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Ciguatoxin extracted from poisonous moray eels Gymnothorax javanicus triggers acetylcholine release from Torpedo cholinergic synaptosomes via reversed Na+ –Ca2+ exchange
65
Neuroscience Letters, 160 (1993) 65-68 C 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
NSL09806
Ciguatoxin extracted from poisonous moray eels Gymnothorax javanicus triggers acetylcholine release from Torpedo cholinergic synaptosomes via reversed N a + -Ca2+ exchange lordi Molg6 a , Yvette Morot Gaudry-Talarmain b , Anne Marie Legrand'" and Nathalie Moulian b Deparrements de "Neuropharmacologie et de bNeurochimie. Laboratoire de Neurobiologie Cellulaire et Moieculaire. Centre National de la Recherche Scientifique. Gif-sur- Yvette (France) (Received 6 April 1993; Accepted 2 June 1993)
Key words:
Ciguatoxin; Acetylcholine release; Torpedo cholinergic synaptosome; Tetrodotoxin; Na+ channel; Lt; Na+-Ca 2+ exchange 2
Ciguatoxin (CTX) (0.1 pM to 10 nM) added to a suspension of Torpedo synaptosomes incubated in Ca +-free medium caused no detectable acetylcholine (ACh) release. However, subsequent addition ofCa2 • caused a large ACh release that depended on time of exposure, dose ofCTX and on [Ca2+). Tetrodotoxin completely prevented CTX-induced Ca2+-dependent ACh release. Simultaneous blockade of Ca 2' channel subtypes by FTX, a tOXin extracted from the venom of the spider Agelenopsis aperta. ro-conotoxin and Gd-'+ did not prevent ACh release caused by CTX, upon addition 2 orCa2+. These results suggest that CTX activates the reversed operation of the Na+/Ca ' exchange system allowing the entry ofCa 2• in exchange for Na·. It is concluded that Torpedo synaptosomes are endowed with NaT channels sensitive to pico- to nanomolar concentrations ofCTX.
Ciguatoxin (CTX) is one of the principal ichtyotoxins inVolved in a complex human food poisoning known as ciguatera. characterized mainly by gastrointestinal and neUrological disorders [1-16]. Ciguatera fish poisoning has been associated with the consumption of many species of tropical and subtropical fishes which acquired the toxicity from their diet and transferred it to other fish through the marine food chain and ultimately to man (1,14. 16]. Recent studies have disclosed that CTX extracted from poisonous moray eels (Gymnothorax javanicus or l..ycodontisjavanicus) named CTX-Ib [14] or CTX-l [8]. has a brevetoxin-Iike polyether structure and a molecular formula of C6QH 860 J9 • CTX exerts its effects primarily by altering the membrane properties of excitable cells in such a way that actiVates voltage-gated Na + channels at normal membrane resting potential causing tetrodotoxin-sensitive membrane depolarization and repetitive or spontaneous ac-
-
Correspondence: J. Molg6, Laboratoire de Neurobiologie Cellulaire et Moleculaire, Centre National de la Recherche Scientifique, 91198 GlFSUR-YVETTE CEDEX, France. Fax: (33) (I) 69-829466. 'Permanent address: A.M. Legrand, Institut Territorial de Recherches MlXiicales Louis Malarde, Associe aI'Institut Pasteur, BP 30, Papeete, Tahiti, polynesie Fran~aise.
