European Journal of Pharmacology, 33 (1975) 337--344 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
FURTHER EVIDENCE THAT EXTRINSIC ACETYLCHOLINE ACTS PREFERENTIALLY ON EXTRAJUNCTIONAL RECEPTORS IN THE CHICK BIVENTER CERVICIS MUSCLE C. CHIUNG CHANG and M. J A I SU
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China Received 4 February 1975, revised MS received 2 May 1975, accepted 27 May 1975
C.C. CHANG and M.J. SU, Further evidence that extrinsic acetylcholine acts preferentially on extrajunctional receptors in the chick biventer cervicis muscle, European J. Pharmacol. 33 (1975) 337--344: The specificity of action of extrinsic acetylcholine on extrajunctional and junctional receptors in the chick biventer cervicis muscle was studied by determining its ability to protect the responses e v o k e d by acetylcholine and by tetanic nerve stimulation from the blockade by ~-bungarotoxin, an irreversible binding agent of acetylcholine receptors. At concentrations of 50--100 pg/ml, acetylcholine caused a desensitization to extrinsic acetylcholine but not to nerve stimulation and protected only the contractile response to extrinsic acetylcholine from the toxin blockade whereas neither the response to tetanic nerve stimulation nor the endplate potentials were protected. For the protection of the latter, higher concentrations of acetylcholine were needed. In the presence of physostigmine, a concentration of acetylcholine as low as 10 pg/ml protected the endplate potentials from the toxin blockade. By contrast, d-tubocurarine protected the tetanic contraction and the endplate potentials induced by nerve stimulation at a concentration which produced the same protection of acetylcholine-induced contraction as that produced by 50--100 pg/ml acetylcholine. These results indicate that in contrast to d-tubocurarine, extrinsic acetylcholine at low concentrations acts preferentially on the extrajunctional receptors in the absence of an anticholinesterase. Extrajunctional acetylcholine receptor Skeletal muscle
Extrinsic acetylcholine d-Tubocurarine
1. Introduction In physiological and pharmacological studies of neuromuscular transmission, it has generally been implied that the junctional receptors are similarly affected whether acetylcholine is released from motor nerve terminals or applied from an extrinsic source because d-tubocurafine antagonizes the responses elicited either by nerve stimulation or by extrinsic acetylcholine to the same extent (Del Castillo and Katz, 1957). It has been reported, however, that barbiturates and desipramine preferent~idlly block endplate depolarization and contraction elicited by extrinsic acetylcholine (Quilliam, 1955; Thesleff, 1956; Adams et al., 19'70; Chang and Tang, 1974). a-Bungarotoxin, a
~-Bungarotoxin
specific and irreversible binding agent of the acetylcholine receptors in skeletal muscle (Chang and Lee, 1963; Miledi and Potter, 1971; Barnard et al., 1971; Berg et al., 1972; Hartzell and Fambrough, 1972; Chang et al., 1973), was also found to cause a more rapid blockade of the action of extrinsic acetylcholine (Chang and Tang, 1974). Furthermore, after complete blockade of the responses to both intrinsic and extrinsic acetylcholine, only the response to extrinsic acetylcholine was restored on removal of this neurotoxin. These authors have proposed that extrinsic acetylcholine, particularly at low concentrations and in the absence of anticholinesterase, acts preferentially on extrajunctional receptors because of rapid hydrolysis by acetylcholinesterase in the synaptic cleft
338 in the chick biventer cervicis muscle. Arguments may be raised, however, that the difference observed in the blockade by a-bungarotoxin of the contractile responses to nerve stimulation and to extrinsic acetylcholine may be due to slow diffusion of the toxin into deeper layers of muscle fibres since different groups of muscle fibres may be involved in the responses to different modes of stimulation, i.e., superficial fibres for extrinsic acetylcholine (Lfillmann et al., 1974) and whole fibres for nerve stimulation. In the present communication, extrinsic acetylcholine was tested for its ability to specifically protect the junctional and extrajunctional acetylcholine receptors from the blockade by a-bungarotoxin. Its specificity of action was compared with that of d-tubocurarine. The results revealed that, whereas d-t~bocurarine protected both the responses to intrinsic and to extrinsic acetylcholine, low concentrations of acetylcholine did not protect the contractile as well as the endplate potential responses to nerve stimulation against the blockade by a-bungarotoxin b u t did protect the response to extrinsic acetylcholine.
2. Materials and methods 2.1. Recording o f contraction Experiments were performed on the biventer cervicis nerve--muscle preparation (Ginsborg and Warriner, 1960) isolated from 4--10 day old male white Leghorn chicks. The organ bath contained 20 ml modified Tyrode solution (composition in mM: NaCI 137, KC1 2.7, CaC12 2.7, MgC12 1.1, NaHCO~ 11.9, NaH2PO4 0.33 and glucose 11.2) at 37°C under oxygenation with 95% 02 + 5% CO2. The distal end of the biventer muscles was connected to a Grass force displacement transducer (FT.03) via a silver spring of compliance 1.0 g/cm. This kind of semi-isotonic recording was found to give results quantitatively different from those obtained by an isometric recording especially when marked shortening of muscle occurred. However, this procedure was necessary to avoid
c.c. CHANG, M.J. SU drastic stretching of the muscle during prolonged contracture which may result in damage of the preparation (Rang and Ritter, 1970). The resting tension on the muscle was 0.5 g. Single or train (100 Hz) electrical stimulation with supramaximal rectangular pulses of 0.5 msec width was applied to the nerve in the tendon. The response to extrinsic acetylcholine was evoked by adding acetylcholine for I min. 2.2. Recording o f endpla te po ten tials The biventer muscle was bathed in 27 ml modified T y r o d e solution containing 15.4-16.5 mM MgC12 and 2.7 mM CaC12 in order to prevent contraction of the muscle u p o n nerve stimulation. The temperature was 33--35°C. Endplate potentials were elicited every 2 sec by single supramaximal rectangular pulses during the period of observation, and recorded intracellularly with a glass microelectrode (Fatt and Katz, 1951) filled with 3 M KC1. The resistance of the microelectrodes was 10--20 MYZ. Since the amplitudes of endplate potentials at one endplate varied greatly because of reduction of the quantum number in the presence of high Mg 2÷ concentration (Del Castillo and Katz, 1954), 8 successive endplate potentials from each endplate were averaged on a signal averager (Atac 201, Nihon Koden Ltd.). 2.3. Chemicals a-Bungarotoxin was isolated from the venom of Bungarus multicinctus as described by Mebs et al. (1972). Acetylcholine chloride (Sigma Chem. Co.), physostigmine sulfate (Sigma Chem. Co) and d-tubocurarine chloride (Astawerke AG ) were used.
