0361-9230/92 $5.00 + .OO
Brain Research Bulletin. Vol. 29, pp. 179-187, 1992
Copyright 0 1992 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
Role of Muscle Fasciculations in the Generation of Myopathies in Mammalian Skeletal Muscle MICHAEL ADLER,*’
DONALD HINMAN* AND C. SUE HUDSON
*Neurotoxicology Branch, Pathophysiology Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010 Department of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
ADLER, M., D. HINMAN AND C. S. HUDSON. Role qf rnuscle,fasciculations in the generation of myopathies in mammalian skeletal muscle. BRAIN RES BULL 29(2) 179-I 87, 1992.-The myotoxicity of pyridostigmine bromide was investigated on rat diaphragm nerve-muscle preparations in vitro. Within 2 h of exposure to pyridostigmine (2 PM), diaphragm muscles exhibited
ultrastructural alterations characterized by swelling of subjunctional mitochondria and disorganization of contractile proteins. These alterations developed both in the absence and presence of electrical stimulation of the phrenic nerve, and were accompanied by continuous muscle fasciculations. Pretreatment by tetrodotoxin suppressed both the muscle fasciculations and the appearance of myopathies. These findings suggest that fasciculations may be an important contributing factor in the development of anticholinesterase-induced myopathies. Rat diaphragm Tetrodotoxin
Neuromuscular junction
Pyridostigmine
EXPOSURE of skeletal muscle to acetylcholinesterase (AChE) inhibitors leads to localized and reversible ultrastructural alterations characterized by dilation of sarcoplasmic reticular membranes, mitochondrial swelling, dissolution of Z-disks, and disruption of myofibrillar organization (2,4-6,1 I, 13). These ultrastructural alterations have been observed in the presence of a large number of organophosphorus and carbamate AChE inhibitors, including paraoxon (I 1,12,2 I), diisopropylfluorophosphate (DFP) ( 13,20), sat-in (10, I5), neostigmine (4,9), physostigmine (IO, l8), and pyridostigmine (56). Based on the similarities in the efficacies but diversities in the molecular structures of the aforementioned inhibitors, it is likely that the myopathies are mediated by accumulation of excess acetylcholine (ACh) rather than by direct actions of the individual compounds. ACh mediation was suggested by early investigators based on findings that organophosphate-induced lesions could be prevented by prior denervation, prompt oxime therapy, or coadministration of the receptor blocker d-tubocurarine (2,21). These procedures serve, respectively, to remove the source of ACh, regenerate active AChE, or prevent prolonged receptor activation. Agonist-induced
Cholinesterase inhibition
Myopathy
mediation of the lesions was substantiated by Leonard and Salpeter ( I3,14), who showed that the nonhydrolyzable cholinomimetic carbamylcholine produced alterations similar to those observed with DFP in the mouse extensor digitorum longus neuromuscular junction. The myopathies have been reported to be more pronounced in slow than in fast twitch muscles ( I I, 1516) and to be exacerbated by nerve stimulation (2). Although most of the studies of anti AChE-induced myopathies have been performed in vivo, evaluation of the alterations under this condition may be complicated by the normal electrical and contractile activity of the muscle and by the uncertainty in the concentration of the cholinesterase inhibitors at their target sites. To overcome such difficulties, we examined the actions of pyridostigmine on neuromuscular ultrastructure under carefully controlled in vitro conditions in rat diaphragm muscle. The results indicate that when AChE is inhibited, myopathies occur in both the absence and presence of nerve stimulation. Myopathies observed in the absence of nerve stimulation may be promoted by spontaneous fasciculations. No ultrastructural alterations were observed in control preparations with nerve or muscle stimulation.
The opmtons or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the U.S. Army or the Department of Defence. In conducting the research described in this article, we adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources,National Research
Council. ’ Requests for reprints should be addressed to Michael Adler. 2Present address: NJC Enterprises, Ltd., 700 Washington Street, New York, NY 100 14.
179
180
ADLER. HINMAN AND HlJDSQN METHOD
Adult male rats (Charles River,) weighing 180-250 g were killed by CO* narcosis followed by decapitation. The left and right hemidiaphragms were removed and placed in standard twitch baths. The bathing medium was a Krebs-Ringer solution consisting of (mM): NaCI, 137; KCI. 2.7: NaHCOl, 11.9: NaH2P04, 0.3; CaC&, 1.8: MgC&, 0.8; glucose, 5.6. The pH of the solution when aerated with 95% 02/5% CO1 and maintained at 32°C was 7.4. To determine the effects of muscle activity on pyridostigmineinduced ultrastructural alterations, hemidiaphragm preparations were incubated in 2 bM pyridostigmine for 2 h in the absence or presence of stimulation. Supramaximal nerve stimulation was delivered by bipolar platinum electrodes at either 20 Hz for I s every 30 s or continuously at 0.67 Hz; both methods producing approximately 4,800 pulses over the 2 h time period. Two groups of muscles were used to assess the effect of fasciculations on the development of myopathies. In one, muscles were exposed to 2 PM pyridostigmine and allowed to fasciculate for 2 h in the absence of electrical stimulation; in the other. 0.5 PM tetrodotoxin (TTX) was administered 30 min before and during the 2 h pyridostigmine exposure to prevent muscle fasciculations. Control muscles were subjected to the same treatments but were incubated in pyridostigmine-free Krebs-Ringer solution, Muscle tensions were measured with Grass FT.03 transducers and displayed on a Grass model 7P1 chart recorder (Grass Instruments, Quincy. MA). Miniature endplate currents (MEPCs) were recorded with a standard 2-microelectrode voltage-clamp technique using an Axoclamp-2A amplifier (Axon Instruments, Foster City. CA).
At the completion of pyridostigmine exposures, muscles were pinned slightly stretched to the bottom of Sylgard-coated dishes and fixed in a solution containing 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4). Neuromuscular junctions were identified by localization of AChE activity using the procedure of Karnovsky and Roots (7). Endplate regions were removed by careful dissection, postfixed in 1%0~0~ and stained en block in aqueous uranyl acetate. Samples were then dehydrated and embedded in an Epon-Araldite mixture (10% Polybed 8 12, 20% Araldite 6005, 70% dodecenyl succinic anhydride and 1.5% DMP-30 as catalyst) and polymerized at 70°C for 24 h. Ultrathin sections were cut with a diamond knife, using an LKB III or IV ultramicrotome, poststained with lead citrate and aqueous or methanolic uranyl acetate, and examined with a JEOL 2000 or 100 CX electron microscope. CONTROL
PY RIDOSTIGMINE (2 /AM)
FIG. I. MEPCs recorded at 32°C from a rat diaphragm muscle at a holding potential of -80 mV under control conditions and in the presence of 2 FM pyridostigmine.