tion potentials in myelinated axons, neuroblastoma cells and skeletal muscle fibres [2, 3, 11]. Binding studies showed that CTX inhibits the binding of eH]brevetoxin3 to rat brain membranes and that CTX and brevetoxins share a common binding site on the neuronal voltagedependent Na+ channel protein [8, 9]. Previous studies using purified CTX revealed that CTX increases the rate of release of CH]r-aminobutyric acid and CH]dopamine from rat brain synaptosomes [3]. CTX-induced transmitter release was sensitive to blockade by tetrodotoxin (TTX) but was unaffected by Ca2+ channel antagonists like nitrendipine and 0-600 [3]. Since CTX was reported to have no action on the activity of the Na+-K+-ATPase, it was suggested that the enhancement of neurotransmitter release may be d~e to the depolarization-induced Ca2+ influx caused by the activation of voltage-dependent Na+ channels of the synaptosomal membrane· [3]. CTX was reported also to enhance spontaneous quantal release of acetylcholine (ACh) from motor nerve terminals. measured electrophysiologically as an increase in miniature end plate potential frequency [II]. TTX completely abolished or prevented CTX action on quantal release. suggesting that CTX effect depends upon Na+ entry into the motor nerve terminals [11]. The aim of the present work was to further character-
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logical medium consisting of (mM): NaCI, 280; KCI; 3; MgCI 2 • 1.8; sucrose. 400; glucose, 5.5; and Tris buffer. SO (pH = 8.6), as required for the choline oxidase-chemiluminescent reaction and 20.u1 of the materials used for the chemiluminescent assay of ACh: choline oxidase (WakO, Japan), 8 .ul of a solution containing 250 U/ml; horseradish peroxidase (EC 1.11.1.7. type III; Sigma. USA). 4 .ul of a solution containing 2 mglml; and luminol (Merck, Germany), 8.u1 of a solution I mM in 0.2 M Tris buffer. pH 8.6. In some experiments, the NaCI was completely replaced by LiCl. Synaptosomal preparations contained sufficient amounts of ACh-esterase for the hydrolysis of released ACh so that no exogenous enzyme was necessary to be added to the mixture of solutions. The light emitted by the chemiluminescent reaction was detected by a photomultiplier unit, recorded and calibrated by the addition of known amounts of standard ACh. Total ACh content of synaptosomes was determined by addition of the detergent Triton X-lOO (0.01 %) [5, 6]. CTX-I b was extracted from G. javanicus viscera as previously described [7. 14]. TTX, synthetic w-conotoxin GVIA and Triton X-I00 (Sigma) stock solutions were dissolved into bi-distilled water. Stock solutions of the Ca ionophore A23187 (Boehringer, Germany) and gramicidin-D were made in dimethylsulfoxide and further diluted I :40 and I :400 in bi-distilled water. FTX, a low molecular weight toxin purified from the venom of the American funnel-web spider Age/enopsis aperta, waS kindly provided by R. Llimis and used at a dilution I: 100,000 [13], Gd chloride was obtained from (Ventro n, Germany). All reagents were added directly to the synaptosomal suspension. CTX (0.1 pM to 10 nM) added to a diluted suspension of Torpedo synaptosomes incubated in a nominally Ca2+free medium caused no detectable change in the light emission, that permits to quantify ACh release, for periods of up to 15 min (Fig. lA). These results indicate that CTX, in the absence of external Ca2+, neither triggers detectable ACh release nor causes leakage of ACh. When synaptosomes were exposed to CTX (10 nM) for 15 min in a Ca 2+ -free medium, subsequent addition of 4 mM Ca 2+ caused an immediate large release of ACh. As shown in Fig. lA, the light emission resulting from the chemiluminescent detection of ACh decayed in -3 min to a steady basal level similar to that observed before Ca 2+ addition. The amount of ACh released, under this condition, expressed as percentage of total content corresponded to 18.7 ± 2.7 (mean ± S.E.M., n =5). Further addition either of high K + (60 mM) or the Ca ionophore A23187 (4 .uM) could still trigger Ca2+ -dependent ACh release (Figs. lA, 3A). However, the amount of ACh released was much less than measured in synaptosomes not exposed to CTX. The decrease in the stimulatory
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Fig. I. Representative recordings of effects of CTX (10 nM) on ACh release (A and B) and its interaction with I ,uM TTX (B) in synap· tosomes incubated either in Na+· (A and B) or Li+.containing medium (C). Arrows and numbers indicate addition of Cal + (4 mM). A23187 (4 ,uM). standard amounts of ACh (pmol) or Triton X·IOG (0.01 %) to medium.
