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Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes
Neuroscience Vol. 54, No. 4, pp. 103S-1041, 1993 Printed in Great Britain
0306-4522/93 $6.00 + 0.00 Pqamon Press Ltd 0 1993 IBRO
AGELENOPSIS APERTA VENOM AND FTX, A PURIFIED TOXIN, INHIBIT ACETYLCHOLINE RELEASE IN TORPEDO SYNAFTOSOMES N. MOULIAN and Y. MOROT GAUDRY-TALARMAIN Dkpartement
de Neurochimie, Laboratoire de Neurobiologie Cellulaire et Moleculaire, C.N.R.S., 91198 Gif-sur Yvette Ccdex, France
Ahtdraet-The presence of P-type calcium channels in synaptosomes prepared from electric organ of Torpedo marmorata was investigated by using the venom of Agelenopsis aperta, a toxin purified from it, FIX, and its synthetic analog. We analysed the action of these agents on acetylcholine release which was continuously followed using a chemiluminescent assay. Agelenopsis aperta venom, FIX and synthetic FIX inhibit acetylcholine release from synaptosomes induced by a presynaptic membrane depolarization with 60 mM KCl. A stronger inhibition of acetylcholine release was observed with the venom than with FIX (70 and 50%, respectively). Another way of triggering acetylcholine release from Torpedo synaptosomes is to insert in the presynaptic membrane a calcium ionophore A23187 which allows the bypass of the natural calcium channels. The venom of Agelenopsis aperta inhibits A23187-evoked acetylcholine release. Purified and synthetic FIX does not possess this property, suggesting that this inhibition of acetylcholine release was due to other toxins of the venom. Another type of pharmacological sensitivity of Torpedo calcium channels was also demonstrated using o-conotoxin GVIA. At a concentration of 20 PM, this toxin was able to inhibit about 35% of KCl-evoked acetylcholine release. When FIX + o-conotoxin GVIA were applied together, the inhibitory effect on KCI-evoked acetylcholine release was not significantly increased in comparison with the one observed with FIX alone. In conclusion, we examined the effect of different agents on acetylcholine release from Torpedo marmorata electric organ synaptosomes; acetylcholine release was elicited with KC1 depolarization and followed continuously with a chemiluminescent assay. It allowed us to distinguish different calcium fluxes in Torpedo synaptosomes. The existence of P-type voltage-dependent calcium channels was supposed upon observing the inhibitory effect on KCl-evoked acetylcholine release of the venom of Agelenopsis aperta, of FIX (a purified toxin), and of a synthetic FIX. Moreover acetylcholine release from Torpedo synaptosomes was moderately inhibited by o-conotoxin GVIA, a blocker of N-type calcium channels.
The increase of calcium concentration in the nerve terminal cytoplasm is the necessary trigger for release of neurotransmitter from all neurons during stimulation. Different kinds of voltage-operated calcium channels have recently been described in the membrane of excitable cells;‘*~‘9~28~30~“~3s when activated, they allow calcium to enter the nerve ending along its concentration gradient. Low-threshold T-type and high-threshold N-, L-, and P-type channels have been described on the basis of their pharmacological sensitivity. It is possible to study this stimulation-release coupling with a rich cholinergic preparation of nerve endings isolated from the electric organ of Torpedo marmorata.‘0*23 Indeed Torpedo synaptosomes are very useful for studies on cholinergic mechanisms since they constitute a homogeneous preparation with respect to the neurotransmitter; they are isolated with low contamination from postsynaptic membranes and other structures; they maintain their membrane potential; and they can be kept in a good metabolic state for more than 24 h after isolation.
Abbreviations: ACh, acetylcholine; FfX, funnel-web spider toxin; o-CTX, omegaconotoxin.
Moreover, a chemiluminescent assay using choline oxidase allows continuous monitoring of the release of acetylcholine (ACh). 8,9 These tools allow the characterization, using pharmacological agents, of the calcium channels present at the presynaptic level of a synapse which is embryologically related to a neuromuscular type. The pharmacological characterization of calcium channels has been done in cholinergic synapses of central or peripheral origin.‘4*32*38*39 To identify the calcium channels which possibly support these activities in Torpedo synaptosomes, we used agents previously described to act on calcium channel subtypes. Using a toxin extracted from the venom of the American funnel-web spider Agelenopsti aperta (FTX), a new type of calcium channel (the P-type) was recently described by LlinPs et aLI Putative P-type channels have been identified pharmacologically in different types of neurons.” As the goal of this study was to investigate the existence of these P channels on the Torpedo synaptosomes, we decided to test the effects of the crude venom of Agelenopsis aperta on evoked ACh release. Further studies were done with boiled venom, with FTX, a purified toxin fraction with a low molecular mass in the 2(l0-400 mol. wt range, and with synthetic FTX.4
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and Y.
MOKOT GAWKY-TALARMAIN
For comparison, the effect of w-conotoxin GVIA (w-CTX), a blocker of N-type calcium channels,15.24.26,41 was included in the study. o-CTX inhibits evoked ATP and ACh releases from Torpedo synaptosomes,2.7.40 suggesting that N-type calcium channel may also be present in Torpedo presynaptic membranes. Two types of stimuli were used to evoke ACh release: a depolarization of the presynaptic membrane by application of high external KC1 concentration or an insertion in the bilayer of a calcium ionophore A23187. The two procedures allow the discrimination between drugs which modify the last step of ACh release after the increase of intracellular calcium concentration (using A23 187 + calcium) and those which affect the calcium uptake through natural calcium channels (using KC1 stimulation). EXPERIMENTAL PROCEDURES
Preparation of synaptosomes
were isolated does not modify the-KCl-evoked ACh release. In some experiments FTX and wCTX were successfully preincubated with the synaptosomes during 2 and 3 min. respectively. Then 4~1 of 0.5 M CaCl, (final concentration 4 mM) was injected into the release medium. Evoked ACh release was triggered by a further addition of 10 111of a 3 M KCI solution (final concentration 69‘mM). Light responses corresponding to ACh release in the first minutes were calibrated by the addition of known amounts of ACh. tosomes
Chemicals
The material used for the chemiluminescent assay of ACh, choline oxidase (Wako Company, Japan), horseradish peroxidase (EC 1.11.1.7) type III from Sigma (St Louis, MO, U.S.A.), and luminol from Merck (Germany) were prepared as described previously. 8,9 Ionophore A23187 came from Boehringer (Germany); stock solutions (20 mM) were made in dimethylsulfoxide and diluted at I:40 in distilled water. Crude venom from the American funnel web spider Agelenopsis aperta was obtained from Spider Pharm, Black Canyon City, AZ, U.S.A. Dr R. Lhnas provided us with FTX, a toxin fraction, and with synthetic FTX (3:3).4 Different F’TX dilutions from the suider venom fraction were used. w-CTX GVIA was from Bachem, Bubendorf, Switzerland. All stock toxin solutions were dissolved into
Pure choline@ nerve endings were isolated from electric organ of the fish Torpedo marmorata. Fish were purchased from the marine station of Arcachon (France) and were kept in oxygenated artificial sea water tanks. The synaptosomes of Torpedo electric organ were prepared as previously described.iO,*r Nerve endings derived from 25 g of electric organ were collected from a discontinuous iso-osmotic sahne-sucrose gradient in 40-50ml of a synaptosomal fraction containina: 280mM NaCl. 3 mM KCI. 5 mM NaHCO,, 1.2 mM sodium-phosphate buffer, pH 7.2, 5.5 mM glucose, 300 mM sucrose and 100 mM urea. The medium is iso-osmotically similar to the elasmobranch plasma medium and differs from the one proposed by Israel et al.” only in the absence of divalent cations in discontinuous sucrose gradients. Thirty to fifty microlitres of this synaptosomal suspension corresponding to IO-30 mg of wet tissue were used for the ensuring ACh release experiments.
