J. Physiol. (1978), 274, pp. 593-600 Printed in Great Britain
593
THE EFFECT OF DISUSE ON CHOLINERGIC ENZYMES
BY I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG From the Neuromuscular Unit, Department of Neurology, Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland 21205, and the Department of Environmental Health Sciences, Division of Toxicology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, U.S.A.
(Received 4 July 1977) SUMMARY
1. The effects of disuse on the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) have been investigated in the sciatic nerve and leg muscles of the rat. 2. Disuse was produced by blockade of nerve conduction by repeated subperineurial injection of tetrodotoxin (TTX), and the effects were compared to those of denervation. 3. After 8 days of disuse there was no change in neural ChAT activity in the sciatic nerves, anterior roots or intramuscular terminals. By contrast, surgical section of the sciatic nerve resulted in a marked decrease of ChAT in the nerve terminals. 4. Similarly, after 8 days of disuse there was no change in neural AChE activity in the sciatic nerves or anterior roots. 5. AChE activity in the disused muscles decreased by more than 50%, which was comparable to the effect of surgical denervation. 6. These results indicate that disuse produced by TTX blockade of nerve conduction does not affect the cholinergic enzymes ChAT and AChE in nerves, but does lead to a significant decrease in muscle AChE. INTRODUCTION
The enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) have important functions in the processes of synaptic transmission at neuromuscular junctions. Within skeletal muscle, ChAT is localized to innervated regions (Hebb, Krnjevi6d & Silver, 1964; Emmelin, Nordenfelt & Perec, 1966; McCaman, Stafford & Skinner, 1967; Flood, Fonnum & Storm-Mathisen, 1970), and serves to catalyse the synthesis of acetylcholine (ACh) at motor nerve terminals (Potter, 1970; Fonnum, 1973). AChE is present in both nerves and muscles (Friedenberg & Seligman, 1972; Fonnum, Frizell & Sj6strand, 1973; Kasa, Mann, Karcsu, Toth & Jordan, 1973). Its major known function is the hydrolysis of ACh at the post-synaptic membrane, rapidly terminating the transmission event (Wurzel, 1967). Since ACh plays a pivotal role in neuromuscular transmission, it seemed possible that the two enzymes directly associated with its metabolism might respond to changes in functional activity of the motor nerves. Previous attempts to study the effects of disuse
594 I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG on ChAT and AChE have employed methods such as tenotomy (Diamond, Franklin & Milfay, 1974) or pinning of animals' limbs (Snyder, Rifenberick & Max, 1973; Tudek, KostifovA & Gutmann, 1976), which produce incomplete disuse, and have additional complex neurological effects (Fischbach & Robbins, 1969; Estavillo, Yellin, Sasaki & Eldred, 1973; Drachman, 1976). In the present investigation, we have utilized a new method of blocking nerve conduction with tetrodotoxin (TTX) which has been shown to be both complete and highly specific (Pestronk, Drachman & Griffin, 1976), and have compared its effect on the enzymes with those of surgical denervation. The results indicate that muscle disuse does not alter neural levels of ChAT and AChE, but produces a marked fall of muscle AChE. METHODS Male Sprague-Dawley rats (Charles River Breeding Laboratories) weighing 250-370 g were used throughout these experiments. It was necessary to use mature animals because of the reported increase in the levels of ChAT during growth in younger rats (Tucek, 1972; Diamond et at. 1974). Before each procedure, the rats were anaesthetized with i.p. chloral hydrate (400
mg/kg).
Tetrodotoxin injections (Pestronk et al. 1976). The right sciatic nerve was exposed by an incision in the thigh, and a subperineurial injection of TTX was made using a glass micropipette coupled to a micrometer-driven syringe, mounted on a micromanipulator. Injection of 2 jag TTX in 2 #1. Ringer solution produces complete paralysis, beginning within 10 min, and lasting usually for more than 48 hr. A total of four intraneural injections of TTX were given at 48 hr intervals to each rat, in order to maintain paralysis for 8 days. The animals were observed daily for the toe spreading reflex (Blunt & Vrbova', 1975). Nerve blockade was tested before each repeat injection of TTX, and at the termination of each experiment, by electrical stimulation of the sciatic nerve above and below the injection site. Animals were discarded if (a) the toe spreading reflex was present at any time, (b) blockade of nerve conduction was incomplete, or (c) the contractile response on distal nerve stimulation was impaired. Control animals were given four subperineurial injections of Ringer solution at 48 hr intervals, and all showed brisk muscle responses on electrical stimulation of the sciatic nerves. The animals were killed at 8 days, and the extensor digitorum longus (EDL) and soleus (SOL) muscles of both legs were rapidly removed and frozen on dry ice. Both sciatic nerves and their lumbar roots were exposed by dissection and laminectomy. Segments of the fourth and fifth lumbar roots, 2 cm long, and a 1 cm long segment of the sciatic nerve distal to the injection sites were removed from both sides and rapidly frozen. Denerwtion. Denervation of rat leg muscles was performed by exposure and section of the sciatic nerve in mid-thigh, and avulsion of the proximal nerve stump.
