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Effects of the sulphydryl inhibitor N-ethyl-maleimide on the phrenic nerve and diaphragm muscle of the rat
Neuropharmacology Vol. 28, No. 8, pp. 765-773, 1989 Printed in Great Britain. All rights reserved
Copyright 0
0028-3908/89 $3.00 + 0.00 1989 Maxwell Pergamon Macmillan plc
EFFECTS OF THE SULPHYDRYL INHIBITOR N-ETHYL-MALEIMIDE ON THE PHRENIC NERVE AND DIAPHRAGM MUSCLE OF THE RAT A. RCXD Department of Physiology and Biochemistry, Dental Faculty, University of Oslo, P.O. Box 1052, Blindern, 0316 Oslo 3, Norway (Accepted 16 February
1989)
Summary-N-Ethyl-maleimide (NEM, 2.5 x 10msM) inhibited the compound action potential of the phrenic nerve and increased the spontaneous release of transmitter from the nerve terminals, recorded as miniature endplate potentials. The first effect was the cause of a blockade of the phrenic nerve diaphragm preparation, during indirect stimulation. The left phrenic nerve was more susceptible to inhibition than the right. An increase of the threshold was observed during the progression of the inhibition. The inhibition was not use-dependent and there was no synergistic interaction with the local anaesthetic drug, tetracaine. The inhibition was partly antagonized by di-thio-threitol (3.0 x 10e3 M). The increase of spontaneous release of transmitter was not accompanied by an increase of the stimulus-evoked release since the amplitude of the endplate potential was not increased and partial inhibition caused by d-tubocurarine or magnesium chloride was not antagonized. When the concentration of NEM was increased to 2.75 x 10m4M, the directly-elicited twitches were inhibited, and the baseline tension was increased. This increase of tension was slightly reduced in a preparation depolarized with potassium chloride; a small depolarization could partly explain this effect. It was not reduced by dantrolene or in a calcium-free solution. The inhibition of the twitch and the increased baseline tension (probably a rigor) might be caused by a reduced sensitivity of the contractile proteins for calcium ions and an inhibition of the myosin ATPase activity, respectively. Ke)’ words--N-ethyl-maleimide,
di-thio-threitol,
phrenic
nerve, excitation
threshold,
diaphragm
muscle,
rigor tension.
Proteins containing sulphydryl groups are widely distributed in cell membranes as well as in intracellular structures. Therefore, it is not surprising that agents which react with sulphydryl groups affect several cellular functions. N-Ethyl-maleimide (NEM), which inactivates sulphydryl groups by alkylation, readily permeates cell
membranes. In the somatic neuromuscular system, NEM may affect membrane excitability in the nerve and muscle cells, pre- and post-synaptic aspects of the neuromuscular transmission process, as well as the intracellular excitation-contraction coupling and contractile mechanism. Previous experiments have shown that NEM affects several of these sites. An inhibition of excitation in nerve has been observed by Huneeus-Cox, Fernandez and Smith (1966) and by Chang, Lu, Wang and Chuang (1970). An interaction with membrane excitability, comparable to that found with local anaesthetic drugs, has been suggested by Shrager (1977), based on his experiments on the giant axon of the squid and on the observation by Marquis and Mautner (1974a,b) that NEM caused a use-dependent inhibition, a characteristic feature of inhibition by local anaesthetics (Courtney, 1975; Strichartz, 1976). At the neuromuscular junction, a marked increase of spontaneous release of transmitter has been demonstrated by Chang et al. (1970). The sarcolemma is known to be depolarized by NEM
(Chang et al., 1970). An intracellular interaction between NEM and the release and reuptake of calcium ions by the sarcoplasmic reticulum (Kirsten and Kuperman, 1970; Saito-Nakatsuka, Yamashita, Kubota and Kawakita, 1987), and with the contractile proteins (Crowder and Cooke, 1984), have been suggested. These interactions could be the cause of the development of tension caused by NEM (Chang et al., 1970). The present experiments were planned to investigate some aspects of the effects of NEM on the neuromuscular preparation, which have not been fully elucidated earlier. (1) At a smaller concentration than that leading to an immediate inhibition, the possibility of use-dependence and synergistic interaction with a local anaesthetic was investigated to describe the inhibition of excitability more closely. Moreover, the inhibition of compound nerves with different fibre populations (phrenic nerve and sciaticperoneal nerve) was compared. (2) The relationship between evoked and spontaneous release of transmitter was investigated in a cut preparation and by pharmacological interaction with d-tubocurarine, magnesium chloride, neostigmine and tetraethylammonium. (3) The development of tension by a larger concentration of NEM was characterized more closely by comparing the tension in control, potassium chloride-depolarized and calcium-free preparations and by testing the effect of dantrolene, an
166
A. &SD
inhibitor of excitation-contraction coupling (Ellis and Bryant, 1972; Putney and Bianchi, 1974; Morgan and Bryant, 1977).
METHODS
Materials
Wistar albino rats of either sex, weighing 100-150 g, were used in the experiments. (Rats greater than 150 g were avoided because they showed a reduced sensitivity to some of the effects of NEM.) The rats were anaesthetized with diethylether and decapitated. Each hemidiaphragm with its phrenic nerve was dissected as described by Biilbring (1946). The preparations were suspended in a Tyrode solution containing (in mM): Na+ 150; K+ 2.7; Ca2+ 1.8; Mg2+ 0.1; Cl- 140; HCO; 12; H,PO; 0.4; dextrose 11. In one series of experiments the concentration of MgCl, was increased to 5.1 mM. In another series the solution was calcium-free. The volume of the organ bath was 200ml. The temperature of the bath was kept at 37°C and the solution was continuously gassed with 95% 02+ 5% C02, which gave a pH = 7.4. The following chemical agents and drugs were used: N-ethyl-maleimide (NEM), di-thio-threitol (DTT), tetraethylammonium bromide, tetracaine HCl, neostigmine bromide and d-tubocurarine HCl. All drugs and agents were Sigma products.
the amplitude of the twitch prior to the addition of NEM. Recording of compound action potential
The phrenic nerve was dissected, transferred to the bath and connected to stimulating and recording electrodes of the Ag/AgCl suction type, as described by Lilleheil (1970). In one series of experiments, the sciatic-peroneal nerve was applied. The stimulating pulses were delivered through a Grass Model SIU 5 stimulus isolation unit. The voltage was set at the threshold for maximum amplitude of the compound action potential and the duration was 50psec. The compound action potential was amplified by a Grass Model P 16 a.c./d.c. microelectrode amplifier, displayed on a Tektronix Model 5030 dual beam oscilloscope in the a.c. mode and photographed with a Grass Model C4N kymograph camera. Intracellular microelectrode recording
Conventional microelectrodes, filled with 3 M KCl, were used for intracellular recording of the membrane potential, endplate potentials and miniature endplate potentials in muscle cells. Endplate potentials were recorded in preparations which were cut to avoid action potentials and twitching, as described by Barstad and Lilleheil (1968). Potentials were amplified, displayed in the d.c. mode and photographed as the compound nerve action potentials. Statistics
Contraction recording
The diaphragm preparations were suspended with the rib end of the fibres pushed into a perspex holder with Ag/AgCl wires, serving as electrodes for direct stimulation. The electrode used for indirect stimulation of the phrenic nerve was of the type described by Bulbring (1946). The square wave pulses for stimulation were delivered by a Grass Model S 48 stimulator. The voltage for indirect stimulation was usually set at the threshold for maximum contraction of the muscle and the pulse duration was 50 psec. The voltage for direct stimulation was 150 V and the pulse duration was 0.5 msec. During direct stimulation, the pulses were delivered through a Grass Model CCU 1 constant-current unit. The frequency during twitch stimulation was 0.1 Hz. In some experiments tetanic stimulation was performed with pulse trains of 50 Hz for 10 sec. The tendinous end of the muscle was tied to a Grass force displacement transducer (Model FT03C or FTlOC), which was connected to a Grass Model 7 polygraph for recording of contraction of the muscle. The effects on the nerve and neuromuscular transmission were usually investigated at a concentration of NEM of 2.5 x lo-‘M and the effects on muscle at a concentration of 2.75 x 10m4M. The time periods to the initiation and completion of the inhibition were measured in min. The stimulus-independent tension at the large concentration of NEM was expressed as a percentage of
The observations are presented as mean + SEM (No. of observations). Tests of statistical significance were performed with the Student’s t-test for unpaired observations. A P-value of P = 0.05 or less was considered to be statistically significant. RESULTS
Effects on the indirectly-stimulated preparation
The effect of NEM (2.5 x IO-’ M-7.5 x 10m6M) on the twitch contractions of the indirectlystimulated left phrenic nerve hemidiaphragm preparation of the rat at 0.1 Hz is shown in Fig. IA-C. After a delay period, NEM caused a rapidly progressing inhibition. The time periods to initiation of the inhibition and to complete inhibition were dose-dependent. The values at 2.5 x lo-‘M are presented in Table 1. In the same rat, the preparations from the left and right side showed a different susceptibility to the inhibitory effect; the inhibition was initiated later and progressed more slowly in the preparation from the right-side (Table 1). Direct stimulation of the completely inhibited preparation gave a normal twitch response, showing that the inhibitory mechanism was located either in the phrenic nerve or in the neuromuscular transmission process. In many experiments, the amplitude of the twitch varied considerably during the inhibitory phase. This
767
Effects of NEM on nerve and muscle
NEM, 2.5x IO-’
M+
NEM. .
