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The effect of aprotinin on the harmine-induced tremor in lymphostatic encephalopathic and normal rats
European Journal of Pharmacology, 32 (1975) 365--369 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication THE E F F E C T OF APROTININ ON THE HARMINE-INDUCED T R E M O R IN LYMPHOSTATIC ENCEPHALOPATHIC AND NORMAL RATS G U N T E R BACK and G E R H A R D SEIDEL
Abteilung fiir Pharmakologie der Medizinischen Hochschule Liibeck, D-2400 Liibeck, Germany Received 19 March 1975, accepted 15 April 1975
G. BACK and G. SEIDEL, The effect of aprotinin on the harmine-induced tremor in lymphostatic encephalopathic and normal rats, European J. Pharmacol. 32 (1975) 365--369. In normal and lymphostatic encephalopathic rats, aprotinin pretreatment alters the duration of the harmine tremor and the cerebral concentration of the alkaloid; low doses shorten the tremor and decrease the concentration, a high dose causes the opposite. Aprotinin may decrease or increase the blood--brain barrier permeability of harmine. Because the findings are similar in both groups of rats the blood--brain barrier for harmine is assumed to be intact in lymphostatic encephalopathy. However, sick rats possibly suffer from an impaired cerebral elimination of harmine. Lymphostatic encephalopathy Blood--brain barrier
Aprotinin
1. Introduction Lymphostatic encephalopathy is a typical disease which, in animals as well as in man, develops after blockade of the cervical lymph flow {review by Ob~l et al., 1973). In lymphostatic encephalopathic rats treated with harmine, tremor duration is prolonged and there are elevated concentrations of the alkaloid in brain. In sick animals, pretreatment with antiinflammatory agents of different potency {e.g. acetylsalicylic acid, alclofenac, aprotinin, cellulose sulfate, coumarin, indomethacin, pentosan polysulfate, rutin) shortens the harmine tremor and decreases the alkaloid concentration in brain. The same is found in normal animals. Therefore, it was assumed that lymphostatic encephalopathic rats suffer from an inhibited lymphatic elimination of harmine from brain w i t h o u t impairment of the blood--brain barrier for the alkaloid. Furthermore, it was concluded that the above cited agents reduce the blood-brain barrier permeability for harmine in lym-
Harmine
Permeability
phostatic encephalopathic as well as in normal rats (Back and Seidel, 1975). As aprotinin reduces the permeability of the blood--brain barrier for 13 l iodine.labelle d albumin in rabbits (Blfimel, 1969) the effects of this polypeptide on the harmine tremor and the time--concentration curves of the alkaloid in brain of lymphostatic encephalopathic rats were studied. A parallel study was done with normal rats in order to analyse the effect of aprotinin on the underlying disturbance in lymphostatic encephalopathy.
2. Materials and methods The cervical lymph nodes of male Wistar rats (140--200 g) were extirpated using techniques similar to those of FSldi et al. (1963). For the controls, sham-operations were performed. All operations were done in the morning using pentobarbital (40--50 mg/kg i.p.) anaesthesia.
366
G. BACK, G. SEIDEL
3 days later, after i.p. pretreatment with aprotinin, harmine was injected into a tail vein within 10 sec. The tremor began 10 sec later. The duration of the tremor was observed by placing each rat in a transparent plastic cylinder. Once every min, a noise was produced by rattling a perforated plastic disc to arouse hidden tremor activity. Control experiments were performed in parallel. For the estimation of harmine in brain the rats were killed by decapitation. The control experiments were done the next or the preceding day. Harmine was extracted from brain and plasma and measured fluol~ometrically (Zetler et al., 1974). The binding of harmine to the rat plasma proteins was determined by means of the Sephadex gel filtration technique used by Zetler et al. (1974).
pretteatmentw g
4 x 10000 U/k 9
Drugs and chemicals: aprotinin (Trasylol ®), Bayer, Leverkusen, 10,000 U/ml; harmine hydrochloride, Fluka, Buchs/Switzerland, solution in 0.1 N H2SO4, 3.33 mg/ml; pentobarbital sodium (Nembutal ®), Abbott, Ingelheim, 50 mg/ml; Sephadex v G 25 M, Pharmacia, Uppsala/Sweden. Other reagents of analytical grade, Merck, Darmstadt.