3. Results
3.1. Responses to extrinsic acetylcholine and nerve stimulation When the nerve was repetitively stimulated, the contraction height was maximal at 100 Hz,
E X T R I N S I C ACh R E C E P T O R
339
5rain
r'-------i
ACh 100
ACh 200
Fig. 1. E f f e c t of acetylcholine on the contractile response to nerve stimulation. Control twitch responses to single nerve stimulation are s h o w n on the left in each tracing. Top, middle and b o t t o m tracings show the effects of 100, 200 and 500 p g / m l acetyicholine, respectively. Nerve stimulation was reinstituted in the, presence of acetylcholine when the peak o f contracture was over.
and no increase in its amplitude was obtained by increasing the pulse frequency to 200 Hz. Addition of acetylcholine to the organ bath caused concentration dependent contractures. In the following experiments, the control response to nerve stimulation at 100 Hz was elicited for each preparation and was taken as 100% for comparison with the responses to acetylcholine.
3.2. Desensitization by extrinsic acetylcholine If the chick biventer cervicis was continuously incubated with 100/~g/ml of acetylcholine, a contracture of a b o u t 75% of the maximal amplitude was induced which then declined gradually within 15--20 min. A marked desensitization to extrinsic acetylcholine occurred as described by Rang and Ritter (1970). The response to single nerve stimulation during
incubation with acetylcholine, however, regained its original amplitude as the muscle relaxed (fig. 1). At higher concentrations of acetylcholine, the decline of the contracture to the baseline was slower and the twitch responses to nerve stimulation were also depressed (fig. 1). This result suggests that, in contrast to the receptor that responds to extrinsic acetylcholine, the junctional receptor was little affected by acetylcholine at concentrations lower than 100 pg/ml.
3.3. Protection of contractile responses by acety lcholine a-Bungarotoxin at 0.25 ~#g/ml completely blocked the responses to both nerve stimulation (100 Hz) and actylcholine (10--500 pg/ml) in 60 min. No recovery of the response to nerve stimulation was observed 60 min after
100
340
C.C. C H A N G , M.J. SU 100
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washout of the toxin, whereas a slight restoration of the response to acetylcholine was observed as previously reported (Chang and Tang, 1974). If the muscle was treated with a-bungarotoxin in the presence of acetylcholine ( 5 0 - - 5 0 0 pg/ml) for 60 min and then washed for another 60 min, various degrees of protection were observed (fig. 2). A comparison with fig. 3 shows that the response to nerve stimulation was not significantly protected when the concentration of acetylcholine fo~ protection was 100 pg/ml or lower. Protection became evident only at 200 pg/ml and increased as the concentration of acetylcholine increased. By contrast, the response to extrinsic acetylcholine was markedly protected already by 50 pg/ml of acetylcholine. These results clearly show that there is a discrepancy between the protection of responses to extrinsic acetylcholine and that of responses to nerve stimulation. Interestingly, the response to a low concentration of acetylcholine (10 pg/ml) was protected to the same extent independent of the acetylcholine concentration used for protection ( 5 0 - - 5 0 0 pg/ml). On the other hand, the pro-
2;o
%o-
Acetylcholine OJg/m[ )
Acety[choline (~cj/rn{) Fig. 2. Dose--response o f a c e t y l c h o l i n e after treatm e n t with ~-bungarotoxin in the presence of acetylcholine. Chick biventer cervicis muscles were incubated with e - b u n g a r o t o x i n (0.25 p g / m l ) for 60 min in the absence (~) or in the presence of acetylcholine, 100 pg/ml (o) or 500 p g / m l (A), and washed for another 60 min. (o): c o n t r o l responses before treatm e n t ; ("): treated with the toxin alone w i t h o u t wash. All responses were expressed as per cent of control response to tetanic nerve stimulation (100 Hz) for each preparation. Mean 2 S.E. n = 4--6.
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Fig. 3. P r o t e c t i o n by acetylcholine from the blockade by a-bungarotoxin. The same e x p e r i m e n t s as s h o w n in fig. 2. Abscissa: c o n c e n t r a t i o n of acetylcholine used for p r o t e c t i o n ; ordinate: percent protection, corrected for the s p o n t a n e o u s recovery during washing. (A): Response to tetanic nerve stimulation, (o): response to 10 /2g/ml acetylcholine, ($): response to 500/.tg/ml acetylcholine. Mean ± S.E. n = 4--6.
tection of the response to a high concentration of acetylcholine (500 pg/ml), the effect of which involved deeper muscle layers, appeared to be dependent on the concentration of acetylcholine used for protection (fig. 3). 3.4. Protection of contractile responses by d-tu bocurarine
The protection afforded by various concen100
80
--------------4
~ 60 ~
40
-
13_
20 0 1'0
20
50
d-Tu bocurarine (uglml)
Fig. 4. P r o t e c t i o n by d-tubocurarine from the blockade by ~-bungarotoxin. Similar e x p e r i m e n t s as in figs. 2 and 3 using d-tubocurarine as protecting agent. Abscissa: c o n c e n t r a t i o n of d-tubocurarine; ordinate: the p e r c e n t p r o t e c t i o n , c o r r e c t e d for spontaneous recovery during washing. (A): Response to tetanic nerve stimulation, (o): response to 10 p g / m l acetylcholine, ($): response to 500 p g / m l acetylcholine.
