Local anaesthetic activity of vesamicol in the electric organ of Torpedo

EuropeaR Journal of Pharmticoloa. 195 (1991) 1-9 0 1991 Elsevier Science Publishers B.V. OOU-2999/91,/$03.50

ADONIS ~14299~1#2lSL EJP 51760

cal anaesthetic activity of vesa

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Romain Girod, Frarqoise Loctin and Yves Dunant DSpartement de Phurmaco~ogie,Centre Mgdical Universitaire, I21 i Gendve 4, Switzerland Received 25 September 1990. revised MS received 5 December 1990, accepted 2 January1991

Synaptic trans~ssion in intact pieces of the Torpedo electric organ treated with vesamicol(2-(4-phenyipipe~diRo~yciohexano1, formerly AH5183) was elicited by trains of repetitive electrical stimulation at different frequencies. When the frequency of stimulation was increased from 10 to 50 or 100 Hz, micromolar concentrations of vesaxnicol enhanced the tetanic rundown of the successive tissue responses. This effect was already detectable with 10 FM vesamicol. It was dramatically potentiated with concentrations of 50 or 100 PM vesticol, which caused complete failure of transmission after usualty less than 10 responses. The drug was unequivocally demonstrated to act by depressing the evoked release of acetylcholine as a consequence of a highly frequency- and concentration-dependent impairement of Na+ channel function in afferent axons. It is concluded that, in the electric organ, vesamicol blocks transmission by acting as a local anaesthetic. This action of micromolar concentrations of vesamicol must be taken into account especially during high-rate nerve activity. Vesamicol (AH5183); Synaptic tr~ns~ssion;

1. Introduction The cholinergic neurotoxin, vesamicol (2-(6phenylpiperidino)cyclohexanol, formerly AH5183), decreases the active transport of labelled acetylcholine (ACh) into purified synaptic vesicles isolated from the Torpedo electric organ (Anderson et al., 1983; 1986; Diebler and Morot Gaudry-Talarmain, 1989). In intact pieces (prisms) of the electric organ (Suszkiw and Manalis, 1987; Girod et al., in press) and in synaptosomes isolated from this preparation (~ichaelson and Burstein, 1985; Michaelson et al., 1986; Morot Gaudry-Talarmin et al., 1989) the drug reduces the uptake of newly synthesized ACh into synaptic vesicles and completely blocks its access to the releasable pool of transmitter. Despite these alterations of the intraterminal metabolism of ACh, vesamicol does not modify the evoked release of endogenous ACh, either in synaptosomes of the electric organ depolarized biochemically (Michaelson and Burstein, 1985; Michaelson et al., 1986; Morot Gaudry-Talarmin et al., 1989) or in prisms stimulated electrically (Girod et al., in press). Moreover, even during extensive electrical stimulation at 1 or 10 Hz, the drug does not accelerate synaptic depression in the

Correspondence lo: R. Girod, DPpartement de Pharmacologic, C.M.U., 1211 Geneve 4. Switzerland.

Anaesthetics (local); Acetylcholine release

electric organ and leaves unaltered the capacity of the tissue to recover from such stimulation (Suszkiw and Manalis, 1987; Girod et al., in press). These results are well consistent with those obtained with PC12, a clonal line of rat pheochromocytoma cells (Melega and Howard, 1984), and with synaptosomes of the electric organ of Ncwcine brasifiensis (Unsworth and Johnson, 1990). They contrast, however, with the reports showing that in several preparations of central orgin, vesamicol diminishes the release of both endogenous and newly synthesized ACh (Carroll, 1985; Jope and Johnson, 1986; Otero et al., 1985; Ricny and Collier, 1986; Suszkiw and Toth, 1986; Suszkiw and Manalis, 1987), and that at the neuromuscular junction it induces depression of synaptic tr~s~ssion which is frequency-dependent and usually occurs after a period of normal activity (Brittain et al., 1969; Marshall, 1970; Gandiha and Marshall, 1973; Enomoto, 1988; Estrella et al., 1988; Lupa, 1988; Maeno and Shibuya, 1988). Consistent with these effects of vesamicol on cholir.ergic function, the drug decreases the evoked release of ACh in sympathetic ganglia (Collier et al., 1986; Cabeza and Collier, 1988). To further investigate whether synaptic transmission in the Torpedo electric organ is insensitive to vesamicol action. we applied higher frequencies of stimulation to the tissue in order to reproduce the natural working of the electric organ (Balbenoit, 1970), and to study in more detail the use-dependence of the drug action.

