Naunyn-Schmiedeberg's

Naunyn-Schmiedeberg's Arch. Pharmacol. 306, 135-146 (1979)

Archivesof

Pharmacology 9 by Springer-Verlag 1979

Effects of Tetrahydropapaveroline and Salsolinol on Cerebral Monoamine Metabolism and Their Interactions with Psychopharmacological Drugs N. Awazi and H. C. Guldberg Department of Pharmacology and Clinical Pharmacology with Toxicology, University of Trondheim, Regional Hospital, N-7000 Trondheim, Norway

Summary. The effects of the tetrahydroisoquinoline alkaloids: tetrahydropapaveroline (THP) and salsolinol alone or in combination with psychopharmacological drugs on cerebral monoamine metabolism in the rat were studied. 1. D,L-THP administered intraventricularly to rats in doses of 70-2501ag induced a sustained fall in striatal dopamine concentrations with a maximum depletion occurring at 3 - 6 h after drug administration. The striatal homovanillic acid (HVA) concentration was increased significantly at 3 h after THP administration. Striatal 5-hydroxytryptamine (5-HT) and diencephalic noradrenaline or 5-HT levels were decreased following the THP administration. THP was not found to induce any in vivo alterations in monoamineoxidase (MAO) or catechol-O-methyl transferase (COMT) activities in rat brain. 2. Salsolinol (250 gg intraventricularly) caused a delayed rise in striatal dopamine concentration which at 6h after the drug administration rose to about 3 times the control value. The effects of salsolinol on striatal HVA showed a fall 3 h after drug administration. Diencephalic noradrenaline and 5-HT concentrations were lower than controls 6h after drug administration. 3. In order to investigate the mechanisms underlying the biochemical effects of THP or salsolinol on brain catecholamines and 5-HT, psychopharmacological drugs were given to rats together with THP or salsolinol (250 lag intraventricularly). Haloperidol (5mg/kg, i.p.) pretreatment 45min before THP partially prevented the striatal dopamine reduction and the HVA increase induced by THP alone; furthermore, haloperidol reversed the THP effect on diencephalic noradrenaline but not 5-HT. Send offprint requests to H. C. Guldberg at the above address

Desmethylimipramine (25mg/kg, i.p.) administered 45 min before THP prevented the THP induced depletions of the monoamines. On the other hand, gamma-hydroxybutyrate pretreatments of the rats were not found to affect the THP induced alterations in catecholamines. The salsolinol induced rise in striatal dopamine was prevented by ~-methyl-p-tyrosine (300 mg/kg, i.p., administered twice in 24 h) pretreatment while the salsolinol induced fall in diencephalic noradrenaline was not affected. Reserpine (5 mg/kg, s.c.) pretreatment of rats prevented the salsolinol induced rise in striatal dopamine and similarly haloperidol (5mg/kg, i.p.) affected the dopamine but not the noradrenaline changes caused by salsolinol. 4. For the mode of action of THP it is likely that the alkaloid is taken up in catecholaminergic, and possibly to a small extent serotonergic, neurones and thereby displacing dopamine or noradrenaline from storage sites. THP or a metabolite may act as a false transmitter and/or affecting catecholaminergic receptors directly and independently of a presynaptic release. THP and salsolinol appear to have some basic differences in their mode of action on dopaminergic, but probably not on noradrenergic, mechanisms. For salsolinol the concept of a false transmitter seems most unlikely for the effect on dopaminergic neurones and, in fact, it may be that salsotinol in addition to actually causing increased synthesis of dopamine, has agonistic, while THP has antagonistic, properties in acting on dopamine receptors. 5. Salsolinol was found to cause hypothermia while THP caused hyperthermia in the rat. THP and salsolinol induced abnormal motor behaviour in rats in the form of gnawing, licking, chewing, head-neck rocking and increased motor activity and the motor dysfunctions were most apparent for THP.

0028-1298/79/0306/0135/$02.40

136 These dyskinetic p h e n o m e n a are discussed briefly in relation to dopaminergic mechanisms. Key words: Tetrahydropapaveroline - Salsolinol Catecholamines - B o d y temperature - M o t o r functions.

