ACTH and Neuromuscular Transmission: Electrophysiological in Vitro Investigation of the Effects of Corticotropin and an ACTH Fragment on Neuromuscular Transmission K. L. Birnberger, M D , R. Riidel, PhD, and A. Struppler, M D The effects of corticotropin (ACTH) and the polypeptide fragment ACTH 4-10 on neuromuscular transmission were studied in rat phrenic nerve-diaphragm preparations in vitro. ACTH decreased the quantum content of end-plate potentials (epps) and increased the transmission failure rate. The frequency of miniature end-plate potentials (mepps) was increased. The results indicate that ACTH acts directly on the presynaptic terminal. The findings could explain the transient decrease in muscle strength often observed during corticotropin therapy for myasthenia gravis, and they suggest that the therapeutic effcct of ACTH is indirectly mediated. The only marked effect produced by the ACTH fragment was an increase in the mepp frequency; quantum content and failure rate of epps remained unchanged. Since ACTH 4- 10 is unable to stimulate cortisone release, its clinical efficacy seems unlikely. Birnberger KL, Rude1 R, Struppler A: ACTH and neurornuscular transmission: electrophysiological in vitro investigation of t h e effects of corticotropin and an ACTH fragment o n neuromuscular transmission. Ann Neurol 1:270-275, 1977

Neuromuscular transmission is impaired in myastheniagravis [ 1,2], with evidence for both presynaptic and postsynaptic abnormalities. Although the pathological mechanism of the disease still is not fully understood, new experimental results point to the postsynaptic membrane as the main site of pathological change [3]. Therapy for myasthenia gravis became possible with the introduction of prostigmine [4]. Management of myasthenic patients was further improved by the use of corticotropin [5,61 and cortisone [ 7 , 81 as well as by immunosuppressive drugs like atathioprine [9].The rational basis for immunosuppressive therapy, which was initially introduced under the assumption that an autoimmune mechanism underlies myasthenia [ l o , 111, has been strengthened by the demonstration of antibodies against acetylcholine receptor protein [12] and by the possibility of producing experimental my asthenia by immunization wirh end-plate protein [ 13, 141. It has never been clearly established whether adrenocorticotropic hormone (ACTH) and the corticosteroids act by immunosuppression alone or if they also have a direct effect o n neuromuscular transmission. Recent animal experiments both in vivo and in vitro, however, have disclosed a direct action of glucocorticoids o n mammalian motor nerve terminals [ 15,161.

From the Neurological Clinic and the Physiological Institute, Technical Universiry of iMunich, Federal Rcpu blic of Germany.

Accepted for publication Sept 13, 1976.

270

Investigation of the effects of A C T H on neuromuscular transmission are sparse and only indirect [17, 181. We therefore attempted a direct analysis of acetylcholine release at rest and during stimulation i n the presence of ACTH. Experiments with corticosteroids will be reported in a later paper. Behavioral studies o n rats [ 191 and experiments with an isolated nerve-muscle preparation [20] have indicated that the polypeptide fragment A C T H 4-10, which lacks adrenocortical stimulating activity [2 11, is as capable of inducing behavioral changes and delaying neuromuscular fatigue as is the whole molecule. A C T H 4-10 therefore seemed to be the part of A C T H which is active on nervous tissue. For this reason we also studied neuromuscular transmission in the presence of A C T H 4-10 in a comparative series of experiments. Portions of this paper have been presented previously [22].

Methods Wisrar rats weighing 150 to 200 gm were anesthetized with intraperitoneally applied pentobarbital, and thoracotornies were performed. T h e left phrenic nerve-hemidiaphragm preparation was quickly dissected, washed in oxygenated solution, and pinned to the borrorn of a muscle perfusion chamber. T h e perfusion fluid, modified after Liley [231, had the following composition (in millirnols per liter): sodium

Address reprint requests to Dr Birnberger, Neurologische Klinik der Technischen Universitiit Munchen, Mohlstrasse 28, D-8000 Miinchen 80, Federal Republic of Germany.