COHTRZJL
1 .mln
AGELENWSIS
APERTA
VENOM
Acetylcholine release experiments using a chemiluminescent assay
The release of endogenous ACh from Torpedo synaptosomes was continuously followed using the choline oxidase chemiluminescent method previously described.‘,’ In summary, this technique uses the specificity of two enzymes: acetylcholinesterase (present in the synaptosomal fraction) hydrolyses released ACh into choline and acetate and then choline oxi&se converts the newly formed choline into betaine and H,O,. H,O, is then converted into light using luminol and peroxidase. This method allowed us to monitor in real time the release of neurotransmitter in a physiological solution at room temperature. Aliquots of the synaptosomal suspension were mixed with 450-470 pl of a physiological medium made with: 280 mM NaCl. 3 mM KCl. 400 mM sucrose. 5.5 mM glucose and 50 mM Tris base/Tris-HCI buffer at pH 8.6 as required for the luminol + peroxidase chemiluminescent reaction. The medium was put in a glass tube and was continuously stirred by a small magnetic bar. Agelenopsis aperta venom and toxins were added directly to the synaptosomal suspension. Preincubations with each of them were carried out during 5min. The effect of w-CTX on neurotransmitter release such as noradrenaline5.6 or [‘H)ACh’9 was shown to be very sensitive and inversely related to the presence of external cations, hence we tested the effect of all the toxins on evoked ACh release after an incubation with no calcium or magnesium. In preliminary experiments, we checked that the omission of divalent cations in the medium in which synap-
DILtJTIOEl lj200
DILUTION
l/20
am
a00
DILUTTQN
l/l60
DILiTI$lN
1;25
000
Ooo
Fig. 1. Effect of Agelenopsis aperta venom on evoked ACh release. Synaptosomes were diluted (1: 16 to 1: zo) in a medium (28OmM NaCl, 400 mM sucrose, 3 mM KCl, 5.5 mM glucose and 90mM Tris, pH 8.6) cont&ng enzymes for chemihuninescont detection of ACh release (see Experimental Procedurea) and with various dilut&ms of Agelenopsis aperta venom during a S-min period. Then calcium was added to reach 4mM fmal conoentration. Release. of ACh was induced either by 6OmM KCl (left traces) or bv 4 uM A23187 (ri&t traces). Representative illustrations-of ACh relase e&&ments are shown in the absence of venom (control) or in the presence of various concentrations of spider venom. ACh release was calibrated by injecting an internal ACh standard. Note that at the highest concentrations of venom, it was necessary to increase the sensitivity of the recorder.
FTX inhibits acetylcholine release in Torpedo synaptosomes
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distilled water; aliquots were stored at -80°C prior to use at the appropriate dilution.
9 8
RFSUL’IS
Efict of venom of Agelenopsis aperta on KCI-evoked acetylcholine release
Synaptosomes were preincubated with aliquots of the venom during a 5-min period. The application of the venom does not induce any significant basal ACh release. Then the release of ACh from synaptosomes was examined (in the presence of 4 mM calcium) after a KC1 depolarization which opens voltage-dependent calcium channels naturally present in the presynaptic membrane. KCl-evoked release was inhibited in a concentration-dependent manner by Agelenopsis aperta venom, as shown in Fig. 1 in recordings of the light emission. Half-inhibition was obtained at the 1/200,OflO dilution of crude venom in the release medium (Fig. 2). Inhibition was observed between l,OOO,OOO-and 10,000-fold-dilution and was never complete. About 30% of ACh release was resistant to the action of the venom. Venom contains components which interfere and decrease the sensitivity of the chemiluminescent assay. At the highest concentrations it was necessary to increase the amplification to compensate for this effect. Venom that had been boiled for 5 min in order to denaturate its proteic components still inhibited KCIevoked ACh release with no loss of potency (data not shown).
i
io
lb0
AGEENCPMAFSRE4DLUllON(x166)
Fig. 2. Dose-response curve of the effect of Agelenopsis apertu venom on evoked ACh release from spaptosomes. Aliquots of synaptosomes diluted in an alkaline iso-osmotic medium (see Experimental Rocedures) were incubated with different concentrations of venom during a 5-min period. Calcium was then added; ACh release was induced either by a membrane depolarization with 60 mh4 KC1 (filled circles; four independent experiments) or with 4 gM A23187 (filled triangles; two independent experiments). In each experiment, individual data or mean of duplicate were expressed as percentages of their own ACh control release (mean of two to five determinations obtained in the absence of toxin). Results were pooled and the mean value is presented (*S.E.M.).
ti
I cwmRol.
10 FTX
DILUTION
15
20
(x10+)
Fig. 3. Effect of purified FTX on ACh release induced by KC1 depolarization or by calcium ionophore A23187. FIX was applied during a S-min period at various toxin dilutions in the conditions described in the kgend of Fig. 2. In presence of 4mM external calcium concentration, AC% release was triggered by addition of 60mM KC1 (tilled circles) or 4mM calcium ionophore A23187 (filled triangles). Dam are mean values f. S.E.M. obtained from three to nine different synaptosome preparations (KC1 depolarization) and three separate experiments (A23187 stimulation). Data were fitted using the computer programme GraphPad Inplot 4.0. E&t of ITX on KCl-evoked release is illustrated in right traces which were obtained in a representative experiment in the absence of toxin and with a 1/200,000 dilution of FfX.
