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Ciguatoxins, a group of polyether neurotoxins which interact with sodium channels
Report
717
and Abstracts
sites (Kd, = 5 nM; B,,,, = 6 pmole/mg protein), together with a much larger amount of a low-affinity component (K,, > 80 nM; Bmarz = 20 pmole/mg protein). Sensitivity of ‘2sI-DbTx binding to purified whole synaptosomes was characterized by both equilibrium and kinetic analyses. The synaptosomal binding site for ‘251-DbTx was heat-sensitive and inactivated by trypsin, and thus most likely is a protein in nature. Binding was strongly dependent on Caz+, but not Mg*+. A polyclonal rabbit antiserum raised against DbTx completely blocked the specific binding of ‘251-DbTx to whole synaptosomes. Our results show for the first time the existence of specific synaptosomal binding sites for DbTx, a major PLA, toxin of Dab& r. siamensis. Tandem mass spectrometry of conus peptides. T. Krishnamurthy, M. Prabhakaran RD&E Center, Aberdeen Proving Ground, MD 21010, U.S.A.).
and S. R. Long (Edgewood,
Small biologically active and toxic peptides (l-2 pmole) and their derivatives were ionized under continuous Row-FAB, LSIMS. electrospray and MALDI conditions. The ions were then subjected to either low- or high-energy collisionally induced dissociations. The recorded low- and high-resolution daughter spectra provided the valuable amino acid sequential information for the investigated peptides. In the process, a now one-step method for the reduction of the disulfide bonds and derivatization with vinylpyridine was developed. The derivatives were obtained in better yields in comparison with the conventional methods. The adapted strategy could be applied to analyze these peptides when present in subpicomole quantities and characterize the unknowns using only l-2 nmoles of the intact molecules. Mapping of the ol-nemotoxin-binding site of the nicotinic acetylcholine receptor. F. Hucho,’ J. Machold,’ Y. N. Utkit? and V. I. Tsetlin2 (‘Freie Universitat Berlin, 14195 Berlin, Germany; and ‘Shemyakin/Ovchinnikow Institute, Moscow, Russia). a-Neurotoxins from the venoms of poisonous snakes are a group of structurally similar, highly specific competitive antagonists of the nicotinic acetylcholine receptor (AChR) (Chang and Lee. 1963). The tertiary structures of several members of this group are known from X-ray diffraction (Low et al.. 1976) and NMR (Golovanov et al.,1993) analysis of the protein in the crystalline state or in solution. Our approach, therefore’ is to map the toxin-binding surface of the AChR by covalently attaching a-neurotoxin molecules to the recepto E which contain photoactivatable groups in specified positions. Receptor domains cross-linked in this way can subsequently be identified by protein chemical means. Furthermore, knowing the dimensions of the toxin and of the cross-linking groups one can measure distances on the receptor surface. First results of this approach, using photo activatable a-neurotoxin derivatives as yardsticks, will be reported. Chang, C. C. and Lee, C. Y. (1963) Arch. Int. Pharmacodyn. 144, 316-332. Golovanov et af. (1993) Eur. J. Biochem. 213, 1213-1223. Low, B., Preston, H. S., Sato, A., Rosen, L. S., Sear], J. E., Rudko, D. and Richardson. Acad. Sri. U.S.A. 73, 2991-2994.
J. S. (1976) Proc. natn.
Effect of neurotoxin MPTP on the energy-related enzymes in brain of mice. H. K. Mangat (Department of Zoology, Guru Nanak Dev University, Amritsar 143 005, Punjab. India).