EXTRINSIC ACh RECEPTOR
341
trations of d-tubocurarine against the blocking action of ~-bungarotoxin is illustrated in fig. 4. In contrast to the protective effect of acetylcholine, d-tubocurarine protected the response to nerve stimulation even better than that to acetylcholine. By increasing the d-tubocurarine concentration to 20/~g/ml, the protection of the response to nerve stimulation was markedly increased although the protection of the acetylcholine response was not more than that afforded by 50--100 pg/ml acetylcholine. It is thus apparent that, unlike the protection by acetylcholine, d-tubocurarine protected both responses against the blockade by ~-bungarotoxin in a parallel way.
3.5. Protection o f endplate poten rials It may be argued that the difference in the protection afforded by acetylcholine and by d-tubocurarine might be due mainly to a poor penetration of acetylcholine into deeper muscle layers. Acetylcholine might thus protect only the receptors on the superficial muscle layers and consequently give a better apparent protection of the response to acetylcholine than to nerve stimulation which involves whole
muscle fibres. In order to clarify this point the protection of endplate potentials recorded from the superficial muscle fibres was investigated. In the control preparations, ~-bungarotoxin at 0.2 pg/ml was found to completely abolish the endplate potential in 25--35 min. No restoration of the potential occurred 3 0 - 6 0 min after washout of the toxin. In table 1, the protection afforded by 20 pg/ml d-tubocurarine and by 100 pg/ml acetylcholine is compared. Although the protection of the contractile response to 10 pg/ml acetylcholine by 100 pg/ml acetylcholine was somewhat greater than that by 20 pg/ml d-tubocurarine (figs. 3,4), the protection of endplate potentials by acetylcholine appeared to be much smaller than that afforded by d-tubocurarine (7 vs. 63%). If physostigmine (1 #g/ml)was present in the organ bath, acetylcholine protected the endplate potentials by 39% at 10 pg/ml. Since the endplate potentials were recorded only from the first or second outermost layers of the muscle fibres, the drugs employed should have easily reached the target sites by diffusion if there was no enzymatic destruction.
TABLE 1 Protection of endplate potentials from blockade by a-bungarotoxin. Tyrode solution contained 15.4--16.5 mM MgCI2 at 35°C. 3 preparations were used for each group of experiments. The chick muscle was treated with 0lbungarotoxin (0.2 pg/ml) for 35 min and then washed 4 times during 30 min. Endplate potentials were recorded before addition of protecting agents and the toxin and 30--60 rain after washout of these agents (except physostigmine which was present throughout). Mean (mV) + S.D. are shown, n indicates the number of endplates studied. 8 endplate potentials were averaged for each endplate. Protecting agent
Before (~-bungarotoxin
After ~-bungarotoxin
Resting potential
End-plate potential
Resting potential
End -plate potential
None
58.1 -+ 10.3
57.4 +
7.6
d-Tubocurarine (20 pg/ml)
56.5+
9.2
59.2+
7.9
Acetylcholine (100 pg/ml)
55,2+
9.3
Acetylcholine (10 pg/ml) + physostigmine (1 pg/ml)
65.5 + 11.8
2.84 + 1.28 (n = 40) 2.33+1.40 (n = 48) 2.17+0.91 (n -- 31) 4.47 + 2.85 (n -- 21)
0 (n = 32) 1.47+1.10 (n = 51) 0.16+-0.26 (n = 49) 1.73 -+ 1.15 (n = 32)
55.3+11.0 58.8 + 10.5
Percent protection
63 7 39
342 4. Discussion Several lines of evidence in the present experiments indicate that in the baby chick biventer cervicis muscle, acetylcholine at concentrations up to 100 #g/ml, affects only the extrajunctional receptors while leaving the junctional receptors unaffected. For example, on prolonged exposure to acetylcholine at 100 pg/ml or less, the response to nerve stimulation was not depressed in spite of desensitization to extrinsic acetylcholine. Furthermore, the irreversible blocking effect of ~-bungarotoxin on the contractile response to extrinsic acetylcholine was protected in the presence of low concentration of acetylcholine, whereas the response to intrinsic acetylcholine released by nerve stimulation was n o t protected. For the protection of the response to intrinsic acetylcholine, higher concentrations of acetylcholine were needed. In contrast to the protection b y acetylcholine, the protection afforded by dtubocurarine against the blockade b y a-bungarotoxin was shown to run parallel for the responses to extrinsic and to intrinsic acetylcholine, d-Tubocurarine by itself has been shown to depress both responses equally well (Del Castillo and Katz, 1957; Chang and Tang, 1974). It is unlikely that slow diffusion of the polypeptide neurotoxin contributed to the difference b e t w e e n the protecting effects of acetylcholine and d-tubocurarine, since sufficient time was allowed for the toxin to act on all muscle fibres. It might still be argued that this difference was entirely due to inability of low concentrations of acetylcholine to penetrate into the deeper muscle layers. Thus only the superficial muscle fibres, on which extrinsic acetylcholine acts to induce contracture (L[illmann et al., 1974), would be protected while the blockade of the response to nerve stimulation, which involves all muscle fibres, would remain unprotected. This seems unlikely since acetylcholine at 100 pg/ml induced a contracture of an amplitude about 75% of the maximum, indicating that n o t just the superficial muscle fibres were involved. Therefore, the inability of acetylcholine to protect the tetanic