Introduction Tetrahydroisoquinolines (TIQs) are c o m p o u n d s that, under certain circumstances, can be f o r m e d in vitro and p r o b a b l y also in vivo in nervous tissue by the condensation o f catecholamines with intermediary aldehydic metabolites. Salsolinol (l-methyl-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline), the cyclization p r o d u c t o f dopamine with acetaldehyde, has been demonstrated in rat brain during ethanol metabolism ( Y a m a n a k a et al., 1970; Davies and Walsh, 1970; Collins and Bigdeli, 1975) and in the urine o f parkinsonian patients during treatment with L - D o p a (Sandler et al., 1973). T e t r a h y d r o p a p a v e r o l i n e (THP, 1,2,3,4-tetrahydro6,7-dihydroxy-l-(3',4'-dihydroxybenzyl)-isoquinoline), the condensation p r o d u c t o f d o p a m i n e with its aldehyde metabolite dopacetaldehyde, was f o u n d in rat brain after chronic treatment with L - D o p a (Turner et al., 1974) and in urine during treatment with L - D o p a o f parkinsonian patients (Sandler et al., 1973). The p r o p o s e d mechanisms o f action o f the T I Q s on adrenergic neurons are: I. that they m a y act on catecholamine u p t a k e systems (Heikkila et al., 1971 ; Cohen et al., 1972, 1974; T e n n y s o n et al., 1973; Locke et al., 1973; Alpers et al., 1975), II. that they m a y possess the properties o f false adrenergic neurotransmitters (Tennyson et al., 1973; Locke et al., 1973; Mytillineou et al., 1974), III. that they m a y have agonist/antagonist receptor activity (Sourkes, 1971; Sheppard and Burghardt, 1974; Simpson, 1975; Feller et al., 1975; D o u g a n et al., 1975) and IV. that they m a y inhibit catechol-O-methyl transferase, C O M T (Rubenstein and Collins, 1973; G u l d b e r g and Marsden, 1975; Giovine et al., 1976) and monoamineoxidase, M A O (Cohen and Katz, 1975; Giovine et al., 1976). Salsolinol, T H P or some o f their metabolites have been shown to be pharmacologically active compounds. These alkaloids m a y exert b o t h sympathomimetic and sympatholytic activities (Hjort et al., 1942; Santi et al., 1967) and induce certain effects on C N S functions (Hjort et al., 1942; Trepanier and Sunder, 1973; Costall et al., 1975). There is some evidence f r o m animal experiments that T I Q alkaloids m a y modify the central actions o f d o p a m i n e and Hornykiewicz (1974) briefly reviewed

Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979) findings that point to the possibilities that these condensation products could potentially either facilitate or inhibit the actions o f striatal dopamine. However, there are few data concerning the effects o f these alkaloids on m o n o a m i n e metabolism in brain in vivo (Collins and Bigdeli, 1975; Livrea et al., 1976). The present investigation was undertaken to study the effects o f T H P and salsolinol on cerebral mon o a m i n e metabolism and some gross behavioural parameters. The results m a y throw some light on the role o f these alkaloids in functions o f m o n o a m i n e r g i c systems.

Material and Method Animals. Albino rats (Wistar) of the male (unless stated specifically

for the experiment) sex and weighing 180- 250 g were used. During the period of drug action the animals were kept in a room with thermostatically controlled temperature (26-27~ The body temperature of the animals was measured with a clinical rectal thermometer. For the intraventricular injection of drugs or vehicle the animals were lightly anaesthetized with ether or sodium pentobarbitone (50 mg/kg i.p.). The drugs in volumes of 1 - 10 gl were injected into the lateral ventricle using a David Kopf stereotaxic instrument. Control animals received an equal volume of 0.9 % W/V NaC[ solution (saline) administered by the same route. The intraventricular injection was made at a rate of 1 gl/min and the needle was left in place for about 1 min after the injection. The animals were killed by decapitation following the period of drug study. D,L-tetrahydropapaveroline (1,2,3,4-tetrahydro-6,7-dihydroxy-l-(3",4'-dihydroxybenzyl)-isoquinoline), salsolinol (1-methyl6,7-dihydroxy-tetradroisoquinoline) and isosalsolinol (l-methyl7,8-dihydroxy-tetrahydroisoquinoline) as the hydrochloride salts were synthesized in our laboratory by the method of the PictetSpengler condensation of dopamine with an aldehyde (Pyman, 1909; Cohen et al., 1972). The other drugs were obtained commercially: reserpine (Serpasil, Ciba), desmethylimipramine (Geigy), c~-methyl-p-tyrosinemethylester HCI (Labkemi A.B.), haloperidol (Mekos) and sodium gammahydroxybutyrate (Sigma). These drugs were injected i.p., except reserpine which was given s.c., and the control animals received an equal volume of vehicle by comparable routes. The drugs were dissolved in 0.9 % W/V NaC1 solution which also was used for the vehicle. Biochemical Procedures. The brains were removed quickly and the brain regions dissected as described by Lorens and Guldberg (1974). The tissues were wrapped in aluminium foil and placed on dry ice and stored at - 20~C except for those used for the assays of catechol-Omethyl transferase and monoamine oxidase which were cooled to 0~C and assayed immediately. The tissue was weighed and homogenized in a glass homogenizer in 3 ml 1N hydrochloric acid in the presence of 50 mg/g sodium metabisulfite. The homogenate was centrifuged at 15.000x g for 20 min at 0~C and the protein precipitate reextracted with 1 ml of l N hydrochloric acid. The combined supernatant was transferred to a glass centrifuge tube and the pH brought to 4 by the addition of 5 N NaOH. The sample was centrifuged at 1,000 • g for 5 min and the clear supernatant was applied for the separation of acids and amines to a Dowex 1 x2 column (200-400mesh, anion exchange resin, Fluka A.G.) for homovanillic acid (HVA) and an aluminium oxide" column (neutral activity, grade 1, Merck) for dopamine and noradrenaline. The amine fraction was eluted with 4ml of 0.05N perchloric Drugs.