chloride, 107.7;potassium chloride, 3.48; calcium chloride, 1.53; magnesium sulfate, 0.69; sodium bicarbonate, 26.2; sodium dihydrogen phosphate, 1.67; sodium gluconate, 9.64; glucose, 5.55; sucrose, 7.6. The perfusion fluid was infused with a mixture of 95% oxygen and 5 9 f carbon dioxide, and its temperature was kept constant at 31°C with a thermostat. For stimulation, the stump of the phrenic nerve (approximately 5 mm) was placed in a suction electrode and supramaximal shocks of 0.2 msec duration were applied. In order to prevent muscle contraction, neuromuscular transmission was blocked by raising the MgSO, concentration to 10 mmol per liter prior to nerve stimulation. Intracellular potentials were recorded in the usual way by glass microelectrodes filled with 3 mol per liter of KCI (5-15 megohm) using a WPI-microprobe system in combination with aTektronix D13 storage oscilloscope and a Polaroid camera. A Siemens Mingograph pen recorder parallel to the oscilloscope registered the potential continuously o n a slow time base. Each experiment was started in physiological perfusion fluid. When it had been established by nerve stimulation and visual inspection that muscle contraction was normal, neuromuscular transmission was blocked by admission of high-Mg" solution. Then single cells in the end-plate region were impaled using a Leitz dissecting scope at 5 0 mag~ nification. O n l y cells with resting potentials negative to - 65 mv were used for experimentation. The main criterion for focal impalement was the rise time of the miniature endplace potentials (mepps);cells in which impalement failed to yield a mepp rise time of less than 1 msec were discarded. After mepp amplitude and frequency were determined, t h e nerve was stimulated for 4 seconds at 30 Hz for the registration of end-plate potentials (epps). When at least 20 cells had been investigated in this manner, the perfusate was changed to a high-Mg" solution containing, in addition, ACTH (Hoechst, Germany) in a concentration of 10 - 7 rnol per liter. In four experiments ACTH was applied only in a concentration of lo-' rnol per liter. ACTH 4-10 (supplied by Organon, Oss, Holland), was applied in a concentration of I V 7 rnol per liter. After each change of solution the preparation was allowed to equilibrate for 30 minutes, then the same set of measurements was repeated. All mepps and epps were corrected adopting -80 mv as a standard resting potential [24]. Quantum size (9)was calculated as the mean amplitude of at least 50 mepps. This method yields values that differ by no more than 10% from values calculated using the variance method [25].Because of initial potentiation, only epps recorded during the last two seconds of stimulation were taken for determination of the mean epp amplitude (V). From q and V, quantum content (m) was calculated according to Boyd and Martin's [261 formula m = V/q.

values are in good agreement with results reported in t h e literature [271.

Results

Effects of A C T H on NeiAromuscufar Transmission I n general, neuromuscular transmission was depressed by A C T H . End-plate potentials recorded during 3 0 Hz stimulation after the preparation had been equilibrated in a solution containing lo-' m o l p e r liter o f A C T H were reduced in amplitude. I n 11 o u t of 12 experiments we found a decrease in V (Fig 1);in only o n e experiment was there n o change. T h e mean decrease was 2 4 p (p < 0.005). T h e resting potential of t h e muscle fibers was not altered in the presence of ACTH, nor was there a change in the mean mepp amplitude. Altered resting potential or m e p p amplitude would hint at postsynaptic changes. Since neither was observed, it follows that A C T H affects m. Mean quantum content was only 1.44 in the presence of A C T H , compared with 1.72 in controls ( p < 0.01). T h e percentage of failures-ie, the number of stimuli that resulted in no detectable epp-was increased in the presence of A C T H by a factor of 1.5, from 18 to 27% (p < 0.02). T h e frequency (f) of mepps was increased in the presence of A C T H in eight o u t of 12 experiments. I n o n e experiment i t was decreased by 1574; in the remaining three experiments it was not significantly altered. Taking all experiments together, mepp frequency rose by 26% in the presence of ACTH. In control studies, however, w e found that f tended to increase with time even in normal solution; therefore, the effect of A C T H might be smaller than this figure suggests. rnol per liter of All results obtained with A C T H are compiled in Figure 2 and Table 1. I n four experiments A C T H was applied in a concentration of 1 0 F rnol p e r liter with similar results: mean decrease in V and m was about 20cJ,, and increase in failures was about 30%. All measurements concerning A C T H action were made 30 minutes after A C T H was applied to the preparation. Therefore, little can be said about t h e time course of A C T H action. In five experiments w e followed the time course, taking the failure rate as an indicator. In these experiments it seemed that the depressing effect of A C T H was already present after 10 minutes and did not change appreciably for at least an hour. T h e effect persisted for some 30 minutes after the solution was changed back to normal perfusion fluid, control values being approached o n e hour after continuous washout.

