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Some effects of bradykinin on the central nervous system
SOME EFFECTS OF BRADYKININ ON THE CENTRAL NERVOUS SYSTEM RADAN ~APEK
Pharmacological Laboratory, Institute o] Organic Chemistry and Biochemistry, Czechoslovak Academy o] Science, Prague, Czechoslovakia
ALMOST no attention has been paid to the actions of b r a d y k i n i n on the central nervous s y s t e m in intact animals. Considering the importance of substance P, a polypeptide isolated f r o m tissue in the central nervous system, it seems to us t h a t a comparison of some k n o w n effects of substance P with b r a d y k i n i n would be of interest. In this paper we are going to summarize o u r results concerning the effects observed after intracerebral injection of b r a d y k i n i n and the influence of this substance on various kinds of convulsions. I n a parallel w a y the effects of substance P were checked in most experiments. B r a d y k i n i n was prepared by the m e t h o d of Rocha e Silva et al. 1 and A n d r a d e et al. 2 As the standard preparation was not available, the activity of our preparation could not be expressed in units*. Therefore the doses are p r e s e n t e d in milligrams. Our preparation caused contractions of the isolated guinea-pig ileum, in concentration of 10--~ - 10-5 and of the isolated rat uterus, in concentrations of 10--+ - 10-7 in the bath. S u b s t a n c e P was prepared by the m e t h o d of Pernow. 3 Standardization was not performed for the reason mentioned above. It was active on the isolated guinea-pig ileum in the concentration of 10-4 in the bath. I n t r a v e n t r i c u l a r i n j e c t i o n s were p e r f o r m e d on 8 cats. The cannulae were implanted in the lateral ventricle 3 - 7 days before the experiment, according to Fektberg and Sherwood,4 using some technical modifications.~ Doses of 50 mg b r a d y k i n i n and 10 mg substance P were injected, dissolved in 0.5 ml. Tyrode soluticn. B r a d y k i n i n was injected intraventricu l a r l y also in mice, according to Haley's t e c h n i q u e ? using an i n s t r u m e n t for semi-automatic application. 7 A c t i o n on Metrazol and S t r y c h n i n e S e i z u r e and on E l e c t r o s h o c k The m e d i a n effective dose (EDs0) of metrazol was d e t e r m i n e d by the probit m e t h o d of Litchfield and Wilcoxon 8 on mice. The positive reaction was a tonic extension of the hind limbs. Groups of 50 mice were used. The * After the Meeting Prof. Rocha e Silva kindly performed in our Laboratory an a s s a y a n d d e t e r m i n e d t h e p o t e n c y of o u r p r e p a r a t i o n w h i c h w a s 0.1 u n i t p e r m g . We are most grateful to him.
[61]
62
R. ~APEK
first group was given only metrazol intravenously. To the second group 5 rain before metrazol administration, a dose of 7 mg/20 g body w e i g h t of b r a d y k i n i n was injected, which corresponds to 1/2 of the m e d i a n lethal dose (LDs0) of our preparation. The third group was injected with 5 mg/20 g of substance P. This corresponds also to 1/2 LDs0 of our preparation. The action of bradyk-inin and substance P on s t r y c h n i n e seizure and t h a t of b r a d y k i n i n on electroshock was e x a m i n e d by the same m e t h od. Electroshock was carried out by means of two electrodes fixed on the ears, using a sinusoidal c u r r e n t of 50 c/sec, and 0.2 sec duration. The resistance of the whole circuit including the animal was in every experi m e n t carefully a d j u s t e d to 60 k~Q. The EDs0 was expressed in volts.
Fro. 1. The balance impairment 2 rain after intracercbral injection of bradykinin. A f t e r intracerebral injection of b r a d y k i n i n w i t h i n 1 min all cats miaouw. Muscular twiches beginning at the ears, move down the neck to dorsal muscles. The balance is impaired (Fig. 1). The cat falls to the side and cannot right itself. The forelegs are stretched with the claws outstretched. The hindlegs are u n d e r the body. In most cases, opisthotonus (Fig. 2) and convulsions develop. The animal m a y take bizzare positions. Autonomic s y m p t o m s are marked. Tachypnoea is always present f r o m the first m i n u t e s a f t e r administration. The respiration is shallow, the f r e q u e n c y more t h a n 100 per minute. The m o u t h is open, the tongue hanging out. Salivation is m o s t l y present; micturition, and sometimes defaecation could be observed. G r a d u a l l y , m a x i m a l mydriasis occurs (Fig. 3). Although the clinical picture was severe, all animals recovered and t h e i r behaviour and autonomic functions r e t u r n e d to normal a f t e r 60 - 90 min. With substance P some vegetative disturbances were observed, but the general behaviour was strikingly different, the animals m a k i n g an impression of deep sedation. Interesting were the results in mice. There is a clear picture showing an excitation of the central nervous system. The animals are v e r y active, t h e y r u n o f t e n to the left side (i.e. the side of injection). The move their
BRADYKININ AND CENTRAL NE~tVOUS SYSTEM
63
FIG. 2. Opisthotonus 12 min after intracerebral injection of bradykinin.