I-ABLE I EFFECTS OF PYRIDOSTIGMINE BROMIDE AND I IX ON MEPCs AND AChE ACTIVITIES IN RAT DIAPHRAGM M1JSCLt __ MEPC
hChE Actlwty
Conditions
Amp (nA)
Tau (msec)
Control Pyridostigmine* TTX t Pyridostigmine t TTX
3.51 + .29 4.39 + .51$ 3.82 + .34
1.41 rf- .I3 4.63 f ,489
I .38 +-.I1
4.28 + .59 0.94 k I og S.Oht .h4
4.48 k .45$
4.79 t ,535
0.76 +- .09Q:
nmol/min/mg
protein
Values for MEPC amplitude (Amp) and decay time constant (Tau) were obtained at 32°C from at least 25 MEPCs per muscle fiber. The holding potential was -80 mV. A total of six fibers from two muscles were sampled for MEPCs and 3-5 muscle strips for AChE activities. Substrate concentration for AChE assay was I mM. * Pyridostigmine concentration was 2 FM: recordings were from 0.52 h. t TTX concentration was 0.5 FM. 4 Differs significantly from control, p 5 0.05 (Student’s I test). 3 Differs significantly from control. p 5 0.0 I.
AChE activity was determined by the radiometric assay of Siakotos et al. (19) using 1 mM [14C]ACh as substrate (specific activity = 1 &i/mmol). Intact hemidiaphragms were exposed to 2 FM pyridostigmine for 2 h at 32°C. The muscles were washed to remove excess inhibitor, minced, and homogenized in phosphate-buffered saline (PBS; pH 7.4) containing I% Triton@ X-100 to solubilize AChE. The detergent also prevented decarbamylation (D. M. Maxwell, personal communication) so that pyridostigmine was not required in the subsequent steps. The homogenate was centrifuged for IO min at 10,000 X g and aliquotes of the supernatant were incubated in PBS containing [14C]ACh and 1% Triton@ X-100 for 10 min. Tetraisopropylpyrophosphoramide (10 @M) was included to inhibit butyrylcholinesterase. The reaction was terminated by addition of Amberlite CC-120 suspended in I ,4-dioxane to remove unhydrolyzed substrate. The samples were centrifuged briefly to remove the Amberlite and the radioactivity in the supernatant was counted in a Beckman LS-7800 liquid scintillation counter. RESULTS
Within 5-10 min of pyridostigmine administration, diaphragm muscles exhibited the characteristic signs of AChE inhibition, consisting of spontaneous fasciculations (3) and prolongation in the MEPC decay (Fig. I). When fully equilibrated (- 30 min). 2 PM pyridostigmine produced a 3-4-fold slowing in the MEPC decay rate but only small increases in the MEPC amplitude (Table 1). Larger increases in the amplitude were not observed due presumably to the direct inhibitory action of pyridostigmine on the ACh receptor and/or channel ( 17). Figure 2 shows a representative section through a neuromuscular junction from an unstimulated control rat diaphragm muscle. The ultrastructure of control muscle fibers was well preserved in these immersion fixed preparations. Nerve terminals contained abundant synaptic vesicles; mitochondria with normal diameters and condensed matrices were observed both pre- and postsynaptically. Muscles exposed to 2 PM pyridostigmine underwent a depression in AChE activity of 78% (Table I) and exhibited
PYRIDOSTIGMINE-INDUCED
MYOPATHIES
181
FIG 2. Electron micrograph of a diaphragm muscle fixed after a 2 h incubation in control physiological solution at 32°C: (n) nerve terminal. 0-l junc:tional folds, (m) mitochondria. Note the well preserved organelles and precise alignment of myofilaments.
spontaneous fasciculations for the entire incubation period. The ultrastructural alterations observed in these hemidiaphragm preparations were similar to those reported in muscles exposed to pyridostigmine in vivo (5,6). After a 2 h incubation with pyridostigmine, most muscles had supercontracted sarcomeres near the endplate region. The subjunctional areas were also marked by disorganization of the myofibrillar apparatus, streaming of Z-bands (Fig. 3a) and mitochondrial swelling (Fig. 3b). The most severely affected mitochondria were observed proximal to the junctional folds with progressively less severe alterations occurring away from the junction. The focal nature of the pyridostigmine-induced myopathy is shown clearly in Fig. 4. From this micrograph it is evident that the regions closest to the synaptic cleft contain the most severely damaged organelles. Essentially all of the mitochondria immediately adjacent to the neuromuscular junction exhibited marked intracristal swelling and some mitochondria showed an apparent absence of matrix. The contractile filaments were supercontracted with only remnants of Z-line material visible. The ultrastructural damage was reduced considerably within a few pm from this region; by 12-14 Km from the subjunctional membrane, it was possible to detect precisely aligned sarcomeres and “normal” intracellular organelles.
Antagonism by TTX Although the pyridostigmine-treated muscles just described were not stimulated, they did undergo pronounced mechanical activity in the form of spontaneous fasciculations. The fasciculations represent periodic spontaneous discharges of phrenic motor units (3). To determine whether this activity was responsible for triggering myopathic changes, 0.5 pm TTX was added prior to, as well during, exposure to 2 PM pyridostigmine to prevent fasciculations.
The effects on basal tension of pyridostigmine, administered alone or coadministered with TTX are shown in Fig. 5. Examination of the tension records reveals that 2 PM pyridostigmine produced intense muscle fasciculations (Fig. SA). These contractions occurred spontaneously after 3 min of pyridostigmine addition and persisted for the entire 2 h exposure period. The fasciculations appeared to have a burst-like pattern with an average frequency of 1.9 f 0.2 Hz and amplitude of 2.6 f 0.3 g (mean f SEM, n = 5). Due to the relatively small size of the unit events, the fasciculation rates may have been underestimated. This is suggested by the findings of Sket et al. (20) who recorded in vivo electromyographic discharge rates of nearly 40 Hz during exposure to DFP. Addition of TTX prior to pyridostigmine prevented fasciculations from developing (Fig. 5B). Examination of the cytoarchitecture of muscle treated with TTX alone revealed no ultrastructural alterations (Fig. 6a). In muscle paralyzed by TTX, pyridostigmine failed to produce its characteristic anti AChE-mediated myopathic changes (Fig. 6b). Endplate regions from such muscles exhibited normal pre- and postjunctional morphology and the myofibrils showed no disorganization. The protection by TTX was dramatic and as complete as that observed with ACh receptor blockers (2) or botulinum toxin (20). To investigate the underlying basis for the efficacy of the TTX, we examined its actions alone and in combination with pyridostigmine, on AChE activities and spontaneous quanta1 transmitter release in diaphragm muscles. The data on MEPCs and AChE activities are presented in Table 1. In the presence of 2 FM pyridostigmine, the MEPC amplitude increased by 23% and the time constant of the decay was prolonged by 328%. These alterations were accompanied by a 78% inhibition of AChE activity. TTX did not alter the characteristics of the MEPC and was without effect on AChE activity (Table 1). These results indicate that the beneficial effect of TTX did not result from
182
ADLER,
HINMAN
AND
HUDSON
FIG. 3. Muscle fibers showing ultrastructural alterations resulting from a 2 h incubation in 2 PM pyridostigmine. (a) The nuclear envelope (Nu) is dilateA, subjunctional mitochondria have rarefied matrices or swelling of intracristal areas. Z-lines are damaged (arrowheads) and sarcomeres lack distir et striations; (n) nerve terminal, (f) junctional folds. (b) The mitochondria of this muscle show the range of damage including those that are dilated and/or lysed (ml) and others with intracristal swelling (m2).
