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Acetylcholine in Intercostal Muscle from Myasthenia Gravis Patients and in Rat Diaphragm after Blockade of Acetylcholine Receptors
Acetylcholine in Intercostal Muscle fi-om Myasthenia Gravis Patients and in Rat Diaphragm after Blockade of Acetylcholine Receptors P.C. MOLENAAR, R.L. POLAK, R. MILEDI, S. ALEMA * , A . VINCENT and J . NEWSOM-DAVIS (P.C.M.)Department of Pharmacology, Sylvius Laboratories, Leiden University Medical Centre, 2333 A L Leiden, (R.L.P.) Medical Biological Laboratory/T.N.O., Rijswijk-Z.H. (The Netherlands); (R.M. and S . A . ) Department of Biophysics, University College London, London WClE 6BTand (A. V. and J.N.D.) Department of Neurological Science, Royal Free Hospital, London NW3 2QC (United Kingdom)
INTRODUCTION My asthenia gravis (MG) is a disease in which neuromuscular transmission to skeletal muscle is impaired. In their study on myasthenic muscle Elmqvist et al. (1964) found that MEPPS - caused by the release of single quanta of acetylcholine (ACh) from motor nerve terminals - were abnormally small. More recently, it was discovered that ACh receptors (AChRs) are reduced in number at myasthenic end-plates (Fambrough et al., 1973; Green et al., 1975a). Further, an immunoglobulin has been demonstrated in the sera of myasthenic patients which binds to solubilized AChRs in vitro (Almon et al., 1974; Mittag et al., 1976; Lindstrom et al., 1976). These findings indicate that MG is an autoimmune disease in which the sensitivity of the muscle fibre membrane to ACh is reduced by a specific antibody. In fact, immunization of animals against AChR protein purified from various sources leads to MGlike symptoms (see for instance: Patrick and Lindstrom, 1973; Sugiyama et al., 1973; Heilbronn and Mattson, 1974; Green et al., 1975b) presumably by cross reaction of antibodies raised against AChR with endogenous receptors in striated muscle. In our studies we have investigated whether the reduction in the number of AChRs in MG is accompanied by changes in the synthesis and release of ACh. We have also investigated rat diaphragms, whose AChRs were blocked in vitro by a-bungarotoxin (Chang and Lee, 1963; Lee et al., 1967; Miledi and Potter, 1971), or by immunization with AChR purified from Torpedo electroplax (Patrick and Lindstrom, 1973; Green et al., 1975b; Alema et al., 1978). Parts of this work have been reported elsewhere (It0 et al., 1976; Miledi et al., 1978; Ito et al., 1978b; Cull-Candy et al., 1978b).
* Present address: Laboratory of Cellular Biology C.N.R., Roma (Italy).
450
METHODS Human muscle. The biopsies were performed under general anaesthesia except in a few cases (congenital MG) in which local anaesthesia was used. Parasternal intercostal muscle was obtained from 15 patients with MG during thymectomy. In all “control” patients, in 5 patients with congenital MG and in TABLE 1 ACETYLCHOLINE CONTENT OF HUMAN INTERCOSTAL MUSCLE ~
~~
Patient
~
ACh content fresh muscle
Sex
(b)
( p m o l . g-l)
Acquired myasthenia 1 2 3 4 5 6 7 8 9 10 11 12 13 Mean f S.E.M. e
gravis 7 19
Congenital myasthenia gravis 1
160*
2 3 4 5
Mean +- S.E.M.f Controls 1
2 3 4
5
6 7 8 9 Mean f S.E.M.
0.59 C 0.13 0.26 0.19 0.23 C 0.44 C 0.26 0.33 C 0.17 0.24 C 0.18 0.15 c 0.25 c
28 27 18 27 18 54 54 36 48 41 27 27 32
M M M
16(4) 4(4) 16(4) 22(3) 44(2) 34
0.07
C
0.15 0.33 0.46
c
13 25 17 13 17
M M M M M
25(2) lO(2) 8(2) 40(2) 60(6) 16(6) 33(2) 12(2) 20(4) 20
1.03 C 0.70 0.64 0.64 0.94 C 1.06 c
56 56 64 41 63 65
-
57 61
(1) 73(2) 3 6 0 2 45(2) l o o * 15(2) 2 8 0 2 37(6) 290* 33(6) 3 4 0 + 55(2) 710 f 127 (3) 280* 16(4) 4 6 0 k 53(2) 210% 33(3) a 360f 19(2) a 840 (1) 420* 61 500*
130* 130+ 240f 300* 190f 250f 120* 170f 190* 2402 150*
l8O+
120* 290f 190*
Clinical state
*
-
-
-
C
57
F
M M F F M M M M F
IV I1 B I1 B I1 B 111
IV IV I1 B I1 B I1 A IV IV I1 A
I1 B I1 B I1 A I1 A I1 B
carcinoma carc. of bronchus carc. of bronchus achalasia carcinoma carcinoma carc. of bronchus carc. of bronchus neurofibroma
~
ACh was determined in 1-6 groups of tendon-to-tendon muscle bundles; each group normally consisted of 3-4 bundles (40-100 mg). a Lateral biopsy; b clinical state in MG was classified ocular (I); mild generalized (I1 A); moderate generalized (I1 B); acute (111); severe acute (IV); severe chronic (V); for control patients the diagnosis is stated (cf. Ito et al., 1976, Table I). c MEPP amplitude in neostigmine bromide ( 5 pM); d after DFP (1 0 pM). e Significantly different from control (Pz < 0.005); f significantly different from acquired MG (P2< 0.005);Welch’s mdtiple test.
45 1
2 cases of MG, external intercostal muscle which had been taken through a lateral incision, was used instead of parasternal intercostal muscle. Muscle from “control” patients - without clinical signs of muscle disease - was obtained during thoracotomy. For clinical details, see Table I. Rat diaphragm. AChR was purified from the electric organ of Torpedo rnarrnorata by affinity chromatography either on a curare-column (Green et al., 1975b) or on a cobratoxin-Sepharose derivative (Alema et al., 1978). Wistar or PVG/C (hooded) rats (1 50-200 g) were injected subcutaneously o n multiple sites with AChR (75-150 pg) in complete Freund adjuvant; animals were injected two to three times at three weeks intervals and killed about three weeks after the last injection. Hemidiaphragms were dissected together with the phrenic nerve. MEPPs were recorded from portions of muscle from the same animal as used for the estimations of ACh. ACh estimation. ACh was extracted from human and rat muscle and subsequently estimated by mass fragmentography; deuterated ACh was used as an internal standard (for details see: Miledi et al., 1977).
