91
J. Physiol. (1979), 294, pp. 91-103 With 14 text-figure. Printed in Great Britain
INHIBITORY EFFECTS OF CHOLINERGIC AGENTS ON THE RELEASE OF TRANSMITTER AT THE FROG NEUROMUSCULAR JUNCTION
By C. J. DUNCAN AND S. J. PUBLICOVER From the Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX
(Received 11 January 1979) SUMMARY
1. The cholinesterase inhibitors neostigmine, edrophonium and eserine (7 x 10-7 M) reduced m.e.p.p. frequency by some 50 % at the frog neuromuscular junction. Neostigmine also produced a small reduction in quantal content. 2. Tetraisopropylpyrophosphoramide, with a high specificity for non-specific cholinesterase, has a similar effect on m.e.p.p. frequency but ambenonium, with a high specificity for acetylcholinesterase, was markedly less effective in this respect. 3. Carbachol (10-5 M) and the muscarinic agonists muscarine and metacholine (7 x 10-7 M) also reduced the rate of spontaneous release. 4. The action of neostigmine was antagonized by atropine, but not by D-tubocurarine. Muscarine did not have any further effect when m.e.p.p. frequency was reduced with neostigmine. 5. Experiments with reduced extracellular Ca2+ concentration suggest that the cholinergic agents reduce Ca2+ permeability directly at the presynaptic terminals and that they do not act via a change in PNa or PK. It is suggested that the consequent reduction in Ca2+ entry causes a fall in both evoked release and in intracellular Ca2+ concentration, thereby reducing m.e.p.p. frequency. 6. It is concluded that non-specific cholinesterases present on the presynaptic terminals can act as inhibitory muscarinic cholinergic receptors. This form of presynaptic inhibition at the amphibian neuromuscular junction contrasts with that described in the mammalian preparation in which the sites are blocked by Dtubocurarine and are excitatory. INTRODUCTION
The histochemical localization of cholinesterases (and, in particular, acetylcholinesterases) at presynaptic terminals led Koelle (1961, 1962) to propose that the acetylcholine (ACh) liberated by the action potential acts initially at the same cholinergic terminal to cause the liberation of additional quanta of ACh. It is the secondarily released, increased amount of ACh that, it is suggested, acts postsynaptically to effect transmission. On this hypothesis, ACh receptors are located at the presynaptic terminals (as well as post-synaptically) and the transmitter has a positive feed-back action. On the evidence available at that time, it was suggested (Duncan, 1967) that cholinergic agents could also cause presynaptic inhibition and 0022-3751/79/5180-0956 $01.50 © 1979 The Physiological Society
92 C. J. DUNCAN AND S. J. PUBLICOVER that ACh could have a negative feed-back effect. The theory has been criticized (Hubbard, 1970), but supporting evidence has accumulated in the intervening years (see Miyamoto, 1978). At the mammalian neuromuscular junction cholinergic agonists and anticholinesterases stimulate spontaneous release (Boyd & Martin, 1956; Blaber & Christ, 1967; Laskowski & Dettbarn, 1975) the action of carbachol being reversible and antagonized by curare (Miyamoto & Volle, 1974). There is considerable evidence that cholinoceptive sites are present on these presynaptic terminals (Riker & Okamoto, 1969) and cholinesterase inhibitors are able to initiate antidromic impulses in the mammalian motor nerve, this effect again being abolished by treatment with curare (Masland & Wigton, 1940; Riker, Werner, Roberts & Kuperman, 1959; Okamoto & Riker, 1969; Blaber, 1972). In this paper we report on the properties of the muscarinic ACh receptors on the presynaptic terminals of the frog neuromuscular junction and show in contrast first how they can produce a form of presynaptic inhibition and secondly how their properties differ from those found in mammalian preparations. METHODS
Electrophysiological recordings were made on the isolated cutaneous pectoris nerve-muscle preparation of the frog Rana temporaria. Frogs were maintained in the laboratory at 10 'C. All salines in which the preparations were bathed contained NaCl 111 mm, KCl 1 9 mM, NaHCO3 4-76 mm and NaH2PO4 0-064 mm, glucose 2 g 1.-i at pH 7-1. Normal saline contained 1-8 mM-CaCl2. Extracellular Ca2+ concentration was maintained constant in some salines by the use of Ca2+-EGTA buffers (pH 7-1; 0-5 mM-EGTA plus the appropriate volume of AnalaR standard volumetric solution of CaCl2). Free Ca2+ concentrations were calculated following the method of Portzehl, Caldwell & Ruegg (1964). In 'Ca2+-free' salines, Ca2+ was simply omitted and the solutions were prepared in distilled water which had been passed through an ion-exchange column and were stored in plastic bottles; all plastic and glassware were acid-washed. Salines with low Na+ concentration were maintained at isosmocity by substituting 1-824 mM-sucrose for 1-0 mM-NaCl (Birks, Burstyn & Firth, 1968). The muscle was excised and equilibrated in the appropriate control saline for 45 min at 10 0C. It was then pinned out in the experimental bath, the micro-electrode inserted in the end-plate region and the temperature adjusted to the experimental value. Records of miniature end-plate potentials (m.e.p.p.s) began after a further 10 min. Electrophysiological recordings were made using conventional glass electrodes; the temperature of the bath was controlled at 22-5 0C ( ± 0-5 'C) by a Peltier device. In any one experiment, the m.e.p.p.s were monitored in a single fibre at the intervals shown in the Figures and at least 100 m.e.p.p.s were counted. Studies on evoked release were carried out on a sartorius nerve-muscle preparation using conventional techniques. Ca2+ in the saline was reduced to 0-2 mm, and 0-5 mMMg2+ was added so that the quantal content (m) was reduced to a mean value of 2-3. The nerve was stimulated at 2 sec-1 for 90 sec and m was estimated by the method of failures from m = log, (N/NO), where N = number of trials; No = number of failures. S.E. of mean = (m/N)i (Crawford, 1974). All inorganic salts used were AnalaR grade. Neostigmine bromide, atropine sulphate and tetraisopropylpyrophosphoramide (iso-OMPA) were obtained from Koch-Light Ltd, Colnbrook; eserine sulphate, D-tubocurarine chloride, muscarine chloride, metacholine chloride, carbachol and EGTA from Sigma Chemical Co., St Louis; edrophonium from Roche Products Ltd, London. Ambenonium chloride was a gift of Winthrop Laboratories, Surbiton-upon-Thames, Surrey.
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS
93
RESULTS
The control m.e.p.p. frequency varied between different preparations and comparisons of the effects of treatments are therefore usually shown by expressing m.e.p.p. frequency as a ratio of the control frequency (expressed as F1/FO, where Fo is the control frequency and F1 the frequency after treatment). The mean control m.e.p.p. frequency is also shown in the Figure legends. However, only small changes 15
-¢ -I_--
°1~0 05 0
10
20 30 Time (min)
40
Fig. 1. Control experiments showing small fall in m.e.p.p. frequency with time. Mean of five separate experiments ± S.E. of mean where this exceeds the diameter of the points. As in most Figures, ordinate = m.e.p.p. frequency (F1) as a ratio of control frequency (FO) before the beginning of the experiment. Temperature = 22-5 0C (as in all Figures). Mean control m.e.p.p. frequency = 93 3 min- (s.E. = 30-4 min-).
1-5 A
T
0*5
05_
B
20 30 40 50 Time (min) Fig. 2. Action of neostigmine bromide (7 x 10-7 M) on m.e.p.p. frequency (line B). Mean of six separate experiments + s.E. of mean. Line A shows the effect of 7 x 10 -7 Mneostigmine after pre-treatment with 10-6 M-atropine sulphate for 40 min (compare with Fig. 1). Mean of five separate experiments + s.E. of mean. Mean control m.e.p.p. frequency: A = 128-8 min- (s.E. = 23-13 min-'), B = 120-17 min-' (s.E. = 31-5 min-'). 0
10
in m.e.p.p. frequency were found in any one preparation and control records over 50 min showed a small, but consistent, fall (F1/FO 0.9) during this time (see Fig. 1, mean of five experiments + S.E. of mean.)