restoration of the prolonged residence time of ACh. Because TTX prevented both muscle fasciculations and the ultrastructural lesions, it is reasonable to assume that fasciculations may serve to trigger the myopathies. Stimulated Muscles
Under in vivo conditions, nerve-evoked muscle activity may also contribute to myopathies, because such activity would lead to a further accumulation of ACh than that expected from fasciculations alone. To investigate this possibility, muscles were exposed to 2 PM pyridostigmine and stimulated via the phrenic nerve for 2 h. Two stimulation patterns were used: brief intermittent 20 Hz trains (Fig. 7a) and continuous 0.67 Hz pulses (Fig. 7b). These were considered to encompass normal skeletal muscle firing patterns. As illustrated in Fig. 7, stimulated muscles exhibited essentially the same organelle damage and myofibrillar disorganization as that observed in unstimulated muscles (Figs.
3 and 4). However, in stimulated muscles, the damage appeared to be more severe and the affected areas were generally more extensive (Fig. 7), but little or no difference was observed between the two stimulation patterns. In the absence of AChE inhibitors, mechanical activity either via the phrenic nerve (Fig. 8a) or direct muscle stimulation (Fig. 8b) produced no ultrastructural alterations. This is consistent with the findings of Rash and Elmund (18). These authors reported that control muscles exhibited “normal” postsynaptic morphology even after 15 min of continuous stimulation at 20 Hz. DISCUSSION
The results of the present investigation reveal that anti AChEmediated myopathies can be elicited in vitro in both the absence or presence of nerve stimulation. Previous investigators have demonstrated the occurrence of such alterations in vivo, where
PYRIDOSTIGMINE-INDUCED
183
MYOPATHIES
FIG. 4. Synaptic region from a hemidiaphragm exposed to 2 PM pyridostigmine for 2 h to illustrate the focal nature of the ultrastructural damage. Subjunctionally, some mitochondria are swollen and lysed (ml); others show pronounced intracristal swelling (m2). At sites distal from the neuromuscular junction, mitcchondrial are indistinguishable from control (m3). Immediately subjacent to the junctional folds,the myofibrillar components are superconnacted beyond recognition of sarcomeres (s) although remnants of Z-line material are randomly observed (arrowheads): (n) nerve terminal, (f) junction folds.
A the anti AChE concentration and the neural inputs to the muscle are generally not under experimental control. In the present study, the pyridostigmine concentration was maintained at 2 PM throughout, and the stimulation rates were well controlled. The myopathies resembled those described for other cholinesterase inhibitors such as neostigmine (4,9), paraoxon ( 11,12,2 1) and DFP (13,20). The alterations were most pronounced in the immediate vicinity of the subjunctional membrane and were barely detectable 10 pm away (Fig. 4). Among the structures most consistently altered were the postjunctional mitochondria, sarcoplasmic reticulum, Z-line and contractile proteins. The presynaptic mitochondria were less severely affected and the junctional folds were generally unaltered. In agreement with previous studies (4,6,2 l), the organelle damage and myofibrillar disorganization appeared rapidly and were well developed by 30 min of pyridostigmine exposure. A 2 h incubation time was selected because fasciculations from in vivo administration of anti AChE agents were found to persist for 2 h, which coincided with maximal ultrastructural damage (2 1).
6
5g
xiiFlG. 5. (A) Spontaneous fasciculations in the presence of 2 PM pyridostigmine. (B) Absence of fasciculations in a preparation pretreated for 30 mm with 0.5 PM TfX prior to and during addition of 2 PM pyridostigmine.
184
ADLER.
HINMAN
AND
FIG 6. Effect of TTX on skeletal muscle ultrastructure. (a) Muscle incubated for 1.5 h in 0.5 PM TTX illustrating that TTX does mor phological changes. (b) Antagonism of pyridostigmine-induced myopathy in a muscle pretreated for 30 min with TTX prior to in T TX plus 2 PM pyridostigmine: (n) nerve terminal. (f) junctional folds, (m) mitochondria. (Nu) muscle nucleus.
The pyridostigmine-induced myopathies do not appear to compromise muscle function. Thus, Adler et al. (1) demonstrated that rat diaphragm muscles undergo no significant reduction in twitch or tetanic tension during or following a 2-week exposure to pyridostigmine. The absence of functional impairment appears to reflect primarily the limited area of involvement in anti AChEinduced myopathies (11). In agreement with previous investigators (4-6,9- 15.20,2 l), the myopathic alterations observed in the presence of pyridostigmine were confined to the immediate vicinity of the endplate region which comprises a small fraction of the total muscle fiber. Therefore, because the pre- and postjunctional membranes were generally not altered by pyridostigmine, (Fig. 3a) synaptic transmission and action potential generation are expected to remain functional. Decrements in tension due to the abnormal properties of the myopathic regions would presumably be too small to detect. It has often been observed that the myopathies induced by
HUDSON
not produce a 2 h incuba
any tion
can involve the endplate region of nearly all muscle fibers but necrosis at the light microscopic level is detectable in < 10% of fibers sampled even after nearly complete AChE inhibition ( 1 1.12,20,2 1). It is not clear why some fibers degenerate, although the majority are preserved in spite of exhibiting marked ultrastructural lesions. The incidence of necrotic fibers is dose-dependent (4-6, I 1,12,2 1) suggesting that some muscle fibers may exhibit an unusually high sensitivity to AChE inhibitors or to the subsequent myopathies. This high sensitivity may be related to the threshold or firing pattern of the motor unit. Leonard and Salpeter (14) proposed that AChE inhibitors produce myopathic lesions by allowing an accumulation of ACh. The high ACh levels were suggested to promote entry of toxic quantities of Ca*+ at the motor endplate via nicotinic ACh receptor channels (8,l I, 15). The excess levels of cellular Ca*+ were suggested to overload Ca2+ sequestering structures such as the AChE inhibitors
PYRIDOSTIGMINE-INDUCED
185
MYOPATHIES
FIG. 7. Endplate regions of muscle fibers exposed to 2 PM pyridostigmine for 2 h with nerve stimulation at 20 Hz for I s every 30 s (a) or al Hz continuously (b). In (a) mitochondria (m) show severe damage. The Z-line material is discernable but sarcomere banding is indistinct; muscle nucleus, (n) nerve terminal, (f) junctional folds. In (b) subjunctional alterations extend over a relatively large area; mitochondrial da and suyiercontraction (between arrowheads) are extreme. Nerve stimulation exacerbated the myopathies regardless of the pulse pattern.