ize the effects of highly purified CTX (CTX-Ib), extracted from G. javanicus. on ACh release from pure cholinergic synaptosomes. Synaptosomes were isolated from the electric organ of the fish Torpedo marmorata according to described methods [5. 12]. All steps of the isolation procedure were carried out at 4°C. Nerve endings derived from 25 g of electric organ were chopped up and diluted in a oxygenated physiological solution of the following composition (mM): NaC!. 280; KCI, 3; MgCI 2, 1.8; CaCI 2, 3.4; Na phosphate. 1.2; NaHCO). 5; glucose, 5.5; urea, 300; and sucrose. 100 (pH =7.2). Then, synaptosomes were collected from a discontinuous iso-osmotic saline-sucrose density gradient in 40-50 ml of the above-mentioned medium devoided of Ca 2+. Aliquots of this synaptosomal suspension were used at room temperature (20-22°C) for ensuring ACh release using the choline oxidase-chemiluminescent method [5, 6]. Aliquots of the synaptosomal fraction (30--50 ,LII) were added to 470-450,LI1 of a physio-
67
effect of the ionophore A23187 after CTX action may be due to a partial exhaustion of the releasable pool of ACh, which has been reported to represent::= 40% of total ACh Content [6, 12]. As shown in Fig. lA, further addition of l'riton X-loo (0.01%) revealed that synaptosomes still Contained consistent amounts of ACh. Ca2+-induced ACh release by CTX was dependent on the concentration of CTX used. Active concentrations of CTX ranged in the pico- to nanomolar range as shown in a representative dose-response curve (Fig. 2A). In addition, ACh release triggered by Ca2+ in the presence of CTX depended on time. of exposure to the toxin in Na+Containing Ca2+ -free medium (Fig. 2B) and on Ca2+ concentration used to trigger ACh release (Fig. 2C). As shown in Fig. 2B,C, ACh release induced by CTX exposUre for 60 min or triggered by 36 mM Ca2+ reached values close to 40% of the total ACh content. TTX (l .uM), when applied a few minutes before CTX to synaptosomes kept in a nominally Ca2+-free medium, Completely prevented CTX-induced ACh release upon addition of 4 mM Ca2+ (Fig. I B). However, TTX (1 .u M ) neither prevented the action of the ionophore A23187 (4.uM) (Fig. IB) nor antagonized the large ACh release caused by 0.5 .u M gramicidin-D that reached 50.5 ± 3.5% (n =4) of total ACh content (data not shown). This antibiotic induces nonspecific membrane channels for monovalent cations [15], depolarizes the 2 sYnaptosome membrane [10) and triggers a large Ca +dependent ACh release [6). To study further the Na+ requirement for CTX-induced ACh release, extracellular Na+ was replaced, on equimolar basis, by Li+. As shown in Fig. Ie. CTX (10 nM) had no detectable action on ACh release in nominally Ca2+-free medium containing Li+, similarly to what Was shown in Na+-containing medium (Fig. IA). The addition of Ca 2+, to synaptosomes exposed for 15 min to CTX in Li+ -containing medium (Fig. I C), triggered Illuch less ACh release « 2% of the total content, n =3) than in Na+ containing medium and the Ca2 +-induced ACh release did not depend on time of exposure to CTX (Fig. 2B). These results suggest that Li+ can only poorly substitute for Na+ during the action ofCTX. In addition, Our data further suggest that CTX, by increasing Na+ influx into synaptosomes, favours ACh release triggered by Ca2+. CTX has been reported to have no inhibitory action on the Na-K ATPase but to enhance 22Na+ influx through voltage-sensitive Na+ channels of neuroblastoma cells and rat skeletal myoblasts when used in synergy with toxins acting on Na+ channels [3]. If the action of CTX was to depolarize the synaptosomal membrane to levels above that needed to actiVate voltage-gated Ca 2+ channels, then it would be expected that membrane depolarization. via Ca2+ influx,
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Fig. 2. A: dose-response curve of effect of CTX on ACh release. Aliquots of synaptosomes were incubated during 15 min in Na +-containing nominally Ca H-free medium and ACh release was triggered by addition of 4 mM Ca 2 + to medium. B: release of ACh triggered upon addition of 4 mM Ca2+ to synaptosomes incubated during various times with IO nM CTX in a nominally Ca 2+-free medium containing either Na+ (e) or Li+ in replacement of Na+ (_). C: release of ACh from synaptosomes exposed during 15 min to CTX (10 nM) in a Na+-containing nominally CaH-free medium upon addition of different Ca 2+ concentrations (abscissae. logarithmic scale). In A-C, results are expressed as percentage of total ACh content from control synaptosomes incubated in toxin- free medium (A and C) and maintained for 60 min in Na+ or Li+ - containing medium (8).
could contribute to Ca2+-dependent ACh release caused by CTX. As shown in Fig. 3B, simultaneous blockade of P- and N-Ca 2 + channel subtypes in synaptosomes by FTX, co-conotoxin [13] and Gd 3 did not prevent ACh release caused by CTX upon addition of Ca2+. On this basis, it is likely that CTX exerts its effects on ACh release from synaptosomes by increasing Na+ levels which would enhance Ca 2 + influx through the reversed operation of the Na+/Ca 2+ exchange system that normally uses the Na+ gradient to extrude Ca2+. The Na+-Ca2+ exchange has been shown to be a completely electrogenic transport reaction. operation of the carrier is controlled by transmembrane ion gradients and electrical potentials. In this way, the Na+-Ca2+ exchange can act either
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We thank M. Israel for cogent advise and critical reading of the manuscript. This research was supported by the Direction des Recherches Etudes et Techniques (Grants 911090 and 921175) and the French Polynesian Government. N. Moulian was supported by a fellowship frorn Laboratoires Servier.