Action of general cholinesterase inhibitors on m.e.p.p. frequency The three general cholinesterase inhibitors neostigmine, edrophonium and eserine, at a concentration of 7 x 10-7 M, all had a similar effect on the spontaneous rate of release of transmitter (Figs. 2-4). The effect was clearly evident after 10 min exposure
94 C. J. DUNCAN AND S. J. PUBLICOVER and m.e.p.p. frequency fell to almost 50 % of the control value after exposure for some 15-20 min.
Effect of neostigmine on evoked release Since cholinesterase inhibitors are able to reduce the spontaneous release of transmitter, the action of neostigmine on evoked release was tested by standard techniques with reduced extracellular Ca2+ concentration, when the extracellular 1-5
0*5 0
L
0
10
20 30 Time (min)
40
50
Fig. 3. Effect of eserine sulphate (7 x 10-7 M) on m.e.p.p. frequency. Time course of two separate experiments shown. Mean control m.e.p.p. frequency = 223-4 min-'. 15
0 0IAd 0*5I
0
10
20 30 40 Time (min) Fig. 4. Effect of edrophonium (7 x 1O-7 M) on m.e.p.p.
50
frequency. Time course of two
separate experiments shown. Mean control m.e.p.p. frequency
=
143-2 min-'.
Mg2+ was 0 5 mm. The method of estimation of m (Crawford, 1974) was chosen to give comparable results to those obtained by Ciani & Edwards (1963) for the action of ACh on evoked release at the frog neuromuscular junction. The results of four experiments are shown in Table 1; neostigmine produces a small but consistent reduction in the quantal content (ml/m0 = 0.8). Action of specific cholinesterase inhibitors Ambenonium chloride (NN'-bis (2-diethylammoethyl) oxamide bis-2-chlorobenzyl chloride; WIN 8077) was chosen as an inhibitor that has a high specificity for acetylcholinesterase ('specific' cholinesterase, EC 3.1.1.7; Koelle, 1957; Koelle & Gromadzki, 1966) and iso-OMPA was used as a highly specific inhibitor of butyrylcholinesterase ('non-specific' cholinesterase, EC 3. 1. 1.8), as recommended by Pearse (1972). Fig. 5 shows that iso-OMPA (10-5 M) has a similar effect to that des-
95 M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS cribed for the general cholinesterase inhibitors; m.e.p.p. frequency falls significantly after 5 min exposure and F1/FO is reduced to 0'5 after 30 min. Similar results were obtained with 5 x 10-6 M-iso-OMPA. In contrast, ambenonium chloride (104 M) had little effect on m.e.p.p. frequency (Fig. 5), the rate after 20 min exposure being identical with that found in control preparations (Fig. 1). This agent was certainly TABLE 1. The effect of neostigmine (0.73 /SM) on evoked release in the frog sartorius nervemuscle preparation. The preparations were stimulated at 2 sec-, to give a sample size of approximately 150. Extracellular Ca2+ = 0-2 mM; extracellular Mg2+ = 0.5 mM. Quantal content: mO= control, ml = with neostigmine Quantal content _ , Expt. P no. Ml Ml/MO MO < 0-001 1-95 1.23 1 0-63 < 0-01 2 0-81 3-20 2-61 n.s. 0-88 1-53 3 1-34 < 0-01 4 0-83 3-50 2-90
15
0
a
I
I
20 30 40 Time (min) Fig. 5. Effect of 'specific' cholinesterase inhibitors on m.e.p.p. frequency. Line A: amnbenonium chloride (10-6 M), mean of five experiments + s.E. of mean. Line B: isoOMPA (10-5 M), mean of five experiments + s.E. of mean. Mean control m.e.p.p. frequency: A = 111-2 min-' (s.E. 20-22 min-), B = 280-4 min-' (s.E. = 82-63 min-). 0
10
effective at the post-synaptic cholinesterase sites under these conditions since both the amplitude and (particularly) the duration of the m.e.p.p.s were substantially increased. Fig. 5 also shows that, after 30-40 min exposure, m.e.p.p. frequency falls below control rates (F,/FO = 0-75 compared with 0.9). Two explanations are possible; first the binding site may have a limited affinity for this inhibitor, or, secondly, inhibition of the post-synaptic specific cholinesterase may cause a gradual increase in the ACh present in the synaptic cleft which may have an inhibitory effect on the presynaptic terminals (see below). The depolarization of the muscle membrane recorded during exposure to ambenonium during these experiments suggests that the latter explanation may be correct. We therefore conclude that cholinesterase inhibitors are able to reduce both the spontaneous and evoked release at the frog neuromuscular junction and that these agents combine with a non-specific cholinesterase.
96
C. J. DUNCAN AND S. J. PUBLICOVER
Action of cholinergic agonists Application of carbachol (which has both muscarinic and nicotinic agonistic actions) at either 10-5 M or 2 x 10-5M caused a fall in m.e.p.p. frequency which began after 5 min. F1/FO fell to 0 75 after 10 min, but thereafter post-synaptic depolarization made further recording of the m.e.p.p.s impossible. A detailed analysis of the action 1 5
05
0
10
20
30
40
50
Time (min)
Fig. 6. Effect of muscarine chloride (7 x 10-7 M) on m.e.p.p. frequency. Mean of five experiments+ s.E. of mean. Mean control m.e.p.p. frequency = 205-45 min-
(s.o.
= 3329
min-l). 1-5
0-5 0
_
0
10
I
I
I
I
20
30
40
50
Time (min)
Fig. 7. Effect of metacholine chloride (7 x 10-7 M) on m.e.p.p. frequency. Time course of three separate experiments shown. Mean control m.e.p.p. frequency = 157x9 min' (S.E. = 34x3 min-).
of this agent on the spontaneous release of transmitter is, therefore, difficult. However, the muscarinic agonists, muscarine chloride and metacholine chloride at 7 x 10-7 M, both also cause a significant fall in m.e.p.p. frequency (Figs. 6, 7). Muscarine was more potent; its effect was evident after 5 min exposure and mean F1/FO fell to 0-6 after 40 min treatment.
Presynaptic muscarinic sites Inhibitors of non-specific cholinesterase and muscarinic agonists therefore both produce a very similar depression of m.e.p.p. frequency. Such observations suggest that muscarinic receptors are present on the presynaptic terminals and that drugreceptor interaction can modify the spontaneous release of transmitter. The following experiments confirm these conclusions. Pre-treatment of the preparation for 40 min with D-tubocurarine (10-7 M, 2 x 10-7 M or 3 x 10-7 M; Fig. 8) had no marked effect on the subsequent action of neostigmine in reducing m.e.p.p. frequency. These concentra-
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS 97 tions of D-tubocurarine completely blocked the increase in m.e.p.p. frequency produced by carbachol at the mammalian neuromuscular junction (Miyamoto & Volle, 1974). Pre-treatment of the frog preparation with 10-6 M-atropine for 40 min, on the other hand, significantly antagonized neostigmine action (Fig. 2); the cholinesterase inhibitor now produced no greater effect than that seen in control preparations 15
LCA
XosL
J
0
10
20
30
40
50
Time (min)
Fig. 8. Action of neostigmine (7 x 10-7 M) on m.e.p.p. frequency after pre-treatment with D-tubocurarine for 40 min. Concentrations of D-tubocurarine, A: 01 pm; B: 0-2 1sm; C: 0-3 /SM. Mean control m.e.p.p. frequency= 793 min-L (s.E.= 1341 min-). 15
1.0
0-5 0
I
0
I
I
I
10
20 30 40 50 Time (min) Fig. 9. Effect of atropine sulphate (10-6 M) on m.e.p.p. frequency. Mean of four experiments ± s.E. of mean. Mean control m.e.p.p. frequency = 87-88 min-L (s.E. = 18 184 min').