E&t of Agelenopsis aperta venom on A23187 + calcium -evoked acetylcholine release
As previously shown,**9calcium ionophore can be used to elicit ACh release by Torpedo synaptosomes. It provides a direct pathway for calcium influx which bypasses activation of native presynaptic calcium channels. Synaptosomes were preincubated during a S-min period with the venom (Fig. 1). Calcium ionophore A23187 was added in the presence of 4 mM external calcium concentration. We tested the effect of various dilutions of the crude venom on evoked ACh release as shown in Fig. 2. The pattern of the inhibition curve was clearly different from the one obtained with KCl-evoked ACh release. No inhibition of the release was obtained before a 500,000-fold dilution. Half-inhibition was obtained at about l/30,000. Inhibition was maximal at lO,OOOfold dilution and was near 70% of control ACh release (without any venom). With boiled crude venom, the inhibitory effect was slightly reduced; the inhibition curve was displaced to the right (not shown). Eflect of FTX, a toxin purified from the venom, and a synthetic FTX on KC1 and A23187-evoked release RX, a low-molecular weight toxin purified from Agelenopsh aperta venom, was tested on KCl-evoked
ACh release and recordings are presented in Fig. 3. An inhibition of ACh release was obtained between 0.4 x 10m6and 20 x 10m6dilution. It was never greater than 40-SO%, showing a decreased inhibitory effect of the toxin in comparison with the crude venom.
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N.MOULIANand Y. MOROTGAIJDRY-TALAKMAIN 50%. FTX and its synthetic analog were also tested
on A23 187-evoked ACh release. Figures 3 and 4 show that both natural toxin and synthetic FTX were unable to inhibit ACh release during this way of stimulating synaptosomes whatever the concentration of toxin used. eflect of co-conotoxin on acetylcholine release induced by KC1 depolarization and A23187 ionophore -+ c&&n
G
01 0
1
2
sFTX
3
(mM)
Fig. 4. Effect of synthetic FTX on evoked-ACh release as a function of its concentration. Conditions are those presented in the legends of Figs 1 and 2. Data from four independent experiments were obtained for KCI stimulation (filled circles); results from two preparations in A23187evoked ACh release (filled triangles). Mean or mean + S.E.M. values are presented. Data were fitted with the GraphPad Inplot 4.0 programme.
The synthetic FTX, described as an argininepolyamine analogue (FTX 3: 3) of the natural FTX,4 was also tested on KCl-evoked ACh release. The concentration dependence of the effect of synthetic toxin is presented in Fig. 4. Synthetic FTX inhibits KCl-evoked ACh release: at 350 PM, evoked neurotransmitter release was slightly inhibited (about 80% of control). A concentration of 3 mM of the synthetic FTX was required to inhibit the release of about
CONOlOXlN
ColirmL
cry-CTX is a potent inhibitor of calcium influx in some but not all synaptosomal preparations.‘4.‘s,3’,3P We confirmed that o-CTX inhibits a fraction of KCl-evoked ACh release in Torpedo synaptosomes. Synaptosomes were pre-incubated for 5min with o-CTX in the absence of divalent cations since low divalent concentrations enhance the efficacy of (o-CTX.5,6,3yMoreover, we observed in preliminary experiments that o-CTX did not have any effect on KCl-induced ACh release when calcium and magnesium were added in the isolation medium. The toxin does not trigger by itself any leakage of ACh from the synaptosomes. ACh release was then stimulated in the presence of 4 mM calcium by depolarization with 60mM KCI. The concentration dependence of the effect of the toxin is presented in Fig. 5. (u-CTX has no effect under I /lM. ACh release inhibition was observed between 2 and 20 PM (about 35% of control at this concentration). A23187 + calcium ACh release was not inhibited (data not shown). Simultaneous uction of’ CO-conotox~n und FTX KCl-stimulated aretylcholine reieaw
0
on
To test whether the two toxms have addinvc effects, we successively applied FTX (10 x 10 ’ dilution) and 2 min later (I,-CTX (20 p M) over 3 min, then we stimulated synaptosomes with KC1 (final concentration 60 mM) in the presence of 4 mM extracellular calcium. In comparison with the effect observed with FTX alone, ACh release inhibition was not significantly enhanced with both toxins (Table 1). A fraction of KCl-evoked ACh release was resistant to both toxins applied simultaneously.
Table I. Effect of simultaneous action of FTX -L(9.
conotoxin Toxins
5
4
01 0
5
10
W-CONOTOXIN
15
20
(LAM)
Fig. 5. Effect of w-CTX on ACh release evoked with 60 mM KC1 depolarization. o-CTX was applied during a 5-mm period under the conditions described in Fig. 2. Data (individual or mean of duplicate) obtained from two to seven synaptosomal preparations were pooled and means + S.E.M. are presented. Data were fitted with the GraphPad Inplot programme.
FTX (l/loO,OoO) o-CTX GVIA (20 PM) FlX (l/100,000) + w-Cl-X GVIA (20 PM)
ACh release (% of control) 45.3 + 3.6 65.1 + 7.2 35.4 + 4.3
In three separate experiments, FTX (dilution l/lOO,W, 5 min), o_CTX (2OpM, 3 mm), and FTX (l/l~,~, 2 min) + w-CTX (20 PM, 3 min) were applied to synaptosomes under the conditions described in Fig. 2. ACh release was induced by KC1 addition. Results are mean + S.E.M. of individual or mean data obtained in each experiment.
