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The action of cholinergic antagonists on spontaneous excitatory potentials recorded from the body wall muscles of the leech Hirudo medicinalis
Comp. Biochem. Physiol., 1970, Vol. 32, pp. 691 to 701. Pergamon Press. Printed in Great Britain
T H E A C T I O N OF C H O L I N E R G I C A N T A G O N I S T S O N SPONTANEOUS EXCITATORY POTENTIALS RECORDED F R O M T H E B O D Y W A L L M U S C L E S OF T H E L E E C H H I R UDO M E D I C I N A L I S R. J. WALKER, G. N. WOODRUFF and G. A. KERKUT Department of Physiology and Biochemistry, The University of Southampton, Southampton SO9 5NH (Received 30 M a y 1969)
Intracellular recordings were made from the body musculature of the leech Hirudo medicinalis. Resting potential values ranged from - 2 2 to - 6 4 mV. Excitatory potentials ranged in amplitude from 100/*V to 30 mV. 2. Atropine, benzoquinonium, decamethonium, gallamine, hexamethonium and tubocurarine were applied to the excitatory potentials by means of diffusion electrodes with tip diameters of between 2 and 10/z. 3. Benzoquinonium, decamethonium, gallamine and tubocurarine all decreased the amplitude of the spontaneous excitatory potentials without altering their frequency. 4. It is concluded that all four antagonists exerted their action mainly postsynaptically on the muscle. Abstract--1.
INTRODUCTION THE ACETYLCHOLINE-INDUCED contractions of the body wall musculature of the leech are antagonized by a n u m b e r of cholinergic antagonists. D-Tubocurarine is a potent blocker of these contractions (Minz, 1932; M a c i n t o s h & Perry, 1952; Flacke & Yeoh, 1968). Gallamine will also antagonize the effect of acetylcholine while atropine, hexamethonium, mecamylamine and procaine have no effect (Flacke & Yeoh, 1968). It is possible to record intracellular excitatory potentials from leech muscle (Washizu, 1967; Walker et al., 1968). It has been suggested that acetylcholine may be responsible for excitatory transmission at the leech neuromuscular junction (Bacq & Coppee, 1937). If this is the case then one would expect that compounds which antagonized the action of acetylcholine would also depress the excitatory potentials. T h e present study investigates the effect of a n u m b e r of cholinergic antagonists on these excitatory potentials. MATERIALS AND METHODS
All experiments were performed on the medicinal leech Hirudo medicinalis. The dissection and recording system were similar to that described by Walker et al. (1968). The cholinergic antagonists were applied to the muscle by means of diffusion electrodes. The glass electrodes were filled with water and the water removed from the shank of the 691
692
R. J. WALKER, G. N. WOODRUFFAND G. A. KERKUT
electrode. The compound was then introduced into the shank using a fine pipette. The electrodes were viewed under a microscope and the tip was slightly broken with a glass rod to give a tip diameter of between 2 and 10/~. The concentrations of the compounds used were as follows: atropine, 20mg/ml; benzoquinonium, 10mg/ml; decamethonium, 10 mg/ml; gallamine, 40 mg/ml; hexamethonium, 10 mg/ml; tubocurarine, 1 mg/ml. The following compounds were used: atropine sulphate--British Drug Houses; decamethonium iodide, hexamethonium bromide--Koch-Light; benzoquinonium (Mytolon) chloride-Bayer Products ; gallamine (Flaxedil) triethiodide--May & Baker; o-tubocurarine chloride-Burroughs Wellcome. RESULTS T h e best recordings in terms of the amplkude of the excitatory potentials and in the time for which the electrode remained in the fibre were obtained w k h microelectrodes with a resistance of between 30 and 60 Mfl. Recordings made with electrodes with a resistance of less than 20 M f l usually declined within a few minutes, the fibre resting potential depolarizing slowly towards zero while the excitatory potentials rapidly declined in amplitude. Using high-resistance electrodes k was possible to remain in a fibre for up to 3 hr. On many occasions during experiments the muscle would contract and the electrode come out of the fibre. The excitatory potentials were very sensitive to magnesium, 5-10 m M MgC12 abolished most of the activity. Excitatory potentials could be obtained from a number of sites along a given fibre. T h e excitatory potentials ranged in amplitude from 100 tLV to 30 mV w k h a duration of up to 100 msec (Fig. la, b). In addition to the excitatory potentials, inhibitory potentials can also be obtained (Fig. lc). These inhibitory potentials are most frequently obtained from the circular muscle. T h e y range in size from 100 t~V to 4 mV, w k h a duration of up to 150 msec. This present study is only concerned with the excitatory potentials and the effect on them of various cholinergic blocking agents. The diffusion electrode containing the blocking agent once lined up close to the recording electrode was left in position for between 30 and 60 sec. If an effect was going to occur it always did within a minute of bringing up the diffusion electrode.