2.3. Focal recording s and chemicals he fish. Torpedo marmorata. were supplied by the on de Biologic Marine (Areachon, France). The substances used were: tetrodotoxin (TTX) obtained from Calbiochem; tricaine (MS 222) obtained from Sandoz (Switzerland); benzocaine and lidocaine obtained from (Switzerland); %a, specific radioactivity 17 g obtained from Medipro S.A. (Belgium); cetate, specific radioactivity 53 mCi/mmol, obtained from tbe Radiochemical Center, Amersham, (U.K.): 2-(4-phenylpiperidino)cyclohexanol (AH5183) as HCl salt was a gift from Dr. D.E. Bays, Pharmaceutical Research Dept., Glaxo Group Research, Ware (UK.). The drug was dissolved in ethanol (final concentration: O-l-2.0%). Slices of electric organ were excised under anaesthesia with tricaine at a concentration of 0.33 g 1-l. Cylinders of 2-3 mm in diameter and about 2 cm long (prisms) were carefully dissected from the slices and allowed to recover in an elasmobranch saline medium of the following composition (mM): NaCl 280; KC1 7; CaCl, 4-k MgCl, 1.3; HEPES buffer 20; NaHCO, 5; urea glucose 5.5; pH 7.1. The medium was gassed with O2 and 5% COz. All experiments were carried out at room temperature (18-20 o C). 2.2. Field stimulation of prisms The effects of vesamicol were usually first analysed after 4-5 h of incubation by stimulating whole prisms, using single or repetitive ‘field’ shocks. Prisms are columns of about 500 superposed electroplaques that generate electrical discharges in response to field stimuli. In Torpedo, the electroplaques themselves are not capable of producing propagating regenerative action potentials, so that field shock stimulation excites only nerve fibres in the tissue. The postsynaptic response is the summation of many electroplaques potentials (EPPs), which are similar in many aspects to the well known end-plate po;entials of neuromuscular junctions. Control and vesamicol-treated prisms were placed on nylon cloth between two stimulating platinum electrodes. They were constantly moistened with physiological medium that did or did not contain the drug. Recording stainless-steel electrodes were placed close to the dorsal ard ventral ends of the prisms. The tissue was then given short trains of supramaximal field shocks at different frequencies (lo-100 Hz, lo-20 impulses). The postsynaptic responses of the prisms were monitored on a digital storage oscilloscope (Gould, OS 4200) and, when needed, were recorded on magnetic tape. The data were digitized and analysed using a micro-computer (Intei 802286, IBM AT).

In a second type of electrophysiological experiment, the action of vesamicol on synaptic transmission was investigated in a restricted zone of the synaptic area with a loose patch clamp system, as described by Dunant and Muller (1986). The great advantage of this technique is that it allows both to inject depolarizing current pulses and to record the evoked postsynaptic responses using a single extracellular electrode. Briefly, a thin slice of the electric organ was sectioned transversely from a small fragment containing four to five prisms, and was positioned with the innervated face uppermost. A glass microelectrode with a tip opening of 5-10 pm was filled with the saline medium and delicately placed against the most superficial electroplaque. To improve the stability of recordings and the signal-to-noise ratio, slight suction was applied to the inside of the pipette. Bursts of depolarizing current pulses were injected through the microelectrode, and the postsynaptic responses were monitored on a digital storage oscilloscope (Gould, OS 4200) and recorded on magnetic tape. The data were digitized and analysed using a micro-computer (Intel 80286, IBM AT). 2.4. A Ch release Spontaneous and stimulation-induced ACh release was measured as described by Dunant et al. (1980). Tissue ACh was labelled by incubating whole prisms with [14C]acetate (1 @i/ml) to distinguish between the release of endogenous ACh and that of newly synthesized [i4C]ACh. Only one radiolabelled precursor was used in the present experiments since both external acetate and choline are utilized to the same extent for the synthesis of Ach in this organ (Israel and TuEek, 1974; Dunant et al., 1980). After 3 h of labelling, the prisms were washed for 1 h in radioactivity-free medium. When present, vesamicol was added to the incubating saline 1 h before initiation of radiolabelling and was kept present throughout the rest of the procedure. At the end of the treatment, prisms were mounted on the electrodes for stimulation under continuous perfusion. The superfusing medium was then collected at regular intervals, before and after a train of 10 field shocks given at 100 Hz. No anticholinesterase was present in the fluid since choline and acetate are released in a one-to-one ratio in this tissue (Dunant et al., 1980). Furthermore, anticholinesterase drugs inhibit evoked ACh release in the Torpedo electric organ (Dunant and Walker, 1982). Thus, ACh was assayed as choline, using a luminescence reaction (Israel and Lesbats, 1981), and [“‘C]ACh was determined as [t4C]acetate counted in the fluid. Boih measurements were performed simultaneously in the same collection samples.

