Neuromuscular blocking action of two brevetoxins from the Florida red tide organism Ptychodiscus brevis

Tadcon. Vol . ]2, No . 1, pp. 75-84, 1984.

0041-0101/84 53 .00+ .00 ® 1984 Perproon Prag Ltd .

Printed in Great Britain.

NEUROMUSCULAR BLOCKING ACTION OF TWO BREVETOXINS FROM THE FLORIDA RED TIDE ORGANISM PTYCHODISCUS BREVIS DANIEL G . BADEN,' GEORGE BIKHAZI, 2 SUSAN J . DECKER,' FRANCIS F. FOLDES 3 and IGNATIUS LEUNG2 Departments of 'Biochemistry, 'Anesthesiology and 'Anatomy, University of Miami School of Medicine, LIMED P .O . Box 016129, Miami, Florida 33101, U .S .A.

(Acceptedfor publication 26 July 1983) D . G . BADEN, G. BIKHAzi, S . J . DEcxn, F. F. FOLDEs, and I . LEuNO. Neuromuscular blocking action of two brevetoxins from the Florida red tide organism, Ptychodiscus brevis. Toxicon 22, 75-84, 1984. - The action of Ptychodiscus brevis "brevetoxins" T17 and T34 on rat phrenic nerve-stimulated hemidiaphragm contraction is reported. The potency of T34 is greater than the potency of T17, but both cause a complete block of neuromuscular transmission in the nM to pM concentration ranges . Preparations exposed to low concentrations of T17 can recover in the presence of the toxin, whereas the effects of T34 are irreversible . The initial contracture produced by each is prevented by tetrodotoxin or curare. Neuromuscular block does not appear to be due to acetylcholine depletion, as determined by electron microscope examination of the neuromuscular junctions of blocked preparations . Persistent nerve depolarization is believed to be responsible for the neuromuscular block .

INTRODUCTION

has been interested in the toxinology of lethal compounds isolated from Florida's red tide organism, Ptychodiscus brevis, and we have purified and crystallized two very similar potent agents, named T17 and T34 (BADEN et al., 1979, 1981 ; BADEN and MENDE, 1982). Toxins from Florida red tides cause a variety of environmental effects including extensive fish kills (STEIDINGER et al., 1973), neurotoxic shellfish poisoning in man (BADEN, 1983) and human respiratory irritation (Music et al., 1973). Both T17 and T34 are potent ichthyotoxins (BADEN et al., 1979) and thus fish kills in situ are likely the concerted effects of at least these two toxins (BADEN et al., 1981). However, only T17 is potent when orally administered, indicating it as a likely oral intoxicant encountered in neurotoxic shellfish poisoning (BADEN and MENDE, 1982). Likewise, only T17 can be linked to the non-productive coughing and sneezing which occurs upon inhalation of P. brevis red tide seaspray. This agent causes a bronchoconstriction in anesthetized guinea pigs (BADEN et al., 1982). The neuromuscular effects of P. brevis extract have been examined in detail by others (GALLAGHER and SHINNICK-GALLAGHER, 1980) utilizing the in vitro rat phrenic nerve - hemidiaphragm preparation. Their studies confirmed earlier work regarding the neuromuscular blocking action of the P. bnevis toxins, the greater sensitivity of nerve to the toxin's action and the presence of both pre- and postsynaptic actions (SASNER et al., 1972; ABBOTT et al., 1975). An accompanying paper (SHINNICK-GALLAGHER, 1980) provided several possible mechanisms by which these toxins could exert their effects. The availability of purified toxins prompted us and others (VOGEL et al., 1982; WU et OUR LABORATORY

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D. G. BADEN, G. BIKHAZI, S. J. DECKER, F. F. FOLDES and 1. LEUNG

al., 1983) to examine the neuromuscular blocking action of T17 and T34 to ascribe specific actions to each potent fraction. With some minor exceptions as to potency and reversibility, T17 and T34 act in identical manners, and the results presented here and elsewhere (VOGEL et al., 1982 ; Wu et al., 1983) correlate well with the observations and conclusions made by GALLAGHER and SHINNICK-GALLAGHER (1980) .