Tubocurarine T h e diffusion electrodes were filled with a solution of I mg/ml D-tubocurarine. This concentration depressed the amplitude of the excitatory potentials (Fig. 2) but had little effect on their frequency. The recovery time after this dose varied from 10 to 30 min, though often the electrode became dislodged from the fibre before the activity had recovered. Although after 30 min activity could be obtained from other fibres in the preparation. In the experiment shown in Fig. 2, tubocurarine treatment resulted in the hyperpolarization of the membrane potential from an initial value of - 32 mV to - 42 mV over a period of about 90 see. Gallamine Gallamine was applied via the diffusion electrode at a concentration of 40 mg/ml. Gallamine decreased the amplitude of the excitatory potentials (Fig. 3), though it
EFFECT OF C H O L I N E R G I C A N T A G O N I S T S ON LEECH M U S C L E P O T E N T I A L S
693
had little effect on the frequency. However, at this concentration the amplitude was so depressed as to make the potentials almost merge into the background noise for a few seconds during the maximum effect of the compound. Gallamine often appeared to depolarize the membrane and transiently increase the amplitude
S / FIG. 1. Examples of excitatory and inhibitory potentials recorded from leech muscle. A. Excitatory potentials recorded from leech muscle; resting potential, -40mV; voltage scale, 5 mV, time scale, 150msec; B. Excitatory potentials recorded from leech muscle; resting potential, -55 mV; voltage scale, 10 mV, time scale, 100 msec; C. Inhibitory potentials and action potentials recorded from leech muscle; resting potential, - 30 mV; voltage scale, 4 mV, time scale, 150 reset.
of the excitatory potentials prior to hyperpolarizing the membrane. In Fig. 3, following the application of the gallamine, the resting potential fell from - 22 mV to - 1 8 mV and then rose to - 2 5 mV by the end of trace B. Six rain later when trace C was taken the resting potential was - 2 6 mV. Two and a half rain later, trace D, the resting potential was still - 26 mV, but the amplitude of the excitatory potentials was beginning to recover. The excitatory potentials in Fig. 3 recovered to normal after about 12 min.
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FIG. 2. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing tubocurarine (1 mg/ml) was positioned close to the recording electrode at the point marked J' on trace A. The resting potential in trace A was - 3 2 mV, in trace B, - 3 4 mV; in trace C, - 3 7 mV which hyperpolarized further to - 4 2 mV; in trace D, - 3 5 mV. Traces A, B and C are continuous; there is a gap of 20 rain between traces C and D. The time scale is marked in seconds.
Benzoquinonium Benzoquinonium was applied via the diffusion electrode at a concentration of 10 mg/ml. At this concentration there was no indication of a depolarization and block. T h e c o m p o u n d greatly reduced the amplitude of the excitatory potentials without altering their frequency (Fig. 4). Even during the m a x i m u m effect of the c o m p o u n d the frequency remained remarkedly constant as can be seen by comparing traces A and C in Fig. 4. T h e benzoquinonium increased the resting
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FzG. 3. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing gallamine (40 mg/ml) was positioned close to the recording electrode at the point marked ~ on trace A. The resting potential in trace A was - 22 mV; in trace B, - 18 mV and then it rose to - 25 mY; in trace C, --26 mV; in trace D, - 2 6 inV. Traces A and B are continuous; there is a gap of 6 min between traces B and C; and a gap of 150 sec between traces C and D. The time scale is marked in seconds. potential f r o m - 2 8 m V to - 3 0 mV. T h e r e is a gap of 25 rain between traces C and D. Figure 5 shows the effect of benzoquinonium on the m a x i m u m amplitude of the excitatory potentials. T h e m a x i m u m excitatory potential height was 6 mV, b u t this was depressed to less t h a n 1 m V by the benzoquinonium. After approximately 30 min, the excitatory potentials had returned to their previous amplitude of 6 mY.
Decamethonium T h e diffusion electrodes contained decamethonium at a concentration of 10 mg/ml. T h i s c o m p o u n d reduced the amplitude of the excitatory potentials b u t had no effect on their frequency (Fig. 6). I n this experiment the decamethonium
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FIG. 4. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing benzoquinonium (10 mg/ml) was positioned close to the recording electrode at the point marked t on trace A. The resting potential in trace A was - 28 mV and in trace C, - 30 mV. Traces A and B are continuous; there is a gap of 60 sec between traces B and C and a gap of 25 min between traces C and D. increased the resting potential f r o m - 6 4 m V to - 6 6 mV. T h e effect on the frequency of excitatory potentials over 4 m V in amplitude following decamethon i u m is shown in Fig. 7. T h e rate of these large potentials was 12-14/15 sec, but following the application of the decamethonium the rate of these large excitatory potentials was reduced to zero. T h e excitatory potentials returned to their previous amplitude after about 3 min. T h e resting potential prior to the application of the d e c a m e t h o n i u m was - 50 mV. Following the application of the decamethonium, the resting potential hyperpolarized to - 6 0 m V and then returned to - 55 m V 4 min after the decamethonium.