N. Awazi and H. C. Guldberg: TIQ Alkaloids and Cerebral Monoamines acid and the eluate was applied to a Dowex 50 x 4 (200- 400 mesh, cation exchange resin, Fluka A.G.) column after the addition of 0.25 ml of 10 M potassium acetate. The column was washed with 10mI of distilled water and dopamine and noradrenaline wereelua~ed with 4ml of 0.4N and 4N HCl respectively. Noradrenaline and dopamine were estimated spectrophotofluorometricallyby the method of Anton and Sayre (1965). HVA was estimated by the method of Juorio et al. (1966). 5-hydroxytryptamine (5-HT) was estimated according to the method of Bertler (1961) with the modification of Ahtee et al. (1970). It was found that THP, salsolinol or isosalsolinol did not interfere with the analytical procedures used for amine or HVA estimations. Recoveriesof compounds added to the homogenate were (means _+S.E.M., no. of experiments): 5-HT 60 _+1.5 ~ (8); dopamine 80 • 1.5 % (10); noradrenaline 75 +_ 2.0 ~ (10) and HVA 65 _+ 2.1% (8). The results have not been corrected for these recoveries. Catechol-O-methyl transferase (COMT) and monoamineoxidase (MAO) activities were measured by the method of Broch and Guldberg (1971) and Snyder and Hendley (1968) respectively. Statistical analyses were done using the Student's t-test.

Results

Effects of TIQs on Cerebral Monoamine Metabolism in the Rat Tetrahydropapaveroline and Monoamines. D,L-THP (250lag intraventricularly) caused a significant and sustained fall in rat striatal dopamine concentrations (Fig. l). The m a x i m u m depletion to about 50% of control values for dopamine (P < 0.001) occurred at 3 - 6 h and returned to normal values at about 18 h after drug administration. HVA, a major end-product of dopamine metabolism, was altered in that the striatal concentration was increased significantly (P < 0.05) at 3h (control: 0.43 • 0.09, after THP" 0.58 • 0.19; means • S.E.M. of 4 determinations). Diencephalic noradrenaline concentrations fell markedly (P < 0.001) at 3 - 6 h after T H P injection to values of about 40 % of controls and even at 18 h after T H P noradrenMine levels were significantly lowered (Fig. 1). 5-HT levels both in the striatum and the diencephalon were slightly, but significantly (P < 0.05), decreased following the T H P administration (Fig. 1). A comparison of the relative effectiveness of graded doses ( 1 0 - 2 5 0 l a g ) of D,L-THP on dopamine, HVA, noradrenaline and 5-HT in brain 3 h after the intraventricular injection of the drug is shown in Table 1. After T H P in a dose of 10 lag no significant change in striatal dopamine was detected while marked reductions were found after higher doses (70, 150 and 250,g) The maximal dopamine depletion was observed with a dose of 250ug of THP, while higher doses were found to be too toxic for the rats. The rise in the striatal H V A concentrations after T H P were of the same magnitude for the higher dose levels. Dience-

137

phalic noradrenaline concentrations were reduced with T H P doses of 150 and 2 5 0 , g by about 50~o and 80% respectively of control values. 5-HT in the diencephalon fell after T H P while striatal 5-HT was not significantly decreased (Table 1).