1.0~10-~
M
NEM. 7.5x lo-’ v
M
2.5x 1O-4 M
B
12v 6
D 2.5x lo+
M
3.0x lo-”
hi
Fig. 1. (A) The effect of N-ethyl-maleimide (NEM) on the phrenic nerve diaphragm preparation of the rat. After a delay period, a complete inhibition ensued during indirect twitch (0.1 Hz) stimulation. Thereafter, the preparation was stimulated directly and NEM was added to give an additional 10 times greater concentration. An increase of the baseline tension and an inhibition of the twitches followed. (B) At a smaller concentration of NEM, the delay period before the inhibition increased and the progression of the inhibition was slowed. Note the irregular twitch profile during the inhibitory phase. (C) When the stimulating current was increased during the inhibitory phase, a partial and transient reversal of the inhibitory effect was obtained. (D) Addition of dithio-threitol (DTT) caused recovery of the completely inhibited twitches. Note the irregular twitch profile in the recovery phase.
was most easily observed at a smaller concentration of NEM (Fig. 1B). When the stimulating current to the phrenic nerve was increased, a transient antagonism of the inhibition was obtained (Fig. 1C). Both observations indicated an increase of the threshold of the phrenic nerve. In some experiments, the frequency of stimulation was increased to a greater twitch frequency (1 Hz) and in other experiments, 10 set periods with tetanic stimulation at 50 Hz, were included both during the delay period and during the progression of the inhibition. No evidence of an additional frequencydependent inhibition similar to that found with tetracaine (see Fig. 6D) was observed in these experiments. The inhibitory effect was irreversible upon extensive washing. The twitch response could, however, be partially recovered by addition of DTT (3.0 x lo-’ M). The amplitude of the recovered twitches varied considerably (Fig. ID), showing that many of the fibres were stimulated around their threshold values. In 9 experiments, a maximum recovery of 30.7 + 5.3% was obtained after exposure to DTT for 30-60min. Efects on the directly-stimulated preparation Increasing the concentration of NEM to 2.75 x 10-j M caused a depression of the twitch of the directly-stimulated preparation. Simultaneously, a slow monophasic development of tension was observed as an elevation of the base-line (Fig. 1A; Table
2). The inhibitory effect on the twitches and the elevation of the baseline tension were irreversible upon washing and were not antagonized by DTT. The stimulus-induced twitch tension was not changed by varying the intensity of the directly-applied stimulating current. The slow stimulus-inde~ndeni development of tension was also observed in preparations which were not stimulated electrically and it was
Table 1. inhibitory effect of NEM (2.5 x IO-* M) on the indirectlystimulated diaphragm and on the isolated phrenic nerve Time period to Initial inhibition (An) Left Right Phrenic nerve; Left Right dTC-depressed diaphragm Left MgCI,-depressed diaphragm Left TEA + tetanus-depressed diaphragm Left Right Tetracaine-depressed diaohranm _ Left Diaphragm;
Complete inhibition
(min)
N
15.6 i 0.7 19.8 * 1.5’ 13.7 t 1.0 17.1 *0.9*
19.9 & 32.7 + 29.9 + 40.7 *
1.0 3.t* 2.5 3.4.
12 9 7 7
16.7 k 1.0
21.0+ 1.4
6
18.2 + 0.8**
23.4 + 0.9”
6
10.3 + 1.1** 9.8 5 IA*’
13.2 k 1.1
Values are given as mean rt SEM.
11 II
17.2rtl.4
6
N = number of experiments. *Significant differences between left and right preparations. **Significant differences from effects of NEM on control pnpara-
tions (two upper rows). Level of significance: P d 0.05.
A.
768
Table 2. Development of an increased baseline tension caused by NEM (2.75 x IO-‘M) on the diaphragm Maximum tension Control diaphragm KCI-depolarized diaphragm Diaphragm in Cal+-free solution
(%)
Time to maximum tension (min)
N
97.9 k 6.8 69.0 + 5.3. 79.4 + 7.9
36.9 + I .9 42.8 5 2.8 40.0 + 3.3
12 6 5
Maximum tension in percentage of’ twitch tension, prior to addition of NEM. Values are given as mean f SEM. N = number of experiments, *Significant difference from control diaphragm. Level of significance: P $0.05.
not significantly changed in calcium-free solution (Table 2). In some experiments, NEM (2.75 x 10m4M) was added to preparations which were depolarized by potassium chloride (1 .O x lo-* M) to give a transient potassium chloride-induced contracture. The NEMinduced tension was then moderately, but significantly, reduced (Table 2). In other experiments, the effect of the musclerelaxing drug, dantrolene (7.5 x 10-j M), on the slow baseline tension was tested by addition of dantrolene at the time of the maximum tension. Dantrolene did not antagonize the increased baseline tension. Effects on the isolated phrenic nerve
The compound action potential of the phrenic nerve was inhibited by NEM (2.5 x 10-j M), in the same way as the indirectly-stimulated preparation (Fig. 2A). After a delay period, a rapidly progressing inhibition ensued and the inhibition was transiently antagonized by increasing the stimulating current. The left nerve was more susceptible to inhibition than the right one (Table 1). The different times for the initiation and completion of the inhibition between the nerves and the neuromuscular preparations may be explained by: (1) a margin of safety between nerve action potentials and neuromuscular transmission and (2) an unequal representation in the compound action potential of superficial and deep fibres. The frequency-dependence of the inhibition was tested at
&ED
increased twitch (1 Hz) and tetanic (50 Hz) stimulation; the frequency of stimulation did not affect the inhibition. In some experiments the inhibitory effect on the sciatic-peroneal nerve, which contains both A alpha and A beta fibres, was tested for comparison with the phrenic nerve, which contains preferentially A beta fibres (Fernand and Young, 1951). Only the second phase of the biphasic compound action potential of the sciatic-peroneal nerve, was inhibited by NEM (2.5 x lo-’ M) (Fig. 2B). Effects on the sarcolemma and the neuromuscular junction
The effect of NEM (2.75 x 10m4M) on the membrane potential of the sarcolemma was investigated with intracellular microelectrode recordings. A slowly developing depolarization was found (Fig. 3). Microelectrode penetrations at the motor endplate were ascertained by the recording of miniature endplate potentials. After a delay period of about 10min (which was shorter than the delay period of the inhibition of the twitches during indirect stimulation), NEM (2.5 x lO_jM) caused a very marked increase of the frequency of miniature endplate potentials, showing that the spontaneous release of transmitter was dramatically increased (Figs 4 and 5A). In spite of the high frequency, miniature endplate potentials with increased amplitude were not usually observed. There was thus little evidence of clustering together of individual miniature endplate potentials. The increase in frequency lasted for a considerable time beyond the time of the complete stimulus-induced inhibition. Some experiments in cut preparations were performed to investigate the effect on the endplate potential. No increase of the amplitude of the endplate potentials was observed, in spite of the enormous increase in spontaneous release, and the ap$earance of the endplate potentials was unchanged. During periods of tetanic (50 Hz) stimulation, there was no marked reduction of the amplitude of the endplate potential (Fig. 5B).
Fig. 2. The effect of NEM (2.5 x low5 M) on the compound action potential of isolated nerves. (A) Effect on the single-peaked potential of the left phrenic nerve. Note that the rapid inhibitory phase occurred after a delay period. Calibration: vertical, 0.5 mV; horizontal, 1 msec. (B) Effect on the two-peaked
potential of the sciatic-peroneal nerve. Note that only the second phase (probably corresponding to the single phase of the phrenic nerve) was inhibited. Calibration: vertical, I mV; horizontal, 1msec.