C.C. CHANG, M.J. SU contraction by nerve stimulation cannot be accounted for by poor penetration. The experiments with endplate potentials recorded from superficial muscle fibres provide evidence on this point. In contrast to d-tubocurarine which gave effective protection of the endplate potentials from blockade by a-bungarotoxin, acetylcholine up to 100 pg/ml, which protected the contractile response to extrinsic acetylcholine better than d-tubocurafine, did not significantly protect the endplate potentials. This result clearly indicates that even the junctional receptors of superficial muscle fibres were not affected by 100 pg/ml acetylcholine. The relative insensitivity of junctional acetylcholine receptors to extrinsic acetylcholine in comparison with extrajunctional ones is apparently n o t due to their low affinity b u t rather to rapid hydrolysis by acetylcholinesterase of extrinsic acetylcholine during diffusion into the synaptic cleft. Indeed, in the presence of physostigmine, endplate potentials were protected b y a much lower concentration of acetylcholine. Since it is not a substrate for the enzyme, d-tubocurarine appeared to affect both types of receptors equally well. The chick biventer cervicis muscle contains both focally and multi-innervated muscles (Ginsborg, 1960). In view of its widespread senstivity to acetylcholine (Fedde, 1969), there may be a substantial a m o u n t of extrajunctional acetylcholine receptors along the whole length of the multi-innervated muscle fibre. By labelling with tritiated a-bungarotoxin, we have found that about one half of the total receptors can be protected by acetylcholine (Chang et al., 1975). Extrinsic acetylcholine may be acting preferentially on these extrajunctional receptors. The question may then be raised whether junctional or extrajunctional receptors are involved when extrinsic acetylcholine is focally applied to an endplate b y iontophoresis in focally innervated muscles. In the latter muscle, the density of extrajunctional receptors is quite low at some distance from the endplate (Hartzell and Fambrough, 1972). The sensitivity to acetylcholine at the endplate is also much higher than at non-end-
EXTRINSIC ACh RECEPTOR
plate sites. The efficacy of intrinsic acetylcholine and of extrinsic acetylcholine focally applied to the endplate in inducing endplate depolarization was compared by Albuquerque et al. (1974). They found that the extrinsic acetylcholine has a 10 times lower efficacy in a diaphragm muscle. This result is in support of the present view that extrinsic acetylcholine may not reach the junctional receptors to a significant extent. It remains to be elucidated whether the evoked potential induced by iontophoretic acetylcholine results mainly from extrajunctional receptors surrounding the endplate rather than from an action on the synaptic junctional receptors. If this were the case, the low efficacy of extrinsic acetylcholine might be explained on the basis of a low density of extrajunctional receptors. The preferential block by barbiturates of the endplate depolarization caused by iontophoretic acetylcholine (Thesleff, 1956; Adams et al., 1970) is in support of the above inference that even the extrinsic acetylcholine focally applied on the endplate affects only the extrajunctional receptors. Despite the much higher density of acetylcholine receptors at endplates (Hartzell and Fambrough, 1972), the even distribution of acetylcholine sensitivity along the length of muscle fibres in denervated muscles (Miledi, 1960; and many others) can be accounted for on the same basis.
Acknowledgement The authors wish to thank prof. E. BrochmannHanssen for helpful discussion during the revision of the manuscript.
References Adams, P.R., H.C. Cash and J.P. Quilliam, 1970, Extrinsic and intrinsic acetylcholine and barbiturate effects on frog skeletal muscle, Brit. J. Pharmacol. 40, 552. Albuquerque, E.X., E.A. Barnard, C.W. Porter and J.E. Warnick, 1974, The density of acetylcholine receptors and their sensitivity in the postsynaptic membrane of muscle endplates, Proc. Nat. Acad. Sci. U.S.A. 71, 2818.
343 Barnard, E.A., J. Wieckowski and T.H. Chiu, 1971, Cholinergic receptor molecules and cholinesterase molecules at mouse skeletal muscle junctions, Nature 234, 207. Berg, D.K., R.B. Kelly, P.B. Sargent, P. William and Z.W. Hall, 1972, Binding of ~-bungarotoxin to acetylcholine receptors in mammalian muscle, Proc. Nat. Acad. Sci. U.S.A. 69, 147. Del Castillo, J. and B. Katz, 1954, The effect of magnesium on the activity of m o t o r nerve endings, J. Physiol. (London) 124, 553. Del Castillo, J. and B. Katz, 1957, The identity of intrinsic and extrinsic acetylcholine receptors in the m o t o r end-plate, Proc. Roy. Soc. B146, 357. Chang, C.C., T.F. Chen and S.T. Chuang, 1973, N,ODi- and N,N,O-tri-[ 3 H ] -acetyl ~-bungarotoxins as specific labeling agents of cholinergic receptors, Brit. J. Pharmacoh 47, 147. Chang, C.C. and C.Y. Lee, 1963, Isolation of neurotoxins from the venom o f Bungarus multicinctus and their modes of neuromuscular blocking action, Arch. Intern. Pharmacodyn. 144, 241. Chang, C.C., M.J. Su and M.C. Lee, 1975, A quantification of acetylcholine receptors of the chick biventer cervicis muscle, J. Pharm. Pharmacol. 27, 454. Chang, C.C. and S.S. Tang, 1974, Differentiation between intrinsic and extrinsic acetylcholine receptors of the chick biventer cervicis muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 282, 379. Fatt, P. and B. Katz, 1951, An analysis of the endplate potential recorded with an intra-cellular electrode, J. Physiol. (London) 115,320. Fedde, M.R., 1969, Electrical properties and acetylcholine sensitivity of singly and multiply innervated avian muscle fibers, J. Gen. Physiol. 53, 624. Ginsborg, B.L., 1960, Spontaneous activity in muscle fibres of the chick, J. Physiol. (London) 150, 707. Ginsborg, B.L. and J. Warriner, 1960, The isolated chick biventer eervicis nerve--muscle preparation, Brit. J. Pharmacol. 15, 410. Hartzell, H.C. and D.M. Fambrough, 1972, Acetylcholine receptors, distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity, J. Gen. Physiol. 60, 248. L~llmann, H., J. Preuner and H. Schaube, 1974, A kinetic approach for an interpretation of the acetylcholine--d-tubocurarine interaction on chronically denervated skeletal muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 281,415. Mebs, D., K. Narita, S. Iwanaga, Y. Samejima and C.Y. Lee, 1972, Purification, properties and amino acid sequence of a-bungarotoxin from the venom of Bungarus multicinctus, Hoppe--Seyler's Z. Physiol. Chem. 353, 243. Miledi, R., 1960, The acetylcholine sensitivity of
344 frog muscle fibres after complete or partial denervation, J. Physiol. (London) 151, 1. Miledi, R. and L.T. Potter, 1971, Acetylcholine receptors in muscle fibres, Nature 233,599. Quilliam, J.P., 1955, The action of hypnotic drugs on frog skeletal muscle, Brit. J. Pharmacol. 10, 133.
C.C. CHANG, M.J. SU Rang, H.P. and J.M. Ritter, 1970, On the mechanisms of desensitization at cholinergic receptors, Mol. Pharmacol. 6, 357. Thesleff, S., 1956, The effect of anesthetic agents on skeletal muscle membrane, Acta Physiol. Scand. 37,335.
