - 3 days while BTX-induced paralysis was present for approximately 6 - 7 days. Animals treated with colchicine did not show as m a r k e d leg immobilization as those treated with B T X or T T X and, in addition, paralysis developed after one day and remained for approximately 6 days. Animals were housed under s t a n d a r d conditions and were sacrificed by decapitation. A nerve segment at the injection site 1.5 cm long was immediately r e m o v e d and placed on ice. For each experiment toxin0304-3940/81/0000-0000/$ 02.50 © Elsevier/North-Holland Scientific Publishers Ltd.
262
pretreated and control nerves from 3 animals were pooled separately and homogenized in a micro-glass homogenizer with 600 #1 cold 0.3 M sucrose, 0.1% Triton X - 100 to give a 3% w / v homogenate [7]. The following enzymes were assayed in triplicate: acid protease employing [14C-methyl]methemoglobin as substrate [4, 19] as well as acid phosphatase and N-acetylglucosaminidase using the fluorescent substrates described previously [7, 16, 17]. The mean value (expressed per mg non-collagen protein) obtained from each experiment in which the nerve was treated with toxin was divided by the mean of the control nerve to give a ratio. A pooled sample t-test was used to determine if there was a significant difference between treated and control mean values. There was a small increase in hydrolase activity within 12 h of injecting TTX into the nerve (Fig. 1). N-acetylglucosaminidase activity responded with the greatest increase (P < 0.01). The enzyme activity, however, decreased to normal values by the second day and remained unaltered up to day 8 (Fig. 1). Subepineural injection of BTX resulted in a similar initial small increase in nerve hydrolase activity, which again was most marked with N-acetylglucosaminidase ( P < 0.001 at 12 h). In contrast to TTX, however, BTX produced a secondary larger increase (Fig. 2a)
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DAYS Fig. 1. Lysosomal enzyme activity present in the sciatic nerve following subepineural administration of TTX. Acid protease, II; acid plaosphatase, A; N-acetylglucosaminidase, ®. Each point represents the mean + S.E.M. of 4 experiments in which the ratio between toxin-treated and control nerve for 3 animals/experiment was determined.
263 u.I > n,u.I Z .J 0 GC I-Z 0 tO
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Fig, 2. Lysosomal enzyme activity present in the sciatic nerve following subepineural administration of BTX (a) or colchicine (b). Acid protease, II; acid phosphatase, A; N-acetylglucosaminidase, e . Each point represents the mean _+ S.E.M. of 4 experiments in which the ratio between toxin-treated and control nerve for 3 animals/experiment was determined.
which reached a maximum between 15 and 30 days and had not returned completely to normal values by day 45. N-acetylglucosaminidase activity increased more than 9fold (P < 0.001) above control while acid protease increased 4-fold (P < 0.001) and acid phosphatase activity doubled (P < 0.01). Subepineural injection of colchicine also resulted in an increase in nerve hydrolase activity similar to that seen after BTX (Fig. 2b). A 7-fold increase in N-acetylglucosaminidase ( P < 0 . 0 0 1 ) and a 5-fold increase in acid protease activity (P < 0.001) were detected as late as 16 days after neural application of the drug. The return to normal N-acetylglucosaminidase activity was more rapid following subepineural injection of colchicine than BTX. The response of neural acid phosphatase activity to toxin insult was dramatically less than that observed with acid protease and N-acetylglucosaminidase.