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FTX Gd 3 + Fig. 3. A: CTX-induced Ca2+ -dependent ACh release did not prevent action of 60 mM K+ on subsequent ACh release. B: simultaneous blockade of Ca l + channels subtypes by 2 JIM ltJ-conotoxin. FTX (I: 100.000) and 0.25 mM Gd)+. respectively. did not prevent synaptosomal ACh release caused by 10 nM CTX upon addition of 4 mM Ca2+. In A and B. synaptosomes were incubated in Na+-containing Cal-free medium for 15 min before 4 mM Ca 2 + addition.
as a Ca 2+ efflux pathway or promote a net Ca 2 + influx. depending on prevailing ionic conditions. Na+-Ca2+ exchange activity in synaptic plasma membranes from Torpedo electric organ has a high-exchange reaction velocity [17]. The marked reduction of ACh release caused by CTX upon addition of Ca 2+ to the Li+ -containing medium. as compared with the Na+-containing medium. is consistent with the fact that Li+ can not replace Na+ in the Na+-Ca 2+ exchange process [4]. Thus. the time-dependent effect of CTX on ACh release, upon Ca2+ addition in the presence ofNa+. might be due to activation of the Na+-Ca 2 + exchange so that Ca2+ enters the synaptosomes in exchange for Na+ that exits. Finally. the present results indicate that pure cholinergic synaptosomes are endowed with voltage-dependent Na+ channels sensitive to the action of both CTX and TTX.
I Anderson. D.M. and Lobel, P.S .• The continuing enigma of ciguatera, BioI. BUll.. 172 (1987) 89-107. 2 Benoit. E., Legrand, A.M. and Dubois, J.M., Effects of ciguatoxin on current and voltage clamped frog myelinated nerve fibre, Toxicon. 24 (1986) 357-364. 3 Bidard, J.N., Vijverberg, H.P.M., Frelin, C., Chungue, E., Legrand, A.M., Bagnis, R. and Lazdunski, M., Ciguatoxin is a novel type of Na+ channel toxin, J. BioI. Chern., 259 (1984) 8353-8357. 4 Hermoni, M., Barzilai, A. and Rahamimoff. H., Modulation of the Na+-Ca l + antiport by its ionic environment: the effect of lithium, Isr. J. Med. Sci., 23 (1987) 44-48. 5 Israel. M. and Lesbats. B., Chemiluminescent determination of acetylcholine and continuous detection of its release from Torpedo electric organ and synaptosomes, Neurochem. Int., 3 (1981) 81-90. 6 Israel, M. and Lesbats, B., Continuous determination by a chemiluminescent method of acetylcholine release and compartimentation in Torpedo electric organ synaptosomes, J. Neurochem., 37 (1981) 1476-1483. 7 Legrand, A.M .• Litaudon. M, Genthon, J.N .. Bagnis, R. and Yasumoto, T., Isolation and some properties of ciguatoxin, J. APpl. Phycol., I (1989) 183-188. 8 Lewis, RJ •. Sellin, M., Polin M.A., Norton, R.S., MacLeod, J.l{. and Sheil. M.M .• Purification and characterization of ciguato xins from moray eel (Lycodontis javanicus, Muraenidae), Toxicon, 29 (1991) 1115-1127. 9 Lombet, A .. Bidard. J.N. and Lazdunski. M .• Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltagedependent Na+ channel, FEBS Lett., 219 (1987) 355-359. 10 Meunier, F.M .. Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ, J. Physiol. (London), 354 (1984) 121-137. II Molg6. J .. Comella. J.x. and Legrand, A.M., Ciguatoxin enhances quantal transmitter release from frog motor nerve terminals. Br. J. Pharmacol.. 99 (\ 990) 695-700. 12 Morel, N., Israel, M .• Manaranche, R. and Mastour-Frachon, P., Isolation of pure cholinergic nerve endings from Torpedo electriC organ. J. Cell BioI.. 75 (1977) 43-55. 13 Moulian. N. and Morot-Gaudry-Talarmain. P.. Agelenopsis aperlQ venom and FTX. a purified toxin. inhibit acetylcholine release in Torpedo synaptosomes, Neuroscience. (1993) 1035-1041. 14 Murata. M .. Legrand A.M .. Ishibashi. Y.. Fukui, M. Yasumoto. T .. Structures and configurations of ciguatoxin from the moray eel Gymnolhorax javanicus and its likely precursor from the dinoflagellate Gambierdiscus toxicus. J. Am. Chern. Soc" 112 (\ 990) 43804386. 15 Pressman. B.c.. Biological applications of ionophores. Annu. Rev. Biochem .. 45 (1976) 501-530. 16 Russell. F.E. and Egen. N.B., Ciguateric fishes. ciguatoxin (CTX) and Ciguatera poisoning. J. Toxicol. Toxin Rev .. 10 (1991) 37-62. 17 Tessari. M. and Rahamimoff. H .. Na+-Ca 2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata. Biochim. Biophys. Acta. 1066 (1991) 208-218.
