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Cholinergic and adrenergic effects of tityustoxin
Gen. Pharmac., 1976, Vol. 7. pp. 115 to 121. Pergamon Press, Printed in Great Britain
CHOLINERGIC AND ADRENERGIC EFFECTS OF TITYUSTOXIN L. FREIRE-MAIA, J. R. CUNHA-MELO, H. A. FUTURO-NETO, A. D. AZEVEDO and J. WEINBERG Department of Physiology and Biophysics, Institute of Biological Sciences, Box 2486, Belo Horizonte, Brazil (Received 15 December 1975) Abstract--The effects of purified scorpion toxin (Tityustoxin or TsTX) were investigated on the isolated guinea-pig and rat ileum, rat spleen strip and hen rectal caecum. The contraction of the ileum was due only in part to the release of acetylcholine, whereas the contraction of the spleen strip and the relaxation of the rectal caecum were due to the release of catecholamines.
INTRODUCTION THE COma~CTION of the isolated guinea-pig or rat ileum produced by the venom of the Brazilian scorpion Tityus serrulatus or its purified toxin (Tityustoxin) was explained by the release of ac~tylcholine and probably substance P (Diniz & Gon~alves, 1956; Diniz & Valeri, 1959; Diniz & Torres, 1968; CunhaMelo et al., 1973; De Paula, 1973; Freire-Maia et al., 1975a). On the other hand, the relaxation of the atropine-treated rat duodenum seemed to be related to an adrenergic mechanism (Cunha-Melo et al., 1973; Freire-Maia et al., 1975, in press). To study further the cholinergic and adrc~ergic effects of Tityustoxin, experiments were conducted using isolated smooth muscle preparations, such as fragments of rat and guinea-pig ileum, rat spleen strips and fragments of hen rectal caecum. The spleen was used because of its totally sympathetic innervation (Fillenz, 1970; Davies & Withrington, 1973~ whereas the rectal caecum was used because of its high sensitivity to catecholamines (Euler, 1956). Preliminary reports have been published elsewhere (Cunha-Melo et al., 1975; Futuro-Neto & Freire-Maia, 1975). MATERIAL AND METHODS Smooth muscle preparations The experiments performed upon the isolated strips of rat and guinea-pig ileum were done in the usual way, in an organ bath containing aerated Tyrode solution, at 35°C. The rat spleen strips were immersed in l0 ml of a Tyrode solution, at 37°C. The fluid was aerated with a mixture of 95% 02 and 5% CO2. The isolated fragments of hen rectal caecum were immersed in 10 ml of an aerated Tyrode solution, at 38°C, according to the method described by Euler (1956). Recording of the contractions or relaxations The contractions of the isolated strips of rat and guineapig intestines were registered on a smoked drum, with an auxotonic lever. The contractions of the rat spleen strips
or the relaxations of the hen rectal caecum were registered by means of a method described by one of us (A. D. Azevedo), using a displacement photoelectric transducer. This transducer is composed of an aluminium blade with a window of proper conformation, placed between a light source and a light dependent resistor (LDR). The resistor is part of a Wheatstone bridge, which is connected to a direct current source and a rectilinear recorder (Texas Instruments Company, U.S.A.). With this method the sensitivity of the recording was increased 5 times, in comparison to the usual method. Drug-receptor dissociation constants (K~) Dissociation constants for acetylcholine were calculated with the help of a computer program described by MaresGuia & Figueiredo (1972), using Clark's equation in the double reciprocal form. Solution used. The Tyrode solution had the following compositions: NaC1 136.8mM; KCI 2.7mM; CaC12 1.4mM; NaHCO3 12.0mM; glucose 5.5mM; MgCl2 1.0raM; Nail2 PO4 0.4mM. For the experiments with the spleen strips and rectal caecum, EDTA 0.04 mM and ascorbic acid 0.11 mM were added to the solution (modified Tyrode solution). Drugs used. Acetylcholine chloride (Sigma), adrenaline hydrochloride (Parke-Davis), noradrenaline bitartrate (Sigma), atropine sulphate (Sigma), phenoxybenzamine (Smith, Kline and French Lab.), propranolol (Sigma)~ guanethidine (Ciba), hexamethonium chloride (City Chemical Corp.), bretilium tosylate (Wellcome), procaine hydrochloride (Sigma), cocaine hydrochloride (Sigma) and tyramine hydrochloride (Sigma). The doses of these substances refer to the weights of the salts. Bradykinin (Sandoz, BRS-640, Batch No. 69055), nicotine (Sigma) and ouabain (Strophantin G, Sigma) were also used. Tityustoxin and tetrodotoxin. Purified Tityustoxin (TsTX) used in this paper was obtained from the venom of the Brazilian scorpion Tityus serrulatus by a combination of extraction and chromatographic techniques (Gomez & Diniz, 1966). The toxin concentration was calculated by using a Beckman spectrophotometer fitted with silica cells of 1.0cm pathway. An extinction coefficient of 1.00.D. unit. mg-1 ml at 280 nm was assumed. The concentration of protein is expressed in #g. Tityustoxin fractions were kindly given by Prof. M. V. Gomez. 115
116
L. FREIRE-MAIA, J. R. CUNHA-MELO, H. A. FUTURO-NETO, A. D. AZEVEDOAND J. WEINBERG
8O
g
o Raf ileum
determinations of K, for a rat and for a guinea-pig (10 exps).
• Guinea-pig ileum
Influence of atropine on the contractions of the ileum produced by TsTX. To avoid tachyphylaxis Tyrode
6.0
solution was used t h r o u g h o u t the experiments (FreireMaia et al., 1975b). The results (5 exps.) show that atropine did not prevent the contraction of the rat ileum produced by the first addition of TsTX to the perfusion chamber, but abolished totally the effects of subsequent doses, added at 30 min intervals (Fig.
if_ 40 20 I
I
0.4
0.7
I
i 1.3
1.0
3).
I 1,6
The effect of atropine o n the guinea-pig ileum was unexpected, since the first dose of TsTX induced an average contraction of 2.48 _+ 0.69 cm and the second dose produced a contraction higher than that evoked by the first, that is, 3.32 _ 0.65 cm (N = 5, P < 0.05). Figure 4 (upper tracing) shows one of these experiments.
tog dose, p.g TsTX
Fig. 1. Dose-response curves of Tityustoxin (TsTX) on isolated rat and guinea-pig ileum, immersed in Tyrode solution, at 35°C. Each point represents the average of the contractions in 5 different fragments. Tetrodotoxin A grade (TTX) was obtained from Calbiochem, La Jolla, California, U.S.A. Statistical analysis of the data was performed by using the Student's t-test, with P < 0.05 indicating significance.
6,C
.h
RESULTS
>'- 4,0
Rat and 9uinea-pi 9 ileum Dose-response curves of Tityustoxin ( Ts T X ). Preliminary data showed that in some experiments the second dose of TsTX added to the organ bath induced a contraction of the guinea-pig a n d rat ileum higher than that produced by the first one. If this fact is kept in mind, it is possible to get dose-response curves for TsTX, as shown in Fig. 1. The data also show that the rat ileum is more sensitive to TsTX than the guinea-pig ileum. K , ofacetylcholine. The acetylcholine-receptor dissociation constants for the guinea-pig ileum have a smaller value than those for the rat ileum (P < 0.01). Figure 2 shows calculated regression lines for the
.\ Aa'~k/'S 'I-
2.0
Kn =O,77xlO-7M
a
J
0.5
1.0
10-7/
l
l
Guinea-pig
[Acefylcholine]
Fig. 2. Dose-response curves and determination of the acetylcholine-receptor dissociation constant (K,), in a rat and a guinea-pig ileum. The regression lines were calculated with the help of a computer program.