and
H. Mann
Parkinson’s syndrome produced by I-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP) in humans and monkeys arises from an impairment of mitochondrial respiration. To evaluate this mitochondrial dysfunction. the effect of MPTP on basal ganglia was studied in male albino mice (20 mg/kg body wt) by estimating ATPase, SDH, NADH-dehydrogenase and GLDH. The animals were given three i.p. injections of MPTP (20 mg/kg body wt) at an interval of 1 hr each. The untreated animals were used as controls. The animals were killed by decapitation at 0.5, 1, 2 and 4 hour intervals after each injection. The brains were dissected out and divided into two parts, i.e. basal ganglia (caudate, putamen, globus pallidus and striatum) and the rest of the brain. In the crude mitochondrial fraction, ATPase and NADH-dehydrogenase showed a mixed response whereas SDH and GLDH had an overall decrease. Thus, the results suggest that MPTP probably caused interference in the energy-related enzymes of crude mitochondrial fraction. thereby interrupting the pathways of carbohydrade metabolism of mice, which has been considered as an animal model of Parkinson’s disease in the present investigation. Ciguatoxins, Q group of polyether neurotoxins which interact with sodium channels. A.-M. Legrand,’ E. Benoit,’ M. Chinain,’ S. Pauillac’ and J. Molgo) (’ Institut Territorial de Recherches Medicales Louis Malarde, Papee e Tahiti, French Polynesia; 2Laboratoire de Physiologie Cellulaire, URA CNRS 1121, Universitie Paris II, Orsa : . France; and ‘Laboratoire Neurobiologie Cellulaire et Moleculaire, CNRS, Gif/Yvette, France). Ciguatoxins are lipid-soluble polyether marine toxins originally produced in coral reef areas by the benthlc dinoflagellate Gambierdiscus toxicus, Adachi and Fukuyo. They concentrate in a variety of coral reef fish transferred first to herbivorous fish and subsequently to carnivores through the food chain. The toxins are responsible for a typical fish poisoning named ciguatera. Ciguatoxin (CTX) and congeners have been isolated either from toxic fish or from G. toxicus. More than 20 ciguatoxin analogues have been found; however, oniy six of them have so far been fully characterized with regard to structure. These polyether toxins exert a very potent
718
Report and Abstracts
action on mammals (i.p. LD~ on mouse near I pg/kg). Their toxic effects depend primarily on a shift activation of sodium channels to more negative potentials causing tetrodotoxine-sensitive membrane depolarization and repetitive or spontaneous action potentials in myelinated axons and skeletal muscle fibres. In isolated frog neuromuscular preparations, CTX in the nanomolar range markedly increases the spontaneous quanta] release of acetylcholine from motor terminals. CTX and some analogues were tested for their action on sodium channels by competitive binding between CTXs and a tritiated brevetoxin, PbTx-3, which is known to interact with the receptor site 5 associated with the a-subunit of the voltage-sensitive sodium channel. The effects of toxin extracts from Malaysian marine fauna on the membrane potential of tonically autoactive neurone (TAN) of the African giant snail (Achantina fulica ferussac). K. H. Kim, M. A. Muhamad and A. Raman (Department of Physiology, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia). In Malaysia, some species of marine fauna have been shown to have toxic effects following accidental ingestion and bites. The number of these cases has been increasing annually. In most cases, there is no rational treatment available since the mechanism of action of these toxins is not clear. However, there are strong indications that some of these act on the nervous system. The present study investigates the effects of toxins extracted from jellyfish, puffertish, catfish, and scombotoxic fish on the membrane potential of TAN. The jellyfish toxin extract was shown to have no effect on the recorded TAN intracellular membrane potential. This suggests that the toxicity expressed is not due to its effect on the central nervous system. Puffetlish toxin was found to inhibit TAN action potentials rapidly and markedly and this is very similar to the action of tetrodotoxin. However, unlike tetrodotoxin the effect persisted even after I hr of continuous washing away of toxin. Catfish toxin extract was found to have a depolarizing effect on TAN membrane potential, leading to increased frequency of spiking. This depolarizing effect was slightly decreased in the presence of 50% calcium and it completely disappeared in calcium-free media. The effect was not altered in the presence of IOm4M isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. The depolarizing effect of the extract could possibly be attributed to the involvement of extracellular or intracellular calcium without involving CAMP. Scombotoxic fish toxin extract induced a slow inhibition on TAN action potential generation. The effect was slightly reduced in the presence of 200% potassium, whereas the effect was slightly increased in the presence of SO% potassium or IBMX in normal saline. The observed effects may be due to a direct effect of the toxin on potassium channels or calcium-activated potassium channels. Stimulation of non-adrenergic non-cholinergic (NANC) nerve by the venom of the scorpion Buthotus hottentota. M. C. E. Gwee,’ L. S. Cheah’ and P. Gopalakrishnakone’ (Venom and Toxin Research Group, Departments of ‘Pharmacology, and *Anatomy, Faculty of Medicine, National University of Singapore, Singapore). Venoms and toxins from several species of scorpions can enhance peripheral neurotransmitter release by causing neuronal membrane depolarization. We have investigated the venom (BHV) from