Neuroscience Letters, 160 (1993) 65-68 C 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
NSL09806
Ciguatoxin extracted from poisonous moray eels Gymnothorax javanicus triggers acetylcholine release from Torpedo cholinergic synaptosomes via reversed N a + -Ca2+ exchange lordi Molg6 a , Yvette Morot Gaudry-Talarmain b , Anne Marie Legrand'" and Nathalie Moulian b Deparrements de "Neuropharmacologie et de bNeurochimie. Laboratoire de Neurobiologie Cellulaire et Moieculaire. Centre National de la Recherche Scientifique. Gif-sur- Yvette (France) (Received 6 April 1993; Accepted 2 June 1993)
Key words:
Ciguatoxin; Acetylcholine release; Torpedo cholinergic synaptosome; Tetrodotoxin; Na+ channel; Lt; Na+-Ca 2+ exchange 2
Ciguatoxin (CTX) (0.1 pM to 10 nM) added to a suspension of Torpedo synaptosomes incubated in Ca +-free medium caused no detectable acetylcholine (ACh) release. However, subsequent addition ofCa2 • caused a large ACh release that depended on time of exposure, dose ofCTX and on [Ca2+). Tetrodotoxin completely prevented CTX-induced Ca2+-dependent ACh release. Simultaneous blockade of Ca 2' channel subtypes by FTX, a tOXin extracted from the venom of the spider Agelenopsis aperta. ro-conotoxin and Gd-'+ did not prevent ACh release caused by CTX, upon addition 2 orCa2+. These results suggest that CTX activates the reversed operation of the Na+/Ca ' exchange system allowing the entry ofCa 2• in exchange for Na·. It is concluded that Torpedo synaptosomes are endowed with NaT channels sensitive to pico- to nanomolar concentrations ofCTX.
Ciguatoxin (CTX) is one of the principal ichtyotoxins inVolved in a complex human food poisoning known as ciguatera. characterized mainly by gastrointestinal and neUrological disorders [1-16]. Ciguatera fish poisoning has been associated with the consumption of many species of tropical and subtropical fishes which acquired the toxicity from their diet and transferred it to other fish through the marine food chain and ultimately to man (1,14. 16]. Recent studies have disclosed that CTX extracted from poisonous moray eels (Gymnothorax javanicus or l..ycodontisjavanicus) named CTX-Ib [14] or CTX-l [8]. has a brevetoxin-Iike polyether structure and a molecular formula of C6QH 860 J9 • CTX exerts its effects primarily by altering the membrane properties of excitable cells in such a way that actiVates voltage-gated Na + channels at normal membrane resting potential causing tetrodotoxin-sensitive membrane depolarization and repetitive or spontaneous ac-
-
Correspondence: J. Molg6, Laboratoire de Neurobiologie Cellulaire et Moleculaire, Centre National de la Recherche Scientifique, 91198 GlFSUR-YVETTE CEDEX, France. Fax: (33) (I) 69-829466. 'Permanent address: A.M. Legrand, Institut Territorial de Recherches MlXiicales Louis Malarde, Associe aI'Institut Pasteur, BP 30, Papeete, Tahiti, polynesie Fran~aise.
