NEUROMUSCULAR NEUROTOXINS

BLOCKING

ISOLATED NAJA

NAJA ---A

Yasushi

FROM AND

LATICAUDA NAJA

COMPARATIVE

KURAISHI,

ACTIONS

Yoshimi

NAJA

OF

SEMIFASCIATA, ATRA

ASSAY--

MISU,

Hiroshi

TAKAGI

and Kyozo HAYASHI* Department of Pharmacologv and'-- Department of Biological Chemistry, faculty of Phurnt(wen tica/ Scieucev, Kveto University, Kvoto 606, Japan Accepted December '_3, 1976

Extensive

pharmacological

and toxicological

that the venoms of most snakes belonging

studies

on snake venoms

have revealed

to the family of Elapidac and Hvdrophiidae

produce

flaccid paralysis and respiratory neurotoxic from

polypeptides,

failure in animals (1). We compared

including

3 types of snake venoms

nerve-diaphragm

preparations

Male Wistar

preparations

maintained

at 37'C

To record

post-tetanic

(atratoxin

utilizing

I), isolated

isolated

150 to 250 g, were used and the isolated

as described

and bubbled

lated with supramaximal

component

transmission,

phrenic

of rats.

rats, weighing

diaphragm

a newly fractionated

on neuromuscular

the effects of 10 different

by Bulbring

(2) were suspended

with 95 % 02 and 5 % CO2.

rectangular potentiation

tetanic

nerve

in 50 nil Krebs solution

The phrenic nerve was stimu

pulses of 0.3 msec duration (PTP),

phrenic

at a frequency

stimulation

of 0.1 Hz.

of 30 Hz for 10 sec was

performed as previously described (3, 4). To determine the effects of neurotoxins on acetylcholine (ACh) sensitivity at the motor end-plate, the retrograde injection of ACh (5.0 eg) into the thoracic as described jection

inferior

vena cava was employed.

by Takagi et al. (4) and Paterson

intervals

semi-isotonic

were 5 min.

lever.

Toxin

cobrotoxin,

atratoxin

procedures

were as described

semifasciata

Twitch

responses

Experimental

Injection

volume

were recorded

B, C, D and I were isolated

procedures

on a smoked

from

elsewhere

7,000 for type II neurotoxin VI) with 60 to 62 amino D) with 71 to 74 residues.

(erabutoxin

acid residues,

b was isolated

Molecular

b, toxin 1, cobrotoxin,

I has almost is low (8).

FIG. I. Effect of erabutoxin b on twitch stimulation (A) and effect of atratoxin toxins or physostigmine washed with fresh Krebs

Erabutoxin

atratoxin

the same amino

were expressed

from

Laticauda as

1, CM I1, V and (toxin B, C and

acid sequence

as that

in terms of final

The preparation was At T.S., tetanic stimuli in terms

and

The isolation

responses of diaphragm to phrenic nerve I on post-tetanic potentiation (B). Neuro

All doses

with a

weight was estimated

All doses were expressed

were added at upward arrows. solution at downward arrows.

(30 Hz for 10 see) were applied. centrations in the organ bath.

paper

Naja naja (Elapidae),

and as 8,000 for type 11 neurotoxin

Atratoxin

but the lethal toxicity

(6).

and Arai (7).

were

was 0.05 nil and in

1, CM 11, V and VI were from Naja naja atra (Elapidae).

(Hychrophiiclae) by Tamiya

of cobrotoxin,

(5).

of final con

concentrations

in the organ bath.

As shown caused

in Fig.

1-A, erabutoxin

50'/)o inhibition

responses

40 min after

after 60 min.

twitch responses

twitch responses

inhibition

reappeared

to the initial level following an administration Atratoxin were partially these responses

inhibited

The transmission by washing reversed

inhibited

within

(Fig. 1-A).

these

M, the reduction

50 min.

respec

The abolished

and gradually

The recovery

of

recovered

was facilitated

by

g/ml).

than erabutoxin

b, twitch

by atratoxin

1 (5

responses

of atratoxin

10-1 M).

to nerve stimulation I (2 >; l0-1 M), while

As Summarized

b in both the potency

in Table

1,

and time course

of

action.

failure

the preparation.

by repeated

washings (10

were similar to erabutoxin

blocking

10

responses,

about 20 and 10 min after an administration,

50 min after an administration

were unaffected

twitch

completely

and 5

30 min after the first washing,

repeated

I was less potent

reduced

and

of 2.:

in both cases occurred

about

of physostigmine

8 other neurotoxins neuromuscular

an administration,

In higher concentrations

to 50% was observed

tively, and a complete

b (10-7 M) gradually

washing

induced

by both atratoxin

On the contrary, until at least

I and CM V was readily reversed

the blockade

120 min after

by toxin B and I could not be the first washing,

while physos

TABLE:I . Comparison among the effects of neurotoxins on the rat phrenic nerve diaphragm preparations. Neuromuscular block: indicates complete block within 50 min after the addition of toxins 2 to 5 v 10 M : , partial block. Reversibility: indicates rapid recovery by single washing: , recovery by repeated washing or the addition of physostigmine: , no recovery. In hibition of PTP: indicates specific inhibition of PTP with the pre-tetanic twitches unaffected: -, no inhibition of PTP without depression of pre-tetanic twitches. Inhibition of response to acetylcholine: indicates inhibition of contractile response to injected acetylcholine

tigmine did reverse the blockade were similar to erabutoxin between the abolition first washing.

and the reappearance

produced

of twitch responses

by type I neurotoxin

but that by type lI neurotoxin Effects of neurotoxins

blockade.

