Sea Anemone Anemonia Sulcata Research Articles

Rapid voltage-dependent dissociation of scorpion alpha-toxins coupled to Na channel inactivation in amphibian myelinated nerves.

The voltage-dependent action of several scorpion alpha-toxins on Na channels was studied in toad myelinated nerve under voltage clamp. These toxins slow the declining phase of macroscopic Na current, apparently by inhibiting an irreversible channel inactivation step and thus permitting channels to reopen from a closed state in depolarized membranes. In this article, we describe the rapid reversal of alpha-toxin action by membrane depolarizations more positive than +20 mV, an effect not achieved by extensive washing. Depolarizations that were increasingly positive and of longer duration caused the toxin to dissociate faster and more completely, but only up to a limiting extent. Repetitive pulses had a cumulative effect equal to that of a single pulse lasting as long as their combined duration. When the membrane of a nonperfused fiber was repolarized, the effects of the toxin returned completely, but if the fiber was perfused during the conditioning procedure, recovery was incomplete and occurred more slowly, as it did at lower applied toxin concentrations. Other alpha-type toxins, from the scorpion Centruroides sculpturatus (IVa) and the sea anemone Anemonia sulcata (ATXII), exhibited similar voltage-dependent binding, though each had its own voltage range and dissociation rate. We suggest that the dissociation of the toxin molecule from the Na channel is coupled to the inactivation process. An equivalent valence for inactivation gating, of less than 1 e per channel, is calculated from the voltage-dependent change in toxin affinity.

The voltage-dependent Na + channel of insect nervous system identified by receptor sites for tetrodotoxin, and scorpion and sea anemone toxins

Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na + channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na + channel (K d = 0.03 – 0.08 nM). The γ toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na + channel having a K d of 6.1 pM. The K d value for toxin II from the sea anemone Anemonia sulcata is 0.12 μM. These results show a high degree of conservation of the pharmacological properties of the brain Na + channels between insects and mammals.

Effects of sea-anemone toxin (ATX-II) on the frequency of miniature endplate potentials at rat neuromuscular junctions.

Soleus and extensor digitorum longus muscles were isolated from rats. The muscles were exposed to ATX-II, a toxin isolated from extracts of the sea-anemone Anemonia sulcata . The toxin caused a dose-dependent increase in the frequency of miniature endplate potentials in both types of muscle. The increase in frequency could be reversed by the application of tetrodotoxin (TTX), and could be prevented by prior exposure of the muscles to TTX. It is concluded that ATX-II causes a sodium-dependent depolarization of the nerve-terminal membrane.

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na + channel specific action of ATX II. Na + current inactivation is slowed with the time constant τ h increasing towards positive membrane potentials. The steady state inactivation curve h oo was shifted to more positive potentials and its slope reduced.

Stings by the sea anemone Anemonia sulcata in the Adriatic Sea.

The sea anemone Anemonia sulcata is the clinically most important Actinaria in the Adriatic Sea. Between 1965 and 1980, 55 patients stung by this cnidarian were seen at the Pula Medical Center in Istria, Yugoslavia. The majority of injuries were inflicted upon the upper extremities, chest, or abdomen. Pain and the appearance of small blanched papules surrounded by slightly reddened and edematous bases were the usually initiating manifestations. Linear lesions were sometimes seen. Vesicles, sometimes filled with serous fluid, localized discoloration, and the formation of bullae sometimes followed. Somnolence, dizziness, nausea, vomiting, muscle aches, and lid edema were reported in some cases. The treatment of these injuries in the northern Adriatic Sea and elsewhere is discussed.