tion potentials in myelinated axons, neuroblastoma cells and skeletal muscle fibres [2, 3, 11]. Binding studies showed that CTX inhibits the binding of eH]brevetoxin3 to rat brain membranes and that CTX and brevetoxins share a common binding site on the neuronal voltagedependent Na+ channel protein [8, 9]. Previous studies using purified CTX revealed that CTX increases the rate of release of CH]r-aminobutyric acid and CH]dopamine from rat brain synaptosomes [3]. CTX-induced transmitter release was sensitive to blockade by tetrodotoxin (TTX) but was unaffected by Ca2+ channel antagonists like nitrendipine and 0-600 [3]. Since CTX was reported to have no action on the activity of the Na+-K+-ATPase, it was suggested that the enhancement of neurotransmitter release may be d~e to the depolarization-induced Ca2+ influx caused by the activation of voltage-dependent Na+ channels of the synaptosomal membrane· [3]. CTX was reported also to enhance spontaneous quantal release of acetylcholine (ACh) from motor nerve terminals. measured electrophysiologically as an increase in miniature end plate potential frequency [II]. TTX completely abolished or prevented CTX action on quantal release. suggesting that CTX effect depends upon Na+ entry into the motor nerve terminals [11]. The aim of the present work was to further character-
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logical medium consisting of (mM): NaCI, 280; KCI; 3; MgCI 2 • 1.8; sucrose. 400; glucose, 5.5; and Tris buffer. SO (pH = 8.6), as required for the choline oxidase-chemiluminescent reaction and 20.u1 of the materials used for the chemiluminescent assay of ACh: choline oxidase (WakO, Japan), 8 .ul of a solution containing 250 U/ml; horseradish peroxidase (EC 1.11.1.7. type III; Sigma. USA). 4 .ul of a solution containing 2 mglml; and luminol (Merck, Germany), 8.u1 of a solution I mM in 0.2 M Tris buffer. pH 8.6. In some experiments, the NaCI was completely replaced by LiCl. Synaptosomal preparations contained sufficient amounts of ACh-esterase for the hydrolysis of released ACh so that no exogenous enzyme was necessary to be added to the mixture of solutions. The light emitted by the chemiluminescent reaction was detected by a photomultiplier unit, recorded and calibrated by the addition of known amounts of standard ACh. Total ACh content of synaptosomes was determined by addition of the detergent Triton X-lOO (0.01 %) [5, 6]. CTX-I b was extracted from G. javanicus viscera as previously described [7. 14]. TTX, synthetic w-conotoxin GVIA and Triton X-I00 (Sigma) stock solutions were dissolved into bi-distilled water. Stock solutions of the Ca ionophore A23187 (Boehringer, Germany) and gramicidin-D were made in dimethylsulfoxide and further diluted I :40 and I :400 in bi-distilled water. FTX, a low molecular weight toxin purified from the venom of the American funnel-web spider Age/enopsis aperta, waS kindly provided by R. Llimis and used at a dilution I: 100,000 [13], Gd chloride was obtained from (Ventro n, Germany). All reagents were added directly to the synaptosomal suspension. CTX (0.1 pM to 10 nM) added to a diluted suspension of Torpedo synaptosomes incubated in a nominally Ca2+free medium caused no detectable change in the light emission, that permits to quantify ACh release, for periods of up to 15 min (Fig. lA). These results indicate that CTX, in the absence of external Ca2+, neither triggers detectable ACh release nor causes leakage of ACh. When synaptosomes were exposed to CTX (10 nM) for 15 min in a Ca 2+ -free medium, subsequent addition of 4 mM Ca 2+ caused an immediate large release of ACh. As shown in Fig. lA, the light emission resulting from the chemiluminescent detection of ACh decayed in -3 min to a steady basal level similar to that observed before Ca 2+ addition. The amount of ACh released, under this condition, expressed as percentage of total content corresponded to 18.7 ± 2.7 (mean ± S.E.M., n =5). Further addition either of high K + (60 mM) or the Ca ionophore A23187 (4 .uM) could still trigger Ca2+ -dependent ACh release (Figs. lA, 3A). However, the amount of ACh released was much less than measured in synaptosomes not exposed to CTX. The decrease in the stimulatory