reticulum and mitochondria and lead to their swelling. In addition, Ca2+ was postulated to activate specific proteases to cauSe dissolution of Z-band and contractile proteins ( 14). In skeletal muscle, inhibition of AChE delays the removal of neurally released ACh and leads to its accumulation in the synaptic cleft (8,9,12,18). ACh accumulation may be a consequence of one or more of the following processes: molecular leakage, spontaneous quanta1 release, evoked release, and fasciculations which represent periodic spontaneous discharge of motor units (8). Based on findings that TTX prevented both the fasciculations (Fig. 5) and the myopathic action of pyridostigmine (Fig. 6), it is likely that fasciculations are responsible for most of the myopathic alterations observed in this study. This finding is in agreement with a recent in vivo study in which a local injection of sarcoplasmic
botulinum toxin inhibited both fasciculations and DIP-induced muscle damage (20). Because TTX does not alter molecular leakage (8), the steady nonquantal release of ACh does not appear to lead to sufficient transmitter accumulation to trigger the observed ultrastructural alterations. This finding is of interest because molecular leakage accounts for the major fraction of the ACh that is released at rest and can generate as much as 10 nM ACh in the synaptic cleft when AChE is completely inhibited (8). Likewise, ACh from spontaneous quanta1 release, which occurred at a rate of -2.5 s-’ in both the absence and presence of pyridostigmine, was not sufficient to produce myopathic alterations because this process, like molecular leakage persists in the presence of TTX (8). Nerve evoked ACh release did exacerbate the myopathies but this was minor relative to the damage observed in nonstimulated muscles.
186
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AND
HUDSON
FK2. 8. Endplate regions in control muscle to illustrate that in the absence of py~dostigmin~ the stimulation intensities used in the present study ,do not: produce ultrastructural alterations. (a) Nerve stimulation at 0.67 Hz for 2 h. (b) Direct muscle stimulation at 0.67 Hz for 2 h. The structt tres ind.icated are: (m) mitochondria, (n) nerve terminal, (Nu) muscle nucleus, and (f) junctional folds. ACKNOWLEDGEMENTS
We thank Margaret G. Filbert for invaluable advice on the chohnest era= assay, M. Morita for technical cont~butjons, Susan Cameron.
and Helen Macfarlane for technical assistance and Laura Hudson for her photographic expertise.
REFERENCES I. Adler, M.; Deshpande, S. S.; Foster, R. E.; Maxwell, D. M.; Albuquerque, E. X. Effects of subacute pyridostigmine administration on mammalian skeletal muscle function. J. Appl. Tox. 1991 (in press). 2. Ariens, A. T.; Meeter, E.; Wolthuis, 0. L.; van Benthem, R. J. M. Reversible necrosis at the end-plate region in striated muscles of the rat poisoned with cholinesterase inhibitors. Experentia 25:57-59: 1969. 3. Ferry, C. B. The origin of the anticholinesterase-induced repetitive activity of the phrenic nervediaphragm preparation of the rat in vitro. Br. J. Pharmacol. 94: 169- 179; 1988. 4. Hudson, C. S.; Rash, J. E.; Tiedt, T. N.; Albuquerque, E. X. Neostigmine-induced aberations at the mammalian neuromuscular junction. II, Ultrastructure. J. Pharmacol. Exp. Ther. 205:340-356; 1978.
5. Hudson, C. S.; Foster, R. E.; Kahng, M. W. Neuromuscular toxicity of pyridostigmine bromide in the diaphragm, extensor digitorum longus and soleus muscles of the rat. Fund. Appl. Tox. 5:5260S269; 1985. effects 6. Hudson, C. S.; Foster, R. E.; Kahng, M. W. Uit~tructu~i of pyridostigmine bromide on neuromuscular junctions in rat diaphragm. Neurotox. 7:167-186; 1986. I. Kamovsky, M. J.; Roots, L. A “direct-coloring” thiocholine method for cholinesterases. J. Histochem. Cytochem. 12:2 19-22 1; 1964. 8. Katz, B.; Miledi, R. Transmitter leakage from motor nerve endings. Proc. Roy. Sot. (Lond.) B 19659-72; 1977. 9. Kawabuchi, M. Neostigmine myopathy is a calcium ion-mediated myopathy initially affecting the motor end-plate. J. Neuropath. Exp. Neurol. 41:298-3 14: 1982. IO Kawabuchi, M.; Boyne, A. F.: Deshpande, S. S.; Albuquerque,
PYRIDOSTIGMINE-INDUCED
Il.
12.
13.
14.
15.
MYOPATHIES
E. X. The reversible carbamate (-)physostigmine, reduced the size of synaptic end plate lesions induced by sarin, an irreversible organophosphate. Tox. Appl. Pharmacol. 97:98-106; 1989. Laskowski, M. B.; Oson, W. H.; Dettbam, W.-D. Ultrastructural changes at the motor endplate produced by an irreversible cholinesterase inhibitor. Exp. Neurol. 47:290-306; 1975. Laskowski, M. B.; Olson, W. H.; Dettbarn, W.-D. Initial ultrastructural abnormalities at the motor end plate produced by a cholinesterase inhibitor. Exp. Neurol. 57: 13-33; 1977. Leonard, J. P.; Salpeter, M. M. Agonist-induced myopathy at the neuromuscular junction is mediated by calcium. J. Cell. Biol. 82: 811-819: 1979. Leonard, J. P.; Salpeter, M. M. Calcium-mediated myopathy at neuromuscular junctions of normal and dystrophic muscle. Exp. Neurol. 76:121-138; 1982. Meshul, C. K.; Boyne, A. F.; Deshpande, S. S.; Albuquerque, E. X. Comparison of the ultrastructural myopathy induced by anticholinesterase agents at the end plates of rat soleus and extensor muscles. Exp. Neurol. 89:96-l 14; 1985.
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16. Padykula, H. A.; Gauthier, G. F. The ultrastructure of the neuromuscular junction of mammalian red, white and intermediate skeletal muscle fibers. J. Cell. Biol. 46:27-41: 1970. 17. Pascuzzo, G. J.; Akaike, A.; Maleque, M. A.; Shaw, K.-P.; Aronstam, R. S.; Rickett, D. L.; Albuquerque, E. X. The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptorion channel complex I. Agonist, desensitizing, and binding properties. Mol. Pharmacol. 25:92-101; 1984. 18. Rash. J. E.; Elmund, J. K. Pathophysiology of anticholinesterase agents. USAMRDC Final Report. 1988:249-27 I. 19. Siakotos, A. N.; Filbert, M. G.; Hester, R. A specific radioisotopic assay for acetylcholinesterase and pseudocholinesterase in brain and plasma. Biochem. Med. 3: 1-12; 1969. 20. Sket, D.; Dettbarn, W.-D.; Clinton, M. E.; Sketelj, J.; Cucek, D.; Brzin, M. Prevention of diisopropylphosphorofluoridate-induced myopathy by botulinurn toxin type A blockage of quanta1 release of acetylcholine. Acta Neuropathol. 82: 134-142; 199 I. 21. Wecker, L.; Kiauta, T.; Dettbarn, W.-D. Relationship between acetylcholinesterase inhibition and the development of a myopathy. J. Pharmacol. Exp. Ther. 206:97-104; 1978.