I t
f
q
I rain
!
A
B
T
i
L5
B
T
B
A
Fig. 3. Strip of rat ileum immersed in Tyrode solution, at 35°C. Atropine (1.5 x 10 - 7 M) was present in the bath throughout. Acetylcholine (A, 5.5 × 10 -7 M), bradykinin (B, 9.4 x 10 - 9 M) and Tityustoxin (T, 1.0 #g/ml). The second dose of Tityustoxin was added to ~he bath 30 min after the first one. The arrows indicate that the preparation was rinsed once or twice. Note the absence of response to the second dose of Tityustoxin and potentiation of bradykinin action after the addition of the toxin.
Effects of Tityustoxin
i I~ lll~ ~ ~ 'lmi'n-I~~ ABIB
A
T
iB
Atropine 1.5
iE
~1 ~
x IO-rM
~ I
,rain
[
T
~
__J
Atropine 1.5 x IO-SM
Fig. 4. Strips of guinea-pig ileum immersed in Tyrode solution, at 35°C. Upper tracing: acetylcholine (A, 2.75 x 10-SM before and 1.1 x 10-TM after atropine), bradykinin (B, 9.4 × 10 - 9 M ) and Tityustoxin (T, 3.0/~g/ml). Note that the second dose of Tityustoxin produced a contraction higher than that evoked by the first. Lower tracing: Tityustoxin (T, 5.0 #g/ml); the doses of acetylcholine and bradykinin are the same of the upper tracing. The additions of Tityustoxin were made at 20 min intervals (the tracing shows the effects of the fourth and fifth dose). The arrows indicate that the preparation was rinsed once or twice. Note that atropine decreased the contraction induced by Tityustoxin.
7O
5C
o
3c
tD
IO
I
o
I
0.5
I
In another series of 5 experiments on the guinea-pig ileum, in which the fourth dose of TsTX induced an average contraction of 3.46 _ 0.34 cm, the addition of atropine to the bath decreased but did not abolish the contraction produced by toxin (2.54_ 0.27 cm, P < 0.01). Moreover, the latency between the addition of TsTX to the bath and the beginning of the contraction was increased in the atropine-treated preparations (Fig. 4, lower tracing). The contractions induced by bradykinin were potentiated by a previous addition of TsTX to the isolated rat and guinea-pig ileum treated with 1.5 × 10 -v M of atropine (Figs. 3 and 4).
Rat spleen strips Dose-response curves. The contraction of the rat spleen strips produced by adrenaline and noradrenaline, added to the organ bath at 5 min intervals, were directly related to the logarithm of the dose (Fig. 5). A dose-response curve to TsTX was not obtained because of the already described tachyphylaxis (FreireMaia et al., 1975, in press). Potentiation of the adrenaline effect by TsTX. The addition of adrenaline to a preparation previously treated with TsTX produced contractions higher than those induced by the same dose before TsTX administration. On the other hand, the effects of noradrenaline and tyramine were unchanged and decreased, respectively (Fig. 6). In another series of 5 experiments it was shown that the average contraction ( + S.E.) produced by isoprenaline (1.2 × 10- 3 M) was not significantly changed by TsTX, since the drug induced contractions of 45.7 + 4.8 mm before and 42.0 + 5.2 mm after TsTX administration (P > 0.05). Influence of drugs on the contractions induced by TsTX and noradrenaline. Hexamethonium: blockade of nicotinic receptors with hexamethonium did not change significantly the contractions of the spleen strips induced by TsTX or noradrenaline (Table 1). Procaine: the contractions produced by TsTX were significantly decreased by the local anesthetic agent procaine, in a dose that did not change significantly the effect of noradrenaline (Table 1). Bretilium or guanethidine: the addition of one of these sympatholytic drugs to the bath prevented totally the contractions induced by TsTX, without affecting significantly the responses to noradrenaline (Table 1). Ouabain: the contractions induced either by TsTX or noradrenaline were not significantly changed by ouabain (Table 1). Cocaine: the effect of TsTX was not significantly changed by cocaine. Simultaneously, the effect of noradrenaline was potentiated (Table 1).
1.0
(og IO-;'g rnt-= Fig. 5. Dose response curves of adrenaline (O O) and noradrenaline (," 0) on the isolated rat spleen strips, immersed in Tyrode solution at 37°C. Each point represents the average of the contractions in 3 different strips. G.P. 7 - - 2 / 3 ~
117
Hen rectal caecum Effects of Tityustoxin and adrenaline on the rectal caecum. Additions of Tityustoxin or adrenaline to the bath containing an isolated strip of hen rectal caecum produced relaxation of the preparations, in all the
118
L. FREIRE-MAIA, J. R. CUNHA-MELO, H. A. FUTURO-NETO, A. D. AZEVEDOAND J. WEINBERG
50
50-
40-
E 40 E E o
c o .e-
50
30-
o
g ~
j
E
n~
20
20-
I0-
IO
I 0.4 Ad
N
T
Fig. 6. Influence of a previous addition of Tityustoxin (TsTX, 10#g/ml, kept in the bath during 3 min) on the average contractions of the isolated rat spleen strips induced by adrenaline (Ad, 9.09 × 10 -7 M), noradrenaline (N, 1.50 X 10 - 6 M) and tyramine (T, 8.06 x 10 -5 M). The strips were immersed in a bath containing Tyrode solution, at 37°C (8 exps). *Significantly different from the control (paired t-tests, P < 0.05). experiments. Successive additions of TsTX to the preparation, at 5 min intervals, were followed by a progressive decrease in the magnitude of the relaxation (tachyphylaxis). Simultaneously, the effect of adrenaline was unchanged, the average relaxation Table 1. Influence of drugs on the contractions produced by Tityustoxin and noradrenaline on the isolated rat spleen strips. Values are means ___S.E. N = number of experiments TREATMENT
N
Contractions (mm) produced by Tityustoxina
None Hexamethoniumc Procaine d Bretilium e Guanethidinef
7 5 5 5 5
32.5 + 3.2 31.2 + 1.7 5.0 ± 2.3 i 0 0
Ouabaine g
7
30.0 ± 4 . 5
Cocaineh
7
41.6 ± 4.1
Noradrenaline b 31.4±1.5 29.8~1.5 26.6+l.4 35.2+7.3 35.2~9.8 37.1+3.3 4 8 . 5 ± 4 . 0i
The experiments were done in a lOml chamber
with a modified Tyrode solution, at 370C. a13.0 ug/ml
f1.26 x IO'5M
bl.50 x lO-6M
gl .00 x lO'4M
c3.98 x IO-5M
h2.05 x 10-SM
d2.80 x lO-4M
I S i g n i f i c a n t l y d i f f e r e n t from control (P < .Of).
e5.90 x IO'6M
I 0.7
I i.o
I i.3
I I.s
lOg I0-~ ml -~
Fig. 7. Dose-response curve of Tityustoxin (TsTX) on the isolated hen rectal caecum, immersed in Tyrode solution at 38°C. Each point represents the average of the relaxations in 3 different preparations. TsTX additions were made at 45 min intervals. (+S.E.) being 27.6 ___2.7 mm before the first addition of TsTX and 27.0 ___ 1.9 mm after the sixth dose of TsTX (P > 0.05). Dose-response curves. Additions of TsTX at 45 min intervals produced relaxation, without tachyphylaxis. Under these circumstances, the responses were directly related to the logarithm of the doses (Fig. 7). The effects of adrenaline were also related to the logarithm of the doses. Effects of other agonists on the rectal caecum. Bradykinin induced relaxation of the preparation, whereas nicotine produced a small relaxation (Table 2) followed by an average contraction of 16.9 +__ 1.8 mm (5 exps.).