Preparation

of toxins

METHODS

The two toxins purified from our cultures of P. brevis are obtained by solvent extraction and thin-layer chromatography (BADEN et al ., 1981). Following purification, each toxin migrates as a single fraction in 8 different solvent systems (BADEN and MENDE, 1982). The toxins are designated T17 and T34 based on their migration on silica gel thin-layer plates using a 30 to 70 (v/v) acetone-light petroleum (BP 30-60°C) mixture as developing solvent. T17 (R,0.17) and T34 (Rr 0.34) are resolved from the fraction originally designated fraction IVa (SAsNEIt et al., 1972).

In vitro preparations A total of 92 hemidiaphragms were utilized. The neuromuscular effects of the toxins were observed in the in vitro phrenic nerve-hemidiaphragm preparation of male Sprague-Dawley rats (BuLaBING, 1946) suspended in 70 ml organ baths containing modified mammalian Krebs' solution containing the same [Ca"] and [Mg ] as rat plasma (FoLDEs, 1981), aerated with 95% O,-5% CO,. The temperature was kept at 37°C and the pH was 7.33 - 7.40. Preparations were electrically stimulated through the nerve with supramaximal (8 -10 V) squarewave single impulses of 0.2 msec duration at 0.1 Hz every 10 sec (twitch) (a) or 0.1 sec trains of impulses at 50 Hz every 20 sec (temnusxb) . The twitch or tetanic tensions were continuously recorded with FT03 transducers on a Grass 7D polygraph. Fresh toxin solutions were prepared daily and were administered, one dose per diaphragm, in modified Krebs' solution to individual preparations at concentrations ranging from 1 pM to 1 FM . Inhibition of neuromuscular transmissi on was defined as the depression of nerve-stimulated muscle contraction observed for at least 6 successive twitch stimuli or 3 successive tetanic stimuli (equivalent in either case to 1 min time span). Log dose-response regression lines were determined for T17. This could not be accomplished using T34, which exhibited an exceedingly steep dose-response curve . After development of a complete block to nerve-stimulated diaphragm contraction, one of several alternative protocols was followed . (a) Electrodes were inserted into the muscle and the effects of supramaximal direct electrical stimulation were observed (150 V at 50 Hz, 2 msec duration every 20 sec) . (b) The effects of washing preparations with toxin-free Krebs' solution were investigated. (c) The nerve and muscle were carefully removed from baths from 1-15 min after complete block and were prepared for electron microscopy by immediately immersing them in chilled 2% paraformaldehyde-2.5% glutaraldehyde fixative at pH 7.3 in 0.13 M Millonig's phosphate buffer (MILLGNIG, 1961). Samples were refrigerated in fixative overnight and then post-fixed in 1% osmium tetroxide buffered with Millonig's phosphate buffer at 4°C for 1 hr before an ethanolic dehydration series . Tissues were embedded in Araldite 502 resin and sectioned on a Sorvall Porter-Blum MT-2 ultramicrotome. Sections were mounted on 0.25% formvar coated 1 x 2 slot grids and stained with 4% uranyl acetate (30 min) followed by Reynolds lead acetate (5 min) (REYNOLDS, 1965). Micrographs were obtained with a Philips 300 electron microscope. The effects of pretreatment with either tetrodotoxin (1 x 10i M) or d-tubocurarine (1 x 10-° M) on theinitial contracture were also observed . The effects of neostigmine methyl sulfate (2 x IV M) or 4-aminopyridine (4 x l0i M) were investigated, both during and after the development of a neuromuscular block. Glass organ baths were washed with 6 N NaOH followed by an acetone rinse after each experiment to remove residual toxin .