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ize the effects of highly purified CTX (CTX-Ib), extracted from G. javanicus. on ACh release from pure cholinergic synaptosomes. Synaptosomes were isolated from the electric organ of the fish Torpedo marmorata according to described methods [5. 12]. All steps of the isolation procedure were carried out at 4°C. Nerve endings derived from 25 g of electric organ were chopped up and diluted in a oxygenated physiological solution of the following composition (mM): NaC!. 280; KCI, 3; MgCI 2, 1.8; CaCI 2, 3.4; Na phosphate. 1.2; NaHCO). 5; glucose, 5.5; urea, 300; and sucrose. 100 (pH =7.2). Then, synaptosomes were collected from a discontinuous iso-osmotic saline-sucrose density gradient in 40-50 ml of the above-mentioned medium devoided of Ca 2+. Aliquots of this synaptosomal suspension were used at room temperature (20-22°C) for ensuring ACh release using the choline oxidase-chemiluminescent method [5, 6]. Aliquots of the synaptosomal fraction (30--50 ,LII) were added to 470-450,LI1 of a physio-
67
effect of the ionophore A23187 after CTX action may be due to a partial exhaustion of the releasable pool of ACh, which has been reported to represent::= 40% of total ACh Content [6, 12]. As shown in Fig. lA, further addition of l'riton X-loo (0.01%) revealed that synaptosomes still Contained consistent amounts of ACh. Ca2+-induced ACh release by CTX was dependent on the concentration of CTX used. Active concentrations of CTX ranged in the pico- to nanomolar range as shown in a representative dose-response curve (Fig. 2A). In addition, ACh release triggered by Ca2+ in the presence of CTX depended on time. of exposure to the toxin in Na+Containing Ca2+ -free medium (Fig. 2B) and on Ca2+ concentration used to trigger ACh release (Fig. 2C). As shown in Fig. 2B,C, ACh release induced by CTX exposUre for 60 min or triggered by 36 mM Ca2+ reached values close to 40% of the total ACh content. TTX (l .uM), when applied a few minutes before CTX to synaptosomes kept in a nominally Ca2+-free medium, Completely prevented CTX-induced ACh release upon addition of 4 mM Ca2+ (Fig. I B). However, TTX (1 .u M ) neither prevented the action of the ionophore A23187 (4.uM) (Fig. IB) nor antagonized the large ACh release caused by 0.5 .u M gramicidin-D that reached 50.5 ± 3.5% (n =4) of total ACh content (data not shown). This antibiotic induces nonspecific membrane channels for monovalent cations [15], depolarizes the 2 sYnaptosome membrane [10) and triggers a large Ca +dependent ACh release [6). To study further the Na+ requirement for CTX-induced ACh release, extracellular Na+ was replaced, on equimolar basis, by Li+. As shown in Fig. Ie. CTX (10 nM) had no detectable action on ACh release in nominally Ca2+-free medium containing Li+, similarly to what Was shown in Na+-containing medium (Fig. IA). The addition of Ca 2+, to synaptosomes exposed for 15 min to CTX in Li+ -containing medium (Fig. I C), triggered Illuch less ACh release « 2% of the total content, n =3) than in Na+ containing medium and the Ca2 +-induced ACh release did not depend on time of exposure to CTX (Fig. 2B). These results suggest that Li+ can only poorly substitute for Na+ during the action ofCTX. In addition, Our data further suggest that CTX, by increasing Na+ influx into synaptosomes, favours ACh release triggered by Ca2+. CTX has been reported to have no inhibitory action on the Na-K ATPase but to enhance 22Na+ influx through voltage-sensitive Na+ channels of neuroblastoma cells and rat skeletal myoblasts when used in synergy with toxins acting on Na+ channels [3]. If the action of CTX was to depolarize the synaptosomal membrane to levels above that needed to actiVate voltage-gated Ca 2+ channels, then it would be expected that membrane depolarization. via Ca2+ influx,
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could contribute to Ca2+-dependent ACh release caused by CTX. As shown in Fig. 3B, simultaneous blockade of P- and N-Ca 2 + channel subtypes in synaptosomes by FTX, co-conotoxin [13] and Gd 3 did not prevent ACh release caused by CTX upon addition of Ca2+. On this basis, it is likely that CTX exerts its effects on ACh release from synaptosomes by increasing Na+ levels which would enhance Ca 2 + influx through the reversed operation of the Na+/Ca 2+ exchange system that normally uses the Na+ gradient to extrude Ca2+. The Na+-Ca2+ exchange has been shown to be a completely electrogenic transport reaction. operation of the carrier is controlled by transmembrane ion gradients and electrical potentials. In this way, the Na+-Ca2+ exchange can act either
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We thank M. Israel for cogent advise and critical reading of the manuscript. This research was supported by the Direction des Recherches Etudes et Techniques (Grants 911090 and 921175) and the French Polynesian Government. N. Moulian was supported by a fellowship frorn Laboratoires Servier.