Salsolinol, Isosalsolinol and Monoamines. Salsolinol or isosalsolinol in doses of 250 lag intraventricularly were found to give a delayed rise in striatal dopamine (at 6 h following the drug administration), salsolinol promoting a rise to about 3 times the control value (P < 0.001) which returned to subnormal levels within 24 h (Fig. 2). The striatal dopamine concentration after sals01inol (100 lag) was 15 + 1.74 (4) lag/g which was nearly twice the control values while salsolinol in a dose of 350 lag intraventricularly was found to be too toxic (30 % of the animals died within 3 h). The effects of salsolinol on striatal H V A showed a fall 3h after drug administration. Noradrenaline in the diencephalon after salsolinol or isosalsolinol (250 lag) showed a reduction (P < 0.05) after 6 h which for salsolinol returned to normal values at 24 h while isosalsolinol caused a significant increase (P < 0.01) at 3 h and 24h (Fig.2). Diencephalic 5-HT levels after salsolinol (250 ~ag) were reduced at 3 and 6 h after the drug administration (Fig.2). The in vivo Effects of D,L-THP and Salsolinol on COMT and MAO Activities in Brain T H P (250lag, intraventricularly) was administered to male and female rats and C O M T and M A O activities were determined in homogenates prepared from brain 0.5 h and 3 h after drug administration. T H P was not found to induce any changes in C O M T activities in vivo in the stiatum (control: 127 _+ 4, 0.5h: t25 +_ 3, 3h: 130 + 8; mean activity of 4 determinations in nmole/h/g wet wt. • S.E.M.) or in the thalamus (control' 147 • 8, 0.5h: 151 • 3, 3h: 163 • 12). Similarly, M A O activities in vivo were not changed in the striatum (control: 8.6 • 0.62, 0.5 h" 8.4 • 1.25, 3 h: 7.6 • 0.85; mean activity of 4 determinations in lamole/h/g wet wt. • S.E.M.) or in the hypothalamus (control" 7.6 • 0.35,0.5h: 7.5 +_ 0.24,3h:7.0 • 0.55). Salsolinol (250 lag, intraventricularly) induced no changes in striatal or hypothalamic C O M T activities i h after drug administration (striatum control : 78 _ 5 and hypothalamus control: 120 • 8 and 1 h after salsolinol: 71 • 5 and 118 + 5 respectively; means, nmole/h/g wet wt. • S.E.M. of 3 determinations).

E/Yects of Various Pharmacological CompowTds on the TIQ Induced Changes in Monoamines Tetrahydropapaveroline. Haloperidol (5 mg/kg i.p.) had no significant effects on dopamine, noradrenaline and

I38

Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979)

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Table 1. Effect of various doses of D,L-THP on the dopamine, HVA, noradrenaline and 5-HT levels in rat brain Group

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5-HT levels in striatum or diencephalon but induced an elevation of the striatal HVA concentration (Tables 1 and 2). Haloperidol pretreatment 45min before the T H P (250pg) administration partially reversed the striatal dopamine reduction induced by T H P alone while the effect on diencephalic noradrenaline was more dramatic in that the combined treatment caused a

significant rise (0.01 > P > 0.001) in noradrenaline concentrations to approximately twice normal values (Table 2). Haloperidol was found to prevent the rise in striatal HVA concentration caused by T H P alone. In addition, brain 5-HT appeared not to be affected by the combined treatment with haloperidol and T H P (Table 2).

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140

Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979)

Table 2. Effects of D,L-THP on the dopamine, HVA, noradrenaline and 5-HT levels in rat brain after haloperidol Treatment

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Haloperidol (5 mg/kg i.p.) was administered 45 rain before D,L-THP(250 ~tg,intraventricularly) and the animals were killed 3 h after D,L-THP administration. Each value is the mean _+ S.E.M. of six determinations except for HVA which was assayed in only 3 samples. " Statistically significant differences between THP and THP + haloperidol groups: * P < 0.05; ** P < 0.01 u Statistically significant differences between haloperidol and haloperidol + T}tI' groups: * P < 0.05; ** P < 0.01

Table 3. Effect of D,L-THP on dopamine, noradrenaline and 5-HT in rat brain after desmethylimipramine Treatment

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0.43 • 0.01 (6) 0.30 ,+0.02~** (6) 0.39,+0.01 (4) 0.44_+0.01b* (6)

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Desmethylimipramine(DMI, 25 mg/kg i.p.) was given 1 h before D,L-THP(250 gg intraventricularly) or an equal volume of isotonic saline to controls. Animals were killed 3 h after the THP administration. Each value is the mean + S.E.M. of four to six determinations. " Significant difference between THP and THP + DMI groups: * P < 0.05; ** P < 0.001 b Significant difference between DMI and THP + DMI groups: * P < 0.05; ** P < 0.001

striatal 5 - H T as well as diencephalic 5 - H T a n d nora d r e n a l i n e (Fig. 3).