3. Results In lymphostatic encephalopathic rats (fig. 1) one single dose of 10,000 U/kg of aprotinin was without any effect whereas repeated treatment with 10,000 U/kg of aprotinin reduced the tremor duration by about 20%. 1 min after the harmine injection, the alkaloid concentration in the brains of these animals was 19% lower
~ 100000 U/kg
/. x 40000 U/k¢
rb'~ ~Cm
n,$.
rQ~
rC~
r(:l~
£ 5 mglkg
10 mg/kg
10m g / ~
5mg/kg
10mg/kg
Fig. 1. Duration of harmine tremor (mean + S.E.M.) in lymphostatic encephalopathic rats and sham-operated controls. Effects of pretreatment with aprotinin: 10,000 U/kg (i.p. 2 hr prior to harmine); 4 x 10,000 U/kg (i.p. 10,000 U/kg once a day for 4 days, first dose in the evening of the operation day, other doses in the morning, last dose 2 hr prior to harmine); 100,000 U/kg (i.p. 2 hr prior to harmine). C: Control; T: Treatment. The figure in a given column indicates the number of animals/group.
APROTININ AND HARMINE TREMOR
367
concentrations in the brain of aprotinin pretreated and of untreated rats were 3.80 -+ 0.27 and 3.75 + 0.30/~g/g wet weight respectively (n in each group = 10). Pretreatment with one high dose of 100,000 U/kg of aprotinin increased the harmine-tremor duration by 18% in lymphostatic encephalopathic rats and by 9% in sham-operated rats (fig. 1). The time--concentration curve of hatmine in the brains of normal rats was raised by this pretreatment (fig. 2b). The differences in brain concentrations of harmine were still statistically significant 60 min after harmine administration. Repeated pretreatment of normal rats with 10,000 U/kg of aprotinin or one single dose of 100,000 U/kg did not change the plasma con-
than that in the controls. 60 min after the injection the difference in concentrations was less marked (fig. 2a). The results were similar to those in sick rats, when sham-operated animals were treated repeatedly with 10,000 U/kg of aprotinin; that is tremor duration was shortened by 17% {fig. 1). The time--concentration curve for harmine was also lowered by aprotinin pretreatment of normal animals and the effect of aprotinin was again evident from 1 to 30 min after i.v. injection of harmine. In sham-operated rats, pretreatment with 4 doses of 40,000 U/kg of aprotinin each did not alter the harmine-tremor duration (fig. 1). In this sham-operated group, 33 min after administration of 10 mg/kg of harmine alkaloid
~, lo-
"~ 10.
5.
5"
3
3 ,E o ,a
I i
I o
E
S
8 =-
"~ 0.5
0.1
'
30
60
90
0.1 120 min
30
60
90
120 mln
Fig. 2a,b. T i m e course of harmine c o n c e n t r a t i o n s in the brain of l y m p h o s t a t i c encephalopathic (a) and normal (sham-operated) (b) rats (means and fiduciai limits f o r p = 0.05, n = 5) and duration of t r e m o r (mean -+ S.E.M., n = 10) after i.v. injection o f 10 mg/kg of harmine, o, aprotinin p r e t r e a t m e n t (a: 4 x 10,000 U/kg i.p., b: 100,000 U/kg i.p., see legend to fig. 1); e, controls.
368
centrations of harmine. 30 min after the injection, the harmine concentrations in the pretreated groups were 2.12 + 0.23 and 2.07 + 0.09 pg/ml, respectively, and in the control groups 2.10 + 0.13 and 2.13 + 0.14 pg/ml, respectively (n in each group = 10). Furthermore, the plasma--protein binding of harmine was not changed by the pretreatments: it remained constant at a b o u t 89%. The harmine concentration in brain at the termination of the tremor was estimated 30 min after i.v. injection of the alkaloid. All values, obtained by extrapolation from the tremor times to the time--concentration curves, were between 3.2 and 4.0 pg/g wet weight.