Determination of ChAT The standard lengths of anterior roots (2 cm) and sciatic nerves (1 cm) were homogenized in 25 mm sodium phosphate, pH 7-4, in volumes of 200 and 100 #1. respectively. The whole muscles were hand homogenized in 500 ul. distilled water. The buffer substrates (McCaman & Hunt, 1965) were prepared so that the final concentrations during incubation were 250 [14C]coenzyme A, 20 mM-MgCl2, 300 mM-NaCl, 10 mmi choline, 0-15 mm eserine, 0-025 % bovine plasma albumin and 100 mM-Na2PO4 buffer, pH 7-4. The samples were incubated for either 10 min (muscle) or 5 min (nervous tissue) at 38 'C. The reaction was terminated by the addition of 50 1l. ice-cold 200 mM-NPO4 buffer, pH 8-0, followed by extraction with 150 ul. tetraphenylboron (75 mg/ml.) in 3-heptanone (Fonnum, 1969). The samples were thoroughly mixed and centrifuged at 10,000 g min. A 100 ,ul. aliquot of the 3-heptanone layer was placed in a scintillation counting vial. One ml. hyamine hydroxide (0-3 M in methanol) and 15 ml. toluene containing 5 g diphenyloxazole (PPO) and 0-1 g p-bis [2-(5-phenyloxazolyl)] benzene (POPOP) per litre of toluene were added to each vial, and the radioactivity was determined. For each muscle sample a separate blank was used in which the procedure was carried out identically except that choline was omitted from the incubation medium.
#sM-acetyl-1.-
DISUSE AND CHOLINERGIC ENZYMES
595
The determination of ChAT in tissue containing skeletal muscle requires special care (Hanin & Goldberg, 1976). The pH of the extraction mixture must be 7-0 or higher to minimize extraction of other reaction products in addition to ACh (Fonnum, 1975). Furthermore, the results of reactions run with choline-free substrate-buffer must be subtracted from those of the standard reactions, to exclude spurious measurement of products other than ACh. The use of choline-free blanks revealed that up to half of the radioactivity measured by this method was not in ACh. Paper chromatography identified the other product as acetylcarnitine. Thus, when ChAT is measured in homogenates of muscle, care must be taken to avoid these pitfalls. This caution does not apply to measurements in nerves, where the only significant radioactive product is ACh. The results are expressed as n-moles of ACh synthesized per hour per 5 mm length of nerve, or per whole muscle. The denominators used for nerve (Lubinska & Niemierko, 1971) and muscle were more useful for the present study than those referred to weight or protein content, since they do not depend on changes resulting from atrophy.
Determination of AChE The determination of AChE was essentially as described by Fonnum (1969) using [14C]ACh. Incubations were carried out for 30 min at 30 0C, using a final concentration during incubation of 0-5-1 mM-ACh in 100 mM-Na2PO4, pH 7-0. All samples were assayed in duplicate, and enzyme activity was calculated on the basis of the known specific activity of the labelled substrate. The conditions of assays were such that less than 10 % of the substrate was utilized and the reaction was linear with both time of incubation and amount of tissue. Statistical analysis was carried out by means of the paired t test. TABLE 1. Sciatic nerve and lumbar root choline acetyltransferase (ChAT)
L4 anterior root (9) L5 anterior root (5) Sciatic nerve (6)
TTX injected 13-2 9-8 14-0
S.E. of mean 1-8 0-8 1-2
Opposite control 13-6 11-1 13-6
5.E. of
L4 anterior root (7) L5 anterior root (6) Sciatic nerve (7)
Ringer injected 11-7 17-0 12-2
Opposite control 12-7 18-2 12-0
mean 1-2 1-4
0-9
s.E. of mean 1-5 1-9 1-4 S.E. of mean 1-7 1-8 0-9
Ratio 0-97 0-89 1-03
P value > 0-10 > 0-10 > 0-10
Ratio 0-92 0-94 1-02
P value > 0-10 > 0-10 > 0-10
The results are means of the samples (number of samples in parentheses) and ChAT activity is expressed as n-moles ACh synthesized/hr. 5 mm anterior root or sciatic nerve. Probabilities were calculated by paired t test. RESULTS
TTX treatment of the sciatic nerve for 8 days produced no significant change in ChAT activity of the motor nerve roots, sciatic nerves (Table 1) or intramuscular nerve endings (muscles) (Table 2). By contrast, surgical section of the sciatic nerves resulted in a marked loss of ChAT in intramuscular nerves (Table 2), consistent with previous reports (Hebb et al. 1964; Tucek, 1973). Although ChAT activity in nerves and muscles varied considerably from animal to animal and especially in groups of animals done at different times, side-to-side comparisons gave good agreement. In the control animals the ratio of mean ChAT activity on the Ringer-injected side to ChAT activity on the untreated side was 0-92 for the L4 anterior roots; 0-94 for the L5 anterior roots; 1-02 for the sciatic
I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG
596
TABLE 2. Muscle (intramuscular nerve ending) choline acetyltransferase (ChAT) TTX S.E. of Opposite S.E. of injected mean control mean Ratio P value 13-59 EDL (12) 0-98 11-43 1-47 > 0-10 1-19 7-17 0-77 SOL (13) 8-48 > 0-10 1-33 0-85 Ringer S.E. of Opposite S.E. of injected mean control mean Ratio P value EDL (7) 1-14 939 10-36 0-94 > 0-10 0-91 7*74 0-36 7-10 SOL (7) 1.09 0-92 > 0-10 S.E. of Opposite S.E. of Denervated mean control mean Ratio P value EDL (6) 3-33 12-48 0-49 0-75 0-27 0-10 > 0-10 > 0-10
> 0-10 > 0-10 > 0-10
The results are means of the samples (number in parentheses) and the AChE activity is expressed as n-mole ACh hydrolysed/hr. 5 mm anterior root or sciatic nerve. TABLE 4. Muscle acetylcholinesterase (AChE)
EDL (13) SOL (13)
TTX injected 2-60 2-66
Ringer injected
S.E. of mean 0-28 0-16 s.E. of mean
Opposite control 5-41 5-61
Opposite control
S.E. of mean 0-73
Ratio 0-48
0-46
0-47
P value < 0-01 < 0-01
Ratio 1-10 0-96
P value > 0-10 > 0-10
Ratio 0-27 0-43
P value
S.E.