3

2.5. “‘Ca accumulation The orotocol was adapted from Babel-G&in (1974). In a typical experiment, 12 control prisms were incubated in physiological saline containing 45Ca (1 @i/ml) for 2 h at 16O C. Radiolabelling of 12 other prisms was concomitant with 100 FM vesamicol treatment. For each condition, four prisms were stimulated for 3 min at 10 Hz, four for 36 s at 50 Hz, and four were not stimulated. Thus, each of the stimulated specimens received 1800 impulses. Immediately after stimulation, all were washed for 1 h in an ice-cold medium composed of sucrose (0.5 mM) and urea (0.5 mM). The tissue samples were then dissolved in NaOH 1 N at 60 OC, neutralized with HCl 1 N, and their radioactivity was determined in a scintillation counter.

3. Results 3.1. Response to single shock At a concentration of 10 PM vesamicol is reported not to modify nerve-electroplaque transmission in pieces of electric organ (prisms) stimulated electrically (Suszkiw and Manalis, 1987; Girod et al., in press). Thus, the effects of drug concentrations up to 100 PM were investigated by first applying a single field shock stimulation to intact prisms. The amplitude of the electrical responses to a single shock was rather variable from one prism to another, even when they were dissected from the same Torpedo. To obtain statistically significant results, a large number of samples was therefore needed. In table 1, each value represents the mean amplitude and S.E.M. of 20 prisms dissected from four different Torpedos. The effects of 100 PM vesamicol were tested after a 3-h or longer period of incubation. It appears that at this concentration the drug caused a slight but not significant decrease of the EPP amplitude elicited by such stimulation. In the rest of the present work, differences of the absolute amplitude of the tissue elecTABLE 1 Electrical response (in volts) to single stimulus after 3 h or more of application of 100 pM vesamicol. Intact pieces of Torpedo electric organ (prisms) were allowed to recover from dissection in physiological saline for at least 1 h. Half were then incubated with 100 PM vesamicd. The amplitude of the electrical discharges elicited by single ‘field shock stimulation of the tissue was measured at the beginning (Vc) and after 3 h or more (Vi) of treatment with the drug. The results are mean amplitudes& S.E.M. of the responses of 20 prisms from four different Torpedos. The small decrease in amplitude caused by the drug was not statistically significant.

Control Vesamicol, 100 CM

Vo

vi

P

3.5 f 0.3 3.6f0.3

x5*0.3 3.OkO.4

n.s. n.s.

trical response will therefore not be considered. The pattern of fatigue of this response during repetitive activity will be analysed instead. 3.2. Frequency- and concentration-dependent blockade of transmission TO detect a possible use-dependent action of vesamicol on transmission in the electric organ short bursts of ten or twenty impulses were applied at high frequencies to intact prisms. This pattern of activity is close to that generated by Torpedo in its natural attack or defensive behaviour (Balbenoit, 1970). The results obtained with frequencies of 10 and 100 Hz are presented in fig. 1. During such brief repetitive stimulation, there is a progressive decline of the postsynaptic response amplitude (fig. 1A). This depression of transmission has been found to be due to impairement of ACh release during the course of the tetanic activity (Dunant et al., 1980). At a concentration of 10 PM vesamicol did not induce any changes in the profile of the successive discharges at 10 Hz (fig. 1A). On the other hand, analysis of the graphs in fig. 1B reveals that the same dose of drug caused a slight acceleration of the transmission fatigue when the frequency was raised to 100 Hz. This use-dependent action of vesamicol was greatly potentiated with larger concentrations. As is obvious from both the electrophysiological records and graphs in fig. 1A and B, the decline of the EPP amplitude was significantly increased when field shocks were given at 10 Hz to tissue treated with 100 PM vesamicol. With a stimulation frequency of 100 Hz, transmission usually failed completely within 70-100 ms of synaptic activity. Similar results were obtained with a frequency of 50 Hz (not illustrated). The detailed dose dependence of this action of vesamicol on transmission during a 100 Hz train is depicted in the right hand-side graph of fig. 1B. A maximal effect was usually achieved after 1 or 2 h of treatment with vesamicol, that is, the time required for the drug to be evenly distributed in the block of tissue by diffusion (see Dunant et al., 1972). Near complete recovery to a normal pattern of transmission occurred after about 2 h of washing-out in drug-free medium. 3.3. Synaptic delay during dynamic block of transmission In high frequency trains, 100 PM vesamicol gradually increased the synaptic delay, defined as the latency between the stimulus artifact and the beginning of the response. This is illustrated in fig. 2, which shows that, in controls, the synaptic delay tended to be shorter in the successive responses of a lOO-Hz train. At 10 PM, vesamicol did not alter this behaGour but 100 /.tM vesamicol markedly increased the delay of the response. This activity-dependent action of 100 PM vesamicol can