EFFECT OF C H O L I N E R G I C ANTAGONISTS ON LEECH MUSCLE P O T E N T I A L S
697
Atropine and hexamethonium Atropine when applied via the diffusion electrode at a concentration of 20 mg/ml failed to alter the excitatory potential height. H e x a m e t h o n i u m at the same concentration likewise had little effect on excitatory activity. Large doses added to the bath reduced the amplitude of the excitatory potentials but also depolarized the membrane potential to zero. In some experiments the effect of several blocking agents was tested in turn via diffusion electrodes on the same fibre. U n d e r these conditions it was found that while, for example, benzoquinonium would reversibly reduce the amplitude of the excitatory potentials, atropine and hexamethonium failed to have an effect. -x-x-x-x-x-x--x--x-x-xox
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FIG. 5. A graph to show the effect of benzoquinonium (10 mg/ml applied via a diffusion electrode) on the maximum amplitude of excitatory potentials recorded from leech muscle. The maximum height of the potentials was depressed from 6 mV to a minimum of 0"8 mV. After about 30 rain the amplitude of the excitatory potentials had returned to 6 mV. Benzoquinonium was applied at the point marked 1'. The ordinate is the maximum amplitude of the excitatory potentials recorded per 15-sec period; the abscissa is the time in 1-min periods. DISCUSSION It is generally accepted that cholinergic antagonists such as tubocurarine act on the postsynaptic membrane to block competitively the effect of acetylcholine. For example, it has been shown by Katz & Miledi (1965) that tubocurarine acts postsynaptically on the miniature end-plate potential and not presynaptically on the nerve terminal in the frog. However Riker (1966) and Standaert (1964) have
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FIG. 6. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing decamethonium (10 mg/ml) was positioned close to the recording electrode at the point marked ~' on trace A. The resting potential in trace A was - 6 4 mV and in trace B, - 6 6 mV. Traces A, B and C are continuous ; there is a gap of 40 sec between traces C and D. presented evidence for a presynaptic role for tubocurarine at the m o t o r nerve terminal in the cat. Antagonists which depolarize and block, such as decamethonium, also act on the postsynaptic m e m b r a n e , though here again a presynaptic role has been suggested. Roberts & Thesleff (1965), using the presynaptic failure m e t h o d of Krnjevic & Miledi (1958), have detected a prejunctional action of decamethonium. Standaert & A d a m s (1965) have found that decamethonium depolarizes nerve endings. Benzoquinonium would also appear to have both p r e - a n d postsynaptic actions. Blaber & Bowman (1962, 1963) stress that
EFFECT OF C H O L I N E R G I C ANTAGONISTS ON LEECH MUSCLE P O T E N T I A L S
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FIG. 7. A graph to show the effect of decamethonium (10 mg/ml applied via a diffusion electrode) on the frequency of excitatory potentials with amplitudes in excess of 4 mV. Decamethonium was applied at point marked JL The ordinate is the frequency of excitatory potentials with amplitudes in excess of 4 mV recorded per 15-sec period; the abscissa is. time in l-rain periods. benzoquinonium exhibits potent blocking actions at the nerve terminal in addition to the accepted postsynaptic anticholinergic action (Luduena & Brown, 1952; Webb, 1965). There is clearly a problem in trying to determine, in a given preparation, if the antagonist is acting postsynaptically, presynapticaUy or possibly at both sites. It is normally assumed that because a compound decreases the amplitude of miniature end-plate potentials (mepps) or end-plate potentials (epps) that it is acting postsynaptically. However, the postsynaptic event is the last in a sequence of events. If the compound acts presynaptically then this will alter the postsynaptic response. A widely used method of assessing drug action stems from the effect of compounds on the frequency of mepps (Fatt& Katz, 1952). If a compound alters the frequency of the mepps then it can be concluded that it has a presynaptic action. An increase in the frequency indicates a depolarizing effect on nerve terminals. In the present study tubocurarine, gaUamine, decamethonium and benzoquinonium all depress the amplitude of the excitatory potentials, but there appears to be no effect on the frequency of the excitatory potentials. Since these are spontaneous, if the antagonists were acting presynaptically, then one would expect to see a decrease in their frequency since a reduction in the number of m e p p s would mean that some of the excitatory potentials would disappear as they are compound mepps.