264 Our data show that BTX and colchicine, two drugs known to block axonal transport, cause a marked increase in hydrolytic enzyme activity in the sciatic nerve. In contrast, TTX, which prevents Na influx, had a minimal effect on lysosomal enzyme activity. BTX- and colchicine-induced paralysis lasts for approximately twice as long as that found with TTX. The increase in hydrolase activity, however, lasts proportionately much longer and is of greater magnitude with BTX and colchicine, suggesting that the duration of paralysis is not related to the increase in enzyme activity. Hydrolases have been identified in lysosomes in the nervous system and appear to be located predominantly in the cell body [8, 12-15]. Increases in the number of lysosomes following a nerve crush injury have been observed in the axon as well as in the surrounding Schwann cells [12, 14]. The influx of Na and H20 caused by BTX [1] could be responsible for the biochemical changes observed. Colchicine inhibits the saltatory movement of intra-axonal particles at a concentration of 0.1 mM [11]. However, concentrations as high as 30 mM are required for complete blockade of axoplasmic transport [10]. Neural application of colchicine has been reported to result in axonal damage with multiplication of Schwann cells, nuclear swelling, and the appearance of endoneurial phagocytes [3]. The changes described above occur 24 h after application of the drug, which coincides with the onset of limb paralysis observed in our study. The recovery phase in the study above corresponds to the time period in our experiments where marked decreases in the hydrolytic enzyme activity were observed. Morphological changes in intra-axonal mitochondria and endoplasmic reticulum have been reported in both the CNS [24] and peripheral nerves [3, 10] following application of a similar dose of colchicine as that used in our study. The increase in lysosomal enzyme activity following neural application of BTX or colchicine could be explained as an initial inhibition of axonal transport of lysosoreal vesicles followed by the appearance of autophagic vacuoles and macrophage infiltration, the morphological changes having already been observed in both BTX [18] and colchicine [3, 24] treated preparations. This is supported by the apparent biphasic increase in acid protease and N-acetylglucosaminidase activity found in both BTX- and colchicine-treated nerves. The damage caused to the nerve by minute amounts of BTX and colchicine thus appears to have a relatively long-lasting biochemical effect. Supported by the Muscular Dystrophy Association of Canada. BTX was a gift from Dr. E.X. Albuquerque. 1 Albuquerque, E.X., The made of action of batrachotoxin, Fed. Proc., 31 (1972) 1133-1138. 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 muscle of the rat, Exp. Neurol., 37 (1972) 607-634.
265 3 Angevine, J.B., Nerve destruction by colchicine in mice and golden hamsters, J. exp. Zool., 136 (1957) 363-391. 4 Anson, M.l., The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin, J. gen. Physiol., 22 (1938) 79-89. 5 Boegman, R.J., Deshpande, S.S. and Albuquerque, E.X., Consequences of axonal transport blockade induced by batrachotoxin on mammalian neuromuscular function, I. Early pre- and postsynaptic changes, Brain Res., 187 (1980) 183-196. 6 Boegman, R.J. and Riopelle, R.J., Batrachotoxin blocks slow and retrograde axonal transport in vivo, Neurosci. Lett., 8 (1980) 143-147. 7 Boegman, R.J. and Oliver, T.W., Neural influence on muscle hydrolase activity, Life Sci., 27 (1980) 1339-1344. 8 Broadwell, R.D., Oliver, C. and Brightman, M.W., Neuronal transport of acid hydrolases and peroxidase within the lysosomal system of organelles: involvement of agranular reticulum-like cisterns, J. comp. Neurol., 190 (1980) 519-532. 9 Dingle, J.T., Lysosomes, A Laboratory Handbook, North-Holland Publ., Amsterdam, 1972. l0 Fink, B.R., Byers, M.R. and Middaugh, M.E., Dynamics of colchicine effects on rapid axonal transport and axonal morphology, Brain Res., 56 (1973) 299-311. l 1 Hammond, G.R. and Smith, R.S., Inhibition of the rapid movement of optically detectable axonal particles by colchicine and vinblastine, Brain Res., 128 (1977) 227-242. 12 Holtzman, E., Lysosomes in the physiology and pathology of neurons. In J.T. Dingle and H.B. Fell (Eds.), Lysosomes in Biology and Pathology, North-Holland Publ., Amsterdam, 1969. 13 Holtzman, E., Cytochemical studies of protein transport in the nervous system, Phil. Trans. B, 261 (1971) 407-421. 14 Holtzman, E. and Novikoff, A.B., Lysosomes in the rat sciatic nerve following crush, J. Cell Biol., 27 (1965) 651-669. 15 Koenig, H., Lysosomes in the nervous system. In J.T. Dingle and H.B. Fell (Eds.), Lysosomes in Biology and Pathology, North-Holland Publ., Amsterdam, 1969. 16 Libelius, R., Jirmanova, I., Lundquist, I. and Thesleff, S., Increased endocytosis with lysosomal activation in skeletal muscle of dystrophic mouse, J. Neuropath. exp. Neurol., 37 (1978) 387-400. 17 Libelius, R. and Lundquist, I., Lysosomal activation in mouse skeletal muscle induced by protamine in vitro, Cell Tiss. Res., 186 (1978) 1-11. 18 Moore, G.R.W., Boegman, R.J., Robertson, D.M. and Riopelle, R.J., Batrachotoxin induced axonal necrosis in peripheral nerve, Brain Res., 207 (1981) 481-485. 19 New England Nuclear Proteolytic Enzyme Assay Information Sheet. 20 Ochs, S. and Worth, R., Batrachotoxin block of fast axoplasmic transport in mammalian nerve fibers, Science, 187 (1975) 1087-1089. 21 Prestronk, A., Drachman, D.B. and Griffin, J.W., Effect of muscle disuse on acetylcholine receptors, Nature (Lond.), 260 (1976) 352-353. 22 Sjostrand, J., Frizell, M. and Hasselgren, P.O., Effects of colchicine on axonal transport in peripheral nerves, J. Neurochem., 17 (1970) 1563-1570. 23 Tiedt, T.N., Lewis Wisler, P. and Younkin, S.G., Neurotrophic regulation of resting membrane potential and acetylcholine sensitivity in rat extensor digitorum longus muscle, Exp. Neurol., 57 (1977) 766-791. 24 Wisniewski, H. and Terry, R.D., Experimental colchicine encephalopathy, Lab. Invest., 17 (1967) 577-587.
