The site of action of Ry&o&cus ~FW& toxin within the parasympathetic axonal sodium channel h gate in airway smooth muscle

Sadahiro Asai, M.D.,* Joseph J. Krzanowski, Ph.D., Richard F. Lackey, M.D., Wayne H. Anderson, Ph.D., Dean F. Martin, Ph.D., James B. Polson, Ph.D., Samuel C. Bukantz, M.D., and Andor Szentivanyi, M.D. 7’mp, Fla.

The red tide toxin produced by Ptychodiscus brevis (PBTX) may cause cough, sneezing, and asthma. Previous in vitro studies with isolated canine tracheal smooth muscle demonstrated that PBTX stimulates sodium channels of parasympathetic nerve endings and thus causes a contractile response. The present study investigated the mechanism of the PBTX effect on canine tracheal smooth muscle. Repeated exposure of the muscle strip to PBTX (final concentration 46 ~glml} followed by washout of the toxin resulted in reestablishment of baseline tension but a

failure of contraction on further addition of PBTX. However, veratridine and scorpion toxin (ET), which are voltage-sensitive sodium channel activators, still induced contraction. Furthermore, the contraction caused by veratridine was enhanced by a high dose of PBTX, whereas contraction caused by SCT was not. Responses to veratridine and SCT as well as PBTX (previously reported’) were blocked by tetrodotoxin (a sodium channel blocker), while acetyleholine responsiveness remained intact. These results indicate that PBTX receptors in parasympathetic nerves influence Naf jkx at the h gate, that these receptors differ from the veratridine and SCT receptors, and that the conformational change in the receptors induced by PBTX afiects the tissue response to veratridine. (J ALLERGY CLIN IMMVNOL 73:824-S, 1984.)

Pzychodiscus brevis, previously known as Gym- nodinium breve, is a unicellular sea alga classified as a special dinoflagellate. 2, 3 Its toxin causes massive

From the Department of Internal Medicine, Division of Allergy and Immunology, and the Department of Pharmacology and Thera- peutics, College of Medicine, and CHEMS Center, Department of Chemistry, College of Natural Sciences, University of South Florida; and Department of Internal Medicine, Section of Allergy and Immunology, James A. Haley Veterans Administration Hospital, Tampa, Fla.

Supported in part by National Institutes of Health grant I-U 23658. Presented in part at the American Academy of Allergy and Immu-

nology Meeting, March 1982, and XI International Congress of Allergology and Clinical Immunology, October 1982, London, England.

Received for publication Aug. 10, 1983. Accepted for publication Dec. 20, 1983. Reprint requests: Sadahiro Asai, M.D., Department of Internal

Medicine, Division of Allergy and Immunology, University of South Florida College of Medicine, 12901 N. 30th St., Tampa, FL 33612.

*Dr. Asai is a faculty member of the Second Department of Internal Medicine, Nagasaki University School of Medicine, S-to- machi, Nagasaki, Japan.

Abbreviations used PBTX: Ptychodiscus brevis toxin ACH: Acetylcholine XT: Scorpion toxin

fish fatality, neurotoxic shellfish poisoning, and con- tact irritation of the human skin.4-‘2 The aerosol in- duces in man such symptoms as nonproductive cough, shortness of breath, tearing of the eyes, a burning sensation of the conjunctiva, rhinorrhea, and sneezing and may cause wheezing in persons predis- posed to asthma.8, ‘O-E An earlier study revealed that PBTX causes canine tracheal smooth muscle contrac- tion by stimulating the axonal sodium channels result- ing in release of ACH at postganglionic parasym- pathetic efferent nerve endings, an effect which is eliminated by treatment with tetrodotoxin.’ Dose- response data indicate that tracheal smooth muscle strips contract at a threshold concentration of 0.3 pg/ml PBTX. Consecutive additions of PBTX to

824

VOLUME 73 NUMBER 6

Site of action of Ptychodiscus brevis toxin 825

H 2 mln

PBiX 20

ffi. 1. Effect of repeated exposure of ACH-responsive (IO” mollL) canine tracheal smooth mu&e to aerial ad- d bath concentrat ion of 46 II tion of the toxin is ineffec- ti the toxin. Data presented are from a reptww&ftive experiment. Control response to PBTX (6 ~glrnl) is 120 5 36 gmlgm of t issue (X rt SE), wherws the response after high doses of PBTX is absent as Marmined in t&sues from seven dogs.

canine tracheal smooth muscle result in a maximal canrsaCtile nspollse at a final concentration cif 1.5 to 3.6 pghni PI5TX.l This investigation was designed to d&he the mechanism by which PBTX affects the parasympathetic axond sodium channel.