FURTHER EVIDENCE THAT EXTRINSIC ACETYLCHOLINE ACTS PREFERENTIALLY ON EXTRAJUNCTIONAL RECEPTORS IN THE CHICK BIVENTER CERVICIS MUSCLE C. CHIUNG CHANG and M. J A I SU
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China Received 4 February 1975, revised MS received 2 May 1975, accepted 27 May 1975
C.C. CHANG and M.J. SU, Further evidence that extrinsic acetylcholine acts preferentially on extrajunctional receptors in the chick biventer cervicis muscle, European J. Pharmacol. 33 (1975) 337--344: The specificity of action of extrinsic acetylcholine on extrajunctional and junctional receptors in the chick biventer cervicis muscle was studied by determining its ability to protect the responses e v o k e d by acetylcholine and by tetanic nerve stimulation from the blockade by ~-bungarotoxin, an irreversible binding agent of acetylcholine receptors. At concentrations of 50--100 pg/ml, acetylcholine caused a desensitization to extrinsic acetylcholine but not to nerve stimulation and protected only the contractile response to extrinsic acetylcholine from the toxin blockade whereas neither the response to tetanic nerve stimulation nor the endplate potentials were protected. For the protection of the latter, higher concentrations of acetylcholine were needed. In the presence of physostigmine, a concentration of acetylcholine as low as 10 pg/ml protected the endplate potentials from the toxin blockade. By contrast, d-tubocurarine protected the tetanic contraction and the endplate potentials induced by nerve stimulation at a concentration which produced the same protection of acetylcholine-induced contraction as that produced by 50--100 pg/ml acetylcholine. These results indicate that in contrast to d-tubocurarine, extrinsic acetylcholine at low concentrations acts preferentially on the extrajunctional receptors in the absence of an anticholinesterase. Extrajunctional acetylcholine receptor Skeletal muscle
Extrinsic acetylcholine d-Tubocurarine
1. Introduction In physiological and pharmacological studies of neuromuscular transmission, it has generally been implied that the junctional receptors are similarly affected whether acetylcholine is released from motor nerve terminals or applied from an extrinsic source because d-tubocurafine antagonizes the responses elicited either by nerve stimulation or by extrinsic acetylcholine to the same extent (Del Castillo and Katz, 1957). It has been reported, however, that barbiturates and desipramine preferent~idlly block endplate depolarization and contraction elicited by extrinsic acetylcholine (Quilliam, 1955; Thesleff, 1956; Adams et al., 19'70; Chang and Tang, 1974). a-Bungarotoxin, a
~-Bungarotoxin
specific and irreversible binding agent of the acetylcholine receptors in skeletal muscle (Chang and Lee, 1963; Miledi and Potter, 1971; Barnard et al., 1971; Berg et al., 1972; Hartzell and Fambrough, 1972; Chang et al., 1973), was also found to cause a more rapid blockade of the action of extrinsic acetylcholine (Chang and Tang, 1974). Furthermore, after complete blockade of the responses to both intrinsic and extrinsic acetylcholine, only the response to extrinsic acetylcholine was restored on removal of this neurotoxin. These authors have proposed that extrinsic acetylcholine, particularly at low concentrations and in the absence of anticholinesterase, acts preferentially on extrajunctional receptors because of rapid hydrolysis by acetylcholinesterase in the synaptic cleft
338 in the chick biventer cervicis muscle. Arguments may be raised, however, that the difference observed in the blockade by a-bungarotoxin of the contractile responses to nerve stimulation and to extrinsic acetylcholine may be due to slow diffusion of the toxin into deeper layers of muscle fibres since different groups of muscle fibres may be involved in the responses to different modes of stimulation, i.e., superficial fibres for extrinsic acetylcholine (Lfillmann et al., 1974) and whole fibres for nerve stimulation. In the present communication, extrinsic acetylcholine was tested for its ability to specifically protect the junctional and extrajunctional acetylcholine receptors from the blockade by a-bungarotoxin. Its specificity of action was compared with that of d-tubocurarine. The results revealed that, whereas d-t~bocurarine protected both the responses to intrinsic and to extrinsic acetylcholine, low concentrations of acetylcholine did not protect the contractile as well as the endplate potential responses to nerve stimulation against the blockade by a-bungarotoxin b u t did protect the response to extrinsic acetylcholine.
2. Materials and methods 2.1. Recording o f contraction Experiments were performed on the biventer cervicis nerve--muscle preparation (Ginsborg and Warriner, 1960) isolated from 4--10 day old male white Leghorn chicks. The organ bath contained 20 ml modified Tyrode solution (composition in mM: NaCI 137, KC1 2.7, CaC12 2.7, MgC12 1.1, NaHCO~ 11.9, NaH2PO4 0.33 and glucose 11.2) at 37°C under oxygenation with 95% 02 + 5% CO2. The distal end of the biventer muscles was connected to a Grass force displacement transducer (FT.03) via a silver spring of compliance 1.0 g/cm. This kind of semi-isotonic recording was found to give results quantitatively different from those obtained by an isometric recording especially when marked shortening of muscle occurred. However, this procedure was necessary to avoid
c.c. CHANG, M.J. SU drastic stretching of the muscle during prolonged contracture which may result in damage of the preparation (Rang and Ritter, 1970). The resting tension on the muscle was 0.5 g. Single or train (100 Hz) electrical stimulation with supramaximal rectangular pulses of 0.5 msec width was applied to the nerve in the tendon. The response to extrinsic acetylcholine was evoked by adding acetylcholine for I min. 2.2. Recording o f endpla te po ten tials The biventer muscle was bathed in 27 ml modified T y r o d e solution containing 15.4-16.5 mM MgC12 and 2.7 mM CaC12 in order to prevent contraction of the muscle u p o n nerve stimulation. The temperature was 33--35°C. Endplate potentials were elicited every 2 sec by single supramaximal rectangular pulses during the period of observation, and recorded intracellularly with a glass microelectrode (Fatt and Katz, 1951) filled with 3 M KC1. The resistance of the microelectrodes was 10--20 MYZ. Since the amplitudes of endplate potentials at one endplate varied greatly because of reduction of the quantum number in the presence of high Mg 2÷ concentration (Del Castillo and Katz, 1954), 8 successive endplate potentials from each endplate were averaged on a signal averager (Atac 201, Nihon Koden Ltd.). 2.3. Chemicals a-Bungarotoxin was isolated from the venom of Bungarus multicinctus as described by Mebs et al. (1972). Acetylcholine chloride (Sigma Chem. Co.), physostigmine sulfate (Sigma Chem. Co) and d-tubocurarine chloride (Astawerke AG ) were used.