Brain Research Bulletin. Vol. 29, pp. 179-187, 1992
Copyright 0 1992 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
Role of Muscle Fasciculations in the Generation of Myopathies in Mammalian Skeletal Muscle MICHAEL ADLER,*’
DONALD HINMAN* AND C. SUE HUDSON
*Neurotoxicology Branch, Pathophysiology Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010 Department of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
ADLER, M., D. HINMAN AND C. S. HUDSON. Role qf rnuscle,fasciculations in the generation of myopathies in mammalian skeletal muscle. BRAIN RES BULL 29(2) 179-I 87, 1992.-The myotoxicity of pyridostigmine bromide was investigated on rat diaphragm nerve-muscle preparations in vitro. Within 2 h of exposure to pyridostigmine (2 PM), diaphragm muscles exhibited
ultrastructural alterations characterized by swelling of subjunctional mitochondria and disorganization of contractile proteins. These alterations developed both in the absence and presence of electrical stimulation of the phrenic nerve, and were accompanied by continuous muscle fasciculations. Pretreatment by tetrodotoxin suppressed both the muscle fasciculations and the appearance of myopathies. These findings suggest that fasciculations may be an important contributing factor in the development of anticholinesterase-induced myopathies. Rat diaphragm Tetrodotoxin
Neuromuscular junction
Pyridostigmine
EXPOSURE of skeletal muscle to acetylcholinesterase (AChE) inhibitors leads to localized and reversible ultrastructural alterations characterized by dilation of sarcoplasmic reticular membranes, mitochondrial swelling, dissolution of Z-disks, and disruption of myofibrillar organization (2,4-6,1 I, 13). These ultrastructural alterations have been observed in the presence of a large number of organophosphorus and carbamate AChE inhibitors, including paraoxon (I 1,12,2 I), diisopropylfluorophosphate (DFP) ( 13,20), sat-in (10, I5), neostigmine (4,9), physostigmine (IO, l8), and pyridostigmine (56). Based on the similarities in the efficacies but diversities in the molecular structures of the aforementioned inhibitors, it is likely that the myopathies are mediated by accumulation of excess acetylcholine (ACh) rather than by direct actions of the individual compounds. ACh mediation was suggested by early investigators based on findings that organophosphate-induced lesions could be prevented by prior denervation, prompt oxime therapy, or coadministration of the receptor blocker d-tubocurarine (2,21). These procedures serve, respectively, to remove the source of ACh, regenerate active AChE, or prevent prolonged receptor activation. Agonist-induced
Cholinesterase inhibition
Myopathy
mediation of the lesions was substantiated by Leonard and Salpeter ( I3,14), who showed that the nonhydrolyzable cholinomimetic carbamylcholine produced alterations similar to those observed with DFP in the mouse extensor digitorum longus neuromuscular junction. The myopathies have been reported to be more pronounced in slow than in fast twitch muscles ( I I, 1516) and to be exacerbated by nerve stimulation (2). Although most of the studies of anti AChE-induced myopathies have been performed in vivo, evaluation of the alterations under this condition may be complicated by the normal electrical and contractile activity of the muscle and by the uncertainty in the concentration of the cholinesterase inhibitors at their target sites. To overcome such difficulties, we examined the actions of pyridostigmine on neuromuscular ultrastructure under carefully controlled in vitro conditions in rat diaphragm muscle. The results indicate that when AChE is inhibited, myopathies occur in both the absence and presence of nerve stimulation. Myopathies observed in the absence of nerve stimulation may be promoted by spontaneous fasciculations. No ultrastructural alterations were observed in control preparations with nerve or muscle stimulation.
The opmtons or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the U.S. Army or the Department of Defence. In conducting the research described in this article, we adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources,National Research
Council. ’ Requests for reprints should be addressed to Michael Adler. 2Present address: NJC Enterprises, Ltd., 700 Washington Street, New York, NY 100 14.
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ADLER. HINMAN AND HlJDSQN METHOD
Adult male rats (Charles River,) weighing 180-250 g were killed by CO* narcosis followed by decapitation. The left and right hemidiaphragms were removed and placed in standard twitch baths. The bathing medium was a Krebs-Ringer solution consisting of (mM): NaCI, 137; KCI. 2.7: NaHCOl, 11.9: NaH2P04, 0.3; CaC&, 1.8: MgC&, 0.8; glucose, 5.6. The pH of the solution when aerated with 95% 02/5% CO1 and maintained at 32°C was 7.4. To determine the effects of muscle activity on pyridostigmineinduced ultrastructural alterations, hemidiaphragm preparations were incubated in 2 bM pyridostigmine for 2 h in the absence or presence of stimulation. Supramaximal nerve stimulation was delivered by bipolar platinum electrodes at either 20 Hz for I s every 30 s or continuously at 0.67 Hz; both methods producing approximately 4,800 pulses over the 2 h time period. Two groups of muscles were used to assess the effect of fasciculations on the development of myopathies. In one, muscles were exposed to 2 PM pyridostigmine and allowed to fasciculate for 2 h in the absence of electrical stimulation; in the other. 0.5 PM tetrodotoxin (TTX) was administered 30 min before and during the 2 h pyridostigmine exposure to prevent muscle fasciculations. Control muscles were subjected to the same treatments but were incubated in pyridostigmine-free Krebs-Ringer solution, Muscle tensions were measured with Grass FT.03 transducers and displayed on a Grass model 7P1 chart recorder (Grass Instruments, Quincy. MA). Miniature endplate currents (MEPCs) were recorded with a standard 2-microelectrode voltage-clamp technique using an Axoclamp-2A amplifier (Axon Instruments, Foster City. CA).
At the completion of pyridostigmine exposures, muscles were pinned slightly stretched to the bottom of Sylgard-coated dishes and fixed in a solution containing 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4). Neuromuscular junctions were identified by localization of AChE activity using the procedure of Karnovsky and Roots (7). Endplate regions were removed by careful dissection, postfixed in 1%0~0~ and stained en block in aqueous uranyl acetate. Samples were then dehydrated and embedded in an Epon-Araldite mixture (10% Polybed 8 12, 20% Araldite 6005, 70% dodecenyl succinic anhydride and 1.5% DMP-30 as catalyst) and polymerized at 70°C for 24 h. Ultrathin sections were cut with a diamond knife, using an LKB III or IV ultramicrotome, poststained with lead citrate and aqueous or methanolic uranyl acetate, and examined with a JEOL 2000 or 100 CX electron microscope. CONTROL
PY RIDOSTIGMINE (2 /AM)
FIG. I. MEPCs recorded at 32°C from a rat diaphragm muscle at a holding potential of -80 mV under control conditions and in the presence of 2 FM pyridostigmine.