RESULTS
Human muscle ACh content. We investigated two types of the disease: acquired and congenital MG; in congenital MG antibodies against AChR have not been found (Cull-Candy et al., 1978b). As shown in Table I muscle from acquired MG patients contained about twice as much ACh (420 pmol/g) as muscle from control patients (190 pmol/g) or muscle from patients suffering from congenital MG ( 190 pmol/g). The difference was statistically significant not-withstanding a considerable variation between different patients or even between bundles of one biopsy. In one patient with Eaton-Lambert syndrome *, the muscle contained 190 pmol/g ACh (not. shown in Table I), which is within the range of control values. Table I further shows that the MEPP amplitudes were reduced in muscles from patients suffering from acquired as well as from congenital MG . Release of ACh. To measure the release of ACh it is necessary to prevent its enzymic hydrolysis; for this purpose DFP was used. However, when cholinesterase activity is blocked, muscles may show spontaneous “fasciculations”, due to the generation of nerve impulses in some terminals (Masland and Wigton, 1940). Since the liberation of ACh by such activity might contribute appreciably to the apparent spontaneous release of ACh from the muscle into the medium, we measured the ACh released in the presence and in the absence of TTX, which is known to block nerve impulse activity without interfering with spontaneous ACh release (cf. Katz and Miledi, 1967).
* Also named myasthenic syndrome, since it is characterized by muscular weakness; it is often associated with carcinoma.
452 TABLE I1 SPONTANEOUS RELEASE O F ACh FROM HUMAN INTERCOSTAL MUSCLE ~~
Acetylcholine release (pmol g-' +
~
No TTX
With TTX (1.6 /.hi)
-~
. min-')
~
Control
Acquired MG
1.7 f 0.5(4) 1.4 f 0.6 (3)
2.8 f 1.1 (4) 0.9 0.1 (4)
Muscle bundles (100-200 mg) were incubated in Ringer (for details see legends of Fig. 1 and Table I). The rates of spontaneous ACh release were averaged in each case from four incubation periods. The spontaneous release, though varying considerably between different patients, remained a t about the same level in each individual case (values are somewhat different from those in Table 11, I t o et al., 1976, due t o additional data). Mean f S.E.M. with number of patients in brackets.
As shown in Table 11, TTX seemed to reduce the spontaneous release of ACh from human muscle, but this reduction was not significant. Moreover, no significant difference was observed between the amounts of ACh released spontaneously from myasthenic and control muscle. However, the situation was different when the release of ACh was stimulated by raising the KC1 concentration in the incubation medium. In the absence of TTX, KC1 initially produced a significantly higher release of ACh from myasthenic than from control muscle. During prolonged incubation the ACh release from myasthenic muscle decreased with time, whereas that from control muscle increased (Fig, 1A). Surprisingly, in the presence of TTX the KCl induced release of ACh from myasthenic muscle was equal to that from control muscle, and distinctly less than in the absence of TTX (Fig. 1B). 6L
6
T
A
B
4
1 0
4 I
1 30
I
A
I 60
0
30
60
timo (min)
Fig. 1. KC1-induced release of ACh in intercostal muscle from acquired MG (circles) and control (squares) patients. Muscle bundles (100-200 mg) were incubated a t about 2OoC in 2 ml Ringer (pH 7.3) consisting of (in mM): NaCl 116; KCI 4.5; NaHC03 23; NaH2P04 1; MgS04 I ; CaCI2 2; glucose 11 ; DFP 0.02. The medium was kept under a n atmosphere of 95% 0 2 and 5% C 0 2 . After 90 min (at time = 0 in the figure) the KCI was raised t o 50 mM and, subsequently, the ACh was collected in 15-min periods. The release of ACh into the medium in the presence of 50 mM KC1 has been corrected for the spontaneous release of ACh in normal Ringer which was determined in two preceding periods (not shown), Incubation was i n the absence (A) or in the presence (B) of 1.6 pM TTX. Means f S.E.M. with number of patients in brackets, t Value significantly different from control (Pz< 0.05, Wilcoxon's Rank Sum Test).
TABLE 111 EFFECT OF ACUTE AND CHRONIC RECEPTOR BLOCKADE ON ACh RELEASE AND CONTENT IN RAT DIAPHRAGM Evoked release (3Isec. %) b
a-Bungarotoxin Immunization with AChR
a
170 f 38 (2) 170 f 26 (9) ___
~
_
_
_
Evoked release a (50 mM-KCI, %) b
Spontaneous release
2 0 0 f 18(11)C
l O O f 13 (14)
1 7 0 + 26(12)
110 f 15 (14)
(%I
Content
(%I
MEPP amplitude /%!
Undetectable 100 f 6 (1 2)
37 f 1.7 (17)
~~
Rat hemi-diaphragms were incubated at about 2OoC in 5 ml Ringer containing 20 p M DFP. After 9 0 min the preparations were rinsed and the incubation was continued for 60 min either in normal Ringer under supramaximal stimulation of the nerve at 3/sec or in Ringer containing 1.6 p M TTX and 50 mM KC1. When added, a-bungarotoxin ( 5 pglml) was present in the medium during the first 60-90 min of incubation. ACh was collected in 15- or 30-min periods. In the experiments with rats immunized against AChR-protein, littermates, most of which had been sham-immunized with Freund’s adjuvant, served as controls. In the experiments with a-bungarotoxin the contralateral hemidiaphragms were used for controls. MEPPs were recorded in fibres form diaphragms not treated with DFP. Means f S.E.M.with number of animals in brackets. a Evoked release of ACh was corrected for spontaneous release in two preceding 30-min periods. b Percentages of the control values; the controls were (mean values): 1.0 (3/sec), 3.2 ( 5 0 mM KC1) and 0.4 pmol . min-’ (spontaneous release), 300 pmol (tissue content) and 0.7 mV (MEPP amplitude). C Significantly different from controls (P2 < 0.05, Student’s t-test).