Salsolinol. ~-Methyl-p-tyrosine (~-MT methyl ester HC1) in doses o f 300 m g / k g i.p. was administered twice with 12h intervals to two groups of rats. Eight hours after the last dose o f c~-MT, the groups of a n i m a l s were given salsolinol (250~rg) or vehicle intraventricularly. F r o m the results in Table 4 it can be seen that c~-MT caused a m a r k e d fall in striatal d o p a m i n e a n d diencephalic n o r a d r e n a l i n e c o n c e n t r a t i o n s (0.01 > P > 0.001). Moreover, e - M T prevented the salsolinol i n d u c e d rise in striatal d o p a m i n e c o n c e n t r a t i o n which was double of the control while the salsolinol i n d u c e d fall in n o r a d r e n a l i n e was n o t affected significantly by e - M T . 5 - H T was n o t affected by ~ - M T a n d the salsolinol i n d u c e d fall in b r a i n 5 - H T was u n a l t e r e d by e - M T p r e t r e a t m e n t (Table 4). Reserpine (5 m g / k g s.c.) caused a m a r k e d reduction in striatal d o p a m i n e levels a n d a significant rise in H V A

levels 2 4 h after drug a d m i n i s t r a t i o n (Table 5). Salsolinol (250 gg), injected intraventricularly to rats pretreated with reserpine for 18 h a n d killed 6 h thereafter, reduced the m a g n i t u d e o f the reserpine-induced fall of d o p a m i n e a n d the salsolinol i n d u c e d rise in dop a m i n e was prevented by reserpine. Reserpine also prevented the salsolinol i n d u c e d rise in striatal H V A c o n c e n t r a t i o n s (Table 5). H a l o p e r i d o l (5 mg/kg i.p.) given to rats h a d n o significant effects o n the c o n c e n t r a t i o n s of b r a i n d o p a m i n e , n o r a d r e n a l i n e a n d 5-HT (Table 6). P r e t r e a t m e n t with haloperidol caused a blockade of the increase in striatal d o p a m i n e induced by salsolinol (250 gg intraventricularly) while the c o m b i n e d drug t r e a t m e n t h a d n o effects o n b r a i n levels of n o r a d r e n aline a n d 5-HT (Table 6).

Effects ~?f TIQs on Some CNS Functions of Rats. D j T H P injected intraventricularly to male a n d female rats caused a dose-related h y p e r t h e r m i a which was m a x i m a l

N. Awazi and H. C. Guldberg: TIQ Alkaloids and Cerebral Monoamines

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5- HT

NA

Fig. 3. Effect of gamma-hydroxybutyric acid (GHB)on the THP-induced changes of dopamine, HVA, noradrenaline and 5-HT levels in rat brain. GHB in doses of 350 mg/kg or 750 mg/kg i.p. were given 1.5 h after the intraventricular injection of D,L-THP (250 gg). Control animals received the same volume of saline by comparable routes. All animals were killed 3 h after the DJ_-THP administration. Each bar is the mean +_ S.E.M. of three to four determinations. * Statistically significant difference (P < 0.05) between THP and THP + GHB groups

Table 4. The effect of ~-methyl-p-tyrosine on the salsolinol induced changes in cerebral monoamines of the rat Time schedule of injection

Drug schedule

Treatment

Concentration gg/g wet. wt

Day 1

Day 2

Striatum

Diencephalon

19.00 h

07.00 h

10.00 h

Dopaminc

5-HT

Noradrenaline

5-HT

c~-MT ct-MT saline saline

c~-MT ~-MT saline saline

saline salsolinol saline salsolinol

1.74_+0.37 3-29_+0,77 *~ 8.68 _+0,43 16.64_+ 1.57 *b

0.31 _ + 0 . 0 3 0.28_+0.01 0.32 _+0.03 0.24_+0.02

0.84_+0.08 1.80_+0.05 *a 2.42 _+0.03 1.61 _+0.13 *b

0.41 _+0.03 0.41_+0.01 0.43 _+0.02 0.42_+0.02

Salsolinol or saline was injected intraventricularly 3 h after the administration of the last dose of c~-methyl-p-tyrosine (300 mg/kg i.p.) or saline. All animals were killed at 15.00h. Values are means based on 3 or 4 determinations _+ S.E.M. Significant difference between c~-MT and c~-MT + salsolino[ groups: * P < 0.02 b Significant difference between saline control and c~-MT + salsolinol groups: * P < 0.05

Table 5. Effect of salsolinol on striatal dopamine and HVA of reserpinized rats Treatments

HVA pg/g wet. wt.

Dopamine pg/g wet. wt.

Reserpine + saline Reserpine + salsolinol Saline control Salsolinol control

0.71 -+ 0.06 0.69• a* 0.37_+ 0,03 0.47 • 0.03

0.79 _+0.30 3,06_+1.26

Eighteen hours after the reserpine administration (5mg/kg s.c.), salsolinol (250 ~g) or isotonic saline were injected intraventricularly. All animals were killed 6 h after the intraventricular injection. Values are means based on 3 or 4 determinations __+ S.E.M. Significant difference between salsolinol and reserpine + salsolinol groups of rats: * P < 0.05

at 1 h and persisted for 3 - 5 h after the drug administration. The intraventricular injections of comparable volumes ( 1 - 1 0 gl) of 0.9 % W/V NaC1 solution were without effects on rectal temperature. The lowest dose (10 gg) tested of THP had no detectable effect on body temperature while the highest dose (250 gg) induced a mean rise of body temperature of 3 +_ 0.5 ~ C (mean + S.E.M. of 6 animals) at 1 h which was highly significantly different from controls (P < 0.001). Salsolinol and isosalsolinol administrations to rats caused a dose related hypothermia. The intraventricular injections of the highest doses (250 gg) of salsolinol or isosalsolinol caused a significant fall (0.01 > P > 0.001) in rectal temperatures at 3h after drug