3. Results
3.1. Responses to extrinsic acetylcholine and nerve stimulation When the nerve was repetitively stimulated, the contraction height was maximal at 100 Hz,
E X T R I N S I C ACh R E C E P T O R
339
5rain
r'-------i
ACh 100
ACh 200
Fig. 1. E f f e c t of acetylcholine on the contractile response to nerve stimulation. Control twitch responses to single nerve stimulation are s h o w n on the left in each tracing. Top, middle and b o t t o m tracings show the effects of 100, 200 and 500 p g / m l acetyicholine, respectively. Nerve stimulation was reinstituted in the, presence of acetylcholine when the peak o f contracture was over.
and no increase in its amplitude was obtained by increasing the pulse frequency to 200 Hz. Addition of acetylcholine to the organ bath caused concentration dependent contractures. In the following experiments, the control response to nerve stimulation at 100 Hz was elicited for each preparation and was taken as 100% for comparison with the responses to acetylcholine.
3.2. Desensitization by extrinsic acetylcholine If the chick biventer cervicis was continuously incubated with 100/~g/ml of acetylcholine, a contracture of a b o u t 75% of the maximal amplitude was induced which then declined gradually within 15--20 min. A marked desensitization to extrinsic acetylcholine occurred as described by Rang and Ritter (1970). The response to single nerve stimulation during
incubation with acetylcholine, however, regained its original amplitude as the muscle relaxed (fig. 1). At higher concentrations of acetylcholine, the decline of the contracture to the baseline was slower and the twitch responses to nerve stimulation were also depressed (fig. 1). This result suggests that, in contrast to the receptor that responds to extrinsic acetylcholine, the junctional receptor was little affected by acetylcholine at concentrations lower than 100 pg/ml.
3.3. Protection of contractile responses by acety lcholine a-Bungarotoxin at 0.25 ~#g/ml completely blocked the responses to both nerve stimulation (100 Hz) and actylcholine (10--500 pg/ml) in 60 min. No recovery of the response to nerve stimulation was observed 60 min after
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340
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washout of the toxin, whereas a slight restoration of the response to acetylcholine was observed as previously reported (Chang and Tang, 1974). If the muscle was treated with a-bungarotoxin in the presence of acetylcholine ( 5 0 - - 5 0 0 pg/ml) for 60 min and then washed for another 60 min, various degrees of protection were observed (fig. 2). A comparison with fig. 3 shows that the response to nerve stimulation was not significantly protected when the concentration of acetylcholine fo~ protection was 100 pg/ml or lower. Protection became evident only at 200 pg/ml and increased as the concentration of acetylcholine increased. By contrast, the response to extrinsic acetylcholine was markedly protected already by 50 pg/ml of acetylcholine. These results clearly show that there is a discrepancy between the protection of responses to extrinsic acetylcholine and that of responses to nerve stimulation. Interestingly, the response to a low concentration of acetylcholine (10 pg/ml) was protected to the same extent independent of the acetylcholine concentration used for protection ( 5 0 - - 5 0 0 pg/ml). On the other hand, the pro-
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Acetylcholine OJg/m[ )
Acety[choline (~cj/rn{) Fig. 2. Dose--response o f a c e t y l c h o l i n e after treatm e n t with ~-bungarotoxin in the presence of acetylcholine. Chick biventer cervicis muscles were incubated with e - b u n g a r o t o x i n (0.25 p g / m l ) for 60 min in the absence (~) or in the presence of acetylcholine, 100 pg/ml (o) or 500 p g / m l (A), and washed for another 60 min. (o): c o n t r o l responses before treatm e n t ; ("): treated with the toxin alone w i t h o u t wash. All responses were expressed as per cent of control response to tetanic nerve stimulation (100 Hz) for each preparation. Mean 2 S.E. n = 4--6.
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Fig. 3. P r o t e c t i o n by acetylcholine from the blockade by a-bungarotoxin. The same e x p e r i m e n t s as s h o w n in fig. 2. Abscissa: c o n c e n t r a t i o n of acetylcholine used for p r o t e c t i o n ; ordinate: percent protection, corrected for the s p o n t a n e o u s recovery during washing. (A): Response to tetanic nerve stimulation, (o): response to 10 /2g/ml acetylcholine, ($): response to 500/.tg/ml acetylcholine. Mean ± S.E. n = 4--6.
tection of the response to a high concentration of acetylcholine (500 pg/ml), the effect of which involved deeper muscle layers, appeared to be dependent on the concentration of acetylcholine used for protection (fig. 3). 3.4. Protection of contractile responses by d-tu bocurarine
The protection afforded by various concen100
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40
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20
50
d-Tu bocurarine (uglml)
Fig. 4. P r o t e c t i o n by d-tubocurarine from the blockade by ~-bungarotoxin. Similar e x p e r i m e n t s as in figs. 2 and 3 using d-tubocurarine as protecting agent. Abscissa: c o n c e n t r a t i o n of d-tubocurarine; ordinate: the p e r c e n t p r o t e c t i o n , c o r r e c t e d for spontaneous recovery during washing. (A): Response to tetanic nerve stimulation, (o): response to 10 p g / m l acetylcholine, ($): response to 500 p g / m l acetylcholine.
EXTRINSIC ACh RECEPTOR
341
trations of d-tubocurarine against the blocking action of ~-bungarotoxin is illustrated in fig. 4. In contrast to the protective effect of acetylcholine, d-tubocurarine protected the response to nerve stimulation even better than that to acetylcholine. By increasing the d-tubocurarine concentration to 20/~g/ml, the protection of the response to nerve stimulation was markedly increased although the protection of the acetylcholine response was not more than that afforded by 50--100 pg/ml acetylcholine. It is thus apparent that, unlike the protection by acetylcholine, d-tubocurarine protected both responses against the blockade by ~-bungarotoxin in a parallel way.