700
R. J. WALKER,G. N. WOODRUFFANO G. A. KERKUT
T h e observation that atropine and h e x a m e t h o n i u m had little or no effect on the excitatory potentials agrees with the findings of Flacke & Yeoh (1968) that neither c o m p o u n d antagonized acetylcholine action on leech muscle. One surprising finding was that d e c a m e t h o n i u m at the concentration used in the diffusion electrode did not depolarize the resting potential of the leech muscle. Gallamine on several occasions depolarized the muscle with a slight potentiation of the amplitude of the excitatory potentials but this was only a transient effect, the m e m b r a n e repolarizing to a value several m V negative to the resting potential of the fibre prior to addition of the gallamine. T h e duration of the block lasted for approximately 3 min in the case of decamethonium. T h e block lasted for m u c h longer in the case of the other three compounds, recovery taking about 30 m i n after tubocurarine and benzoquinonium. I t is difficult to assess the relative potencies of the four antagonists due to differences in the tip diameter of the diffusion electrode and the proximity of the diffusion electrode to the recording electrode. However, it would appear that tubocurarine and benzoquinonium were the two most potent antagonists. Acknowledgements--A gift of Mytolon (benzoquinonium chloride) from Bayer Products is gratefully acknowledged,
REFERENCES
BACQ Z. M. & COPPEE G. (1937) R6action des vers et des mollusques ~ l'6s6rine. Existence de nerfs cholinergiques chez les vers. Archs int. Physiol. 45, 310-324. BLABER L. C. & BOWMAN W. C. (1962) The interaction between benzoquinonium and anticholinesterases in skeletal muscle. Archs int. Pharmacodyn. 138, 90-104. BLnBER L. C. & BOWMANW. C. (1963) The effects of some drugs on the repetitive discharges produced in nerve and muscle by anticholinesterases. Int..7. Neuropharmac. 2, 1-16. FATT P. & KATZ B. (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. FLACKE W. & YEOH T. S. (1968) Differentiation of acetylcholine and succinylcholine receptors in leech muscle. Br. ft. Pharmac. Chemother. 33, 154-161. KaTZ B. & MILEDI R. (1965) Propagation of electrical activity in motor nerve terminals. Proc. R. Soc. Lond. Series B, 161, 453-482. KRNJEVIC K. & MILEDXR. (1958) Failure of neuromuscular propagation in rats. J. Physiol. 140, 440-461. LUDUENA F. P. & BROWN T. G. (1952) Mytolon and related compounds as antagonists of acetylcholine on the heart of Venus mercenaria, ft. Pharrnac. exp. Ther. 105, 232-239. MACINTOSHF. C. & PERRYW. L. M. (1950) Biological estimation of acetylcholine Methods in MedicalResearch (Edited by GERARDR. W.), Vol. 3, pp. 78-92. Year Book Publishers, Chicago. MINZ B. (1932) Pharmakologische Untersuchungen am Blutegelpr~iparat, zugleich eine Methode zum biologischen Naehweis von Acetylcholin bei Anwesenheit anderer pharmakologisch wirksamer k6rperiegener Stoffe. Arch. exp. Path. Pharmak. 168, 292-304. Rn~ER W. F., JR. (1966) Actions of acetylcholine on mammalian motor nerve terminal. J. Pharmac. exp. Ther. 152, 397-416. ROBF-RTSD. V. & TH~.SLEFFS. (1965) Neuromuscular transmission in vivo and the actions of decamethonium: a micro-electrode study. Acta anaesthesiol, scand. 9, 165-172.
EFFECT OF C H O L I N E R G I C ANTAGONISTS ON LEECH MUSCLE POTENTIALS
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STANDAERT F. G. (1964) -The action of D-tubocurarine on the motor nerve terminal. .~. Pharmac. exp. Ther. 143, 181-186. STANDAERT F. G. & ADAMS J. E. (1965) T h e actions of succinylcholine on the mammalian motor nerve terminal..7. Pharmac. Exp. Ther. 149, 113-123. WALKER R. J., WOODRUFF G. N. & ~ G. A. (1968). T h e effect of acetylcholine and 5-hydroxytryptamine on electrophysiological recordings from muscle fibres of the leech, Hirudo medicinalis. Comp. Biochem. Physiol. 24, 987-990. WASHIZU Y. (1967) Electrical properties of leech dorsal muscle. Comp. Biochem. Physiol. 20, 641-646. WEBB G. D. (1965) Affinity of benzoquinonium and ambenonium derivatives for the acetylcholine receptor, tested on the electroplax, and for acetylcholinesterase in solution. Biochim. biophys. Acta 102, 172-184.