FTX inhibits acetylcholine release in Torpedo synaptosomes DISCUSSION Some physiological characteristics of the channels which control the voltage-dependent influx of calcium in Torpedo synaptosomes were previously studied.i6*” The present results show that ACh release from Torpedo synaptosomes (used as a model of neuromuscular junctions) is mediated by different calcium influxes defined from a pharmacological point of view. The existence of a voltage-operated calcium channel of the P-type can be supposed upon observing the inhibitory effect on KCl-evoked ACh release of the venom of Agelenopsis uperta (inhibition of 70%), both native and after 5 min of boiling; of FTX, a purified fraction of the venom (inhibition of 50%); and of a synthetic FTX. These criteria correspond to some of the characteristics of P-type channels first described in dendritic and somatic membranes of cerebellar Purkinje cells and in squid giant synapse. 4~‘2The present results are also in accordance with recent electrophysiological data of Uchitel et ~1.~“ obtained with the levator auris muscle of mouse, which is used to study the presynaptic motor nerve terminals,’ and the neuromuscular junction of the mouse diaphragm. In this last preparation, Uchitel et ~1.~~ obtained a half-inhibition of the quanta1 content of evoked transmitter release for a FTX dilution of about l/200,000. In two preparations from different species, mammalian neuromuscular junctions and nerve terminals of elasmobranch electric organ, half-inhibition of FTX on neurotransmitter release mediated by two different stimuli is obtained in a similar range of concentrations. In both cases, neurotransmitter release is also inhibited by synthetic FTX. Thus, Torpedo synaptosomes seem to possess voltage-operated Ptype calcium channels sensitive to a low-molecular weight toxin of Agelenopsis upertu venom that mediates a significant part of evoked ACh release. However, the inhibition of neurotransmitter release by FTX observed by Uchitel et ~1.~~on the neuromuscular junction of the mouse diaphragm was almost total, while in Torpedo synaptosomes the maximal inhibition of ACh release with FTX we measured was about 50% of control. Furthermore L-type calcium channel blockers or agonists were previously shown not to act on KCl-induced ACh release from Torpedo synaptosomes.’ So it was interesting to examine whether the FTX-resistant KCl-evoked ACh release was supported by an w-CTX-sensitive calcium flux. We observed that a moderate part (35%) of ACh release is sensitive to w-CTX (at 20 PM). These results confirm the data of Farinas et ~1.’ who showed that ACh and ATP releases from Torpedo synaptosomes were differentially inhibited by o-CTX. Sensitivity of ATP release to w-CTX had been also described in previous works.*@’ As in these studies, the concentrations used here are rather high (in the micromolar range). We cannot exclude the possibility
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that o-CTX exerts non-selective effects, as was previously suggested by Kasai and Neher,” on calcium channels in NGlOS-15, a mammalian neuroblastoma-glioma cell line. PTX and o-CTX were applied simultaneously on Torpedo synaptosomes. We chose FIX and wCTX concentrations at which a maximal inhibition of each toxin was observed. FTX +w-CTX do not induce a notable increase of the inhibition of ACh release in comparison to the effect of FTX alone. Similar results were obtained at lower FTX and w-CTX concentrations and when o-CTX was applied before FTX on Torpedo synaptosomes (data not shown). Therefore, w-CTX + FTX do not exert additive effects on KCl-induced ACh release in Torpedo synaptosomes. This fact could be related to the hypothesis of non-selective effects of o-CTX.” According to Kasai and Neher,” o-CTX could first combine with a site common to many calcium channels followed by a transition to a specific binding site. This may explain the fact that o-CTX + FTX inhibition is similar to FTX inhibition. We plan to investigate the presence of N-type calcium channels using other criteria than the effect of w-CTX on ACh release, such as the study of binding properties of iodinated w-CTX on synaptosomal presynaptic plasma membranes. A lower efficacy of natural FTX in comparison with the crude Agelenopsis upertu venom (50 and 70%, respectively) suggested that other components present in the venom could act on ACh release triggered by a membrane depolarization. Indeed previous studies have shown that the crude venom of Agelenopsis upertu contains different toxins which were studied and classified;‘*27~31 they have higher molecular weights than FTX. Among these toxins, the w-agatoxins are able to inhibit calcium currents in some preparations2’~2s*29J7 in the nanomolar range and o-agatoxin IVA was recently shown to target P-type channels. *‘z The presence of calcium fluxes sensitive to toxins different from FTX in Torpedo synaptosomes could explain the stronger inhibition exerted by Agelenopsis venom on KCl-evoked ACh release than FTX. In a parallel set of experiments we studied the effects of Agelenopsis aperta venom and purified and synthetic FTX on ACh release triggered by the addition of a calcium ionophore in the release medium. The interest of using such a stimulus is to bypass the natural presynaptic calcium channel and to study the effect of pharmacological agents on the final step of ACh release. The A23187 + calciumevoked ACh release was shown to be sensitive to a high concentration of the crude venom and was inhibited to a maximum of 70%. FTX and synthetic FTX do not possess this property which could be related to other toxins present in the funnel spider venom. A target of these toxins could be the final step of ACh release and other processes which take place after the calcium entry.
N.MOULIAN
IO40
and Y. MOKOTGAIJDKY-TALAKMAIN
CONCLUSIONS
Torpedo
synaptosomes are a very good tool for studying the relationship between the entry of calcium through voltage-dependent channels and the release of ACh. In the present work, at least two types of calcium influxes can be revealed from a pharmacological point of view (FTX and U-CTX sensitive). Further studies are required to know if and how two types of calcium channels co-exist in the presynaptic membrane of Torpedo synaptosomes; it will allow a
more precise analysis of the physiological interactions between calcium channel entry and the release of neurotransmitter. A~,knowledgemunts--This work was made possible by the generous gift of purified and synthetic FTX by Dr R. Llin$s. We are sincerely grateful to him for his help and advice. We thank Dr M. Israel in whose laboratory this work was performed. Financial support was from CNRS and Servier pharmaceutical firm. N. Moulian is a thesis student of IFSBM (Institut de Formation Sup&ieure Bio-M&kale)
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FTX inhibits acetylcholine release in Torpedo synaptosomes
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29. Scott R. H., Dolphin A. C., Bindokas V. P. and Adams M. E. (1990) Inhibition of neuronal Ca*+ channel currents by the funnel web spider toxin w-Aga-IA. hfolec. Pharmac. 38, 71 I-718. 30. Sher E., Biancardi E., Pasafaro M. and Clementi F. (1991) Physiopathology of neuronal voltage operated calcium channels. Fedn Am. Sots exp. biol. J. 5, 2677-2683. 31. Skinner W. S., Adams M. E., Quistad G. B., Kataoka H., Cesarin B. J., Enderlin F. E. and Schooley D. A. (1989) Purification and characterixation of two classes of neurotoxins from the funnel web spider Agelenopsis apertu. J. biol. Chem. 264, 215&2155. 32. Stanley E. F. (1991) Single calcium channels on a cholinergic presynaptic nerve terminal. Neuron 7, 585-591. 33. Suskiw J. B.. Murawsky M. M. and Fortner R. C. (1987) Heterogeneity of presynaptic calcium channels revealed by species differences in the sensitivity of synaptosomal‘45Ca.entry to;conotoxin. kochem. biophys. Res. Commun. 145, 1283-1286. 34. Swandulla D., Carbone E. and Lux H. D. (1991) Do calcium channel classifications account for neuronal calcium channel diversity? Trends Neurosci. 14, 4651. 35. Tsien R. W., Ellinor P. T. and Home W. A. (1991) Molecular diversity of voltage-dependent Ca2+ channels. Trends Pharmac. Sci. 12, 349-354. 36. Uchitel 0. D., Protti D. A., Sanchez V., Cherksey B. D., Sugimori M. and Llinls R. (1992) P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses. Proc. natn. Acod. Sci. U.S.A. 89, 3330-3333. 37. Venema V. J., Swiderek K. M., Lee T. D., Hathaway G. M. and Adams M. E. (1992) Antagonism of synaptosomal calcium channels by subtypes of w-agatoxins. J. biol. Chem. 267, 261&2615.