3.5. Protection o f endplate poten rials It may be argued that the difference in the protection afforded by acetylcholine and by d-tubocurarine might be due mainly to a poor penetration of acetylcholine into deeper muscle layers. Acetylcholine might thus protect only the receptors on the superficial muscle layers and consequently give a better apparent protection of the response to acetylcholine than to nerve stimulation which involves whole
muscle fibres. In order to clarify this point the protection of endplate potentials recorded from the superficial muscle fibres was investigated. In the control preparations, ~-bungarotoxin at 0.2 pg/ml was found to completely abolish the endplate potential in 25--35 min. No restoration of the potential occurred 3 0 - 6 0 min after washout of the toxin. In table 1, the protection afforded by 20 pg/ml d-tubocurarine and by 100 pg/ml acetylcholine is compared. Although the protection of the contractile response to 10 pg/ml acetylcholine by 100 pg/ml acetylcholine was somewhat greater than that by 20 pg/ml d-tubocurarine (figs. 3,4), the protection of endplate potentials by acetylcholine appeared to be much smaller than that afforded by d-tubocurarine (7 vs. 63%). If physostigmine (1 #g/ml)was present in the organ bath, acetylcholine protected the endplate potentials by 39% at 10 pg/ml. Since the endplate potentials were recorded only from the first or second outermost layers of the muscle fibres, the drugs employed should have easily reached the target sites by diffusion if there was no enzymatic destruction.
TABLE 1 Protection of endplate potentials from blockade by a-bungarotoxin. Tyrode solution contained 15.4--16.5 mM MgCI2 at 35°C. 3 preparations were used for each group of experiments. The chick muscle was treated with 0lbungarotoxin (0.2 pg/ml) for 35 min and then washed 4 times during 30 min. Endplate potentials were recorded before addition of protecting agents and the toxin and 30--60 rain after washout of these agents (except physostigmine which was present throughout). Mean (mV) + S.D. are shown, n indicates the number of endplates studied. 8 endplate potentials were averaged for each endplate. Protecting agent
Before (~-bungarotoxin
After ~-bungarotoxin
Resting potential
End-plate potential
Resting potential
End -plate potential
None
58.1 -+ 10.3
57.4 +
7.6
d-Tubocurarine (20 pg/ml)
56.5+
9.2
59.2+
7.9
Acetylcholine (100 pg/ml)
55,2+
9.3
Acetylcholine (10 pg/ml) + physostigmine (1 pg/ml)
65.5 + 11.8
2.84 + 1.28 (n = 40) 2.33+1.40 (n = 48) 2.17+0.91 (n -- 31) 4.47 + 2.85 (n -- 21)
0 (n = 32) 1.47+1.10 (n = 51) 0.16+-0.26 (n = 49) 1.73 -+ 1.15 (n = 32)
55.3+11.0 58.8 + 10.5
Percent protection
63 7 39
342 4. Discussion Several lines of evidence in the present experiments indicate that in the baby chick biventer cervicis muscle, acetylcholine at concentrations up to 100 #g/ml, affects only the extrajunctional receptors while leaving the junctional receptors unaffected. For example, on prolonged exposure to acetylcholine at 100 pg/ml or less, the response to nerve stimulation was not depressed in spite of desensitization to extrinsic acetylcholine. Furthermore, the irreversible blocking effect of ~-bungarotoxin on the contractile response to extrinsic acetylcholine was protected in the presence of low concentration of acetylcholine, whereas the response to intrinsic acetylcholine released by nerve stimulation was n o t protected. For the protection of the response to intrinsic acetylcholine, higher concentrations of acetylcholine were needed. In contrast to the protection b y acetylcholine, the protection afforded by dtubocurarine against the blockade b y a-bungarotoxin was shown to run parallel for the responses to extrinsic and to intrinsic acetylcholine, d-Tubocurarine by itself has been shown to depress both responses equally well (Del Castillo and Katz, 1957; Chang and Tang, 1974). It is unlikely that slow diffusion of the polypeptide neurotoxin contributed to the difference b e t w e e n the protecting effects of acetylcholine and d-tubocurarine, since sufficient time was allowed for the toxin to act on all muscle fibres. It might still be argued that this difference was entirely due to inability of low concentrations of acetylcholine to penetrate into the deeper muscle layers. Thus only the superficial muscle fibres, on which extrinsic acetylcholine acts to induce contracture (L[illmann et al., 1974), would be protected while the blockade of the response to nerve stimulation, which involves all muscle fibres, would remain unprotected. This seems unlikely since acetylcholine at 100 pg/ml induced a contracture of an amplitude about 75% of the maximum, indicating that n o t just the superficial muscle fibres were involved. Therefore, the inability of acetylcholine to protect the tetanic
C.C. CHANG, M.J. SU contraction by nerve stimulation cannot be accounted for by poor penetration. The experiments with endplate potentials recorded from superficial muscle fibres provide evidence on this point. In contrast to d-tubocurarine which gave effective protection of the endplate potentials from blockade by a-bungarotoxin, acetylcholine up to 100 pg/ml, which protected the contractile response to extrinsic acetylcholine better than d-tubocurafine, did not significantly protect the endplate potentials. This result clearly indicates that even the junctional receptors of superficial muscle fibres were not affected by 100 pg/ml acetylcholine. The relative insensitivity of junctional acetylcholine receptors to extrinsic acetylcholine in comparison with extrajunctional ones is apparently n o t due to their low affinity b u t rather to rapid hydrolysis by acetylcholinesterase of extrinsic acetylcholine during diffusion into the synaptic cleft. Indeed, in the presence of physostigmine, endplate potentials were protected b y a much lower concentration of acetylcholine. Since it is not a substrate for the enzyme, d-tubocurarine appeared to affect both types of receptors equally