142

Naunyn-Schmiedeberg'sArch. Pharmacol. 306 (1979)

Table6. The effectsof haloperidol on the salsolinol induced changes in cerebral monoamines of the rat Drug schedule

Saline Saline Hatoperidol Haloperidol

Striatum

saline salsolinol saline salsolinol

Diencephaton

Dopamine

5-HT

Noradrenaline

5-HT

8.68 + 0.43 17.65 _+1.56 9.22 _+0.31 8.22 _+0.69*

0.25 _+0.02 0.18 _+0.02 0.24 +_0.01 0.22 + 0.01

2.42 + 0.32 1.61 + 0.13 2.33 + 0.11 1.85 + 0.11

0.41 • 0.03 0.32 • 0.02 0.37 • 0.01 0.43 _+0.04

Haloperidol(5 mg/kgi.p.) was given 15 rain beforethe intraventricularinjectionof salsolinol(250gg) or saline and the animals werekilled6 h later. Amine concentrationsare expressedin gg/g wet tissue. Means + S.E.M. of 3 or 4 determinations. Significantly different from salsolinol control: * P < 0.05

administrations. The maximal falls in body temperature were noted at 3 h and were of the magnitude of 2.1~ + 0.2 (mean _+ S.E.M. of 6 animals). D,t-THP caused changes in motor functions of the rat which were recorded by observation. T H P (250 lag intraventricularly) induced dyskinetic phenomena of the repetitive type: chewing, licking, gnawing and headneck rocking which developed within 2 . 5 - 3 h after drug administration and lasted for at least 4h. Increased motor activity was also apparent in approximately 80 ~ of the animals. In rats treated with lower doses ( 5 0 - 1 0 0 lag) of THP, the abnormal movements were less pronounced and the lowest dose (10 lag) did not cause any abnormal behaviour. If haloperidol (5mg/kg, i.p.) was given 45min before D,L-THP (250 lag) the dyskinetic phenomena were prevented completely. In contrast to the behaviour of animals treated with haloperidol alone, the animals treated with haloperidol in combination with D,L-THP seemed to be more active. Salsolinol or isosalsolinol, in doses of 250 tag intraventricularly, also induced some abnormal movements in the form of gnawing (salivation/ licking/chewing) but these were transient (lasting 20rain) and occurred about 2 . 5 - 3 h after drug administration. In reserpinized rats the salsolinol induced gnawing movements were markedly reduced while rats pretreated with ~-methyl-p-tyrosine still exhibited compulsive gnawing behaviour after salsolinol.

Discussion

Effects of TIQs on Cerebral Monoamine Metabolism The present results have demonstrated that TIQs under certain experimental conditions may excert profound effects on cerebral cateeholamines in the rat. Moreover, there were striking differences in the effects on striatal

dopamine metabolism of the two TIQs tested: tetrahydropapaveroline (THP) and salsolinol. In relatively high doses (from about 100-250~tg, intraventricularly), T H P induced a sustained depletion of striatal dopamine with an initial rise in its acid metabolite HVA while salsolinol caused a somewhat delayed and marked increase in dopamine with a tendency to a fall in HVA. However, the effects of these TIQs were not confined to dopaminergic neurones in that diencephalic noradrenaline was depleted by T H P and also to some extent by salsolinol. Furthermore, it appeared from our results that serotonergic neurones could also be affected since falls were occassionally observed in brain 5-HT, especially after T H P administration. There are no reports, as far as we are aware, in the literature on the effects of exogenously administered TIQs on steady-state levels of cerebral monoamines. In pyrogallol pretreated rats with acute ethanol intoxication, the in vivo occurrence of salsolinol was demonstrated and, in addition, it was reported that brain levels of dopamine and noradrenaline were depleted (Collins and Bigdeli, t975). As regards brain dopamine, but not noradrenaline, this study is apparently at variance with our results but the experimental conditions were different and the presence of high amounts of acetaldehyde might have affected brain catecholamines. Recently, it was reported that following the systemic administration of THP, brain levels of HVA rose initially, which is in keeping with the present results, and also 5-HT metabolism was reported to be affected in that 5-hydroxyindoleacetic acid levels rose initially in rat brain after T H P (Livrea et al., 1976). T H P and salsolinol were reported to be inhibitors of C O M T in vitro (Rubenstein and Collins, 1973; Guldberg and Marsden, 1975) but the current study indicates that these catechol-containing alkaloids do not inhibit significantly COMT in vivo. The reason for this is unknown but it is observed for several COMT inhibitors that they adequately inhibit the enzyme in vitro but apparently are poor inhibitors in vivo