38. Vickroy T. W., Schneider C. J. and Hildreth J. M. (1992) Pharmacological heterogeneity among channels that subserve acetylcholine release in vertebrate forebrain. Neuropharmacology 31, 307-309. 39. Wessler I., Dooley D. J., Werhand J. and Schlemmer F. (1990) Differentially effects of calcium channel antagonists (co-conotoxin GVIA, nifedipine, verapamil) on the electrically-evoked release of [‘I-I)acetylcholine from the myenteric plexus, phrenic nerve and neocortex of rats. Naunyn-Schmiedeberg’s Arch. Phurmac. 341, 288-294. 40. Yeager R. E., Yoshikami D., Rivier J., Crux L. J. and Miljanich G. P. (1987) Transmitter release from presynaptic terminals of electric organ: inhibition by the calcium channel antagonist omega conus toxin. J. Neurosci. 7.2390-2396. 41. Yoshikami D., Bagaboldo Z. and Olivera B. M. (1989) The inhibitory effects of omega-conotoxins on Ca channels and synapses. Ann. N.Y. Acad. Sci. 560, 23&248. (Accepted 20 January 1993)
0306-4522/93 $6.00 + 0.00 Pqamon Press Ltd 0 1993 IBRO
AGELENOPSIS APERTA VENOM AND FTX, A PURIFIED TOXIN, INHIBIT ACETYLCHOLINE RELEASE IN TORPEDO SYNAFTOSOMES N. MOULIAN and Y. MOROT GAUDRY-TALARMAIN Dkpartement
de Neurochimie, Laboratoire de Neurobiologie Cellulaire et Moleculaire, C.N.R.S., 91198 Gif-sur Yvette Ccdex, France
Ahtdraet-The presence of P-type calcium channels in synaptosomes prepared from electric organ of Torpedo marmorata was investigated by using the venom of Agelenopsis aperta, a toxin purified from it, FIX, and its synthetic analog. We analysed the action of these agents on acetylcholine release which was continuously followed using a chemiluminescent assay. Agelenopsis aperta venom, FIX and synthetic FIX inhibit acetylcholine release from synaptosomes induced by a presynaptic membrane depolarization with 60 mM KCl. A stronger inhibition of acetylcholine release was observed with the venom than with FIX (70 and 50%, respectively). Another way of triggering acetylcholine release from Torpedo synaptosomes is to insert in the presynaptic membrane a calcium ionophore A23187 which allows the bypass of the natural calcium channels. The venom of Agelenopsis aperta inhibits A23187-evoked acetylcholine release. Purified and synthetic FIX does not possess this property, suggesting that this inhibition of acetylcholine release was due to other toxins of the venom. Another type of pharmacological sensitivity of Torpedo calcium channels was also demonstrated using o-conotoxin GVIA. At a concentration of 20 PM, this toxin was able to inhibit about 35% of KCl-evoked acetylcholine release. When FIX + o-conotoxin GVIA were applied together, the inhibitory effect on KCI-evoked acetylcholine release was not significantly increased in comparison with the one observed with FIX alone. In conclusion, we examined the effect of different agents on acetylcholine release from Torpedo marmorata electric organ synaptosomes; acetylcholine release was elicited with KC1 depolarization and followed continuously with a chemiluminescent assay. It allowed us to distinguish different calcium fluxes in Torpedo synaptosomes. The existence of P-type voltage-dependent calcium channels was supposed upon observing the inhibitory effect on KCl-evoked acetylcholine release of the venom of Agelenopsis aperta, of FIX (a purified toxin), and of a synthetic FIX. Moreover acetylcholine release from Torpedo synaptosomes was moderately inhibited by o-conotoxin GVIA, a blocker of N-type calcium channels.
The increase of calcium concentration in the nerve terminal cytoplasm is the necessary trigger for release of neurotransmitter from all neurons during stimulation. Different kinds of voltage-operated calcium channels have recently been described in the membrane of excitable cells;‘*~‘9~28~30~“~3s when activated, they allow calcium to enter the nerve ending along its concentration gradient. Low-threshold T-type and high-threshold N-, L-, and P-type channels have been described on the basis of their pharmacological sensitivity. It is possible to study this stimulation-release coupling with a rich cholinergic preparation of nerve endings isolated from the electric organ of Torpedo marmorata.‘0*23 Indeed Torpedo synaptosomes are very useful for studies on cholinergic mechanisms since they constitute a homogeneous preparation with respect to the neurotransmitter; they are isolated with low contamination from postsynaptic membranes and other structures; they maintain their membrane potential; and they can be kept in a good metabolic state for more than 24 h after isolation.
Abbreviations: ACh, acetylcholine; FfX, funnel-web spider toxin; o-CTX, omegaconotoxin.
Moreover, a chemiluminescent assay using choline oxidase allows continuous monitoring of the release of acetylcholine (ACh). 8,9 These tools allow the characterization, using pharmacological agents, of the calcium channels present at the presynaptic level of a synapse which is embryologically related to a neuromuscular type. The pharmacological characterization of calcium channels has been done in cholinergic synapses of central or peripheral origin.‘4*32*38*39 To identify the calcium channels which possibly support these activities in Torpedo synaptosomes, we used agents previously described to act on calcium channel subtypes. Using a toxin extracted from the venom of the American funnel-web spider Agelenopsti aperta (FTX), a new type of calcium channel (the P-type) was recently described by LlinPs et aLI Putative P-type channels have been identified pharmacologically in different types of neurons.” As the goal of this study was to investigate the existence of these P channels on the Torpedo synaptosomes, we decided to test the effects of the crude venom of Agelenopsis aperta on evoked ACh release. Further studies were done with boiled venom, with FTX, a purified toxin fraction with a low molecular mass in the 2(l0-400 mol. wt range, and with synthetic FTX.4
1035
IO.36
N.MOULIAN
and Y.