CUMULATEDRELATIVE AMPLITUDE 8

i0

I

/ I

1

5 RESPONSE NUMBER

10

1

5 RESPONSE NUMBER

10 .

_

Fig. 1. Concentration and use dependence of synaptic blockade by vesamicol. The electrophysiologial records in (A) show the responses of pnms ot Torpedo electroplaques to 20 stimuh given at 10 and 100 Hz, in controls or in the presence of 10 and 100 nM vesamicol. Calibration bars are 200 ms and 1 V on the left and 20 ms and 1 V on the right. Due to the time scale used at 10 Hz, the discharges, or electroplaque potentials (EPPs), appear as upward thin spikes. The ‘cumulated relative responses’ graphs in (B) allow precise analysis of transmission fatigue in control (open symbols) and drug-treated prisms (filled symbols): the amplitudes of the first 10 successive electroplaque potentials were normalized for a given train of stimulation by the amplitude of the initial EPP. The normalized responses to the successive stimuli were then added to each other, from the first to the tenth stimulus. Thus, the dotted diagonal lines would correspond to a constant discharge amplitude during the train. Any depression of transmission is visualized as a clockwise rotating of the curve. Each point represents the mean of the cumulated relative responses of 4-13 prismsfS.EM. With a stimulation frequency of 10 Hz. 10 nM vesamicol (A) did not affect the successive responses of the tissue, as compared to the control (0) but at a dose of 100 PM (0) however it induced enhanced fatigue. This effect was clearly frequency-dependent: at 100 Hz, 100 I.~M vesamicol caused complete blockade of nerve-electroplaque transmission when no more than 7-10 discharges had been elicited. The detailed concentration dependence of transmission blockade at 100 Hz is shown in the right hand graph in (B) (0. controls; A, 10 PM; q, 50 PM; @, 100 CM; a. 200 nM vesamicol). This blockade, already visible in the presence OC10 pM vesamicol, increased as a function of drug concentration.

be visualized in records like those illustrated in fig. 1 (100 pM vesamicol, 100 Hz). The first discharge (V,) of both control and drug-treated prisms represents typical electrical responses of prisms stimulated by field shocks. There is often a rebound of this response, which can be seen clearly in these records. It reflects restimulation of the fibers in the tissue by the first electrical response.

3.4. Rapid recovery of transmission after complete blockade When successive trains of 10 impulses at 100 Hz delivered at 10-s intervals were given to pieces of electric organ treated with 100 @vI vesamicol, synaptic transmission failed within each train but recovered

5

r-KtOrrMVESAMCOL

1.