(Fig. 1). Atropine alone reduced mean F1/F0 to 0 7 after 10 min exposure but, after 20 min, m.e.p.p. frequency slowly rose again, returning to the control rate after 35 min (Fig. 9). The subsequent action of neostigmine was not tested until this initial depressive action of atropine was completed. Are cholinesterase inhibitors and muscarinic agonists acting at the same site? These experimental findings are explicable in a number of ways that are discussed below, but the evidence suggests that the cholinesterase inhibitors and the muscarinic agonists are acting at the same presynaptic receptor site. This hypothesis was tested by treating the preparation with neostigmine (7 x 10-7 M) for 25 min and then adding muscarine. The agonist was not effective in promoting any further fall in m.e.p.p. frequency. 4
PHlY
294
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C. J. DUNCAN AND S. J. PUBLICOVER
Changes in Ca2+ permeability caused by cholinergic agents Since m.e.p.p. frequency has been shown to be markedly modified by intracellular Ca2+ concentration at the presynaptic terminals (Duncan & Statham, 1977), it seems probable that the interaction of muscarinic agonists or cholinesterase inhibitors with presynaptic receptors produces a reduction in both Ca2+ permeability and Ca2+ influx. The action of neostigmine was tested in modified salines. Ca2+ was omitted 1.5
05
10
20 30 40 50 Time (min) Fig. 10. Effect of extracellular Ca2+ concentration on the action of neostigmine (7 x 10-7 M) on m.e.p.p. frequency. Line A: saline contained 5 x 10-7 M Ca2+, buffered with a Ca2+-EGTA buffer; mean of five experiments + S.E. of mean. Line B: 'Ca2+-free' saline; mean of four experiments+ s.E. of mean. Mean control m.e.p.p. frequency: A = 22-05 min- (s.E. = 3-67 min'), B = 26-27 min- (s.E. = 6-21 min-). 0
in 'Ca2+-free' saline and precautions were taken to minimize contamination (see Methods), but free Ca2+ was probably about 10- M. Under these conditions the initial, control m.e.p.p. frequency is low, as previously reported (Duncan & Statham, 1977) and neostigmine was now markedly less effective than in normal extracellular Ca2+ concentrations; F1/F0 fell to 0 75 after 40 min (Fig. 10; compare also with Fig. 1) compared with 0.5 when the extracellular Ca2+ was 1-8 mm (Fig. 2). However, when extracellular Ca2+ was reduced to 5 x 10-7 M, using a Ca2+-EGTA buffer, neostigmine produced a small but significant rise in m.e.p.p. frequency. Fj1F0 rose to 1-4 after 50 min (Fig. 10). We believe that in buffered salines with extracellular Ca2+ maintained low, not only does m.e.p.p. rate fall, but the EGTA seems to promote an outward CaO+ flux (Shimoni, Alnaes & Rahamimoff, 1977; Erulkar, Rahamimoff & Rotshenker, 1978). If neostigmine reduces Pca (and hence, under these conditions, minimizes Ca2+ loss from the presynaptic terminals), its application may effectively cause a rise in intracellular CaO+ concentration, thus accounting for the rise in m.e.p.p. frequency in the presence of EGTA (Fig. 10). This hypothesis was tested as follows. The preparation was equilibrated in EGTAbuffered saline (extracellular Ca2+ = 5 x 10-7 M) and was then slightly depolarized by raising the extracellular K+ to 20 mm and replacing Na+ isosmotically. M.e.p.p. frequency rose some three to seven-fold during the first minute, possibly due to Ca2+ leaving the terminals and crossing key sites at the plasma membrane, but thereafter consistently fell to below 50 % of the initial rate (Fig. 11). Returning the extracellular K+ to normal values (with extracellular Ca2+ buffered at 5 x 10-7 M) caused the m.e.p.p. rate to rise again; a second depolarization by elevation of the extracellular
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS 99 K+ once more caused a fall in the rate of spontaneous release (Fig. 11) and the brief, dramatic rise in m.e.p.p. frequency was not observed on this second depolarization. We conclude, in agreement with earlier studies (Shimoni et al. 1977; Erulkar et al. 1978), that EGTA can promote Ca2+ efflux at the terminals. This action is seen most clearly when PCa is raised by depolarization. We suggest, however, that when EGTA [KJ0
140
__-
_ |
t
\J
l
I
100
E
~ If C_
:\
II. \
I:
1-5
I~~~~~~~~~~~~~~
20,
10
20
61 30 Time (min)
50
Fig. II. Action of Ca2+-EGTA buffer system in promoting CO2+ efflux from the presynaptic terminals. Three separate experiments in which the preparation was equilibrated in saline containing .5 x 10-7 M Ca2+ and then slightly depolarized by raising extracellular K+ concentration to 20 mm (shown by black bars) which caused a brief, marked rise in m.e.p.p. frequency (300s400 min-', indicated by arrow). Spontaneous release then fell to less than 50 % of control rate, rose again when K+ was returned to normal values, and fell once more when depolarized again. The stimulated release found initially on raising K+ is not an ' on-off ' response associated with depolarization since it was not found when the depolarization was subsequently repeated.
I I
I~~~~~~~~~~~~~~~~~~ is having this effect at normal extracellular K+ concentration, with a consequently low intracellular Ca2+ concentration and m.e.p.p. frequency, a marked reduction in Pca produced by neostigmine (Fig. 10) would reduce the efflux of Ca2+ and the intracellular Ca2+ concentration would rise, being now largely determined by leakage from intracellular Ca2+ storage sites. In turn, m.e.p.p. frequency would be expected to rise (Fig. 10). Possible involvement of changes in permeability to monovalent cations We conclude that cholinesterase inhibitors produce a reduction in Ca2+ permeability at the presynaptic terminals (see Fig. 14). This reduction of Ca2+ permeability 4-2
C. J. DUNCAN AND S. J. PUBLICOVER could either be produced directly or, perhaps, by hyperpolarization of the terminals. Hyperpolarization could be effected either by a decrease in PNa or an increase in PK, the resulting hyperpolarization producing, in turn, a change in Ca2+ permeability. The action of neostigmine was therefore tested when the extracellular Na+ was reduced to 55 mm (extracellular Ca2+ = 1-8 mM). M.e.p.p. frequency is raised under these conditions, as previously reported (Statham & Duncan, 1977), but neostigmine 100
15
0*5 0
0
10
0
10
20 40 30 50 Time (min) Fig. 12. Effect of neostigmine (7 x 1O-7 M) when terminals equilibrated in saline in which extracellular Na+ was reduced to 55 mm by replacing with sucrose. Time course of two separate experiments. Mean control m.e.p.p. frequency = 458-25 min". 15
05 20 30 Time (min)
40
50
Fig. 13. Effect of neostigmine (7 x 1O-7 M) when terminals equilibrated in K+-free saline. Time course of three separate experiments. Mean control m.e.p.p. frequency = 204-8 min- (s.E. = 78.85 min-).