of
mean
EDL (7) SOL (7)
9-95
0-58
9-05
5-37
0-26
5-59
0-45 0-34
5.E. of mean
Opposite
S.E. of
control
mean
EDL (7) SOL (7)
Denervated 1-34 1-99
0-10
4.93 4.59
0-87 0-46
0-24
< 0-01 < 0.01
The results are means of the samples (number in parentheses) and AChE activity is expressed in molee ACh hydrolysed/hr.whole muscle.
DISUSE AND CHOLINERGIC ENZYMES 597 nerves; 0-91 for the EDL muscles and 1.09 for the SOL muscles. There were no significant differences from side-to-side (P > 0410, paired t test). Acetylcholinesterase. AChE activity in the anterior roots and sciatic nerves showed no significant differences after 8 days of TTX treatment (P > 0.10) (Table 3). As with ChAT measurements, side-to-side comparison were used to assess the effects of the experimental procedure. In the control animals, the ratios of AChE activity of the Ringer-injected over the untreated side were close to unity (Table 3). By contrast, there was a highly significant (P < 0.01) decrease in muscle AChE on the TTX-treated side to 47 % of control in the SOL, and 48 % of control in the EDL. The ratio for the SOL was not significantly different from that seen with denervation. However, denervation produced a greater fall of AChE activity in the EDL than did TTX treatment of the nerve (Table 4). DISCUSSION
There is considerable evidence that activity or usage of nerves may play a role in regulation of the neurotransmitter-related enzymes (Thoenen, 1974; Schmitt, Dev & Smith, 1976). This has been shown particularly clearly in the sympathetic nervous system, where the adrenergic enzymes, dopamine-/J-hydroxylase and tyrosine hydroxylase, as well as ChAT in the presynaptic neurones, increased promptly in response to stimulation (Mueller, Thoenen & Axelrod, 1969; Goldberg & Welch, 1972; Oesch & Thoenen, 1973; Ben-Ari & Zigmond, 1975). By analogy, it might be expected that cholinergic enzyme levels of motor nerves would also respond to usage. In this study, we have evaluated the effects of decreased neuromuscular activity on the cholinergic enzymes ChAT and AChE. We have utilized a highly specific new method to block impulse conduction in the sciatic nerves of rats by means of local injections of TTX (Pestronk et al. 1976). TTX interferes with conduction of electrical impulses in excitable membranes by blocking Na conductance channels (Hille, 1968). Its effect is localized to a short segment of nerve at the level of the injection site, while the remaining length of nerve remains fully functional proximal and distal to the block. TTX-blocked nerves continue to release ACh spontaneously, giving rise to miniature end-plate potentials which are normal in frequency and amplitude. Furthermore, the TTX injection method does not alter the rapid axonal transport of radioactively labelled proteins and does not damage the nerve morphologically (Pestronk et al. 1976). As used in the present experiments, TTX thus produces total 'disuse' of the muscle and blockade of nerve conduction at the level of the sciatic nerve, while allowing all other neuromuscular functions tested to remain unaltered. Disuse brought about by this method had no effect on the levels of ChAT or AChE within motor nerves. Since intraneural ChAT and AChE are synthesized in the nerve cell bodies and are carried centrifugally by axonal transport (Fonnum et al. 1973), change in enzyme levels might be expected to occur earlier in the proximal portions of motor nerves than in the periphery. We therefore measured enzyme activities in the nerves and motor nerve roots as well as in the intramuscular nerve endings, but found no decrease after disuse. Our results are at variance with previous reports of decreased ChAT levels in muscles subjected to various types of 'disuse' (Snyder et al. 1973; Diamond et al.
