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)