I-ABLE I EFFECTS OF PYRIDOSTIGMINE BROMIDE AND I IX ON MEPCs AND AChE ACTIVITIES IN RAT DIAPHRAGM M1JSCLt __ MEPC
hChE Actlwty
Conditions
Amp (nA)
Tau (msec)
Control Pyridostigmine* TTX t Pyridostigmine t TTX
3.51 + .29 4.39 + .51$ 3.82 + .34
1.41 rf- .I3 4.63 f ,489
I .38 +-.I1
4.28 + .59 0.94 k I og S.Oht .h4
4.48 k .45$
4.79 t ,535
0.76 +- .09Q:
nmol/min/mg
protein
Values for MEPC amplitude (Amp) and decay time constant (Tau) were obtained at 32°C from at least 25 MEPCs per muscle fiber. The holding potential was -80 mV. A total of six fibers from two muscles were sampled for MEPCs and 3-5 muscle strips for AChE activities. Substrate concentration for AChE assay was I mM. * Pyridostigmine concentration was 2 FM: recordings were from 0.52 h. t TTX concentration was 0.5 FM. 4 Differs significantly from control, p 5 0.05 (Student’s I test). 3 Differs significantly from control. p 5 0.0 I.
AChE activity was determined by the radiometric assay of Siakotos et al. (19) using 1 mM [14C]ACh as substrate (specific activity = 1 &i/mmol). Intact hemidiaphragms were exposed to 2 FM pyridostigmine for 2 h at 32°C. The muscles were washed to remove excess inhibitor, minced, and homogenized in phosphate-buffered saline (PBS; pH 7.4) containing I% Triton@ X-100 to solubilize AChE. The detergent also prevented decarbamylation (D. M. Maxwell, personal communication) so that pyridostigmine was not required in the subsequent steps. The homogenate was centrifuged for IO min at 10,000 X g and aliquotes of the supernatant were incubated in PBS containing [14C]ACh and 1% Triton@ X-100 for 10 min. Tetraisopropylpyrophosphoramide (10 @M) was included to inhibit butyrylcholinesterase. The reaction was terminated by addition of Amberlite CC-120 suspended in I ,4-dioxane to remove unhydrolyzed substrate. The samples were centrifuged briefly to remove the Amberlite and the radioactivity in the supernatant was counted in a Beckman LS-7800 liquid scintillation counter. RESULTS
Within 5-10 min of pyridostigmine administration, diaphragm muscles exhibited the characteristic signs of AChE inhibition, consisting of spontaneous fasciculations (3) and prolongation in the MEPC decay (Fig. I). When fully equilibrated (- 30 min). 2 PM pyridostigmine produced a 3-4-fold slowing in the MEPC decay rate but only small increases in the MEPC amplitude (Table 1). Larger increases in the amplitude were not observed due presumably to the direct inhibitory action of pyridostigmine on the ACh receptor and/or channel ( 17). Figure 2 shows a representative section through a neuromuscular junction from an unstimulated control rat diaphragm muscle. The ultrastructure of control muscle fibers was well preserved in these immersion fixed preparations. Nerve terminals contained abundant synaptic vesicles; mitochondria with normal diameters and condensed matrices were observed both pre- and postsynaptically. Muscles exposed to 2 PM pyridostigmine underwent a depression in AChE activity of 78% (Table I) and exhibited
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181
FIG 2. Electron micrograph of a diaphragm muscle fixed after a 2 h incubation in control physiological solution at 32°C: (n) nerve terminal. 0-l junc:tional folds, (m) mitochondria. Note the well preserved organelles and precise alignment of myofilaments.
spontaneous fasciculations for the entire incubation period. The ultrastructural alterations observed in these hemidiaphragm preparations were similar to those reported in muscles exposed to pyridostigmine in vivo (5,6). After a 2 h incubation with pyridostigmine, most muscles had supercontracted sarcomeres near the endplate region. The subjunctional areas were also marked by disorganization of the myofibrillar apparatus, streaming of Z-bands (Fig. 3a) and mitochondrial swelling (Fig. 3b). The most severely affected mitochondria were observed proximal to the junctional folds with progressively less severe alterations occurring away from the junction. The focal nature of the pyridostigmine-induced myopathy is shown clearly in Fig. 4. From this micrograph it is evident that the regions closest to the synaptic cleft contain the most severely damaged organelles. Essentially all of the mitochondria immediately adjacent to the neuromuscular junction exhibited marked intracristal swelling and some mitochondria showed an apparent absence of matrix. The contractile filaments were supercontracted with only remnants of Z-line material visible. The ultrastructural damage was reduced considerably within a few pm from this region; by 12-14 Km from the subjunctional membrane, it was possible to detect precisely aligned sarcomeres and “normal” intracellular organelles.
Antagonism by TTX Although the pyridostigmine-treated muscles just described were not stimulated, they did undergo pronounced mechanical activity in the form of spontaneous fasciculations. The fasciculations represent periodic spontaneous discharges of phrenic motor units (3). To determine whether this activity was responsible for triggering myopathic changes, 0.5 pm TTX was added prior to, as well during, exposure to 2 PM pyridostigmine to prevent fasciculations.
The effects on basal tension of pyridostigmine, administered alone or coadministered with TTX are shown in Fig. 5. Examination of the tension records reveals that 2 PM pyridostigmine produced intense muscle fasciculations (Fig. SA). These contractions occurred spontaneously after 3 min of pyridostigmine addition and persisted for the entire 2 h exposure period. The fasciculations appeared to have a burst-like pattern with an average frequency of 1.9 f 0.2 Hz and amplitude of 2.6 f 0.3 g (mean f SEM, n = 5). Due to the relatively small size of the unit events, the fasciculation rates may have been underestimated. This is suggested by the findings of Sket et al. (20) who recorded in vivo electromyographic discharge rates of nearly 40 Hz during exposure to DFP. Addition of TTX prior to pyridostigmine prevented fasciculations from developing (Fig. 5B). Examination of the cytoarchitecture of muscle treated with TTX alone revealed no ultrastructural alterations (Fig. 6a). In muscle paralyzed by TTX, pyridostigmine failed to produce its characteristic anti AChE-mediated myopathic changes (Fig. 6b). Endplate regions from such muscles exhibited normal pre- and postjunctional morphology and the myofibrils showed no disorganization. The protection by TTX was dramatic and as complete as that observed with ACh receptor blockers (2) or botulinum toxin (20). To investigate the underlying basis for the efficacy of the TTX, we examined its actions alone and in combination with pyridostigmine, on AChE activities and spontaneous quanta1 transmitter release in diaphragm muscles. The data on MEPCs and AChE activities are presented in Table 1. In the presence of 2 FM pyridostigmine, the MEPC amplitude increased by 23% and the time constant of the decay was prolonged by 328%. These alterations were accompanied by a 78% inhibition of AChE activity. TTX did not alter the characteristics of the MEPC and was without effect on AChE activity (Table 1). These results indicate that the beneficial effect of TTX did not result from
182
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FIG. 3. Muscle fibers showing ultrastructural alterations resulting from a 2 h incubation in 2 PM pyridostigmine. (a) The nuclear envelope (Nu) is dilateA, subjunctional mitochondria have rarefied matrices or swelling of intracristal areas. Z-lines are damaged (arrowheads) and sarcomeres lack distir et striations; (n) nerve terminal, (f) junctional folds. (b) The mitochondria of this muscle show the range of damage including those that are dilated and/or lysed (ml) and others with intracristal swelling (m2).