598 I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG 1974; Tucek et al. 1976). The discrepancies may be accounted for by two procedural differences in the experimental methods. (1) In some studies (Snyder et al. 1973; Tucek et al. 1976) precautions were not taken to ensure the specificity of the ChAT determinations, as noted above (Methods). Contamination by substances other than ACh may be great enough to explain an apparent 50 % fall in enzyme levels. (2) Although the 'disuse' methods employed in different experiments result in decreased muscle activity, their actual physiological effects differ greatly. The method of nerve block (TTX) used in our experiments completely eliminates nerve evoked muscle action potentials and contraction. However, its direct effects are confined to the periphery; so far as is known, it does not produce changes in synaptic or electrical activity of the cell bodies. By contrast, 'disuse' brought about by pinning or tenotomy does not directly interfere with peripheral nerve activity. The effects of these procedures are complex, being attributed in part to mechanical interference with normal contraction of muscle, and in part to secondary alterations of reflex mechanisms (Fischbach et al. 1969; Estavillo et al. 1973). Our data suggest that peripheral neuromuscular quiescence does not result in decreased synthesis and/or delivery of ChAT and AChE by motor nerves. However, the possibility that synaptic activity at the level of the nerve cell body might regulate cholinergic enzyme levels has not been tested by the present exeriments, and could conceivably explain the observation of a decrease in ChAT levels with tenotomy (Diamond et al. 1974). In the present study, the marked effect of disuse on muscle AChE contrasts sharply with its lack of effect on the nerve enzymes. The decrease of muscle AChE activity with TTX-induced disuse is similar to that occurring after denervation. This suggests that the motor nerve's trophic role in regulation of muscle AChE depends in large measure on its ability to trigger usage of muscle. It is consistent with our previous observations on the effects of botulinum treatment on muscle AChE (Drachman, 1972). It should be emphasized that levels of ChAT and AChE in motor nerves are not immutable, but are capable of responding to changes in the environment. For example, injury produces alterations of enzyme levels in both the proximal and distal portions of the damaged nerve. The decrease of ChAT levels in the distal segment of the nerves proceeds rapidly, with a marked reduction by 8 days (Tucek, 1974; Table 2). However, the changes in ChAT levels occurring in the proximal stump after a reversible nerve lesion (crush or freeze) are more complex (Frizell & Sj6strand, 1974; Butler, Drachman & Goldberg, 1975). There is an initial rise, presumably due to accumulation of transported ChAT proximal to the lesion. This is followed by a fall to subnormal levels during the phase of nerve regeneration. A return to normal levels occurs at later times, presumably reflecting re-connexion of the regenerated nerves with the periphery. A similar rise of ChAT levels has been observed in cocultured nerve and muscle about the time of junction formation (Giller, Schrier, Shainberg, Fisk & Nelson, 1973), and in the ciliary ganglion when synapses are formed (Chiappinelli, Giacobini, Pilar & Uchimura, 1976). Thus it appears that connexion with the periphery results in enhancement of neural ChAT levels. Whether thi3 increase requires neuromuscular transmission has not yet been tested, but our evidence implies that functional activity may not be necessary. More likely other kinds of signals serve to modulate the activities of ChAT and AChE in motor nerves.
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We thank Ms Andrea Lentz for technical assistance and Ms Christine Barlow for assistance. Supported by N.I.H. Grants 5 PO1 NS10920, 5 ROI HD04817, NIEHS 00454, NIEHS 00034 and a grant from the Dysautonomia Foundation. REFERENCES BEN-ARI, Y. & ZIGMOND, R. E. (1975). Delayed increase in tyrosine hydroxylase activity in rat superior cervical ganglion after electrical stimulation of the pre-ganglionic nerve. J. Phyaiol. 248, 48P-49P. BLUNT, R. J. & VRBOVA, G. (1975). The use of local anaesthetics to produce prolonged motor nerve block in the study of denervation hypersensitivity. Pfluigera Arch. 357,189-199. BUTLER, I. J., DRACHMAN, D. B. & GOLDBERG, A. M. (1975). Selective decrease of enzyme transport in regenerating motor nerves. Neurology 25, 367. CHIAPPINELLI, V., GiAcOBINI, E., PILAR, G. & UCHIMURA, H. (1976). Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation. J. Physiol. 257, 749-766. DIAMoND, I., FRANKLIN, G. M. & MILFAY, D. (1974). The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function. J. Phyeiol. 236, 247-257. DRACHMAN, D. B. (1972). Neurotrophic regulation of muscle cholinesterase: effects of botulinum toxin and denervation. J. Phyqiol. 226, 619-627. DRACHMAN, D. B. (1976). Trophic interactions between nerves and muscles: the role ofcholinergic transmission (including usage) and other factors. In Biology of Cholinergic Function, ed. GOLDBERG, A. M. & HANIN, I., pp. 161-186. New York: Raven. EMMELIN, N., NORDENFELT, I. & PEREC, C. (1966). Rate of fall in choline acetyltransferase activity in denervated diaphragms as dependent on the length of the degenerating nerve. Experientia 22, 725-726. ESTAVILLO, J., YELLIN, H., SASAKI, Y. & ELDRED, E. (1973). Observations on the expected decrease in proprioceptive discharge and purported advent of non-proprioceptive activity from the chronically tenotomized muscle. Brain Res. 63, 75-91. FISCHBACH, G. D. & ROBBINS, N. (1969). Changes in contractile properties of disused soleus muscles. J. Phyriol. 201, 305-320. FLOOD, P. R., FONNUm, F. & STORM-MATHISEN, J. (1970). Choline acetyltransferase and acetylcholinesterase in myo-tendinous and neuromuscular junctions of mouse skeletal muscle. Experientia 26, 964-965. FoNNum, F. (1969). Radiochemical microassays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J. 115, 465-472. FONNUM, F. (1973). Recent developments in biochemical investigation of cholinergic trans. mission. Brain Rea. 62, 497-507. FoNNUM, F. (1975). A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24, 407-409. FoNNum, F., FRIZELL, M. & SJoSTRAND, J. (1973). Transport, turnover and distribution of choline acetyltransferase and acetylcholinesterase in the vagus and hypoglossal nerves of the rabbit. J. Neurochem. 21, 1109-1120. FRIEDENBERG, R. M. & SELIGMAN, A. M. (1972). Acetylcholinesterase at the myoneural junction: cytochemical ultrastructure and some biochemical considerations. J. Hietochem. Cytochem. 20, 771-792. FRIZELL, M. & SJOSTRAND, J. (1974). Transport of proteins, glycoprotein and cholinergic enzymes in regenerating hypoglossal neurons. J. Neurochem. 22, 845-850. GILLER, E. L., JR., SCKRIER, B. K., SHAINBERG, A., FISK, H. R. & NELSON, P. G. (1973). Choline acetyltransferase activity is increased in combined cultures of spinal cord and muscle cells from mice. Science, N.Y. 182, 588-589. GOLDBERG, A. M. & WELCH, B. L. (1972). Adaptation of the adrenal medulla: sustained increase in choline acetyltransferase by psychosocial stimulation. Science, N.Y. 178, 319-320. HANIN, I. & GOLDBERG, A. M. (1976). Appendix I: Quantitative assay methodology for choline, acetylcholine, choline acetyltransferase and acetylcholinesterase. In Biology of Cholinergic Function, ed. GOLDBERG, A. M. & HANIN, I., pp. 647-654. New York: Raven.