"as

irreversible

vvere 45 to 75 min after the

abolished

twitches.

On the other hand, as summarized

Erabutoxin twitch responses

PTP in a concentration

h, cobrotoxin,

responses.

as origin of PTP has been considered

10

of 5

ACh.

Atratoxin

to its I

10-` yl vvhick did not affect pre-tetanic

in Table 1, 9 other neurotoxins

1vl vvhich inhibited

Their time courses

I (3

to

in Fig. 1-13, atratoxin

pre-tetanic

10

were faster than

10-- N1) also inhibited

did not inhibit

twitches.

toxin B, C and I in the range from 0.5 to 1

to injected

washing,

effect of a drug on PTP has been attributed

reversibly

of 0.5 to I

by Lee et al. (9)

in most preparations.

nerve terminal (3, 10. 1 1). As demonstrated

PTP in concentrations

The periods

could be reversed by repeated

on PTP were examined,

events and the depressive

effect on the presynaptic

mediated

of the transmission

These results are in fairly good parallel with those reported

in that the blockade

be presynaptic

by toxin t, but not that by toxin 13. The other 5 neurotoxins

b in the reversibility

tit inhibited

those

the twitch response

of nerve to injected

ACh. Effect of atratoxin than

I on neuromuscular

the effects of the other 9 neurotoxins;

as did d-tubocurarine, pre-tetanic

twitches

have a similar amino differences on PTP.

junction resembled atratoxin

which has been demonstrated (1 1).

Among

acid sequence

in the potency

(8).

Nevertheless

and the reversibility

investigations

tested, atratoxin

of neuromuscular

The present results suggest that atratoxin

and physiological

I produced to abolish

the 10 neurotoxins

that of d-tubocurarine

more

the same action on PTP PTP with no influence

atratoxin

on

I and cobrotoxin

I and cobrotoxin blockade,

showed

and the effect

I will he a useful tool for pharmacological

of the neuromuscular

junction,

particularly

the presynaptic

site. l cAnorvlcdgc'ntents: Faculty supported Culture,

of Science,

The authors

Tohoku

by a Scientific

Univ., Research

arc grateful Sendai Fund

to Prof. N. Tarniya,

for the gift of erabutoxin from

the

Dept. of Chemistry, b.

'I"bis study

.Ministry of Education.

Science

vvas and

.Japan.

RC:I-L_RF.NCLS I ) Li-r, C.Y.: P/,armacolovrr card tlic Futios of Man. Proc. 5th Int. ( onyr. Pluurntac•olo,_rly, San Francisco 1972. Vol. 2, p. 210, Karger, Basel 11971): 2) Ri`usrzr~G. L.: Brit. .1. Ph(7rnracol. 1, 38 (1946): 3) SiGAvyA. T., KOJISIA,M. AM) TAKA(,i, H.: Japu):. J. Plrarmaco~. 17, 450 (1967): 4) TAKAGI, H., KOJlh1A, N1., NAGArA, M. A'sD Ki,rvovu. I1.:..c-rnophurntacol. 9, 359 (1970): 5) Part.xsoN, G.: J. Phurnr. Pharmucol. 17, '81 (1965): F,) NAKar, K.. SAS.AK 1, T. AND HAYA snr, K.: I3iochem. hiophv.s. Res. Comnrun. 44, 893 (1971 ): 7) TA v1IYA,N. AyD ?u.A1,1-1.:Bioclrem. .1.99, 624 (1966): 8) On r.A, M., HAYASnr,K., Kur?nrsrrr, V., -1-.SKAGI, H. AyD S_vsAKi,7 .: ,.llh.vtract of the 48th animal 5Ic'ctirr, of Biochcmistr-Y at Iukaol.a. 47, 708 (1975) (iii Japanese); 9) Lr.r , C.Y., CHANG, C.C. AND CHIN, V. NI.: J. I hrnro.vun rnsst. A,s. 71. 344 (19 721: 10) KOISIA, M. A,,,o TAKAGI, H.: I ranop. .1. Pharmacol. 5, f bI (I969): 11) RhKrtz, WA .: 1Invc/c Rc/u.vants, Edited by K.ATZ, R.L., p. 59, North-Holland Publishing Co. (1975)

Neuromuscular blocking actions of neurotoxins isolated from Laticauda semifasciata, Naja naja and Naja naja atra--a comparative assay--.

NEUROMUSCULAR NEUROTOXINS BLOCKING ISOLATED NAJA NAJA ---A Yasushi FROM AND LATICAUDA NAJA COMPARATIVE KURAISHI, ACTIONS Yoshimi NAJA OF...
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