Influence of drugs on the relaxation induced by TsTX. Phenoxybenzamine and propanolol: phenoxybenzamine, kept in the bath during 30 min, decreased significantly the magnitude of the relaxations induced by adrenaline or TsTX. On the other hand, additions of propranolol to the phenoxybenzamine-treated preparations prevented totally the effects of adrenaline or TsTX, without affecting significantly the relaxation induced by bradykinin (Table 2). Guanethidine: this drug, kept in the perfusion bath during 15 min, prevented totally the relaxations induced by nicotine and TsTX, but did not change significantly the relaxation induced by adrenaline (Table 2). On the other hand, the average contraction produced by nicotine 6.10 x 1 0 - 6 M was not significantly changed by guanethidine (15.6+ 1.8mm before and 15.5 ___ 1.2 mm after the drug). In 3 out of 5 experiments addition of TsTX to a preparation, previously treated with guanethidine, induced an average contraction of 1 6 . 0 _ 3.3mm, which was abolished by atropine at a concentration of 1.4 x 10 -7 M.
Effects of Tityustoxin
119
Table 2. Influence of drugs on the relaxation produced by Tityustoxin and agonists on the isolated hen rectal caecum. Values are means +__S.E. N = number of experiments Relaxation (mm) produced by TREATMENT
N Tityustoxina
Adrenaline b
Bradykinifl c
32.4 + 5.8 16.2 + 2.1 i
25.8 + 3.0 16.0 + 1.4 i
28.0 + 1.8 --
5
0
0
30.8 + 2.3
5 5
0 0
None 5 )henoxybenzamine e 5 ~henoxybenzam~ne + Propranolol Guanethidine g Tetrodotoxin h
Nicotine d
8.5±2.5
29.2 + 3.7
--
0
23.0 + 1.2
--
0
The experiments were done in a lOml chamber with a modified Tyrode s o l u t i o n , at 38°C.
a2.0 ~g/ml
f1.69 x lO-7M
b6.01 x lO-9M
gi.26 x IO-5M
c9.18 x lO'gM
h2.05 x IO-7M
d6.10 x lO-6M
i S i g n i f i c a n t l y d i f f e r e n t from control (P < .01).
e5.89 x lO'6M
Tetrodotoxin: addition of tetrodotoxin to the bath abolished totally the relaxation produced by TsTX, but did not change significantly the effect of adrenaline (Table 2). In 5 experiments, nicotine 6.10 x 1 0 - 6 M induced a relaxation of 6.8 +__0.6 mm, followed by a contraction of 15.0 + 2.8 mm. These effects were totally abolished by tetrodotoxin (2.05 x 10-7 M).
DISCUSSION
We have shown in this paper that it is possible to obtain a dose-response curve by addition of tityustoxin (TsTX) to an organ bath containing strips of guinea-pig or rat ileum. The tachyphylaxis previously described (Cunha-Melo et al., 1973) was avoided by additions of TsTX at 30 min intervals, to a bath containing Tyrode solution. The dose-response curves also showed that the rat ileum is much more sensitive to TsTX than the guinea-pig ileum, confirming results obtained with a single dose of TsTX (Freire-Maia et al., 1975a). Considering that the contraction of the guinea-pig and rat ileum is due, at least in part, to the release of acetylcholine (Diniz & Torres, 1968; TazieffDepierre, 1972; De Paula, 1973; Freire-Maia et al., 1974; Freire-Maia et al., 1975a) it would be possible to assume that the rat ileum is more sensitive to TsTX as a consequence of a greater affinity of the preparation to acetylcholine. However, this hypothesis was not supported by our experiments, which showed
that the rat ileum had smaller affinity for acetylcholine than the guinea-pig ileum. We have cOnfirmed in this paper that atropine did not prevent the contraction of the rat ileum induced by the first dose of TsTX. However, the effects of subsequent doses were abolished by atropine (Fig. 3). The contraction of the atropine-treated preparation was explained by the release of a second mediator, which could be substance P (Cunha-Melo et al., 1973; Tafuri et al., 1974). On the other hand, atropine did not prevent the contraction of the guinea-pig ileum induced by the first dose of Tityustoxin. Moreover, the second dose produced a contraction higher than that induced by the first one (Fig. 4, upper tracing). These results are different from those reported by Diniz & Gonqalves (1956) and Diniz & Valeri (1959), who showed that atropine abolished the contraction induced by scorpion venort~ The contraction of the atropine-treated guinea-pig strip could also be due to the release of a second mediator. However, experiments were not done to test this hypothesis. The mechanism whereby the first dose of TsTX induced a potentiation of the effect produced by the second dose was not investigated. Based on our experiments we cannot rule out the possibility of a direct effect of Tityustoxin on the intestinal smooth muscle, to explain the contractions of the atropine-treated preparations. We have also shown that TsTX increases the sensibility of the atropine-treated guinea-pig and rat ileum to bradykinin. This effect could be explained, at least in part, by the presence in our purified Tityustoxin
120
L. FREIRE-MA1A, J. R. CUNHA-MELo,H. A. FUTURO-NETO,A. D. AZEVEDOAND J. WEINBERG
of small amounts of the bradykinin potentiating factor, described by Araujo & Gomez (1974). As far as the experiments of the spleen strips are concerned, the data obtained with the sympatholytic drugs bretilium and guanethidine and the local anesthetic agent procaine seem to confirm our hypothesis (Futuro-Neto et al., 1974; Freire-Maia et al., in press) that the contraction of the preparation produced by TsTX is due to the release of catecholamines from nerve endings. As the action of TsTX is also abolished by tetrodotoxin it seems that the release of catecholamines could be mediated by a depolarization of the axonal membrane. This hypothesis is supported by experiments performed on different preparations (Adam et aI., 1966; Koppenh6fer & Schmidt, 1968; Narahashi et al., 1972). On the other hand, our previous data seem to indicate that the catecholamines are not released from the cytoplasmic mobile pool, but from the granular depots (Tafuri et al., 1971; Calixto & Freire-Maia, 1972; Tafuri et al., 1974; Freire-Maia et al., 1975, in press). We have observed a potentiation by TsTX of the adrenaline effects on the spleen strips. Several investigators reported potentiation by crude scorpion venom or its purified toxin (TsTX) of the effects of catecholamines on other preparations (Calixto et al., 1971; Calixto & Freire-Maia, 1972, Corrado et al., 1974; Melito & Corrado, 1975; Langer et aI., 1975). However, the mechanism of this potentiation is not yet known. The experiments performed on the isolated hen rectal caecum showed that Tityustoxin induced relaxation of the preparation, which is an unusual result, since it is well known that the toxin produces contraction of the intestine of several species. As the relaxation was prevented by teti'odotoxin or guanethidine, and also by the addition of propranolol to a preparation previously treated with phenoxybenzamine, it seems likely that the effect could be due to the release of catecholamines from nerve endings by the toxin. The experiments also showed that the relaxation of the hen rectal caecum produced by adrenaline depended on alpha and beta receptors, what is in accordance with results in other preparations (Alhquist & Levy, 1959; Levy, 1959). In the guanethidine-treated preparation TsTX produced a contraction, which was abolished by atropine. Thus, is seems likely that TsTX could release not only catecholamines but also acetylcholine from the isolated rectal caecum. The high sensitivity of the preparation to catecholamines (Euler, 1956) would explain the relaxation always induced by Tityustoxin. SUMMARY AND CONCLUSIONS 1. The effects of Tityustoxin (TsTX) on the isolated guinea-pig and rat ileum, rat spleen strips and hen rectal caecum were investigated. 2. D o s ~ r e s p o n s e curves showed that the rat ileum is more sensitive to TsTX than the guinea-pig ileum.