Fla. 3. Mydriasis 14 rain after intracerebral injection of bradykinin. tails and j u m p to r e m a r k a b l e heights. T h e y m a y also h a v e convulsions. A typical sign is piloerection so t h a t t h e y give t he impression of a ball.9 As m a y be seen f r o m Table I, b r a d y k i n i n in the doses used decreases significantly the seizure t hr e s hol d to metrazol, s t r y c h n i n e and electroshock in d u ced convulsions, whereas, as k n o w n f r o m t h e l i t e r a t u r e 10, 11, 12 and c o n f i r m e d b y us, substance P has the opposite effect. Von E u l e r and P e r n o w 13 saw a f t e r i n t r a c e r e b r a l injection of substance P the same p a t t e r n s w i t h the p r e d o m i n a n t m ot ori c sedation. Rocha e SiN va 14 also described m ydr i a s i s in d i f f e r e n t e x p e r i m e n t s with b r a d y k i n i n a d m i n i s t r e d i n t r a v e n t r i c u l a r l y . A s ,we h a v e seen t h a t o u r p r e p a r a t i o n s , although of d i f f e r e n t p r o v e n i e n c e and pu ri t y, have t he same effects as described b y others, w e m a y assume t hat also the n e w l y described effects are reliable.
64
R. CAPEK
T h e a c t i o n o f b r a d y k i n i n o n s m o o t h m u s c l e s a n d c i r c u l a t i o n is t h e object of main interest. These actions of both bradykinin and substance P are similar. In our experiments
these p e p t i d e s h a d a n opposite effect.
I t s e e m s to us, a c c o r d i n g to o u r r e s u l t s , t h a t m o r e a t t e n t i o n s h o u l d b e p a i d to e f f e c t s o f b r a d y k i n i n o n t h e c e n t r a l n e r v o u s s y s t e m d i r e c t l y , o r to its i n t e r a c t i o n w i t h d r u g s w h i c h i n f l u e n c e t h e c e n t r a l n e r v o u s s y s t e m . TABLE I
T h e a c t i o n of b r a d y k i n i n a n d s u b s t a n c e P on m e t r a z o l , s t r y c h n i n and electroshock induced convulsions Figures represent the relative changes in median effective dose (EDs0) in per cent with confidence limits (P = 0.05) after b r a d y k i n i n or substance P pretreatment. Control
EDso
Bradykinin
Substance P
metrazol
100
57"3 (49.0-67" 0)
133 "3 (115'7-153.3
strychnin
I00
59"0 (43-I-80.7)
131.4 (105-1-164-2)
electroshock
100
83.7
(73" 9-94' 7)
REFERENCES 1. M. ROCHA E SILVA, W. T. BERALDO and G. ROSNNFELD; Amer. J. Physiol. 156 261 (1949). 2. A. S. ANDRADE and M. ROCHA E SILVA; Biochem. J. 64 701 (1956). 3. B. PERNOW; Acta Physiol. Scand. 29 suppl. 105 (1953). 4. W. FELDBERG and S. L. SHF.RVOOD; J. Physiol. 102 3P (1953). 5. V. KR~:BS and J. VAN~OEK; Physiol. bohemoslov. 8 167 (1959) (in Russian). 6. T. J. HALEY and W. G. McCoRMICK; Brit. J. Pharmacol. 12 12 (1957). 7. J. VAN~CEK, V. KREBS, E. SCHEER and T. BIELEKE; J. Amer. Pharm. Ass., Sci. Ed. 49 178 (1960). 8. J. T. LITCHFIELD and F. W. WILCOXON; J. Pharmacol. 96 99 (1949). 9. R. ~APEK; CS. ]ysiol. 9 283 (1960) (in Czech). 10. G. ZETLER; Arch. Exp. Path. Pharmakol. 228 513 (1956). 11. G. ZETLER; Arch. Exp. Path. Pharmakol. 237 11 (1959). 12. P. STERN and V. DosRiC; In Psychotropis Drugs, p. 448 (Edited by S. GARATTINI and V. GHET'rI) Elsevier, 1957. 13. U. S. EULER and B. PERNOW; Nature, 174 184 (1954). 14. M. ROCHA E SILVA; XXI International CongYess oi Physiological Sciences Buenos Aires 1959, Symposia and Special Lectures, p. 50.
