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Electrophysiological studies of myotoxin a isolated from prairie rattlesnake (Crotalus viridis viridis) venom on murine skeletal muscle
Abstract
Myotoxin a reduced the resting membrane potential of mouse and rat diaphragms from about -80 mV to -60 mV, induced spontaneous repetitive firing and enhanced the contractile force in response to single stimulations. The depolarizing effect was reversed noncompetitively by tetrodotoxin, local anesthetics or low Na+ solution, but was augmented by ouabain or low Cl-solution while being unaffected by high K+ solution or electrical stimulation of the muscle. The duration of muscle action potential was prolonged by only 20-30%, whereas the rate of rise (dV/dt) was unaffected. About a 40% increase of membrane conductance was observed, be abolished by the Na+-channel blocker tetrodotoxin. By contrast, K+ conductance was unaffected. Effects on caffeine-induced contracture, quantal release of neurotransmitter and the amplitude of miniature endplate potential were not appreciably affected. These effects of myotoxin a indicate that the toxin affects the muscle, but not the nerve, by acting specifically on the Na+-channel of the sarcolemma or T-tubule, like crotamine, rather than on the sarcoplasmic reticulum. The effects of sea anemone toxin II on the Na+-channel (marked depolarization and prolongation of action potential) could not be prevented by saturating the muscle with myotoxin a. On the other hand, the effect of veratridine, a member of another group of toxins acting on the Na+-channel, was enhanced. These results suggest that myotoxin a acts on the Na+-channel at a site which is discrete from those of tetrodotoxin, veratridine and sea anemone toxin II.
... Demnach führt eine erhöhte Membranleitfähigkeit für Natriumionen zu einem geringeren Ruhepotential und somit letztlich zur Depolarisation der Skelettmuskelzellen (Chang et al. 1983). Da es sich hierbei um einen TTX-sensitiven Effekt zu handeln scheint, wird vermutet, dass diese Wirkungsweise von Crotamin über entsprechende Natriumkanäle der Plasmamembran vermittelt wird (Filho et al. 1978;Tsai et al. 1981;Hong et al. 1985;Brazil et al. 1993). (Furukawa et al. 1994;Ohkura et al. 1994;Ohkura et al. 1995). ...
... Für die Sequenzierung mittels Nano-LC-ESI-MS/MS wurden zunächst die in den Fraktionen Aminosäuren entstehen können, waren zu klein, um sie mit dieser Methode zu detektieren. (Filho et al. 1978;Tsai et al. 1981;Hong et al. 1985;Brazil et al. 1993 (Pereira et al. 2011;Leon et al. 2011;Nascimento et al. 2012;Macedo et al. 2015). ...
Für das Verständnis der pharmakologischen Aktivität von Proteinen und Peptiden ist es wichtig, den Zusammenhang zwischen Molekülstruktur und Wirkungsweise zu untersuchen. Schlangengifte setzen sich aus einer Vielzahl von Komponenten zusammen, die in unterschiedlichster Weise den Organismus der Beutetiere beeinflussen und schädigen. Sie stellen daher eine reiche Quelle für Substanzen mit biologischer Wirksamkeit dar. Die Isolierung der einzelnen Komponenten bietet somit eine effektive Möglichkeit, pharmakologisch aktive Proteine und Peptide zu gewinnen. Der Fokus der vorliegenden Arbeit lag auf der Isolierung und Charakterisierung von Einzelkomponenten aus dem Gift der südamerikanischen Klapperschlange Crotalus durissus terrificus. Es konnten zwei der Hauptbestandteile, Crotamin, sowie eine Mischung an Crotoxin B Isoformen, mittels RP-HPLC isoliert und durch massenspektrometrische Untersuchungen identifiziert werden. Im Weiteren lag der Schwerpunkt auf der Charakterisierung der Interaktion von Crotamin mit künstlichen Membranen. Crotamin ist ein amphiphatisches, hoch basisches Polypeptid mit einer Größe von 4,9 kDa und stark positiver Oberflächenladung. Zunächst als Neurotoxin beschrieben, sind für Crotamin mittlerweile eine Vielzahl weiterer Wirkungen berichtet worden. Zu diesen gehören myotoxische, analgetische, antimikrobielle und antitumorale Effekte. Die Wirkungsweise beruht teilweise auf einer direkten Interaktion mit den Lipiden der Membran. Crotamin werden dabei sowohl membranbeeinflussende Eigenschaften, als auch die Fähigkeit zur Translokation in die Zelle zugeschrieben. Die vorliegende Arbeit zeigt deutlich den Einfluss der Lipidkomposition von Membranen auf die membranbeeinflussenden Eigenschaften von Crotamin. In Monolayern aus Asolektin führte die Anwesenheit von Cholesterin zu einem langsameren Einbau von Crotamin. Zudem verursachte sowohl die Anwesenheit bivalenter Kationen, als auch die Erhöhung des lateralen Drucks im Monolayer, einen beschleunigten Einbau von Crotamin. Des Weiteren konnte gezeigt werden, dass Crotamin die Fluidität von Vesikelmembranen verändert. Bei reinen Asolektinvesikeln führte Crotamin zu festeren, bei DOPC-Vesikeln zu fluideren Membranen. Bei Vesikeln, deren Membran zusätzlich Cholesterin enthielt, führte Crotamin unabhängig von der gewählten Grundsubstanz (Asolektin oder DOPC) zu fluideren Membranen. Das Ausmaß der Fluidisierung war jedoch abhängig von der jeweiligen Cholesterinkonzentration. Je mehr Cholesterin die Vesikelmembranen enthielten, desto geringer die Wirkung von Crotamin. Generell war hierbei der Einfluss der Cholesterinkonzentration bei asolektinhaltigen Vesikeln größer als bei DOPC-haltigen. Demnach wirkte Cholesterin den membranbeeinflussenden Eigenschaften von Crotamin entgegen. Vermutlich beruhte dies unter anderem auf der geringeren Fluidität von cholesterinhaltigen Membranen. Zudem könnte für die polaren Kopfgruppen der Lipide die Wahrscheinlichkeit zur Interaktion mit kationischen Molekülen wie Crotamin durch die Anwesenheit von Cholesterin verringert sein. Der stärkere Einfluss von Crotamin auf die Fluidität von asolektinhaltigen Membranen könnte auf den, in Asolektin enthaltenen, Lipiden mit negativer Ladung beruhen. Diese könnten, auf Grund einer negativeren Oberflächenladung der Membran, zu einer stärkeren Anziehung von Crotamin führen. Außerdem könnte Crotamin, durch die Interaktion mit negativ geladenen Lipiden, die Bildung von Domänen verursachen. Die Untersuchungen dieser Arbeit lassen somit den Schluss zu, dass sowohl Cholesterin als auch Lipide mit negativer Ladung die Wirkungsweise von Crotamin auf Membranen verändern. Zudem wird sie durch die Anwesenheit bivalenter Kationen in der umgebenden Lösung und dem lateralen Membrandruck beeinflusst. Darüber hinaus konnte gezeigt werden, dass Crotamin die Kalziumhomöostase von neuronalen Zellen beeinflusst. Hierbei verursachte Crotamin einen konzentrationsabhängigen Anstieg der intrazellulären freien Kalziumkonzentration.
... OwNBY et al. (1976) suggested that this dilatation could result from alteration of the permeability of the sarcolemma to sodium . This hypothesis has been supported by CHANG and TSENG (1978) using crotamine, and by HONG and CHANG (1985) using myotoxin a. Others, however, propose that these myotoxins act directly on the calcium channels of the sarcoplasmic reticulum Clu and MoRrrA, 1983;VOLPE et al., 1986). Although the basic mechanism by which the toxin acts is unresolved, its first microscopically observable effect on muscle cells, i.e. vacuolation, is clear. ...
... Our observations on mice that received a lethal dose suggest that death may result from respiratory failure, perhaps caused by paralysis of the diaphragm and skeletal musculature controlling movement of the rib cage . Such a conclusion would appear to be supported by the effects of myotoxin a on the resting membrane potential of mouse and rat diaphragms (HONG and CHANG, 1985). Myotoxins from C. v. concolor do not appear to induce death by cardiac failure. ...
Myotoxins from C. v. concolor venom were isolated by gel filtration. This crude myotoxin peak was subfractionated into either two or four subfractions by cation exchange FPLC, depending upon the source of the venom. When injected at 2 micrograms/g, crude concolor myotoxin caused vacuolation of mouse muscle cells typical of myotoxin a from C. v. viridis and crotamine from C. d. terrificus. All four subfractions showed qualitatively identical myotoxin activity. In double immunodiffusion studies, myotoxin a antiserum produced lines of identity when reacted with myotoxin a, crude concolor myotoxin and the four concolor subfractions. A second batch of material showed two major components when subfractionated by cation exchange FPLC. The more basic of these two components displayed approximately twice the i.v. lethality of the more acidic component. The LD50 for the basic component lies between 0.625 and 0.75 microgram/g while that of the acidic component falls between 1.00 and 1.25 micrograms/g.