769
Effects of NEM on nerve and muscle 0 >
E
-20 [
s
-40
i ..g
NEM, 2.5~1O-~hi
+
2.5x1d4M
B -80 D B 5 f
/ -8O-
I +
-1ooL;
IO
20
30
40
50
80
o
min
Fig. 3. Intraeell~ar recording of the membrane potential of muscle cells of the diaphragm. The points represent recordings from 12-30 cells in the preceding 5 min period; SEM values are indicated. A depolarization occurred after the large concentration of NEM was added.
In some of the experiments with recording of endplate potentials during tetanic stimulation, the frequency of miniature endplate potentials before and after this period of stimulation was observed. The tetamc stimulation caused a further increase of the high frequency miniature endplate potentials in the post-tetanic period, both in control and NEM-exposed preparations (Fig. 93). E#ects on ~re~urutjons pretreated with drugs uflecting the neuromuscular junction
The microelectrode
recordings
obviously
repre-
sented a few cells in the superficial cell layer. Therefore, the effects on all the endplates of the whole preparation were also monitored by recording the effects on contractions of combining NEM with drugs known to affect the neuromuscular junction. In the NEM-treated preparation, no sign of the increased frequency of miniature endplate potentials was observed on the tension recordings of a resting preparation (Fig. 6A). Addition of neostigmine (7.0 x 10e6 M) to inhibit the cholinesterase and prolong the individual miniature endplate potentials, caused an irregular twitching of the preparation. However, this twitching was not more extensive than that caused by neostigmine, in a preparation without NEM (Fig. 6A). This confirmed the observation that the miniature endplate potentials were evenly distributed along the time axis with no marked tendency to clustering. d-Tubocurarine (7.3 x IO-‘M) or an increased concentration of magnesium chloride (5.1 x 1O-3 M) was used to partially inhibit some preparations, so that many of the endplate potentials were probably closely beyond the fringe to elicit action potentials. Therefore, a reduced tension ensued. If NEM caused an increase of the amplitude of the endplate potential, an increase of the amplitude of the twitch should ensue, but that was never observed (Fig. 6B). The observation with microelectrodes of an unchanged
NEM, 2.5~lO’~M
. .
l .
l .
.
i
.
t . .
l
:
.
. l .
I
0
,
I
I
I
20
40
SO
80
mln
&
100
I
120
Fig. 4. The NEM-induced increase of the frequency of miniature endplate potentials (MEPPs). Frequencies greater than 500 Hz could not be counted and were recorded as 500 Hz. Note that the period with high frequency lasted beyond the time of complete inhibition of the indir~tly~licit~ twitch contractions.
110
A. Control
-5 M
kZ9ED
A
B
c
NEM. 2.5x10-’
dTC. 7,3xlo-‘M .
M
NW. 7.0x10-eM
NW& 7.0x10-6M
NEM. 2.5x10-JM .
TEA. 2.6x 1,O‘3 M_
I
NEM. 2.5X
NEM. 2.5x10-5M .
D
L Fig. 5. Recordings from the endplate of control and preparations exposed to NEM. Downward deflection represents depolarization. Recording on moving film; recording started from top. (A) Miniature endplate potentials in one control and two preparations exposed to NEM showing a moderate (middle trace) and a marked (lower trace) increase of frequency caused by NEM. Calibration: vertical, 16mV; horizontal, 20msec. (B) Endplate potentials evoked at about 50 Hz stimulation in one control and one NEMexposed preparation. Note the post-tetanic potentiation of the frequency of miniature endplate potentials in both preparations. Calibration: vertical, 30 mV; horizontal, 8 msec.
amplitude of the endplate potential was thus confirmed for the whole preparation. The times to initial and complete inhibition were unchanged withpretreatment with dTC, but significantly increased with pretreatment with magnesium chloride. Experiments with an increased concentration of calcium chloride should be performed to test if this was an unspecific effect of divalent cations. In another series of experiments the transmitter stores were depleted by periods of tetanic stimulation of a preparation treated with TEA (2.6 x lo-’ M), as described by Rnred (1989). When the twitches were markedly reduced, NEM (2.5 x 10-j M) caused an inhibition which was initiated after a significantly shorter delay than the NEM-induced inhibition of the untreated preparation (Fig. 6C). This inhibition appeared after the same delay time in left and right preparations (Table 1) and was not antagonized by increased stimulating current.
TetraCMW.
NEM.
e.7x10-eM r v
2.5x10-5M .
I
Fig. 6. Effects of NEM, in combination with other agents affecting neuromuscular transmission or membrane excitability. Calibration: vertical, 10 g at twitch stimulation; the amplifier sensitivity was halved during the first 5 tetanic contractions in (C) and during all tetanic contractions in (D). Horizontal, 5min at twitch stimulation; the paper speed was increased during the tetanic contractions of 10 set duration. (A) In a resting preparation, neostigmine (Neo) prolonged and increased the amplitude of the miniature endplate potentials so that some of them exceeded the threshold for excitation of the sarcolemma. Increasing the frequency of miniature endplate potentials by pretreatment with NEM did not increase the number of miniature endplate potentials exceeding the threshold. (B) Preparations were pretreated with d-tubocurarine (dTC) or MgCl, to give a partial inhibition; NEM did not antagonize this inhibition, indicating that the amplitude of the endplate potentials was not increased together with the frequency of miniature endplate potentials. (C) The store of transmitter was reduced by a combination of tetraethylammonium (TEA) and tetanic stimulation and NEM caused a slow additional inhibition due to depletion of transmitter prior to the inhibition of the phrenic nerve. Note the complete use-dependent inhibition of the 4 latest tetanic contractions. (D) Pretreatment with tetracaine did not accelerate the inhibitory effect of NEM and the tetracaine-induced usedependent inhibition was not enhanced by NEM.
Effects on preparations
pretreated
with tetracaine
By itself NEM did not cause any use-dependent inhibition. In some experiments the preparations were pretreated with the local anaesthetic drug, tetracaine (6.7 x 10e6 M), to cause a partial useNEM inhibition. Addition of dependent (2.5 x 10m5 M) did not potentiate the use-dependent inhibition (Fig. 6D). The tetanic tension at the end of the second 10 set period of stimulation, after addition of tetracaine, was (in % of predrug twitch tension) 78.0 + 5.2 (5) without NEM and 72.3 f 1.7 (4) with NEM. The difference was not significant.
Effects of NEM on nerve and muscle There was no significant change in the time to initiation or completion of the NEM-induced inhibition in the preparations which were pretreated with tetracaine (Table 1). DISCtKSION
The present experiments disclosed effects of NEM on the myelinated nerve axons as well as on the naked nerve terminals, and on the muscle cells at sites distal to the neuromuscular junction. Efsects on the phrenic neme axons Obviously, the nerve axons were much more susceptible to effects of NEM than the muscle ceils. Probably, the myelin of the axons serves to concentrate the highly lipid soluble NEM, close to the excitable axolemma. The delay period may be in accordance with this suggestion; a certain concentration of NEM might be achieved before the interference with the excitability of the membrane was initiated. The inhibitory effect on the compound action potential was obviously the cause of the inhibition during indirect stimulation of the neuromuscular preparation, since the inhibition of the nerve, as well as the indirectly stimulated preparation, could be transiently antagonized by increasing the stimulating current. Moreover, the inhibitory effect on the compound action potential, and during indirect stimulation, both showed a greater susceptibility of the left-side than of the right-side preparations. The lesser susceptibility to inhibition of the thick fast-conducting A alpha fibres, than of the thinner A beta fibres of the sciatic-peroneal nerve, shows that the diameter of the fibre affected the ability of NEM to inhibit axons. In accordance with this, the left phrenic nerve may have a population of A beta libres with a smaller diameter than the right one. Unfortunately, a comparison of the fibre populations of left and right phrenic nerves with histological methods is not yet available. Alternatively, a greater amount of fat around the right nerve may explain the reduced effect of NEM; it was more difficult to clear the right than the left nerve from the surrounding tissue during dissection. An inhibitory effect of NEM on the phrenic nerve was observed by Chang et al. (1970). They used a larger concentration of NEM and observed neither the time delay before initiation of the inhibition, nor the increase in threshold during the inhibitory period. The inhibition of the compound action potential was characterized by an increase in the threshold. This is a well known effect of local anaesthetic drugs, which inhibit sodium currents specifically (Taylor, 1959). Another effect of these drugs is the usedependence of their inhibitory effect (Courtney, 1975; Strichartz, 1976). Use-dependence has also been implied in the effect of sulphydryl inhibitors on squid giant axons (Marquis and Mautner, 1974a, b) and
771
NEM has been shown to act specifically on sodium currents in crayfish axons (Shrager, 1977). In the present experiments with rat preparations, there was no frequency dependence, neither in the deiay period before the inhibitory effect occurred, nor during the inhibitory period and there was no synergistic interaction between tetracaine and NEM. Accordingly, there is no necessary relationship between a druginduced increase in threshold and use-dependence. Therefore, the possible correlation between sulphydryl inhibitors and local anaesthetics may be limited to some aspects of their action, as suggested by Shrager (1977). The interaction of NEM with sulphydryl groups in proteins is usually regarded as an irreversible alkylation and the inhibition of the phrenic nerve was completely irreversible upon washing. However, DTT partly reversed the inhibitor effect of NEM. Di-thiol-threitol is capable of reducing disulphides quantitatively and of maintaining the sulphydryl groups in the reduced state (Cleland, 1964). The partial reversibility caused by DTT showed that the binding of NEM to sulphydryl groups of proteins may not be completely irreversible, as suggested by Beutler, Srivastava and West (1970) for binding to reduced glutathione of red cells. The incomplete reversibility may also indicate that NEM inhibits with other mechanisms, in addition to interaction with sulphydryl groups, as suggested by Marquis (1978) for the interaction of thiol with lobster giant axons. EA;ects ofNEM on the nerve terminals Another possible mechanism of inhibition might be depletion of transmitter, due to the enormous increase in spontaneous release of transmitter. However, the high frequency of miniature endplate potentials continued after the complete inhibition had occurred, and lack of transmitter caused an inhibition, prior to the block of the phrenic nerve, only if the stores of transmitter were depleted by treatment with TEA, combined with periods of tetanic stimulation. The increase of the frequency of miniature endplate potentials caused by NEM was reported previously by Chang et ai. (1970) and by Carmody (1978). In their experiments, the increase in frequency was transient and did not reach the large values reported here. The lower temperature, applied in the previous experiments, may explain the difference. Toxins acting on the motor nerve terminal, e.g. black widow spider venom (Longenecker, Hurlbut, Mauro and Clark, 1970), diamide (Kosower and Werman, 1971) and batrachotoxin (Albuquerque, Warnick and Sansone, 1971) can cause a dramatic increase of the frequency of miniature endplate potentials. A comparison with batrachotoxin is of special interest, since Albuquerque (1972) suggested that this toxin interacts with sulphydryl groups of membrane proteins. With both NEM and batra-
A.