P
VI
w
454
Synthesis ofACh. Since KCl stimulates the release of ACh, one might expect it to cause a reduction of the ACh content of the tissue. However, it was found that after incubation with 50 mM KCl the ACh content was virtually unchanged or even increased. Apparently synthesis must have occurred in the tissue, replacing the amounts of ACh lost to the medium. In the absence of TTX the synthesis (change in tissue ACh + ACh released into the medium) was about 600 pmol/g, both in myasthenic and control muscle.
Rat muscle In isolated rat diaphragms after treatment with a-bungarotoxin end-plate potentials and MEPPs were completely abolished. On the other hand, in diaphragms from rats after immunization with AChR the MEPPs were not abolished, but their amplitude was reduced (see Table 111). Furthermore, neuromuscular transmission was practically unaffected in immunized Wistar rats, though weakness was sometimes observed in PVG/C rats at a later stage of immunization (weak rats have not been used for the experiments in Table 111). As shown in Table 111 both a-bungarotoxin and immunization with AChR caused about a 2-fold increase in the amount of ACh released under the influence of electrical (3/sec) or chemical (50 mM KCl) stimulation. Receptor blockade by a-bungarotoxin or by anti-AChR antibodies did not alter the spontaneous release of ACh, nor did it affect the ACh content of the muscles. '
DISCUSSION It was not possible to obtain control intercostal muscle from the same (parastemal) site as in MG patients; we therefore used external intercostal muscle taEen from a lateral site in patients who were often ill and who were generally older than the MG patients. If we accept these limitations, the present findings appear to contradict the suggestion that a defect in transmitter synthesis or release is the cause of transmission failure in MG (see for instance: Desmedt, 1958; Elmqvist et al., 1964). In fact, we found that the ACh content of myasthenic muscle was double that of control muscle (see also Ito et al., 1976). This difference does not appear to be due to a difference in the number of end-plates in myasthenic and control muscle, which was about lSOOO/g of wet tissue in both types of muscle. Probably it was also not caused by the treatment with anticholinesterase drugs given to MG patients since increased ACh levels were also found in two patients who did not receive such drugs (patients Nos. 6 and 9 in Table I). Moreover, it should be noted that Chang et al. (1973) found that the ACh content of rat diaphragm was not altered after 7-days' treatment with neostigmine. The severity of the clinical condition in MG patients was not clearly related to the ACh content of their muscles. A similar lack of correlation has been found between the clinical condition and MEPP amplitudes (It0 et al., 1978b; this paper), the number of AChRs or the plasma levels of anti-AChR antibodies (Lindstrom et al., 1976; Ito et al., 1978a). It has been demonstrated (Miledi et al., 1977) that frog muscle contains a
45 5
non-neural, probably muscular, store of ACh, apart from a larger store situated in the nerve terminals. It is possible that human muscle also contains a nonneural store of ACh, and that it is this store alone which is increased in MG. However, preliminary experiments in which the fibres were divided into endplate containing and end-plate free segements, suggest that the increased total content of ACh of myasthenic muscle is due to an increase in the ACh content of the nerve terminals. The finding that the high rate of KC1-induced release of ACh from myasthenic muscle is not sustained for 60 min although the ACh content of the muscle has not decreased at the end of this period, suggests that the ACh in the preparation is present in at least two compartments: a small compartment in which the ACh seems to be available for release and a larger one containing ACh which is unavailable for release; exhaustion of the small pool might not become apparent when the total ACh content is measured. An alternative possibility is that the release mechanism becomes “fatigued” during stimulation with KCl. The finding that the KC1-induced release of ACh from myasthenic muscle was poorly sustained, is probably not related to the “decremental response” in MG since this is due to a phenomenon occurring at both normal and myasthenic end-plates. It is clinically evident in MG only because of the reduced sensitivity of the muscle membrane to ACh. Whether the exhaustion of ACh release is related to the aggravation of muscle weakness in MG during exercise, remains to be seen. The finding that TTX depressed the KC1-induced release of ACh from my asthenic muscle suggests that Na’-influx resulting from depolarization by K’ may in some way influence ACh release, for instance by mobilizing internal calcium ions or by accelerating the synthesis of ACh, which is known to be dependent on Na’ ions (Quastel, 1962; Birks, 1963; Bhatnagar and MacIntosh, 1967). Our finding that the ACh content of muscle from patients with congenital MG (whose symptoms are indistinguishable from acquired MG) is within the normal range, and about half that of myasthenic muscle, raises the question whether this type of the disease is fundamentally different from acquired MG. This view is supported by the finding that congenital MG patients do not have elevated anti-AChR antibody titres (Cull-Candy et al., 1978b) and that their condition is not improved by total plasma exchange (Pinching et al., 1976), a procedure which brings about a considerable improvement in most patients with acquired MG (Newsom-Davis et al., 1978; Dau et al., 1977). In diaphragms from immunized rats the MEPP amplitude was decreased to almost the same extent as in human myasthenic muscle. In spite of this practically all the fibres responded with an action potential to nerve stimulation. This contrasts with human myasthenic muscle and immunized rabbit muscle in which nerve impulses fail to trigger action potentials in a proportion of the fibres (cf. Green et al., 1975b). This difference may be due to a higher safety margin for neuromuscular transmission in rats than in rabbits or human muscle (see also Lambert et al., 1976), but it could also be that there is a more adequate adaptation (e.g. increase of ACh release, cf. Table 111) to the loss of AChRs in the rat than in the other species.