the scorpion B. hottentota using the rat isolated anococcygeus muscle (Acm) in which nitric oxide (NO) is strongly implicated as the inhibitory NANC neurotransmitter involved in mediating relaxant responses of the Acm. The Acm was mounted under I g tension in 6 ml Krebs solution containing phentolamine (5 PM), maintained at 37°C and aerated with 5% CO, in 02. Relaxant responses of the carbachol precontracted Acm to BHV were recorded isometrically either without or during electrical field stimulation (EFS: 20-30 V. I-2 Hz x IO set, I msec pulse width, every 2 min) which produces relaxant responses. BHV produced rapid, marked and fairly persistent decrease in tone (i.e. relaxation) of the Acm which was inhibited by either 50pM L-NAME (NG-L-nitro-L-arginine-methylester), a stereoselective NO synthase (NOS) inhibitor, or I PM tetrodotoxin (TTx), a selective neuronal sodium channel blocker. Thus, the results strongly suggest that relaxant responses of the Acm to BHV are mediated via stimulation of NANC nerves involving the L-arginine-NOS pathway. Supported by grants (RP870357 and RP940304) from NUS. Adrenergic nerve stimulation by venom of the scorpion Buthotus hottentota. M. C. E. Gwee,’ L. S. Cheah’ and P. Gopalakrishnakone2 (Venom and Toxin Research Group, Departments of ‘Pharmacology, and 2Anatomy, Faculty of Medicine, National University of Singapore, Singapore). Several scorpion venoms and toxins can depolarize peripheral nerves resulting in enhancement of adrenergic neurotransmitter release which can be potentially lethal in scorpion envenomation. The venom (BHV) from the scorpion Euthotus hottentota was therefore investigated using the rat isolated anococcygeus muscle (Acm) which is densely innervated by adrenergic fibres. The Acm was mounted under 1 g tension in 6ml Krebs solution, maintained at 37°C and aerated with 5% CO2 in 0,. Contractile responses of the Acm to electrical field stimulation (EFS: 25-30 V, 10 Hz x IO set, I msec pulse width, every 2 mitt), noradrenaline (NA: 3 PM), BHV (1.5 pg/ml) and tyramine (Tyr: IOPM) were recorded isometrically before (control) and after the addition of either phentolamine (5 PM) or guanethidine (5 PM) or after reserpine pretreatment (5 mg/kg S.C.and 21 hr later S mg/kg i.p.; rat sacrificed 3 hr after second dose). Contractile responses of the Acm to: EFS, BHV, Tyr and NA were completely blocked by phentolamine; EFS and BHV were completely blocked by guanethidine which potentiated the responses to NA, but generally did not affect the Tyr responses; EFS, BHV, and Tyr were absent
717
and Abstracts
sites (Kd, = 5 nM; B,,,, = 6 pmole/mg protein), together with a much larger amount of a low-affinity component (K,, > 80 nM; Bmarz = 20 pmole/mg protein). Sensitivity of ‘2sI-DbTx binding to purified whole synaptosomes was characterized by both equilibrium and kinetic analyses. The synaptosomal binding site for ‘251-DbTx was heat-sensitive and inactivated by trypsin, and thus most likely is a protein in nature. Binding was strongly dependent on Caz+, but not Mg*+. A polyclonal rabbit antiserum raised against DbTx completely blocked the specific binding of ‘251-DbTx to whole synaptosomes. Our results show for the first time the existence of specific synaptosomal binding sites for DbTx, a major PLA, toxin of Dab& r. siamensis. Tandem mass spectrometry of conus peptides. T. Krishnamurthy, M. Prabhakaran RD&E Center, Aberdeen Proving Ground, MD 21010, U.S.A.).
and S. R. Long (Edgewood,
Small biologically active and toxic peptides (l-2 pmole) and their derivatives were ionized under continuous Row-FAB, LSIMS. electrospray and MALDI conditions. The ions were then subjected to either low- or high-energy collisionally induced dissociations. The recorded low- and high-resolution daughter spectra provided the valuable amino acid sequential information for the investigated peptides. In the process, a now one-step method for the reduction of the disulfide bonds and derivatization with vinylpyridine was developed. The derivatives were obtained in better yields in comparison with the conventional methods. The adapted strategy could be applied to analyze these peptides when present in subpicomole quantities and characterize the unknowns using only l-2 nmoles of the intact molecules. Mapping of the ol-nemotoxin-binding site of the nicotinic acetylcholine receptor. F. Hucho,’ J. Machold,’ Y. N. Utkit? and V. I. Tsetlin2 (‘Freie Universitat Berlin, 14195 Berlin, Germany; and ‘Shemyakin/Ovchinnikow Institute, Moscow, Russia). a-Neurotoxins from the venoms of poisonous snakes are a group of structurally similar, highly specific competitive antagonists of the nicotinic acetylcholine receptor (AChR) (Chang and Lee. 1963). The tertiary structures of several members of this group are known from X-ray diffraction (Low et al.. 1976) and NMR (Golovanov et al.,1993) analysis of the protein in the crystalline state or in solution. Our approach, therefore’ is to map the toxin-binding surface of the AChR by covalently attaching a-neurotoxin molecules to the recepto E which contain photoactivatable groups in specified positions. Receptor domains cross-linked in this way can subsequently be identified by protein chemical means. Furthermore, knowing the dimensions of the toxin and of the cross-linking groups one can measure distances on the receptor surface. First results of this approach, using photo activatable a-neurotoxin derivatives as yardsticks, will be reported. Chang, C. C. and Lee, C. Y. (1963) Arch. Int. Pharmacodyn. 144, 316-332. Golovanov et af. (1993) Eur. J. Biochem. 213, 1213-1223. Low, B., Preston, H. S., Sato, A., Rosen, L. S., Sear], J. E., Rudko, D. and Richardson. Acad. Sri. U.S.A. 73, 2991-2994.