RESULTS

The structures for both T17 and T34 have been reported by others (CHOU and et al., 1981), although there has unfortunately been a lack of standardized nomenclature . It can now be stated that T34 is the toxin BTX-B (LIN et al., 1981) and GB-2 (CHOU and SHIMIZU, 1982) (Fig . IA) and T17 is the toxin GB-3 (CHOU and SHIMIZU, 1982) (Fig . 1B). Recent chemical modifications of T34 and BTX-B and correlation with literature values for GB-2 have confirmed their identical natures (Baden, Mende and Block, unpublished results). Therefore, the experiments described below SHIMIZU, 1982 ; LIN

Neuromuscular Action of Brevetoxins

77

FIG. 1. STRUCTURES OF TOXINS T34 AND T17. The chemical structures for "brevetoxins" T34 (BTX-B, LIN et al., 1981) (A) and T17 (GB-3, CHOU and SHIMIZU, 1982) (B) illustrate the polyether rigid ladder nature of these potent materials.

illustrate the effects of purified P. brevis toxins T17 (GB-3) and T34 (BTX-B, GB-2) on neuromuscular transmission . Addition of T17 to the bath caused a transient concentration-related increase in resting tension of the muscle as well as a concentration-related neuromuscular block. The time to peak contracture and the delay time from the time of toxin administration to initiation of contracture were shorter at higher toxin concentration, and the contracture always waned spontaneously within 5 (1 .5 x 10-° M) to 10 (2 x 109 M) min. The initial contracture was inhibited by either 1 x 10-6 M tetrodotoxin or 1 x 1V M d-tubocurarine and was enhanced by 4x 105 M 4-aminopyridine or 2 x 1V M neostigmine. Toxin treated preparations which did not proceed to a complete neuromuscular block recovered to produce nerve-stimulated muscle contraction strengths 75 -100% of initial strengths . Per cent recovery within that range was not always concentration-dependent. The time required to completely recover under twitch stimulation conditions was 15 t 6 min for 2.Ox109 MT17,29t9minfor3 .0 x 109 MT17,30t8minfor4.0 x 10-9 MT17 and 45 t 16 min for 1.5 x 10-° M T17 (n=12) (Fig. 2A). The neuromuscular block dose - response curve for T17 was steep, and the concentration of toxin necessary to cause 50% inhibition of neuromuscular transmission (ic) was lower during stimulation with short trains of tetani (icso 1.1 t 0.4 x 10-1 ° M; ic9, 1 .2 t 0.3 x 10-1° M; n=28) than during stimulation with single impulses (icso 5.0 t 1 .0 x 109 M; ic9, 6.5 t 1 .5 x 109 M; n = 36) (Fig. 3). Electrical stimulation was not necessary for the contracture response, nor was it necessary for the production of the neuromuscular block, but each toxin was more potent under conditions of nerve stimulation . Once nerve-stimulated contraction was blocked, it was not antagonized by neostigmine or 4-aminopyridine. There was a partial spontaneous recovery of neuromuscular transmission that could be hastened by washout (Fig. 2B). Fluid removed from the bath after partial recovery from the T17-induced neuromuscular block caused complete blockage of neuromuscular transmission in fresh preparations . Recovered preparations that had been washed with fresh Krebs' solution, when reintroduced to the original T17-containing solution removed prior to washing, again underwent initial diaphragm contracture, neuromuscular block

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F50. 2. EFFEmOF T17 ON NEUROMUSCULARTRAMIONL?V t17RO . Rat phrenic nerve- hemidiaphragm preparations, suspended in modified Krebs' solution at 37°C and pH 7.4 and aerated with 9354 0,-5% C0 2, were each exposed to a single concentration of T17 dissolved in modified Krebs' solution . Preparations were stimulated electrically through the nerve with either supramaximal impulses of 0.2 msec duration at 0.1 Hz (twitch, panel A) or with 0.1 sec trains of impulses at 50 Hz every 20 sec (tetanus, panel B) . Panel A illustratestheconcentration-related initial contractureof the muscle during stimulation . As thetoxinconcentration wasincreased from 2 x 10 -° Mto 1 .5 x 10M, the initial height increasedandthe time to recovery of individual preparations increased from 15 to 45 min. Per cent recovery ranged from 75 to 100% . Panel B illustrates the concentration-dependence of the rate of development of the neuromuscular block. Note the increased potency of T17 under conditions of tetanic stimulation. At low concentrations, preparations often spontaneously recovered in the presence of toxin. Recovery could be hastened by washing the preparations with toxin-free Krebs' solution (compare panel B, middle andlowertracings). 2A is representative of 3 replicates at each indicated concentration. 2B is representative of 5 replicates each .