restoration of the prolonged residence time of ACh. Because TTX prevented both muscle fasciculations and the ultrastructural lesions, it is reasonable to assume that fasciculations may serve to trigger the myopathies. Stimulated Muscles
Under in vivo conditions, nerve-evoked muscle activity may also contribute to myopathies, because such activity would lead to a further accumulation of ACh than that expected from fasciculations alone. To investigate this possibility, muscles were exposed to 2 PM pyridostigmine and stimulated via the phrenic nerve for 2 h. Two stimulation patterns were used: brief intermittent 20 Hz trains (Fig. 7a) and continuous 0.67 Hz pulses (Fig. 7b). These were considered to encompass normal skeletal muscle firing patterns. As illustrated in Fig. 7, stimulated muscles exhibited essentially the same organelle damage and myofibrillar disorganization as that observed in unstimulated muscles (Figs.
3 and 4). However, in stimulated muscles, the damage appeared to be more severe and the affected areas were generally more extensive (Fig. 7), but little or no difference was observed between the two stimulation patterns. In the absence of AChE inhibitors, mechanical activity either via the phrenic nerve (Fig. 8a) or direct muscle stimulation (Fig. 8b) produced no ultrastructural alterations. This is consistent with the findings of Rash and Elmund (18). These authors reported that control muscles exhibited “normal” postsynaptic morphology even after 15 min of continuous stimulation at 20 Hz. DISCUSSION
The results of the present investigation reveal that anti AChEmediated myopathies can be elicited in vitro in both the absence or presence of nerve stimulation. Previous investigators have demonstrated the occurrence of such alterations in vivo, where
PYRIDOSTIGMINE-INDUCED
183
MYOPATHIES
FIG. 4. Synaptic region from a hemidiaphragm exposed to 2 PM pyridostigmine for 2 h to illustrate the focal nature of the ultrastructural damage. Subjunctionally, some mitochondria are swollen and lysed (ml); others show pronounced intracristal swelling (m2). At sites distal from the neuromuscular junction, mitcchondrial are indistinguishable from control (m3). Immediately subjacent to the junctional folds,the myofibrillar components are superconnacted beyond recognition of sarcomeres (s) although remnants of Z-line material are randomly observed (arrowheads): (n) nerve terminal, (f) junction folds.
A the anti AChE concentration and the neural inputs to the muscle are generally not under experimental control. In the present study, the pyridostigmine concentration was maintained at 2 PM throughout, and the stimulation rates were well controlled. The myopathies resembled those described for other cholinesterase inhibitors such as neostigmine (4,9), paraoxon ( 11,12,2 1) and DFP (13,20). The alterations were most pronounced in the immediate vicinity of the subjunctional membrane and were barely detectable 10 pm away (Fig. 4). Among the structures most consistently altered were the postjunctional mitochondria, sarcoplasmic reticulum, Z-line and contractile proteins. The presynaptic mitochondria were less severely affected and the junctional folds were generally unaltered. In agreement with previous studies (4,6,2 l), the organelle damage and myofibrillar disorganization appeared rapidly and were well developed by 30 min of pyridostigmine exposure. A 2 h incubation time was selected because fasciculations from in vivo administration of anti AChE agents were found to persist for 2 h, which coincided with maximal ultrastructural damage (2 1).
6
5g
xiiFlG. 5. (A) Spontaneous fasciculations in the presence of 2 PM pyridostigmine. (B) Absence of fasciculations in a preparation pretreated for 30 mm with 0.5 PM TfX prior to and during addition of 2 PM pyridostigmine.
184
ADLER.
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AND
FIG 6. Effect of TTX on skeletal muscle ultrastructure. (a) Muscle incubated for 1.5 h in 0.5 PM TTX illustrating that TTX does mor phological changes. (b) Antagonism of pyridostigmine-induced myopathy in a muscle pretreated for 30 min with TTX prior to in T TX plus 2 PM pyridostigmine: (n) nerve terminal. (f) junctional folds, (m) mitochondria. (Nu) muscle nucleus.
The pyridostigmine-induced myopathies do not appear to compromise muscle function. Thus, Adler et al. (1) demonstrated that rat diaphragm muscles undergo no significant reduction in twitch or tetanic tension during or following a 2-week exposure to pyridostigmine. The absence of functional impairment appears to reflect primarily the limited area of involvement in anti AChEinduced myopathies (11). In agreement with previous investigators (4-6,9- 15.20,2 l), the myopathic alterations observed in the presence of pyridostigmine were confined to the immediate vicinity of the endplate region which comprises a small fraction of the total muscle fiber. Therefore, because the pre- and postjunctional membranes were generally not altered by pyridostigmine, (Fig. 3a) synaptic transmission and action potential generation are expected to remain functional. Decrements in tension due to the abnormal properties of the myopathic regions would presumably be too small to detect. It has often been observed that the myopathies induced by
HUDSON
not produce a 2 h incuba
any tion
can involve the endplate region of nearly all muscle fibers but necrosis at the light microscopic level is detectable in < 10% of fibers sampled even after nearly complete AChE inhibition ( 1 1.12,20,2 1). It is not clear why some fibers degenerate, although the majority are preserved in spite of exhibiting marked ultrastructural lesions. The incidence of necrotic fibers is dose-dependent (4-6, I 1,12,2 1) suggesting that some muscle fibers may exhibit an unusually high sensitivity to AChE inhibitors or to the subsequent myopathies. This high sensitivity may be related to the threshold or firing pattern of the motor unit. Leonard and Salpeter (14) proposed that AChE inhibitors produce myopathic lesions by allowing an accumulation of ACh. The high ACh levels were suggested to promote entry of toxic quantities of Ca*+ at the motor endplate via nicotinic ACh receptor channels (8,l I, 15). The excess levels of cellular Ca*+ were suggested to overload Ca2+ sequestering structures such as the AChE inhibitors
PYRIDOSTIGMINE-INDUCED
185
MYOPATHIES
FIG. 7. Endplate regions of muscle fibers exposed to 2 PM pyridostigmine for 2 h with nerve stimulation at 20 Hz for I s every 30 s (a) or al Hz continuously (b). In (a) mitochondria (m) show severe damage. The Z-line material is discernable but sarcomere banding is indistinct; muscle nucleus, (n) nerve terminal, (f) junctional folds. In (b) subjunctional alterations extend over a relatively large area; mitochondrial da and suyiercontraction (between arrowheads) are extreme. Nerve stimulation exacerbated the myopathies regardless of the pulse pattern.