112
chotoxin there was a delay before the onset of action and the effects were irreversible upon washing. However, the increase in frequency of miniature endplate potentials lasted longer with NEM, and batrachotoxin depolarized the muscle cells simultaneously with the fall of frequency of miniature endplate potentials. N-Ethyl-maleimide therefore showed a greater affinity towards the nerve cell membrane than towards muscle; a ten times greater concentration was necessary to affect the muscle cells. Agents which increase the frequency of miniature endplate potentials often cause a simultaneous increase in the evoked release of transmitter (Albuquerque et al., 1971; Longenecker et al., 1970). Balnave and Gage (1974) found that 1.0 x 10e4 M NEM caused a parallel increase in the frequency of miniature endplate potentials and amplitude of endplate potentials in the toad sartorius. The increase of evoked release may be observed as an increase of the amplitude of the endplate potential. However, in the present experiments, NEM did not cause a parallel increase of the evoked release of transmitter, as ascertained by microelectrode recording of endplate potentials in cut preparations and confirmed by the lack of antagonism by NEM of a partial curarization or magnesium-induced inhibition. This suggested that spontaneous and evoked release were partly independent. An increased osmotic pressure is also reported to increase the frequency of miniature endplate potentials, without affecting the stimulusinduced output of acetylcholine (Hubbard, Jones and Landau, 1968; Kita, Narita and Van der Kloot, 1982). The fact that the evoked output of transmitter was not increased by NEM might be the cause of the persistence of the stores of transmitter in spite of the increased spontaneous output of transmitter. Even a period of tetanic stimulation did not reduce the frequency of miniature endplate potentials. As in the control preparation (del Castillo and Katz, 1954; Liley, 1956), an increase in the frequency of miniature endplate potentials after tetanic stimulation was observed when the frequency had been previously increased by NEM. When the frequency of miniature endplate potentials was increased by depolarization with potassium chloride, tetanic stimulation induced a reduction of the frequency of miniature endplate potentials (Ohta and Kuba, 1980). Therefore, the NEM-induced increase in frequency was probably not caused by a depolarization of the nerve terminal. Efects
on the muscle cells
The effects of NEM on nerve and muscle cells differed markedly. Whereas, in the nerve cells, the excitability was affected, the effects on the muscle cells were probably caused by an intracellular interference with the sarcoplasmic reticulum which controls the release and reuptake of calcium ions, or with the contractile proteins. The small depolarizing effect on the sarcolemma,
&ED
which might be caused by the inhibitory effect of NEM on Na+, K+-ATPase (Skou and Hilberg, 1965), could only partly explain the increase in baseline tension, since only a small reduction of the NEM-induced tension was found in preparations depolarized with potassium. The simultaneously developing depression of the twitch did not show the characteristics of a depolarization-induced depression (as seen in high K+ solutions): it was not preceded by a potentiation of the twitch. It might be due to a NEM-induced loss of sensitivity of the regulatory proteins to calcium ions (Yasui, Fuchs and Briggs, 1968). A transient NEM-induced initial contracture, similar to that observed by Okamoto and Kuperman (1966) in frog sartorius muscle, was never observed in the present experiments. Kirsten and Kuperman (1970) found a slow increase of tension in frog sartorius muscle which they ascribed to an increased release of calcium ions from the sarcoplasmic reticulum. They did not investigate the effect of dantrolene on this tension. An increase in the baseline tension, induced either by depolarization or by an increased release of calcium from the sarcoplasmic reticulum, should have been inhibited by dantrolene. This drug interferes with excitation
author wishes to thank Mrs Inger
Haehre and assistance.
Steen
Mrs
Elsa
for
their
skilful
technical
Effects of NEM on nerve and muscle REFERENCES
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Barstad J. A. B. and Lilleheil G. (1968) Transversally cut diaphragm preparation from rat. An adjuvant tool in the study of the physiology and pharmacology of the myoneural junction. Arch. int. Pharmacodyn. 175: 373-390. Beutler E., Srivastava S. K. and West C. (1970) The reversibility of N-ethylmaleimide (NEM) alkylation of red cell glutathione. Biochem. biophys. Res. Commun. 38: 341-347
Bulbring E. (1946) Observations of the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmac. 1: 38-61. Burke M., Reisler E. and Harrington W. F. (1976) Effect of bridging the two essential thiols of myosin on its spectral and actin-bindine urooerties. Biochemisrrv 15: 1923-1927. Carmody J. J. (19%) Enhancement of acetylcholine secretion by two suhhydryl reagents. Eur. J. Pharmac. 47: 457460
de1 Castilh J. and Katz B. (1954) Statistical factors involved in neuromuscular facilitation and depression. J. Physiol. 124: 574-585. Chang C. C., Lu S. E., Wang P. N. and Chuang S. T. (1970) A comparison of the effects of various sulfhydryl reagents on neuromuscular transmission. Eur. J. Pharmac. 11: 195-203. Cleland W. W. (1964) Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3: 480-482. Courtney K. R. (1975) Mechanism of frequency-dependent inhibition of sodium currents in frog myelinated nerve by the lidocaine derivative GEA 968. J. Pharmac. exp. Ther. 195: 225-236.
Crowder h4. S. and Cooke R. (1984) The effect of myosin sulphydryl modification on the mechanics of fibre contraction. J. muscle Res. cell Motil. 5: 131-146. Ellis K. 0. and Bryant S. H. (1972) Excitationcontraction uncoupling in skeletal muscle by dantrolene sodium. Naunyn-Schmiedebergs
Arch. Pharmac. 274: 107-109.
Fernand V. S. V. and Young J. Z. (1951) The sizes of nerve fibres of muscle nerves. Proc. R. Sot. Med. B. 139: 38-58. Hubbard J. I., Jones S. F. and Landau E. M. (1968) An examination of the effects of osmotic pressure changes upon transmitter release from mammalian motor nerve terminals. J. Physiol. 197: 639-657. Huneeus-Cox F., Fernandez H. L. and Smith B. H. (1966) Effects of redox and sufhydryl reagents on the bioelectric properties of the giant axon of the squid. Biophys. J. 6: 675-689.
Kirsten E. B. and Kuperman A. S. (1970) Effects of sulphydryl inhibitors on frog sartorius muscle: Nethylmaleimide. Br. J. Pharmac. 40: 827-835. Kita H., Narita K. and Van der Kloot W. (1982) The relation between tonicity and impulse-evoked transmitter release in the frog. J. Physiol. 325: 213-222. Kosower E. M. and Werman R. (1971) New step in
113
transmitter release at the myoneural junction. Nature New Biol. 233: 121-123. Liley A. W. (1956) An investigation of spontaneous activity at the neuromuscular junction of the rat. J. Physiol. 132: 65&666. Lilleheil G. (1970) A simple arrangement for in vitro testing of local anaesthetic properties of drugs. Acra pharmac. toxic. 28: 63.