It seems likely that this effect is not due to a higher affinity of the rat ileum to acetylcholine. 3. Atropine did not prevent the contraction of the rat ileum produced by the first dose of TsTX, but abolished the effects of subsequent doses. 4. In the atropine-treated guinea-pig ileum the contraction produced by the second dose of TsTX was higher than that induced by the first one. The contraction of the ileum induced by bradykinin was also potentiated by TsTX. 5. The contraction of the rat spleen strips induced by adrenaline was potentiated by a previous addition of TsTX to the preparation. 6. Guanethidine, bretilium or procaine prevented the contraction of the spleen strips induced by TsTX, confirming our previous hypothesis that this contraction is due to the release of catecholamines from the adrenergic nerve endings. 7. TsTX induced relaxation of the isolated hen rectal caecum, which was directly related to the logarithm of the dose. 8. The relaxation of the caecum is prevented by tetrodotoxin, guanethidine and phenoxybenzamine plus propranolol, and seems to be due to the release of catecholamines from nerve endings by TsTX.
REFERENCES
ADAM K. R., SCHMIDTH., STAMPFUR. & WEISS C. (1966) The effect of scorpion venom on single myelinated nerve fibres of the frog. Br. J. Pharmac. Chemother. 26, 666-677. AHLQUIST R. P. & LEVY B. (1959) Adrenergic receptive mechanism of canine ileum. J. Pharmac. exp. Ther. 127, 146-149. ARAmO R. L. & GOMEZ MARCUS V. (1974) Bradykinin potentiating activity in scorpion venom--Tityus serrulatus. Proc. Int. Union Physiol. Sci. XI, p. 266. CALIXTO S. L. & FREIRE-MAtA L. (1972) Mechanism of action of purified scorpion toxin on the isolated rat duodenum. Ci~nc. Cult., S~zo Paulo 24, (Suppl.) 287-288. CAUXTO S. L., CUNHA-MELO J. R. & FREIRE-MAIA L. (1971) Estudo sobre o mecanismo de a~ao da toxina purificada de escorpi~o sobre a musculatura lisa isolada. Ci~nc. Cult., S~o Paulo 23, (Suppl.) 333-334. CORRADOA. P., RICCIOPPONETO F. & ANTON10 A. (1974) The mechanism of the hypertensive effect of Brazilian scorpion venom. (Tityus serrulatus Lutz e Mello). Toxicon 12, 145-150. 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. CUNHA-MELOJ. R., WEINBERGJ. & FREIRE-MAIAL. (1975) Estudo comparativo dos efeitos da tityustoxina e da acetilcolina sobre segmentos isolados de ileo de rato e de cobaia. Ci~nc. Cult., Sgto Paulo 27, (Suppl.) 337. DAWES B. N. & WITHRINGTONP. G. (1973) The action of drugs on the smooth muscle of the capsule and blood vessels of the spleen. Pharmac. Rev. 25, 373-413. DE PAULA M. C. H. G. (1973) Libera~5,o de acetilcolina por tityustoxina em sistema nervoso perif6rico. Thesis. Belo Horizonte, Brasil, 108 pp.
Effects of Tityustoxin DINIZ C. R. & GON~ALVES J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In Venoms (Edited by BUCKLEYE. E. & PORGES N.) pp. 131-144. Am. Ass. Adv. Sci., Washington. DIN1Z C. R. & TORRESJ. M. (1968) Release of an acetylcholine-like substance from guinea-pig ileum by scorpion venom. Toxicon 5, 277-281. DINIZ C. R. & VALER1V. (1959) Effects of a toxin present in a purified extract of telsons from the scorpion T. serrulatus on smooth muscle preparation and in mice. Archs Int. Pharmacodyn. Ther. 71, 1-13. EULER U. S. VON (1956) Noradrenaline. Charles C. Thomas, Springfield, Ill. FmLENZ M. (1970) The innervation of the cat spleen. Proc. R. Soc. Lond. B. 174, 459-468. FREIRE-MAIAL., CUNHA-MELoJ. R., GOMEZ M. V., TAFURI W. L., MARIA T. A. & FU~RO-NETO H. A. (1975a) Studies on the mechanism of action of tityustoxin. Toxicon 13, 93. FREIRE-MAIAL., CUNnA-MELOJ. R., GOMEZ M. V., TAFVRI W. L., MARIA T. A., CALIXTO S. L. & FU~JRo-NETo H. A. (in press) Studies on the mechanism of action of tityustoxin. In Animal, Plant and Microbial Toxins (Edited by OHSAKA A., HAVSASHI K. & SAWAY Y.). Plenum, London. FRE1RE-MAIA L., MELO J. R. C. & GOMEZ M. V. (1974) Mecanismo de aqao da tityustoxina. Ci~nc. Cult., S~o Paulo 26, (Suppl.) 326. FUTURO-NETO H. A. & FREIRE-MAIA L. (1975) Efeito da tityustoxina sobre fatias isoladas de ba~o de rato. Ci~nc. Cult., S~o Paulo 27, (Suppl.) 332-333. FUTURO-NETO H. A., MELO J. R. C. & FRE1RE-MAIA L. (1974) Efeito adrenergico perifgrico da tityustoxina. Ci~nc. Cult., Sfw Paulo 26, (Suppl.) 319. GOMEZ M. V. & DINIZ C. R. (1966) Separation of toxic components from the Brazilian scorpion Tityus serrulatus venom. Mere. Inst. Butantan Simp. Int. 33, 899-902.
12I
KOPPENHOFER E. & SCHMIDTH. (1968) Incomplete sodium inactivation in nodes of Ranvier treated with scorpion venom. Experientia, Basel 24, 41-42. LANGER S. Z., ADLER-GRASCHINSKYE., ALMEIDAA. P. & DINIZ C. R. (1975) Prejunctional effect to a purified toxin from the scorpion Tityus serrulatus. Naunyn-Schmiedeberg's Arch. Pharmac. 287, 243-259. LEVY B. (1959) Adrenergic blockade produced by the dichloro analogs of epinephrine, arterenol and isoproterenol. J. Pharmac. exp. Ther. 127, 150-156. MARES-GUIA M. & FIGUEIREDO A. F. S. (1972) Trypsinorganic solvent interaction. The simultaneous operation of competitive inhibition and dielectric effect. Biochemistry 11, 2091-2098. MELITO I. & CORRADO A. P. (1975) Supersensibilidade induzida pelo veneno do escorpi~to (Tityus serrulatus) aos efeitos farmacol6gicos causados por agonistas no canal deferente de cobaia. Ci~nc. Cult., Sdo Paulo 27, (Suppl.) 568. 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. TAFUR1 W. L., MARIA T. A., FREIRE-MAIA L. & CUNHAMELO J. R. (1971) Effect of purified scorpion toxin on vesicular components in the myoenteric plexus of the rat. Toxicon 9, 427-428. TAFURI W. L., MARIA Z. A., FREIRE-MAIA L. & CUNHAMELO J. R. (1974) Effect of the scorpion toxin on the granular vesicles in the Auerbach's plexus of the rat ileum. J. Neural Trans. 35, 233-240. TAZIEFF-DEPIERRE F. (1972) Venin de scorpion, calcium et 6mission d'acetylcholine par les fibres nerveuses dans l'il6on de cobaye. C.r. hebd. sdanc. Acad. Sci. Ser. D. 275, 3021-3024.