produces the normal reduction in m.e.p.p. frequency (Fig. 12). Further reductions in extracellular Na+ made the detection of m.e.p.p.s difficult but we conclude that cholinergic agents do not act presynaptically by hyperpolarizing the terminals via a decrease in PNa. Equally, equilibration in K+-free saline (which causes a substantial hyperpolarization of the muscle membrane) does not affect the inhibitory action of neostigmine on m.e.p.p. frequency (Fig. 13) and we again conclude that cholinergic agents probably do not operate at the terminals via an increase in PK. DISCUSSION
The evidence summarized in the Introduction (see also Miyamoto, 1978) seems to show that ACh receptors are located presynaptically at the mammalian neuromuscular junction and that drug-receptor interaction can initiate antidromic firing
s tfig. Curae
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS 101 and promote the spontaneous release of transmitter, these effects being antagonized by curare. In contrast, cholinergic agents clearly have an inhibitory effect on the release system of the frog neuromuscular junction. The average number of quanta released by each nerve impulse in the Mg2+-blocked preparation is decreased by ACh (Ciani & Edwards, 1963). Our studies show that both cholinesterase inhibitors and muscarinic agonists reduce m.e.p.p. frequency; the action is antagonized by atropine Intracellular Ca stores
BChE
Ca
gat
Atropine
Atropine
gate
v
Muscarine
*@. EGTA
I
|
~
F
~ } Metacholine
iso-OMPA
Neostigmine
Esersitnieg'rni
_
rci [C a]
Edropnoniumr
Ambenoniumn
AChE
I
Carbachol
AChR
Fig. 14. Diagram of the synaptic cleft to illustrate the proposed site of action at the frog neuromuscular junction of the agents tested in the present study. Acetylcholine (ACh) is shown with an excitatory (+) action post-synaptically and with an inhibitory (-) action presynaptically. Acetylcholine receptors (AChR) are shown as triangles; the presynaptic non-specific cholinesterase (BChE) is shown acting as an inhibitory ACh receptor, linked to the Ca2+ channels. The action of competitive inhibitors is shown by parallel bars.
but not by curare. The results with carbachol are the reverse of those obtained with a mammalian preparation; at the same concentration this agent stimulates m.e.p.p. frequency at the rat neuromuscular junction and the effect is antagonized by curare (Miyamoto & Volle, 1974). Furthermore, the amphibian preparation differs from that of the mammal in that cholinesterase inhibitors do not generate antidromic nerve impulses (Riker & Okamoto, 1969). We therefore conclude that cholinergic receptors are present on the presynaptic motor terminals of both mammals and amphibians but that the sites differ in their properties in these two classes of vertebrates. Drugreceptor interaction is stimulatory in the mammal and is blocked by curare, whereas the sites are muscarinic in the frog and they serve to reduce the spontaneous release of transmitter (Fig. 14). However, the way in which ACh itself acts at the frog
C. J. DUNCAN AND S. J. PUBLICOVER presynaptic terminals is not yet clear. Katz & Mfiledi (1972) report that ACh increases the rate of spontaneous release at some junctions and suggest that the variability is related to the initial level of Em at the terminals, whereas Ciani & Edwards (1963) report that the transmitter reduces evoked release. It could be suggested that the cholinesterase inhibitors act post-synaptically in frog, thereby increasing the concentration of ACh which then acts at the presynaptic sites. We consider this hypothesis unlikely since: (i) atropine antagonizes the effect of both muscarine and neostigmine, (ii) ambenonium has an obvious post-synaptic effect (augmenting m.e.p.p. amplitude and duration) but has little presynaptic effect (compare the effect of iso-OMPA), (iii) cholinesterase is known to be localized presynaptically (Koelle, 1961, 1962). We believe that the general cholinesterase inhibitors, the muscarinic agonists and iso-OMPA all act at the same presynaptic site since: (i) they produce a similar pattern of response, (ii) the muscarinic antagonist atropine blocks neostigmine action and (iii) neostigmine pre-treatment blocks the action of muscarine. It is probable that the cholinesterase identified histochemically at the presynaptic terminals constitutes this muscarinic receptor, as previously suggested (Duncan, 1967); the receptor appears to be a non-specific cholinesterase (EC 3. 1. 1 .8) which is inhibited by iso-OMPA. The results summarized in Figs. 10 and 11 show that these presynaptic effects are dependent on extracellular Ca2+; the results suggest that cholinesterase inhibitors reduce Ca2+ permeability at the terminals. Evidence is accumulating that intracellular Ca2+ concentration at the terminals has a marked effect on m.e.p.p. frequency, although reductions in Ca2+ fluxes across the plasma membrane do not produce dramatic effects (Statham & Duncan, 1975; Duncan & Statham, 1977; Publicover & Duncan, 1979). The reduction of the mean F1/F0 to 0-5 in the present studies therefore probably represents a substantial reduction in Ca2+ influx. It could be argued that cholinergic agents act by hyperpolarizing the terminals via changes in PNa or PK which in turn causes a reduction in Pc. We believe that this hypothesis is unlikely since: (i) m.e.p.p. frequency in the frog is largely insensitive to hyperpolarization (Del Castillo & Katz, 1954) and (ii) the action of neostigmine is not affected by changes in the concentrations of extracellular Na+ (Fig. 12) or K+ (Fig. 13). We conclude that cholinergic agents have a direct effect on the Ca2+ channels and markedly reduce Ca2+ influx. 102
We thank Miss S. Scott for assistance in the preparation of the manuscript. REFERENCES BTxs, R. I., ButSTYN, P. G. R. & FIRTH, D. R. (1968). The form of sodium-calcium competition at the frog myoneural junction. J. gen. Phy8iol. 52, 887-907. BLABER, L. C. (1972). The mechanisms of the facilitatory action of edrophonium in cat skeletal muscle. Br. J. Pharmac. 46, 498-507. BLABER, L. C. & CHRIST, D. D. (1967). The action of facilitatory drugs on the isolated tenuissimus muscle of the cat. Int. J. Neuro-pharmacol. 6, 473-484. BOYD, I. A. & MARTIN, A. R. (1956). Spontaneous subthreshold activity at mammalian neuromuscular junctions. J. Physiol. 132, 61-73.