P. brevis was cultured in enriched seawater media (B-5) described previo~sly.‘~ The toxic fraction was isolated by acidifying cultures, extracting twice with redistilled chloro- form under a nitrogen atmosphere, and evaporating the dried, combined extracts under reduced pressure.‘, I’ This isohttikm proeednre was used throughout the study and the combined extracted fraction is referred to as PBTX.

The methods used in this laboratory have been described previously. I5 Mixed-breed dogs weighing 15 to 22 kg were anesthetized (pentobarbital, 30 mg/kg), and the trachea was excised to the bifurcation and placed in aerated (95% O2 to 5% CO,) Krebs-Ringer solution of the following composi- tion (mM): NaCf, 117.0; KCI, 4.0; NaHCO,, 25.0; MgS04, 2.4; NaH,PO,, 1.2; Cat&, 2.5; and dextrose, I 1 .O. A smooth muscle 5trip dissected from the cartilage was mounted in the isolated bath (10 ml of Krebs-Ringer solu- tion, pH 7.4,38’ C, oxygenated with 95% Oz to 5% CO2).‘6 Contra&e tension was measured isometrically with a Grass FTO3 transducer coupled to a Grass Model 7 (Grass Instru- ment Co., Quincy, Mass.) polygraph. The tissue was ini- tiatfy loaded with 2 gm of tension and allowed to equilibrate for 3Q min before beginning an experiment. By use of these conditfons, resting tension is less than 10% of the maximum active tension generated to ACH (0.85 kg/cm2). This is consistent with the length at which maximum active tension develops (Lt,) for canine tracheal smooth muscle as reported by Stepbens16 and as retested in the current protocol.

b) I

ACH c&ride, veratr@ine, ark& fE?T were, Sigma Chemical Co., St. Lds, I+&. tamed from CaIMoehem-Behriug Corp., San Diego, F&if.

828 Asai et al. J. ALLERGY CLIN. IMMUNOL. JUNE 1984

$

o--. + i

(extracallular) /TTX“‘;/

-$yq$

400

; fj 300

e P

200

100

0 Vwat (10 pg/ml) W& (10 rg/ml) SCT (3 pg/ml) ,:X:,(3 pg/ml)

PSTX PBTX

FIG. 4. Analysis of effect of high doses of PBTX on con- tractile response of veratridine and scorpion toxin. These data are a graphic presentation of data obtained after the same protocol illustrated in Figs. 2 and 3. Refer to these figures for PBTX concentrations.

utor 00 MTX-vorat p

400 -

\/

d / *A

\ 200 -

‘J f’ Control

0 @ @ 8 I n=7

lJ 0 1 1 1 1 ,o-9 -a -7 -9 -5 -4

Y Ach

ro-9 -9 -7 -6 -5 -4 Y

Ach

FIG. 5. ACH responsiveness of canine tracheal smooth muscle after exposure to PBTX and veratridine or SCT.

Measurement of contractile response After equilibration, muscle strips were washed, and a

dose-response curve for ACH was obtained at the beginning of each study to establish physiologic responsiveness of the tissues. PBTX was added from a stock solution containing 3 mg/ml. All contractile events were calculated in terms of grams of tension generated per gram of tissue.

Statistical methods The Wilcoxon ranked sum test was used.”

RESULTS Contractions of canine tracheal smooth muscle

strips occurred after initial exposure to 6 wg/ml of PBTX and a subsequent addition of 20 pg/ml, but a second addition of 20 pg/ml failed to elicit a contrac- tion (Fig. 1). The tissue remained unresponsive after washout of the PBTX and reexpoaure to 6 &HEflcIf PBTX (Fig. 1). The concentration of .6 &g/ml of PBTX was selected to insure a maximrim response and is a concentration blocked by tetrodotoxin.’

I Na+ I

(Introeotlular) Na+ \

h gate

FIG. 8. Hypothetical model of the site of action of PBTX on the sodium channel.