The Social Structure of Inachus phalangium, a Spider Crab Associated with the Sea Anemone

Abstract and SummaryThe behaviour of the spider crab Inachus phalangium (Fabricius, 1775), which lives in association with the sea anemone Anemonia sulcata (Pennant), was studied in the field.The crab was found in the littoral zone of the Mediterranean Sea near Banyuls sur Mer, France, in the whole depth range studied (0.5–25 m). The crabs had a long‐lasting association with individual Anemonia sulcata, occasionally with Aiptasia mutabilis. Most crabs were found in association with the same anemone for several days, some crabs were found in association with the same anemone for longer than one month.In the areas studied, on average 65 Inachus phalangium were found on 100 anemones.Crabs released in the vicinity of anemones moved towards them and entered them. Inachus phalangium could walk between the tentacles of Anemonia sulcata and Aiptasia mutabilis without eliciting feeding reactions of the anemone. The crabs left the anemones for moulting. After moulting masking material was removed from the exuvia and used again. The animals returned into an anemone while still soft. Material used for masking, usually algae, could be picked off the body and eaten. Masking material may be a food reservoir in addition to providing camouflage.Anemones were left only during night‐time. The crabs left their anemone to moult, to feed in the vicinity, fleeing from larger conspecifics, and to migrate to a different anemone.Outside the anemone's protection Inachus was eaten by several species of fish.Individuals appeared to avoid each other. 57% of all animals were found alone on an anemone. Large males and females were more frequently found alone than were small males and females. Fights were observed between members of the same and of the opposite sex. During fights, legs and claws could be torn off.Adult males migrated more often between anemones and moved over larger distances when migrating than did adult females. Adult males probably migrated in search of sexually mature females. Such a roving strategy is evolutionarily stable only when the higher costs (in terms of energy expenditure and mortality) are compensated for by a higher number of offspring than produced in the alternative, pair‐bonding strategy.

Botulinum toxin type A: kinetics of calcium dependent paralysis of the neuromuscular junction and antagonism by drugs and animal toxins

The effect of botulinum Toxin (BoTx), which blocks the mechanism of release of acetylcholine at neuromuscular junctions and induces paralysis of muscles stimulated by nerves, is known to be Ca 2+-dependent. Amplitude of muscular contractions evoked by nerve impulse was studied in BoTx poisoned preparations. The present report notes that an increase in Ca 2+ concentration in vitro delays paralysis of muscular contractions of the frog evoked by nerve impulse. The restorative effect of different drugs on this paralysis has been tested: 4-aminopyridine, ATXII (toxin isolated and purified from the sea anemone Anemonia sulcata tentacles) and a crude venom isolated from the scorpion Androctonus australis antagonize the BoTx induced paralysis at physiological concentrations of Ca 2+ (Ca 2+ 0 = 2 mM), whereas the restorative effect observed with tetra-ethylammonium or guanidine occurs at higher concentrations of Ca 2+ (Ca 2+ 0 = 4 mM), as in mammals. ATXII restores in vivo the activity of a BoTx paralysed muscle of guinea pig and this effect is more efficient if the interval between the injection of BoTx and ATXII is shortened. These results on the frog and guinea pig are in agreement with those obtained on other biological preparations by several investigators. Moreover it is suggested that the antagonism of BoTx induced paralysis is a consequence of the increase in Ca 2+ at the nerve ending. The efficiency of 4-aminopyridine and animal toxins is explained by an action on the nerve ending, by increasing Ca 2+ from an interval compartment of the cell, whereas antagonism produced by guanidine and tetraethylammonium involves uptake of Ca 2+ from the external medium. The bathing medium must be at a higher concentration of Ca 2+ than usual. This explains the differences in antagonism obtained by these drugs and toxins in vitro and in vivo.

Studies on the mechanism of the positive inotropic effect of ATX II (Anemonia sulcata) on isolated guinea pig atria.

The basic polypeptide ATX II (MW 4,770) isolated from the sea anemone Anemonia sulcata evokes a pronounced and dose-dependent positive inotropic effect in different mammalian heart preparations. The mechanism of this effect is so far unknown. (a) Investigations on isolated guinea pig atria indicate that changes of the steady state cellular Na, K and Ca concentrations cannot account for the positive inotropic effect. (b) An increase of the surface pressure of phospholipid monolayers was observed only at cardiotoxic ATX II concentrations. However, the 45Ca binding to phosphatidylserine, as the essential Ca-binding phospholipid, was not changed even at cardiotoxic ATX II concentrations. (c) Neither the enzymatic activity nor the ouabain inhibition kinetic of an isolated Na/K-ATPase preparation was affected by ATX II. (d) In intact electrically stimulated (1 Hz) guinea pig atria the binding of [3H]ouabain increases by about 50% at a positive inotropic ATX II concentration. The results suggest that the positive inotropic effect of ATX II is not caused by an unspecific membrane damaging action or by a direct interaction with the Na/K-ATPase. The increased binding of [3H]ouabain to intact heart muscles indirectly reflects an increased pump activity of the Na/K-ATPase, which is caused by an elevated Na transient due to the electrophysiologically well-established mechanism of the ATX II action on fast Na channel, i.e., delayed inactivation of the fast Na flux. However, the exact mechanism of the ATX II induced positive inotropic effect remains unknown.