Longenecker H. E. Jr, Hurlbut W. P., Mauro A. and Clark A. W. (1970) Effects of black widow spider venom on the frog neuromuscular junction. Nature 225: 701-705. Marquis J. K. (1978) Analysis of the nerve-blocking action of mercurochrome, a fluorescent thiol reagent. Neuropharmacology 17: 631635. Marquis J. K. and Mautner H. G. (1974a) The binding of thiol reagents to axonal membranes: the effect of electrical stimulation. Biochem. biophys. Res. Commun. 57: 154-161.
Marquis J. K. and Mautner H. G. (1974b) The effect of electrical stimulation on the action of sulfhydryl reagents in the giant axon of squid: suggested mechanisms for the role of thiol and disulfide arouos in electricallv-induced conformational changes. J.‘;Membrane Biol. 15r0249-260. Morgan K. G. and Bryant S. H. (1977) The mechanism of action of dantrolene sodium. J. Pharmac. exp. Ther. 201: 138-147.
Murphy A. J. (1976) Sulfhydryl group modification of sarcoplasmic reticulum membranes. Biochemistry 15: 44924496. Ohta Y. and Kuba K. (1980) Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in high K+ solution. Pfriigers Arch. 386: 29-34. Okamoto M. and Kuperman A. S. (1966) Muscle contraction produced by sulphydryl inhibitors. Nufure 210: 1062-1063. Putney J. W. Jr and Bianchi C. P. (1974) Site of action of dantrolene in frog sartorius muscle. J. Pharmac. exp. Ther. 189: 202-212. Reisler E., Burke M. and Harrington W. F. (1974) Cooperative role of two sulfhydryl groups in myosin adenosine triphosphatase. Biochemistry 13: 2014-2022. Reed A. (1989) The effects of tetraethylammonium during twitch and tetanic stimulation of the phrenic nerve diaphragm preparation in the rat. Neuropharmacology 2s: 585-592.
Saito-Nakatsuka K., Yamashita T., Kubota I. and Kawakita M. (1987) Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide. J. Biochem. 101: 3655376. Shrager P. (1977) Slow sodium inactivation in nerve after exposure to sulfhydryl blocking reagents. J. gen. Physiol. 69: 183-202.
Skou J. C. and Hilberg C. (1965) The effect of sulphydrylblocking reagents and of urea on the (Na+-K+)-activated enzyme system. Biochem. biophys. Acra 110: 359-369. Strichartz G. (1976) Molecular mechanism of nerve block by local anesthetics. Anesthesiology 45: 42141. Taylor R. E. (1959) Effect of procaine on electrical properties of squid axon membrane. Am. J. Physiol. 196: 1071&1078.
Yasui B., Fuchs F. and Briggs F. N. (1968) The role of sulfhydryl groups of tropomyosin and troponin in the calcium control of actomyosin contractility. J. biol. Chem. 243: 735-742.
Copyright 0
0028-3908/89 $3.00 + 0.00 1989 Maxwell Pergamon Macmillan plc
EFFECTS OF THE SULPHYDRYL INHIBITOR N-ETHYL-MALEIMIDE ON THE PHRENIC NERVE AND DIAPHRAGM MUSCLE OF THE RAT A. RCXD Department of Physiology and Biochemistry, Dental Faculty, University of Oslo, P.O. Box 1052, Blindern, 0316 Oslo 3, Norway (Accepted 16 February
1989)
Summary-N-Ethyl-maleimide (NEM, 2.5 x 10msM) inhibited the compound action potential of the phrenic nerve and increased the spontaneous release of transmitter from the nerve terminals, recorded as miniature endplate potentials. The first effect was the cause of a blockade of the phrenic nerve diaphragm preparation, during indirect stimulation. The left phrenic nerve was more susceptible to inhibition than the right. An increase of the threshold was observed during the progression of the inhibition. The inhibition was not use-dependent and there was no synergistic interaction with the local anaesthetic drug, tetracaine. The inhibition was partly antagonized by di-thio-threitol (3.0 x 10e3 M). The increase of spontaneous release of transmitter was not accompanied by an increase of the stimulus-evoked release since the amplitude of the endplate potential was not increased and partial inhibition caused by d-tubocurarine or magnesium chloride was not antagonized. When the concentration of NEM was increased to 2.75 x 10m4M, the directly-elicited twitches were inhibited, and the baseline tension was increased. This increase of tension was slightly reduced in a preparation depolarized with potassium chloride; a small depolarization could partly explain this effect. It was not reduced by dantrolene or in a calcium-free solution. The inhibition of the twitch and the increased baseline tension (probably a rigor) might be caused by a reduced sensitivity of the contractile proteins for calcium ions and an inhibition of the myosin ATPase activity, respectively. Ke)’ words--N-ethyl-maleimide,
di-thio-threitol,
phrenic
nerve, excitation
threshold,
diaphragm
muscle,
rigor tension.
Proteins containing sulphydryl groups are widely distributed in cell membranes as well as in intracellular structures. Therefore, it is not surprising that agents which react with sulphydryl groups affect several cellular functions. N-Ethyl-maleimide (NEM), which inactivates sulphydryl groups by alkylation, readily permeates cell
membranes. In the somatic neuromuscular system, NEM may affect membrane excitability in the nerve and muscle cells, pre- and post-synaptic aspects of the neuromuscular transmission process, as well as the intracellular excitation-contraction coupling and contractile mechanism. Previous experiments have shown that NEM affects several of these sites. An inhibition of excitation in nerve has been observed by Huneeus-Cox, Fernandez and Smith (1966) and by Chang, Lu, Wang and Chuang (1970). An interaction with membrane excitability, comparable to that found with local anaesthetic drugs, has been suggested by Shrager (1977), based on his experiments on the giant axon of the squid and on the observation by Marquis and Mautner (1974a,b) that NEM caused a use-dependent inhibition, a characteristic feature of inhibition by local anaesthetics (Courtney, 1975; Strichartz, 1976). At the neuromuscular junction, a marked increase of spontaneous release of transmitter has been demonstrated by Chang et al. (1970). The sarcolemma is known to be depolarized by NEM
(Chang et al., 1970). An intracellular interaction between NEM and the release and reuptake of calcium ions by the sarcoplasmic reticulum (Kirsten and Kuperman, 1970; Saito-Nakatsuka, Yamashita, Kubota and Kawakita, 1987), and with the contractile proteins (Crowder and Cooke, 1984), have been suggested. These interactions could be the cause of the development of tension caused by NEM (Chang et al., 1970). The present experiments were planned to investigate some aspects of the effects of NEM on the neuromuscular preparation, which have not been fully elucidated earlier. (1) At a smaller concentration than that leading to an immediate inhibition, the possibility of use-dependence and synergistic interaction with a local anaesthetic was investigated to describe the inhibition of excitability more closely. Moreover, the inhibition of compound nerves with different fibre populations (phrenic nerve and sciaticperoneal nerve) was compared. (2) The relationship between evoked and spontaneous release of transmitter was investigated in a cut preparation and by pharmacological interaction with d-tubocurarine, magnesium chloride, neostigmine and tetraethylammonium. (3) The development of tension by a larger concentration of NEM was characterized more closely by comparing the tension in control, potassium chloride-depolarized and calcium-free preparations and by testing the effect of dantrolene, an
166
A. &SD
inhibitor of excitation-contraction coupling (Ellis and Bryant, 1972; Putney and Bianchi, 1974; Morgan and Bryant, 1977).