... And these effects have 423 been explained by their action on ions fluxes, producing an imbalance in electrolytes 424 and membrane potentials (Baker et al. 1993). 425 Some hypothesis found in the literature to explain the biological effects of 426 myotoxins is mainly based on the promotion of vacuolization that is dependent of 427 the action on sodium and potassium channels (Hong and Chang 1985). The sodium 428 channels are fundamental for cell despolarization and some authors described that 429 crotamine [50 μM] is able to increase the cell despolarization peak in 50 % and also 430 the maximum sodium conductance in 58 %, suggesting that this mechanism could be 431 responsible for the vacuolization effect (Matavel et al. 1998). ...
Crotamine is a basic, 42-residue polypeptide from snake venom that has been shown to possess cell-penetrating properties. Here we describe the preparation, purification, biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine. We also describe the formation and characterization of crotamine–DNA and crotamine–RNA nanoparticles; and the delivery of these nanoparticles into cells and animals. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine–plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in living organisms such as mice, Plasmodium, and worms. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. We also describe here the design and characterization of crotamine-functionalized gold nanoparticles, and the delivery of these nanoparticles into cells. We also evaluated the viability of using the combination of crotamine with silica nanoparticles in animal models, aiming to provide slow delivery, and to decrease the crotamine doses needed for the biological effects. In addition, the efficacy of administering crotamine orally was also demonstrated.
... Some hypothesis found in the literature to explain the biological effects of 426 myotoxins is mainly based on the promotion of vacuolization that is dependent of 427 the action on sodium and potassium channels (Hong and Chang 1985). The sodium 428 channels are fundamental for cell despolarization and some authors described that 429 crotamine [50 μM] is able to increase the cell despolarization peak in 50 % and also 430 the maximum sodium conductance in 58 %, suggesting that this mechanism could be 431 responsible for the vacuolization effect (Matavel et al. 1998). ...
The snake venom is composed by a complex combination of components (namely, proteins and/or enzymes, peptides, nucleic acids, among others) known as toxins, which are designed in principle to act essentially on prey’s cardiovascular system or on specific tissues as, for instance, muscles (skeletal or smooth muscles) and central nervous system (CNS). Crotamine is one of the most abundant components of the venom of the South American rattlesnake Crotalus durissus terrificus, corresponding to about 12–25 % of the dry weight of the crude venom. Several data reported by the group in the last decade suggested that this polypeptide may also be a promising tool for biotechnological and biomedical applications. In addition, it may also represent a potential structural model for the development of new drugs. Most significant evidences of crotamine versatility for diverse applications will be discussed in the present work, including: (1) the ability to translocate biological membranes and penetrate into highly proliferative cells; (2) the specificity for intracellular compartments, particularly nucleus and lysosomes; (3) the ability to carry nucleic acids, and other molecules, into cells, which could make it useful as a transfection vector; (4) the antimicrobial property with remarkable activity against some yeasts and fungus; and (5) the cytotoxic activity against cancer cells with the ability to stop the growth of certain tumors in vivo. In addition, other recently described biological activities of crotamine as the antiparasitic (antimalarial and anthelmintic) activity, as well its action on CNS, are described here.
... And these effects have 423 been explained by their action on ions fluxes, producing an imbalance in electrolytes 424 and membrane potentials (Baker et al. 1993). 425 Some hypothesis found in the literature to explain the biological effects of 426 myotoxins is mainly based on the promotion of vacuolization that is dependent of 427 the action on sodium and potassium channels (Hong and Chang 1985). The sodium 428 channels are fundamental for cell despolarization and some authors described that 429 crotamine [50 μM] is able to increase the cell despolarization peak in 50 % and also 430 the maximum sodium conductance in 58 %, suggesting that this mechanism could be 431 responsible for the vacuolization effect (Matavel et al. 1998). ...
The snake venom is composed by a complex combination of components (namely, proteins and/or enzymes, peptides, nucleic acids, among others) known as toxins, which are designed in principle to act essentially on prey’s cardiovascular system or on specific tissues as, for instance, muscles (skeletal or smooth muscles) and central nervous system (CNS). Crotamine is one of the most abundant components of the venom of the South American rattlesnake Crotalus durissus terrificus, corresponding to about 12–25 % of the dry weight of the crude venom. Several data reported by the group in the last decade suggested that this polypeptide may also be a promising tool for biotechnological and biomedical applications. In addition, it may also represent a potential structural model for the development of new drugs. Most significant evidences of crotamine versatility for diverse applications will be discussed in the present work, including: (1) the ability to translocate biological membranes and penetrate into highly proliferative cells; (2) the specificity for intracellular compartments, particularly nucleus and lysosomes; (3) the ability to carry nucleic acids, and other molecules, into cells, which could make it useful as a transfection vector; (4) the antimicrobial property with remarkable activity against some yeasts and fungus; and (5) the cytotoxic activity against cancer cells with the ability to stop the growth of certain tumors in vivo. In addition, other recently described biological activities of crotamine as the antiparasitic (antimalarial and anthelmintic) activity, as well its action on CNS, are described here.
... Studies analyzing Crotalus venom effects were performed with isolated fraction (toxins) of this venom, and it has been shown that these toxins cause a reduction in the resting potential of the membrane and an increase in membrane conductance. 45,46 Rangel-Santos et al 25 showed in their assays that Crotalus d. terrificus venom induced higher inhibition of the cellular proliferative response, in cultures stimulated by Con A, in both crude venom and crotoxin (isolated fraction). However, their cell viability test did not show significant results. ...
Snake venom is a complex biological mixture used for immobilization and killing of prey for alimentation. Many effects are inflicted by this venom, such as coagulation, necrosis, bleeding, inflammation, and shock. This study aimed to evaluate the inflammatory activity promoted by Bothrops erythromelas and Crotalus durissus cascavella snake venom. It was observed that both B. erythromelas and C. d. cascavella venom induced higher interferon- gamma and interleukin-6 production. Nitric oxide (NO) was significantly produced only by B. erythromelas venom, which also showed a higher rate of cell death induction when compared with C. d. cascavella. Results showed that B. erythromelas and C. d. cascavella venom induced distinct response in vitro through cytokines and NO production. However, B. erythromelas induces a proinflammatory response and a higher rate of cell death in relation to C. d. cascavella venom. © 2011 Luna et al, publisher and licensee Dove Medical Press Ltd.
... channels [55,59] but may be similar in mechanism of action to myotoxin a [60]. Myotoxin a, isolated from the prairie rattlesnake, induces local skeletal muscle contracture followed by myofibril degeneration and a vacuolisation pattern similar to crotamine [61][62][63]. Both crotamine and myotoxin a cause strong Ca 2? release from heavy sarcoplasmic reticulum through the ryanodine receptor [64] and in the case of myotoxin a, the mechanism may also involve the 30 kDa protein [65]. ...
Venom as a form of chemical prey capture is a key innovation that has underpinned the explosive radiation of the advanced snakes (Caenophidia). Small venom proteins are often rich in disulfide bonds thus facilitating stable molecular scaffolds that present key functional residues on the protein surface. New toxin types are initially developed through the venom gland over-expression of normal body proteins, their subsequent gene duplication and diversification that leads to neofunctionalisation as random mutations modify their structure and function. This process has led to preferentially selected (privileged) cysteine-rich scaffolds that enable the snake to build arrays of toxins many of which may lead to therapeutic products and research tools. This review focuses on cysteine-rich small proteins and peptides found in snake venoms spanning natriuretic peptides to phospholipase enzymes, while highlighting their three-dimensional structures and biological functions as well as their potential as therapeutic agents or research tools.
... The molecular mechanism of action of these toxins is not completely established. Both crotamine and myotoxin a have been shown to act on a Na + channel, increasing the membrane permeability to this ion, on rat and mouse skeletal muscle preparations (Chang and Tseng, 1978;Hong and Chang, 1985). This action would allow the influx of Na + and water, which in turn would cause swelling of the endoplasmic reticulum, and eventually, necrosis of the muscle cell (Mebs and Ownby, 1990). ...
Some characteristics of the local tissue damage and inflammatory reactions
induced by snake venoms were analyzed in a mouse model. Tissue damage was
studied by intravital, light, and electron microscopic techniques, and by the use of
biochemical markers. Detailed information on the early development and dynamics
of local tissue damage was obtained by intravital microscopy. Main alterations were
microvascular plasma leakage, hemorrhage, blood flow disturbances, thrombosis,
and myonecrosis. A new technique for the quantification of myonecrosis in vivo was
established, based on the principle of MTT reduction. The method was tested for its
usefulness in the evaluation of antibody-mediated neutralization of myotoxicity.
The inflammatory response to venom included early lymphopenia and
neutrophilia, thrombocytopenia, with edema and leukocyte extravasation at the site
of injection. A rapid plasma peak of IL-6 was induced by venoms, as well as by
purified muscle-damaging and hemorrhagic toxins, in response to cellular damage.