456
Experimental MG in animals induced by immunization against AChR-protein has been proposed as an animal model for MG (see for review: Vincent, 1978). Notwithstanding several differences between the results obtained with human and rat muscle, we found that the evoked release of ACh was increased in muscles from both myasthenics and immunized rats. In either preparation the increase may have been due to an adaptation process compensating for the chronic loss of AChRs. In this connection it is of interest to note that CullCandy et al. (1978a) found that the number of quanta released by an action potential at low calcium concentrations was about six times normal in myasthenic intercostal muscle. The increase in the evoked release of ACh from the rat diaphragm caused by a-bungarotoxin could also result from a compensatory mechanism which, of course, need not be identical to that in the immunized preparation: in the experiments with a-bungarotoxin the AChRs were blocked acutely and completely, in the immunized rats chronically and partially. SUMMARY ACh was extracted from intercostal muscles from patients with acquired and congenital myasthenia gravis (MG) and from control patients with no clinical signs of muscle disease. ACh was estimated by mass fragmentography. It was found that the concentration of ACh was about two times higher in muscle from acquired MG than from congenital MG or control patients, Muscle from acquired MG and control patients was also incubated in Ringer with 50 mM KCl in order to stimulate the release of ACh. During the first 15 min of incubation with KCl more ACh was released from myasthenic than from control muscle, but this difference was not sustained on prolonged incubation. Further, it was found that tetrodotoxin depressed the amount of ACh released into the mkdium in the presence of 50 mM KCI. Diaphragms from normal rats were treated with a-bungarotoxin, or taken from animals which had been immunized against the ACh receptor protein from Torpedo marmorata. It was found that from these preparations KCl released about twice as much ACh as from control diaphragms. It is suggested that in myasthenic muscle and in diaphragms from immunized rats there is an adaptation process in the transmitter release compensating for the chronic loss of ACh receptors. If there is such a mechanism, it might be more efficient in rat than in man. ACKNOWLEDGEMENTS We are grateful to the patients for their collaboration and to Mr. M.F. Sturridge and Mr. J.R. Belcher for their help in obtaining the biopsies, to Drs. Y. Ito, S.G.Cull-Candy and O.D. Uchitel for measuring MEPPs and to Mrs. P. Braggaar-Schaap for technical assistance. Financial support by the Medical Research Council (to R.M.), the foundations I.B.R.O. and PROMESO (to P.C.M.) and FUNGO-ZWO (to R.L.P. and P.C.M.) is gratefully acknowledged.
457
S.A. was a recipient of a long-term fellowship from the European Molecular Biology Organization.
REFERENCES Alema, S., Miledi, R. and Vincent, A. (1978) Nicotinic acetylcholine receptors and the role of specific antibodies in experimental myasthenia neuromuscular block. In Organ Specific Immunity, P.A. Miescher (Ed.), Menarini series on immunopathology, Vol. I, Schwabe, Basel, in press. Almon, R.R., Andrew, C.G. and Appel, S.H. (1 974) Serum globulin in myasthenia gravis: inhibition of a-bungarotoxin binding to acetylcholine-receptors. Science, 186, 55-57. Bhatnagar, S.P. and Machtosh, F.C. (1967) Effect of quaternary bases and inorganic cations on acetylcholine synthesis in nervous tissue. Canad. J. Physiol. Pharmacol., 45,249268. Birks, R.I. (1963) The role of sodium ions in the metabolism of acetylcholine. Canad. J. Biochem. Physiol., 41, 2573-2597. Chang, C.C. and Lee, C.Y. (1963) Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch. Int. Pharmacodyn., 144,241-257. Chang, C.C., Chen, T.F. and Chuang, S.-T. (1973) Influence of chronic neostigmine treatment on the number of acetylcholine receptors and the release of acetylcholine from the rat diaphragm. J. Physiol. (Lond.), 230,613-618. CullCandy, S.G., Miledi, R. and Trautmann, A. ( 1978) Acetylcholine-induced channels and transmitter release at human end-plates. Nature (Lond.), 271, 74-75. CullCandy, S.G., Miledi, R., Molenaar, P.C., Newsom-Davis, J., Polak, R.L.,Trautmann, A. and Vincent, A. (1978), in preparation. Dau, P.C., Lindstrom, J.M., Cassel, J.K., Denys, E.H., Shev, E.E. and Spitter, L.E. (1977) Plasmapheresis and immunosuppressive drug therapy in myasthenia gravis. New Engl. J. Med., 297, 1134- 1140. Desmedt, J.E. ( 1958) Myasthenia-like features of neuromuscular transmission after administration of an inhibitor of acetylcholine synthesis. Nature (Lond.), 182, 1973- 1974. Elmqvist, D., Hofmann, W.W., Kugelberg, J . and Quastel, D.M.J. (1964) An electrophysiological investigation of neuromuscular transmission in myasthenia gravis. J . Physiol. (Lond.), 174,417-434. Fambrough, D.M., Drachman, D.B. and Satyamurti, S. (1973) Neuromuscular junction in myasthenia gravis: Decreased acetylcholine receptors. Science, 182, 293-295. Green, D.P.L., Miledi, R., Perez de la Mora, M. and Vincent, A. (1975a) Acetylcholine receptors. Phil. Trans. roy. SOC. Lond. B, 270, 551-559. Green, D.P.L., Miledi, R. and Vincent, A. (1975b) Neuromuscular transmission after immunization against acetylcholine receptors, Proc. roy. SOC.Lond. B, 189,57-68. Heilbronn, E. and Mattson, Ch. (1 974) The nicotinic cholinergic receptor protein: improved purification method, preliminary amino acid composition and observed auto-immuno response. J. Neurochem., 2 2 , 3 15-3 17. Ito, Y., Miledi, R., Molenaar, P.C., Vincent, A., Polak, R.L., van Gelder, M. and NewsomDavis, J. (1976) Acetylcholine in human muscle, Proc. roy. SOC. Lond. B, 192,475480. Ito, Y., Miledi, R., Molenaar, P.C., Newsom-Davis, J., Polak, R.L. and Vincent, A. (1978a) Neuromuscular transmission in myasthenia gravis and the significance of anti-acetylcholine antibodies. In Biochemistry of myasthenia gravis and muscular dystrophy, R. Marchbanks and J . Lunt (Eds.), Academic Press, pp. 89- 109. Ito, Y.,Miledi, R., Vincent, A. and Newsom-Davis, J. (1978b) Acetylcholine receptors and electrophysiological function in myasthenia gravis. Brain, 101, 345-368. Katz, B. and Miledi. R. (1 967) Tetrodoxin and neuromuscular transmission. Proc. roy. SOC. Lond. B, 167, 8-22. Lambert, E.H., Lindstrom, J.M. and Lennon, V.A. (1976) End-plate potentials in experimental autoimmune myasthenia gravis in rats. Ann. N.Y. Acad. Sci., 274, 300-318. Lee, C.Y., Tseng, L.F. and Chiu, T.H. (1967) Influence of denervation o n localization of neurotoxins from elapid venoms in rat diaphragms. Nature (Lond.), 21 5, 1 177-1 178. Lindstrom, J.M., Lennon, V.A., Seybold, M.E. and Whittingham, S. (1976) Experimental
458 autoimmune myasthenia gravis and myasthenia gravis: biochemical and immunochemical aspects. Ann. N.Y. Acad. Sci., 274, 254-274. Masland, R.L. and Wigton, R.S. ( 1940) Nerve activity accompanying fasciculation produced by neostigmine. J. Neurophysiol., 3, 269-275. Miledi, R. and Potter, L.T. (1971) Acetylcholine receptors in muscle fibres. Nature (Lond.), 233,599-603. Miledi, R., Molenaar, P.C. and Polak, R.L. ( 1 977) An analysis of acetylcholine in frog muscle by mass fragmentography. Proc. roy. SOC.Lond. B, 197, 285-297. Miledi, R., Molenaar, P.C.and Polak, R.L. (1978) a-Bungarotoxin enhances transmitter ‘released’ at the neuromuscular junction. Nature (Lond.), 272, 641 -643. Mittag, T., Kornfeld, P., Tormay, A. and Woo, B. (1976) Detection of anti-acetylcholine receptor factors in serum and thymus from patients with myasthenia gravis. New. Engl. J. Med., 294, 691 -694. Newsom-Davis, J., Pinching, A.J., Vincent, A. and Wilson, S.C. (1978) Functioning of circulating antibody to acetylcholine receptor in myasthenia gravis investigated by plasma exchange. Neurol. (Minneap.), 28,266-272. Patrick, J. and Lindstrom, J . ( 1 973) Autoimmune response t o acetylcholine receptor. Science, 180,87 1-872. Pinching, A.J., Peters, D.K. and Newsom-Davis, J. (1976) Remission of myasthenia gravis following plasma exchange. Lancet, ii, 1373- 1376. Quastel, D.M.J. (1962) Ph.D. Thesis, McGill University, Montreal, Quebec. Sugiyama, H.,Benda, P., Meunier, J.C. and Changeux, J.-P. (1973) Immunological characterisation of the cholinergic receptor protein from Electrophorus electricus. FEBS Lett., 35,124-128. Vincent, A. (1 978) Experimental autoimmune myasthenia gravis. In Neuroimmunology, F.C. Rose (Ed.), Blackwell, Oxford, in press.
INTRODUCTION My asthenia gravis (MG) is a disease in which neuromuscular transmission to skeletal muscle is impaired. In their study on myasthenic muscle Elmqvist et al. (1964) found that MEPPS - caused by the release of single quanta of acetylcholine (ACh) from motor nerve terminals - were abnormally small. More recently, it was discovered that ACh receptors (AChRs) are reduced in number at myasthenic end-plates (Fambrough et al., 1973; Green et al., 1975a). Further, an immunoglobulin has been demonstrated in the sera of myasthenic patients which binds to solubilized AChRs in vitro (Almon et al., 1974; Mittag et al., 1976; Lindstrom et al., 1976). These findings indicate that MG is an autoimmune disease in which the sensitivity of the muscle fibre membrane to ACh is reduced by a specific antibody. In fact, immunization of animals against AChR protein purified from various sources leads to MGlike symptoms (see for instance: Patrick and Lindstrom, 1973; Sugiyama et al., 1973; Heilbronn and Mattson, 1974; Green et al., 1975b) presumably by cross reaction of antibodies raised against AChR with endogenous receptors in striated muscle. In our studies we have investigated whether the reduction in the number of AChRs in MG is accompanied by changes in the synthesis and release of ACh. We have also investigated rat diaphragms, whose AChRs were blocked in vitro by a-bungarotoxin (Chang and Lee, 1963; Lee et al., 1967; Miledi and Potter, 1971), or by immunization with AChR purified from Torpedo electroplax (Patrick and Lindstrom, 1973; Green et al., 1975b; Alema et al., 1978). Parts of this work have been reported elsewhere (It0 et al., 1976; Miledi et al., 1978; Ito et al., 1978b; Cull-Candy et al., 1978b).
* Present address: Laboratory of Cellular Biology C.N.R., Roma (Italy).
450
METHODS Human muscle. The biopsies were performed under general anaesthesia except in a few cases (congenital MG) in which local anaesthesia was used. Parasternal intercostal muscle was obtained from 15 patients with MG during thymectomy. In all “control” patients, in 5 patients with congenital MG and in TABLE 1 ACETYLCHOLINE CONTENT OF HUMAN INTERCOSTAL MUSCLE ~
~~
Patient
~
ACh content fresh muscle
Sex
(b)
( p m o l . g-l)
Acquired myasthenia 1 2 3 4 5 6 7 8 9 10 11 12 13 Mean f S.E.M. e
gravis 7 19
Congenital myasthenia gravis 1
160*
2 3 4 5
Mean +- S.E.M.f Controls 1
2 3 4
5
6 7 8 9 Mean f S.E.M.