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HEBB, C. O., KRNJEvf6, K. & SILVER, A. (1964). Acetylcholine and choline acetyltransferase in the diaphragm of the rat. J. Phy8iol. 171, 504-513. HuiLE, B. (1968). Pharmacological modifications of the sodium channels of frog nerve. J. gen. Phy8iol. 51, 199-219. KASA, P., MANN, S. P., KARcsu, S., T6TH, L. & JORDAN, S. (1973). Transport of choline acetyltransferase and acetylcholinesterase in the rat sciatic nerve: a biochemical and electron histochemical study. J. Neurochem. 21, 431-436. LUBIN'SxA, L. & NIEMIERKO, S. (1971). Velocity and intensity of bidirectional migration of acetylcholinesterase in transacted nerves. Brain Re8. 27, 329-342. MCCAMAN, M., STAFFORD, M. L. & SiNNER, E. C. (1967). Choline acetyltransferase and cholinesterase activities in muscle of dystrophic mice. Am. J. Phy8iol. 212, 228-232. McCAMAN, R. E. & HuNT, J. M. (1965). Microdeterminations of choline acetylase in nervous tissue. J. Neurochem. 12, 253-259. MUELLER, R. A., THOENEN, H. & AxELROD, J. (1969). Increase in tyrosine hydroxylase activity after reserpine administration. J. Pharmac. exp. Ther. 169, 74-79. OESCH, F. & THOENEN, H. (1973). Increased activity of the peripheral sympathetic nervous system: induction of choline acetyltransferase in the preganglionic cholinergic neurone. Nature, Lond. 242, 536-537. PEsTRONK, A., DRACHMAN, D. B. & GRIFFIN, J. W. (1976). Effect of muscle disuse on acetylcholine receptors. Nature, Lond. 260, 352-353.
POTTER, L. T. (1970). Synthesis, storage and release of [14C]acetylcholine in isolated rat diaphragm muscles. J. Physiol. 206, 145-166. SCHMITT, F. O., DEV, P. & SMITH, B. H. (1976). Electronic processing of information by brain cells. Science, N.Y. 193, 114-120. SNYDER, D. H., RIFENDERICK, D. H. & MAx, S. R. (1973). Effects of neuromuscular activity on choline acetyltransferase and acetylcholinesterase. Expl Neurol. 40, 36-42. THOENEN, H. (1974). Trans-synaptic enzyme induction. Life Sci. Oxford 14, 223-235. TU6EK, S. (1968). Motor nerve and the activity of choline acetyltransferase in the skeletal muscle. Biochim. biophy8. Acta 170, 457-458. TU6EK, S. (1972). Choline acetyltransferase activity in rat skeletal muscles during postnatal development. Expl Neurol. 36, 378-388. TU6EK, S. (1973). Choline acetyltransferase activity in skeletal muscles after denervation. Expl Neurol. 40, 23-25. TuEK, S. (1974). Transport and changes of activity of choline acetyltransferase in the peripheral stump of an interrupted nerve. Brain Re8. 82, 249-261. TU6EK, S., KO§TrfOvA, D. & GUTmANN, E. (1976). Effects of castration, testosterone and immobilization on the activities of choline acetyltransferase and cholinesterase in rat limb muscles. J. neurol. Sci. 27, 363-372. WURZEL, M. (1967). The physiological role of cholinesterase at cholinergic receptor sites. Ann. N.Y. Acad. Sci. 144, 694-704.
593
THE EFFECT OF DISUSE ON CHOLINERGIC ENZYMES
BY I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG From the Neuromuscular Unit, Department of Neurology, Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland 21205, and the Department of Environmental Health Sciences, Division of Toxicology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, U.S.A.