The effects of a purified hemorrhagic toxin on cultured endothelial cells were
studied. The toxin was not directly lytic to these cells even at high concentrations,
and caused moderate cell detachment due to its metalloproteinase activity,
suggesting that the endothelial cell damage in vivo occurs via an indirect mechanism,
probably initiated by proteolytic degradation of the basal lamina of microvessels.
Myotoxin II from Bothrops asper venom was shown to lyse a variety of cell
types in culture, including myoblasts and endothelial cells. This property was
exploited in a cytotoxicity assay for the evaluation of myotoxin neutralization.
Heparin was found to be a potent inhibitor of its cytolytic activity, by forming
complexes, held at least in part by electrostatic interactions. The ability of heparin to
neutralize several related non neurotoxic phospholipase A2 myotoxins present in
crotalid venoms, was not dependent on its anticoagulant effect. Thus, nonanticoagulant
heparin has a potential as a therapeutic aid, which should be further
evaluated. The structural characteristic of the binding interaction of heparin with
myotoxin II were analyzed, indicating that a hexasaccharide is the minimal heparin
chain size required, and that both N-sulfate and O-sulfate groups participate in the
binding.
... The molecular mechanism of action of these toxins is not completely established. Both crotamine and myotoxin a have been shown to act on a Na + channel, increasing the membrane permeability to this ion, on rat and mouse skeletal muscle preparations (Chang and Tseng, 1978;Hong and Chang, 1985). This action would allow the influx of Na + and water, which in turn would cause swelling of the endoplasmic reticulum, and eventually, necrosis of the muscle cell (Mebs and Ownby, 1990). ...
ABSTRACT
Some characteristics of the local tissue damage and inflammatory reactions
induced by snake venoms were analyzed in a mouse model. Tissue damage was
studied by intravital, light, and electron microscopic techniques, and by the use of
biochemical markers. Detailed information on the early development and dynamics
of local tissue damage was obtained by intravital microscopy. Main alterations were
microvascular plasma leakage, hemorrhage, blood flow disturbances, thrombosis,
and myonecrosis. A new technique for the quantification of myonecrosis in vivo was
established, based on the principle of MTT reduction. The method was tested for its
usefulness in the evaluation of antibody-mediated neutralization of myotoxicity.
The inflammatory response to venom included early lymphopenia and
neutrophilia, thrombocytopenia, with edema and leukocyte extravasation at the site
of injection. A rapid plasma peak of IL-6 was induced by venoms, as well as by
purified muscle-damaging and hemorrhagic toxins, in response to cellular damage.
The effects of a purified hemorrhagic toxin on cultured endothelial cells were
studied. The toxin was not directly lytic to these cells even at high concentrations,
and caused moderate cell detachment due to its metalloproteinase activity,
suggesting that the endothelial cell damage in vivo occurs via an indirect mechanism,
probably initiated by proteolytic degradation of the basal lamina of microvessels.
Myotoxin II from Bothrops asper venom was shown to lyse a variety of cell
types in culture, including myoblasts and endothelial cells. This property was
exploited in a cytotoxicity assay for the evaluation of myotoxin neutralization.
Heparin was found to be a potent inhibitor of its cytolytic activity, by forming
complexes, held at least in part by electrostatic interactions. The ability of heparin to
neutralize several related non neurotoxic phospholipase A2 myotoxins present in
crotalid venoms, was not dependent on its anticoagulant effect. Thus, nonanticoagulant
heparin has a potential as a therapeutic aid, which should be further
evaluated. The structural characteristic of the binding interaction of heparin with
myotoxin II were analyzed, indicating that a hexasaccharide is the minimal heparin
chain size required, and that both N-sulfate and O-sulfate groups participate in the
binding.
ISBN 91-628-1162-2
... It is expected that carnivorous insects use enzymes as collagenase to exploit this rich source of protein. The enzyme has been studied in snakes, scorpions, and spiders, and it is known as an important component of venom (Hong and Chang 1985;Atkinson and Wright 1992). Some studies on arthropods have indicated that collagenase is not sensitive to EDTA and so collagenase is to be considered a serine protease [e.g. the flesh flies, Hypoderma lineatum and Lucilia cuprina (Boulard and Garrone 1978;Bowles et al. 1988), the crab Uca pugilator (Eisen et al. 1973;Grant et al. 1983)]. ...
Podisus maculiventris (Say) is a generalist predator attacking many insect species from different orders. The bug injects saliva into its prey's body. The ingested hemolymph and liquefied internal tissues pass through the bug's alimentary tract. Collagenase working on peptide bonds of collagen and basement membrane proteins, leads to the disintegration of the prey's internal organs. As yet, there is an almost complete lack of knowledge on the collagenase activity in P. maculiventris. The collagenase activity of the salivary glands and midgut was optimum at pH 8.0 which was congruent with the optimal pH of the total proteolytic activity of the salivary glands. More collagenolytic activity was determined in the posterior lobe of the salivary glands and anterior midgut. Significant inhibition of collagenolytic activity by ethylenediaminetetraacetic acid (EDTA) revealed the enzyme is a metalloproteinase. The collagenase activity notably decreased when the bug went hungry. The salivary gland collagenase is a vital enzyme in extra-oral digestion and facilitates the action of other digestive enzymes. The midgut collagenase may be involved in the digestion of the ingested muscle fibers. The collagenase probably acts as an intoxicating agent in the saliva (venom) of P. maculiventris. Paralysing toxins are present in the salivary gland secretion.
... The answer is 'yes' for Na + but 'no' for Ca 2+ channels. Many crotalus venoms contain crotamine (also called myotoxin a) which specifically attacks muscle-type (but not nerve) Na + channels [16,24]. Fortunately, various ˆ-toxins blocking Pand N-type Ca2 + channels (such as ˆ-conotoxin and ˆagatoxin) have been isolated from other sources of venom and contributed greatly to our knowledge of these ion channels. ...
This review is a personal narration by a retiring pharmacologist from Taiwan who looks back at his discovery of α-bungarotoxin from the historical perspective of Taiwan during the last 50 years, with accounts of his experiences and his efforts to overcome hardship. How the α-toxin was isolated and characterized as an irreversible specific nicotinic acetylcholine (ACh) receptor antagonist, and how it subsequently became a useful experimental probe are presented here. The dilemma of differentiating the actions of tubocurarine and α-bungarotoxin is analyzed. The author also outlines findings based on work done in his laboratory using α-bungarotoxin as a tool on particular aspects of synaptic transmission. These include presynaptic receptor for positive feedback of transmitter release, explosive release of ACh, up- and downregulation of ACh receptors after chronic drug treatment, autodesensitization of junctional ACh receptors, differences in action between natural transmitter and exogenous agonists and that between junctional and extrajunctional ACh receptors. Some experimental pitfalls, in which biomedical scientists are frequently trapped, are raised. Finally, some anecdotes are appended from which the reader may further understand scientific life in the 20th century, including its joys and regrets.
... For example, snake venom neurotoxins, mainly α–Btx, played a leading role in myasthenia gravis research (Samson et al., 2001). Crotamine, myotoxin a, and homologous peptides are also seem to be useful candidates to study the muscle cell membrane and their sodium channels (Hong and Chang, 1985). Dendrotoxin (a toxin derived from Dendroaspic venom) and their homologues, are becoming extremely valuable tools to characterize K + channels which are involved in the regulation mechanisms of cell excitability and synaptic transmission (Harvey and Anderson, 2004). ...
Snake venom is a highly toxic secretion produced and stored in specialized salivary glands of snakes
which constitutes a vast array of biologically-active compounds, such as enzymes, proteins, peptides and low
molecular weight compounds. These substances target an immense number of receptors and membrane proteins
as well as coagulation proteins with high affinity, selectivity and potency, and can serve as potential drugs or
scaffolds for drug design. During the recent years, much attention has been given to understand the mechanism of
action of complex venom proteins for the development of novel drugs and therapeutic agents to treat life-threatening
diseases such as cardiovascular diseases, cancer, thrombosis, arthritis, microbial infections and hypertension etc.
Further, snake venom components have found uses in the diagnosis of haemostatic disorders. This paper reviews
the various biomedical applications of snake venom proteins in terms of therapeutic and diagnostic values.
... The myotoxicity mechanisms of crotamine and the closely related myotoxin-a have been both unraveled in part by electrophysiological and biochemical studies carried out in different isolated muscle preparations or cultured muscle cells. It has been shown that these toxins cause reduction in the resting membrane potential and increase in membrane conductance by a tetrodotoxin-sensitive, i.e., Na þ -channel mediated mechanism (Hong and Chang, 1985; Brazil and Fontana, 1993). However, recently described electrophysiological experiments showed that crotamine is inactive on sodium channels, suggesting that the actual mechanism of action of this toxin is still a matter of debate (Rizzi et al., 2007). ...
Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 in after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.