261
Elsevier/North-Holland Scientific Publishers Ltd.
NEUROTOXIN-INDUCED NERVE
H Y D R O L A S E A C T I V I T Y IN P E R I P H E R A L
ROLAND J. BOEGMAN and BRENDA SCARTH Department of Pharmacology, Queen "s University, Kingston, Ontario K7L 3N6 (Canada)
(Received December 22nd, 1980; Revised version received April 6th, 1981; Accepted April 15th, 1981)
The effect of neurotoxins which block axonal transport or impulse transmission was examined on sciatic nerve hydrolase activity. Subepineural injection of batrachotoxin (BTX) or colchicine resulted in a 4-9-fold increase in acid protease and N-acetylglucosaminidase activity with a smaller increase in acid phosphatase activity. The enzyme activity remained elevated for up to 48 days. Similar injections of tetrodotoxin (TTX) led to a relatively small increase in the activity of these enzymes which returned to normal values within two days. The prolonged increase in hydrolase activity produced by BTX and colchicine suggests that blockade of axonal transport may be mediated by nerve necrosis.
Neurotoxins such as b a t r a c h o t o x i n (BTX), colchicine and tetrodotoxin (TTX), w h e n applied to peripheral nerve in vivo and in vitro, has been used to interrupt axonal transport a n d / o r impulse transmission [2, 5, 6, 10, 11, 20, 21, 23]. The physiological action o f these drugs has been t h o u g h t to be r e m a r k a b l y selective and readily reversible [20, 21]. In this c o m m u n i c a t i o n we show that the hydrolase activity present in peripheral nerve increases following subepineural injections o f neurotoxin and with certain neurotoxins (BTX, colchicine) remains elevated for an extended time period. U n d e r pentobarbital anesthesia 1 #1 o f each o f the following in 10% dextrosesaline was injected subepineurally via a thin glass capillary, into the right sciatic nerve at the sciatic n o t c h o f 35 g male Swiss White mice: B T X (9 x 10- ~2 mol), colchicine (1 x 10 -a mol), T T X (6.3 × 10 -9 mol). E a c h animal received a similar injection o f vehicle alone into the left sciatic nerve to serve as internal c o n t r o l [5, 7]. All surgical procedures were carried out as aseptically as possible. L i m b paralysis immediately following injection o f T T X or B T X was present and was used as an index o f successful toxin application. Paralysis following T T X lasted for 2 - 3 days while BTX-induced paralysis was present for approximately 6 - 7 days. Animals treated with colchicine did not show as m a r k e d leg immobilization as those treated with B T X or T T X and, in addition, paralysis developed after one day and remained for approximately 6 days. Animals were housed under s t a n d a r d conditions and were sacrificed by decapitation. A nerve segment at the injection site 1.5 cm long was immediately r e m o v e d and placed on ice. For each experiment toxin0304-3940/81/0000-0000/$ 02.50 © Elsevier/North-Holland Scientific Publishers Ltd.