(Received 4 July 1977) SUMMARY
1. The effects of disuse on the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) have been investigated in the sciatic nerve and leg muscles of the rat. 2. Disuse was produced by blockade of nerve conduction by repeated subperineurial injection of tetrodotoxin (TTX), and the effects were compared to those of denervation. 3. After 8 days of disuse there was no change in neural ChAT activity in the sciatic nerves, anterior roots or intramuscular terminals. By contrast, surgical section of the sciatic nerve resulted in a marked decrease of ChAT in the nerve terminals. 4. Similarly, after 8 days of disuse there was no change in neural AChE activity in the sciatic nerves or anterior roots. 5. AChE activity in the disused muscles decreased by more than 50%, which was comparable to the effect of surgical denervation. 6. These results indicate that disuse produced by TTX blockade of nerve conduction does not affect the cholinergic enzymes ChAT and AChE in nerves, but does lead to a significant decrease in muscle AChE. INTRODUCTION
The enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) have important functions in the processes of synaptic transmission at neuromuscular junctions. Within skeletal muscle, ChAT is localized to innervated regions (Hebb, Krnjevi6d & Silver, 1964; Emmelin, Nordenfelt & Perec, 1966; McCaman, Stafford & Skinner, 1967; Flood, Fonnum & Storm-Mathisen, 1970), and serves to catalyse the synthesis of acetylcholine (ACh) at motor nerve terminals (Potter, 1970; Fonnum, 1973). AChE is present in both nerves and muscles (Friedenberg & Seligman, 1972; Fonnum, Frizell & Sj6strand, 1973; Kasa, Mann, Karcsu, Toth & Jordan, 1973). Its major known function is the hydrolysis of ACh at the post-synaptic membrane, rapidly terminating the transmission event (Wurzel, 1967). Since ACh plays a pivotal role in neuromuscular transmission, it seemed possible that the two enzymes directly associated with its metabolism might respond to changes in functional activity of the motor nerves. Previous attempts to study the effects of disuse
594 I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG on ChAT and AChE have employed methods such as tenotomy (Diamond, Franklin & Milfay, 1974) or pinning of animals' limbs (Snyder, Rifenberick & Max, 1973; Tudek, KostifovA & Gutmann, 1976), which produce incomplete disuse, and have additional complex neurological effects (Fischbach & Robbins, 1969; Estavillo, Yellin, Sasaki & Eldred, 1973; Drachman, 1976). In the present investigation, we have utilized a new method of blocking nerve conduction with tetrodotoxin (TTX) which has been shown to be both complete and highly specific (Pestronk, Drachman & Griffin, 1976), and have compared its effect on the enzymes with those of surgical denervation. The results indicate that muscle disuse does not alter neural levels of ChAT and AChE, but produces a marked fall of muscle AChE. METHODS Male Sprague-Dawley rats (Charles River Breeding Laboratories) weighing 250-370 g were used throughout these experiments. It was necessary to use mature animals because of the reported increase in the levels of ChAT during growth in younger rats (Tucek, 1972; Diamond et at. 1974). Before each procedure, the rats were anaesthetized with i.p. chloral hydrate (400
mg/kg).
Tetrodotoxin injections (Pestronk et al. 1976). The right sciatic nerve was exposed by an incision in the thigh, and a subperineurial injection of TTX was made using a glass micropipette coupled to a micrometer-driven syringe, mounted on a micromanipulator. Injection of 2 jag TTX in 2 #1. Ringer solution produces complete paralysis, beginning within 10 min, and lasting usually for more than 48 hr. A total of four intraneural injections of TTX were given at 48 hr intervals to each rat, in order to maintain paralysis for 8 days. The animals were observed daily for the toe spreading reflex (Blunt & Vrbova', 1975). Nerve blockade was tested before each repeat injection of TTX, and at the termination of each experiment, by electrical stimulation of the sciatic nerve above and below the injection site. Animals were discarded if (a) the toe spreading reflex was present at any time, (b) blockade of nerve conduction was incomplete, or (c) the contractile response on distal nerve stimulation was impaired. Control animals were given four subperineurial injections of Ringer solution at 48 hr intervals, and all showed brisk muscle responses on electrical stimulation of the sciatic nerves. The animals were killed at 8 days, and the extensor digitorum longus (EDL) and soleus (SOL) muscles of both legs were rapidly removed and frozen on dry ice. Both sciatic nerves and their lumbar roots were exposed by dissection and laminectomy. Segments of the fourth and fifth lumbar roots, 2 cm long, and a 1 cm long segment of the sciatic nerve distal to the injection sites were removed from both sides and rapidly frozen. Denerwtion. Denervation of rat leg muscles was performed by exposure and section of the sciatic nerve in mid-thigh, and avulsion of the proximal nerve stump.
Determination of ChAT The standard lengths of anterior roots (2 cm) and sciatic nerves (1 cm) were homogenized in 25 mm sodium phosphate, pH 7-4, in volumes of 200 and 100 #1. respectively. The whole muscles were hand homogenized in 500 ul. distilled water. The buffer substrates (McCaman & Hunt, 1965) were prepared so that the final concentrations during incubation were 250 [14C]coenzyme A, 20 mM-MgCl2, 300 mM-NaCl, 10 mmi choline, 0-15 mm eserine, 0-025 % bovine plasma albumin and 100 mM-Na2PO4 buffer, pH 7-4. The samples were incubated for either 10 min (muscle) or 5 min (nervous tissue) at 38 'C. The reaction was terminated by the addition of 50 1l. ice-cold 200 mM-NPO4 buffer, pH 8-0, followed by extraction with 150 ul. tetraphenylboron (75 mg/ml.) in 3-heptanone (Fonnum, 1969). The samples were thoroughly mixed and centrifuged at 10,000 g min. A 100 ,ul. aliquot of the 3-heptanone layer was placed in a scintillation counting vial. One ml. hyamine hydroxide (0-3 M in methanol) and 15 ml. toluene containing 5 g diphenyloxazole (PPO) and 0-1 g p-bis [2-(5-phenyloxazolyl)] benzene (POPOP) per litre of toluene were added to each vial, and the radioactivity was determined. For each muscle sample a separate blank was used in which the procedure was carried out identically except that choline was omitted from the incubation medium.
#sM-acetyl-1.-
DISUSE AND CHOLINERGIC ENZYMES
595
The determination of ChAT in tissue containing skeletal muscle requires special care (Hanin & Goldberg, 1976). The pH of the extraction mixture must be 7-0 or higher to minimize extraction of other reaction products in addition to ACh (Fonnum, 1975). Furthermore, the results of reactions run with choline-free substrate-buffer must be subtracted from those of the standard reactions, to exclude spurious measurement of products other than ACh. The use of choline-free blanks revealed that up to half of the radioactivity measured by this method was not in ACh. Paper chromatography identified the other product as acetylcarnitine. Thus, when ChAT is measured in homogenates of muscle, care must be taken to avoid these pitfalls. This caution does not apply to measurements in nerves, where the only significant radioactive product is ACh. The results are expressed as n-moles of ACh synthesized per hour per 5 mm length of nerve, or per whole muscle. The denominators used for nerve (Lubinska & Niemierko, 1971) and muscle were more useful for the present study than those referred to weight or protein content, since they do not depend on changes resulting from atrophy.