... PII: S 0 1 6 7-4 8 8 9 ( 9 9 ) 0 0 0 8 2-8 acid residues, including three disul¢de bonds [19]. Intramuscular injection of MYTX caused immediate and sustained local skeletal muscle contracture, followed by vacuolation of the SR after 12^24 h and degeneration of the myo¢brils without damage of the membranes of sarcolemma and SR after 48^72 h [18,20,21]. Recently, we found that MYTX strongly induced Ca 2 release from the heavy fraction of SR (HSR) containing RyR [22]. ...
The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.
... The mechanism by which crotamine exerts its toxicity is similar to that of its most studied counterpart, myotoxin-a (Cameron and Tu, 1978; Fletcher et al., 1996). It is known to cause reduction in resting membrane potential and increase in membrane conductance by a tetrodotoxin-sensitive, i.e., Na + -channel mediated mechanism (Hong and Chang, 1985; Brazil and Fontana, 1993). It also induces Ca 2+ release from the heavy fraction of the sarcoplasmic reticulum, by interacting with calsequestrin (Furukawa et al., 1994; Ohkura et al., 1994 Ohkura et al., , 1995), resulting in swelling of the sarcoplasmic reticulum and necrosis of skeletal muscle. ...
A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by Laure (1975), and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon. Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.
... ne, batrachotoxin and grayanotoxin I (Cheymol et al., 1964Cheymol et al., , 1971a Chang and Tseng, 1978; Tsai et al., 1981; Hong and Chang, 1983 Vital Brazil, 1990; Vital Brazil and Fontana, 1993). Chang et al. (1983) and Hong and Chang (1983) concluded that the small myotoxins act directly upon sodium channels on the sarcolemma and Ttubule system. Chang (1985) and Hong et al. (1994) later claimed that crotamine and its homologs act speci®cally at neurotoxin receptor site 3 of the sarcolemma Na 1 channel, but that they do not bind to the SR. In contrast, Maurer et al. (1987) observed that myotoxin a inhibits the formation of two-dimensional SR membrane crystals and that it disrupts previously form ...
Snake envenomation employs three well integrated strategies: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion. Purines (adenosine, guanosine and inosine) evidently play a central role in the envenomation strategies of most advanced snakes. Purines constitute the perfect multifunctional toxins, participating simultaneously in all three envenomation strategies. Because they are endogenous regulatory compounds in all vertebrates, it is impossible for any prey organism to develop resistance to them. Purine generation from endogenous precursors in the prey explains the presence of many hitherto unexplained enzyme activities in snake venoms: 5'-nucleotidase, endonucleases (including ribonuclease), phosphodiesterase, ATPase, ADPase, phosphomonoesterase, and NADase. Phospholipases A(2), cytotoxins, myotoxins, and heparinase also participate in purine liberation, in addition to their better known functions. Adenosine contributes to prey immobilization by activation of neuronal adenosine A(1) receptors, suppressing acetylcholine release from motor neurons and excitatory neurotransmitters from central sites. It also exacerbates venom-induced hypotension by activating A(2) receptors in the vasculature. Adenosine and inosine both activate mast cell A(3) receptors, liberating vasoactive substances and increasing vascular permeability. Guanosine probably contributes to hypotension, by augmenting vascular endothelial cGMP levels via an unknown mechanism. Novel functions are suggested for toxins that act upon blood coagulation factors, including nitric oxide production, using the prey's carboxypeptidases. Leucine aminopeptidase may link venom hemorrhagic metalloproteases and endogenous chymotrypsin-like proteases with venom L-amino acid oxidase (LAO), accelerating the latter. The primary function of LAO is probably to promote prey hypotension by activating soluble guanylate cyclase in the presence of superoxide dismutase. LAO's apoptotic activity, too slow to be relevant to prey capture, is undoubtedly secondary and probably serves principally a digestive function. It is concluded that the principal function of L-type Ca(2+) channel antagonists and muscarinic toxins, in Dendroaspis venoms, and acetylcholinesterase in other elapid venoms, is to promote hypotension. Venom dipeptidyl peptidase IV-like enzymes probably also contribute to hypotension by destroying vasoconstrictive peptides such as Peptide YY, neuropeptide Y and substance P. Purines apparently bind to other toxins which then serve as molecular chaperones to deposit the bound purines at specific subsets of purine receptors. The assignment of pharmacological activities such as transient neurotransmitter suppression, histamine release and antinociception, to a variety of proteinaceous toxins, is probably erroneous. Such effects are probably due instead to purines bound to these toxins, and/or to free venom purines.
... Single administration of sodium channel-blocking toxins produces no morphological changes as part of their physiological action (Love et al., 1986;Freitas et al., 2001;Cruz-Höfling et al., 2002). In contrast, extensive morphological alterations in skeletal muscle occur with small basic peptides toxins, such as crotamine and myotoxin a from Crotalus durissus terrificus and Crotalus viridis viridis snake venoms, respectively (Cameron and Tu, 1978;Ownby et al., 1982), which activate or delay the inactivation of Na þ channels (Chang and Tseng, 1978;Hong and Chang, 1985). ...
The aim of this study was to investigate if the Na(+)-channel activating alkaloid veratrine is able to change the oxidative and m-ATPase activities of a fast-twitch glycolytic muscle (EDL, extensor digitorum longus) and slow-twitch oxidative muscle (SOL, soleus) in mice. Oxidative fibers and glycolytic fibers were more sensitive to veratrine than oxidative-glycolytic fibers 15, 30 and 60 min after the i.m. injection of veratrine (10 ng/kg) with both showing an increase in their metabolic activity in both muscles. In EDL, the m-ATPase reaction revealed a significant (p < 0.001) decrease (50%) in the number of type IIB fibers after 30 min while the number of type I fibers increased by 550%. Type I fibers decreased from 34% in control SOL to 17% (50% decrease) in veratrinized muscles, with a 10% decrease in type IIA fibers within 15 min. A third type of fiber appeared in SOL veratrinized muscle, which accounted for 28% of the fibers. Our work gives evidence that the changes in the percentage of the fiber types induced by veratrine may be the result, at least partially, from a direct effect of veratrine on muscle fibers and else from an interaction with the muscle type influencing distinctively the response of a same fiber type. Based on the results obtained in the present study the alterations in EDL may be related to the higher number of Na(+) channels present in this muscle whereas those in SOL may involve an action of veratrine on mitochondria. Although it is unlikely that the shift of enzymes activities induced by veratrine involves genotypic expression changes an alternative explanation for the findings cannot be substantiated by the present experimental approach.
Snake venom myotoxins can be categorized into three types: small, basic polypeptides such as myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A(2) toxins such as crotoxin and notexin. All three types of myotoxins induce depolarization and contraction of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide myotoxins is different from that induced by the cardiotoxins and phospholipase A(2) myotoxins in that the former do not appear to lyse the sarcolemma whereas the latter two types cause lysis of the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they are all highly basic proteins, and a large portion of their surface charge is positive. Also, they all have considerable beta-sheet structure which may be involved in interaction with the membrane. The purpose of this review is to describe the structure and function of these myotoxins and to evaluate features they might share which could shed light on their mechanism of myotoxic action.
Snake venom myotoxins can be categorized into three types: small, basic polypeptides such as myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A2, toxins such as crotoxin and notexin. All three types of myotoxins induce depolarization and contraction of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide myotoxins is different from that induced by the cardiotoxins and phospholipase A2 myotoxins in that the former do not appear to lyse the sarcolemma whereas the latter two types cause lysis of the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they are all highly basic proteins, and a large portion of their surface charge is positive Also, they all have considerable β-sheet structure which may be involved in interaction with the membrane The purpose of this review is to describe the structure and function of these myotoxins and to evaluate features they might share which could shed light on their mechanism of myotoxic action
Abstract Myotoxin a and a group of closely related, small, basic toxins cause myonecrotic destruction of muscle tissue upon envenomation. The sarcoplasmic reticulum swells and eventually breaks down to small vesicles. Degeneration of myofibrils and myofilaments ensues and loss of the classic striation pattern is apparent. These toxins exhibit high sequence similarity as well as sequence microheterogeneity. A conformational heterogeneity was recently discovered in myotoxin a. The existence of myotoxin a in two forms in equilibrium in solution hinders the generation of a well defined three-dimensional structure. The difference, if any, in the biological activity of the two forms has not been established yet. Recent biochemical studies indicate that myotoxin a is a potent Ca2+ releasing agent that binds to calsequestrin in the lumen of the sarcoplasmic reticulum.