Mean control values from each preparation used for experiments with ACTH and ACTH 4- 10 are listed in Tables 1 and 2. T h e grand mean of all resting potentials was - 78.9 mv, mean m e p p amplitude was 0.66 mv, and mean frequency was 1.8 p e r second. These

Effects of A C T H 4-20 o n Neuromusczcfar Transmission I n contrast to whole A C T H , the 4-10 fragment, when applied in a concentration of lo-' rnol p e r liter, did

Birnberger, Riidel, and Struppler: ACTH and Neuromuscular Transmission 271

Table 1 . ReJdtJ Obtained with ACTH on 240 End-Plate.ia t Illarci

RMP (mv) Darr

Control

ACTH

Courrol

ACTH

845+17 80.4 2 I 3 81.0 L I I

YJO * 1 1

2.0i- 0 22 1.1 = 0.16

2.5

i0

1 7

+

1 0 18 1.3 c 0 18 2.4 L 0 22 1 . 7 ? 0.09 2.4 i 0.25 20I026 1.11+ 0.20 1.7 i 0.13

2.4 i 0.28

x2.n 2 0.9

1.1

0 + 1.3 79.8 f 1.1

843215 76'+09

86.X F 1.3 794r06

82.8 2 0.q

77.0

=

86.0

+ 17 71!1+13 72.8

= 1.3

'5.9 2 1.3 735206

-7.7

2

81./l+II

82.4 t I I

79.3 + 1.1

78.9

0.C)

73.4 t

I.'

75

?LlFl?

>E

q (mv)

0.9

7>.42 1

V

+ 0.9

1.1

- 0.11

3.0

+ 0.21

l.8:O.lO

3.2 + 0.30 1.4 1; 0.3li 2.3 = 0 20 1.8 2 11.16 1.6 0.11

1.6 I 0 . 1 6

1

ACIH

Conrroi

18 020

1 8 2 0 I6

35202'

19+011

2.4 z 0

18"

'; Failure

m

lmv)

cL>n:rol

AC.1 H

Conrrol

ACTH

Cmrrnl

7!04/' 5

0.97 * 0 06

112

I 04

21

' i 2 2 ~ 5

1.51

L

0.18

0.75 t 0.02 I 13 L 0.04

2.3

1.68

12.'

7/29/75 7i30!7 5

I (I

116

0 6X 2 0.09

1.1

(1 89

65 35 5

1DO

n.x5

0.119

1.06 + O.0h

I'

16

18

32

I1 2

14.1

lhk

1.02

8/07/75 5

1.37 t 0.00 1.83 + 0.11 1.02 + 0.0') 1 I 3 i 0 20 0 83 2 0.0~9

5

10i28/75

I i t 3 I!'> 11/0'!'5

0 84 ,Mean

3

SE

"ACTH cancenrrarion. 10.'

0.75 F

0.119

14

1.1

1.2

0 11

24

22 1.5

+

=

X

10

I45

1'8

0.95 i 0 09

164

131

0.63 f 0.06

1.21

1.11

I75

19 1

11

19

0 Oh

1.i8

0.9 2 I).r)6,

?