J. S. (1976) Proc. natn.
Effect of neurotoxin MPTP on the energy-related enzymes in brain of mice. H. K. Mangat (Department of Zoology, Guru Nanak Dev University, Amritsar 143 005, Punjab. India).
and
H. Mann
Parkinson’s syndrome produced by I-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP) in humans and monkeys arises from an impairment of mitochondrial respiration. To evaluate this mitochondrial dysfunction. the effect of MPTP on basal ganglia was studied in male albino mice (20 mg/kg body wt) by estimating ATPase, SDH, NADH-dehydrogenase and GLDH. The animals were given three i.p. injections of MPTP (20 mg/kg body wt) at an interval of 1 hr each. The untreated animals were used as controls. The animals were killed by decapitation at 0.5, 1, 2 and 4 hour intervals after each injection. The brains were dissected out and divided into two parts, i.e. basal ganglia (caudate, putamen, globus pallidus and striatum) and the rest of the brain. In the crude mitochondrial fraction, ATPase and NADH-dehydrogenase showed a mixed response whereas SDH and GLDH had an overall decrease. Thus, the results suggest that MPTP probably caused interference in the energy-related enzymes of crude mitochondrial fraction. thereby interrupting the pathways of carbohydrade metabolism of mice, which has been considered as an animal model of Parkinson’s disease in the present investigation. Ciguatoxins, Q group of polyether neurotoxins which interact with sodium channels. A.-M. Legrand,’ E. Benoit,’ M. Chinain,’ S. Pauillac’ and J. Molgo) (’ Institut Territorial de Recherches Medicales Louis Malarde, Papee e Tahiti, French Polynesia; 2Laboratoire de Physiologie Cellulaire, URA CNRS 1121, Universitie Paris II, Orsa : . France; and ‘Laboratoire Neurobiologie Cellulaire et Moleculaire, CNRS, Gif/Yvette, France). Ciguatoxins are lipid-soluble polyether marine toxins originally produced in coral reef areas by the benthlc dinoflagellate Gambierdiscus toxicus, Adachi and Fukuyo. They concentrate in a variety of coral reef fish transferred first to herbivorous fish and subsequently to carnivores through the food chain. The toxins are responsible for a typical fish poisoning named ciguatera. Ciguatoxin (CTX) and congeners have been isolated either from toxic fish or from G. toxicus. More than 20 ciguatoxin analogues have been found; however, oniy six of them have so far been fully characterized with regard to structure. These polyether toxins exert a very potent
718
Report and Abstracts
action on mammals (i.p. LD~ on mouse near I pg/kg). Their toxic effects depend primarily on a shift activation of sodium channels to more negative potentials causing tetrodotoxine-sensitive membrane depolarization and repetitive or spontaneous action potentials in myelinated axons and skeletal muscle fibres. In isolated frog neuromuscular preparations, CTX in the nanomolar range markedly increases the spontaneous quanta] release of acetylcholine from motor terminals. CTX and some analogues were tested for their action on sodium channels by competitive binding between CTXs and a tritiated brevetoxin, PbTx-3, which is known to interact with the receptor site 5 associated with the a-subunit of the voltage-sensitive sodium channel. The effects of toxin extracts from Malaysian marine fauna on the membrane potential of tonically autoactive neurone (TAN) of the African giant snail (Achantina fulica ferussac). K. H. Kim, M. A. Muhamad and A. Raman (Department of Physiology, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia). In Malaysia, some species of marine fauna have been shown to have toxic effects following accidental ingestion and bites. The number of these cases has been increasing annually. In most cases, there is no rational treatment available since the mechanism of action of these toxins is not clear. However, there are strong indications that some of these act on the nervous system. The present study investigates the effects of toxins extracted from jellyfish, puffertish, catfish, and scombotoxic fish on the membrane potential of TAN. The jellyfish toxin extract was shown to have no effect on the recorded TAN intracellular membrane potential. This suggests that the toxicity expressed is not due to its effect