Binding of sea anemone toxin to receptor sites associated with gating system of sodium channel in synaptic nerve endings in vitro.

Iodination of toxin II from the sea anemone Anemonia sulcata gives a labeled monoiododerivative that retains 80% of the original neurotoxicity. This derivative binds specifically to rat brain synaptosomes at 20 degrees C and pH 7.4 with a second-order rate constant of association ka = 4.6 x 10(4) M-1 sec-1 and a first-order rate constant of dissociation kd = 1.1 x 10(-2) sec-1. The binding occurs on the Na+ channel at a binding site distinct from that of other gating system toxins like batrachotoxin, veratridine, grayanotoxin, aconitine, and pyrethroids. The maximal binding capacity Bmax is 3.2 pmol/mg of protein (i.e., about two sea anemone toxin binding sites per tetrodotoxin binding site) and the Kd is 240 nM for the monoiododerivative and 150 nM for the native toxin. Corresponding binding parameters for the association of a 125I-labeled derivative of toxin II from the scorpion Androctonus australis Hector are Bmax = 0.3 pmol/mg of protein and Kd = 1 nM, whereas the Kd of the unmodified scorpion toxin is 0.6 nM. Competition experiments involving scorpion toxins, sea anemone toxins, and synaptosomes demonstrate that, although the sea anemone toxin is able to displace the scorpion toxin bound to synaptosomes, the scorpion toxin does not displace the sea anemone toxin. The sea anemone toxin but not the scorpion toxin binds to depolarized synaptosomes. Differences between binding properties of the two polypeptide toxins are analyzed in the discussion.

Scorpion toxin: Specific binding to rat synaptosomes

Summary The protein neurotoxin II from the venom of the scorpion Androctonus australis Hector was labeled with 125I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non interacting binding sites of high affinity (KD = 0.20 – 0.35 nM) and low capacity (30 – 60 fmoles/mg protein) to rat synaptosomes. This binding is reversible: the values of the rate association and dissociation constants k1 and k−1 are respectively 1.5×107 M−1s−1 and 1.6×10−3 s−1 and are in good agreement with the equilibrium constant. Moreover the bound labeled scorpion toxin II was displaced by the toxin II from the sea anemone Anemonia Sulcata .

Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore.

Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.

Polyvalent Proteinase Inhibitor from the Sea Anemone Anemonia Sulcata: Effect on Coagulation and Fibrinolysis

Polyvalent Proteinase Inhibitor from the Sea Anemone Anemonia Sulcata: Effect on Coagulation and Fibrinolysis

Amino acid sequence of toxin III from Anemonia sulcata.

Toxin III, the smallest toxin component of the poison of the sea anemone Anemonia sulcata, is a polypeptide with 27 amino acids. Its structure is stabilized by three disulfide bridges. The amino acid sequence was determined by solid-phase Edman degradation of the aminoethylated derivative. The peptide was coupled to the carrier, porous glass, by thiourea bridges between the alpha-amino group of arginine-1 and the epsilon-amino group of lysine-26 and the isothiocyanate groups of the carrier. Another fraction of the polypeptide was bound by an acid-amide condensation of the C-terminal valine-27 with the aminopropyl group of the carrier. The sequence of toxin III has no regions homologous to the 47-residue toxin II. Comparison with the known partial sequence of toxin I, which contains 46 amino acids (Wunderer, G. & Eulitz, M., in preparation) also fails to reveal homologies.

The action of a toxin from the sea anemone Anemonia sulcata upon Mammalian heart muscles.