* 0 IX

-2

I51

I31

211

2 0'1

1.72 i 0 I 1

I 44 t 0 . 1 1 ~

6.5)

2.3

2 11 18.6 1 1.4

27.1

L

2.6"

rnol per lirer. Values are mean i srandard error. f o r p c 0 003. fur p < 0 01, and

Mean value5 which difter sigmhcanrly from conrrols are denoted

RhlP

41.8

1.73

0 67 1.21

0.118

28 14

0.91i O.!l

i- 0.0'1 1.61 + 0.22 2

ACTH

resring membrane potenrial; f - mepp frequent) imepps per second), q

=

"

forp < 0 05

quanrum size, V = avcragc epp amplitude at 30 H L stirnulatinn. m = quantum cnunr during 30 Hz srimulaoon

Table 2. Results Obtained with A C l ' H 4-10 on 160 Ed-Phtes" f ( I isecr

M I ' (mv) Date

ACTH 4-10

Control

ll/l'/'5

75 5

+ 0.86

1.9

11: I W 5

79.2 z o 80

81.8 2 1 0 8

1.7

11/25/75

7-.ll f 0.86

78 5

11/28/75 12: 1l P 5

8.2 8 '3.2

l 2 I 18:'s

77

12/iO/75

802?108

i!i)-!76

81.3 f 0.64

Mean 2

SF

9

78.4

2

o 86

-6.5

f

?

0 86

79 9 5

+

0 86

7-1 3 z 76.9 t 78 3 76.2 3

+ 0.6

+

+ 0.64

'7

x

I 08 0 64 1 30

0.86

I 08 1 OF1

i 11.44

3

n.ii

16

2 18 f 0.20

I 9 5 + 0.20 1.29 f 0.16

2.62 L 0.211 2.76 + 0.22

I 36

0 oil

2.03

0.13

2 99 z 0 24

1.24

~

?

I 5 1 z 0.16

2 29

1 5 1 1 0.06

+ 0.1) f

0.1 1 "

C.c.,Xi)illustrated in Figure 4 are not significant. The percentage of failures was not increased in the presence of the fragment, although a rise had been observed with the whole molecule. T h e mean failure rate in the presence of mol per liter of A C T H 4-10 turned out to be even lower than in the control solution; however, this decrease (19%) was not significant (see Fig 4). Like whole ACTH, the 4-10 fragment had no effect o n resting potential and mean mepp amplitude.

The only change observed was in the mean mepp frequency, which was increased by 50% (see Fig 4 , Table 2).

Discussion The major finding of our investigations was the direct and rapid effect of corticotropin on neuromuscular transmission, indicating that ACTH affects more than just its proper target organ, the adrenal correx. In the nonstimulated nerve-muscle preparation A C T H leads to a rise in mepp frequency, and during stimulation ACTH causes a decline in the epp quantum content as well as an increase in the failure rate, thus impairing acetylcholine ( ACh) release. Quantum size and the resting potential of the muscle fibers were not affected by ACTH. These findings together indicate that the site of action is presynaptic, since postsynaptic changes, such as decreased input resistance of the muscle fiber or decreased ACh sensitivity, should both change the quantum size. O n e hypothesis to explain our results is that A C T H might decrease the resting membrane potential of the presynaptic terminal. In adrenal cells A C T H does in fact depolarize the membrane [ 2 8 ] . Probably the hormone combines with a receptor in the membrane, resulting in a permeability change for one or several ionic species [29].Depolarization of the nerve terminal would increase mepp frequency and decrease ACh release during activity 1301, although we are not sure to what extent this mechanism applies at the Mg++ concentration used in our experiments [30]. A C T H also may increase the number of presynaptic nerve blocks that occur at a stimulus frequency of 30 Hz. Nerve blocks are influenced by a number of factors, one of which is depolarization of the nerve [31]. Presynaptic nerve block would increase the number of failures and decrease the average epp amplitude, but it should not influence mepp frequency. Interestingly, other polypeptides have been shown also to interfere with neuromuscular transmission; eg, some antibiotics produce reversible myasthenic syndromes [32, 331. A reduction in quantum content has

Birnberger, Rudel, and Struppler: ACTH and Neuromuscular Transmission 273

mv

1.6r 1.

1. 1 0.

0. 0.