MOKOT GAWKY-TALARMAIN
For comparison, the effect of w-conotoxin GVIA (w-CTX), a blocker of N-type calcium channels,15.24.26,41 was included in the study. o-CTX inhibits evoked ATP and ACh releases from Torpedo synaptosomes,2.7.40 suggesting that N-type calcium channel may also be present in Torpedo presynaptic membranes. Two types of stimuli were used to evoke ACh release: a depolarization of the presynaptic membrane by application of high external KC1 concentration or an insertion in the bilayer of a calcium ionophore A23187. The two procedures allow the discrimination between drugs which modify the last step of ACh release after the increase of intracellular calcium concentration (using A23 187 + calcium) and those which affect the calcium uptake through natural calcium channels (using KC1 stimulation). EXPERIMENTAL PROCEDURES
Preparation of synaptosomes
were isolated does not modify the-KCl-evoked ACh release. In some experiments FTX and wCTX were successfully preincubated with the synaptosomes during 2 and 3 min. respectively. Then 4~1 of 0.5 M CaCl, (final concentration 4 mM) was injected into the release medium. Evoked ACh release was triggered by a further addition of 10 111of a 3 M KCI solution (final concentration 69‘mM). Light responses corresponding to ACh release in the first minutes were calibrated by the addition of known amounts of ACh. tosomes
Chemicals
The material used for the chemiluminescent assay of ACh, choline oxidase (Wako Company, Japan), horseradish peroxidase (EC 1.11.1.7) type III from Sigma (St Louis, MO, U.S.A.), and luminol from Merck (Germany) were prepared as described previously. 8,9 Ionophore A23187 came from Boehringer (Germany); stock solutions (20 mM) were made in dimethylsulfoxide and diluted at I:40 in distilled water. Crude venom from the American funnel web spider Agelenopsis aperta was obtained from Spider Pharm, Black Canyon City, AZ, U.S.A. Dr R. Lhnas provided us with FTX, a toxin fraction, and with synthetic FTX (3:3).4 Different F’TX dilutions from the suider venom fraction were used. w-CTX GVIA was from Bachem, Bubendorf, Switzerland. All stock toxin solutions were dissolved into
Pure choline@ nerve endings were isolated from electric organ of the fish Torpedo marmorata. Fish were purchased from the marine station of Arcachon (France) and were kept in oxygenated artificial sea water tanks. The synaptosomes of Torpedo electric organ were prepared as previously described.iO,*r Nerve endings derived from 25 g of electric organ were collected from a discontinuous iso-osmotic sahne-sucrose gradient in 40-50ml of a synaptosomal fraction containina: 280mM NaCl. 3 mM KCI. 5 mM NaHCO,, 1.2 mM sodium-phosphate buffer, pH 7.2, 5.5 mM glucose, 300 mM sucrose and 100 mM urea. The medium is iso-osmotically similar to the elasmobranch plasma medium and differs from the one proposed by Israel et al.” only in the absence of divalent cations in discontinuous sucrose gradients. Thirty to fifty microlitres of this synaptosomal suspension corresponding to IO-30 mg of wet tissue were used for the ensuring ACh release experiments.
COHTRZJL
1 .mln
AGELENWSIS
APERTA
VENOM
Acetylcholine release experiments using a chemiluminescent assay
The release of endogenous ACh from Torpedo synaptosomes was continuously followed using the choline oxidase chemiluminescent method previously described.‘,’ In summary, this technique uses the specificity of two enzymes: acetylcholinesterase (present in the synaptosomal fraction) hydrolyses released ACh into choline and acetate and then choline oxi&se converts the newly formed choline into betaine and H,O,. H,O, is then converted into light using luminol and peroxidase. This method allowed us to monitor in real time the release of neurotransmitter in a physiological solution at room temperature. Aliquots of the synaptosomal suspension were mixed with 450-470 pl of a physiological medium made with: 280 mM NaCl. 3 mM KCl. 400 mM sucrose. 5.5 mM glucose and 50 mM Tris base/Tris-HCI buffer at pH 8.6 as required for the luminol + peroxidase chemiluminescent reaction. The medium was put in a glass tube and was continuously stirred by a small magnetic bar. Agelenopsis aperta venom and toxins were added directly to the synaptosomal suspension. Preincubations with each of them were carried out during 5min. The effect of w-CTX on neurotransmitter release such as noradrenaline5.6 or [‘H)ACh’9 was shown to be very sensitive and inversely related to the presence of external cations, hence we tested the effect of all the toxins on evoked ACh release after an incubation with no calcium or magnesium. In preliminary experiments, we checked that the omission of divalent cations in the medium in which synap-
DILtJTIOEl lj200
DILUTION
l/20
am
a00
DILUTTQN
l/l60
DILiTI$lN
1;25
000
Ooo
Fig. 1. Effect of Agelenopsis aperta venom on evoked ACh release. Synaptosomes were diluted (1: 16 to 1: zo) in a medium (28OmM NaCl, 400 mM sucrose, 3 mM KCl, 5.5 mM glucose and 90mM Tris, pH 8.6) cont&ng enzymes for chemihuninescont detection of ACh release (see Experimental Procedurea) and with various dilut&ms of Agelenopsis aperta venom during a S-min period. Then calcium was added to reach 4mM fmal conoentration. Release. of ACh was induced either by 6OmM KCl (left traces) or bv 4 uM A23187 (ri&t traces). Representative illustrations-of ACh relase e&&ments are shown in the absence of venom (control) or in the presence of various concentrations of spider venom. ACh release was calibrated by injecting an internal ACh standard. Note that at the highest concentrations of venom, it was necessary to increase the sensitivity of the recorder.
FTX inhibits acetylcholine release in Torpedo synaptosomes
1037
distilled water; aliquots were stored at -80°C prior to use at the appropriate dilution.
9 8
RFSUL’IS
Efict of venom of Agelenopsis aperta on KCI-evoked acetylcholine release
Synaptosomes were preincubated with aliquots of the venom during a 5-min period. The application of the venom does not induce any significant basal ACh release. Then the release of ACh from synaptosomes was examined (in the presence of 4 mM calcium) after a KC1 depolarization which opens voltage-dependent calcium channels naturally present in the presynaptic membrane. KCl-evoked release was inhibited in a concentration-dependent manner by Agelenopsis aperta venom, as shown in Fig. 1 in recordings of the light emission. Half-inhibition was obtained at the 1/200,OflO dilution of crude venom in the release medium (Fig. 2). Inhibition was observed between l,OOO,OOO-and 10,000-fold-dilution and was never complete. About 30% of ACh release was resistant to the action of the venom. Venom contains components which interfere and decrease the sensitivity of the chemiluminescent assay. At the highest concentrations it was necessary to increase the amplification to compensate for this effect. Venom that had been boiled for 5 min in order to denaturate its proteic components still inhibited KCIevoked ACh release with no loss of potency (data not shown).
i
io
lb0
AGEENCPMAFSRE4DLUllON(x166)
Fig. 2. Dose-response curve of the effect of Agelenopsis apertu venom on evoked ACh release from spaptosomes. Aliquots of synaptosomes diluted in an alkaline iso-osmotic medium (see Experimental Rocedures) were incubated with different concentrations of venom during a 5-min period. Calcium was then added; ACh release was induced either by a membrane depolarization with 60 mh4 KC1 (filled circles; four independent experiments) or with 4 gM A23187 (filled triangles; two independent experiments). In each experiment, individual data or mean of duplicate were expressed as percentages of their own ACh control release (mean of two to five determinations obtained in the absence of toxin). Results were pooled and the mean value is presented (*S.E.M.).
ti
I cwmRol.