4. Discussion The effects of aprotinin pretreatment may be interpreted as effects on the blood--brain barrier permeability in normal as well as in lymphostatic encephalopathic rats. This view seems to be supported by the altered cerebral harmine concentration just after the alkaloid injection and by the unchanged half-life times for harmine in brain. The prolonged actions of harmine in lymphostatic encephalopathic rats may be considered a consequence of the impaired lympathic elimination of the alkaloid from the brain (Back and Seidel, 1975). This interpretation of the present results seems to be supported by the finding of constant plasma concentrations of harmine witho u t any change of the protein binding of the alkaloid by aprotinin pretreatment. Furthermore, cerebral susceptibility to harmine remained constant as the harmine concentrations at the termination of the tremor did not change and agree with those found previously (Back and Seidel, 1975; Zetler et al., 1974). The possible decrease of blood--brain barrier permeability for harmine after low doses of aprotinin could be a consequence of the antiphlogistic actions of the polypeptide (Kaller et al., 1966). This interpretation is in agreement with that of very similar effects of other anti-
G. BACK, G. SEIDEL
inflammatory agents (Back and Seidel, 1975). High doses of aprotinin possibly open the blood--brain barrier for harmine (see fig. 1, 100,000 U/kg). This is reminiscent of the ineffectiveness of high doses of aprotinin in reducing the increased Evans blue content in brains of lymphostatic encephalopathic rats (Wendel and Seidel, 1972) and of noxious effects of other substances on the blood--brain barrier (Jeppsson and Olin, 1972; RSssner and Nussstein, 1972; Pardridge et al., 1973). High doses of acetylsalicylic (ASA) acid, may also lead to a lesion of the blood--brain barrier since pretreatment of rats with ASA, 400 mg/kg p.o. 2 hr prior to i.v. harmine, increased the harmine tremor (Seidel and Back, unpublished results). Aprotinin may change the blood--brain barrier permeability for other substances besides harmine. Increase or decrease of the blood-brain barrier permeability by aprotinin may be of clinical relevance when the agent is administered in combination with other drugs.
References Back, G. and G. Seidel, 1975, Effect of antiphlogistics on blood--brain barrier in lymphostatic encephalopathic and in healthy rats, Agents and Actions 5 (in press.). Bliimel, G., 1969, Uber die Beeinflussung yon Permeabilit~/tsstSrungen im Gehirn durch natiirliche ProteinasenhemmkSrper, Langenbecks Arch. Klin. Chir. 325, 310. FSldi, M., E. Csanda, F. Ob~l, I. Madar~sz, G. Szeghy and O.T. Zolt~in, 1963, llber Wirkungen der Unterbindung der Lymphgefi~sse und Lymphknoten des Halses auf das Zentralnervensystem im Tierversuch, Z. Ges. Exptl. Med. 137, 483. Jeppsson, P.G. and T. Olin, 1972, Lesions of the blood--brain barrier following selective injection of contrast media into the vertebral artery in rabbits, Acta Radiol. (Stockholm) 12, 271. Kaller, H., F. Hoffmeister and G. Kroneberg, 1966, Die Wirkung yon Trasylol auf verschiedene Odemformen der Rattenpfote, Arch. Intern. Pharmacodyn. 161,398. Ob~l, F., I. Madar~isz, O.T. Zolt~n, E. Csanda and M. FSldi, 1973, Effect of the disturbance of cervical lymph drainage (lymphostatic encephalopathy) upon the EEG and cerebral function, Recent De-
APROTININ AND HARMINE TREMOR velopments of Neurobiology in Hungary IV, 215. Pardridge, U.M., I.L. Crawford and J.D. Connor, 1973, Permeability changes in the blood--brain barrier induced by nortriptylin and chlorpromazin, Toxicol. Appl. Pharmacol. 26, 49. R6ssner, W. and R. Nussstein, 1972, Untersuchung des Verlaufes der Wirkung von Bis-tri~ithylzinnsulfat auf die Permeabilit~it der Blut--Hirn-Schranke, Arzneim. Forsch. 22, 1372.
369 Wendel, U. and G. Seidel, 1972, Kinins -- evidence for the involvement in lymphostatic encephalopathy in rats, Pharmacology 7, 17. Zetler, G., G, Back and H. Iven, 1974, Pharmacokinetics in the rat of the hallucinogenic alkaloids harmine and harmaline,.Naunyn-Schmiedeb. Arch. Pharmacol. 285, 273.