0.

been shown to underlie the myasthenic syndrome produced by colisrin [ 2 5 ] .Thymopoietin, still being discussed as to whether it causes myasthenia, is also a polypeptide. Small doses of thymopoietin produce a myasthenic syndrome in mice in vivo [34].In patients with myasthenic syndrome induced by antibiotics, intravenous injection of calcium produces rapid clinical improvement [321. This observation indicates another possible basis for t h e ACTH effect: experiments with squid axons have shown that nerve tissue takes up calcium in proportion to the ambient Ca++ concentration 1351. If A C T H interfered with presynaptic Cat+ channels, the e p p would be decreased, and mepp frequency might rise because of calcium accumulating outside the terminal [36]. During long-lasting (2- to 30-minute) supramaxima1 stimulation of nerve-muscle preparations, ACTH has been shown to reduce the decline of muscle action potential and of muscular force, thus antagonizing fatigue [17, 181. O u r results might explain these findings. A C T H could reduce ACh release enough to delay exhaustion but not enough to prevent action potential generation in a nonblocked synapse. Another possibility is that polypeptide hormones, besides their direct effects on membrane properties, also have indirect intracellular effects via secondary messengers like Ca++or cyclic adenosine monophosphate [301. Such an intracellular effect could be increased ACh production, which would also explain the observed long-term improvement in neuromuscular transmission in a nonblocked synapse [ 171. The experiments with A C T H 4-10 were started with the hypothesis that the fragment acts similarly to the whole molecule. This assumption was based o n a study in which ACTH 4-10 showed the same antifatigue effect as A C T H [20]. T h e effects of the fragment were similar to those of the whole molecule only insofar as the mepp frequency of nonstimulated endplates was increased. However, contrary to whole ACTH, the fragment had no depressing action o n ACh release during repetitive stimulation. Neither epp amplitude and quantum content nor percentage of transmission failures was significantly altered in the presence of ACTH 4- 10. These findings suggest that 274 Annals of Neurology Vol 1 No 3 March 1977

F i g 3 , Effect of ACTH 4-10 r w d per liter) (solid columns) on epp amplitude driving S O H t .stimuiatIoti. E a c h pair of c o f ~ i m n srepyesetits t h e ui,erdge of at /east 20 cells from one prepmution.

150%

100%

50%

RMP

f

W

Y

rn

XF

4.Meatr remlts o f d l experinrerzts Ti’ithACTN 4-10 (solid columns) Coiitvolr (striped columns) taken as I 0 O C r iAbbrei Zattom same a J in Figure 2 I

Fig

the fragment has little effect on nerve terminals, just as it fails to affect adrenal cortical cells. This conclusion is corroborated by clinical experiments (to be reported elsewhere) in which patients receiving ACTH 4-10 showed n o change in muscle strength. The increase of mepp frequency by A C T H 4-10 in the absence of other influences on ACh release is difficult to explain. Possibly it indicates a dosedependent phenomenon rather than any basic difference in action of the two polypeptide molecules. This problem might be solved by studying the effects of ACTH 4-10 over a wide range of concentrations. In our experiments neuromuscular transmission was partially blocked by high Mg++.This may be an appropriate model for a myasthenic end-plate because in myasthenia gravis, neuromuscular transmission is characterized by a decreased safety factor [ 3 7 ] .ACTH concentrations of l o p 7 and l o p x mol per liter were chosen because similar blood levels may be reached during therapeutic application [38]. It is well known that the clinical condition of patients frequently deteriorates during A C T H therapy and improves only

after termination of ACTH treatment [6]. This could be based on impaired ACh release in the presence of ACTH. The basis for the therapeutic effect of ACTH therefore does not seem to be its direct effect on neuromuscular transmission. More likely it acts through the release of cortisone, which has been demonstrated to reduce antibody production [39].In light of this observation one should consider whether or not cortisone therapy is superior to ACTH treatment in myasthenic patients, because the same therapeutic goals can be reached with a lesser degree of initial clinical deterioration. Christiane Schlegel provided technical assistance; Lotte Bauer gave secretarial help; and Doris Burg, MD, Josef Dudel, MD, and Hanns Hart, PhD, reviewed the manuscript. A gift of A C T H 4-10 by Organon ( 0 s )is acknowledged.