reticulum and mitochondria and lead to their swelling. In addition, Ca2+ was postulated to activate specific proteases to cauSe dissolution of Z-band and contractile proteins ( 14). In skeletal muscle, inhibition of AChE delays the removal of neurally released ACh and leads to its accumulation in the synaptic cleft (8,9,12,18). ACh accumulation may be a consequence of one or more of the following processes: molecular leakage, spontaneous quanta1 release, evoked release, and fasciculations which represent periodic spontaneous discharge of motor units (8). Based on findings that TTX prevented both the fasciculations (Fig. 5) and the myopathic action of pyridostigmine (Fig. 6), it is likely that fasciculations are responsible for most of the myopathic alterations observed in this study. This finding is in agreement with a recent in vivo study in which a local injection of sarcoplasmic
botulinum toxin inhibited both fasciculations and DIP-induced muscle damage (20). Because TTX does not alter molecular leakage (8), the steady nonquantal release of ACh does not appear to lead to sufficient transmitter accumulation to trigger the observed ultrastructural alterations. This finding is of interest because molecular leakage accounts for the major fraction of the ACh that is released at rest and can generate as much as 10 nM ACh in the synaptic cleft when AChE is completely inhibited (8). Likewise, ACh from spontaneous quanta1 release, which occurred at a rate of -2.5 s-’ in both the absence and presence of pyridostigmine, was not sufficient to produce myopathic alterations because this process, like molecular leakage persists in the presence of TTX (8). Nerve evoked ACh release did exacerbate the myopathies but this was minor relative to the damage observed in nonstimulated muscles.
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FK2. 8. Endplate regions in control muscle to illustrate that in the absence of py~dostigmin~ the stimulation intensities used in the present study ,do not: produce ultrastructural alterations. (a) Nerve stimulation at 0.67 Hz for 2 h. (b) Direct muscle stimulation at 0.67 Hz for 2 h. The structt tres ind.icated are: (m) mitochondria, (n) nerve terminal, (Nu) muscle nucleus, and (f) junctional folds. ACKNOWLEDGEMENTS
We thank Margaret G. Filbert for invaluable advice on the chohnest era= assay, M. Morita for technical cont~butjons, Susan Cameron.
and Helen Macfarlane for technical assistance and Laura Hudson for her photographic expertise.
REFERENCES I. Adler, M.; Deshpande, S. S.; Foster, R. E.; Maxwell, D. M.; Albuquerque, E. X. Effects of subacute pyridostigmine administration on mammalian skeletal muscle function. J. Appl. Tox. 1991 (in press). 2. Ariens, A. T.; Meeter, E.; Wolthuis, 0. L.; van Benthem, R. J. M. Reversible necrosis at the end-plate region in striated muscles of the rat poisoned with cholinesterase inhibitors. Experentia 25:57-59: 1969. 3. Ferry, C. B. The origin of the anticholinesterase-induced repetitive activity of the phrenic nervediaphragm preparation of the rat in vitro. Br. J. Pharmacol. 94: 169- 179; 1988. 4. Hudson, C. S.; Rash, J. E.; Tiedt, T. N.; Albuquerque, E. X. Neostigmine-induced aberations at the mammalian neuromuscular junction. II, Ultrastructure. J. Pharmacol. Exp. Ther. 205:340-356; 1978.
5. Hudson, C. S.; Foster, R. E.; Kahng, M. W. Neuromuscular toxicity of pyridostigmine bromide in the diaphragm, extensor digitorum longus and soleus muscles of the rat. Fund. Appl. Tox. 5:5260S269; 1985. effects 6. Hudson, C. S.; Foster, R. E.; Kahng, M. W. Uit~tructu~i of pyridostigmine bromide on neuromuscular junctions in rat diaphragm. Neurotox. 7:167-186; 1986. I. Kamovsky, M. J.; Roots, L. A “direct-coloring” thiocholine method for cholinesterases. J. Histochem. Cytochem. 12:2 19-22 1; 1964. 8. Katz, B.; Miledi, R. Transmitter leakage from motor nerve endings. Proc. Roy. Sot. (Lond.) B 19659-72; 1977. 9. Kawabuchi, M. Neostigmine myopathy is a calcium ion-mediated myopathy initially affecting the motor end-plate. J. Neuropath. Exp. Neurol. 41:298-3 14: 1982. IO Kawabuchi, M.; Boyne, A. F.: Deshpande, S. S.; Albuquerque,
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MYOPATHIES
E. X. The reversible carbamate (-)physostigmine, reduced the size of synaptic end plate lesions induced by sarin, an irreversible organophosphate. Tox. Appl. Pharmacol. 97:98-106; 1989. Laskowski, M. B.; Oson, W. H.; Dettbam, W.-D. Ultrastructural changes at the motor endplate produced by an irreversible cholinesterase inhibitor. Exp. Neurol. 47:290-306; 1975. Laskowski, M. B.; Olson, W. H.; Dettbarn, W.-D. Initial ultrastructural abnormalities at the motor end plate produced by a cholinesterase inhibitor. Exp. Neurol. 57: 13-33; 1977. Leonard, J. P.; Salpeter, M. M. Agonist-induced myopathy at the neuromuscular junction is mediated by calcium. J. Cell. Biol. 82: 811-819: 1979. Leonard, J. P.; Salpeter, M. M. Calcium-mediated myopathy at neuromuscular junctions of normal and dystrophic muscle. Exp. Neurol. 76:121-138; 1982. Meshul, C. K.; Boyne, A. F.; Deshpande, S. S.; Albuquerque, E. X. Comparison of the ultrastructural myopathy induced by anticholinesterase agents at the end plates of rat soleus and extensor muscles. Exp. Neurol. 89:96-l 14; 1985.
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16. Padykula, H. A.; Gauthier, G. F. The ultrastructure of the neuromuscular junction of mammalian red, white and intermediate skeletal muscle fibers. J. Cell. Biol. 46:27-41: 1970. 17. Pascuzzo, G. J.; Akaike, A.; Maleque, M. A.; Shaw, K.-P.; Aronstam, R. S.; Rickett, D. L.; Albuquerque, E. X. The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptorion channel complex I. Agonist, desensitizing, and binding properties. Mol. Pharmacol. 25:92-101; 1984. 18. Rash. J. E.; Elmund, J. K. Pathophysiology of anticholinesterase agents. USAMRDC Final Report. 1988:249-27 I. 19. Siakotos, A. N.; Filbert, M. G.; Hester, R. A specific radioisotopic assay for acetylcholinesterase and pseudocholinesterase in brain and plasma. Biochem. Med. 3: 1-12; 1969. 20. Sket, D.; Dettbarn, W.-D.; Clinton, M. E.; Sketelj, J.; Cucek, D.; Brzin, M. Prevention of diisopropylphosphorofluoridate-induced myopathy by botulinurn toxin type A blockage of quanta1 release of acetylcholine. Acta Neuropathol. 82: 134-142; 199 I. 21. Wecker, L.; Kiauta, T.; Dettbarn, W.-D. Relationship between acetylcholinesterase inhibition and the development of a myopathy. J. Pharmacol. Exp. Ther. 206:97-104; 1978.