Short communication THE E F F E C T OF APROTININ ON THE HARMINE-INDUCED T R E M O R IN LYMPHOSTATIC ENCEPHALOPATHIC AND NORMAL RATS G U N T E R BACK and G E R H A R D SEIDEL
Abteilung fiir Pharmakologie der Medizinischen Hochschule Liibeck, D-2400 Liibeck, Germany Received 19 March 1975, accepted 15 April 1975
G. BACK and G. SEIDEL, The effect of aprotinin on the harmine-induced tremor in lymphostatic encephalopathic and normal rats, European J. Pharmacol. 32 (1975) 365--369. In normal and lymphostatic encephalopathic rats, aprotinin pretreatment alters the duration of the harmine tremor and the cerebral concentration of the alkaloid; low doses shorten the tremor and decrease the concentration, a high dose causes the opposite. Aprotinin may decrease or increase the blood--brain barrier permeability of harmine. Because the findings are similar in both groups of rats the blood--brain barrier for harmine is assumed to be intact in lymphostatic encephalopathy. However, sick rats possibly suffer from an impaired cerebral elimination of harmine. Lymphostatic encephalopathy Blood--brain barrier
Aprotinin
1. Introduction Lymphostatic encephalopathy is a typical disease which, in animals as well as in man, develops after blockade of the cervical lymph flow {review by Ob~l et al., 1973). In lymphostatic encephalopathic rats treated with harmine, tremor duration is prolonged and there are elevated concentrations of the alkaloid in brain. In sick animals, pretreatment with antiinflammatory agents of different potency {e.g. acetylsalicylic acid, alclofenac, aprotinin, cellulose sulfate, coumarin, indomethacin, pentosan polysulfate, rutin) shortens the harmine tremor and decreases the alkaloid concentration in brain. The same is found in normal animals. Therefore, it was assumed that lymphostatic encephalopathic rats suffer from an inhibited lymphatic elimination of harmine from brain w i t h o u t impairment of the blood--brain barrier for the alkaloid. Furthermore, it was concluded that the above cited agents reduce the blood-brain barrier permeability for harmine in lym-
Harmine
Permeability
phostatic encephalopathic as well as in normal rats (Back and Seidel, 1975). As aprotinin reduces the permeability of the blood--brain barrier for 13 l iodine.labelle d albumin in rabbits (Blfimel, 1969) the effects of this polypeptide on the harmine tremor and the time--concentration curves of the alkaloid in brain of lymphostatic encephalopathic rats were studied. A parallel study was done with normal rats in order to analyse the effect of aprotinin on the underlying disturbance in lymphostatic encephalopathy.
2. Materials and methods The cervical lymph nodes of male Wistar rats (140--200 g) were extirpated using techniques similar to those of FSldi et al. (1963). For the controls, sham-operations were performed. All operations were done in the morning using pentobarbital (40--50 mg/kg i.p.) anaesthesia.
366
G. BACK, G. SEIDEL
3 days later, after i.p. pretreatment with aprotinin, harmine was injected into a tail vein within 10 sec. The tremor began 10 sec later. The duration of the tremor was observed by placing each rat in a transparent plastic cylinder. Once every min, a noise was produced by rattling a perforated plastic disc to arouse hidden tremor activity. Control experiments were performed in parallel. For the estimation of harmine in brain the rats were killed by decapitation. The control experiments were done the next or the preceding day. Harmine was extracted from brain and plasma and measured fluol~ometrically (Zetler et al., 1974). The binding of harmine to the rat plasma proteins was determined by means of the Sephadex gel filtration technique used by Zetler et al. (1974).
pretteatmentw g
4 x 10000 U/k 9
Drugs and chemicals: aprotinin (Trasylol ®), Bayer, Leverkusen, 10,000 U/ml; harmine hydrochloride, Fluka, Buchs/Switzerland, solution in 0.1 N H2SO4, 3.33 mg/ml; pentobarbital sodium (Nembutal ®), Abbott, Ingelheim, 50 mg/ml; Sephadex v G 25 M, Pharmacia, Uppsala/Sweden. Other reagents of analytical grade, Merck, Darmstadt.