The cardiac activity of toxin II, a basic polypeptide (m.w.: 4770) from the sea anemone Anemonia sulcata, was investigated in isolated electrically driven guinea-pig and rat auricles, Langendorff heart preparations of guinea-pigs and cat heart-lung preparations. Low concentrations of toxin II (2-100 nM) evoked a dose-dependent positive inotropic effect in the three different heart muscle preparations investigated. Higher concentrations of toxin II produced toxic symptoms like contracture and arrhythmia in auricles and atria (about 25 nM). In isolated cat hearts high toxin II concentrations (about 160 nM) caused unusual toxic symptoms such as long periods of ventricular fibrillation alternating with periods of normal cardiac activity. In rat and guinea-pig auricles as well as in Langendorff hearts of guinea-pigs the extent and rate of the positive inotropic effect induced by toxin II depended on the extracellular calcium concentration (0.45 to 2.7 mM). Toxin II did not alter the heart rate in spontaneously beating isolated cat hearts. In electrically driven guinea-pig auricles, the rate of the inotropic effect induced by toxin II was accelerated by increasing stimulation frequencies. Toxin II did not change the coronary flow in Langendorff heart preparations of guinea-pigs.

Electromechanical studies of an Anemonia sulcata toxin in mammalian cardiac muscle.

1. In guinea-pig papillary muscles toxin II isolated from the sea anemone Anemonia sulcata (ATX) enhanced the force of contraction without changing the time-to-peak tension. ATX increased the relaxation time. The positive inotropic effect was accompanied by a prolongation in action potential duration. The action potential amplitude and the resting membrane potential remained essentially unchanged. The velocity of depolarization (dV/dtmax) decreased slightly. The effects werenot modified by pretreatment of the animals with reserpine. 2. In preparations partially depolarized by elevated extracellular potassium (14.7 mM KCl) a concentration of 1 X 10(-8) M ATX was ineffective with respect to increase in force of contraction or delay of repolarization. The effects of 1 X 10(-8) M ATX could be reversed by 5 X 10(-7) M tetrodotoxin. 3. Our results suggest that ATX affects the action potential duration by delaying the inactivation of the fast sodium permeability change which initiates the action potential. It is not clear, however, in which manner this effect is related to the increase in force of contraction.

The effect of toxins fromAmeonia sulcata (Coelenterata) on neuromuscular transmission and nerve action potentials in the crayfish (Astacus leptodactylus)

1. The effects of three toxins (ATX I, II and III) from the sea anemoneAnemonia sulcata have been investigated on neuromuscular transmission and nerve action potentials in the crayfisch. 2. In the neuromuscular preparations the toxins produce a gradual increase in the amplitude of the excitatory junction potentials until they reach the threshold for the generation of electrically excitable membrane responses. This is followed by repetitive activity of the motor axons, first on single stimuli, later occurring spontaneously. 3. The effects of the three toxins differ only with regard to the effective concentrations: threshold concentrations amount to 5×10−9 M for ATX I and II, to 10−7 M for ATX III. 4. The toxins show no postsynaptic effects on the muscle fibre membrane and on their electrical responses upon direct stimulation. 5. All toxins prolong the action potentials of the giant axons in the abdominal nerve cord. The duration may reach more than one second. The rate of rise of the action potentials is not affected. In several cases, however, an increase of the amplitude of the action potential was observed. 6. The effects of ATX I and II are irreversible in contrast to those of ATX III. 7. It is suggested that the high potency of the toxins in the nanomolar range, their small size, the known composition and, in the case of ATX II, sequence of amino acids together with the specificity of action on the sodium channels, proved in different preparations, make these substances to important tools in the study of excitable membranes.

Isolation and characterization of four toxic protein fractions from the sea anemone Anemonia sulcata

A simple and rapid method is described for the isolation from the sea anemone Anemonia sulcata of four protein fractions of low molecular weight which have lethal activity against crabs. These proteins have been partially purified on ion exchange resins Dowex 50 W and CM cellulose and have been examined by zonal electrophoresis and micro isoelectric focusing. The stability of the fractions was determined under various experimental conditions. The fractions have strong paralytic action on Carcinus maenas. The toxic activity, however, greatly differs from one fraction to another.