N. Awazi and H. C. Guldberg: TIQ Alkaloids and Cerebral Monoamines (Guldberg and Marsden, 1975). There are few reports in the literature on the effects of TIQs on MAO activity but they appeared to be weak MAO inhibitors in vitro (Collins et al., 1973). From the indirect evidence that mice treated with reserpine and TIQs showed a decrease in 3H-deaminated products in peripheral nerves compared to reserpine controls, it was suggested that TIQs may inhibit partially MAO in vivo (Cohen and Katz, 1975). In the present study, no evidence was found for THP being an inhibitor of rat brain MAO in vivo.

On the Mode of Action of TIQ Alkaloids on Cerebral Monoamines Tetrahydropapaveroline. For the mode of action of TIQ alkaloids on central adrenergic mechanisms, the attention of many investigators has been focused on the catecholaminergic mechanisms of uptake, storage, release, false transmitter or receptor phenomena. It has been shown that TIQs are taken up in catecholaminergic neurones and this occurs most likely by a process of competing for the transport systems for catecholamines (Cohen, 1973; Tennyson et al., 1973; Locke et al., 1973; Mytillineou et al., 1974; Alpers et al., 1975) and thereby displacing noradrenaline or dopamine from storage sites. The fact that 5-HT also was affected slightly in the present experiments suggests that this process of TIQ uptake is not entirely specific for catecholaminergic neurones. Our results that THP caused marked falls in diencephalic noradrenaline and striatal dopamine are compatible with such an action of the alkaloid and, moreover, the rise in striatat HVA may represent metabolism of the displaced dopamine. The present results indicate that desmethylimipfamine (DMI) blocked the THP induced depletions of diencephalic noradrenaline as well as that of striatal dopamine. The effect on noradrenaline is probably due to the inhibition of neuronal uptake by THP but this is not likely to be so for dopamine since DMI affects noradrenergic neurones, but dopaminergic neurones very little (Carlsson, 1966; Nyb/ick et al., 1968). That interference with neuronal uptake mechanisms for catecholamines by THP cannot be the only explanation for our results, was indicated by some pharmacological evidence. First of all DMI may have a dopamine receptor blocking effect (Carlsson, 1966) but more interesting were the results from the present experiments using haloperidol. Haloperidol pretreatment of rats partially prevented the striatal dopamine depletion and fully prevented the increase in HVA induced by THP. Even more remarkable was the effect on diencephalic noradrenaline caused by the combination of haloperidol with THP: the THP fall was

143

converted to a significant rise when the rats were treated with haloperidol before THP. The effect of haloperidol is probably related to blockade of catecholamine receptors with a positive feed-back on syntheses of the amines (Nyb~ick et al., 1968; And+n et al., 1969). Thus, the haloperidol induced blockade of the catecholamine responses to THP is proposed to be due to competition between haloperidol and THP for catecholamine receptors with the affinity of the receptors being greater for haloperidol than for THP. Replenishment of the catecholamine stores with the combination of haloperidol and THP might be due to an accelerated turnover by the receptor blockade. THP has been reported to have no stimulant property on the dopamine-sensitive adenyl-cyclase of the rat striatum (Sheppard and Burghardt, 1974; Miller et al., 1974) and recently it was reported that L-THP was a potent dopamine antagonist (Dougan et al., 1975). This hypothesis would be compatible with the postulate that many of the TIQs possess the properties of false adrenergic transmitters (Cohen, 1973; Tennyson et al., 1973; Locke et al., 1973; Mytillineou et al., 1974). Gamma-hydroxybutyric acid (GHB) has been reported to block the release of dopamine without affecting noradrenaline or 5-HT (Roth and Suhr, 1970; Walters and Roth, 1972; Menon et al., 1974). The results from our experiments indicate that GHB did not affect the dopamine depletion induced by THP which is in accordance with THP affecting the granular stores and displacing dopamine from these storage sites. No conclusions from the present experiments on the effect of GHB on synaptic release of dopamine can be made. Menon et al. (1974) found that GHB did not antagonize the disruption of the granular uptake and storage of dopamine by tetrabenezine and this would be in keeping with the proposed action of THP. The reason why GHB in the present experiment affected the noradrenaline and 5-HT induced changes by THP is not known. Salsolinol. Salsolinol or isosalsolinol caused a depletion of noradrenaline but a rise in striatel dopamine with a tendency to a fall in HVA concentrations. This action of salsolinol on the brain dopamine was partially blocked by reserpine or c~-methyl-p-tyrosine (e-MT) and fully prevented by haloperidol. Reserpine acts by preventing the normal intraneuronal storage of catecholamines in central monoaminergic neurones (Bertler, 1961 ; Glowinski et al., 1966) but there are catecholamine stores which are resistent to its depleting action (H~iggendal and Lindqvist, 1964). The salsolinol induced retardation of the dopamine increase after reserpine is in all likelihood due to diminished storage capacity possibly without affecting the storage for newly synthesized dopamine.