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CIANI, S. & EDWARDS, C. (1963). The effect of acetylcholine on neuromuscular transmission in the frog. J. Pharmac. exp. Ther. 142, 21-23. CRAWFORD, A. C. (1974). The dependence of evoked transmitter release on external calcium ions at very low mean quantal contents. J. Physiol. 240, 255-278. DEL CASTILLO, J. & KATZ, B. (1954). Changes in end-plate activity produced by pre-synaptic polarization. J. Physiol. 124, 586-604. DUNCAN, C. J. (1967). The Molecular Properties and Evolution of Excitable Cells. Oxford: Pergamon Press. DUNCAN, C. J. & STATHAM, H. E. (1977). Interacting effects of temperature and extracellular calcium on the spontaneous release of transmitter at the frog neuromuscular junction. J. Physiol. 268, 319-333. ERULKAR, S. D., RAHAMIMOFF, R. & ROTSHENKER, S. (1978). Quelling of spontaneous transmitter release by nerve impulses in low extracellular calcium solutions. J. Physiol. 278, 491-500. HUBBARD, J. I. (1970). Mechanism of transmitter release. Prog. Biophys. 21, 33-124. KATZ, B. & MILEDI, R. (1972). The statistical nature of the acetylcholine potential and its molecular components. J. Physiol. 224, 665-699. KOELLE, G. B. (1957). Histochemical demonstration of reversible anti-cholinesterase action at selective cellular sites in vivo. J. Pharmac. exp. Ther. 120, 488-503. KOELLE, G. B. (1961). A proposed dual neurohumoral role of acetylcholine: its functions at the pre- and post-synaptic sites. Nature, Lond. 190, 208-211. KOELLE, G. B. (1962). A new general concept of the neurohumoral functions of acetylcholine and acetylcholinesterase. J. Pharm. Pharmac. 14, 65-90. KOELLE, G. B. & GROMADZKI, C. G. (1966). Comparison of the gold-thiocholine and goldthiolacetic acid methods for the histochemical localization of acetylcholinesterase and cholinesterases. J. Histochem. Cytochem. 14, 443-454. LASKOWSKI, M. B. & DETTBARN, W. D. (1975). Presynaptic effects of neuromuscular cholinesterase inhibition. J. Pharmac. exp. Ther. 194, 351-361. MASLAND, R. L. & WIGTON, R. S. (1940). Nerve activity accompanying fasciculation produced by prostigmin. J. Neurophysiol. 3, 269-275. MrYAMOTO, M. D. (1978). The actions of cholinergic drugs on motor nerve terminals. Pharmac. Rev. 29, 221-247. MIYAMOTO, M. D. & VOLLE, R. L. (1974). Enhancement by carbachol of transmitter release from motor nerve terminals. Proc. natn. Acad. Sci. U.S.A. 71, 1489-1492. OKAMOTO, M. & RIKER, W. F. JR. (1969). Sub-acute denervation: a means of disclosing mammalian motor nerve terminals as critical sites of acetylcholine and facilitatory drug actions. J. Pharmac. exp. Ther. 166, 217-224. PEARSE, A. G. E. (1972). Histochemistry Theoretical and Applied, 3rd ed., vol. 2. London: Churchill. PORTZEHL, H., CALDWELL, P. C. & RtEGG, J. C. (1964). The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta 79, 581-591. PUBLICOVER, S. J. & DUNCAN, C. J. (1979). The action of verapamil on the rate of spontaneous release of transmitter at the frog neuromuscular junction. Eur. J. Pharmacol. 54, 119-127. RIKER, W. F. JR. & OKAMOTO, M. (1969). Pharmacology of motor nerve terminals. A. Rev. Pharmac. 9, 173-208. RIKER, W. F. JR., WERNER, G., ROBERTS, J. & KUPERMAN, A. (1959). Pharmacologic evidence for the existence of a presynaptic event in neuromuscular transmission. J. Pharmac. exp. Ther. 125, 150-158. SHIMONI, Y., ALNAEs, E. & RAHAMIMOFF, R. (1977). Is hyperosmotic neurosecretion from motor nerve endings a calcium-dependent process? Nature, Lond. 267, 170-172. STATHAM, H. E. & DUNCAN, C. J. (1975). The action of ionophores at the frog neuromuscular junction. Life Sci. 17, 1401-1406. STATHAM, H. E. & DUNCAN, C. J. (1977). The effect of sodium ions on MEPP frequency at the frog neuromuscular junction. Life Sci. 20, 1839-1846.
J. Physiol. (1979), 294, pp. 91-103 With 14 text-figure. Printed in Great Britain
INHIBITORY EFFECTS OF CHOLINERGIC AGENTS ON THE RELEASE OF TRANSMITTER AT THE FROG NEUROMUSCULAR JUNCTION
By C. J. DUNCAN AND S. J. PUBLICOVER From the Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX
(Received 11 January 1979) SUMMARY
1. The cholinesterase inhibitors neostigmine, edrophonium and eserine (7 x 10-7 M) reduced m.e.p.p. frequency by some 50 % at the frog neuromuscular junction. Neostigmine also produced a small reduction in quantal content. 2. Tetraisopropylpyrophosphoramide, with a high specificity for non-specific cholinesterase, has a similar effect on m.e.p.p. frequency but ambenonium, with a high specificity for acetylcholinesterase, was markedly less effective in this respect. 3. Carbachol (10-5 M) and the muscarinic agonists muscarine and metacholine (7 x 10-7 M) also reduced the rate of spontaneous release. 4. The action of neostigmine was antagonized by atropine, but not by D-tubocurarine. Muscarine did not have any further effect when m.e.p.p. frequency was reduced with neostigmine. 5. Experiments with reduced extracellular Ca2+ concentration suggest that the cholinergic agents reduce Ca2+ permeability directly at the presynaptic terminals and that they do not act via a change in PNa or PK. It is suggested that the consequent reduction in Ca2+ entry causes a fall in both evoked release and in intracellular Ca2+ concentration, thereby reducing m.e.p.p. frequency. 6. It is concluded that non-specific cholinesterases present on the presynaptic terminals can act as inhibitory muscarinic cholinergic receptors. This form of presynaptic inhibition at the amphibian neuromuscular junction contrasts with that described in the mammalian preparation in which the sites are blocked by Dtubocurarine and are excitatory. INTRODUCTION
The histochemical localization of cholinesterases (and, in particular, acetylcholinesterases) at presynaptic terminals led Koelle (1961, 1962) to propose that the acetylcholine (ACh) liberated by the action potential acts initially at the same cholinergic terminal to cause the liberation of additional quanta of ACh. It is the secondarily released, increased amount of ACh that, it is suggested, acts postsynaptically to effect transmission. On this hypothesis, ACh receptors are located at the presynaptic terminals (as well as post-synaptically) and the transmitter has a positive feed-back action. On the evidence available at that time, it was suggested (Duncan, 1967) that cholinergic agents could also cause presynaptic inhibition and 0022-3751/79/5180-0956 $01.50 © 1979 The Physiological Society
92 C. J. DUNCAN AND S. J. PUBLICOVER that ACh could have a negative feed-back effect. The theory has been criticized (Hubbard, 1970), but supporting evidence has accumulated in the intervening years (see Miyamoto, 1978). At the mammalian neuromuscular junction cholinergic agonists and anticholinesterases stimulate spontaneous release (Boyd & Martin, 1956; Blaber & Christ, 1967; Laskowski & Dettbarn, 1975) the action of carbachol being reversible and antagonized by curare (Miyamoto & Volle, 1974). There is considerable evidence that cholinoceptive sites are present on these presynaptic terminals (Riker & Okamoto, 1969) and cholinesterase inhibitors are able to initiate antidromic impulses in the mammalian motor nerve, this effect again being abolished by treatment with curare (Masland & Wigton, 1940; Riker, Werner, Roberts & Kuperman, 1959; Okamoto & Riker, 1969; Blaber, 1972). In this paper we report on the properties of the muscarinic ACh receptors on the presynaptic terminals of the frog neuromuscular junction and show in contrast first how they can produce a form of presynaptic inhibition and secondly how their properties differ from those found in mammalian preparations. METHODS
Electrophysiological recordings were made on the isolated cutaneous pectoris nerve-muscle preparation of the frog Rana temporaria. Frogs were maintained in the laboratory at 10 'C. All salines in which the preparations were bathed contained NaCl 111 mm, KCl 1 9 mM, NaHCO3 4-76 mm and NaH2PO4 0-064 mm, glucose 2 g 1.-i at pH 7-1. Normal saline contained 1-8 mM-CaCl2. Extracellular Ca2+ concentration was maintained constant in some salines by the use of Ca2+-EGTA buffers (pH 7-1; 0-5 mM-EGTA plus the appropriate volume of AnalaR standard volumetric solution of CaCl2). Free Ca2+ concentrations were calculated following the method of Portzehl, Caldwell & Ruegg (1964). In 'Ca2+-free' salines, Ca2+ was simply omitted and the solutions were prepared in distilled water which had been passed through an ion-exchange column and were stored in plastic bottles; all plastic and glassware were acid-washed. Salines with low Na+ concentration were maintained at isosmocity by substituting 1-824 mM-sucrose for 1-0 mM-NaCl (Birks, Burstyn & Firth, 1968). The muscle was excised and equilibrated in the appropriate control saline for 45 min at 10 0C. It was then pinned out in the experimental bath, the micro-electrode inserted in the end-plate region and the temperature adjusted to the experimental value. Records of miniature end-plate potentials (m.e.p.p.s) began after a further 10 min. Electrophysiological recordings were made using conventional glass electrodes; the temperature of the bath was controlled at 22-5 0C ( ± 0-5 'C) by a Peltier device. In any one experiment, the m.e.p.p.s were monitored in a single fibre at the intervals shown in the Figures and at least 100 m.e.p.p.s were counted. Studies on evoked release were carried out on a sartorius nerve-muscle preparation using conventional techniques. Ca2+ in the saline was reduced to 0-2 mm, and 0-5 mMMg2+ was added so that the quantal content (m) was reduced to a mean value of 2-3. The nerve was stimulated at 2 sec-1 for 90 sec and m was estimated by the method of failures from m = log, (N/NO), where N = number of trials; No = number of failures. S.E. of mean = (m/N)i (Crawford, 1974). All inorganic salts used were AnalaR grade. Neostigmine bromide, atropine sulphate and tetraisopropylpyrophosphoramide (iso-OMPA) were obtained from Koch-Light Ltd, Colnbrook; eserine sulphate, D-tubocurarine chloride, muscarine chloride, metacholine chloride, carbachol and EGTA from Sigma Chemical Co., St Louis; edrophonium from Roche Products Ltd, London. Ambenonium chloride was a gift of Winthrop Laboratories, Surbiton-upon-Thames, Surrey.