Studies were conducted with veratridine and SCT to identify the site of action of PBTX on the sodium channel (Figs. 2 and 3). Veratridine, a known sodium channel h gate stimulator, caused contraction of canine tracheal smooth muscle after the tissue was no longer reactive to PBTX (Fig. 2, panel b). Similar results were obtained with SCT, another established channel h gate stimulator. After cumulative adminis- tration to a high concentration (46 pg/ml) of PBTX, SCT still induced canine tracheal smooth muscle contraction, whereas PBTX did not (Fig. 3, panel b).

The contraction induced by veratridine was en- hanced (p < 0.05) by previous exposure of the tissue to PBTX (46 fig/ml), whereas the response obtained with SCT was not (p > 0.05) (Fig. 4). The contractile dose response of the smooth muscle after ACH re- mained intact after all experimental sequences (Fig. 5). The effect of veratridine or SCT was completely reversed by tetrodotoxin (1 OT7M), indicating that both substances induce contraction via their influence on the sodium channel.

DISCUSSKJN The association of respiratory irritation with expo-

sure to Florida red tide toxin was reported in 19171s and was subsequently confirmed by other inves- tigators . *, lo-l2 We previously reported that PBTX causes smooth muscle contraction by stimulating axonal sodium channels. 1 By use of the voltage clamp method, three components of the sodium channel have been identified: voltage-dependent activation (m gate), voltagedependent,inactivation (h gate), and se- lective ion transport (selectivity filter). lD, 2o Tetrodo- toxin blocks the selectivity filter.21

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Site of action of Ptychodiscus brevis toxin 827

Veratridine and SCT each impair the h-gate inacti- vation of the sodium channel and keep it in an open position.2z These toxins influence the h gate of the sodium channel through a cooperative interactionz2 that indicates that they affect separate receptor sites in the h gate.

The present study reveals that high concentrations of PBTX (up to 46 pg/ml) result in an initial contrac- tion of canine tracheal smooth muscle followed by a return to baseline tension. Further administration of PBTX is ineffective even after washout of the high concentration of PBTX. However, both veratridine and SCT induce contractions of canine tracheal smooth muscle after administration of high concen- trations of PBTX. The exposure to high concentra- tions of PBTX enhances the veratridine-induced con- traction but not that induced by SCT. Tetrodotoxin, which blocks the selectivity filter of the sodium chan- nel, eliminates the contraction caused by veratridine and SCT even in the presence of high concentrations of PBTX. Thus these smooth muscle contractions are apparently caused by an influx of sodium ions via the sodium channels. The ineffectiveness of PBTX after exposing the tissue to high concentration of this toxin may indicate that in this situation all PBTX-receptive sites are saturated. However, veratridine and scorpion toxin are still able to induce contraction of the PBTX- saturated muscle.

These observations indicate that the site of PBTX action is in the sodium channel but differs from the veratridine and scorpion toxin sites. There is appar- ently cooperative interaction between PBTX and ver- atridine sites suggesting that the PBTX site is near the veratridine-receptor site. Catterall and Risk’” tested P. hrevis toxin on neuroblastoma cells, and they sug- gest that the toxin may stimulate new receptor sites. Our data support their hypothesis and suggest that the receptor sites of the neuroblastoma cells and of the parasympathetic nerve endings on canine tracheal smooth muscle cells possess sodium channels with PBTX-sensitive sites at the h gate area (Fig. 6).

W e thank Saber S. Samaan, Ph.D., for his useful sug- gestions, Mrs. Lolita Binford and Ms. Judith Smith for their excellent technical assistance, and Mrs. G inger Montuoro for her secretarial help.

REFkWEfUCES

1. Asai S, Krzanowski JJ, Anderson WH, Martin DF, Polsoo JB, Hockey RF, Bukantz SC, Szentivanyi A: Effects of the toxin of red tide, Pryckodiscus brevis, on canine tracheal smooth mus- cle: A possible new asthma-triggering mechanism. J Allergy Clin Immunol 69:418, 1982

2. Steidinger KA, Truby EW, Dawes CJ: Ultrastructure of the red

tide dinoflagellate G~mnadinium hrt*t,~. 1. General descrip- tion J Physiol 1472, 1978

3. Steidinger KA: Collection, enumerat ion, and identification of free living marine dinotlagellates. In Taylor DL. Seliger HH, editors: Toxin dinoflagellate blooms. New York, 1979, ElsevieriNorth Holland, Inc.. p 435