1 RESPONSE NUMBER

CONTROL

Fig. 2. Effect of vesamicol on the synaptic delay. During a IOB-Hx stimulation of whole prisms, vesamicol caused the latency of the postsynaptic response to be prolonged during transmission blockade. The graph shows the successive synaptic delays (mean delays of four prisms& S.E.M.) of the first 10 discharges (response number l-10) in a lOB-Hz tram. In the controls (o), the delay tended to be slightly shortened, whereas in presence of 100 CM vemmicol (6) it was progressively increased up to 200% of its initial value. Vesamicol 10 yM (A) did not induce such an effect. The eleetrophysiological records illustrate the postsynaptic responses to the first (Ve) and to the tenth stimulus (Vre) of a control and of a 100 phi vesamicol-treated prism. Calibration bars are 2.5 ms and 0.5 V.

readily during each resting period. To further analyse this aspect, experiments of the type illustrated in fig. 3 were carried out: a 100-ms burst of electrical shocks at 100 Hz was applied to prisms of electroplaques incubated with 100 FM vesamicol to completely inhibit transmission. Recovery was studied by measuring the amplitude of a test EPP (Vi) evoked by a single pulse at intervals form 20 ms to 5 s after the end of the tetanus (the graph of fig. 3 shows test EPPs at intervals up to 1 s). The drug was present during the entire procedure. During the short burst of activity, the amplitude of the discharge was decreased by 35% in the control and by 90% in the drug-treated prisms. After stimulation was stopped, the block caused by vesamicol was very quickly overcome. After some 200 ms, the EPP amplitude was 50% of that of the first EPP (Vc) in the train. A recovery comparable to that of the control tissue, which was 95% after about 300-500 ms, was achieved within l-2 s following the tetanic burst. 3.5. Blockade of A Ch release by uesamieol To test whether vesamicol acted by inhibiting the evoked release of ACB, we measured the amount of transmitter overflowing from intact prisms before and after nerve stimulation. Tissue ACh was labelled with [14C]acetate so that the evoked release of endogenous and of newly synthesized transmitter was analyzed separately. A typical experiment is illustrated in fig. 4. The amounts of choline and [14C]acetate released prior to stimulation were slightly but not significantly decreased by 100 PM vesamicol. After a single burst of 10 im-

pulses at 100 Hz, a marked increase of both choline and [14C]acetate release took place in the controls. Concomitant with failure of transmission, there was a significant 70% reduction (P ( 0.001). induced by 100 PM vesamicol, of the amount of choline released by a similar burst. Similar results were obtained in two other experiments. In addition, in accordance with the action of vesamiccl on the metabolism of newly synthesized transmitter in the electric organ (Michaelson and Burstein, 1985; Michaelson et al., 1986; Suszkiw and Manalis, 1987; Girod et al., in press), the evoked release of [‘4C]acetate was ~mpletely ~~~sh~ since synthesis took place in the continuous presence of the drug. 3.6. *‘Ca accumulation in the neroe terminals during tetanic stimulation In an attempt to define the presynaptic origin of the transmission blockade caused by vesamicol, we analysed, in the presence and absence of the drug, the accumulation of “Ca that occurs in the tissue following stimulation. In the Torpedo electric organ, accumulation occurs

Vi (percent

Of VO 1

A

i

Ol_

C

0

500 INTERVAL(ms)

lcm

D

10Oj~M VESAMICOL

CONTROL

Fig. 3. Relief of vesamicol-induced block of transmission after short periods of rest. Vesamicol 100 gM was used and bursts of 10 impulses were given at 100 Hz to whole prisms. At the end of the train, the last EPP amplitude was 65 f 4% of the first EPP amplitude in the control (A) and g f 2% in the drug-treated tissue (A). Recovery of transmission was then studied by giving a single test pulse at increasing tune intervals after the stimulation. In the ordinate, the test EPP amplitude (Vi) is expressed as percent of the initial discharge amplitude (V’s). The abscissa indicates the time intervals between the end of the train and the test pulses. Each point is the meanf S.E.M. of the values obtained with six to nine different prisms. In normal conditiOm (0). maximal recovery was 95% and was achieved 300-500 ms after the end of the stim~ation. The v~~~l-indu~ block of transmission was relieved by 50% after 200 ms of rest; near complete recovery had occurred after 1 s (0). Record traces illustrate transmission recovery after a resting period of 20 ms (A), 50 ms (B), 100 ms (C) and 300 ms (D) for the drug-treated tissue, and of 300 ms for the control. Calibration bars are 50 ms and 1 V.