0.59 C 0.13 0.26 0.19 0.23 C 0.44 C 0.26 0.33 C 0.17 0.24 C 0.18 0.15 c 0.25 c
28 27 18 27 18 54 54 36 48 41 27 27 32
M M M
16(4) 4(4) 16(4) 22(3) 44(2) 34
0.07
C
0.15 0.33 0.46
c
13 25 17 13 17
M M M M M
25(2) lO(2) 8(2) 40(2) 60(6) 16(6) 33(2) 12(2) 20(4) 20
1.03 C 0.70 0.64 0.64 0.94 C 1.06 c
56 56 64 41 63 65
-
57 61
(1) 73(2) 3 6 0 2 45(2) l o o * 15(2) 2 8 0 2 37(6) 290* 33(6) 3 4 0 + 55(2) 710 f 127 (3) 280* 16(4) 4 6 0 k 53(2) 210% 33(3) a 360f 19(2) a 840 (1) 420* 61 500*
130* 130+ 240f 300* 190f 250f 120* 170f 190* 2402 150*
l8O+
120* 290f 190*
Clinical state
*
-
-
-
C
57
F
M M F F M M M M F
IV I1 B I1 B I1 B 111
IV IV I1 B I1 B I1 A IV IV I1 A
I1 B I1 B I1 A I1 A I1 B
carcinoma carc. of bronchus carc. of bronchus achalasia carcinoma carcinoma carc. of bronchus carc. of bronchus neurofibroma
~
ACh was determined in 1-6 groups of tendon-to-tendon muscle bundles; each group normally consisted of 3-4 bundles (40-100 mg). a Lateral biopsy; b clinical state in MG was classified ocular (I); mild generalized (I1 A); moderate generalized (I1 B); acute (111); severe acute (IV); severe chronic (V); for control patients the diagnosis is stated (cf. Ito et al., 1976, Table I). c MEPP amplitude in neostigmine bromide ( 5 pM); d after DFP (1 0 pM). e Significantly different from control (Pz < 0.005); f significantly different from acquired MG (P2< 0.005);Welch’s mdtiple test.
45 1
2 cases of MG, external intercostal muscle which had been taken through a lateral incision, was used instead of parasternal intercostal muscle. Muscle from “control” patients - without clinical signs of muscle disease - was obtained during thoracotomy. For clinical details, see Table I. Rat diaphragm. AChR was purified from the electric organ of Torpedo rnarrnorata by affinity chromatography either on a curare-column (Green et al., 1975b) or on a cobratoxin-Sepharose derivative (Alema et al., 1978). Wistar or PVG/C (hooded) rats (1 50-200 g) were injected subcutaneously o n multiple sites with AChR (75-150 pg) in complete Freund adjuvant; animals were injected two to three times at three weeks intervals and killed about three weeks after the last injection. Hemidiaphragms were dissected together with the phrenic nerve. MEPPs were recorded from portions of muscle from the same animal as used for the estimations of ACh. ACh estimation. ACh was extracted from human and rat muscle and subsequently estimated by mass fragmentography; deuterated ACh was used as an internal standard (for details see: Miledi et al., 1977).
RESULTS
Human muscle ACh content. We investigated two types of the disease: acquired and congenital MG; in congenital MG antibodies against AChR have not been found (Cull-Candy et al., 1978b). As shown in Table I muscle from acquired MG patients contained about twice as much ACh (420 pmol/g) as muscle from control patients (190 pmol/g) or muscle from patients suffering from congenital MG ( 190 pmol/g). The difference was statistically significant not-withstanding a considerable variation between different patients or even between bundles of one biopsy. In one patient with Eaton-Lambert syndrome *, the muscle contained 190 pmol/g ACh (not. shown in Table I), which is within the range of control values. Table I further shows that the MEPP amplitudes were reduced in muscles from patients suffering from acquired as well as from congenital MG . Release of ACh. To measure the release of ACh it is necessary to prevent its enzymic hydrolysis; for this purpose DFP was used. However, when cholinesterase activity is blocked, muscles may show spontaneous “fasciculations”, due to the generation of nerve impulses in some terminals (Masland and Wigton, 1940). Since the liberation of ACh by such activity might contribute appreciably to the apparent spontaneous release of ACh from the muscle into the medium, we measured the ACh released in the presence and in the absence of TTX, which is known to block nerve impulse activity without interfering with spontaneous ACh release (cf. Katz and Miledi, 1967).
* Also named myasthenic syndrome, since it is characterized by muscular weakness; it is often associated with carcinoma.
452 TABLE I1 SPONTANEOUS RELEASE O F ACh FROM HUMAN INTERCOSTAL MUSCLE ~~
Acetylcholine release (pmol g-' +
~
No TTX
With TTX (1.6 /.hi)
-~
. min-')
~
Control
Acquired MG
1.7 f 0.5(4) 1.4 f 0.6 (3)
2.8 f 1.1 (4) 0.9 0.1 (4)
Muscle bundles (100-200 mg) were incubated in Ringer (for details see legends of Fig. 1 and Table I). The rates of spontaneous ACh release were averaged in each case from four incubation periods. The spontaneous release, though varying considerably between different patients, remained a t about the same level in each individual case (values are somewhat different from those in Table 11, I t o et al., 1976, due t o additional data). Mean f S.E.M. with number of patients in brackets.
As shown in Table 11, TTX seemed to reduce the spontaneous release of ACh from human muscle, but this reduction was not significant. Moreover, no significant difference was observed between the amounts of ACh released spontaneously from myasthenic and control muscle. However, the situation was different when the release of ACh was stimulated by raising the KC1 concentration in the incubation medium. In the absence of TTX, KC1 initially produced a significantly higher release of ACh from myasthenic than from control muscle. During prolonged incubation the ACh release from myasthenic muscle decreased with time, whereas that from control muscle increased (Fig, 1A). Surprisingly, in the presence of TTX the KCl induced release of ACh from myasthenic muscle was equal to that from control muscle, and distinctly less than in the absence of TTX (Fig. 1B). 6L
6
T
A
B
4
1 0
4 I
1 30
I
A
I 60
0
30
60
timo (min)
Fig. 1. KC1-induced release of ACh in intercostal muscle from acquired MG (circles) and control (squares) patients. Muscle bundles (100-200 mg) were incubated a t about 2OoC in 2 ml Ringer (pH 7.3) consisting of (in mM): NaCl 116; KCI 4.5; NaHC03 23; NaH2P04 1; MgS04 I ; CaCI2 2; glucose 11 ; DFP 0.02. The medium was kept under a n atmosphere of 95% 0 2 and 5% C 0 2 . After 90 min (at time = 0 in the figure) the KCI was raised t o 50 mM and, subsequently, the ACh was collected in 15-min periods. The release of ACh into the medium in the presence of 50 mM KC1 has been corrected for the spontaneous release of ACh in normal Ringer which was determined in two preceding periods (not shown), Incubation was i n the absence (A) or in the presence (B) of 1.6 pM TTX. Means f S.E.M. with number of patients in brackets, t Value significantly different from control (Pz< 0.05, Wilcoxon's Rank Sum Test).