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Each point was determined by a single exposure of individual diaphragms to concentrations of T17 in modified Krebs' solution . Per cent neuromuscular block is defined in the text . The neuromuscular potency of T17 was greater during stimulation with abort trains of tetani (triangles) than during stimulation with single impulses (circles) . The tc and IC,e values were derived from data on 64 diaphragms . Per cent neuromuscular block is indicated t S.E .

and recovery. The effect diminished after the S - 6th repetition of washing and recovery . Fresh T17 solution still elicited the original response, indicating neuromuscular preparation integrity had not diminished during the procedure. The neuromuscular potency of T34 was about 100 times greater than that of T17, during stimulation with either single impulses or short trains of impulses . In the 1 .0 -1 .5 x 10'`i M (n =10) and 1 .3 - 4.5 x 10-` 2 M (n =10) concentration ranges, T34 caused a complete block of neuromuscular transmission during stimulation with short trains of tetani or single impulses, respectively . Because of the extreme potency of T34 and problems associated with its solubility, the lc,, and 1Cyo values could not be determined precisely. Analogous to the situation with T17, the block induced by T34 was not antagonized by neostigmine or 4-aminopyridine, but the initial increase in muscle tension could be enhanced by treatment with 4-aminopyridine or neostigmine. Curare or tetrodotoain prevented the initial contracture. In contrast to T17, however, there was no spontaneous recovery of neuromuscular transmission even if bath fluid was changed at hourly intervals for 4 hr. Muscles could be stimulated directly after complete block of nerve-stimulated contracture, even when the concentrations of T17 (1 .3 x 10-° M; n = 32) and T34 (1 x 101 M; n =10) approached the limits of solubility . Electron microscopy of the neuromuscular junction of toxin-inhibited preparations (n =16), when compared to control preparations (n = 8), showed a complete complement of clear-core acetylcholine vesicles (Fig. 4), regardless of toxin concentration or time to preparation fixation following block, up to a limit of 15 min. General deterioration of the preparation was evident when incubated with 1 x 10-' M or higher toxin concentrations for longer than 60 min (n=8). Disruption of the myelin sheath and mitochondrial swelling were the most predominant and reproducible features noted at longer time intervals, but since the neuromuscular block was complete long before such changes took place, these effects were not studied further .

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D. G. BADEN, G. BIKHAZI, S. J. DECKER, F. F. FOLDES and I. LEUNG DISCUSSION