4. Iizuka S: On occurence of similar organisms to G?mnrM’inium breve Davis in Omura Bay. Bulletin of Plankton Society of Japan 21:45, 1975

5. l izuka S: Occurences of a toxic dinogagellate, G~mrzorl inium breve, in the coastal waters of Japan. In Okaichi T, editor: Studies on toxic phytoplankton. Project 236019 Monbusho Kagaku Kenkyuhi Hojokin Sogokenkyo (At, 1977, p 14

6. Quick JA Jr: Evidence of new ichthyointoxicative phenomena in Gymnodimum breve red tides. In LoCicero YR. editor: Pro- ceedings of the 1st International Conference on toxic di- noflagellate blooms. Nov. 1974. Wakefield, Mass., 1975. Massachusetts Science and Technology Foundation, p 414

7. Quick JA Jr, Henderson GE: Effects of G~nrtrodiniurrr breve red tide on fishes and birds: A preliminary report on behavior, anatomy, hematology, and histopathology. 111 Miller R, editor: Proceedings of the gulf coast regional symposium on diseases of aquatic animals. Sea Grant, 1974, Louisiana State Univer.. sity, p 85

8. Martin DF. h4artin BB: Red tide, red terror. Effects of red tide and related toxins. J Chem Ed 53:414, 1976

9. McFarren EF, Tanabe H, Silva FJ, Wilson WR, Campbell JE, Lewis KH: The occurrence of a ciguatera-like poison in oys- ters, clams, and Gwnnodiniurn brc~~~ culturc%. Toxicon 3:lll. 1965

10. Hemmert W I-I: The public health implications ofC;ymnadiniunr breve red tides, a review of the literature and recent events. In LoCicero VR, editor: Proceedings of the 1st International Con- ference on toxic dinoflapllate blooms. Wakefield, Mass.. 1975, Massachusetts Science and Technology Foundation, p 489

11. Woodcock AH: Note concerning human respiratory irritation associated with high concentrations of plankton and mass mor- tality of marine organisms. J Marine Res 7:56, 1948

12. Music SI, Howell JT, Brumback CL: Red tide, its public health implifcations. J Florida Med Assoc 60:27, 1973

13 Brydon GA, Martin DF, Olander WK: Laboratory culturing of the Florida red tide organism, G~mmxfinium hrrve. Environ Iat 1:235, 1971

14. Doig M T III, Martin DF: Physical and chemical stability of ichthyotoxins produced by G~mntxiinitrm breve. Environ Lett 3~279, 1972

15. Anderson WH, Krzanowski JJ, Poison JR, Szentivanyi A: Characteristics of h&mine tachyphylaxis in canine tracheal smooth muscle: A possibre ~r~~~ n~$&& mocha- nism Naunyn-Schmiedebargs Arch Islarmtaeo1308:7 17, 1979

16. Stephens NIL: The mechanies of isolated airway sn%ootb mus- cle. In Bouhuys A, editor: Airway dynantics: Physiology and pharmacology. Springfield, Iii., 1970, Charles C Thomas, Publisher, p 191

17. Snedecor GW: Statistical methods. ed 5. Ames, Iowa, 1956, Iowa State University Press

18. Taylor HF: Mortality of fishes on the west coast of Florida. Report of the US Commissioner of Fisheries, Document 848, 1917, p 5

19. Catterall WA: Neurotoxins that act on volta8esensitive so- dium charnels in excitable membranes. Annu Rev Pharmacol Toxic01 20~15. 1980

828 Asai et al. J. ALLERGY CLIN. IMMUNOL. JUNE 1984

20. Hille B: Local anaesthetics: Hydrophilic and hydrophobic Na+ ionophore by neurotoxins. Proc Nat1 Acad Sci USA pathways for drug-receptor reaction. J Gen Physiol 69:497, 72~1782, 1975 1977 23. Catterall WA, Risk M: Toxin T4s from Ptychodiscus brevis

21. Narahashi T: Mechanism of action of tetrodotoxin and saxi- (formerly Gymnodinium breve) enhances activation of voltage- toxin on excitable membranes. Fed Proc 3131124, 1972 sensitive sodium channels by veratridine. Mol Pharmacol 19:

22. Catterall WA: Cooperative activation of the action potential 345, 1981

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