Myonecrosis induced in vivo by cardiotoxin, melittin, and Asp49 and Lys49 phospholipase A2 (PLA2) myotoxins involves rapid lysis of the sarcolemma, myofibril clumping, and hypercontraction of sarcomeres. In contrast, skeletal muscle necrosis induced by crotamine and myotoxin a is much slower, consisting of mitochondrial and sarcoplasmic reticulum swelling, myofibril degeneration, and lack of sarcolemma or transverse tubule damage. The mechanisms contributing to the myonecrosis induced by these peptides were evaluated. Two cardiotoxins and two Lys49 PLA2 myotoxins lysed primary cultures of human skeletal muscle within 24 hr at a concentration of 0.25 μM, while melittin, crotamine, and myotoxin a, and an Asp49 PLA2 myotoxin were non-cytolytic at concentrations up to 5.0 μM, suggesting that cytolysis is not a good measure of myotoxicity. Crotamine and the Lys49 PLA2 myotoxin altered Ca2+ ion flux in human heavy sarcoplasmic reticulum by opening the ryanodine receptor. Whole-cell patch-clamp studies demonstrated that administrating crotamine intracellularly increased Na+ currents. Free fatty acids, liberated by activation of tissue phospholipase C or by the PLA2 activity of the myotoxins, were monitored for crotamine, myotoxin a and a Lys49 PLA2 myotoxin in cell cultures in which the lipids had been radiolabeled. Only the Lys49 myotoxin produced significant amounts of fatty acid in cell cultures, supporting a potential role for fatty acid production only in the mechanism of sarcolemma-destroying myotoxins. These findings, coupled with those in the literature, support a hypothesis in which the myotoxins and/or products of lipase activity (e.g. fatty acids) are acting at a site existing on both the Na+ channel and a protein involved in Ca2+ release and probably serving a modulatory function for ion regulation. Based on the similarities in mechanisms between the toxins and fatty acids, the most likely site would be a fatty acid binding site on the protein (either similar to that on fatty acid binding proteins, or an acylated cysteine residue) or in the membrane.
M. L. Yudkowsky, J. Beech and J. E. Fletcher. Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle. Toxicon32, 273–278, 1994.—Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, induces necrosis in skeletal muscle. In isolated organelles, it has been reported that myotoxin a reduces Ca2+ uptake into the sarcoplasmic reticulum. The effects of the toxin on Ca2+ regulation were examined in heavy sarcoplasmic reticulum fractions from human and equine skeletal muscle. Ca2+ uptake and release (the threshold of Ca2+-induced Ca2+ release) were examined by dual wavelength spectrophotometry. The toxin lowered the threshold of Ca2+-induced Ca2+ release in a dose-dependent manner (1–10 μM) and this effect was antagonized by ruthenium red, a Ca2+ release channel blocker. Ca2+ uptake into equine heavy sarcoplasmic reticulum was not decreased by myotoxin a (10 μM) when Ca2+ release was blocked by ruthenium red. [3H]Ryanodine binding to equine heavy sarcoplasmic reticulum was converted from a relatively low affinity state to a higher affinity state by myotoxin a. These results suggest that the dominant effect of myotoxin a is to increase the Ca2+ sensitivity for the opening of the calcium release channel (ryanodine receptor). Myotoxin a may prove to be a useful tool to probe the modulation of calcium release in sarcoplasmic reticulum fractions.
Pseudechis colletti is an Australian elapid snake with a range limited to central Queensland, Australia. The venom of this snake, as well as that of several other Australian elapids, has been shown to contain a phospholipase A2 (PLA2) which can cause a marked myoglobinuria in mice. Few studies have described the histopathologic and ultrastructural changes that result from myotoxic PLA2-induced damage. Our investigation demonstrated that the isolated PLA2 induced myodegeneration and necrosis in myocardium in a dose-related manner, with subsequent myoglobinuria and myoglobinuric nephropathy.
Cultured myoblasts and moytubes were used to study the effects of purified myotoxins from rattlesnake venoms. Standard cell culture techniques were used to establish and maintain primary cultures derived from neonatal rat tissue and two clonal cell lines, rat RMo cells and mouse C2 cells. Toxin concentrations, ranging from 0.04 to 1.0 microM, were added to the cultures at various times under distinct, well-defined conditions. Addition of myotoxin alpha to primary myoblast cultures did not appear to affect the fusion process, whereas similar experiments with two clonal cell lines produced larger myotubes when contrasted with untreated control cultures, particularly with RMo cells. The myotubes derived from primary cell cultures twitched spontaneously but the twitching ceased when the medium was replaced with a serum-free chemically defined incubation medium. Addition of myotoxin alpha to the primary myotubes incubated with serum-free defined medium caused the myotubes to twitch again. Derivatives of myotoxin alpha were prepared by reactions with tetranitromethane and with iodoacetic acid, the latter under reducing and non-reducing conditions. The resulting products, purified but not chemically characterized, were nearly devoid of activity when primary cultures were used to test activity.
Necrosis of skeletal muscle is produced by two types of snake venom components: single chain peptides consisting of 42-44 amino acid residues and phospholipases A2 representing either single chain proteins or existing as complexes of several enzyme subunits or combined with other nonenzymatic proteins. Vacuolation, lysis and necrosis of skeletal muscle cells are the major pathological effects of these myotoxins. Although the exact mode of action of these toxins is not clear, interactions with the plasma membrane leading to permeability changes for ions and to their complete destruction is evident. The high specificities of some venom phospholipases A2 for skeletal muscle cells suggest a specific binding to certain membrane receptors; however, an enzymatic action on membranes may also be involved.
Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.
Some snake venoms contain toxins that are reported to be selective for damaging muscle. This specificity can be used to design experiments intended to eliminate muscle. We studied the small myotoxins and fractions IV and V of Bothrops nummifer venom to evaluate their direct effect on cultured muscle cells, neurons, macrophages, and a fibroblast cell line. The small myotoxins, at 100 micrograms/ml for 2 h, had no effect in vitro, contrary to the in vivo applications. Fractions IV and V were both myotoxic and, at 100 micrograms/ml, destroyed all cell types. However, at 10 micrograms/ml the effects of fraction IV were more selective for muscle. Vacuolation of the sarcoplasmic reticulum and T-tubules was first seen in the poisoned muscles, without initial lesions in the nuclei, sarcolemma, mitochondria, and rough endoplasmic reticulum. Fractions IV and V have different toxic activity in cells other than muscles and are a mixture of two basic proteins (i and ii). Protein ii is predominant in fraction IV and protein i is predominant in fraction V. The toxic effects may be mediated by the formation of nonspecific ionic pores in the sarcolemma and/or T-tubule muscle membrane.
The binding of radiolabeled myotoxin a to various cultured cell lines was evaluated. One rat skeletal muscle-derived cell line, L8, bound substantially more myotoxin a than did all all other cell lines examined. Several biophysical parameters of myotoxin a-L8 binding were determined. Binding was saturable with a moderate binding affinity. Scatchard analysis and Hill plots indicated a single class of binding sites. The binding was reversible, as demonstrated by chase experiments. Radiolabeled myotoxin a bound to the cell surface at a site inaccessible to the general protease, pronase. Specificity and biological relevance of the binding was suggested by competition with unlabeled toxin and various peptides derived from the toxin. Biologically active peptides, corresponding to the N- and C-terminal sequence of myotoxin a, competed with radiolabeled toxin for L8 binding. It was concluded that the L8 system is a suitable cell model to study myotoxin a mechanism of action.
Over 5000 Americans suffer from snake bites annually, and of these, nearly one quarter are from poisonous species. Although these cases are undeniably reported, death appears to occur in only a few cases each year, and often reflects delay in obtaining medical care. Two families of venomous snake indigenous to the United States account for most envenomations: Crotalidae (pit vipers or new world vipers) and Elapidae. This article focuses on the snakes of the Crotalidae family.
Previous studies showed that toxic phospholipases A2 (Pa-8 and Pa-10F) from the venom of Pseudechis australis, the Australian king brown snake, reduced acetylcholine release at mouse neuromuscular junctions and depressed motor nerve terminal action potentials [Fatehi et al. (1994a), Toxicon 32, 1559-1572], and it was postulated that these toxins induced their effect on the action potential waveforms through nerve terminal depolarisation. To test this hypothesis, the effects of Pa-11 (another phospholipase A2 from the venom of Pseudechis australis), and the known depolarising agents. myotoxin a, from the venom of the rattlesnake. Crotalus viridis viridis, and ouabain on these waveforms were compared with the changes induced in the nerve terminal action potentials by Pa-8 and Pa-10F. The experiments were performed on the isolated mouse triangularis sterni preparation, using extracellular recordings. Pa-11 (0.1 microM) decreased the component of nerve terminal action potential related to Na+ and K+ currents to about 80% and 40% of control, respectively, after 60 min. Myotoxin alpha (5 microM) and ouabain (50 microM) produced similar, time-dependent changes in the nerve terminal action potential. These effects are similar to those produced by Pa-8 and Pa-10F, and are consistent with a slow but partial loss of membrane potential at the nerve terminal. In addition, whole-cell patch-clamp recording was employed to investigate possible direct actions of Pa-8. Pa10F and Pa-11 on Na+ and K+ currents in NG108 and PC12 cells in culture. None of these toxins (0.8 microM) reduced the Na+ and K+ currents in these cells. There was also no displacement of [125I]alpha-dendrotoxin bound to voltage-sensitive potassium channels on rat synaptosomal membranes induced by Pa-8, Pa-10F and Pa-11 (up to 100 microM). These results support the hypothesis that the alteration of nerve terminal waveforms by these toxic phospholipases A2 is mediated by nerve terminal depolarisation.