262
pretreated and control nerves from 3 animals were pooled separately and homogenized in a micro-glass homogenizer with 600 #1 cold 0.3 M sucrose, 0.1% Triton X - 100 to give a 3% w / v homogenate [7]. The following enzymes were assayed in triplicate: acid protease employing [14C-methyl]methemoglobin as substrate [4, 19] as well as acid phosphatase and N-acetylglucosaminidase using the fluorescent substrates described previously [7, 16, 17]. The mean value (expressed per mg non-collagen protein) obtained from each experiment in which the nerve was treated with toxin was divided by the mean of the control nerve to give a ratio. A pooled sample t-test was used to determine if there was a significant difference between treated and control mean values. There was a small increase in hydrolase activity within 12 h of injecting TTX into the nerve (Fig. 1). N-acetylglucosaminidase activity responded with the greatest increase (P < 0.01). The enzyme activity, however, decreased to normal values by the second day and remained unaltered up to day 8 (Fig. 1). Subepineural injection of BTX resulted in a similar initial small increase in nerve hydrolase activity, which again was most marked with N-acetylglucosaminidase ( P < 0.001 at 12 h). In contrast to TTX, however, BTX produced a secondary larger increase (Fig. 2a)
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DAYS Fig. 1. Lysosomal enzyme activity present in the sciatic nerve following subepineural administration of TTX. Acid protease, II; acid plaosphatase, A; N-acetylglucosaminidase, ®. Each point represents the mean + S.E.M. of 4 experiments in which the ratio between toxin-treated and control nerve for 3 animals/experiment was determined.
263 u.I > n,u.I Z .J 0 GC I-Z 0 tO
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Fig, 2. Lysosomal enzyme activity present in the sciatic nerve following subepineural administration of BTX (a) or colchicine (b). Acid protease, II; acid phosphatase, A; N-acetylglucosaminidase, e . Each point represents the mean _+ S.E.M. of 4 experiments in which the ratio between toxin-treated and control nerve for 3 animals/experiment was determined.
which reached a maximum between 15 and 30 days and had not returned completely to normal values by day 45. N-acetylglucosaminidase activity increased more than 9fold (P < 0.001) above control while acid protease increased 4-fold (P < 0.001) and acid phosphatase activity doubled (P < 0.01). Subepineural injection of colchicine also resulted in an increase in nerve hydrolase activity similar to that seen after BTX (Fig. 2b). A 7-fold increase in N-acetylglucosaminidase ( P < 0 . 0 0 1 ) and a 5-fold increase in acid protease activity (P < 0.001) were detected as late as 16 days after neural application of the drug. The return to normal N-acetylglucosaminidase activity was more rapid following subepineural injection of colchicine than BTX. The response of neural acid phosphatase activity to toxin insult was dramatically less than that observed with acid protease and N-acetylglucosaminidase.
264 Our data show that BTX and colchicine, two drugs known to block axonal transport, cause a marked increase in hydrolytic enzyme activity in the sciatic nerve. In contrast, TTX, which prevents Na influx, had a minimal effect on lysosomal enzyme activity. BTX- and colchicine-induced paralysis lasts for approximately twice as long as that found with TTX. The increase in hydrolase activity, however, lasts proportionately much longer and is of greater magnitude with BTX and colchicine, suggesting that the duration of paralysis is not related to the increase in enzyme activity. Hydrolases have been identified in lysosomes in the nervous system and appear to be located predominantly in the cell body [8, 12-15]. Increases in the number of lysosomes following a nerve crush injury have been observed in the axon as well as in the surrounding Schwann cells [12, 14]. The influx of Na and H20 caused by BTX [1] could be responsible for the biochemical changes observed. Colchicine inhibits the saltatory movement of intra-axonal particles at a concentration of 0.1 mM [11]. However, concentrations as high as 30 mM are required for complete blockade of axoplasmic transport [10]. Neural application of colchicine has been reported to result in axonal damage with multiplication of Schwann cells, nuclear swelling, and the appearance of endoneurial phagocytes [3]. The changes described above occur 24 h after application of the drug, which coincides with the onset of limb paralysis observed in our study. The recovery phase in the study above corresponds to the time period in our experiments where marked decreases in the hydrolytic enzyme activity were observed. Morphological changes in intra-axonal mitochondria and endoplasmic reticulum have been reported in both the CNS [24] and peripheral nerves [3, 10] following application of a similar dose of colchicine as that used in our study. The increase in lysosomal enzyme activity following neural application of BTX or colchicine could be explained as an initial inhibition of axonal transport of lysosoreal vesicles followed by the appearance of autophagic vacuoles and macrophage infiltration, the morphological changes having already been observed in both BTX [18] and colchicine [3, 24] treated preparations. This is supported by the apparent biphasic increase in acid protease and N-acetylglucosaminidase activity found in both BTX- and colchicine-treated nerves. The damage caused to the nerve by minute amounts of BTX and colchicine thus appears to have a relatively long-lasting biochemical effect. Supported by the Muscular Dystrophy Association of Canada. BTX was a gift from Dr. E.X. Albuquerque. 1 Albuquerque, E.X., The made of action of batrachotoxin, Fed. Proc., 31 (1972) 1133-1138. 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 muscle of the rat, Exp. Neurol., 37 (1972) 607-634.