Pharmacological Laboratory, Institute o] Organic Chemistry and Biochemistry, Czechoslovak Academy o] Science, Prague, Czechoslovakia
ALMOST no attention has been paid to the actions of b r a d y k i n i n on the central nervous s y s t e m in intact animals. Considering the importance of substance P, a polypeptide isolated f r o m tissue in the central nervous system, it seems to us t h a t a comparison of some k n o w n effects of substance P with b r a d y k i n i n would be of interest. In this paper we are going to summarize o u r results concerning the effects observed after intracerebral injection of b r a d y k i n i n and the influence of this substance on various kinds of convulsions. I n a parallel w a y the effects of substance P were checked in most experiments. B r a d y k i n i n was prepared by the m e t h o d of Rocha e Silva et al. 1 and A n d r a d e et al. 2 As the standard preparation was not available, the activity of our preparation could not be expressed in units*. Therefore the doses are p r e s e n t e d in milligrams. Our preparation caused contractions of the isolated guinea-pig ileum, in concentration of 10--~ - 10-5 and of the isolated rat uterus, in concentrations of 10--+ - 10-7 in the bath. S u b s t a n c e P was prepared by the m e t h o d of Pernow. 3 Standardization was not performed for the reason mentioned above. It was active on the isolated guinea-pig ileum in the concentration of 10-4 in the bath. I n t r a v e n t r i c u l a r i n j e c t i o n s were p e r f o r m e d on 8 cats. The cannulae were implanted in the lateral ventricle 3 - 7 days before the experiment, according to Fektberg and Sherwood,4 using some technical modifications.~ Doses of 50 mg b r a d y k i n i n and 10 mg substance P were injected, dissolved in 0.5 ml. Tyrode soluticn. B r a d y k i n i n was injected intraventricu l a r l y also in mice, according to Haley's t e c h n i q u e ? using an i n s t r u m e n t for semi-automatic application. 7 A c t i o n on Metrazol and S t r y c h n i n e S e i z u r e and on E l e c t r o s h o c k The m e d i a n effective dose (EDs0) of metrazol was d e t e r m i n e d by the probit m e t h o d of Litchfield and Wilcoxon 8 on mice. The positive reaction was a tonic extension of the hind limbs. Groups of 50 mice were used. The * After the Meeting Prof. Rocha e Silva kindly performed in our Laboratory an a s s a y a n d d e t e r m i n e d t h e p o t e n c y of o u r p r e p a r a t i o n w h i c h w a s 0.1 u n i t p e r m g . We are most grateful to him.
[61]
62
R. ~APEK
first group was given only metrazol intravenously. To the second group 5 rain before metrazol administration, a dose of 7 mg/20 g body w e i g h t of b r a d y k i n i n was injected, which corresponds to 1/2 of the m e d i a n lethal dose (LDs0) of our preparation. The third group was injected with 5 mg/20 g of substance P. This corresponds also to 1/2 LDs0 of our preparation. The action of bradyk-inin and substance P on s t r y c h n i n e seizure and t h a t of b r a d y k i n i n on electroshock was e x a m i n e d by the same m e t h od. Electroshock was carried out by means of two electrodes fixed on the ears, using a sinusoidal c u r r e n t of 50 c/sec, and 0.2 sec duration. The resistance of the whole circuit including the animal was in every experi m e n t carefully a d j u s t e d to 60 k~Q. The EDs0 was expressed in volts.
Fro. 1. The balance impairment 2 rain after intracercbral injection of bradykinin. A f t e r intracerebral injection of b r a d y k i n i n w i t h i n 1 min all cats miaouw. Muscular twiches beginning at the ears, move down the neck to dorsal muscles. The balance is impaired (Fig. 1). The cat falls to the side and cannot right itself. The forelegs are stretched with the claws outstretched. The hindlegs are u n d e r the body. In most cases, opisthotonus (Fig. 2) and convulsions develop. The animal m a y take bizzare positions. Autonomic s y m p t o m s are marked. Tachypnoea is always present f r o m the first m i n u t e s a f t e r administration. The respiration is shallow, the f r e q u e n c y more t h a n 100 per minute. The m o u t h is open, the tongue hanging out. Salivation is m o s t l y present; micturition, and sometimes defaecation could be observed. G r a d u a l l y , m a x i m a l mydriasis occurs (Fig. 3). Although the clinical picture was severe, all animals recovered and t h e i r behaviour and autonomic functions r e t u r n e d to normal a f t e r 60 - 90 min. With substance P some vegetative disturbances were observed, but the general behaviour was strikingly different, the animals m a k i n g an impression of deep sedation. Interesting were the results in mice. There is a clear picture showing an excitation of the central nervous system. The animals are v e r y active, t h e y r u n o f t e n to the left side (i.e. the side of injection). The move their
BRADYKININ AND CENTRAL NE~tVOUS SYSTEM
63
FIG. 2. Opisthotonus 12 min after intracerebral injection of bradykinin.