METHODS
Materials
Wistar albino rats of either sex, weighing 100-150 g, were used in the experiments. (Rats greater than 150 g were avoided because they showed a reduced sensitivity to some of the effects of NEM.) The rats were anaesthetized with diethylether and decapitated. Each hemidiaphragm with its phrenic nerve was dissected as described by Biilbring (1946). The preparations were suspended in a Tyrode solution containing (in mM): Na+ 150; K+ 2.7; Ca2+ 1.8; Mg2+ 0.1; Cl- 140; HCO; 12; H,PO; 0.4; dextrose 11. In one series of experiments the concentration of MgCl, was increased to 5.1 mM. In another series the solution was calcium-free. The volume of the organ bath was 200ml. The temperature of the bath was kept at 37°C and the solution was continuously gassed with 95% 02+ 5% C02, which gave a pH = 7.4. The following chemical agents and drugs were used: N-ethyl-maleimide (NEM), di-thio-threitol (DTT), tetraethylammonium bromide, tetracaine HCl, neostigmine bromide and d-tubocurarine HCl. All drugs and agents were Sigma products.
the amplitude of the twitch prior to the addition of NEM. Recording of compound action potential
The phrenic nerve was dissected, transferred to the bath and connected to stimulating and recording electrodes of the Ag/AgCl suction type, as described by Lilleheil (1970). In one series of experiments, the sciatic-peroneal nerve was applied. The stimulating pulses were delivered through a Grass Model SIU 5 stimulus isolation unit. The voltage was set at the threshold for maximum amplitude of the compound action potential and the duration was 50psec. The compound action potential was amplified by a Grass Model P 16 a.c./d.c. microelectrode amplifier, displayed on a Tektronix Model 5030 dual beam oscilloscope in the a.c. mode and photographed with a Grass Model C4N kymograph camera. Intracellular microelectrode recording
Conventional microelectrodes, filled with 3 M KCl, were used for intracellular recording of the membrane potential, endplate potentials and miniature endplate potentials in muscle cells. Endplate potentials were recorded in preparations which were cut to avoid action potentials and twitching, as described by Barstad and Lilleheil (1968). Potentials were amplified, displayed in the d.c. mode and photographed as the compound nerve action potentials. Statistics
Contraction recording
The diaphragm preparations were suspended with the rib end of the fibres pushed into a perspex holder with Ag/AgCl wires, serving as electrodes for direct stimulation. The electrode used for indirect stimulation of the phrenic nerve was of the type described by Bulbring (1946). The square wave pulses for stimulation were delivered by a Grass Model S 48 stimulator. The voltage for indirect stimulation was usually set at the threshold for maximum contraction of the muscle and the pulse duration was 50 psec. The voltage for direct stimulation was 150 V and the pulse duration was 0.5 msec. During direct stimulation, the pulses were delivered through a Grass Model CCU 1 constant-current unit. The frequency during twitch stimulation was 0.1 Hz. In some experiments tetanic stimulation was performed with pulse trains of 50 Hz for 10 sec. The tendinous end of the muscle was tied to a Grass force displacement transducer (Model FT03C or FTlOC), which was connected to a Grass Model 7 polygraph for recording of contraction of the muscle. The effects on the nerve and neuromuscular transmission were usually investigated at a concentration of NEM of 2.5 x lo-‘M and the effects on muscle at a concentration of 2.75 x 10m4M. The time periods to the initiation and completion of the inhibition were measured in min. The stimulus-independent tension at the large concentration of NEM was expressed as a percentage of
The observations are presented as mean + SEM (No. of observations). Tests of statistical significance were performed with the Student’s t-test for unpaired observations. A P-value of P = 0.05 or less was considered to be statistically significant. RESULTS
Effects on the indirectly-stimulated preparation
The effect of NEM (2.5 x IO-’ M-7.5 x 10m6M) on the twitch contractions of the indirectlystimulated left phrenic nerve hemidiaphragm preparation of the rat at 0.1 Hz is shown in Fig. IA-C. After a delay period, NEM caused a rapidly progressing inhibition. The time periods to initiation of the inhibition and to complete inhibition were dose-dependent. The values at 2.5 x lo-‘M are presented in Table 1. In the same rat, the preparations from the left and right side showed a different susceptibility to the inhibitory effect; the inhibition was initiated later and progressed more slowly in the preparation from the right-side (Table 1). Direct stimulation of the completely inhibited preparation gave a normal twitch response, showing that the inhibitory mechanism was located either in the phrenic nerve or in the neuromuscular transmission process. In many experiments, the amplitude of the twitch varied considerably during the inhibitory phase. This
767
Effects of NEM on nerve and muscle
NEM, 2.5x IO-’
M+
NEM. .
1.0~10-~
M
NEM. 7.5x lo-’ v
M
2.5x 1O-4 M
B
12v 6
D 2.5x lo+
M
3.0x lo-”
hi
Fig. 1. (A) The effect of N-ethyl-maleimide (NEM) on the phrenic nerve diaphragm preparation of the rat. After a delay period, a complete inhibition ensued during indirect twitch (0.1 Hz) stimulation. Thereafter, the preparation was stimulated directly and NEM was added to give an additional 10 times greater concentration. An increase of the baseline tension and an inhibition of the twitches followed. (B) At a smaller concentration of NEM, the delay period before the inhibition increased and the progression of the inhibition was slowed. Note the irregular twitch profile during the inhibitory phase. (C) When the stimulating current was increased during the inhibitory phase, a partial and transient reversal of the inhibitory effect was obtained. (D) Addition of dithio-threitol (DTT) caused recovery of the completely inhibited twitches. Note the irregular twitch profile in the recovery phase.
was most easily observed at a smaller concentration of NEM (Fig. 1B). When the stimulating current to the phrenic nerve was increased, a transient antagonism of the inhibition was obtained (Fig. 1C). Both observations indicated an increase of the threshold of the phrenic nerve. In some experiments, the frequency of stimulation was increased to a greater twitch frequency (1 Hz) and in other experiments, 10 set periods with tetanic stimulation at 50 Hz, were included both during the delay period and during the progression of the inhibition. No evidence of an additional frequencydependent inhibition similar to that found with tetracaine (see Fig. 6D) was observed in these experiments. The inhibitory effect was irreversible upon extensive washing. The twitch response could, however, be partially recovered by addition of DTT (3.0 x lo-’ M). The amplitude of the recovered twitches varied considerably (Fig. ID), showing that many of the fibres were stimulated around their threshold values. In 9 experiments, a maximum recovery of 30.7 + 5.3% was obtained after exposure to DTT for 30-60min. Efects on the directly-stimulated preparation Increasing the concentration of NEM to 2.75 x 10-j M caused a depression of the twitch of the directly-stimulated preparation. Simultaneously, a slow monophasic development of tension was observed as an elevation of the base-line (Fig. 1A; Table
2). The inhibitory effect on the twitches and the elevation of the baseline tension were irreversible upon washing and were not antagonized by DTT. The stimulus-induced twitch tension was not changed by varying the intensity of the directly-applied stimulating current. The slow stimulus-inde~ndeni development of tension was also observed in preparations which were not stimulated electrically and it was
Table 1. inhibitory effect of NEM (2.5 x IO-* M) on the indirectlystimulated diaphragm and on the isolated phrenic nerve Time period to Initial inhibition (An) Left Right Phrenic nerve; Left Right dTC-depressed diaphragm Left MgCI,-depressed diaphragm Left TEA + tetanus-depressed diaphragm Left Right Tetracaine-depressed diaohranm _ Left Diaphragm;
Complete inhibition
(min)
N
15.6 i 0.7 19.8 * 1.5’ 13.7 t 1.0 17.1 *0.9*
19.9 & 32.7 + 29.9 + 40.7 *
1.0 3.t* 2.5 3.4.
12 9 7 7
16.7 k 1.0
21.0+ 1.4
6
18.2 + 0.8**
23.4 + 0.9”
6
10.3 + 1.1** 9.8 5 IA*’
13.2 k 1.1
Values are given as mean rt SEM.
11 II
17.2rtl.4
6
N = number of experiments. *Significant differences between left and right preparations. **Significant differences from effects of NEM on control pnpara-
tions (two upper rows). Level of significance: P d 0.05.
A.
768
Table 2. Development of an increased baseline tension caused by NEM (2.75 x IO-‘M) on the diaphragm Maximum tension Control diaphragm KCI-depolarized diaphragm Diaphragm in Cal+-free solution
(%)
Time to maximum tension (min)
N
97.9 k 6.8 69.0 + 5.3. 79.4 + 7.9
36.9 + I .9 42.8 5 2.8 40.0 + 3.3
12 6 5
Maximum tension in percentage of’ twitch tension, prior to addition of NEM. Values are given as mean f SEM. N = number of experiments, *Significant difference from control diaphragm. Level of significance: P $0.05.