This review is a personal narration by a retiring pharmacologist from Taiwan who looks back at his discovery of alpha-bungarotoxin from the historical perspective of Taiwan during the last 50 years, with accounts of his experiences and his efforts to overcome hardship. How the alpha-toxin was isolated and characterized as an irreversible specific nicotinic acetylcholine (ACh) receptor antagonist, and how it subsequently became a useful experimental probe are presented here. The dilemma of differentiating the actions of tubocurarine and alpha-bungarotoxin is analyzed. The author also outlines findings based on work done in his laboratory using alpha-bungarotoxin as a tool on particular aspects of synaptic transmission. These include presynaptic receptor for positive feedback of transmitter release, explosive release of ACh, up- and downregulation of ACh receptors after chronic drug treatment, autodesensitization of junctional ACh receptors, differences in action between natural transmitter and exogenous agonists and that between junctional and extrajunctional ACh receptors. Some experimental pitfalls, in which biomedical scientists are frequently trapped, are raised. Finally, some anecdotes are appended from which the reader may further understand scientific life in the 20th century, including its joys and regrets.
Phoneutria nigriventer (Labidognatha, Ctenidae) is a spider found in the warm regions of South America. Bites by this species cause intense local pain, autonomic dysfunction and paralysis. PhTx2, a neurotoxic fraction of the venom of this species, interferes with the physiology of sodium channel function. The present study describes the morphological changes in mouse phrenic nerve and diaphragm muscle after 15, 30, 45 and 60 min of incubation with 1 microg of PhTx2/ml. Light and transmission electron microscopy showed that PhTx2 caused progressive myonecrosis which involved swelling of the sarcoplasmic reticulum, mitochondrial damage, disorganization of the sarcomeres, zones of hypercontracted myofibrils and rupture of the plasma membrane. The intramuscular fascicles of the phrenic nerve showed vacuolated myelinated axons and Schwann cells. The neuromuscular junctions had vesicle-depleted nerve terminals with swollen mitochondria. The axolema was frequently invaginated and sequestered portions of the axoplasm, or was sometimes interrupted at the site of the synaptic gutter. The post-synaptic junctional folds were shallow and disperse. These morphological alterations in the muscle and nerve fibres were similar to those caused by osmotic disturbances and agree with the ability of PhTx2 to increase the permeability of sodium channels. An increase in sodium influx would probably be accompanied by an influx of water and an elevation in the concentration of cytosolic calcium as a result of calcium release by the sarcoplasmic reticulum and/or mitochondria and the entry of extracellular calcium. The morphological effects caused by PhTx2 were comparable to those seen with Phoneutria nigriventer whole venom which is known to activate and to delay the inactivation of sodium channels. We conclude that PhTx2 is probably the main toxic fraction responsible for such morphological alterations.
We investigated whether veratrine (5 microl, 10 ng/kg) injected into the mouse extensor digitorum longus (EDL) (fast-twitch) and soleus (SOL) (slow-twitch) muscles provokes distinctive ultrastructural disturbances 15, 30 and 60 min later. The mitochondria in SOL were affected earlier (within 15 min) than in EDL. Swelling of the sarcoplasmic reticulum terminal cisternae was more marked in EDL than in SOL and caused distortion of sarcomeres so that fragmentation of myofilaments was more pronounced in EDL. Hypercontracted sarcomeres were seen mainly in SOL and veratrine caused infoldings of the sarcolemma only in this muscle. In both muscles, the T-tubules remained unaffected and by 60 min after veratrine most of the above alterations had reverted to normal. Pretreatment with tetrodotoxin prevented the alterations induced by veratrine. This suggests that most of the alterations resulted from the enhanced influx of Na+ into muscle fibers. These results emphasize the importance of considering the type of muscle when studying the action of myotoxic agents.
The effects of polypeptide neurotoxin from Anemonia sulcata on nerve conduction in crayfish giant axons and on frog myelinated fibers have been analyzed. The main features of toxin action are the following: (i) the toxin acts at very low doses and its action is apparently irreversible. (ii) The toxin selectively affects the closing (inactivation) of the Na+ channel by slowing it down considerably; it does not alter the opening mechanism of the Na+ channel or the steady-state potassium conductance. (iii) The tetrodotoxin-receptor association is unaffected by previous treatment of the axonal membrane with the sea anemone toxin. (iv) Conversely, the sea anemone toxin can only associate with the membrane when the Na+ channel is open for Na+; it does not bind when the channel is previously blocked by tetrodotoxin. (v) Besides its effect on the action potential, the sea anemone toxin displays a veratridine-type depolarizing action at low Ca2+ concentration which can be suppressed by tetrodotoxin. The sea anemone toxin greatly stimulates the release of gamma-[3H]aminobutyric acid from neurotransmitter-loaded rat brain synaptosomes. The apparent dissociation constant of the neurotoxin-receptor complex in this system is 20 nM. The sea anemone toxin effect is antagonized by tetrodotoxin.
Purified ciguatoxin at 0.1 to 10 ng/ml inhibits the net accumulation of neurotransmitters (gamma-aminobutyric acid and dopamine) by brain synaptosomes. This action is due to a stimulation of neurotransmitter release. The half-maximum effect of the toxin is observed at 0.62 ng/ml. The effect of ciguatoxin is completely inhibited by tetrodotoxin (K0.5 = 4 nM). Electrophysiological studies on neuroblastoma cells indicate that ciguatoxin induces a membrane depolarization which is prevented by tetrodotoxin and which is due to an action that increases Na+ permeability. Under appropriate conditions ciguatoxin creates spontaneous oscillations in the membrane polarization level and repeated action potentials. Ciguatoxin stimulates 22Na+ entry through the voltage-dependent Na+ channels of neuroblastoma cells and rat skeletal myoblasts when it is used in synergy with veratridine, batrachotoxin, pyrethroids, sea anemone, or scorpion toxins. The half-maximum effect of ciguatoxin on 22Na+ flux in the presence of veratridine occurs at a concentration of 0.5 ng/ml. Stimulation of 22Na+ flux by ciguatoxin is abolished by tetrodotoxin. These results taken together indicate that ciguatoxin belongs to a new class of toxins acting on Na+ channels.
Regulation of Na+ channels by neurotoxins has been studied in pinched-off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.
Crotamine and rattlesnake venom showed identical effects on phrenic-nerve-diaphragm of the rat or rectus abdominis of the frog. On diaphragm of the rat we observed an immediate contracture followed by other spontaneous and irregular contractures and tachyphylaxis. This effect is inhibited by tetrodotoxin, Ca, Mg and Kions, and partly by chronic denervation or previous curarization. Small doses of venom and toxin sensitized the rectus abdominis of the frog to contracture by K ions. Large doses produced irregular contractions due to instability of the muscle membrane. This action was inhibited by Ca and Mg ions. There was also some sensitization of the preparation to acetylcholine.
1. A comparative study has been performed between Crotalus durissus terrificus var. crotaminicus venom (Brasil) and crotamine, a peptide isolated from this venom testing their lethality in mice and chicks, and neuromuscular effects on chicken and rat.2. The crude venom, more toxic than crotamine, produced a typical contracture and paralysis in mice, only a paralysis in chicks—crotamine induced a contracture in mice, but no effects in chicks.3. In chicken in situ neuromuscular preparations, the crude venom produced, after delay, a slow irreversible paralysis. Crotamine induced neither contracture, nor paralysis.4. In rat in situ neuromuscular preparations, crude venom and crotamine had very similar effects: at small doses a state of contracture impeded relaxation; at high doses, a strong short lasting contracture was produced, generally followed by a reversible paralysis of muscle fibre and potentiation of the maximal twitch. The contracture was prevented or reversed by tetrodotoxin, Ca and Mg ions and exhibited tachyphylaxis. d-Tubocurarine and decamethonium did not prevent the crotaminic contracture but enhanced the secondary paralysis.5. In the rat, crotamine action was on the muscle fibre membrane: a change in Ca or Na ion permeability produced contracture. This contracture induced a potassium ion efflux, responsible for the later phases of the crotamine action. A long lasting interaction with Ca ion sites could explain the appearance of spontaneous contractures and tachyphylaxis.
Quinquestriatus toxin (QTX) isolated from the venom of a scorpion ( Leiurus quinquestriatus ) and sea anemone ( Anemonia sulcata ) toxin II enhanced the twitch response of the rat and mouse diaphragms and like crotamine (isolated from the venom of Crotalus durissus terrificus ) caused spontaneous fasciculation of the muscle.
Trains of action potentials in muscles at 70–250 Hz, which could not be antagonized by (+)‐tubocurarine, were triggered by single stimulation or occurred spontaneously after treatment with these toxins.
QTX and toxin II prolonged the rat muscle action potential 3 to 4 fold whereas crotamine prolonged the action potential by only 30%.