CHOLINERGIC AND ADRENERGIC EFFECTS OF TITYUSTOXIN L. FREIRE-MAIA, J. R. CUNHA-MELO, H. A. FUTURO-NETO, A. D. AZEVEDO and J. WEINBERG Department of Physiology and Biophysics, Institute of Biological Sciences, Box 2486, Belo Horizonte, Brazil (Received 15 December 1975) Abstract--The effects of purified scorpion toxin (Tityustoxin or TsTX) were investigated on the isolated guinea-pig and rat ileum, rat spleen strip and hen rectal caecum. The contraction of the ileum was due only in part to the release of acetylcholine, whereas the contraction of the spleen strip and the relaxation of the rectal caecum were due to the release of catecholamines.
INTRODUCTION THE COma~CTION of the isolated guinea-pig or rat ileum produced by the venom of the Brazilian scorpion Tityus serrulatus or its purified toxin (Tityustoxin) was explained by the release of ac~tylcholine and probably substance P (Diniz & Gon~alves, 1956; Diniz & Valeri, 1959; Diniz & Torres, 1968; CunhaMelo et al., 1973; De Paula, 1973; Freire-Maia et al., 1975a). On the other hand, the relaxation of the atropine-treated rat duodenum seemed to be related to an adrenergic mechanism (Cunha-Melo et al., 1973; Freire-Maia et al., 1975, in press). To study further the cholinergic and adrc~ergic effects of Tityustoxin, experiments were conducted using isolated smooth muscle preparations, such as fragments of rat and guinea-pig ileum, rat spleen strips and fragments of hen rectal caecum. The spleen was used because of its totally sympathetic innervation (Fillenz, 1970; Davies & Withrington, 1973~ whereas the rectal caecum was used because of its high sensitivity to catecholamines (Euler, 1956). Preliminary reports have been published elsewhere (Cunha-Melo et al., 1975; Futuro-Neto & Freire-Maia, 1975). MATERIAL AND METHODS Smooth muscle preparations The experiments performed upon the isolated strips of rat and guinea-pig ileum were done in the usual way, in an organ bath containing aerated Tyrode solution, at 35°C. The rat spleen strips were immersed in l0 ml of a Tyrode solution, at 37°C. The fluid was aerated with a mixture of 95% 02 and 5% CO2. The isolated fragments of hen rectal caecum were immersed in 10 ml of an aerated Tyrode solution, at 38°C, according to the method described by Euler (1956). Recording of the contractions or relaxations The contractions of the isolated strips of rat and guineapig intestines were registered on a smoked drum, with an auxotonic lever. The contractions of the rat spleen strips
or the relaxations of the hen rectal caecum were registered by means of a method described by one of us (A. D. Azevedo), using a displacement photoelectric transducer. This transducer is composed of an aluminium blade with a window of proper conformation, placed between a light source and a light dependent resistor (LDR). The resistor is part of a Wheatstone bridge, which is connected to a direct current source and a rectilinear recorder (Texas Instruments Company, U.S.A.). With this method the sensitivity of the recording was increased 5 times, in comparison to the usual method. Drug-receptor dissociation constants (K~) Dissociation constants for acetylcholine were calculated with the help of a computer program described by MaresGuia & Figueiredo (1972), using Clark's equation in the double reciprocal form. Solution used. The Tyrode solution had the following compositions: NaC1 136.8mM; KCI 2.7mM; CaC12 1.4mM; NaHCO3 12.0mM; glucose 5.5mM; MgCl2 1.0raM; Nail2 PO4 0.4mM. For the experiments with the spleen strips and rectal caecum, EDTA 0.04 mM and ascorbic acid 0.11 mM were added to the solution (modified Tyrode solution). Drugs used. Acetylcholine chloride (Sigma), adrenaline hydrochloride (Parke-Davis), noradrenaline bitartrate (Sigma), atropine sulphate (Sigma), phenoxybenzamine (Smith, Kline and French Lab.), propranolol (Sigma)~ guanethidine (Ciba), hexamethonium chloride (City Chemical Corp.), bretilium tosylate (Wellcome), procaine hydrochloride (Sigma), cocaine hydrochloride (Sigma) and tyramine hydrochloride (Sigma). The doses of these substances refer to the weights of the salts. Bradykinin (Sandoz, BRS-640, Batch No. 69055), nicotine (Sigma) and ouabain (Strophantin G, Sigma) were also used. Tityustoxin and tetrodotoxin. Purified Tityustoxin (TsTX) used in this paper was obtained from the venom of the Brazilian scorpion Tityus serrulatus by a combination of extraction and chromatographic techniques (Gomez & Diniz, 1966). The toxin concentration was calculated by using a Beckman spectrophotometer fitted with silica cells of 1.0cm pathway. An extinction coefficient of 1.00.D. unit. mg-1 ml at 280 nm was assumed. The concentration of protein is expressed in #g. Tityustoxin fractions were kindly given by Prof. M. V. Gomez. 115
116
L. FREIRE-MAIA, J. R. CUNHA-MELO, H. A. FUTURO-NETO, A. D. AZEVEDOAND J. WEINBERG
8O
g
o Raf ileum
determinations of K, for a rat and for a guinea-pig (10 exps).
• Guinea-pig ileum
Influence of atropine on the contractions of the ileum produced by TsTX. To avoid tachyphylaxis Tyrode
6.0
solution was used t h r o u g h o u t the experiments (FreireMaia et al., 1975b). The results (5 exps.) show that atropine did not prevent the contraction of the rat ileum produced by the first addition of TsTX to the perfusion chamber, but abolished totally the effects of subsequent doses, added at 30 min intervals (Fig.
if_ 40 20 I
I
0.4
0.7
I
i 1.3
1.0
3).
I 1,6
The effect of atropine o n the guinea-pig ileum was unexpected, since the first dose of TsTX induced an average contraction of 2.48 _+ 0.69 cm and the second dose produced a contraction higher than that evoked by the first, that is, 3.32 _ 0.65 cm (N = 5, P < 0.05). Figure 4 (upper tracing) shows one of these experiments.
tog dose, p.g TsTX
Fig. 1. Dose-response curves of Tityustoxin (TsTX) on isolated rat and guinea-pig ileum, immersed in Tyrode solution, at 35°C. Each point represents the average of the contractions in 5 different fragments. Tetrodotoxin A grade (TTX) was obtained from Calbiochem, La Jolla, California, U.S.A. Statistical analysis of the data was performed by using the Student's t-test, with P < 0.05 indicating significance.
6,C
.h
RESULTS
>'- 4,0
Rat and 9uinea-pi 9 ileum Dose-response curves of Tityustoxin ( Ts T X ). Preliminary data showed that in some experiments the second dose of TsTX added to the organ bath induced a contraction of the guinea-pig a n d rat ileum higher than that produced by the first one. If this fact is kept in mind, it is possible to get dose-response curves for TsTX, as shown in Fig. 1. The data also show that the rat ileum is more sensitive to TsTX than the guinea-pig ileum. K , ofacetylcholine. The acetylcholine-receptor dissociation constants for the guinea-pig ileum have a smaller value than those for the rat ileum (P < 0.01). Figure 2 shows calculated regression lines for the
.\ Aa'~k/'S 'I-
2.0
Kn =O,77xlO-7M
a
J
0.5
1.0
10-7/
l
l
Guinea-pig
[Acefylcholine]
Fig. 2. Dose-response curves and determination of the acetylcholine-receptor dissociation constant (K,), in a rat and a guinea-pig ileum. The regression lines were calculated with the help of a computer program.