Spectroscopic analysis of each toxin revealed identical structures except that the a, Punsaturated aldehyde in T34 is reduced to the primary alcohol in T17 (LIN et al., 1981 ; CHOU and SHIMIZU, 1982). The profound difference in neuromuscular potency and reversibility may be related to the oxidation state at carbon 42 . T34 is approximately 10-fold less soluble in aqueous solution than is T17. The toxins are thought to solubilize in the lipid component of excitable membranes and so their respective lipid solubilities may reflect their potencies. Both are extremely potent, however, and so their lethal effects must be due to another as yet undetermined portion or portions of the molecule . Spontaneous recovery of preparations occurs in the presence of low concentrations of T17, but not of T34. The recovery does not reflect the stability of T17 in solution, for it is stable in Krebs' solutions for 2 hr at 37°C in the absence of diaphragms . T17-containing solutions taken from recovered preparations cause a complete neuromuscular block in fresh preparations, indicating a retention of potency in the presence of tissue in the bath . Washing the recovered preparation with fresh Krebs' solution and reapplication of the original T17-containing solution produces the characteristic progression through initial contracture to neuromuscular block and recovery . These findings, coupled with an increasingly diminished effect upon repeated application, suggest (1) that the preparations became insensitive to the effects of T17 upon prolonged exposure, (2) that sensitivity could be restored by washing with Krebs' solution and (3) that T17 appeared to be depleted upon repeated application and washout. An inactive bound form of T17 may be preventing access to the toxin's site of action in the recovered preparation. The observations we and others (VOGEL et al ., 1981 ; WU et al., 1983) have made correlate with the muscle twitch and microelectrode data, respectively, collected by GALLAGHER and SHINNICK-GALLAGHER (1980) . Their detailed studies (using GBTX) in the rat phrenic nerve - hemidiaphragm preparation illustrated that the toxin progressively inhibited the contraction of nerve-stimulated muscle and that the directly-stimulated muscle was inhibited only at higher concentrations of toxin. Toxin-induced muscle contractures were effectively inhibited by tetrodotoxin or curare . Based on purification data published elsewhere (Baden, Ph .D . dissertation, University of Miami, Florida, 1977), one mg GBTX contains approximately 7 .5 x 10 -3 mg T34 and 2 x 10-' mg T17. The concentration of GBTX required to block indirectly elicited hemidiaphragm contracture was 2 x 10-3 mg/ml (calculated to contain 1 .7 x 10-8 M T34 and 4.5 x 10-' M T17) and to block contractions elicited by direct stimulation, 1 .0 x 10-' mg/ml 8 M T17) . By these calculations, under (contains 8.5 x 101 M T34 and 2.3 x 10conditions of twitch stimulation T17 and T34 appear more potent in the purified state. This is not likely to be an enhancement of specific toxicity but perhaps reflects the presence of natural antagonists of toxin action in GBTX that are removed during purification . We cannot explain the reversibility of GBTX action by washing that is not reflected in studies utilizing T34 . We can detect no direct effect on muscle, as contraction always resulted during direct electrical stimulation. It is possible that either the toxins have been altered in activity during purification or that other potent fractions exist in GBTX which account for its direct effect on muscle. We favor the latter explanation. GOLicK et al. (1982) have described BTX-C, a chloromethyl ketone derivative of T34, isolated from the same Texas cultures of P. brevis as GBTX, as well as a fraction BTX-A (brevetoxin A) which is more potent than any other P. brevis toxin described to date . Descriptions of the

81

FIG . 4. ELECTRON MICROGRAPHS OF RAT PHRENIC NERVE -HENIIDIAPHRAOM NEUROMUSCULAR JUNCTION FOLLOWING EXPOSURE To CONCENTRATIONS OF T17 OR T34 WHICH CAUSE A COMPLETE BLOCK OF NEUROMUSCULARTRANSNUMON. Preparations were stimulated indirectly under tetanic conditions and were exposed to 1 .0 x 106 M T17 or T34 . Three minutes following a complete block of nerve-stimulated muscle contraction, the

neuromuscular preparations were removed and prepared for electron microscopy as described in the text. Panel A illustrates the integrity of control preparations . Panel B illustrates preparations exposed to T17, panel C preparations exposed to T34 . Acetylcholine-containing vesicles do not appear to be depleted. These results are representative of 24 replicates, 8 each of control, T17 and T34 . Paired diaphragms were utilized in the first 4 experiments for each of T17 and T34 to minimize tissue variation in animals, i .e. one hemidiaphragm served as a control for the toxin-exposed corresponding opposite hemidu ' phragm . The remaining 4 replicates of T17 and T34 toxin-exposed hemidiaphragms were not paired with controls. m mitochondria ; v acetylcholine vesicles ; ntf muscle fiber .