Key Word Index--Acetylcholine; atropine; benzoquinonium; deeamethonium; gallahexamethonium; tubocurarine; leech ; muscle; excitatory potential; Hirudo medicinalis. mine;
T H E A C T I O N OF C H O L I N E R G I C A N T A G O N I S T S O N SPONTANEOUS EXCITATORY POTENTIALS RECORDED F R O M T H E B O D Y W A L L M U S C L E S OF T H E L E E C H H I R UDO M E D I C I N A L I S R. J. WALKER, G. N. WOODRUFF and G. A. KERKUT Department of Physiology and Biochemistry, The University of Southampton, Southampton SO9 5NH (Received 30 M a y 1969)
Intracellular recordings were made from the body musculature of the leech Hirudo medicinalis. Resting potential values ranged from - 2 2 to - 6 4 mV. Excitatory potentials ranged in amplitude from 100/*V to 30 mV. 2. Atropine, benzoquinonium, decamethonium, gallamine, hexamethonium and tubocurarine were applied to the excitatory potentials by means of diffusion electrodes with tip diameters of between 2 and 10/z. 3. Benzoquinonium, decamethonium, gallamine and tubocurarine all decreased the amplitude of the spontaneous excitatory potentials without altering their frequency. 4. It is concluded that all four antagonists exerted their action mainly postsynaptically on the muscle. Abstract--1.
INTRODUCTION THE ACETYLCHOLINE-INDUCED contractions of the body wall musculature of the leech are antagonized by a n u m b e r of cholinergic antagonists. D-Tubocurarine is a potent blocker of these contractions (Minz, 1932; M a c i n t o s h & Perry, 1952; Flacke & Yeoh, 1968). Gallamine will also antagonize the effect of acetylcholine while atropine, hexamethonium, mecamylamine and procaine have no effect (Flacke & Yeoh, 1968). It is possible to record intracellular excitatory potentials from leech muscle (Washizu, 1967; Walker et al., 1968). It has been suggested that acetylcholine may be responsible for excitatory transmission at the leech neuromuscular junction (Bacq & Coppee, 1937). If this is the case then one would expect that compounds which antagonized the action of acetylcholine would also depress the excitatory potentials. T h e present study investigates the effect of a n u m b e r of cholinergic antagonists on these excitatory potentials. MATERIALS AND METHODS
All experiments were performed on the medicinal leech Hirudo medicinalis. The dissection and recording system were similar to that described by Walker et al. (1968). The cholinergic antagonists were applied to the muscle by means of diffusion electrodes. The glass electrodes were filled with water and the water removed from the shank of the 691
692
R. J. WALKER, G. N. WOODRUFFAND G. A. KERKUT
electrode. The compound was then introduced into the shank using a fine pipette. The electrodes were viewed under a microscope and the tip was slightly broken with a glass rod to give a tip diameter of between 2 and 10/~. The concentrations of the compounds used were as follows: atropine, 20mg/ml; benzoquinonium, 10mg/ml; decamethonium, 10 mg/ml; gallamine, 40 mg/ml; hexamethonium, 10 mg/ml; tubocurarine, 1 mg/ml. The following compounds were used: atropine sulphate--British Drug Houses; decamethonium iodide, hexamethonium bromide--Koch-Light; benzoquinonium (Mytolon) chloride-Bayer Products ; gallamine (Flaxedil) triethiodide--May & Baker; o-tubocurarine chloride-Burroughs Wellcome. RESULTS T h e best recordings in terms of the amplkude of the excitatory potentials and in the time for which the electrode remained in the fibre were obtained w k h microelectrodes with a resistance of between 30 and 60 Mfl. Recordings made with electrodes with a resistance of less than 20 M f l usually declined within a few minutes, the fibre resting potential depolarizing slowly towards zero while the excitatory potentials rapidly declined in amplitude. Using high-resistance electrodes k was possible to remain in a fibre for up to 3 hr. On many occasions during experiments the muscle would contract and the electrode come out of the fibre. The excitatory potentials were very sensitive to magnesium, 5-10 m M MgC12 abolished most of the activity. Excitatory potentials could be obtained from a number of sites along a given fibre. T h e excitatory potentials ranged in amplitude from 100 tLV to 30 mV w k h a duration of up to 100 msec (Fig. la, b). In addition to the excitatory potentials, inhibitory potentials can also be obtained (Fig. lc). These inhibitory potentials are most frequently obtained from the circular muscle. T h e y range in size from 100 t~V to 4 mV, w k h a duration of up to 150 msec. This present study is only concerned with the excitatory potentials and the effect on them of various cholinergic blocking agents. The diffusion electrode containing the blocking agent once lined up close to the recording electrode was left in position for between 30 and 60 sec. If an effect was going to occur it always did within a minute of bringing up the diffusion electrode.