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS
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RESULTS
The control m.e.p.p. frequency varied between different preparations and comparisons of the effects of treatments are therefore usually shown by expressing m.e.p.p. frequency as a ratio of the control frequency (expressed as F1/FO, where Fo is the control frequency and F1 the frequency after treatment). The mean control m.e.p.p. frequency is also shown in the Figure legends. However, only small changes 15
-¢ -I_--
°1~0 05 0
10
20 30 Time (min)
40
Fig. 1. Control experiments showing small fall in m.e.p.p. frequency with time. Mean of five separate experiments ± S.E. of mean where this exceeds the diameter of the points. As in most Figures, ordinate = m.e.p.p. frequency (F1) as a ratio of control frequency (FO) before the beginning of the experiment. Temperature = 22-5 0C (as in all Figures). Mean control m.e.p.p. frequency = 93 3 min- (s.E. = 30-4 min-).
1-5 A
T
0*5
05_
B
20 30 40 50 Time (min) Fig. 2. Action of neostigmine bromide (7 x 10-7 M) on m.e.p.p. frequency (line B). Mean of six separate experiments + s.E. of mean. Line A shows the effect of 7 x 10 -7 Mneostigmine after pre-treatment with 10-6 M-atropine sulphate for 40 min (compare with Fig. 1). Mean of five separate experiments + s.E. of mean. Mean control m.e.p.p. frequency: A = 128-8 min- (s.E. = 23-13 min-'), B = 120-17 min-' (s.E. = 31-5 min-'). 0
10
in m.e.p.p. frequency were found in any one preparation and control records over 50 min showed a small, but consistent, fall (F1/FO 0.9) during this time (see Fig. 1, mean of five experiments + S.E. of mean.)
Action of general cholinesterase inhibitors on m.e.p.p. frequency The three general cholinesterase inhibitors neostigmine, edrophonium and eserine, at a concentration of 7 x 10-7 M, all had a similar effect on the spontaneous rate of release of transmitter (Figs. 2-4). The effect was clearly evident after 10 min exposure
94 C. J. DUNCAN AND S. J. PUBLICOVER and m.e.p.p. frequency fell to almost 50 % of the control value after exposure for some 15-20 min.
Effect of neostigmine on evoked release Since cholinesterase inhibitors are able to reduce the spontaneous release of transmitter, the action of neostigmine on evoked release was tested by standard techniques with reduced extracellular Ca2+ concentration, when the extracellular 1-5
0*5 0
L
0
10
20 30 Time (min)
40
50
Fig. 3. Effect of eserine sulphate (7 x 10-7 M) on m.e.p.p. frequency. Time course of two separate experiments shown. Mean control m.e.p.p. frequency = 223-4 min-'. 15
0 0IAd 0*5I
0
10
20 30 40 Time (min) Fig. 4. Effect of edrophonium (7 x 1O-7 M) on m.e.p.p.
50
frequency. Time course of two
separate experiments shown. Mean control m.e.p.p. frequency
=
143-2 min-'.
Mg2+ was 0 5 mm. The method of estimation of m (Crawford, 1974) was chosen to give comparable results to those obtained by Ciani & Edwards (1963) for the action of ACh on evoked release at the frog neuromuscular junction. The results of four experiments are shown in Table 1; neostigmine produces a small but consistent reduction in the quantal content (ml/m0 = 0.8). Action of specific cholinesterase inhibitors Ambenonium chloride (NN'-bis (2-diethylammoethyl) oxamide bis-2-chlorobenzyl chloride; WIN 8077) was chosen as an inhibitor that has a high specificity for acetylcholinesterase ('specific' cholinesterase, EC 3.1.1.7; Koelle, 1957; Koelle & Gromadzki, 1966) and iso-OMPA was used as a highly specific inhibitor of butyrylcholinesterase ('non-specific' cholinesterase, EC 3. 1. 1.8), as recommended by Pearse (1972). Fig. 5 shows that iso-OMPA (10-5 M) has a similar effect to that des-
95 M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS cribed for the general cholinesterase inhibitors; m.e.p.p. frequency falls significantly after 5 min exposure and F1/FO is reduced to 0'5 after 30 min. Similar results were obtained with 5 x 10-6 M-iso-OMPA. In contrast, ambenonium chloride (104 M) had little effect on m.e.p.p. frequency (Fig. 5), the rate after 20 min exposure being identical with that found in control preparations (Fig. 1). This agent was certainly TABLE 1. The effect of neostigmine (0.73 /SM) on evoked release in the frog sartorius nervemuscle preparation. The preparations were stimulated at 2 sec-, to give a sample size of approximately 150. Extracellular Ca2+ = 0-2 mM; extracellular Mg2+ = 0.5 mM. Quantal content: mO= control, ml = with neostigmine Quantal content _ , Expt. P no. Ml Ml/MO MO < 0-001 1-95 1.23 1 0-63 < 0-01 2 0-81 3-20 2-61 n.s. 0-88 1-53 3 1-34 < 0-01 4 0-83 3-50 2-90
15
0
a
I
I
20 30 40 Time (min) Fig. 5. Effect of 'specific' cholinesterase inhibitors on m.e.p.p. frequency. Line A: amnbenonium chloride (10-6 M), mean of five experiments + s.E. of mean. Line B: isoOMPA (10-5 M), mean of five experiments + s.E. of mean. Mean control m.e.p.p. frequency: A = 111-2 min-' (s.E. 20-22 min-), B = 280-4 min-' (s.E. = 82-63 min-). 0
10
effective at the post-synaptic cholinesterase sites under these conditions since both the amplitude and (particularly) the duration of the m.e.p.p.s were substantially increased. Fig. 5 also shows that, after 30-40 min exposure, m.e.p.p. frequency falls below control rates (F,/FO = 0-75 compared with 0.9). Two explanations are possible; first the binding site may have a limited affinity for this inhibitor, or, secondly, inhibition of the post-synaptic specific cholinesterase may cause a gradual increase in the ACh present in the synaptic cleft which may have an inhibitory effect on the presynaptic terminals (see below). The depolarization of the muscle membrane recorded during exposure to ambenonium during these experiments suggests that the latter explanation may be correct. We therefore conclude that cholinesterase inhibitors are able to reduce both the spontaneous and evoked release at the frog neuromuscular junction and that these agents combine with a non-specific cholinesterase.