Determination of AChE The determination of AChE was essentially as described by Fonnum (1969) using [14C]ACh. Incubations were carried out for 30 min at 30 0C, using a final concentration during incubation of 0-5-1 mM-ACh in 100 mM-Na2PO4, pH 7-0. All samples were assayed in duplicate, and enzyme activity was calculated on the basis of the known specific activity of the labelled substrate. The conditions of assays were such that less than 10 % of the substrate was utilized and the reaction was linear with both time of incubation and amount of tissue. Statistical analysis was carried out by means of the paired t test. TABLE 1. Sciatic nerve and lumbar root choline acetyltransferase (ChAT)
L4 anterior root (9) L5 anterior root (5) Sciatic nerve (6)
TTX injected 13-2 9-8 14-0
S.E. of mean 1-8 0-8 1-2
Opposite control 13-6 11-1 13-6
5.E. of
L4 anterior root (7) L5 anterior root (6) Sciatic nerve (7)
Ringer injected 11-7 17-0 12-2
Opposite control 12-7 18-2 12-0
mean 1-2 1-4
0-9
s.E. of mean 1-5 1-9 1-4 S.E. of mean 1-7 1-8 0-9
Ratio 0-97 0-89 1-03
P value > 0-10 > 0-10 > 0-10
Ratio 0-92 0-94 1-02
P value > 0-10 > 0-10 > 0-10
The results are means of the samples (number of samples in parentheses) and ChAT activity is expressed as n-moles ACh synthesized/hr. 5 mm anterior root or sciatic nerve. Probabilities were calculated by paired t test. RESULTS
TTX treatment of the sciatic nerve for 8 days produced no significant change in ChAT activity of the motor nerve roots, sciatic nerves (Table 1) or intramuscular nerve endings (muscles) (Table 2). By contrast, surgical section of the sciatic nerves resulted in a marked loss of ChAT in intramuscular nerves (Table 2), consistent with previous reports (Hebb et al. 1964; Tucek, 1973). Although ChAT activity in nerves and muscles varied considerably from animal to animal and especially in groups of animals done at different times, side-to-side comparisons gave good agreement. In the control animals the ratio of mean ChAT activity on the Ringer-injected side to ChAT activity on the untreated side was 0-92 for the L4 anterior roots; 0-94 for the L5 anterior roots; 1-02 for the sciatic
I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG
596
TABLE 2. Muscle (intramuscular nerve ending) choline acetyltransferase (ChAT) TTX S.E. of Opposite S.E. of injected mean control mean Ratio P value 13-59 EDL (12) 0-98 11-43 1-47 > 0-10 1-19 7-17 0-77 SOL (13) 8-48 > 0-10 1-33 0-85 Ringer S.E. of Opposite S.E. of injected mean control mean Ratio P value EDL (7) 1-14 939 10-36 0-94 > 0-10 0-91 7*74 0-36 7-10 SOL (7) 1.09 0-92 > 0-10 S.E. of Opposite S.E. of Denervated mean control mean Ratio P value EDL (6) 3-33 12-48 0-49 0-75 0-27 0-10 > 0-10 > 0-10
> 0-10 > 0-10 > 0-10
The results are means of the samples (number in parentheses) and the AChE activity is expressed as n-mole ACh hydrolysed/hr. 5 mm anterior root or sciatic nerve. TABLE 4. Muscle acetylcholinesterase (AChE)
EDL (13) SOL (13)
TTX injected 2-60 2-66
Ringer injected
S.E. of mean 0-28 0-16 s.E. of mean
Opposite control 5-41 5-61
Opposite control
S.E. of mean 0-73
Ratio 0-48
0-46
0-47
P value < 0-01 < 0-01
Ratio 1-10 0-96
P value > 0-10 > 0-10
Ratio 0-27 0-43
P value
S.E.
of
mean
EDL (7) SOL (7)
9-95
0-58
9-05
5-37
0-26
5-59
0-45 0-34
5.E. of mean
Opposite
S.E. of
control
mean
EDL (7) SOL (7)
Denervated 1-34 1-99
0-10
4.93 4.59
0-87 0-46
0-24
< 0-01 < 0.01
The results are means of the samples (number in parentheses) and AChE activity is expressed in molee ACh hydrolysed/hr.whole muscle.