Tubocurarine T h e diffusion electrodes were filled with a solution of I mg/ml D-tubocurarine. This concentration depressed the amplitude of the excitatory potentials (Fig. 2) but had little effect on their frequency. The recovery time after this dose varied from 10 to 30 min, though often the electrode became dislodged from the fibre before the activity had recovered. Although after 30 min activity could be obtained from other fibres in the preparation. In the experiment shown in Fig. 2, tubocurarine treatment resulted in the hyperpolarization of the membrane potential from an initial value of - 32 mV to - 42 mV over a period of about 90 see. Gallamine Gallamine was applied via the diffusion electrode at a concentration of 40 mg/ml. Gallamine decreased the amplitude of the excitatory potentials (Fig. 3), though it
EFFECT OF C H O L I N E R G I C A N T A G O N I S T S ON LEECH M U S C L E P O T E N T I A L S
693
had little effect on the frequency. However, at this concentration the amplitude was so depressed as to make the potentials almost merge into the background noise for a few seconds during the maximum effect of the compound. Gallamine often appeared to depolarize the membrane and transiently increase the amplitude
S / FIG. 1. Examples of excitatory and inhibitory potentials recorded from leech muscle. A. Excitatory potentials recorded from leech muscle; resting potential, -40mV; voltage scale, 5 mV, time scale, 150msec; B. Excitatory potentials recorded from leech muscle; resting potential, -55 mV; voltage scale, 10 mV, time scale, 100 msec; C. Inhibitory potentials and action potentials recorded from leech muscle; resting potential, - 30 mV; voltage scale, 4 mV, time scale, 150 reset.
of the excitatory potentials prior to hyperpolarizing the membrane. In Fig. 3, following the application of the gallamine, the resting potential fell from - 22 mV to - 1 8 mV and then rose to - 2 5 mV by the end of trace B. Six rain later when trace C was taken the resting potential was - 2 6 mV. Two and a half rain later, trace D, the resting potential was still - 26 mV, but the amplitude of the excitatory potentials was beginning to recover. The excitatory potentials in Fig. 3 recovered to normal after about 12 min.
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FIG. 2. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing tubocurarine (1 mg/ml) was positioned close to the recording electrode at the point marked J' on trace A. The resting potential in trace A was - 3 2 mV, in trace B, - 3 4 mV; in trace C, - 3 7 mV which hyperpolarized further to - 4 2 mV; in trace D, - 3 5 mV. Traces A, B and C are continuous; there is a gap of 20 rain between traces C and D. The time scale is marked in seconds.
Benzoquinonium Benzoquinonium was applied via the diffusion electrode at a concentration of 10 mg/ml. At this concentration there was no indication of a depolarization and block. T h e c o m p o u n d greatly reduced the amplitude of the excitatory potentials without altering their frequency (Fig. 4). Even during the m a x i m u m effect of the c o m p o u n d the frequency remained remarkedly constant as can be seen by comparing traces A and C in Fig. 4. T h e benzoquinonium increased the resting
EFFECT O F C H O L I N E R G I C A N T A G O N I S T S O N L E E C H M U S C L E P O T E N T I A L S
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FzG. 3. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing gallamine (40 mg/ml) was positioned close to the recording electrode at the point marked ~ on trace A. The resting potential in trace A was - 22 mV; in trace B, - 18 mV and then it rose to - 25 mY; in trace C, --26 mV; in trace D, - 2 6 inV. Traces A and B are continuous; there is a gap of 6 min between traces B and C; and a gap of 150 sec between traces C and D. The time scale is marked in seconds. potential f r o m - 2 8 m V to - 3 0 mV. T h e r e is a gap of 25 rain between traces C and D. Figure 5 shows the effect of benzoquinonium on the m a x i m u m amplitude of the excitatory potentials. T h e m a x i m u m excitatory potential height was 6 mV, b u t this was depressed to less t h a n 1 m V by the benzoquinonium. After approximately 30 min, the excitatory potentials had returned to their previous amplitude of 6 mY.
Decamethonium T h e diffusion electrodes contained decamethonium at a concentration of 10 mg/ml. T h i s c o m p o u n d reduced the amplitude of the excitatory potentials b u t had no effect on their frequency (Fig. 6). I n this experiment the decamethonium
R. J. WALKER, G. N. WOODRUFF/LhTDG. A. KERKUT
696
Benzo¢uinonium
B
C
] 4mV
1
15
i sec
FIG. 4. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing benzoquinonium (10 mg/ml) was positioned close to the recording electrode at the point marked t on trace A. The resting potential in trace A was - 28 mV and in trace C, - 30 mV. Traces A and B are continuous; there is a gap of 60 sec between traces B and C and a gap of 25 min between traces C and D. increased the resting potential f r o m - 6 4 m V to - 6 6 mV. T h e effect on the frequency of excitatory potentials over 4 m V in amplitude following decamethon i u m is shown in Fig. 7. T h e rate of these large potentials was 12-14/15 sec, but following the application of the decamethonium the rate of these large excitatory potentials was reduced to zero. T h e excitatory potentials returned to their previous amplitude after about 3 min. T h e resting potential prior to the application of the d e c a m e t h o n i u m was - 50 mV. Following the application of the decamethonium, the resting potential hyperpolarized to - 6 0 m V and then returned to - 55 m V 4 min after the decamethonium.