on the central nervous system. Puffetlish toxin was found to inhibit TAN action potentials rapidly and markedly and this is very similar to the action of tetrodotoxin. However, unlike tetrodotoxin the effect persisted even after I hr of continuous washing away of toxin. Catfish toxin extract was found to have a depolarizing effect on TAN membrane potential, leading to increased frequency of spiking. This depolarizing effect was slightly decreased in the presence of 50% calcium and it completely disappeared in calcium-free media. The effect was not altered in the presence of IOm4M isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. The depolarizing effect of the extract could possibly be attributed to the involvement of extracellular or intracellular calcium without involving CAMP. Scombotoxic fish toxin extract induced a slow inhibition on TAN action potential generation. The effect was slightly reduced in the presence of 200% potassium, whereas the effect was slightly increased in the presence of SO% potassium or IBMX in normal saline. The observed effects may be due to a direct effect of the toxin on potassium channels or calcium-activated potassium channels. Stimulation of non-adrenergic non-cholinergic (NANC) nerve by the venom of the scorpion Buthotus hottentota. M. C. E. Gwee,’ L. S. Cheah’ and P. Gopalakrishnakone’ (Venom and Toxin Research Group, Departments of ‘Pharmacology, and *Anatomy, Faculty of Medicine, National University of Singapore, Singapore). Venoms and toxins from several species of scorpions can enhance peripheral neurotransmitter release by causing neuronal membrane depolarization. We have investigated the venom (BHV) from the scorpion B. hottentota using the rat isolated anococcygeus muscle (Acm) in which nitric oxide (NO) is strongly implicated as the inhibitory NANC neurotransmitter involved in mediating relaxant responses of the Acm. The Acm was mounted under I g tension in 6 ml Krebs solution containing phentolamine (5 PM), maintained at 37°C and aerated with 5% CO, in 02. Relaxant responses of the carbachol precontracted Acm to BHV were recorded isometrically either without or during electrical field stimulation (EFS: 20-30 V. I-2 Hz x IO set, I msec pulse width, every 2 min) which produces relaxant responses. BHV produced rapid, marked and fairly persistent decrease in tone (i.e. relaxation) of the Acm which was inhibited by either 50pM L-NAME (NG-L-nitro-L-arginine-methylester), a stereoselective NO synthase (NOS) inhibitor, or I PM tetrodotoxin (TTx), a selective neuronal sodium channel blocker. Thus, the results strongly suggest that relaxant responses of the Acm to BHV are mediated via stimulation of NANC nerves involving the L-arginine-NOS pathway. Supported by grants (RP870357 and RP940304) from NUS. Adrenergic nerve stimulation by venom of the scorpion Buthotus hottentota. M. C. E. Gwee,’ L. S. Cheah’ and P. Gopalakrishnakone2 (Venom and Toxin Research Group, Departments of ‘Pharmacology, and 2Anatomy, Faculty of Medicine, National University of Singapore, Singapore). Several scorpion venoms and toxins can depolarize peripheral nerves resulting in enhancement of adrenergic neurotransmitter release which can be potentially lethal in scorpion envenomation. The venom (BHV) from the scorpion Euthotus hottentota was therefore investigated using the rat isolated anococcygeus muscle (Acm) which is densely innervated by adrenergic fibres. The Acm was mounted under 1 g tension in 6ml Krebs solution, maintained at 37°C and aerated with 5% CO2 in 0,. Contractile responses of the Acm to electrical field stimulation (EFS: 25-30 V, 10 Hz x IO set, I msec pulse width, every 2 mitt), noradrenaline (NA: 3 PM), BHV (1.5 pg/ml) and tyramine (Tyr: IOPM) were recorded isometrically before (control) and after the addition of either phentolamine (5 PM) or guanethidine (5 PM) or after reserpine pretreatment (5 mg/kg S.C.and 21 hr later S mg/kg i.p.; rat sacrificed 3 hr after second dose). Contractile responses of the Acm to: EFS, BHV, Tyr and NA were completely blocked by phentolamine; EFS and BHV were completely blocked by guanethidine which potentiated the responses to NA, but generally did not affect the Tyr responses; EFS, BHV, and Tyr were absent