I t
f
q
I rain
!
A
B
T
i
L5
B
T
B
A
Fig. 3. Strip of rat ileum immersed in Tyrode solution, at 35°C. Atropine (1.5 x 10 - 7 M) was present in the bath throughout. Acetylcholine (A, 5.5 × 10 -7 M), bradykinin (B, 9.4 x 10 - 9 M) and Tityustoxin (T, 1.0 #g/ml). The second dose of Tityustoxin was added to ~he bath 30 min after the first one. The arrows indicate that the preparation was rinsed once or twice. Note the absence of response to the second dose of Tityustoxin and potentiation of bradykinin action after the addition of the toxin.
Effects of Tityustoxin
i I~ lll~ ~ ~ 'lmi'n-I~~ ABIB
A
T
iB
Atropine 1.5
iE
~1 ~
x IO-rM
~ I
,rain
[
T
~
__J
Atropine 1.5 x IO-SM
Fig. 4. Strips of guinea-pig ileum immersed in Tyrode solution, at 35°C. Upper tracing: acetylcholine (A, 2.75 x 10-SM before and 1.1 x 10-TM after atropine), bradykinin (B, 9.4 × 10 - 9 M ) and Tityustoxin (T, 3.0/~g/ml). Note that the second dose of Tityustoxin produced a contraction higher than that evoked by the first. Lower tracing: Tityustoxin (T, 5.0 #g/ml); the doses of acetylcholine and bradykinin are the same of the upper tracing. The additions of Tityustoxin were made at 20 min intervals (the tracing shows the effects of the fourth and fifth dose). The arrows indicate that the preparation was rinsed once or twice. Note that atropine decreased the contraction induced by Tityustoxin.
7O
5C
o
3c
tD
IO
I
o
I
0.5
I
In another series of 5 experiments on the guinea-pig ileum, in which the fourth dose of TsTX induced an average contraction of 3.46 _ 0.34 cm, the addition of atropine to the bath decreased but did not abolish the contraction produced by toxin (2.54_ 0.27 cm, P < 0.01). Moreover, the latency between the addition of TsTX to the bath and the beginning of the contraction was increased in the atropine-treated preparations (Fig. 4, lower tracing). The contractions induced by bradykinin were potentiated by a previous addition of TsTX to the isolated rat and guinea-pig ileum treated with 1.5 × 10 -v M of atropine (Figs. 3 and 4).
Rat spleen strips Dose-response curves. The contraction of the rat spleen strips produced by adrenaline and noradrenaline, added to the organ bath at 5 min intervals, were directly related to the logarithm of the dose (Fig. 5). A dose-response curve to TsTX was not obtained because of the already described tachyphylaxis (FreireMaia et al., 1975, in press). Potentiation of the adrenaline effect by TsTX. The addition of adrenaline to a preparation previously treated with TsTX produced contractions higher than those induced by the same dose before TsTX administration. On the other hand, the effects of noradrenaline and tyramine were unchanged and decreased, respectively (Fig. 6). In another series of 5 experiments it was shown that the average contraction ( + S.E.) produced by isoprenaline (1.2 × 10- 3 M) was not significantly changed by TsTX, since the drug induced contractions of 45.7 + 4.8 mm before and 42.0 + 5.2 mm after TsTX administration (P > 0.05). Influence of drugs on the contractions induced by TsTX and noradrenaline. Hexamethonium: blockade of nicotinic receptors with hexamethonium did not change significantly the contractions of the spleen strips induced by TsTX or noradrenaline (Table 1). Procaine: the contractions produced by TsTX were significantly decreased by the local anesthetic agent procaine, in a dose that did not change significantly the effect of noradrenaline (Table 1). Bretilium or guanethidine: the addition of one of these sympatholytic drugs to the bath prevented totally the contractions induced by TsTX, without affecting significantly the responses to noradrenaline (Table 1). Ouabain: the contractions induced either by TsTX or noradrenaline were not significantly changed by ouabain (Table 1). Cocaine: the effect of TsTX was not significantly changed by cocaine. Simultaneously, the effect of noradrenaline was potentiated (Table 1).
1.0
(og IO-;'g rnt-= Fig. 5. Dose response curves of adrenaline (O O) and noradrenaline (," 0) on the isolated rat spleen strips, immersed in Tyrode solution at 37°C. Each point represents the average of the contractions in 3 different strips. G.P. 7 - - 2 / 3 ~
117
Hen rectal caecum Effects of Tityustoxin and adrenaline on the rectal caecum. Additions of Tityustoxin or adrenaline to the bath containing an isolated strip of hen rectal caecum produced relaxation of the preparations, in all the
118
L. FREIRE-MAIA, J. R. CUNHA-MELO, H. A. FUTURO-NETO, A. D. AZEVEDOAND J. WEINBERG
50
50-
40-
E 40 E E o
c o .e-
50
30-
o
g ~
j
E
n~
20
20-
I0-
IO
I 0.4 Ad
N
T
Fig. 6. Influence of a previous addition of Tityustoxin (TsTX, 10#g/ml, kept in the bath during 3 min) on the average contractions of the isolated rat spleen strips induced by adrenaline (Ad, 9.09 × 10 -7 M), noradrenaline (N, 1.50 X 10 - 6 M) and tyramine (T, 8.06 x 10 -5 M). The strips were immersed in a bath containing Tyrode solution, at 37°C (8 exps). *Significantly different from the control (paired t-tests, P < 0.05). experiments. Successive additions of TsTX to the preparation, at 5 min intervals, were followed by a progressive decrease in the magnitude of the relaxation (tachyphylaxis). Simultaneously, the effect of adrenaline was unchanged, the average relaxation Table 1. Influence of drugs on the contractions produced by Tityustoxin and noradrenaline on the isolated rat spleen strips. Values are means ___S.E. N = number of experiments TREATMENT
N
Contractions (mm) produced by Tityustoxina
None Hexamethoniumc Procaine d Bretilium e Guanethidinef
7 5 5 5 5
32.5 + 3.2 31.2 + 1.7 5.0 ± 2.3 i 0 0
Ouabaine g
7
30.0 ± 4 . 5
Cocaineh
7
41.6 ± 4.1
Noradrenaline b 31.4±1.5 29.8~1.5 26.6+l.4 35.2+7.3 35.2~9.8 37.1+3.3 4 8 . 5 ± 4 . 0i
The experiments were done in a lOml chamber
with a modified Tyrode solution, at 370C. a13.0 ug/ml
f1.26 x IO'5M
bl.50 x lO-6M
gl .00 x lO'4M
c3.98 x IO-5M
h2.05 x 10-SM
d2.80 x lO-4M
I S i g n i f i c a n t l y d i f f e r e n t from control (P < .Of).
e5.90 x IO'6M
I 0.7
I i.o
I i.3
I I.s
lOg I0-~ ml -~
Fig. 7. Dose-response curve of Tityustoxin (TsTX) on the isolated hen rectal caecum, immersed in Tyrode solution at 38°C. Each point represents the average of the relaxations in 3 different preparations. TsTX additions were made at 45 min intervals. (+S.E.) being 27.6 ___2.7 mm before the first addition of TsTX and 27.0 ___ 1.9 mm after the sixth dose of TsTX (P > 0.05). Dose-response curves. Additions of TsTX at 45 min intervals produced relaxation, without tachyphylaxis. Under these circumstances, the responses were directly related to the logarithm of the doses (Fig. 7). The effects of adrenaline were also related to the logarithm of the doses. Effects of other agonists on the rectal caecum. Bradykinin induced relaxation of the preparation, whereas nicotine produced a small relaxation (Table 2) followed by an average contraction of 16.9 +__ 1.8 mm (5 exps.).