10 FTX
DILUTION
15
20
(x10+)
Fig. 3. Effect of purified FTX on ACh release induced by KC1 depolarization or by calcium ionophore A23187. FIX was applied during a S-min period at various toxin dilutions in the conditions described in the kgend of Fig. 2. In presence of 4mM external calcium concentration, AC% release was triggered by addition of 60mM KC1 (tilled circles) or 4mM calcium ionophore A23187 (filled triangles). Dam are mean values f. S.E.M. obtained from three to nine different synaptosome preparations (KC1 depolarization) and three separate experiments (A23187 stimulation). Data were fitted using the computer programme GraphPad Inplot 4.0. E&t of ITX on KCl-evoked release is illustrated in right traces which were obtained in a representative experiment in the absence of toxin and with a 1/200,000 dilution of FfX.
E&t of Agelenopsis aperta venom on A23187 + calcium -evoked acetylcholine release
As previously shown,**9calcium ionophore can be used to elicit ACh release by Torpedo synaptosomes. It provides a direct pathway for calcium influx which bypasses activation of native presynaptic calcium channels. Synaptosomes were preincubated during a S-min period with the venom (Fig. 1). Calcium ionophore A23187 was added in the presence of 4 mM external calcium concentration. We tested the effect of various dilutions of the crude venom on evoked ACh release as shown in Fig. 2. The pattern of the inhibition curve was clearly different from the one obtained with KCl-evoked ACh release. No inhibition of the release was obtained before a 500,000-fold dilution. Half-inhibition was obtained at about l/30,000. Inhibition was maximal at lO,OOOfold dilution and was near 70% of control ACh release (without any venom). With boiled crude venom, the inhibitory effect was slightly reduced; the inhibition curve was displaced to the right (not shown). Eflect of FTX, a toxin purified from the venom, and a synthetic FTX on KC1 and A23187-evoked release RX, a low-molecular weight toxin purified from Agelenopsh aperta venom, was tested on KCl-evoked
ACh release and recordings are presented in Fig. 3. An inhibition of ACh release was obtained between 0.4 x 10m6and 20 x 10m6dilution. It was never greater than 40-SO%, showing a decreased inhibitory effect of the toxin in comparison with the crude venom.
1038
N.MOULIANand Y. MOROTGAIJDRY-TALAKMAIN 50%. FTX and its synthetic analog were also tested
on A23 187-evoked ACh release. Figures 3 and 4 show that both natural toxin and synthetic FTX were unable to inhibit ACh release during this way of stimulating synaptosomes whatever the concentration of toxin used. eflect of co-conotoxin on acetylcholine release induced by KC1 depolarization and A23187 ionophore -+ c&&n
G
01 0
1
2
sFTX
3
(mM)
Fig. 4. Effect of synthetic FTX on evoked-ACh release as a function of its concentration. Conditions are those presented in the legends of Figs 1 and 2. Data from four independent experiments were obtained for KCI stimulation (filled circles); results from two preparations in A23187evoked ACh release (filled triangles). Mean or mean + S.E.M. values are presented. Data were fitted with the GraphPad Inplot 4.0 programme.
The synthetic FTX, described as an argininepolyamine analogue (FTX 3: 3) of the natural FTX,4 was also tested on KCl-evoked ACh release. The concentration dependence of the effect of synthetic toxin is presented in Fig. 4. Synthetic FTX inhibits KCl-evoked ACh release: at 350 PM, evoked neurotransmitter release was slightly inhibited (about 80% of control). A concentration of 3 mM of the synthetic FTX was required to inhibit the release of about
CONOlOXlN
ColirmL
cry-CTX is a potent inhibitor of calcium influx in some but not all synaptosomal preparations.‘4.‘s,3’,3P We confirmed that o-CTX inhibits a fraction of KCl-evoked ACh release in Torpedo synaptosomes. Synaptosomes were pre-incubated for 5min with o-CTX in the absence of divalent cations since low divalent concentrations enhance the efficacy of (o-CTX.5,6,3yMoreover, we observed in preliminary experiments that o-CTX did not have any effect on KCl-induced ACh release when calcium and magnesium were added in the isolation medium. The toxin does not trigger by itself any leakage of ACh from the synaptosomes. ACh release was then stimulated in the presence of 4 mM calcium by depolarization with 60mM KCI. The concentration dependence of the effect of the toxin is presented in Fig. 5. (u-CTX has no effect under I /lM. ACh release inhibition was observed between 2 and 20 PM (about 35% of control at this concentration). A23187 + calcium ACh release was not inhibited (data not shown). Simultaneous uction of’ CO-conotox~n und FTX KCl-stimulated aretylcholine reieaw
0
on
To test whether the two toxms have addinvc effects, we successively applied FTX (10 x 10 ’ dilution) and 2 min later (I,-CTX (20 p M) over 3 min, then we stimulated synaptosomes with KC1 (final concentration 60 mM) in the presence of 4 mM extracellular calcium. In comparison with the effect observed with FTX alone, ACh release inhibition was not significantly enhanced with both toxins (Table 1). A fraction of KCl-evoked ACh release was resistant to both toxins applied simultaneously.
Table I. Effect of simultaneous action of FTX -L(9.
conotoxin Toxins
5
4
01 0
5
10
W-CONOTOXIN
15
20
(LAM)
Fig. 5. Effect of w-CTX on ACh release evoked with 60 mM KC1 depolarization. o-CTX was applied during a 5-mm period under the conditions described in Fig. 2. Data (individual or mean of duplicate) obtained from two to seven synaptosomal preparations were pooled and means + S.E.M. are presented. Data were fitted with the GraphPad Inplot programme.
FTX (l/loO,OoO) o-CTX GVIA (20 PM) FlX (l/100,000) + w-Cl-X GVIA (20 PM)
ACh release (% of control) 45.3 + 3.6 65.1 + 7.2 35.4 + 4.3
In three separate experiments, FTX (dilution l/lOO,W, 5 min), o_CTX (2OpM, 3 mm), and FTX (l/l~,~, 2 min) + w-CTX (20 PM, 3 min) were applied to synaptosomes under the conditions described in Fig. 2. ACh release was induced by KC1 addition. Results are mean + S.E.M. of individual or mean data obtained in each experiment.