not significantly changed in calcium-free solution (Table 2). In some experiments, NEM (2.75 x 10m4M) was added to preparations which were depolarized by potassium chloride (1 .O x lo-* M) to give a transient potassium chloride-induced contracture. The NEMinduced tension was then moderately, but significantly, reduced (Table 2). In other experiments, the effect of the musclerelaxing drug, dantrolene (7.5 x 10-j M), on the slow baseline tension was tested by addition of dantrolene at the time of the maximum tension. Dantrolene did not antagonize the increased baseline tension. Effects on the isolated phrenic nerve
The compound action potential of the phrenic nerve was inhibited by NEM (2.5 x 10-j M), in the same way as the indirectly-stimulated preparation (Fig. 2A). After a delay period, a rapidly progressing inhibition ensued and the inhibition was transiently antagonized by increasing the stimulating current. The left nerve was more susceptible to inhibition than the right one (Table 1). The different times for the initiation and completion of the inhibition between the nerves and the neuromuscular preparations may be explained by: (1) a margin of safety between nerve action potentials and neuromuscular transmission and (2) an unequal representation in the compound action potential of superficial and deep fibres. The frequency-dependence of the inhibition was tested at
&ED
increased twitch (1 Hz) and tetanic (50 Hz) stimulation; the frequency of stimulation did not affect the inhibition. In some experiments the inhibitory effect on the sciatic-peroneal nerve, which contains both A alpha and A beta fibres, was tested for comparison with the phrenic nerve, which contains preferentially A beta fibres (Fernand and Young, 1951). Only the second phase of the biphasic compound action potential of the sciatic-peroneal nerve, was inhibited by NEM (2.5 x lo-’ M) (Fig. 2B). Effects on the sarcolemma and the neuromuscular junction
The effect of NEM (2.75 x 10m4M) on the membrane potential of the sarcolemma was investigated with intracellular microelectrode recordings. A slowly developing depolarization was found (Fig. 3). Microelectrode penetrations at the motor endplate were ascertained by the recording of miniature endplate potentials. After a delay period of about 10min (which was shorter than the delay period of the inhibition of the twitches during indirect stimulation), NEM (2.5 x lO_jM) caused a very marked increase of the frequency of miniature endplate potentials, showing that the spontaneous release of transmitter was dramatically increased (Figs 4 and 5A). In spite of the high frequency, miniature endplate potentials with increased amplitude were not usually observed. There was thus little evidence of clustering together of individual miniature endplate potentials. The increase in frequency lasted for a considerable time beyond the time of the complete stimulus-induced inhibition. Some experiments in cut preparations were performed to investigate the effect on the endplate potential. No increase of the amplitude of the endplate potentials was observed, in spite of the enormous increase in spontaneous release, and the ap$earance of the endplate potentials was unchanged. During periods of tetanic (50 Hz) stimulation, there was no marked reduction of the amplitude of the endplate potential (Fig. 5B).
Fig. 2. The effect of NEM (2.5 x low5 M) on the compound action potential of isolated nerves. (A) Effect on the single-peaked potential of the left phrenic nerve. Note that the rapid inhibitory phase occurred after a delay period. Calibration: vertical, 0.5 mV; horizontal, 1 msec. (B) Effect on the two-peaked
potential of the sciatic-peroneal nerve. Note that only the second phase (probably corresponding to the single phase of the phrenic nerve) was inhibited. Calibration: vertical, I mV; horizontal, 1msec.
769
Effects of NEM on nerve and muscle 0 >
E
-20 [
s
-40
i ..g
NEM, 2.5~1O-~hi
+
2.5x1d4M
B -80 D B 5 f
/ -8O-
I +
-1ooL;
IO
20
30
40
50
80
o
min
Fig. 3. Intraeell~ar recording of the membrane potential of muscle cells of the diaphragm. The points represent recordings from 12-30 cells in the preceding 5 min period; SEM values are indicated. A depolarization occurred after the large concentration of NEM was added.
In some of the experiments with recording of endplate potentials during tetanic stimulation, the frequency of miniature endplate potentials before and after this period of stimulation was observed. The tetamc stimulation caused a further increase of the high frequency miniature endplate potentials in the post-tetanic period, both in control and NEM-exposed preparations (Fig. 93). E#ects on ~re~urutjons pretreated with drugs uflecting the neuromuscular junction
The microelectrode
recordings
obviously
repre-
sented a few cells in the superficial cell layer. Therefore, the effects on all the endplates of the whole preparation were also monitored by recording the effects on contractions of combining NEM with drugs known to affect the neuromuscular junction. In the NEM-treated preparation, no sign of the increased frequency of miniature endplate potentials was observed on the tension recordings of a resting preparation (Fig. 6A). Addition of neostigmine (7.0 x 10e6 M) to inhibit the cholinesterase and prolong the individual miniature endplate potentials, caused an irregular twitching of the preparation. However, this twitching was not more extensive than that caused by neostigmine, in a preparation without NEM (Fig. 6A). This confirmed the observation that the miniature endplate potentials were evenly distributed along the time axis with no marked tendency to clustering. d-Tubocurarine (7.3 x IO-‘M) or an increased concentration of magnesium chloride (5.1 x 1O-3 M) was used to partially inhibit some preparations, so that many of the endplate potentials were probably closely beyond the fringe to elicit action potentials. Therefore, a reduced tension ensued. If NEM caused an increase of the amplitude of the endplate potential, an increase of the amplitude of the twitch should ensue, but that was never observed (Fig. 6B). The observation with microelectrodes of an unchanged
NEM, 2.5~lO’~M
. .
l .
l .
.
i
.
t . .
l
:
.
. l .
I
0
,
I
I
I
20
40
SO
80
mln
&
100
I
120
Fig. 4. The NEM-induced increase of the frequency of miniature endplate potentials (MEPPs). Frequencies greater than 500 Hz could not be counted and were recorded as 500 Hz. Note that the period with high frequency lasted beyond the time of complete inhibition of the indir~tly~licit~ twitch contractions.
110
A. Control
-5 M
kZ9ED
A
B
c
NEM. 2.5x10-’
dTC. 7,3xlo-‘M .
M
NW. 7.0x10-eM
NW& 7.0x10-6M
NEM. 2.5x10-JM .
TEA. 2.6x 1,O‘3 M_
I
NEM. 2.5X
NEM. 2.5x10-5M .
D
L Fig. 5. Recordings from the endplate of control and preparations exposed to NEM. Downward deflection represents depolarization. Recording on moving film; recording started from top. (A) Miniature endplate potentials in one control and two preparations exposed to NEM showing a moderate (middle trace) and a marked (lower trace) increase of frequency caused by NEM. Calibration: vertical, 16mV; horizontal, 20msec. (B) Endplate potentials evoked at about 50 Hz stimulation in one control and one NEMexposed preparation. Note the post-tetanic potentiation of the frequency of miniature endplate potentials in both preparations. Calibration: vertical, 30 mV; horizontal, 8 msec.
amplitude of the endplate potential was thus confirmed for the whole preparation. The times to initial and complete inhibition were unchanged withpretreatment with dTC, but significantly increased with pretreatment with magnesium chloride. Experiments with an increased concentration of calcium chloride should be performed to test if this was an unspecific effect of divalent cations. In another series of experiments the transmitter stores were depleted by periods of tetanic stimulation of a preparation treated with TEA (2.6 x lo-’ M), as described by Rnred (1989). When the twitches were markedly reduced, NEM (2.5 x 10-j M) caused an inhibition which was initiated after a significantly shorter delay than the NEM-induced inhibition of the untreated preparation (Fig. 6C). This inhibition appeared after the same delay time in left and right preparations (Table 1) and was not antagonized by increased stimulating current.
TetraCMW.
NEM.
e.7x10-eM r v
2.5x10-5M .
I
Fig. 6. Effects of NEM, in combination with other agents affecting neuromuscular transmission or membrane excitability. Calibration: vertical, 10 g at twitch stimulation; the amplifier sensitivity was halved during the first 5 tetanic contractions in (C) and during all tetanic contractions in (D). Horizontal, 5min at twitch stimulation; the paper speed was increased during the tetanic contractions of 10 set duration. (A) In a resting preparation, neostigmine (Neo) prolonged and increased the amplitude of the miniature endplate potentials so that some of them exceeded the threshold for excitation of the sarcolemma. Increasing the frequency of miniature endplate potentials by pretreatment with NEM did not increase the number of miniature endplate potentials exceeding the threshold. (B) Preparations were pretreated with d-tubocurarine (dTC) or MgCl, to give a partial inhibition; NEM did not antagonize this inhibition, indicating that the amplitude of the endplate potentials was not increased together with the frequency of miniature endplate potentials. (C) The store of transmitter was reduced by a combination of tetraethylammonium (TEA) and tetanic stimulation and NEM caused a slow additional inhibition due to depletion of transmitter prior to the inhibition of the phrenic nerve. Note the complete use-dependent inhibition of the 4 latest tetanic contractions. (D) Pretreatment with tetracaine did not accelerate the inhibitory effect of NEM and the tetracaine-induced usedependent inhibition was not enhanced by NEM.