Fla. 3. Mydriasis 14 rain after intracerebral injection of bradykinin. tails and j u m p to r e m a r k a b l e heights. T h e y m a y also h a v e convulsions. A typical sign is piloerection so t h a t t h e y give t he impression of a ball.9 As m a y be seen f r o m Table I, b r a d y k i n i n in the doses used decreases significantly the seizure t hr e s hol d to metrazol, s t r y c h n i n e and electroshock in d u ced convulsions, whereas, as k n o w n f r o m t h e l i t e r a t u r e 10, 11, 12 and c o n f i r m e d b y us, substance P has the opposite effect. Von E u l e r and P e r n o w 13 saw a f t e r i n t r a c e r e b r a l injection of substance P the same p a t t e r n s w i t h the p r e d o m i n a n t m ot ori c sedation. Rocha e SiN va 14 also described m ydr i a s i s in d i f f e r e n t e x p e r i m e n t s with b r a d y k i n i n a d m i n i s t r e d i n t r a v e n t r i c u l a r l y . A s ,we h a v e seen t h a t o u r p r e p a r a t i o n s , although of d i f f e r e n t p r o v e n i e n c e and pu ri t y, have t he same effects as described b y others, w e m a y assume t hat also the n e w l y described effects are reliable.
64
R. CAPEK
T h e a c t i o n o f b r a d y k i n i n o n s m o o t h m u s c l e s a n d c i r c u l a t i o n is t h e object of main interest. These actions of both bradykinin and substance P are similar. In our experiments
these p e p t i d e s h a d a n opposite effect.
I t s e e m s to us, a c c o r d i n g to o u r r e s u l t s , t h a t m o r e a t t e n t i o n s h o u l d b e p a i d to e f f e c t s o f b r a d y k i n i n o n t h e c e n t r a l n e r v o u s s y s t e m d i r e c t l y , o r to its i n t e r a c t i o n w i t h d r u g s w h i c h i n f l u e n c e t h e c e n t r a l n e r v o u s s y s t e m . TABLE I
T h e a c t i o n of b r a d y k i n i n a n d s u b s t a n c e P on m e t r a z o l , s t r y c h n i n and electroshock induced convulsions Figures represent the relative changes in median effective dose (EDs0) in per cent with confidence limits (P = 0.05) after b r a d y k i n i n or substance P pretreatment. Control
EDso
Bradykinin
Substance P
metrazol
100
57"3 (49.0-67" 0)
133 "3 (115'7-153.3
strychnin
I00
59"0 (43-I-80.7)
131.4 (105-1-164-2)
electroshock
100
83.7
(73" 9-94' 7)
REFERENCES 1. M. ROCHA E SILVA, W. T. BERALDO and G. ROSNNFELD; Amer. J. Physiol. 156 261 (1949). 2. A. S. ANDRADE and M. ROCHA E SILVA; Biochem. J. 64 701 (1956). 3. B. PERNOW; Acta Physiol. Scand. 29 suppl. 105 (1953). 4. W. FELDBERG and S. L. SHF.RVOOD; J. Physiol. 102 3P (1953). 5. V. KR~:BS and J. VAN~OEK; Physiol. bohemoslov. 8 167 (1959) (in Russian). 6. T. J. HALEY and W. G. McCoRMICK; Brit. J. Pharmacol. 12 12 (1957). 7. J. VAN~CEK, V. KREBS, E. SCHEER and T. BIELEKE; J. Amer. Pharm. Ass., Sci. Ed. 49 178 (1960). 8. J. T. LITCHFIELD and F. W. WILCOXON; J. Pharmacol. 96 99 (1949). 9. R. ~APEK; CS. ]ysiol. 9 283 (1960) (in Czech). 10. G. ZETLER; Arch. Exp. Path. Pharmakol. 228 513 (1956). 11. G. ZETLER; Arch. Exp. Path. Pharmakol. 237 11 (1959). 12. P. STERN and V. DosRiC; In Psychotropis Drugs, p. 448 (Edited by S. GARATTINI and V. GHET'rI) Elsevier, 1957. 13. U. S. EULER and B. PERNOW; Nature, 174 184 (1954). 14. M. ROCHA E SILVA; XXI International CongYess oi Physiological Sciences Buenos Aires 1959, Symposia and Special Lectures, p. 50.