EFFECT OF C H O L I N E R G I C ANTAGONISTS ON LEECH MUSCLE P O T E N T I A L S
697
Atropine and hexamethonium Atropine when applied via the diffusion electrode at a concentration of 20 mg/ml failed to alter the excitatory potential height. H e x a m e t h o n i u m at the same concentration likewise had little effect on excitatory activity. Large doses added to the bath reduced the amplitude of the excitatory potentials but also depolarized the membrane potential to zero. In some experiments the effect of several blocking agents was tested in turn via diffusion electrodes on the same fibre. U n d e r these conditions it was found that while, for example, benzoquinonium would reversibly reduce the amplitude of the excitatory potentials, atropine and hexamethonium failed to have an effect. -x-x-x-x-x-x--x--x-x-xox
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FIG. 5. A graph to show the effect of benzoquinonium (10 mg/ml applied via a diffusion electrode) on the maximum amplitude of excitatory potentials recorded from leech muscle. The maximum height of the potentials was depressed from 6 mV to a minimum of 0"8 mV. After about 30 rain the amplitude of the excitatory potentials had returned to 6 mV. Benzoquinonium was applied at the point marked 1'. The ordinate is the maximum amplitude of the excitatory potentials recorded per 15-sec period; the abscissa is the time in 1-min periods. DISCUSSION It is generally accepted that cholinergic antagonists such as tubocurarine act on the postsynaptic membrane to block competitively the effect of acetylcholine. For example, it has been shown by Katz & Miledi (1965) that tubocurarine acts postsynaptically on the miniature end-plate potential and not presynaptically on the nerve terminal in the frog. However Riker (1966) and Standaert (1964) have
R. J, WALKER, G. N. WOODRUFFAND G. A. KERKUT
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Decamethonium
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FIG. 6. A pen recording of excitatory potentials recorded from leech muscle. A diffusion electrode containing decamethonium (10 mg/ml) was positioned close to the recording electrode at the point marked ~' on trace A. The resting potential in trace A was - 6 4 mV and in trace B, - 6 6 mV. Traces A, B and C are continuous ; there is a gap of 40 sec between traces C and D. presented evidence for a presynaptic role for tubocurarine at the m o t o r nerve terminal in the cat. Antagonists which depolarize and block, such as decamethonium, also act on the postsynaptic m e m b r a n e , though here again a presynaptic role has been suggested. Roberts & Thesleff (1965), using the presynaptic failure m e t h o d of Krnjevic & Miledi (1958), have detected a prejunctional action of decamethonium. Standaert & A d a m s (1965) have found that decamethonium depolarizes nerve endings. Benzoquinonium would also appear to have both p r e - a n d postsynaptic actions. Blaber & Bowman (1962, 1963) stress that
EFFECT OF C H O L I N E R G I C ANTAGONISTS ON LEECH MUSCLE P O T E N T I A L S
14
m
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FIG. 7. A graph to show the effect of decamethonium (10 mg/ml applied via a diffusion electrode) on the frequency of excitatory potentials with amplitudes in excess of 4 mV. Decamethonium was applied at point marked JL The ordinate is the frequency of excitatory potentials with amplitudes in excess of 4 mV recorded per 15-sec period; the abscissa is. time in l-rain periods. benzoquinonium exhibits potent blocking actions at the nerve terminal in addition to the accepted postsynaptic anticholinergic action (Luduena & Brown, 1952; Webb, 1965). There is clearly a problem in trying to determine, in a given preparation, if the antagonist is acting postsynaptically, presynapticaUy or possibly at both sites. It is normally assumed that because a compound decreases the amplitude of miniature end-plate potentials (mepps) or end-plate potentials (epps) that it is acting postsynaptically. However, the postsynaptic event is the last in a sequence of events. If the compound acts presynaptically then this will alter the postsynaptic response. A widely used method of assessing drug action stems from the effect of compounds on the frequency of mepps (Fatt& Katz, 1952). If a compound alters the frequency of the mepps then it can be concluded that it has a presynaptic action. An increase in the frequency indicates a depolarizing effect on nerve terminals. In the present study tubocurarine, gaUamine, decamethonium and benzoquinonium all depress the amplitude of the excitatory potentials, but there appears to be no effect on the frequency of the excitatory potentials. Since these are spontaneous, if the antagonists were acting presynaptically, then one would expect to see a decrease in their frequency since a reduction in the number of m e p p s would mean that some of the excitatory potentials would disappear as they are compound mepps.