Influence of drugs on the relaxation induced by TsTX. Phenoxybenzamine and propanolol: phenoxybenzamine, kept in the bath during 30 min, decreased significantly the magnitude of the relaxations induced by adrenaline or TsTX. On the other hand, additions of propranolol to the phenoxybenzamine-treated preparations prevented totally the effects of adrenaline or TsTX, without affecting significantly the relaxation induced by bradykinin (Table 2). Guanethidine: this drug, kept in the perfusion bath during 15 min, prevented totally the relaxations induced by nicotine and TsTX, but did not change significantly the relaxation induced by adrenaline (Table 2). On the other hand, the average contraction produced by nicotine 6.10 x 1 0 - 6 M was not significantly changed by guanethidine (15.6+ 1.8mm before and 15.5 ___ 1.2 mm after the drug). In 3 out of 5 experiments addition of TsTX to a preparation, previously treated with guanethidine, induced an average contraction of 1 6 . 0 _ 3.3mm, which was abolished by atropine at a concentration of 1.4 x 10 -7 M.
Effects of Tityustoxin
119
Table 2. Influence of drugs on the relaxation produced by Tityustoxin and agonists on the isolated hen rectal caecum. Values are means +__S.E. N = number of experiments Relaxation (mm) produced by TREATMENT
N Tityustoxina
Adrenaline b
Bradykinifl c
32.4 + 5.8 16.2 + 2.1 i
25.8 + 3.0 16.0 + 1.4 i
28.0 + 1.8 --
5
0
0
30.8 + 2.3
5 5
0 0
None 5 )henoxybenzamine e 5 ~henoxybenzam~ne + Propranolol Guanethidine g Tetrodotoxin h
Nicotine d
8.5±2.5
29.2 + 3.7
--
0
23.0 + 1.2
--
0
The experiments were done in a lOml chamber with a modified Tyrode s o l u t i o n , at 38°C.
a2.0 ~g/ml
f1.69 x lO-7M
b6.01 x lO-9M
gi.26 x IO-5M
c9.18 x lO'gM
h2.05 x IO-7M
d6.10 x lO-6M
i S i g n i f i c a n t l y d i f f e r e n t from control (P < .01).
e5.89 x lO'6M
Tetrodotoxin: addition of tetrodotoxin to the bath abolished totally the relaxation produced by TsTX, but did not change significantly the effect of adrenaline (Table 2). In 5 experiments, nicotine 6.10 x 1 0 - 6 M induced a relaxation of 6.8 +__0.6 mm, followed by a contraction of 15.0 + 2.8 mm. These effects were totally abolished by tetrodotoxin (2.05 x 10-7 M).
DISCUSSION
We have shown in this paper that it is possible to obtain a dose-response curve by addition of tityustoxin (TsTX) to an organ bath containing strips of guinea-pig or rat ileum. The tachyphylaxis previously described (Cunha-Melo et al., 1973) was avoided by additions of TsTX at 30 min intervals, to a bath containing Tyrode solution. The dose-response curves also showed that the rat ileum is much more sensitive to TsTX than the guinea-pig ileum, confirming results obtained with a single dose of TsTX (Freire-Maia et al., 1975a). Considering that the contraction of the guinea-pig and rat ileum is due, at least in part, to the release of acetylcholine (Diniz & Torres, 1968; TazieffDepierre, 1972; De Paula, 1973; Freire-Maia et al., 1974; Freire-Maia et al., 1975a) it would be possible to assume that the rat ileum is more sensitive to TsTX as a consequence of a greater affinity of the preparation to acetylcholine. However, this hypothesis was not supported by our experiments, which showed
that the rat ileum had smaller affinity for acetylcholine than the guinea-pig ileum. We have cOnfirmed in this paper that atropine did not prevent the contraction of the rat ileum induced by the first dose of TsTX. However, the effects of subsequent doses were abolished by atropine (Fig. 3). The contraction of the atropine-treated preparation was explained by the release of a second mediator, which could be substance P (Cunha-Melo et al., 1973; Tafuri et al., 1974). On the other hand, atropine did not prevent the contraction of the guinea-pig ileum induced by the first dose of Tityustoxin. Moreover, the second dose produced a contraction higher than that induced by the first one (Fig. 4, upper tracing). These results are different from those reported by Diniz & Gonqalves (1956) and Diniz & Valeri (1959), who showed that atropine abolished the contraction induced by scorpion venort~ The contraction of the atropine-treated guinea-pig strip could also be due to the release of a second mediator. However, experiments were not done to test this hypothesis. The mechanism whereby the first dose of TsTX induced a potentiation of the effect produced by the second dose was not investigated. Based on our experiments we cannot rule out the possibility of a direct effect of Tityustoxin on the intestinal smooth muscle, to explain the contractions of the atropine-treated preparations. We have also shown that TsTX increases the sensibility of the atropine-treated guinea-pig and rat ileum to bradykinin. This effect could be explained, at least in part, by the presence in our purified Tityustoxin
120
L. FREIRE-MA1A, J. R. CUNHA-MELo,H. A. FUTURO-NETO,A. D. AZEVEDOAND J. WEINBERG
of small amounts of the bradykinin potentiating factor, described by Araujo & Gomez (1974). As far as the experiments of the spleen strips are concerned, the data obtained with the sympatholytic drugs bretilium and guanethidine and the local anesthetic agent procaine seem to confirm our hypothesis (Futuro-Neto et al., 1974; Freire-Maia et al., in press) that the contraction of the preparation produced by TsTX is due to the release of catecholamines from nerve endings. As the action of TsTX is also abolished by tetrodotoxin it seems that the release of catecholamines could be mediated by a depolarization of the axonal membrane. This hypothesis is supported by experiments performed on different preparations (Adam et aI., 1966; Koppenh6fer & Schmidt, 1968; Narahashi et al., 1972). On the other hand, our previous data seem to indicate that the catecholamines are not released from the cytoplasmic mobile pool, but from the granular depots (Tafuri et al., 1971; Calixto & Freire-Maia, 1972; Tafuri et al., 1974; Freire-Maia et al., 1975, in press). We have observed a potentiation by TsTX of the adrenaline effects on the spleen strips. Several investigators reported potentiation by crude scorpion venom or its purified toxin (TsTX) of the effects of catecholamines on other preparations (Calixto et al., 1971; Calixto & Freire-Maia, 1972, Corrado et al., 1974; Melito & Corrado, 1975; Langer et aI., 1975). However, the mechanism of this potentiation is not yet known. The experiments performed on the isolated hen rectal caecum showed that Tityustoxin induced relaxation of the preparation, which is an unusual result, since it is well known that the toxin produces contraction of the intestine of several species. As the relaxation was prevented by teti'odotoxin or guanethidine, and also by the addition of propranolol to a preparation previously treated with phenoxybenzamine, it seems likely that the effect could be due to the release of catecholamines from nerve endings by the toxin. The experiments also showed that the relaxation of the hen rectal caecum produced by adrenaline depended on alpha and beta receptors, what is in accordance with results in other preparations (Alhquist & Levy, 1959; Levy, 1959). In the guanethidine-treated preparation TsTX produced a contraction, which was abolished by atropine. Thus, is seems likely that TsTX could release not only catecholamines but also acetylcholine from the isolated rectal caecum. The high sensitivity of the preparation to catecholamines (Euler, 1956) would explain the relaxation always induced by Tityustoxin. SUMMARY AND CONCLUSIONS 1. The effects of Tityustoxin (TsTX) on the isolated guinea-pig and rat ileum, rat spleen strips and hen rectal caecum were investigated. 2. D o s ~ r e s p o n s e curves showed that the rat ileum is more sensitive to TsTX than the guinea-pig ileum.