DISUSE AND CHOLINERGIC ENZYMES 597 nerves; 0-91 for the EDL muscles and 1.09 for the SOL muscles. There were no significant differences from side-to-side (P > 0410, paired t test). Acetylcholinesterase. AChE activity in the anterior roots and sciatic nerves showed no significant differences after 8 days of TTX treatment (P > 0.10) (Table 3). As with ChAT measurements, side-to-side comparison were used to assess the effects of the experimental procedure. In the control animals, the ratios of AChE activity of the Ringer-injected over the untreated side were close to unity (Table 3). By contrast, there was a highly significant (P < 0.01) decrease in muscle AChE on the TTX-treated side to 47 % of control in the SOL, and 48 % of control in the EDL. The ratio for the SOL was not significantly different from that seen with denervation. However, denervation produced a greater fall of AChE activity in the EDL than did TTX treatment of the nerve (Table 4). DISCUSSION
There is considerable evidence that activity or usage of nerves may play a role in regulation of the neurotransmitter-related enzymes (Thoenen, 1974; Schmitt, Dev & Smith, 1976). This has been shown particularly clearly in the sympathetic nervous system, where the adrenergic enzymes, dopamine-/J-hydroxylase and tyrosine hydroxylase, as well as ChAT in the presynaptic neurones, increased promptly in response to stimulation (Mueller, Thoenen & Axelrod, 1969; Goldberg & Welch, 1972; Oesch & Thoenen, 1973; Ben-Ari & Zigmond, 1975). By analogy, it might be expected that cholinergic enzyme levels of motor nerves would also respond to usage. In this study, we have evaluated the effects of decreased neuromuscular activity on the cholinergic enzymes ChAT and AChE. We have utilized a highly specific new method to block impulse conduction in the sciatic nerves of rats by means of local injections of TTX (Pestronk et al. 1976). TTX interferes with conduction of electrical impulses in excitable membranes by blocking Na conductance channels (Hille, 1968). Its effect is localized to a short segment of nerve at the level of the injection site, while the remaining length of nerve remains fully functional proximal and distal to the block. TTX-blocked nerves continue to release ACh spontaneously, giving rise to miniature end-plate potentials which are normal in frequency and amplitude. Furthermore, the TTX injection method does not alter the rapid axonal transport of radioactively labelled proteins and does not damage the nerve morphologically (Pestronk et al. 1976). As used in the present experiments, TTX thus produces total 'disuse' of the muscle and blockade of nerve conduction at the level of the sciatic nerve, while allowing all other neuromuscular functions tested to remain unaltered. Disuse brought about by this method had no effect on the levels of ChAT or AChE within motor nerves. Since intraneural ChAT and AChE are synthesized in the nerve cell bodies and are carried centrifugally by axonal transport (Fonnum et al. 1973), change in enzyme levels might be expected to occur earlier in the proximal portions of motor nerves than in the periphery. We therefore measured enzyme activities in the nerves and motor nerve roots as well as in the intramuscular nerve endings, but found no decrease after disuse. Our results are at variance with previous reports of decreased ChAT levels in muscles subjected to various types of 'disuse' (Snyder et al. 1973; Diamond et al.
598 I. J. BUTLER, D. B. DRACHMAN AND A. M. GOLDBERG 1974; Tucek et al. 1976). The discrepancies may be accounted for by two procedural differences in the experimental methods. (1) In some studies (Snyder et al. 1973; Tucek et al. 1976) precautions were not taken to ensure the specificity of the ChAT determinations, as noted above (Methods). Contamination by substances other than ACh may be great enough to explain an apparent 50 % fall in enzyme levels. (2) Although the 'disuse' methods employed in different experiments result in decreased muscle activity, their actual physiological effects differ greatly. The method of nerve block (TTX) used in our experiments completely eliminates nerve evoked muscle action potentials and contraction. However, its direct effects are confined to the periphery; so far as is known, it does not produce changes in synaptic or electrical activity of the cell bodies. By contrast, 'disuse' brought about by pinning or tenotomy does not directly interfere with peripheral nerve activity. The effects of these procedures are complex, being attributed in part to mechanical interference with normal contraction of muscle, and in part to secondary alterations of reflex mechanisms (Fischbach et al. 1969; Estavillo et al. 1973). Our data suggest that peripheral neuromuscular quiescence does not result in decreased synthesis and/or delivery of ChAT and AChE by motor nerves. However, the possibility that synaptic activity at the level of the nerve cell body might regulate cholinergic enzyme levels has not been tested by the present exeriments, and could conceivably explain the observation of a decrease in ChAT levels with tenotomy (Diamond et al. 1974). In the present study, the marked effect of disuse on muscle AChE contrasts sharply with its lack of effect on the nerve enzymes. The decrease of muscle AChE activity with TTX-induced disuse is similar to that occurring after denervation. This suggests that the motor nerve's trophic role in regulation of muscle AChE depends in large measure on its ability to trigger usage of muscle. It is consistent with our previous observations on the effects of botulinum treatment on muscle AChE (Drachman, 1972). It should be emphasized that levels of ChAT and AChE in motor nerves are not immutable, but are capable of responding to changes in the environment. For example, injury produces alterations of enzyme levels in both the proximal and distal portions of the damaged nerve. The decrease of ChAT levels in the distal segment of the nerves proceeds rapidly, with a marked reduction by 8 days (Tucek, 1974; Table 2). However, the changes in ChAT levels occurring in the proximal stump after a reversible nerve lesion (crush or freeze) are more complex (Frizell & Sj6strand, 1974; Butler, Drachman & Goldberg, 1975). There is an initial rise, presumably due to accumulation of transported ChAT proximal to the lesion. This is followed by a fall to subnormal levels during the phase of nerve regeneration. A return to normal levels occurs at later times, presumably reflecting re-connexion of the regenerated nerves with the periphery. A similar rise of ChAT levels has been observed in cocultured nerve and muscle about the time of junction formation (Giller, Schrier, Shainberg, Fisk & Nelson, 1973), and in the ciliary ganglion when synapses are formed (Chiappinelli, Giacobini, Pilar & Uchimura, 1976). Thus it appears that connexion with the periphery results in enhancement of neural ChAT levels. Whether thi3 increase requires neuromuscular transmission has not yet been tested, but our evidence implies that functional activity may not be necessary. More likely other kinds of signals serve to modulate the activities of ChAT and AChE in motor nerves.
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