700
R. J. WALKER,G. N. WOODRUFFANO G. A. KERKUT
T h e observation that atropine and h e x a m e t h o n i u m had little or no effect on the excitatory potentials agrees with the findings of Flacke & Yeoh (1968) that neither c o m p o u n d antagonized acetylcholine action on leech muscle. One surprising finding was that d e c a m e t h o n i u m at the concentration used in the diffusion electrode did not depolarize the resting potential of the leech muscle. Gallamine on several occasions depolarized the muscle with a slight potentiation of the amplitude of the excitatory potentials but this was only a transient effect, the m e m b r a n e repolarizing to a value several m V negative to the resting potential of the fibre prior to addition of the gallamine. T h e duration of the block lasted for approximately 3 min in the case of decamethonium. T h e block lasted for m u c h longer in the case of the other three compounds, recovery taking about 30 m i n after tubocurarine and benzoquinonium. I t is difficult to assess the relative potencies of the four antagonists due to differences in the tip diameter of the diffusion electrode and the proximity of the diffusion electrode to the recording electrode. However, it would appear that tubocurarine and benzoquinonium were the two most potent antagonists. Acknowledgements--A gift of Mytolon (benzoquinonium chloride) from Bayer Products is gratefully acknowledged,
REFERENCES
BACQ Z. M. & COPPEE G. (1937) R6action des vers et des mollusques ~ l'6s6rine. Existence de nerfs cholinergiques chez les vers. Archs int. Physiol. 45, 310-324. BLABER L. C. & BOWMAN W. C. (1962) The interaction between benzoquinonium and anticholinesterases in skeletal muscle. Archs int. Pharmacodyn. 138, 90-104. BLnBER L. C. & BOWMANW. C. (1963) The effects of some drugs on the repetitive discharges produced in nerve and muscle by anticholinesterases. Int..7. Neuropharmac. 2, 1-16. FATT P. & KATZ B. (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. FLACKE W. & YEOH T. S. (1968) Differentiation of acetylcholine and succinylcholine receptors in leech muscle. Br. ft. Pharmac. Chemother. 33, 154-161. KaTZ B. & MILEDI R. (1965) Propagation of electrical activity in motor nerve terminals. Proc. R. Soc. Lond. Series B, 161, 453-482. KRNJEVIC K. & MILEDXR. (1958) Failure of neuromuscular propagation in rats. J. Physiol. 140, 440-461. LUDUENA F. P. & BROWN T. G. (1952) Mytolon and related compounds as antagonists of acetylcholine on the heart of Venus mercenaria, ft. Pharrnac. exp. Ther. 105, 232-239. MACINTOSHF. C. & PERRYW. L. M. (1950) Biological estimation of acetylcholine Methods in MedicalResearch (Edited by GERARDR. W.), Vol. 3, pp. 78-92. Year Book Publishers, Chicago. MINZ B. (1932) Pharmakologische Untersuchungen am Blutegelpr~iparat, zugleich eine Methode zum biologischen Naehweis von Acetylcholin bei Anwesenheit anderer pharmakologisch wirksamer k6rperiegener Stoffe. Arch. exp. Path. Pharmak. 168, 292-304. Rn~ER W. F., JR. (1966) Actions of acetylcholine on mammalian motor nerve terminal. J. Pharmac. exp. Ther. 152, 397-416. ROBF-RTSD. V. & TH~.SLEFFS. (1965) Neuromuscular transmission in vivo and the actions of decamethonium: a micro-electrode study. Acta anaesthesiol, scand. 9, 165-172.
EFFECT OF C H O L I N E R G I C ANTAGONISTS ON LEECH MUSCLE POTENTIALS
701
STANDAERT F. G. (1964) -The action of D-tubocurarine on the motor nerve terminal. .~. Pharmac. exp. Ther. 143, 181-186. STANDAERT F. G. & ADAMS J. E. (1965) T h e actions of succinylcholine on the mammalian motor nerve terminal..7. Pharmac. Exp. Ther. 149, 113-123. WALKER R. J., WOODRUFF G. N. & ~ G. A. (1968). T h e effect of acetylcholine and 5-hydroxytryptamine on electrophysiological recordings from muscle fibres of the leech, Hirudo medicinalis. Comp. Biochem. Physiol. 24, 987-990. WASHIZU Y. (1967) Electrical properties of leech dorsal muscle. Comp. Biochem. Physiol. 20, 641-646. WEBB G. D. (1965) Affinity of benzoquinonium and ambenonium derivatives for the acetylcholine receptor, tested on the electroplax, and for acetylcholinesterase in solution. Biochim. biophys. Acta 102, 172-184.
Key Word Index--Acetylcholine; atropine; benzoquinonium; deeamethonium; gallahexamethonium; tubocurarine; leech ; muscle; excitatory potential; Hirudo medicinalis. mine;