Effects on preparations
pretreated
with tetracaine
By itself NEM did not cause any use-dependent inhibition. In some experiments the preparations were pretreated with the local anaesthetic drug, tetracaine (6.7 x 10e6 M), to cause a partial useNEM inhibition. Addition of dependent (2.5 x 10m5 M) did not potentiate the use-dependent inhibition (Fig. 6D). The tetanic tension at the end of the second 10 set period of stimulation, after addition of tetracaine, was (in % of predrug twitch tension) 78.0 + 5.2 (5) without NEM and 72.3 f 1.7 (4) with NEM. The difference was not significant.
Effects of NEM on nerve and muscle There was no significant change in the time to initiation or completion of the NEM-induced inhibition in the preparations which were pretreated with tetracaine (Table 1). DISCtKSION
The present experiments disclosed effects of NEM on the myelinated nerve axons as well as on the naked nerve terminals, and on the muscle cells at sites distal to the neuromuscular junction. Efsects on the phrenic neme axons Obviously, the nerve axons were much more susceptible to effects of NEM than the muscle ceils. Probably, the myelin of the axons serves to concentrate the highly lipid soluble NEM, close to the excitable axolemma. The delay period may be in accordance with this suggestion; a certain concentration of NEM might be achieved before the interference with the excitability of the membrane was initiated. The inhibitory effect on the compound action potential was obviously the cause of the inhibition during indirect stimulation of the neuromuscular preparation, since the inhibition of the nerve, as well as the indirectly stimulated preparation, could be transiently antagonized by increasing the stimulating current. Moreover, the inhibitory effect on the compound action potential, and during indirect stimulation, both showed a greater susceptibility of the left-side than of the right-side preparations. The lesser susceptibility to inhibition of the thick fast-conducting A alpha fibres, than of the thinner A beta fibres of the sciatic-peroneal nerve, shows that the diameter of the fibre affected the ability of NEM to inhibit axons. In accordance with this, the left phrenic nerve may have a population of A beta libres with a smaller diameter than the right one. Unfortunately, a comparison of the fibre populations of left and right phrenic nerves with histological methods is not yet available. Alternatively, a greater amount of fat around the right nerve may explain the reduced effect of NEM; it was more difficult to clear the right than the left nerve from the surrounding tissue during dissection. An inhibitory effect of NEM on the phrenic nerve was observed by Chang et al. (1970). They used a larger concentration of NEM and observed neither the time delay before initiation of the inhibition, nor the increase in threshold during the inhibitory period. The inhibition of the compound action potential was characterized by an increase in the threshold. This is a well known effect of local anaesthetic drugs, which inhibit sodium currents specifically (Taylor, 1959). Another effect of these drugs is the usedependence of their inhibitory effect (Courtney, 1975; Strichartz, 1976). Use-dependence has also been implied in the effect of sulphydryl inhibitors on squid giant axons (Marquis and Mautner, 1974a, b) and
771
NEM has been shown to act specifically on sodium currents in crayfish axons (Shrager, 1977). In the present experiments with rat preparations, there was no frequency dependence, neither in the deiay period before the inhibitory effect occurred, nor during the inhibitory period and there was no synergistic interaction between tetracaine and NEM. Accordingly, there is no necessary relationship between a druginduced increase in threshold and use-dependence. Therefore, the possible correlation between sulphydryl inhibitors and local anaesthetics may be limited to some aspects of their action, as suggested by Shrager (1977). The interaction of NEM with sulphydryl groups in proteins is usually regarded as an irreversible alkylation and the inhibition of the phrenic nerve was completely irreversible upon washing. However, DTT partly reversed the inhibitor effect of NEM. Di-thiol-threitol is capable of reducing disulphides quantitatively and of maintaining the sulphydryl groups in the reduced state (Cleland, 1964). The partial reversibility caused by DTT showed that the binding of NEM to sulphydryl groups of proteins may not be completely irreversible, as suggested by Beutler, Srivastava and West (1970) for binding to reduced glutathione of red cells. The incomplete reversibility may also indicate that NEM inhibits with other mechanisms, in addition to interaction with sulphydryl groups, as suggested by Marquis (1978) for the interaction of thiol with lobster giant axons. EA;ects ofNEM on the nerve terminals Another possible mechanism of inhibition might be depletion of transmitter, due to the enormous increase in spontaneous release of transmitter. However, the high frequency of miniature endplate potentials continued after the complete inhibition had occurred, and lack of transmitter caused an inhibition, prior to the block of the phrenic nerve, only if the stores of transmitter were depleted by treatment with TEA, combined with periods of tetanic stimulation. The increase of the frequency of miniature endplate potentials caused by NEM was reported previously by Chang et ai. (1970) and by Carmody (1978). In their experiments, the increase in frequency was transient and did not reach the large values reported here. The lower temperature, applied in the previous experiments, may explain the difference. Toxins acting on the motor nerve terminal, e.g. black widow spider venom (Longenecker, Hurlbut, Mauro and Clark, 1970), diamide (Kosower and Werman, 1971) and batrachotoxin (Albuquerque, Warnick and Sansone, 1971) can cause a dramatic increase of the frequency of miniature endplate potentials. A comparison with batrachotoxin is of special interest, since Albuquerque (1972) suggested that this toxin interacts with sulphydryl groups of membrane proteins. With both NEM and batra-
A.
112
chotoxin there was a delay before the onset of action and the effects were irreversible upon washing. However, the increase in frequency of miniature endplate potentials lasted longer with NEM, and batrachotoxin depolarized the muscle cells simultaneously with the fall of frequency of miniature endplate potentials. N-Ethyl-maleimide therefore showed a greater affinity towards the nerve cell membrane than towards muscle; a ten times greater concentration was necessary to affect the muscle cells. Agents which increase the frequency of miniature endplate potentials often cause a simultaneous increase in the evoked release of transmitter (Albuquerque et al., 1971; Longenecker et al., 1970). Balnave and Gage (1974) found that 1.0 x 10e4 M NEM caused a parallel increase in the frequency of miniature endplate potentials and amplitude of endplate potentials in the toad sartorius. The increase of evoked release may be observed as an increase of the amplitude of the endplate potential. However, in the present experiments, NEM did not cause a parallel increase of the evoked release of transmitter, as ascertained by microelectrode recording of endplate potentials in cut preparations and confirmed by the lack of antagonism by NEM of a partial curarization or magnesium-induced inhibition. This suggested that spontaneous and evoked release were partly independent. An increased osmotic pressure is also reported to increase the frequency of miniature endplate potentials, without affecting the stimulusinduced output of acetylcholine (Hubbard, Jones and Landau, 1968; Kita, Narita and Van der Kloot, 1982). The fact that the evoked output of transmitter was not increased by NEM might be the cause of the persistence of the stores of transmitter in spite of the increased spontaneous output of transmitter. Even a period of tetanic stimulation did not reduce the frequency of miniature endplate potentials. As in the control preparation (del Castillo and Katz, 1954; Liley, 1956), an increase in the frequency of miniature endplate potentials after tetanic stimulation was observed when the frequency had been previously increased by NEM. When the frequency of miniature endplate potentials was increased by depolarization with potassium chloride, tetanic stimulation induced a reduction of the frequency of miniature endplate potentials (Ohta and Kuba, 1980). Therefore, the NEM-induced increase in frequency was probably not caused by a depolarization of the nerve terminal. Efects
on the muscle cells
The effects of NEM on nerve and muscle cells differed markedly. Whereas, in the nerve cells, the excitability was affected, the effects on the muscle cells were probably caused by an intracellular interference with the sarcoplasmic reticulum which controls the release and reuptake of calcium ions, or with the contractile proteins. The small depolarizing effect on the sarcolemma,
&ED
which might be caused by the inhibitory effect of NEM on Na+, K+-ATPase (Skou and Hilberg, 1965), could only partly explain the increase in baseline tension, since only a small reduction of the NEM-induced tension was found in preparations depolarized with potassium. The simultaneously developing depression of the twitch did not show the characteristics of a depolarization-induced depression (as seen in high K+ solutions): it was not preceded by a potentiation of the twitch. It might be due to a NEM-induced loss of sensitivity of the regulatory proteins to calcium ions (Yasui, Fuchs and Briggs, 1968). A transient NEM-induced initial contracture, similar to that observed by Okamoto and Kuperman (1966) in frog sartorius muscle, was never observed in the present experiments. Kirsten and Kuperman (1970) found a slow increase of tension in frog sartorius muscle which they ascribed to an increased release of calcium ions from the sarcoplasmic reticulum. They did not investigate the effect of dantrolene on this tension. An increase in the baseline tension, induced either by depolarization or by an increased release of calcium from the sarcoplasmic reticulum, should have been inhibited by dantrolene. This drug interferes with excitation
author wishes to thank Mrs Inger
Haehre and assistance.
Steen
Mrs
Elsa
for
their
skilful
technical
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