Supersensitivity induced by the toxin of Brazilian scorpion venom (Tityus serrulatus Lutz e Mello)

Gen. Pharmac., 1976, Vol. 7, pp. 111 to 114. Pergamon Press. Printed in Great Britain

SUPERSENSITIVITY I N D U C E D BY THE TOXIN OF BRAZILIAN SCORPION VENOM (TITYUS SERRULATUS LUTZ e MELLO)* A. ANTONIO 1, I. MELITO 2, A. P. ALMEIDA3 and A. P. CORRADO 1 1 Faculty of Medicine of Ribeir~o Preto, S.P., Brazil 2 Faculty of Veterinary Medicine and Agronomy of Jaboticabal, S.P., Brazil 3 Institute of Biological Sciences of Belo Horizonte, M.G., Brazil (Received 18 December 1975)

Abstract--The purified toxin of the Brazilian scorpion venom (TsTX) enhances the pharmacological actions of directly and indirectly acting substances on several preparations. The enhancing action of TsTX is more pronounced for the indirectly acting drugs, which is highly suggestive of a prejunctional site of action. Secondarily, TsTX does also produce an unspecific increase of sensitivity at the level of the pharmacological receptor itself. The mechanism by which these changes are brought about remains to be determined.

INTRODUCTION THE VENOM of the Brazilian scorpion, Tityus serrulatus, elicits sympathomimetic (Corrado, Antonio & Diniz, 1968; Cunha Melo, Freire-Maia, Tafuri & Maria, 1973; Corrado, Riccioppo Neto & Antonio, 1974; Corrado, Diniz & Antonio, 1975) and parasympathomimetic (Diniz & Gonqalves, 1956; Diniz & Valeri, 1959; Corrado et al., 1968) effects, which were attributed to the release of endogenous norepinephrine and acetylcholine respectively, through an action at the post-ganglionic nerve endings (Corrado et al., 1968). Scorpion venom also stimulates the neuromuscular junction of the frog (Benoit & Mambrini, 1967; Katz & Edwards, 1972) and of the rat (Vital Brazil, Neder & Corrado, 1973). A net release of acetylcholine has been demonstrated in the guinea-pig ileum (Diniz & Torres, 1968), the rat brain (Gomez, Dai & Diniz, 1973) and the neuromuscular junction (Vital Brazil et al., 1973), while the release of 3H-norepinephrine has been shown in synaptosomes of rat brain (Moss, Colburn & Kopin, 1974a) and other tissues (Moss, Thoa & Kopin, 1974b; Langer, Adler-Graschinsky, Almeida & Diniz, 1975). Dopamine-fl-hydroxylase is also released together with norepinephrine (Moss et al., 1974b) and the deaminated glycol, 3,4-dihydroxyphenyl-glycol, represented the main norepinephrine metabolite following exposure to scorpion toxin (Langer et al., 1975). Moreover, the transmitter release is long-lasting, dependent on the presence of calcium in the external medium and completely blocked by tetrodotoxin, a pattern which resembles the release induced by electrical stimulation. Accordingly, the

venom has the ability to depolarize the nerve membrane and to elicit a prolongation of the plateau phase of the action potential (Narahashi, Shapiro, Deguchi, Scuka & Wang, 1972). It has also been shown that the scorpion toxin increases the synthesis of acetylcholine (Gomez et al., 1973) and norepinephrine (Moss et al., 1974b), thus reinforcing the similarity between the action of scorpion toxin and the nerve impulse. ENHANCING EFFECT OF THE TOXIN

Preliminary experiments (Corrado et of., 1974) have shown that scorpion toxin (TsTX) enhances the hypertensive response due to carotid occlusion as well as to injected norepinephrine. Recently, Langer et aL (1975) demonstrated an enhancing effect of the scorpion toxin on the transmitter overflow elicited by accelerans nerve stimulation. They showed that scorpion toxin increased about 8-fold the 3H-norepinephrine release elicited by accelerans nerve stimulation on the isolated guinea-pig atria (Table 1). The same results were reproduced in the medial muscle of the cat nictitating membrane. As shown in Fig. 1, the tension developed by the isolated medial muscle of the cat nictitating membrane in response to nerve stimulation is increased in the presence of TsTX; on the other hand, the responses to exogenous norepinephrine were practically unchanged by the presence of TsTX. The experiments of Langer et al. (1975) with the cat nictitating membrane practically exclude an action at the post-synaptic site to explain the enhancing action of TsTX. In order to test further this possibility, we compared the inotropic effects of directly and indirectly* Supported in part by Fundaq~o de Amparo/l Pesquisa acting substances in the absence and in the presence do Estado de S~o Paulo (FAPESP). 111.