It seems likely that this effect is not due to a higher affinity of the rat ileum to acetylcholine. 3. Atropine did not prevent the contraction of the rat ileum produced by the first dose of TsTX, but abolished the effects of subsequent doses. 4. In the atropine-treated guinea-pig ileum the contraction produced by the second dose of TsTX was higher than that induced by the first one. The contraction of the ileum induced by bradykinin was also potentiated by TsTX. 5. The contraction of the rat spleen strips induced by adrenaline was potentiated by a previous addition of TsTX to the preparation. 6. Guanethidine, bretilium or procaine prevented the contraction of the spleen strips induced by TsTX, confirming our previous hypothesis that this contraction is due to the release of catecholamines from the adrenergic nerve endings. 7. TsTX induced relaxation of the isolated hen rectal caecum, which was directly related to the logarithm of the dose. 8. The relaxation of the caecum is prevented by tetrodotoxin, guanethidine and phenoxybenzamine plus propranolol, and seems to be due to the release of catecholamines from nerve endings by TsTX.
REFERENCES
ADAM K. R., SCHMIDTH., STAMPFUR. & WEISS C. (1966) The effect of scorpion venom on single myelinated nerve fibres of the frog. Br. J. Pharmac. Chemother. 26, 666-677. AHLQUIST R. P. & LEVY B. (1959) Adrenergic receptive mechanism of canine ileum. J. Pharmac. exp. Ther. 127, 146-149. ARAmO R. L. & GOMEZ MARCUS V. (1974) Bradykinin potentiating activity in scorpion venom--Tityus serrulatus. Proc. Int. Union Physiol. Sci. XI, p. 266. CALIXTO S. L. & FREIRE-MAtA L. (1972) Mechanism of action of purified scorpion toxin on the isolated rat duodenum. Ci~nc. Cult., S~zo Paulo 24, (Suppl.) 287-288. CAUXTO S. L., CUNHA-MELO J. R. & FREIRE-MAIA L. (1971) Estudo sobre o mecanismo de a~ao da toxina purificada de escorpi~o sobre a musculatura lisa isolada. Ci~nc. Cult., S~o Paulo 23, (Suppl.) 333-334. CORRADOA. P., RICCIOPPONETO F. & ANTON10 A. (1974) The mechanism of the hypertensive effect of Brazilian scorpion venom. (Tityus serrulatus Lutz e Mello). Toxicon 12, 145-150. 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. CUNHA-MELOJ. R., WEINBERGJ. & FREIRE-MAIAL. (1975) Estudo comparativo dos efeitos da tityustoxina e da acetilcolina sobre segmentos isolados de ileo de rato e de cobaia. Ci~nc. Cult., Sgto Paulo 27, (Suppl.) 337. DAWES B. N. & WITHRINGTONP. G. (1973) The action of drugs on the smooth muscle of the capsule and blood vessels of the spleen. Pharmac. Rev. 25, 373-413. DE PAULA M. C. H. G. (1973) Libera~5,o de acetilcolina por tityustoxina em sistema nervoso perif6rico. Thesis. Belo Horizonte, Brasil, 108 pp.
Effects of Tityustoxin DINIZ C. R. & GON~ALVES J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In Venoms (Edited by BUCKLEYE. E. & PORGES N.) pp. 131-144. Am. Ass. Adv. Sci., Washington. DIN1Z C. R. & TORRESJ. M. (1968) Release of an acetylcholine-like substance from guinea-pig ileum by scorpion venom. Toxicon 5, 277-281. DINIZ C. R. & VALER1V. (1959) Effects of a toxin present in a purified extract of telsons from the scorpion T. serrulatus on smooth muscle preparation and in mice. Archs Int. Pharmacodyn. Ther. 71, 1-13. EULER U. S. VON (1956) Noradrenaline. Charles C. Thomas, Springfield, Ill. FmLENZ M. (1970) The innervation of the cat spleen. Proc. R. Soc. Lond. B. 174, 459-468. FREIRE-MAIAL., CUNHA-MELoJ. R., GOMEZ M. V., TAFURI W. L., MARIA T. A. & FU~RO-NETO H. A. (1975a) Studies on the mechanism of action of tityustoxin. Toxicon 13, 93. FREIRE-MAIAL., CUNnA-MELOJ. R., GOMEZ M. V., TAFVRI W. L., MARIA T. A., CALIXTO S. L. & FU~JRo-NETo H. A. (in press) Studies on the mechanism of action of tityustoxin. In Animal, Plant and Microbial Toxins (Edited by OHSAKA A., HAVSASHI K. & SAWAY Y.). Plenum, London. FRE1RE-MAIA L., MELO J. R. C. & GOMEZ M. V. (1974) Mecanismo de aqao da tityustoxina. Ci~nc. Cult., S~o Paulo 26, (Suppl.) 326. FUTURO-NETO H. A. & FREIRE-MAIA L. (1975) Efeito da tityustoxina sobre fatias isoladas de ba~o de rato. Ci~nc. Cult., S~o Paulo 27, (Suppl.) 332-333. FUTURO-NETO H. A., MELO J. R. C. & FRE1RE-MAIA L. (1974) Efeito adrenergico perifgrico da tityustoxina. Ci~nc. Cult., Sfw Paulo 26, (Suppl.) 319. GOMEZ M. V. & DINIZ C. R. (1966) Separation of toxic components from the Brazilian scorpion Tityus serrulatus venom. Mere. Inst. Butantan Simp. Int. 33, 899-902.
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KOPPENHOFER E. & SCHMIDTH. (1968) Incomplete sodium inactivation in nodes of Ranvier treated with scorpion venom. Experientia, Basel 24, 41-42. LANGER S. Z., ADLER-GRASCHINSKYE., ALMEIDAA. P. & DINIZ C. R. (1975) Prejunctional effect to a purified toxin from the scorpion Tityus serrulatus. Naunyn-Schmiedeberg's Arch. Pharmac. 287, 243-259. LEVY B. (1959) Adrenergic blockade produced by the dichloro analogs of epinephrine, arterenol and isoproterenol. J. Pharmac. exp. Ther. 127, 150-156. MARES-GUIA M. & FIGUEIREDO A. F. S. (1972) Trypsinorganic solvent interaction. The simultaneous operation of competitive inhibition and dielectric effect. Biochemistry 11, 2091-2098. MELITO I. & CORRADO A. P. (1975) Supersensibilidade induzida pelo veneno do escorpi~to (Tityus serrulatus) aos efeitos farmacol6gicos causados por agonistas no canal deferente de cobaia. Ci~nc. Cult., Sdo Paulo 27, (Suppl.) 568. 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. TAFUR1 W. L., MARIA T. A., FREIRE-MAIA L. & CUNHAMELO J. R. (1971) Effect of purified scorpion toxin on vesicular components in the myoenteric plexus of the rat. Toxicon 9, 427-428. TAFURI W. L., MARIA Z. A., FREIRE-MAIA L. & CUNHAMELO J. R. (1974) Effect of the scorpion toxin on the granular vesicles in the Auerbach's plexus of the rat ileum. J. Neural Trans. 35, 233-240. TAZIEFF-DEPIERRE F. (1972) Venin de scorpion, calcium et 6mission d'acetylcholine par les fibres nerveuses dans l'il6on de cobaye. C.r. hebd. sdanc. Acad. Sci. Ser. D. 275, 3021-3024.