TABLE 111 EFFECT OF ACUTE AND CHRONIC RECEPTOR BLOCKADE ON ACh RELEASE AND CONTENT IN RAT DIAPHRAGM Evoked release (3Isec. %) b
a-Bungarotoxin Immunization with AChR
a
170 f 38 (2) 170 f 26 (9) ___
~
_
_
_
Evoked release a (50 mM-KCI, %) b
Spontaneous release
2 0 0 f 18(11)C
l O O f 13 (14)
1 7 0 + 26(12)
110 f 15 (14)
(%I
Content
(%I
MEPP amplitude /%!
Undetectable 100 f 6 (1 2)
37 f 1.7 (17)
~~
Rat hemi-diaphragms were incubated at about 2OoC in 5 ml Ringer containing 20 p M DFP. After 9 0 min the preparations were rinsed and the incubation was continued for 60 min either in normal Ringer under supramaximal stimulation of the nerve at 3/sec or in Ringer containing 1.6 p M TTX and 50 mM KC1. When added, a-bungarotoxin ( 5 pglml) was present in the medium during the first 60-90 min of incubation. ACh was collected in 15- or 30-min periods. In the experiments with rats immunized against AChR-protein, littermates, most of which had been sham-immunized with Freund’s adjuvant, served as controls. In the experiments with a-bungarotoxin the contralateral hemidiaphragms were used for controls. MEPPs were recorded in fibres form diaphragms not treated with DFP. Means f S.E.M.with number of animals in brackets. a Evoked release of ACh was corrected for spontaneous release in two preceding 30-min periods. b Percentages of the control values; the controls were (mean values): 1.0 (3/sec), 3.2 ( 5 0 mM KC1) and 0.4 pmol . min-’ (spontaneous release), 300 pmol (tissue content) and 0.7 mV (MEPP amplitude). C Significantly different from controls (P2 < 0.05, Student’s t-test).
P
VI
w
454
Synthesis ofACh. Since KCl stimulates the release of ACh, one might expect it to cause a reduction of the ACh content of the tissue. However, it was found that after incubation with 50 mM KCl the ACh content was virtually unchanged or even increased. Apparently synthesis must have occurred in the tissue, replacing the amounts of ACh lost to the medium. In the absence of TTX the synthesis (change in tissue ACh + ACh released into the medium) was about 600 pmol/g, both in myasthenic and control muscle.
Rat muscle In isolated rat diaphragms after treatment with a-bungarotoxin end-plate potentials and MEPPs were completely abolished. On the other hand, in diaphragms from rats after immunization with AChR the MEPPs were not abolished, but their amplitude was reduced (see Table 111). Furthermore, neuromuscular transmission was practically unaffected in immunized Wistar rats, though weakness was sometimes observed in PVG/C rats at a later stage of immunization (weak rats have not been used for the experiments in Table 111). As shown in Table 111 both a-bungarotoxin and immunization with AChR caused about a 2-fold increase in the amount of ACh released under the influence of electrical (3/sec) or chemical (50 mM KCl) stimulation. Receptor blockade by a-bungarotoxin or by anti-AChR antibodies did not alter the spontaneous release of ACh, nor did it affect the ACh content of the muscles. '
DISCUSSION It was not possible to obtain control intercostal muscle from the same (parastemal) site as in MG patients; we therefore used external intercostal muscle taEen from a lateral site in patients who were often ill and who were generally older than the MG patients. If we accept these limitations, the present findings appear to contradict the suggestion that a defect in transmitter synthesis or release is the cause of transmission failure in MG (see for instance: Desmedt, 1958; Elmqvist et al., 1964). In fact, we found that the ACh content of myasthenic muscle was double that of control muscle (see also Ito et al., 1976). This difference does not appear to be due to a difference in the number of end-plates in myasthenic and control muscle, which was about lSOOO/g of wet tissue in both types of muscle. Probably it was also not caused by the treatment with anticholinesterase drugs given to MG patients since increased ACh levels were also found in two patients who did not receive such drugs (patients Nos. 6 and 9 in Table I). Moreover, it should be noted that Chang et al. (1973) found that the ACh content of rat diaphragm was not altered after 7-days' treatment with neostigmine. The severity of the clinical condition in MG patients was not clearly related to the ACh content of their muscles. A similar lack of correlation has been found between the clinical condition and MEPP amplitudes (It0 et al., 1978b; this paper), the number of AChRs or the plasma levels of anti-AChR antibodies (Lindstrom et al., 1976; Ito et al., 1978a). It has been demonstrated (Miledi et al., 1977) that frog muscle contains a
45 5
non-neural, probably muscular, store of ACh, apart from a larger store situated in the nerve terminals. It is possible that human muscle also contains a nonneural store of ACh, and that it is this store alone which is increased in MG. However, preliminary experiments in which the fibres were divided into endplate containing and end-plate free segements, suggest that the increased total content of ACh of myasthenic muscle is due to an increase in the ACh content of the nerve terminals. The finding that the high rate of KC1-induced release of ACh from myasthenic muscle is not sustained for 60 min although the ACh content of the muscle has not decreased at the end of this period, suggests that the ACh in the preparation is present in at least two compartments: a small compartment in which the ACh seems to be available for release and a larger one containing ACh which is unavailable for release; exhaustion of the small pool might not become apparent when the total ACh content is measured. An alternative possibility is that the release mechanism becomes “fatigued” during stimulation with KCl. The finding that the KC1-induced release of ACh from myasthenic muscle was poorly sustained, is probably not related to the “decremental response” in MG since this is due to a phenomenon occurring at both normal and myasthenic end-plates. It is clinically evident in MG only because of the reduced sensitivity of the muscle membrane to ACh. Whether the exhaustion of ACh release is related to the aggravation of muscle weakness in MG during exercise, remains to be seen. The finding that TTX depressed the KC1-induced release of ACh from my asthenic muscle suggests that Na’-influx resulting from depolarization by K’ may in some way influence ACh release, for instance by mobilizing internal calcium ions or by accelerating the synthesis of ACh, which is known to be dependent on Na’ ions (Quastel, 1962; Birks, 1963; Bhatnagar and MacIntosh, 1967). Our finding that the ACh content of muscle from patients with congenital MG (whose symptoms are indistinguishable from acquired MG) is within the normal range, and about half that of myasthenic muscle, raises the question whether this type of the disease is fundamentally different from acquired MG. This view is supported by the finding that congenital MG patients do not have elevated anti-AChR antibody titres (Cull-Candy et al., 1978b) and that their condition is not improved by total plasma exchange (Pinching et al., 1976), a procedure which brings about a considerable improvement in most patients with acquired MG (Newsom-Davis et al., 1978; Dau et al., 1977). In diaphragms from immunized rats the MEPP amplitude was decreased to almost the same extent as in human myasthenic muscle. In spite of this practically all the fibres responded with an action potential to nerve stimulation. This contrasts with human myasthenic muscle and immunized rabbit muscle in which nerve impulses fail to trigger action potentials in a proportion of the fibres (cf. Green et al., 1975b). This difference may be due to a higher safety margin for neuromuscular transmission in rats than in rabbits or human muscle (see also Lambert et al., 1976), but it could also be that there is a more adequate adaptation (e.g. increase of ACh release, cf. Table 111) to the loss of AChRs in the rat than in the other species.