well. The chick biventer cervicis muscle contains both focally and multi-innervated muscles (Ginsborg, 1960). In view of its widespread senstivity to acetylcholine (Fedde, 1969), there may be a substantial a m o u n t of extrajunctional acetylcholine receptors along the whole length of the multi-innervated muscle fibre. By labelling with tritiated a-bungarotoxin, we have found that about one half of the total receptors can be protected by acetylcholine (Chang et al., 1975). Extrinsic acetylcholine may be acting preferentially on these extrajunctional receptors. The question may then be raised whether junctional or extrajunctional receptors are involved when extrinsic acetylcholine is focally applied to an endplate b y iontophoresis in focally innervated muscles. In the latter muscle, the density of extrajunctional receptors is quite low at some distance from the endplate (Hartzell and Fambrough, 1972). The sensitivity to acetylcholine at the endplate is also much higher than at non-end-
EXTRINSIC ACh RECEPTOR
plate sites. The efficacy of intrinsic acetylcholine and of extrinsic acetylcholine focally applied to the endplate in inducing endplate depolarization was compared by Albuquerque et al. (1974). They found that the extrinsic acetylcholine has a 10 times lower efficacy in a diaphragm muscle. This result is in support of the present view that extrinsic acetylcholine may not reach the junctional receptors to a significant extent. It remains to be elucidated whether the evoked potential induced by iontophoretic acetylcholine results mainly from extrajunctional receptors surrounding the endplate rather than from an action on the synaptic junctional receptors. If this were the case, the low efficacy of extrinsic acetylcholine might be explained on the basis of a low density of extrajunctional receptors. The preferential block by barbiturates of the endplate depolarization caused by iontophoretic acetylcholine (Thesleff, 1956; Adams et al., 1970) is in support of the above inference that even the extrinsic acetylcholine focally applied on the endplate affects only the extrajunctional receptors. Despite the much higher density of acetylcholine receptors at endplates (Hartzell and Fambrough, 1972), the even distribution of acetylcholine sensitivity along the length of muscle fibres in denervated muscles (Miledi, 1960; and many others) can be accounted for on the same basis.
Acknowledgement The authors wish to thank prof. E. BrochmannHanssen for helpful discussion during the revision of the manuscript.
References Adams, P.R., H.C. Cash and J.P. Quilliam, 1970, Extrinsic and intrinsic acetylcholine and barbiturate effects on frog skeletal muscle, Brit. J. Pharmacol. 40, 552. Albuquerque, E.X., E.A. Barnard, C.W. Porter and J.E. Warnick, 1974, The density of acetylcholine receptors and their sensitivity in the postsynaptic membrane of muscle endplates, Proc. Nat. Acad. Sci. U.S.A. 71, 2818.
343 Barnard, E.A., J. Wieckowski and T.H. Chiu, 1971, Cholinergic receptor molecules and cholinesterase molecules at mouse skeletal muscle junctions, Nature 234, 207. Berg, D.K., R.B. Kelly, P.B. Sargent, P. William and Z.W. Hall, 1972, Binding of ~-bungarotoxin to acetylcholine receptors in mammalian muscle, Proc. Nat. Acad. Sci. U.S.A. 69, 147. Del Castillo, J. and B. Katz, 1954, The effect of magnesium on the activity of m o t o r nerve endings, J. Physiol. (London) 124, 553. Del Castillo, J. and B. Katz, 1957, The identity of intrinsic and extrinsic acetylcholine receptors in the m o t o r end-plate, Proc. Roy. Soc. B146, 357. Chang, C.C., T.F. Chen and S.T. Chuang, 1973, N,ODi- and N,N,O-tri-[ 3 H ] -acetyl ~-bungarotoxins as specific labeling agents of cholinergic receptors, Brit. J. Pharmacoh 47, 147. Chang, C.C. and C.Y. Lee, 1963, Isolation of neurotoxins from the venom o f Bungarus multicinctus and their modes of neuromuscular blocking action, Arch. Intern. Pharmacodyn. 144, 241. Chang, C.C., M.J. Su and M.C. Lee, 1975, A quantification of acetylcholine receptors of the chick biventer cervicis muscle, J. Pharm. Pharmacol. 27, 454. Chang, C.C. and S.S. Tang, 1974, Differentiation between intrinsic and extrinsic acetylcholine receptors of the chick biventer cervicis muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 282, 379. Fatt, P. and B. Katz, 1951, An analysis of the endplate potential recorded with an intra-cellular electrode, J. Physiol. (London) 115,320. Fedde, M.R., 1969, Electrical properties and acetylcholine sensitivity of singly and multiply innervated avian muscle fibers, J. Gen. Physiol. 53, 624. Ginsborg, B.L., 1960, Spontaneous activity in muscle fibres of the chick, J. Physiol. (London) 150, 707. Ginsborg, B.L. and J. Warriner, 1960, The isolated chick biventer eervicis nerve--muscle preparation, Brit. J. Pharmacol. 15, 410. Hartzell, H.C. and D.M. Fambrough, 1972, Acetylcholine receptors, distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity, J. Gen. Physiol. 60, 248. L~llmann, H., J. Preuner and H. Schaube, 1974, A kinetic approach for an interpretation of the acetylcholine--d-tubocurarine interaction on chronically denervated skeletal muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 281,415. Mebs, D., K. Narita, S. Iwanaga, Y. Samejima and C.Y. Lee, 1972, Purification, properties and amino acid sequence of a-bungarotoxin from the venom of Bungarus multicinctus, Hoppe--Seyler's Z. Physiol. Chem. 353, 243. Miledi, R., 1960, The acetylcholine sensitivity of
344 frog muscle fibres after complete or partial denervation, J. Physiol. (London) 151, 1. Miledi, R. and L.T. Potter, 1971, Acetylcholine receptors in muscle fibres, Nature 233,599. Quilliam, J.P., 1955, The action of hypnotic drugs on frog skeletal muscle, Brit. J. Pharmacol. 10, 133.
C.C. CHANG, M.J. SU Rang, H.P. and J.M. Ritter, 1970, On the mechanisms of desensitization at cholinergic receptors, Mol. Pharmacol. 6, 357. Thesleff, S., 1956, The effect of anesthetic agents on skeletal muscle membrane, Acta Physiol. Scand. 37,335.