3. Results In lymphostatic encephalopathic rats (fig. 1) one single dose of 10,000 U/kg of aprotinin was without any effect whereas repeated treatment with 10,000 U/kg of aprotinin reduced the tremor duration by about 20%. 1 min after the harmine injection, the alkaloid concentration in the brains of these animals was 19% lower
~ 100000 U/kg
/. x 40000 U/k¢
rb'~ ~Cm
n,$.
rQ~
rC~
r(:l~
£ 5 mglkg
10 mg/kg
10m g / ~
5mg/kg
10mg/kg
Fig. 1. Duration of harmine tremor (mean + S.E.M.) in lymphostatic encephalopathic rats and sham-operated controls. Effects of pretreatment with aprotinin: 10,000 U/kg (i.p. 2 hr prior to harmine); 4 x 10,000 U/kg (i.p. 10,000 U/kg once a day for 4 days, first dose in the evening of the operation day, other doses in the morning, last dose 2 hr prior to harmine); 100,000 U/kg (i.p. 2 hr prior to harmine). C: Control; T: Treatment. The figure in a given column indicates the number of animals/group.
APROTININ AND HARMINE TREMOR
367
concentrations in the brain of aprotinin pretreated and of untreated rats were 3.80 -+ 0.27 and 3.75 + 0.30/~g/g wet weight respectively (n in each group = 10). Pretreatment with one high dose of 100,000 U/kg of aprotinin increased the harmine-tremor duration by 18% in lymphostatic encephalopathic rats and by 9% in sham-operated rats (fig. 1). The time--concentration curve of hatmine in the brains of normal rats was raised by this pretreatment (fig. 2b). The differences in brain concentrations of harmine were still statistically significant 60 min after harmine administration. Repeated pretreatment of normal rats with 10,000 U/kg of aprotinin or one single dose of 100,000 U/kg did not change the plasma con-
than that in the controls. 60 min after the injection the difference in concentrations was less marked (fig. 2a). The results were similar to those in sick rats, when sham-operated animals were treated repeatedly with 10,000 U/kg of aprotinin; that is tremor duration was shortened by 17% {fig. 1). The time--concentration curve for harmine was also lowered by aprotinin pretreatment of normal animals and the effect of aprotinin was again evident from 1 to 30 min after i.v. injection of harmine. In sham-operated rats, pretreatment with 4 doses of 40,000 U/kg of aprotinin each did not alter the harmine-tremor duration (fig. 1). In this sham-operated group, 33 min after administration of 10 mg/kg of harmine alkaloid
~, lo-
"~ 10.
5.
5"
3
3 ,E o ,a
I i
I o
E
S
8 =-
"~ 0.5
0.1
'
30
60
90
0.1 120 min
30
60
90
120 mln
Fig. 2a,b. T i m e course of harmine c o n c e n t r a t i o n s in the brain of l y m p h o s t a t i c encephalopathic (a) and normal (sham-operated) (b) rats (means and fiduciai limits f o r p = 0.05, n = 5) and duration of t r e m o r (mean -+ S.E.M., n = 10) after i.v. injection o f 10 mg/kg of harmine, o, aprotinin p r e t r e a t m e n t (a: 4 x 10,000 U/kg i.p., b: 100,000 U/kg i.p., see legend to fig. 1); e, controls.
368
centrations of harmine. 30 min after the injection, the harmine concentrations in the pretreated groups were 2.12 + 0.23 and 2.07 + 0.09 pg/ml, respectively, and in the control groups 2.10 + 0.13 and 2.13 + 0.14 pg/ml, respectively (n in each group = 10). Furthermore, the plasma--protein binding of harmine was not changed by the pretreatments: it remained constant at a b o u t 89%. The harmine concentration in brain at the termination of the tremor was estimated 30 min after i.v. injection of the alkaloid. All values, obtained by extrapolation from the tremor times to the time--concentration curves, were between 3.2 and 4.0 pg/g wet weight.