112

A.

I.

ANTONIO,

MEL1TO,

A. P. ALMEIDAAND A. P. CORRADO

Table 1. Effects of TsTX on 3H-noradrenaline release elicited by nerve stimulation in isolated guinea-pig atria Experimental

group

Fraction released per shock (x i0-5) a

N

SI= l.ll Control

6

TsTX 1.0~g/ml b

4

TsTX 0.3pg/ml

4

Ratio S2/S 1

-+ 0.18

1.03 -+ 0.11

$2= 1.12 -+ 0.22

7.70 -+ 1.22

SI= 1.06 ± 0.16 $2= 7.94 ± 1.52

6.35

SI= 0.85 +- 0.23 TsTX

± 0.94

$2= 5.83 -+ 1.86

1.0#g/ml c

a Fraction

released

per shock:

by the total nCi r e m a i n i n g

total nCi released

per shock divided

in the tissue at the onset of nerve

stimulation. b 30 min before TsTX.

This was

c 40 min before TsTX followed

$2,

the atria were exposed

followed $2,

for i0 min to 1.0~g/ml

by a 20 min w a s h i n g

the atria were exposed

by a 20 min w a s h i n g

Shown are mean values

+ S.E.M.

of TsTX. Figure 2 shows the tension developed by the isolated guinea-pig atria treated with atropine in response to various drugs, before and after the exposure to TsTX. While the effects of histamine and norepinephrine which are directly-acting inotropic sub-

period

for Io min to 0.3~g/ml

period

of

and then by S 2 . of

and then by S 2.

(From Langer

et al.,

1975).

stances were not affected by TsTX, the effects of the norepinephrine-releasing substances, nicotine, D M P P and acetylcholine were greatly enhanced by TsTX. The same results were reproduced in the electrically-

2OO A 180 o~

160

~

140

±

120

p--

[]

Control

[]

After 12jug/ml TsTx

100 ~,

20

-B

n.,-

o

a:

80

--~

60-

--~ I---

40-

o EL

20-

15 10

0

5

n

•

NA

n i

NA i

•

NA •

NA

TsTX

Fig. 1. Effects of TsTX on responses to nerve stimulation and to noradrenaline (NA, 5.9 #M) of the isolated medial muscle of the cat nictitating membrane. Ordinates: tension developed, in g. (A) Controls. (B) Before, during and after exposure to TsTX, 1.0pg/ml (indicated by the horizontal bar). Shown are values obtained from a typical experiment (According to Langer et aL, 1975).

03 40.3003 "1030 50 LO'50LO ]OtO030100 2.0 NOREPINEPHRIN£ H#STAMINE ACETYLCHOLIN£NIC

8.0 D~PP

Fig. 2. Effects of the scorpion venom tityustoxin (TsTX) on responses to norepinephrine, histamine, acetylcholine, nicotine (Nic) and dimethylphenylpiperazine (DMPP) of the isolated guinea-pig atria. Ordinate: tension developed as the percent of the control. Shown are mean values of 6 experiments. Vertical bars represent standard errors. Doses of the agonists are in ,ug/ml. The influence of the toxin was studied as follows: the atria were exposed for 3min to TsTX; this was followed by a 20rain washing period after which the agonist to be tested was added.