The membrane potential was depolarized from about −82 mV to −55 mV by crotamine 2 μg ml ⁻¹ , −41 mV by toxin II 5 μg ml ⁻¹ and to −50 mV by QTX 1 μg ml ⁻¹ . The concentrations to induce 50% maximal depolarization (K 0.5 ) were 0.07, 0.15 and > 0.4 μg ml ⁻¹ , respectively, for QTX, crotamine and toxin II, whereas the rates of depolarization were in the order toxin II > crotamine > QTX. The depolarizing effects of crotamine and QTX, but not of toxin II, were saturable.
The depolarizing effects of all three toxins were irreversible whereas the membrane potential could be restored by tetrodotoxin non‐competitively.
Simultaneous treatment with crotamine and QTX or crotamine and toxin II at concentrations below K 0.5 caused only additive effects on depolarization.
When the muscle was depolarized by pretreating with a saturating concentration of crotamine, the onset of depolarization by QTX was greatly retarded whereas that by toxin II was unaffected. Action potentials were further prolonged in both cases.
It is inferred that all three peptide toxins act at sites on the sodium channel and the binding sites for QTX and crotamine overlap to a considerable extent. On the other hand, the site for toxin II appears not to overlap with that of crotamine but may overlap with that of QTX.
The primary structure of myotoxin a, a myotoxin protein from the venom of the North American rattlesnake Crotalus viridis viridis, was determined and the position of the disulfide bonds assigned. The toxin was isolated, carboxymethylated, and cleaved by cyanogen bromide, and the resultant peptides were isolated. The cyanogen bromide peptides were subjected to amino acid sequence analysis. In order to assign the positions of the three disulfide bonds, the native toxin was cleaved sequentially with cyanogen bromide and trypsin. A two peptide unit connected by one disulfide bond was isolated and characterized, and a three-peptide unit connected by two disulfide bonds was isolated. One peptide in the three-peptide unit was identified as Cys-Cys-Lys. In order to establish the linkages between the peptides and Cys-Cys-Lys, one cycle of Edman degradation was carried out such that the Cys-Cys bond was cleaved. Upon isolation and analysis of the cleavage products, the disulfide bonds connecting the three peptides were determined. The positions of the disulfide bridges of myotoxin a were determined to be totally different from those of neurotoxins isolated from snake venoms. The sequence of myotoxin a was compared with the sequences of other snake venom toxins using the computer program RELATE to determine whether myotoxin a is similar to any other types of toxins. From the computer analysis, myotoxin a did not show any close relationship to other toxins except crotamine from the South American rattlesnake Crotalus durissus terrificus.
Myonecrosis is a serious result of rattlesnake bite and constitutes a persistent clinical problem. In the current study we have isolated crotamine from the venom of Crotalus durissus terrificus to test its ability to cause structural damage to skeletal muscle, and to make direct chemical comparisons with Myotoxin a, a myotoxic polypeptide we recently isolated from prairie rattlesnake (Crotalus viridis viridis) venom. Disc gel electrophoresis, isoelectric focusing, circular dichroic spectroscopy, and amino acid analysis, all indicated a high degree of chemical similarity. Light microscope histology revealed that crotamine caused vacuolizationof skeletal muscle fibers, qualitatively the same as the vacuolization caused by Myotoxin a. The ability of these two basic snake venom polypeptides to cause structural damage to skeletal muscle fibers has significant implications toward more complete understanding of the cause of snake venom-induced myonecrosis.
Crotamine (0.5 μg/ml) augmented the single twitch response of the rat and mouse isolated diaphragm to direct stimulation and prolonged the time course of contraction. At higher doses (10 to 50 μg/ml), contracture was observed with spontaneous fibrillation.
The resting membrane potential of diaphragm was rapidly depolarized to about −50 mV within 5 minutes. No increase of depolarization occurred on prolongation of the incubation time or increase of crotamine concentration from 0.5 μg/ml to 50 μg/ml. The effect was not reversed by washing.
Tetrodotoxin, low Na ⁺ (12 m m ), Ca ²⁺ (10 m m ) and procaine (1 m m ) prevented the crotamine‐depolarization. However, depolarization resumed when crotamine and the antagonists were removed.
Low Cl− (8.5 m m ) and pretreatment with ouabain enhanced depolarization by crotamine.
High K ⁺ (25 to 50 m m ) prevented the further depolarization by crotamine and the membrane potential was restored to normal on washout of crotamine with normal Tyrode solution.
Effective membrane resistance was decreased by about 50% by crotamine.
²⁴ Na‐influx of the rat diaphragm was increased by crotamine. ⁴² K‐influx was slightly increased if tetrodotoxin was also present but was decreased in the absence of tetrodotoxin.
No effect on the miniature and evoked endplate potential of the rat diaphragm was observed. Skeletal muscles from frog and chick were not affected.
It is inferred that crotamine acts on a molecule regulating the Na ⁺ ‐ permeability of the Na ⁺ channel of murine muscles. It is proposed that extracellular K ⁺ depresses the permeability of the Na ⁺ channel by acting on the same regulator molecule.
A previously unknown polypeptide myotoxin, designated myotoxin a, was isolated for the first time from prairie rattlesnake (Crotalus viridis viridis) venom. Electrophoretic homogeneity of myotoxin a was shown in beta-alanine disc gel polyacrylamide gel electrophoresis and in isoelectric focusing gel electrophoresis. Molecular weight and isoelectric point estimates of 4100 and 9.6 were obtained by gel filtration and isoelectric focusing gel electrophoresis, respectively. Amino acid composition showed a total of 39 amino acid residues, with 10 lysine residues and two disulfide bridges. When the two disulfide brides were reduced and alkylated, the myotoxic activity was abolished, indicating that the disulfide bridges of myotoxin a are essential for its biological activity. The loss of the biological activity is probably due to a marked change in secondary structure. The circular dichroic spectrum indicates that the chemically modified, inactive myotoxin exhibits typical random-coil conformation.
The pathogenesis of myonecrosis induced by a purified component of rattlesnake (Crotalus viridis viridis) venom was studied at the light and electron microscopic levels. Crude venom was fractionated by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25). Electrophoretic homogeneity of the isolated myotoxin (Fraction II from C-25 column) was demonstrated in isoelectric focusing and disc gel polyacrylamide gel electrophoresis. White mice were injected intramuscularly with 1.5 mug/g of the purified protein in 0.1 ml of physiologic saline. Light microscopic examination of injected muscle revealed a series of degenerative events including partial vacuolation of muscle cells at 6, 12, and 24 hours and complete vacuolation and loss of striations at 48 and 72 hours. Hemorrhage was not observed. At the electron microscopic level the perinuclear space and sarcoplasmic reticulum were dilated in all samples. By 48 and 72 hours the myofibrils lacked striations and the sarcomeres were disorganized. Plasma membranes and T tubules remained intact in all samples. These results correlated well with the myonecrosis induced by crude Crotalus viridis viridis venom except for several important aspects. The pure component altered skeletal muscle cells specifically, with the sarcoplasmic reticulum being the primary site of action.
Four neurotoxins that activate the action potential Na+ ionophore of electrically excitable neuroblastoma cells interact with two distinct classes of sites, one specific for the alkaloids veratridine, batrachotoxin, and aconitine, and the second specific for scorpion toxin. Positive heterotropic cooperativity is observed between toxins bound at these two classes of sites. Tetrodotoxin is a noncompetitive inhibitor of activation by each of these toxins (KI = 4-8 nM). These results suggest the existence of three functionally separable components of the action potential Na+ ionophore: two regulatroy components, which bind activating neurotoxins and interact allosterically in controlling the activity of a third ion-transport component, which binds tetrodotoxin.
The interaction between batrachotoxin (BTX) and tetrodotoxin (TTX) and local anesthetics (procaine and lidocaine) was examined on rat diaphragm muscle at 37°. At low concentrations TTX shifted the curve for BTX induced membrane depolarization to the right, and at higher concentrations it depressed the maximal membrane depolarization induced by BTX. A plot of log (dose ratio - 1) vs. log TTX concentration gave a straight line with a slope of 1.31. The estimated dissociation constant K(B) of TTX was 30.2 nM. Both procaine and lidocaine inhibited BTX induced membrane depolarization when applied either before or during exposure to BTX; a form of noncompetitive antagonism was disclosed. Although TTX reversed the depolarization induced by BTX, the persistent and irreversible action of BTX became apparent after TTX had been washed from the bath. When procaine and lidocaine were applied in a similar manner the repolarization of the muscle membrane was incomplete; upon washing in drug free physiological solution, the membrane potential and frequency of spontaneous miniature end plate potentials returned toward control values. During the protective action of procaine and lidocaine, sodium activation associated with an action potential was only slightly reduced. The results suggest that TTX and BTX react with different sites along the same sodium channel, but that procaine and lidocaine probably interfere with the binding of BTX to its receptor site.