4. Discussion The effects of aprotinin pretreatment may be interpreted as effects on the blood--brain barrier permeability in normal as well as in lymphostatic encephalopathic rats. This view seems to be supported by the altered cerebral harmine concentration just after the alkaloid injection and by the unchanged half-life times for harmine in brain. The prolonged actions of harmine in lymphostatic encephalopathic rats may be considered a consequence of the impaired lympathic elimination of the alkaloid from the brain (Back and Seidel, 1975). This interpretation of the present results seems to be supported by the finding of constant plasma concentrations of harmine witho u t any change of the protein binding of the alkaloid by aprotinin pretreatment. Furthermore, cerebral susceptibility to harmine remained constant as the harmine concentrations at the termination of the tremor did not change and agree with those found previously (Back and Seidel, 1975; Zetler et al., 1974). The possible decrease of blood--brain barrier permeability for harmine after low doses of aprotinin could be a consequence of the antiphlogistic actions of the polypeptide (Kaller et al., 1966). This interpretation is in agreement with that of very similar effects of other anti-
G. BACK, G. SEIDEL
inflammatory agents (Back and Seidel, 1975). High doses of aprotinin possibly open the blood--brain barrier for harmine (see fig. 1, 100,000 U/kg). This is reminiscent of the ineffectiveness of high doses of aprotinin in reducing the increased Evans blue content in brains of lymphostatic encephalopathic rats (Wendel and Seidel, 1972) and of noxious effects of other substances on the blood--brain barrier (Jeppsson and Olin, 1972; RSssner and Nussstein, 1972; Pardridge et al., 1973). High doses of acetylsalicylic (ASA) acid, may also lead to a lesion of the blood--brain barrier since pretreatment of rats with ASA, 400 mg/kg p.o. 2 hr prior to i.v. harmine, increased the harmine tremor (Seidel and Back, unpublished results). Aprotinin may change the blood--brain barrier permeability for other substances besides harmine. Increase or decrease of the blood-brain barrier permeability by aprotinin may be of clinical relevance when the agent is administered in combination with other drugs.
References Back, G. and G. Seidel, 1975, Effect of antiphlogistics on blood--brain barrier in lymphostatic encephalopathic and in healthy rats, Agents and Actions 5 (in press.). Bliimel, G., 1969, Uber die Beeinflussung yon Permeabilit~/tsstSrungen im Gehirn durch natiirliche ProteinasenhemmkSrper, Langenbecks Arch. Klin. Chir. 325, 310. FSldi, M., E. Csanda, F. Ob~l, I. Madar~sz, G. Szeghy and O.T. Zolt~in, 1963, llber Wirkungen der Unterbindung der Lymphgefi~sse und Lymphknoten des Halses auf das Zentralnervensystem im Tierversuch, Z. Ges. Exptl. Med. 137, 483. Jeppsson, P.G. and T. Olin, 1972, Lesions of the blood--brain barrier following selective injection of contrast media into the vertebral artery in rabbits, Acta Radiol. (Stockholm) 12, 271. Kaller, H., F. Hoffmeister and G. Kroneberg, 1966, Die Wirkung yon Trasylol auf verschiedene Odemformen der Rattenpfote, Arch. Intern. Pharmacodyn. 161,398. Ob~l, F., I. Madar~isz, O.T. Zolt~n, E. Csanda and M. FSldi, 1973, Effect of the disturbance of cervical lymph drainage (lymphostatic encephalopathy) upon the EEG and cerebral function, Recent De-
APROTININ AND HARMINE TREMOR velopments of Neurobiology in Hungary IV, 215. Pardridge, U.M., I.L. Crawford and J.D. Connor, 1973, Permeability changes in the blood--brain barrier induced by nortriptylin and chlorpromazin, Toxicol. Appl. Pharmacol. 26, 49. R6ssner, W. and R. Nussstein, 1972, Untersuchung des Verlaufes der Wirkung von Bis-tri~ithylzinnsulfat auf die Permeabilit~it der Blut--Hirn-Schranke, Arzneim. Forsch. 22, 1372.
369 Wendel, U. and G. Seidel, 1972, Kinins -- evidence for the involvement in lymphostatic encephalopathy in rats, Pharmacology 7, 17. Zetler, G., G, Back and H. Iven, 1974, Pharmacokinetics in the rat of the hallucinogenic alkaloids harmine and harmaline,.Naunyn-Schmiedeb. Arch. Pharmacol. 285, 273.