Toxin of Brazilian scorpion venom

Table 2. The influence of scorpion toxin (TsTX) on the sensitivity of the guinea-pig vas deferens to noradrenaline

A

o ssoI--

[] z

300

113

[]

Control After 1.2~g/ml TsTx

Experimental

TsTX

situation

N

Control

-

13

16.70

Control

+

6

4.35

(2.5 -

4

3,70

(2.3 -

7.2)

4

2,62

(1.6 -

4.3)

5

I1.60

(3.8 - 30.6}

5

3,83

(0.6 - 27.4)

Denervated b

rr

zoo150-

Denervated Reserpinlzed c

+

Reserpinlzed

+

a ED50

is the c o n c e n t r a t i o n

50% of the m a x i m u m

,

.03 .IO-30.03.10.30

were c a l c u l a t e d

I

50-

NOREPINEPHRINE

3010050100

and Jellett b Denervation

2.0

ACETYLCHOLINE N)COTIN£

driven right-ventricular strip of the guinea-pig heart (Fig. 3). On the other hand, in the guinea-pig vas deferens, the venom increases the effects of norepinephrine; the dose-response curve is shifted to the left. The enhancing effect of TsTX upon the isolated vas deferens seems to be unspecific since the acetylcholine-induced contraction (Fig. 4) as well as that of bradykinin is also potentiated. An increase in the maximal response of the vas deferens is observed in the presence of scor-

(6)

ew

120

y

I00

~

.

/

..~ . . . . . (III

'5 x

l/k"

.//

/[

40

20 /j(' , /[ -6

.//,

-5

,

,

-4

-3

7.6)

a contraction which iB

according

95% confidence

to Pleminq,

~stfall,

intervals de la Lande

(1972).

was performed by the method of B i r m i n g h a m

(1970)

7 days before the experiment.

Fig. 3. Effects of TsTX on responses to norepinephrine, acetylcholine and nicotine of the electrically driven rightventricular strip of the guinea-pig heart. Explanations as in Fig. 2. Shown are mean values of 6 experiments.

Z O o..

producing

(9.7 - 30.9)

for the drug in an individual experiraent;

mean ED50 with the c o r r e s p o n d i n g

loo-

~

C.I.}

x 10 -6 M

.

250.

G)

Mean E D 5 0 a ( 9 5 %

0.1~g/ml

log ACETYLCHOLINE CONCENTRATION (M)

Fig. 4. Mean dose-response curves to acetylcholine in control (@. . . . @) and TsTX-treated (0----0) guinea-pig vas deferens. The final concentration of TsTX in the organ bath was 0.1 #g/ml. Vertical bars represent standard errors. In brackets, the number of experiments.

c Reserpine was daily given,

1.0mg/kg,

during

a 5-days p e r i o d

preceding the experiment.

pion toxin. Table 2 shows the influence of TsTX on the ZDso of norepinephrine upon the guinea-pig vas deferens under different conditions. It can be seen that the ratio between the EDso with and without TsTX is greater in normal than in denervated or reserpinetreated preparations. These experiments indicate two components in the enhancing action of TsTX; a presynaptic or pre-junctional one, which is more important than the second, post-synaptic, at the level of the pharmacological receptors.

CONCLUDING REMARKS Scorpion toxin elicits sympathomimetic and cholinomimetic effects which are indirect and due to the release of the neurotransmitter through an action at the post-ganglionic nerve terminals. In addition, TsTX enhances the transmitter overflow and the pharmacological actions elicited by nerve stimulation and by drugs. Both the releasing and enhancing actions of TsTX are long-lasting and still observed 20~0 min after the toxin has been removed from the organ bath, Although the major part of the supersensitivity induced by TsTX is due to a pre-synaptic action, our experiments with the arterial blood pressure and the guinea-pig vas deferens are suggestive of a postsynaptic action as well. The facilitatory action of TsTX occurs, therefore, at both, the nerve endings and the post-synaptic receptor sites. The prejunctional effect which ultimately leads to an increased transmitter overflow, thus explaining the enhancing action of nerve stimulation and indirectly acting substances is probably due to a subthreshold depolarization of the nerve membranes. On the other hand, whatever the mechanism of the facilitatory action at the receptor level might be, it is probably different from the prejunctional one, since TsTX does not seem