456
Experimental MG in animals induced by immunization against AChR-protein has been proposed as an animal model for MG (see for review: Vincent, 1978). Notwithstanding several differences between the results obtained with human and rat muscle, we found that the evoked release of ACh was increased in muscles from both myasthenics and immunized rats. In either preparation the increase may have been due to an adaptation process compensating for the chronic loss of AChRs. In this connection it is of interest to note that CullCandy et al. (1978a) found that the number of quanta released by an action potential at low calcium concentrations was about six times normal in myasthenic intercostal muscle. The increase in the evoked release of ACh from the rat diaphragm caused by a-bungarotoxin could also result from a compensatory mechanism which, of course, need not be identical to that in the immunized preparation: in the experiments with a-bungarotoxin the AChRs were blocked acutely and completely, in the immunized rats chronically and partially. SUMMARY ACh was extracted from intercostal muscles from patients with acquired and congenital myasthenia gravis (MG) and from control patients with no clinical signs of muscle disease. ACh was estimated by mass fragmentography. It was found that the concentration of ACh was about two times higher in muscle from acquired MG than from congenital MG or control patients, Muscle from acquired MG and control patients was also incubated in Ringer with 50 mM KCl in order to stimulate the release of ACh. During the first 15 min of incubation with KCl more ACh was released from myasthenic than from control muscle, but this difference was not sustained on prolonged incubation. Further, it was found that tetrodotoxin depressed the amount of ACh released into the mkdium in the presence of 50 mM KCI. Diaphragms from normal rats were treated with a-bungarotoxin, or taken from animals which had been immunized against the ACh receptor protein from Torpedo marmorata. It was found that from these preparations KCl released about twice as much ACh as from control diaphragms. It is suggested that in myasthenic muscle and in diaphragms from immunized rats there is an adaptation process in the transmitter release compensating for the chronic loss of ACh receptors. If there is such a mechanism, it might be more efficient in rat than in man. ACKNOWLEDGEMENTS We are grateful to the patients for their collaboration and to Mr. M.F. Sturridge and Mr. J.R. Belcher for their help in obtaining the biopsies, to Drs. Y. Ito, S.G.Cull-Candy and O.D. Uchitel for measuring MEPPs and to Mrs. P. Braggaar-Schaap for technical assistance. Financial support by the Medical Research Council (to R.M.), the foundations I.B.R.O. and PROMESO (to P.C.M.) and FUNGO-ZWO (to R.L.P. and P.C.M.) is gratefully acknowledged.
457
S.A. was a recipient of a long-term fellowship from the European Molecular Biology Organization.
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458 autoimmune myasthenia gravis and myasthenia gravis: biochemical and immunochemical aspects. Ann. N.Y. Acad. Sci., 274, 254-274. Masland, R.L. and Wigton, R.S. ( 1940) Nerve activity accompanying fasciculation produced by neostigmine. J. Neurophysiol., 3, 269-275. Miledi, R. and Potter, L.T. (1971) Acetylcholine receptors in muscle fibres. Nature (Lond.), 233,599-603. Miledi, R., Molenaar, P.C. and Polak, R.L. ( 1 977) An analysis of acetylcholine in frog muscle by mass fragmentography. Proc. roy. SOC.Lond. B, 197, 285-297. Miledi, R., Molenaar, P.C.and Polak, R.L. (1978) a-Bungarotoxin enhances transmitter ‘released’ at the neuromuscular junction. Nature (Lond.), 272, 641 -643. Mittag, T., Kornfeld, P., Tormay, A. and Woo, B. (1976) Detection of anti-acetylcholine receptor factors in serum and thymus from patients with myasthenia gravis. New. Engl. J. Med., 294, 691 -694. Newsom-Davis, J., Pinching, A.J., Vincent, A. and Wilson, S.C. (1978) Functioning of circulating antibody to acetylcholine receptor in myasthenia gravis investigated by plasma exchange. Neurol. (Minneap.), 28,266-272. Patrick, J. and Lindstrom, J . ( 1 973) Autoimmune response t o acetylcholine receptor. Science, 180,87 1-872. Pinching, A.J., Peters, D.K. and Newsom-Davis, J. (1976) Remission of myasthenia gravis following plasma exchange. Lancet, ii, 1373- 1376. Quastel, D.M.J. (1962) Ph.D. Thesis, McGill University, Montreal, Quebec. Sugiyama, H.,Benda, P., Meunier, J.C. and Changeux, J.-P. (1973) Immunological characterisation of the cholinergic receptor protein from Electrophorus electricus. FEBS Lett., 35,124-128. Vincent, A. (1 978) Experimental autoimmune myasthenia gravis. In Neuroimmunology, F.C. Rose (Ed.), Blackwell, Oxford, in press.