20Bq

g-1 min-1

2OOpmoles g-1 min-1

1OOuM VESAMICOL

CONTROL

Fig. 4. Inhibition by vesamicol(100 CM) of the release of endogenous ACh and suppression of the release of newly synthesized [14C]ACh when stimulation consisted of 10 shocks given at 100 Hr The records show the postsynaptic responses of the control and drug-treated tissue. Calibration bars are 10 ms and 0.5 V. Intraterminal pools of .4Ch were labelled by incubating prisms with [“Clacetate for 3 h. The

drug was added 1 h before the initiation of labelling and kept present throughoutthe rest of the procedure. No anticholinesterase was used. Tbe released transmitter was therefore assayed as choline (open blocks) and as [‘4Clacetate (filled bars) overflowing in the fluid before and after stimulation (S). Each measure (mean of four samples, SEM. between 8 and 18% of the values, not shown) represents a I-mitt period of collection. At a 100 Hz frequency, vesamicol significantly reduced the evoked release of ACh by 70X, and completely inhibited that of [“C]Ach.

mainly in the presynaptic nerve endings (Babel-GuCrin, 1974; Babel-G&in et al., 1977). In the experiment reported in table 2, repetitive synaptic activation was elicited by field shocks applied at 10 and 50 Hz for 3 miu and 36 s respectively. Thus, the prisms had all received 1800 impulses at the end of the stimulation. At 10 Hz, the drug (100 PM) caused an intermediate bloc-

TABLE 2 Stimulation-induced presynaptic accumulation of 4SCa in the presence of 100 pM vesamicol. Prisms prelabelled with 4SCa (1 @i/ml and 4.4 mhl Ca’+ ) were given 1800 electric shocks at two different frequencies (10 Hz for 3 mitt and 50 Hz for 36 s). The presynaptic accumulatioa of radioactivity before and after the stimulation is shown as meansfS.EM. from four prisms and is given in kBq/g. Control prisms stimulated at 50 Hz accumulated 40% less ‘?a than those stimulated at 10 Hz. The drug did not affect the amount of radioactivity counted in the unstimulated samples. On the other hand, at a frequency of 10 Hz. the drug reduced the accumulation of 4sCa by 43%. At 50 Hz, no 4SCa accumulation above the unstimulated level occurred in the presence of the drug.

Control Vesamicol, P

100 p M

kade of transmission (see fig. l), whereas at 50 Hz no more than four to five discharges were generated by the drug-treated prisms. When 4SCa was assayed, it appeared that the amount of labelled tracer that accumulated in the tissue under resting conditions was not modified by 100 PM vesamicol. After stimulation of controls at both frequencies, a clear accumulation of 4SCa above the unstimulated level was measured in the tissue. This accumulation was however less at 50 than at 10 Hz (P < 0.05). In the presence of 100 FM vesamicol, the extra accumulation of 45Ca was reduced by 43% at 10 Hz (P < 0.05j and was completely abolished at 50 Hz. This experiment suggested that the evoked ACh release was inhibited by 100 PM vesamicol as a consequence of a frequency-dependent impairment of calcium movement into nerve endings during synaptic activity.

Non-stimulated

10 Hz

50 Hz

11.6& 1.0 11.7* 1.4 n.s.

17.7 * 0.7 15.2 f 0.5 < 0.05

15.3kO.4 10.7kO.6 c 0.001

3.7. Effects of vesamicol on transmission evoked by focal depolarization at the nerve-electroplaque junction To further study blockade of transmission by vesamicol, we used a loose patch extracellular microelectrode to analyse the postsynaptic responses induced by focal stimulation at a restricted zone of the membranes, involving a limited number of nerve-electroplaque junctions. Dunant and Muller (1986), using this type of procedure, observed two types of responses in the electric organ: first, the ‘graded’ response, the amplitude of which increases with depolarizing pulses of increasing strength and is insensitive to TTX; second, the ‘all-or-none’ response, which is evoked at a precise threshold level of depolarization, shows constant amplitude and is abolished by TTX. The graded response has been attributed to direct activation of voltage-dependent calcium channels by the depolarizing pulse, whereas the all-or-none response involves a Na+-dependent presynaptic action potential. The stimulation consisted of bursts of 10 depolarizing current pulses delivered at 100 Hz through the electrode. When graded responses were studied, TTX 1 PM was added to the bath, and the strength of the stimulation pulse was adjusted so as to obtain the maximal amplitude. Figure 5A and D show respectively the all-or-none and the graded responses recorded under control conditions. The two types of responses were differently affected by vesamicol. A characteristic blockade of the all-or-none response was observed after 2.5 h of treatment with vesamicol50 PM: its amplitude was progressively depressed and its latency was prolonged during repetitive activity. The appearance of two peak profiles during tetanic run-down of the all-or-none discharge was often observed (fig. 5B). Half an hour later, only two discharges could be elicited at the beginning of the train (fig. 5C). On the other hand, the graded response did not show any sign of impairment when the drug had been present in the saline at 50 PM for 2.5 h (fig. 5E). Even raising the dose