265 3 Angevine, J.B., Nerve destruction by colchicine in mice and golden hamsters, J. exp. Zool., 136 (1957) 363-391. 4 Anson, M.l., The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin, J. gen. Physiol., 22 (1938) 79-89. 5 Boegman, R.J., Deshpande, S.S. and Albuquerque, E.X., Consequences of axonal transport blockade induced by batrachotoxin on mammalian neuromuscular function, I. Early pre- and postsynaptic changes, Brain Res., 187 (1980) 183-196. 6 Boegman, R.J. and Riopelle, R.J., Batrachotoxin blocks slow and retrograde axonal transport in vivo, Neurosci. Lett., 8 (1980) 143-147. 7 Boegman, R.J. and Oliver, T.W., Neural influence on muscle hydrolase activity, Life Sci., 27 (1980) 1339-1344. 8 Broadwell, R.D., Oliver, C. and Brightman, M.W., Neuronal transport of acid hydrolases and peroxidase within the lysosomal system of organelles: involvement of agranular reticulum-like cisterns, J. comp. Neurol., 190 (1980) 519-532. 9 Dingle, J.T., Lysosomes, A Laboratory Handbook, North-Holland Publ., Amsterdam, 1972. l0 Fink, B.R., Byers, M.R. and Middaugh, M.E., Dynamics of colchicine effects on rapid axonal transport and axonal morphology, Brain Res., 56 (1973) 299-311. l 1 Hammond, G.R. and Smith, R.S., Inhibition of the rapid movement of optically detectable axonal particles by colchicine and vinblastine, Brain Res., 128 (1977) 227-242. 12 Holtzman, E., Lysosomes in the physiology and pathology of neurons. In J.T. Dingle and H.B. Fell (Eds.), Lysosomes in Biology and Pathology, North-Holland Publ., Amsterdam, 1969. 13 Holtzman, E., Cytochemical studies of protein transport in the nervous system, Phil. Trans. B, 261 (1971) 407-421. 14 Holtzman, E. and Novikoff, A.B., Lysosomes in the rat sciatic nerve following crush, J. Cell Biol., 27 (1965) 651-669. 15 Koenig, H., Lysosomes in the nervous system. In J.T. Dingle and H.B. Fell (Eds.), Lysosomes in Biology and Pathology, North-Holland Publ., Amsterdam, 1969. 16 Libelius, R., Jirmanova, I., Lundquist, I. and Thesleff, S., Increased endocytosis with lysosomal activation in skeletal muscle of dystrophic mouse, J. Neuropath. exp. Neurol., 37 (1978) 387-400. 17 Libelius, R. and Lundquist, I., Lysosomal activation in mouse skeletal muscle induced by protamine in vitro, Cell Tiss. Res., 186 (1978) 1-11. 18 Moore, G.R.W., Boegman, R.J., Robertson, D.M. and Riopelle, R.J., Batrachotoxin induced axonal necrosis in peripheral nerve, Brain Res., 207 (1981) 481-485. 19 New England Nuclear Proteolytic Enzyme Assay Information Sheet. 20 Ochs, S. and Worth, R., Batrachotoxin block of fast axoplasmic transport in mammalian nerve fibers, Science, 187 (1975) 1087-1089. 21 Prestronk, A., Drachman, D.B. and Griffin, J.W., Effect of muscle disuse on acetylcholine receptors, Nature (Lond.), 260 (1976) 352-353. 22 Sjostrand, J., Frizell, M. and Hasselgren, P.O., Effects of colchicine on axonal transport in peripheral nerves, J. Neurochem., 17 (1970) 1563-1570. 23 Tiedt, T.N., Lewis Wisler, P. and Younkin, S.G., Neurotrophic regulation of resting membrane potential and acetylcholine sensitivity in rat extensor digitorum longus muscle, Exp. Neurol., 57 (1977) 766-791. 24 Wisniewski, H. and Terry, R.D., Experimental colchicine encephalopathy, Lab. Invest., 17 (1967) 577-587.