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17. Torda C, W'olff HG: Effect of pituitary hormones, cortisone and adrenalectomy o n some aspects of neuromuscular function and acetylcholine synthesis. Am J Physiol 169: 140-149, 1952 18. Strand FL, Stoboy H, Cayrr A : A possible direct action o f A C T H on nerve and muscle. Neuroendocrinology 13:l-20, 1973174 19. de Wied D: Effects of peptide hormones on behavior, in Gauong WF (ed): Frontiers in Neuroendocrinology. New York, Oxford University Press, 1969 20. Strand FL, Cayer A: A modulatory effect ofpituitary polypeptides o n peripheral nerve and muscle. Brain 42:187-194, 1975 21. Greven H M , de Wied D: The active sequence in the ACTH molecule responsible for inhibition of the extinction of conditioned avoidance behavior in rats. Eur J Pharmacol2:14-16, 1967 22. Birnberger KL, Struppler A: Einfluss von A C T H und Cortison auf die neuromuskulire Uebercragung, in Hertel G, Mertens H G , Ricker K, e t a1 (eds):Myastheniagravis. Stutcgart, Thieme Verlag, 1977 23. Liley AW: The quanta1 components of the mammalian endplate potential. J Physiol (Lond) 135:571-587, 1956 24. Karz B, Thesleff S: O n the factors which determine the amplitude of the 'miniature endplate potential.' J Physiol (Lond) 137:267-278, 1957 25. McQuillen MP, Engbaek L Mechanism of colistin induced neuromuscular depression. Arch Neurol 32:235-238, 1975 26. Boyd A, Martin A R The endplate potentiaj in mammalian muscles. J Physiol (Lond) 132:74-91, 1956 27. Kuba K: Effects ofcarecholamines on the neuromuscular junctionin the rat diaphragm. J Physiol(Lond) 21 1:55 1-570,1970 28. Matthew EK, Saffran M: Ionic dependence of adrenal steroidogenesis and ACTH-induced changes in the membrane potential of adrenocortical cells. J Physiol (Lond) 234:43-64, 1973 2 9 . Peterson O H : Cell permeability change: an important step in hormone action. Experientia 30:1105-1107, 1974 30. Hubbard JI. Willis WD: The effects ofdepolarization ofmotor nerve terminals upon the release of transmitter by nerve impulses. J Physiol (Lond) 194:381-405, 1968 31. Krnjevit K , Miledi R: Presynaptic failure of neuromuscular propagation in rats. J Physiol (Lond) 149:l-22, 1959 32. Pittinger CB,AdamsonR: Antibiotic blockade ofneuromuscular function. Annu Rev Pharmacol 12:169-184, 1972 33. McQuillen MP, Cantor MA, Rourke J R Myasthenic syndrome associated with antibiotics. Arch Neurol 18:402-415, 1968 34. Goldstein G. Schlesinger DH: Thymopoietin and myasthenia gravis: neostigmine-responsive neuromuscular block produced in mice by a synthetic peptide fragment of thymopoietin. Lancet 2:256-259, 1975 35. Baker PF, Hodgkin AL, Ridgway EB: Depolarization and calcium entry in squid giant axons. J Physiol (Lond) 218:709755,1971 36. Hubbard JI: The effect of calcium and magnesium on the spontaneous release of transmitter from mammalian motor nerve endings. J Physiol (Lond) 159:507-525, 1961 37. Elmqvist D, Hofman WW, Kugelberg J, e t al: An electroph ysiological investigation of neuromuscular transmission in myasthenia gravis. J Physiol (Lond) 174:417-434, 1064 38. Berson SA, Yalow RS: Radioimmunoassay of A C T H in plasma. J Clin Invest 47:2725-2751, 1968 39. Abramsky 0, Aharonov A, Teirelbaum D, et al: Myasthenia gravis and acetylcholine recepcor. Arch Neurol 32:684-687, 1975

B i r n b e r g e r , Rudel, a n d Sttuppler: ACTH a n d N e u r o m u s c u l a r T r a n s m i s s i o n

275