144

Moreover, the increased amount of dopamine caused by salsolinol could either be due to increased synthesis of dopamine or decreased release from the neurones. The fact that ~-MT prevented the effect of salsolinol on dopamine metabolism would tend to favour the hypothesis that salsolinol stimulates dopamine synthesis. The absence of changes in dopamine by salsolinol after hatoperidol treatment may be due to the blockade of the central catecholamine receptors by the latter drug, thereby masking the effect of salsolinol on the dopamine synthesis rate. The antogonistic effect of haloperidol on the salsolinol-induced change in dopamine seems to rule out the hypothesis that salsolinol acts primarily by inhibiting the neuronal release of the amine. The exact mechanism of action of salsolinol on striatal dopamine cannot be established by the present investigation. In all likelihood the TIQ alkaloids THP and salsolinol or isosalsolinol have basic differences in the mode of action on dopaminergic, but probably not on noradrenergic, mechanisms. The hypothesis of ~ transmitter" action is not compatible with the present results for the action of salsolinol on dopaminergic neurones in the rat striatum. In this respect it was interesting to note the results of Dougan et al. (1975) who demonstrated evidence for a different pharmacological activity of the alkaloids in that D- and Lsalsolinol were found to possess agonistic while L-THP was found to have antagonistic properties on dopamine receptors.

Effects of TIQ Alkaloids on Some CArS Functions Salsolinol or isosalsolinol were found to cause hypothermia while THP caused hyperthermia in the rat, thus, demonstrating a complex relation of TIQ alkaloids to thermoregulation. It has been reported previously that 6,7-dihydroxytetrahydroisoquinoline (a condensation product of dopamine with formaldehyde) caused hypothermia in animals (Brezenoff and Cohen, 1973), but hyperthermia has not been reported for any of the TIQ alkaloids. In the present investigations the body temperature changes, occurring early after drug administration, did not correspond well in time with the changes in monoamine metabolism induced by THP or salsolinol. Thus, it may be that the temperature changes are not directly related to changes in dopamine, noradrenaline or 5-HT induced by HTP or salsolinol. Other neuronal systems, like acetylcholine - or GABA-containing neurones, could possibly be affected by the TIQ alkaloids. Tetrahydroisoquinoline derivatives can cause abnormal motor behaviour in rats (Hornkiewicz, 1974;

Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979)

Collins and Bigdeli, 1975). The present observations are largely in agrement with these reports. In the rat, lower doses of THP induced some gnawing while the high doses caused marked gnawing and also licking, chewing, head-neck rocking and increased motor activity. Haloperidol antagonized the dyskinesias caused by THP and this suggests that THP directly or indirectly affects dopamine receptors by the mechanism of "false neurotransmission". Salsolinol was observed also to cause a compulsive gnawing behaviour in the rats. This motor dysfunction was to some extent inhibited by reserpine, but the gnawing was still observed after treatment with ~methyl-p-tyrosine which blocks the synthesis of catecholamines. This suggests that the abnormal behaviour was not mediated directly by dopamine. The fact that a similar behaviour was observed after 'L-Dopa administration (Ernst, 1965; Butcher and Engel, 1969) suggests that the mechanism causing the gnawing behaviour after L-Dopa may be through the formation of some compound other than dopamine. Moreover, haloperidol blocked the increase of the striatal dopamine caused by salsolinol and although it did not stop the gnawing behaviour haloperidol delayed its appearance by approximately 50 rain. This suggests that haloperidol, at least in part, may have antagonized the effects of the alkaloid on dopamine receptors. The gnawing symptoms caused by L-Dopa treatment could be caused by the formation of salsolinol, THP or related compounds. These alkaloids must still be considered in relation to the effects of L-Dopa in the treatment of Parkinson's disease which is supported by the isolation of tetrahydropapaveroline and salsolinol in the urine from parkinsonian patients (Sandler et al., 1973). The fact that the TIQ alkaloids themselves affect profoundly catecholamine metabolism complicates the interpretation of their functional consequences. Acknowledgements. This research was supported by grant from the Norwegian Research Council for Science and Humanities to whom we are sincerely grateful. The authors are indebted to Elfrid Blomdal, Inger Sletten and Halvard Bergesen for their expert technical assistance.

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Received April 4/Aecepted November 15, 1978