96
C. J. DUNCAN AND S. J. PUBLICOVER
Action of cholinergic agonists Application of carbachol (which has both muscarinic and nicotinic agonistic actions) at either 10-5 M or 2 x 10-5M caused a fall in m.e.p.p. frequency which began after 5 min. F1/FO fell to 0 75 after 10 min, but thereafter post-synaptic depolarization made further recording of the m.e.p.p.s impossible. A detailed analysis of the action 1 5
05
0
10
20
30
40
50
Time (min)
Fig. 6. Effect of muscarine chloride (7 x 10-7 M) on m.e.p.p. frequency. Mean of five experiments+ s.E. of mean. Mean control m.e.p.p. frequency = 205-45 min-
(s.o.
= 3329
min-l). 1-5
0-5 0
_
0
10
I
I
I
I
20
30
40
50
Time (min)
Fig. 7. Effect of metacholine chloride (7 x 10-7 M) on m.e.p.p. frequency. Time course of three separate experiments shown. Mean control m.e.p.p. frequency = 157x9 min' (S.E. = 34x3 min-).
of this agent on the spontaneous release of transmitter is, therefore, difficult. However, the muscarinic agonists, muscarine chloride and metacholine chloride at 7 x 10-7 M, both also cause a significant fall in m.e.p.p. frequency (Figs. 6, 7). Muscarine was more potent; its effect was evident after 5 min exposure and mean F1/FO fell to 0-6 after 40 min treatment.
Presynaptic muscarinic sites Inhibitors of non-specific cholinesterase and muscarinic agonists therefore both produce a very similar depression of m.e.p.p. frequency. Such observations suggest that muscarinic receptors are present on the presynaptic terminals and that drugreceptor interaction can modify the spontaneous release of transmitter. The following experiments confirm these conclusions. Pre-treatment of the preparation for 40 min with D-tubocurarine (10-7 M, 2 x 10-7 M or 3 x 10-7 M; Fig. 8) had no marked effect on the subsequent action of neostigmine in reducing m.e.p.p. frequency. These concentra-
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS 97 tions of D-tubocurarine completely blocked the increase in m.e.p.p. frequency produced by carbachol at the mammalian neuromuscular junction (Miyamoto & Volle, 1974). Pre-treatment of the frog preparation with 10-6 M-atropine for 40 min, on the other hand, significantly antagonized neostigmine action (Fig. 2); the cholinesterase inhibitor now produced no greater effect than that seen in control preparations 15
LCA
XosL
J
0
10
20
30
40
50
Time (min)
Fig. 8. Action of neostigmine (7 x 10-7 M) on m.e.p.p. frequency after pre-treatment with D-tubocurarine for 40 min. Concentrations of D-tubocurarine, A: 01 pm; B: 0-2 1sm; C: 0-3 /SM. Mean control m.e.p.p. frequency= 793 min-L (s.E.= 1341 min-). 15
1.0
0-5 0
I
0
I
I
I
10
20 30 40 50 Time (min) Fig. 9. Effect of atropine sulphate (10-6 M) on m.e.p.p. frequency. Mean of four experiments ± s.E. of mean. Mean control m.e.p.p. frequency = 87-88 min-L (s.E. = 18 184 min').
(Fig. 1). Atropine alone reduced mean F1/F0 to 0 7 after 10 min exposure but, after 20 min, m.e.p.p. frequency slowly rose again, returning to the control rate after 35 min (Fig. 9). The subsequent action of neostigmine was not tested until this initial depressive action of atropine was completed. Are cholinesterase inhibitors and muscarinic agonists acting at the same site? These experimental findings are explicable in a number of ways that are discussed below, but the evidence suggests that the cholinesterase inhibitors and the muscarinic agonists are acting at the same presynaptic receptor site. This hypothesis was tested by treating the preparation with neostigmine (7 x 10-7 M) for 25 min and then adding muscarine. The agonist was not effective in promoting any further fall in m.e.p.p. frequency. 4
PHlY
294
98
C. J. DUNCAN AND S. J. PUBLICOVER
Changes in Ca2+ permeability caused by cholinergic agents Since m.e.p.p. frequency has been shown to be markedly modified by intracellular Ca2+ concentration at the presynaptic terminals (Duncan & Statham, 1977), it seems probable that the interaction of muscarinic agonists or cholinesterase inhibitors with presynaptic receptors produces a reduction in both Ca2+ permeability and Ca2+ influx. The action of neostigmine was tested in modified salines. Ca2+ was omitted 1.5
05
10
20 30 40 50 Time (min) Fig. 10. Effect of extracellular Ca2+ concentration on the action of neostigmine (7 x 10-7 M) on m.e.p.p. frequency. Line A: saline contained 5 x 10-7 M Ca2+, buffered with a Ca2+-EGTA buffer; mean of five experiments + S.E. of mean. Line B: 'Ca2+-free' saline; mean of four experiments+ s.E. of mean. Mean control m.e.p.p. frequency: A = 22-05 min- (s.E. = 3-67 min'), B = 26-27 min- (s.E. = 6-21 min-). 0
in 'Ca2+-free' saline and precautions were taken to minimize contamination (see Methods), but free Ca2+ was probably about 10- M. Under these conditions the initial, control m.e.p.p. frequency is low, as previously reported (Duncan & Statham, 1977) and neostigmine was now markedly less effective than in normal extracellular Ca2+ concentrations; F1/F0 fell to 0 75 after 40 min (Fig. 10; compare also with Fig. 1) compared with 0.5 when the extracellular Ca2+ was 1-8 mm (Fig. 2). However, when extracellular Ca2+ was reduced to 5 x 10-7 M, using a Ca2+-EGTA buffer, neostigmine produced a small but significant rise in m.e.p.p. frequency. Fj1F0 rose to 1-4 after 50 min (Fig. 10). We believe that in buffered salines with extracellular Ca2+ maintained low, not only does m.e.p.p. rate fall, but the EGTA seems to promote an outward CaO+ flux (Shimoni, Alnaes & Rahamimoff, 1977; Erulkar, Rahamimoff & Rotshenker, 1978). If neostigmine reduces Pca (and hence, under these conditions, minimizes Ca2+ loss from the presynaptic terminals), its application may effectively cause a rise in intracellular CaO+ concentration, thus accounting for the rise in m.e.p.p. frequency in the presence of EGTA (Fig. 10). This hypothesis was tested as follows. The preparation was equilibrated in EGTAbuffered saline (extracellular Ca2+ = 5 x 10-7 M) and was then slightly depolarized by raising the extracellular K+ to 20 mm and replacing Na+ isosmotically. M.e.p.p. frequency rose some three to seven-fold during the first minute, possibly due to Ca2+ leaving the terminals and crossing key sites at the plasma membrane, but thereafter consistently fell to below 50 % of the initial rate (Fig. 11). Returning the extracellular K+ to normal values (with extracellular Ca2+ buffered at 5 x 10-7 M) caused the m.e.p.p. rate to rise again; a second depolarization by elevation of the extracellular
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS 99 K+ once more caused a fall in the rate of spontaneous release (Fig. 11) and the brief, dramatic rise in m.e.p.p. frequency was not observed on this second depolarization. We conclude, in agreement with earlier studies (Shimoni et al. 1977; Erulkar et al. 1978), that EGTA can promote Ca2+ efflux at the terminals. This action is seen most clearly when PCa is raised by depolarization. We suggest, however, that when EGTA [KJ0
140
__-
_ |
t
\J
l
I
100
E
~ If C_
:\
II. \
I:
1-5
I~~~~~~~~~~~~~~
20,
10
20
61 30 Time (min)
50
Fig. II. Action of Ca2+-EGTA buffer system in promoting CO2+ efflux from the presynaptic terminals. Three separate experiments in which the preparation was equilibrated in saline containing .5 x 10-7 M Ca2+ and then slightly depolarized by raising extracellular K+ concentration to 20 mm (shown by black bars) which caused a brief, marked rise in m.e.p.p. frequency (300s400 min-', indicated by arrow). Spontaneous release then fell to less than 50 % of control rate, rose again when K+ was returned to normal values, and fell once more when depolarized again. The stimulated release found initially on raising K+ is not an ' on-off ' response associated with depolarization since it was not found when the depolarization was subsequently repeated.