7 ALL-or- NONE RESPONSE A

83

aspects. During short trains of stimulation at 100 Hz, the compounds caused a rapid tetanic rundown of the electrical discharge of the tissue and progressive prolongation of the synaptic delay. As with vesmid, transmission recovered within a few 100 ms of rest after the tetanic burst. At a ~ncentration of 100 pM, these agents were usually without effect and larger doses had to be used to block transmission. An example of the action of 500 PM tricaine is given in fig. 6.

most c

GRADED RESPONSE (TTX 1uM) D

E

F

4. Discussion Fig. 5. Postsynaptic currents evoked by 10 depolarizing pulses applied at 100 Hz to restricted zones of eleetroplaques, using a loose patch clamp system. For convenience, negative currents are illustrated as upward traces. Two types of responses were obtained: the all-or-none response (A-C), which involves a Na+-dependent presynaptic action potential, and the graded response (D-F), which reflects direct activation of the calcium channels and is TTX-insensitive. When SO pM vesamicol had been present for 2.5 h, the all-or-none response faded during repetitive activity (B). whereas the graded response remamed unchanged (E). Half an hour later, fading of the all-or-none response was enhanced (C). The graded response remained unaltered even when the drug concentration was increased to 100 pM for an additional 1.5 h (F). Calibration bars are 10 ms and S nA.

up to 100 FM for an additional 1.5-h period of incubation did not affect the graded response (fig. 5F). Thus, vesamicol interferes with excitation and propagation of the presynaptic Na+-dependent action potentials but does not seem to disturb the working of Ca2” channels. 3.8. Comparison with local anaesthetics Use-dependent blockade of nerve conduction is the m~cha~sm postulated to account for the action of local aneasthetics (see Catterall, 1987). Tricaine, benzocaine, and lidocaine were therefore tested for their effects on synaptic transmission in prisms of Torpedo electric organ. These three compounds were found to have a physiolo~cal action very similar to that of vesamicol in

500~4M TRICAINE

L

Fig. 6. Effects of SO6gM tricaine on transmi~ion in an intact prism of electroplaques. Stimulation consisted of a train of ten impulses given at 100 Hz, followed 100 ms later by a single pulse to test recovery of transmission. The amplitude of the discharge decreased rapidly and its delay was prolonged during the train. Near complete recovery of transmission was achieved after the lOO-msperiod of rest. Calibration bars are 20 ms and 0.5 V.