Some effects of the sea‐anemone toxin ATX‐II on mammalian nerve‐muscle preparations have been described
When ATX‐II (10 ⁻⁸ ‐10 ⁻⁶ m) was applied to rat hemidiaphragm preparations, both directly and indirectly generated twitch responses were potentiated and prolonged. At the same time the resting tension of the preparations increased
The increase in resting tension caused by ATX‐II in innervated muscles was not prevented by curarization, but was reversed by exposure to tetrodotoxin. The increase in denervated muscles was not completely reversed by tetrodotoxin
At concentrations exceeding 1 × 10 ⁻⁷ M, ATX‐II caused a sodium‐dependent depolarization of both normal and denervated muscles. The depolarization of the denervated muscles was only partially reversed by tetrodotoxin
In the presence of ATX‐II repetitive endplate potentials (e.p.ps) were evoked by single shocks to the motor nerves in many fibres, and in those in which a single e.p.p. was still observed, the quantum content (m) was increased. Miniature e.p.p. frequency was not increased by ATX‐II, even when muscle fibres were depolarized by 30 mV
The indirectly and directly elicited action potentials of normal and denervated muscle fibres were much prolonged by ATX‐II. The action potentials remained sodium‐dependent. The sodium‐dependent tetrodotoxin‐resistant action potential of the denervated muscle fibre was also prolonged by ATX‐II. 7 It is concluded that ATX‐II both activates, and delays inactivation of, sodium channels in mammalian skeletal muscle fibres, probably by interacting with the channel ‘gate’.
The interactions between crotamine and tetrodotoxin and group II sodium channel toxins, including batrachotoxin, protoveratrine A and grayanotoxin I, were studied on the rat diaphragm muscle. When the diaphragm was pretreated with 0.1 micrograms crotamine/ml for 45 min (a condition known to depolarize the muscle by less than 3 mV, which is only 20% of the maximal depolarization induced by a saturating concentration of crotamine), the rate of depolarization by group II toxins was markedly enhanced and the time to reach the steady state depolarization was greatly shortened. The maximal depolarizations induced by each of the group II toxins, however, were not increased. Pretreatment with saturating concentrations of crotamine also caused no change of the steady state depolarization induced by batrachotoxin or grayanotoxin I. Moreover, pretreatment of the diaphragm with a high concentration of grayanotoxin I, whose effect is reversible, did not impede the depolarizing effect of crotamine. Tetrodotoxin restored the membrane potential, depolarized by crotamine, with 50% restoration at a concentration of 16 ng/ml, no matter whether a high (20 micrograms/ml) or a low (2 micrograms/ml) concentration of crotamine were used. The above results indicate that there is no competition between crotamine and group II toxins or between crotamine and tetrodotoxin. However, crotamine may affect the binding of group II toxins allosterically, increasing their affinity although the intrinsic activity may not be changed.
Neurons process and transmit information in the form of electrical signals. Their electrical excitability is due to the presence
of voltage-sensitive ion channels in the neuronal plasma membrane. In recent years, the voltage-sensitive sodium channel of
mammalian brain has become the first of these important neuronal components to be studied at the molecular level. This article
describes the distribution of sodium channels among the functional compartments of the neuron and reviews work leading to
the identification, purification, and characterization of this membrane glycoprotein.
The effects of several local anesthetics on the binding of ligands to receptors associated with voltage-sensitive sodium channels in rat brain synaptosomes have been examined. In the presence of 0.3 microM scorpion toxin, the 13 local anesthetics tested inhibited the specific binding of [3H]batrachotoxinin A 20 alpha-benzoate [( 3H]BTX-B), a ligand which binds to a receptor site responsible for the activation of sodium channel ion flux, in a dose-dependent fashion, with KD values ranging from 1.2 microM for tetracaine to 1.58 mM for benzocaine. A plot of log KD from these binding experiments against log K0.5 for inhibition of sodium currents by local anesthetics from electrophysiological experiments yielded a regression line with a slope of 0.84 and a correlation coefficient, r, of 0.86, demonstrating that the inhibition of [3H]BTX-B binding by local anesthetics occurs within a concentration range of physiological relevance. Tetracaine had little effect on basal 125I-labeled scorpion toxin binding to synaptosomes in the absence of batrachotoxin. However, in the presence of batrachotoxin, tetracaine inhibited the batrachotoxin-dependent increase in scorpion toxin binding (KD = 2.0 microM) in a dose-dependent manner, suggesting that inhibition of [3H]BTX-B binding by local anesthetics does not occur through binding at the scorpion toxin binding site. The inhibition of [3H]BTX-B binding by lidocaine was reversible within 30 min when samples were diluted from 10(-3)M to 10(-4) M lidocaine. Scatchard analysis of [3H]BTX-B binding to synaptosomes showed that bupivacaine and tetracaine reduced receptor affinity without decreasing maximal binding capacity. This reduction in receptor affinity in the presence of local anesthetics appears to be due, at least in part, to an increased rate of ligand dissociation from the receptor-ligand complex, suggesting an indirect allosteric mechanism for the inhibition of [3H]BTX-B binding by local anesthetics. Analysis of the effects of local anesthetics in terms of an allosteric model of drug action showed that they bind to inactive states of sodium channels with at least a 10-fold higher affinity than active states. A 7-fold difference in KD for inhibition of [3H]BTX-B binding between the local anesthetic stereoisomers RAC 109 I and RAC 109 II was observed. Similarly, the dissociation rate constant for the [3H]BTX-B/receptor complex was increased 9.3-fold in the presence of RAC 109 II and 4.3-fold in the presence of a comparable concentration of RAC 109 I.(ABSTRACT TRUNCATED AT 250 WORDS)
Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus atrox (western diamondback rattlesnake) and is composed of 42 amino acid residues. Earlier electron microscopic observation indicated that the toxin causes extensive swelling of the sarcoplasmic reticulum followed by disorganization of the sarcomers. In the present paper we describe the evidence for the attachment of peroxidase-conjugated myotoxin a to the membrane of sarcoplasmic reticulum of human muscle. It is thus suggested that the attachment of the toxin to the sarcoplasmic reticulum and the subsequent swelling are the first steps in myonecrosis induced by myotoxin a.
Antiserum to myotoxin f isolated from prairie rattlesnake (Crotalus viridis viridis) venom was tested for its ability to neutralize the local myotoxic and lethal activities of myotoxin a and of crude C. v. viridis venom. Wyeth's polyvalent (Crotalidae) antivenin was also tested for its ability to neutralize the crude venom. Using a light microscopic method to quantitate myonecrosis, the effect of myotoxin a, i.e. vacuolation, could be distinguished from total myonecrosis induced by whole venom. The results indicate that anti-myotoxin a serum is more effective in neutralizing local myonecrosis, but polyvalent antivenin is more effective in neutralizing lethality when they are mixed with crude prairie rattlesnake venom prior to injection.
Using a pure myotoxin (myotoxin a) isolated from prairie rattlesnake Crotalus viridis viridis) venom, a comparison between two methods for quantitating myonecrosis in mice was made. Measurement of creatine kinase (CK) levels in the plasma showed two peaks in CK levels after myotoxin injection, whereas measurement by histological assay (vacuolation index) showed only one peak. The first peak in CK levels at 3 hr after injection did not correlate with a high vacuolation index, but did correlate with contraction of muscle induced by the toxin. However, there was good correlation between the two methods at 6, 12, 24, 48 and 72 hr after injection, at which time the muscle cells were necrotic. In the case of the first CK peak this method might be measuring contraction of the muscle, but the second peak was probably measuring altered permeability of the sarcolemma since vacuolation, i.e. swelling of sarcoplasmic reticulum, did follow. High plasma CK levels usually preceded high vacuolation indexes, indicating that CK release due to altered sarcolemma permeability preceded vacuolation. Caution should be taken when using plasma CK levels to estimate the quantity of necrotic muscle cells, so as to insure that pathologic and not physiologic changes are being measured.
In this paper a further study is made of the spontaneous synaptic potentials in frog muscle (Fatt & Katz, 1952a), and their relation to the end-plate response. It has been suggested that the end-plate potential (e.p.p.) at a single nerve-muscle junction is built up statistically of small all-or-none units which are identical in size with the spontaneous 'miniature e.p.p.'s'. The latter, therefore, could be regarded as the least unit, or the 'quantum', of end-plate response. A convenient picture of how hundreds of such quanta, each capable of producing a miniature potential of 0 5-1 0 mV, can build up an e.p.p. of, say, 70-80 mV is provided by the hypothesis that separate parcels of acetyl- choline (ACh), released from discrete spots of the nerve endings, short-circuit the muscle membrane. The unit changes of membrane conductance produced at many parallel spots summate and lead to an intense depolarization of the
Transversely cut diaphragm preparation from rat
- Barstard
Ciguatoxin is a novel type of Na+ channel toxin
- Bldard
Die Primärstruktur des Crotamines
- Laure
Sea anemone toxin: a tool to study molecular mechanisms of nerve conduction and excitation-secretion coupling
- Rgmey