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FTX Gd 3 + Fig. 3. A: CTX-induced Ca2+ -dependent ACh release did not prevent action of 60 mM K+ on subsequent ACh release. B: simultaneous blockade of Ca l + channels subtypes by 2 JIM ltJ-conotoxin. FTX (I: 100.000) and 0.25 mM Gd)+. respectively. did not prevent synaptosomal ACh release caused by 10 nM CTX upon addition of 4 mM Ca2+. In A and B. synaptosomes were incubated in Na+-containing Cal-free medium for 15 min before 4 mM Ca 2 + addition.
as a Ca 2+ efflux pathway or promote a net Ca 2 + influx. depending on prevailing ionic conditions. Na+-Ca2+ exchange activity in synaptic plasma membranes from Torpedo electric organ has a high-exchange reaction velocity [17]. The marked reduction of ACh release caused by CTX upon addition of Ca 2+ to the Li+ -containing medium. as compared with the Na+-containing medium. is consistent with the fact that Li+ can not replace Na+ in the Na+-Ca 2+ exchange process [4]. Thus. the time-dependent effect of CTX on ACh release, upon Ca2+ addition in the presence ofNa+. might be due to activation of the Na+-Ca 2 + exchange so that Ca2+ enters the synaptosomes in exchange for Na+ that exits. Finally. the present results indicate that pure cholinergic synaptosomes are endowed with voltage-dependent Na+ channels sensitive to the action of both CTX and TTX.
I Anderson. D.M. and Lobel, P.S .• The continuing enigma of ciguatera, BioI. BUll.. 172 (1987) 89-107. 2 Benoit. E., Legrand, A.M. and Dubois, J.M., Effects of ciguatoxin on current and voltage clamped frog myelinated nerve fibre, Toxicon. 24 (1986) 357-364. 3 Bidard, J.N., Vijverberg, H.P.M., Frelin, C., Chungue, E., Legrand, A.M., Bagnis, R. and Lazdunski, M., Ciguatoxin is a novel type of Na+ channel toxin, J. BioI. Chern., 259 (1984) 8353-8357. 4 Hermoni, M., Barzilai, A. and Rahamimoff. H., Modulation of the Na+-Ca l + antiport by its ionic environment: the effect of lithium, Isr. J. Med. Sci., 23 (1987) 44-48. 5 Israel. M. and Lesbats. B., Chemiluminescent determination of acetylcholine and continuous detection of its release from Torpedo electric organ and synaptosomes, Neurochem. Int., 3 (1981) 81-90. 6 Israel, M. and Lesbats, B., Continuous determination by a chemiluminescent method of acetylcholine release and compartimentation in Torpedo electric organ synaptosomes, J. Neurochem., 37 (1981) 1476-1483. 7 Legrand, A.M .• Litaudon. M, Genthon, J.N .. Bagnis, R. and Yasumoto, T., Isolation and some properties of ciguatoxin, J. APpl. Phycol., I (1989) 183-188. 8 Lewis, RJ •. Sellin, M., Polin M.A., Norton, R.S., MacLeod, J.l{. and Sheil. M.M .• Purification and characterization of ciguato xins from moray eel (Lycodontis javanicus, Muraenidae), Toxicon, 29 (1991) 1115-1127. 9 Lombet, A .. Bidard. J.N. and Lazdunski. M .• Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltagedependent Na+ channel, FEBS Lett., 219 (1987) 355-359. 10 Meunier, F.M .. Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ, J. Physiol. (London), 354 (1984) 121-137. II Molg6. J .. Comella. J.x. and Legrand, A.M., Ciguatoxin enhances quantal transmitter release from frog motor nerve terminals. Br. J. Pharmacol.. 99 (\ 990) 695-700. 12 Morel, N., Israel, M .• Manaranche, R. and Mastour-Frachon, P., Isolation of pure cholinergic nerve endings from Torpedo electriC organ. J. Cell BioI.. 75 (1977) 43-55. 13 Moulian. N. and Morot-Gaudry-Talarmain. P.. Agelenopsis aperlQ venom and FTX. a purified toxin. inhibit acetylcholine release in Torpedo synaptosomes, Neuroscience. (1993) 1035-1041. 14 Murata. M .. Legrand A.M .. Ishibashi. Y.. Fukui, M. Yasumoto. T .. Structures and configurations of ciguatoxin from the moray eel Gymnolhorax javanicus and its likely precursor from the dinoflagellate Gambierdiscus toxicus. J. Am. Chern. Soc" 112 (\ 990) 43804386. 15 Pressman. B.c.. Biological applications of ionophores. Annu. Rev. Biochem .. 45 (1976) 501-530. 16 Russell. F.E. and Egen. N.B., Ciguateric fishes. ciguatoxin (CTX) and Ciguatera poisoning. J. Toxicol. Toxin Rev .. 10 (1991) 37-62. 17 Tessari. M. and Rahamimoff. H .. Na+-Ca 2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata. Biochim. Biophys. Acta. 1066 (1991) 208-218.