114

A. ANTONIO,I. MEL1TO,A. P. ALMEIDA AND A. P. CORRADO

to p r o d u c e any direct p h a r m a c o l o g i c a l action, i.e. the v e n o m does n o t act at the p h a r m a c o l o g i c a l r e c e p t o r by itself. Acknowledgements--We are grateful to Prof. C. R. Diniz for the generous gift of TsTX. REFERENCES BENOIT P. R. & MAMBRINI J. (1967) Action of scorpion venom on the neuromuscular junction of frogs. J. Physiol., Paris 59. 348. BIRMINGHAM A. T. (1970) Sympathetic denervation of the smooth muscle of the vas deferens. J. Physiol., I~nd. 206, 645-661. CORRADO A. P., ANTONIO A. & DINIZ C. R. (1968) Brazilian scorpion venom (Tityus serrulatus), an unusual sympathetic post-ganglionic stimulant. J. Pharmac. exp. Ther. 164, 253 258. CORRADO A. P., R1CCIOPPO NETO F. t~, ANTONIO A. (1974) The mechanism of the hypertensive effect of Brazilian scorpion venom (Tityus serrulatus Lutz e Mello). Toxicon 12, 145-150. CORRADO A. P., DINIZ C. R. tf¢.:ANTONIO A. (1975) Neurotransmitter release by the toxin of Brazilian scorpion venom (Tityus serrulatus Lutz e Mello). In Concepts of Membranes in Regulation and Excitation. (Edited by ROCHA E SILVA M. & SUAREZ-KURTZ G.). pp. 193-199. Raven Press, New York. CUNHA MELO J. R., FREIRE-MAIA L., TAFURI W. L. & MARIA T. A. (1973) Mechanism of action of purified scorpion toxin on the isolated rat intestine. Toxicon 11, 81 84. DINIZ C. R. & GON(~ALVES J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In Venoms. (Edited by BUCKLEY E. E. & PORGES N.). pp. 131-141. AAAS, Washington, D.C.

DINIZ C. R. & TORRES J. M. (1964) Release of an acetylcholine-like substance from guinea-pig ileum by scorpion venom. Toxicon 5, 277 281. DINIZ C. R. & VALERI V. (1959) Effects of a toxin present in a purified extract of telsons from the scorpion Tityus serrulatus on smooth muscle preparations and in mice. Archs Int. Pharmacodyn. 71, 1 13. FLEMING W. W., WESTFALL D. P., DE LA LANDE I. S. & JELLET L. B. (1972) Log-normal distribution of quieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmac. exp. Ther. 181. 339-345. GOMEZ, N. M., DA1 M. E. M. & DINIZ C. R. (1973) Effect of scorpion venom, Tityustoxin, on the release of acetylcholine from incubated slices of rat brain. J. Neurochem. 20, 1051-1061. KATZ N. L. & EDWARDS C. (1972) The effect of scorpion venom on the neuromuscular junction of the frog. Toxicon 10, 133. LANGER S. Z., ADLER-GRASCHINSKY E., ALMEIDA A. P. & DINIZ C. R. (1975) Prejunctional effects of a purified toxin from the scorpion Tityus serrulatus. Release of aH-noradrenaline and enhancement of transmitter overflow elicited by nerve stimulation. Naunyn-Schmiedeberg's Arch. Pharmak. 287, 243-259. Moss J., COLBURN R. W. & KOPIN I. J. (1974a) Scorpion toxin induced catecholamine release from synaptosomes. J. Neurochem. 22, 217-221. Moss J., THOA N. B. & KOPIN I. J. (1974b) On the mechanism of scorpion toxin-induced release of norepinephrine from peripheral adrenergic neurons. J. Pharmac. exp. Ther. 190, 39-48. NARAHASHI T., SHAPIRO B. I., DEGUCHI T., SCUKA M. & WANG C. M. (1972) Effects of scorpion venom on squid axon membranes. Am. J. Physiol. 222, 850-857. VITAL BRAZIL O., NEDER A. C. & CORRADO A P. (1973) Effects and mechanism of action of Tityus serrulatus venom on skeletal muscle. Pharmac. Res. Commun. 5, 137 150.