I I
I~~~~~~~~~~~~~~~~~~ is having this effect at normal extracellular K+ concentration, with a consequently low intracellular Ca2+ concentration and m.e.p.p. frequency, a marked reduction in Pca produced by neostigmine (Fig. 10) would reduce the efflux of Ca2+ and the intracellular Ca2+ concentration would rise, being now largely determined by leakage from intracellular Ca2+ storage sites. In turn, m.e.p.p. frequency would be expected to rise (Fig. 10). Possible involvement of changes in permeability to monovalent cations We conclude that cholinesterase inhibitors produce a reduction in Ca2+ permeability at the presynaptic terminals (see Fig. 14). This reduction of Ca2+ permeability 4-2
C. J. DUNCAN AND S. J. PUBLICOVER could either be produced directly or, perhaps, by hyperpolarization of the terminals. Hyperpolarization could be effected either by a decrease in PNa or an increase in PK, the resulting hyperpolarization producing, in turn, a change in Ca2+ permeability. The action of neostigmine was therefore tested when the extracellular Na+ was reduced to 55 mm (extracellular Ca2+ = 1-8 mM). M.e.p.p. frequency is raised under these conditions, as previously reported (Statham & Duncan, 1977), but neostigmine 100
15
0*5 0
0
10
0
10
20 40 30 50 Time (min) Fig. 12. Effect of neostigmine (7 x 1O-7 M) when terminals equilibrated in saline in which extracellular Na+ was reduced to 55 mm by replacing with sucrose. Time course of two separate experiments. Mean control m.e.p.p. frequency = 458-25 min". 15
05 20 30 Time (min)
40
50
Fig. 13. Effect of neostigmine (7 x 1O-7 M) when terminals equilibrated in K+-free saline. Time course of three separate experiments. Mean control m.e.p.p. frequency = 204-8 min- (s.E. = 78.85 min-).
produces the normal reduction in m.e.p.p. frequency (Fig. 12). Further reductions in extracellular Na+ made the detection of m.e.p.p.s difficult but we conclude that cholinergic agents do not act presynaptically by hyperpolarizing the terminals via a decrease in PNa. Equally, equilibration in K+-free saline (which causes a substantial hyperpolarization of the muscle membrane) does not affect the inhibitory action of neostigmine on m.e.p.p. frequency (Fig. 13) and we again conclude that cholinergic agents probably do not operate at the terminals via an increase in PK. DISCUSSION
The evidence summarized in the Introduction (see also Miyamoto, 1978) seems to show that ACh receptors are located presynaptically at the mammalian neuromuscular junction and that drug-receptor interaction can initiate antidromic firing
s tfig. Curae
M.E.P.P. FREQUENCY AND CHOLINERGIC AGENTS 101 and promote the spontaneous release of transmitter, these effects being antagonized by curare. In contrast, cholinergic agents clearly have an inhibitory effect on the release system of the frog neuromuscular junction. The average number of quanta released by each nerve impulse in the Mg2+-blocked preparation is decreased by ACh (Ciani & Edwards, 1963). Our studies show that both cholinesterase inhibitors and muscarinic agonists reduce m.e.p.p. frequency; the action is antagonized by atropine Intracellular Ca stores
BChE
Ca
gat
Atropine
Atropine
gate
v
Muscarine
*@. EGTA
I
|
~
F
~ } Metacholine
iso-OMPA
Neostigmine
Esersitnieg'rni
_
rci [C a]
Edropnoniumr
Ambenoniumn
AChE
I
Carbachol
AChR
Fig. 14. Diagram of the synaptic cleft to illustrate the proposed site of action at the frog neuromuscular junction of the agents tested in the present study. Acetylcholine (ACh) is shown with an excitatory (+) action post-synaptically and with an inhibitory (-) action presynaptically. Acetylcholine receptors (AChR) are shown as triangles; the presynaptic non-specific cholinesterase (BChE) is shown acting as an inhibitory ACh receptor, linked to the Ca2+ channels. The action of competitive inhibitors is shown by parallel bars.
but not by curare. The results with carbachol are the reverse of those obtained with a mammalian preparation; at the same concentration this agent stimulates m.e.p.p. frequency at the rat neuromuscular junction and the effect is antagonized by curare (Miyamoto & Volle, 1974). Furthermore, the amphibian preparation differs from that of the mammal in that cholinesterase inhibitors do not generate antidromic nerve impulses (Riker & Okamoto, 1969). We therefore conclude that cholinergic receptors are present on the presynaptic motor terminals of both mammals and amphibians but that the sites differ in their properties in these two classes of vertebrates. Drugreceptor interaction is stimulatory in the mammal and is blocked by curare, whereas the sites are muscarinic in the frog and they serve to reduce the spontaneous release of transmitter (Fig. 14). However, the way in which ACh itself acts at the frog
C. J. DUNCAN AND S. J. PUBLICOVER presynaptic terminals is not yet clear. Katz & Mfiledi (1972) report that ACh increases the rate of spontaneous release at some junctions and suggest that the variability is related to the initial level of Em at the terminals, whereas Ciani & Edwards (1963) report that the transmitter reduces evoked release. It could be suggested that the cholinesterase inhibitors act post-synaptically in frog, thereby increasing the concentration of ACh which then acts at the presynaptic sites. We consider this hypothesis unlikely since: (i) atropine antagonizes the effect of both muscarine and neostigmine, (ii) ambenonium has an obvious post-synaptic effect (augmenting m.e.p.p. amplitude and duration) but has little presynaptic effect (compare the effect of iso-OMPA), (iii) cholinesterase is known to be localized presynaptically (Koelle, 1961, 1962). We believe that the general cholinesterase inhibitors, the muscarinic agonists and iso-OMPA all act at the same presynaptic site since: (i) they produce a similar pattern of response, (ii) the muscarinic antagonist atropine blocks neostigmine action and (iii) neostigmine pre-treatment blocks the action of muscarine. It is probable that the cholinesterase identified histochemically at the presynaptic terminals constitutes this muscarinic receptor, as previously suggested (Duncan, 1967); the receptor appears to be a non-specific cholinesterase (EC 3. 1. 1 .8) which is inhibited by iso-OMPA. The results summarized in Figs. 10 and 11 show that these presynaptic effects are dependent on extracellular Ca2+; the results suggest that cholinesterase inhibitors reduce Ca2+ permeability at the terminals. Evidence is accumulating that intracellular Ca2+ concentration at the terminals has a marked effect on m.e.p.p. frequency, although reductions in Ca2+ fluxes across the plasma membrane do not produce dramatic effects (Statham & Duncan, 1975; Duncan & Statham, 1977; Publicover & Duncan, 1979). The reduction of the mean F1/F0 to 0-5 in the present studies therefore probably represents a substantial reduction in Ca2+ influx. It could be argued that cholinergic agents act by hyperpolarizing the terminals via changes in PNa or PK which in turn causes a reduction in Pc. We believe that this hypothesis is unlikely since: (i) m.e.p.p. frequency in the frog is largely insensitive to hyperpolarization (Del Castillo & Katz, 1954) and (ii) the action of neostigmine is not affected by changes in the concentrations of extracellular Na+ (Fig. 12) or K+ (Fig. 13). We conclude that cholinergic agents have a direct effect on the Ca2+ channels and markedly reduce Ca2+ influx. 102
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