By delivering short trains of impulses at 50 or 100 Hz to intact pieces of Torpedo electric organ, a pattern of activity meant to reproduce the in vivo physiology of the fish (Balbenoit, 1970), we found that vesamicol accelerates the tetanic rundown of transmission during a 20-pulse train. In the presence of micromolar concentrations of vesamicol, transmission was slightly depressed at 10 Hz but completely blocked at 100 Hz, when usually no more than 10 responses had been elicited. The presynaptic origin of the inhibition was demonstrated by the fact that the amount of endogenous ACh released from vesamicol-depressed synapses was decreased with respect to the control in a train of imp&es given at 100 Hz. Inhibition of evoked ACh release by vesamicol has usually been attributed to disturbances of vesicular ACh storage caused by this neurotoxin (for a review see Marshall and Parson, 1987). However, several observations suggest strongly that this explanation does not hold for the electrophysiological effects described here: (a) at 10 PM vesamicol indeed reduced the rate of vesicular AC)? storage in the electric organ, but this did not result in any alteration of transmission, even after prolonged stimulation at 10 Hz and recovery (Girod et al., in press); (b) the fact that at a high rate of activity vesamicol decreased the presynaptic accumulation of calcium, suggests that obsession failure arose at a step prior to the process of transmitter release itself; Cc) finally, the use of a loose patch-clamp technique allowed us to precisely identify the target of the action of vesamicol: the presynaptic propagation of Na+-dependent action potentials down to the terminals. If this i~bition is bypassed by direct gating of the voltage-dependent calcium channels, ACh release will occur normally. This experiment also indicates that, although vesamicol was reported to interfere with the nicotinic receptor at the neuromuscular endplate (Enomoto, I%S), such an action of the drug was probably not a major component of the transmission blockade described here. We conclude that in the Torpedo electric organ vesamicol depresses transmission in a highly use- and concentration-dependent manner by acting as a local

~~~~tbet~c. Its action was almost indistinguishable from taut of tbm well-known local anaesthetics, lidocaine, wine and tetracaine. Several characteristics of the transmission blockade by vesamicol and local anaesthetits need comment. The strong use dependence of their action indicates that they interact mainly with the activated Na+ channel. From the rapid recovery of transmission after the tetanic rundown, one can estimate that these drugs unbind from the channels in a few 100 ms, indicating rapid dissociation from the inactive channel. The highly labile binding of the drug to its target is also emphasized by the transient partial recovery of transmission that was observed during the course of vesamicol-induced blockade (fig. 5B). These characteristics are consistent with the mechanism generally proposed to account for the action of local anaesthetics on nerve conduction (see Catterall, 1987). Moreover, the increase in delay produced by local anaesthetics and vesamicol suggests that these agents depress the velocity with which the action potential is conducted down to the terminals. Consistent with this interpretation is the appearance of multiple peak profiles during the vesamicol-induced tetanic run-down of the all-or-none discharge (Fig. 5B). This might be explained as follows: if several terminal branches are isolated under the tip of the ext.racellular electrode, the drug, by slowing nerve conduction, would amplify the differences in the time taken by action potentials to reach nerve endings in multiple branches, and thus would induce iemporal desynchronisation of ACh release. Fig. 1B shows that a dose of vesamicol as low as 10 PM interfered with the Na+ channel. Previous experiments on the local anaesthetic activity of vesamicol (Marshall, 1970; Estrella et al., 1988) had to be done with concentrations 10-100 times higher than those we used if the amplitude of action potentials recorded from isolated nerve preparations was to be reduced. The discrepancy might arise from the slow frequencies of stimulation (from 0.05 to 5 Hz) used in these experiments. In this respect, it is of interest to note that the three local anaesthetics now tested had to be applied at concentrations five times higher than those of vesamicol to obtain a comparable effect, suggesting that this neurotoxin is the most potent of the four compounds to interact with the activated Naf channel. Vesamicol therefore seems to have several different targets in cholinergic synapses and its mode of action may be different from one preparation to another. In sympathetic ganglia vesamicol inhibited transmitter release without affecting radioactive calcium accumulation (Collier et al., 1986). The drug also altered ACh release evoked by high-potassium depolarization in different preparations of the central nervous system (Carroll. 1985; Gtero et al., 1985; Jope and Johnson, 1986; RcBY and Collier, 1986; Suszkiw and Toth, 1986; SUS-

zkiw and Manalis, 1987), suggesting a target located at a step subsequent to activation of the Na+ channels. Moreover the transmission impairment caused by vesamicol at the neuromuscular junction can be observed at frequencies lower than 10 Hz. has a slow onset and long duration (Brittain et al., 1969; Marshall, 1970; Gandiha and Marshall, 1973; Enomoto, 1988; Estrella et al., 1988; Lupa, 1988; Maeno and Shibuya, 1988). Such patterns of blockade differ from that reported here. On the other hand, in experiments where nervemuscle preparations were given short trains of stimulation at 30-50 Hz (Brittain et al., 1969; Marshall, 1970; Gandiha and Marshall, 1973; Suszkiw and Manalis, 1987; Estrella et al., 1988), an action of vesamicol on the Na+ channel probably was a factor in the failure of transmission. We conclude that in the Torpedo electric organ vesamicol blocks transmission by altering nerve conduction, whereas its action on the intraterrninal metabolism of ACh occurs at lower concentrations and is apparently without detectable functional consequences (Suszkiw and Manalis, 1987; Girod et al., in press). The contribution of the local anaesthetic activity of micromolar doses of vesamicol in blocking transmission in other preparations might not be negligible and must be taken into consideration, especially when high frequencies of stimulation are used.

Acknowledgements The authors would like to thank Mme Nicole Collet and M. Fred Pillone: for help with the manuscript, and Dr. Dominique Muller for valuable discussions. This work was supported by the Swiss FNRS (Grant No. 3.641.0.87).

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