FTX inhibits acetylcholine release in Torpedo synaptosomes DISCUSSION Some physiological characteristics of the channels which control the voltage-dependent influx of calcium in Torpedo synaptosomes were previously studied.i6*” The present results show that ACh release from Torpedo synaptosomes (used as a model of neuromuscular junctions) is mediated by different calcium influxes defined from a pharmacological point of view. The existence of a voltage-operated calcium channel of the P-type can be supposed upon observing the inhibitory effect on KCl-evoked ACh release of the venom of Agelenopsis uperta (inhibition of 70%), both native and after 5 min of boiling; of FTX, a purified fraction of the venom (inhibition of 50%); and of a synthetic FTX. These criteria correspond to some of the characteristics of P-type channels first described in dendritic and somatic membranes of cerebellar Purkinje cells and in squid giant synapse. 4~‘2The present results are also in accordance with recent electrophysiological data of Uchitel et ~1.~“ obtained with the levator auris muscle of mouse, which is used to study the presynaptic motor nerve terminals,’ and the neuromuscular junction of the mouse diaphragm. In this last preparation, Uchitel et ~1.~~ obtained a half-inhibition of the quanta1 content of evoked transmitter release for a FTX dilution of about l/200,000. In two preparations from different species, mammalian neuromuscular junctions and nerve terminals of elasmobranch electric organ, half-inhibition of FTX on neurotransmitter release mediated by two different stimuli is obtained in a similar range of concentrations. In both cases, neurotransmitter release is also inhibited by synthetic FTX. Thus, Torpedo synaptosomes seem to possess voltage-operated Ptype calcium channels sensitive to a low-molecular weight toxin of Agelenopsis upertu venom that mediates a significant part of evoked ACh release. However, the inhibition of neurotransmitter release by FTX observed by Uchitel et ~1.~~on the neuromuscular junction of the mouse diaphragm was almost total, while in Torpedo synaptosomes the maximal inhibition of ACh release with FTX we measured was about 50% of control. Furthermore L-type calcium channel blockers or agonists were previously shown not to act on KCl-induced ACh release from Torpedo synaptosomes.’ So it was interesting to examine whether the FTX-resistant KCl-evoked ACh release was supported by an w-CTX-sensitive calcium flux. We observed that a moderate part (35%) of ACh release is sensitive to w-CTX (at 20 PM). These results confirm the data of Farinas et ~1.’ who showed that ACh and ATP releases from Torpedo synaptosomes were differentially inhibited by o-CTX. Sensitivity of ATP release to w-CTX had been also described in previous works.*@’ As in these studies, the concentrations used here are rather high (in the micromolar range). We cannot exclude the possibility
1039
that o-CTX exerts non-selective effects, as was previously suggested by Kasai and Neher,” on calcium channels in NGlOS-15, a mammalian neuroblastoma-glioma cell line. PTX and o-CTX were applied simultaneously on Torpedo synaptosomes. We chose FIX and wCTX concentrations at which a maximal inhibition of each toxin was observed. FTX +w-CTX do not induce a notable increase of the inhibition of ACh release in comparison to the effect of FTX alone. Similar results were obtained at lower FTX and w-CTX concentrations and when o-CTX was applied before FTX on Torpedo synaptosomes (data not shown). Therefore, w-CTX + FTX do not exert additive effects on KCl-induced ACh release in Torpedo synaptosomes. This fact could be related to the hypothesis of non-selective effects of o-CTX.” According to Kasai and Neher,” o-CTX could first combine with a site common to many calcium channels followed by a transition to a specific binding site. This may explain the fact that o-CTX + FTX inhibition is similar to FTX inhibition. We plan to investigate the presence of N-type calcium channels using other criteria than the effect of w-CTX on ACh release, such as the study of binding properties of iodinated w-CTX on synaptosomal presynaptic plasma membranes. A lower efficacy of natural FTX in comparison with the crude Agelenopsis upertu venom (50 and 70%, respectively) suggested that other components present in the venom could act on ACh release triggered by a membrane depolarization. Indeed previous studies have shown that the crude venom of Agelenopsis upertu contains different toxins which were studied and classified;‘*27~31 they have higher molecular weights than FTX. Among these toxins, the w-agatoxins are able to inhibit calcium currents in some preparations2’~2s*29J7 in the nanomolar range and o-agatoxin IVA was recently shown to target P-type channels. *‘z The presence of calcium fluxes sensitive to toxins different from FTX in Torpedo synaptosomes could explain the stronger inhibition exerted by Agelenopsis venom on KCl-evoked ACh release than FTX. In a parallel set of experiments we studied the effects of Agelenopsis aperta venom and purified and synthetic FTX on ACh release triggered by the addition of a calcium ionophore in the release medium. The interest of using such a stimulus is to bypass the natural presynaptic calcium channel and to study the effect of pharmacological agents on the final step of ACh release. The A23187 + calciumevoked ACh release was shown to be sensitive to a high concentration of the crude venom and was inhibited to a maximum of 70%. FTX and synthetic FTX do not possess this property which could be related to other toxins present in the funnel spider venom. A target of these toxins could be the final step of ACh release and other processes which take place after the calcium entry.
N.MOULIAN
IO40
and Y. MOKOTGAIJDKY-TALAKMAIN
CONCLUSIONS
Torpedo
synaptosomes are a very good tool for studying the relationship between the entry of calcium through voltage-dependent channels and the release of ACh. In the present work, at least two types of calcium influxes can be revealed from a pharmacological point of view (FTX and U-CTX sensitive). Further studies are required to know if and how two types of calcium channels co-exist in the presynaptic membrane of Torpedo synaptosomes; it will allow a
more precise analysis of the physiological interactions between calcium channel entry and the release of neurotransmitter. A~,knowledgemunts--This work was made possible by the generous gift of purified and synthetic FTX by Dr R. Llin$s. We are sincerely grateful to him for his help and advice. We thank Dr M. Israel in whose laboratory this work was performed. Financial support was from CNRS and Servier pharmaceutical firm. N. Moulian is a thesis student of IFSBM (Institut de Formation Sup&ieure Bio-M&kale)
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Adams M. E., Bindokas V. P., Hasegawa L. and Venema V. J. (1990) w-Agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis Aperta) venom. J. biol. Chem. 265, 861-867. 2. Ahmad S. N. and Miljanich G. P. (1988) The calcium channel antagonist, o-conotoxin and electric organ nerve terminals: binding and inhibition of transmitter release and calcium influx. Bruin Res. 453, 247 256. 3. Angaut-Petit D., Molgo J., Connold A. L. and Faille L. (1987) The levator auris longus muscle of the mouse: a convenient preparation for studies of short and long-term presynaptic effects. Neurosci. Let?. 82, 83-88. 4. Cherksey B. D., Sugimori M. and Llinas R. (1991) Properties of calcium channels isolated with spider toxin, FTX. Ann. N.Y. Acad. Sci. 635, 80-89. 5. Clasbrummel B., Osswald H. and Illes P. (1989) Inhibition of noradrenaline tail artery. Br. J. Pharmac. %, 101-l 10.
release by w-conotoxin GVIA m the rat
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