Neuromuscular Action of Brevetoxins

83

neuromuscular effects of these fractions are not available in the literature, but ichthyopotency is of the same order of magnitude as T17 and T34. Intracellular recording at the rat neuromuscular junction illustrated that GBTX increased miniature endplate potential (m.e.p.p.) frequency and at low concentration increased or at high concentration decreased m.e .p.p. amplitude (GALLAGHER and SHINNICK-GALLAGHER, 1980). Analogous work utilizing T17 at bath concentrations of 3-20 x 109 M illustrated a concentration-dependent increase in in.e.p .p. frequency without alteration in m.e.p.p. amplitud e (VOGEL et al., 1982; Wu et al., 1983) . Spontaneous e.p.p .s were seen with T17, but not with GBTX . In both cases it was concluded that the principal mechanism of action involved sodium channel-mediated depolarization, as the toxins' effects were antagonized by tetrodotoxin . Nerve terminal depolarization was postulated to be sufficient to inhibit nerve-stimulated transmitter release (SHINNICK-GALLAGHER, 1980). Subsequent neurotransmitter depletion was proposed as a mechanism for neuromuscular block, as a result of sodium channel depolarization (VOGEL et al., 1981). Our electron micrographs showed no depletion of clear-core vesicles, regardless of which P. brevis toxin or what concentration was used. Therefore, although the concentration-dependent initial contracture is inhibited by curare and tetrodotoxin, and enhanced by acetylcholinesterase inhibitors or 4-aminopyridine (suggestive of nerve-stimulated neurotransmitter release), we conclude that nervestimulated acetylcholine release is only transient and does not produce the neuromuscular block as a consequence of neurotransmitter depletion. Supporting studies conducted with T17 and T34 have shown a dose-dependent depolarization of crayfish or squid axon, respectively (HUANG et al., 1983 ; Wu et al., 1983). Thus, we feel that persistent depolarization of the nerve and nerve terminal are responsible for the neuromuscular block caused by T17 and T34 and that initial release of acetylcholine accounts for many of the observed toxicological effects. Acknowledgement - The authors wish to thank J . HuANG, C . Wu and K. NAKANISHI for disclosure of unpublished results . Portions of this research were funded by NIH grant number ES02299 . REFERENCES ABBOTT, B . C ., SIGER, A. and SPImiELsTEIN, M . (1974) Toxins from the blooms of Gymnodinium breve. In: Proc. Fyrs1 Int. Conf. on Toxic Dino,Jlagellate Blooms, p. 355 (Lo CIcERo, V . R ., Ed .) . Wakefield : Masse hussetts Science and Technology Foundation . BADEN, D . G . (1983) Marine food-borne dinoflagellate toxins . In: International Review of Cytology V, Vol. 82, p . 99 (BouRNE, G . H . and DANtELLi, J . F ., Eds .) . New York : Academic Press . BADEN, D . G . and MENDE, T . J . (1982) Toxicity of two toxins from the Florida red tide dinoflagellate (Ptychodiscus brevis) Toxicon 20, 457 . BADEN, D . G ., MENDE, T . J . and BLOCK, R . E . (1979) Two similar toxins isolated from Gymnodinium breve. In : Toxic Dinoflagellate Blooms, p. 327 (TAYLOR, D . L . and SELIGER, H . H ., Eds .) . New York : Elsevier/North-Holland . BADEN, D . G ., MENDE, T . J ., LICHTER, W . and WELLHAM, L . (1981) Crystallization and toxicology of T34 : a major toxin from Florida's red tide organism, (Ptychodiscus brevis). Toxlcon 19, 455 . BADEN, D . G ., MENDE, T. J ., BIKHAm, G. and LEuNG, 1 . (1982) Bronchoconstriction caused by Florida red tide toxins . Toxicon 20, 929 . BULBRING, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat . Br. J. Pharmac. Chemother. 1, 38 . CHou, H . N. and SHrntizu, Y . (1982) A new polyether toxin from Gymnodinium breve Davis . Tetrahedron Lett . 23, 5521 . FOLDES, F. F., CHAuNDRY, I ., OHTA, Y ., AmAKi, Y ., NAGASHimA, H . and DuNcAu, D . (1981) The influence of stimulation parameters on potency and reversibility of neuromuscular blocking agents . J. Neural 7Ynnsm . 52,227 . GALLAGHER, J . P . and SHINNICK-GALLAGHER, P . (1980) Effect of Gymnodinium breve toxin in the rat phrenic nerve diaphragm preparation . Br. J. Pharmac. 69, 367.

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MILLONIG, G. (1961) Advantages of a phosphate buffer for OsO, solutions in fixation . J. appl. Phys. 32, 1637 . Music, S . I ., HOWELL, J . T . and BRUMBACK, C . L . (1973) Red tide: its public health implications . J. Fla Med.

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