Effect of pentoxifylline and aminophylline on biogenic amine metabolism of the rat brain

European Journal o f Pharmacology, 47 (1978) 239--243

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© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n E F F E C T OF P E N T O X I F Y L L I N E AND AMINOPHYLLINE ON BIOGENIC AMINE METABOLISM OF THE RAT BRAIN DANIEL P. CARDINALI Centro de Estudios Farmacoldgicos y de Principios Naturales (Cefaprin), Consejo Nacional de Investigaciones Cientificas y Tdcnicas de la Rep~blica Argentina, Obligado 2490, 1428 Buenos Aires, Argentina

Received 28 October 1977, accepted 24 November 1977

D.P. CARDINALI, Effect o f penloxifylline and aminophylline on biogenic amine metabolism o f the rat brain, European J. Pharmacol. 47 (1978) 239--243. In the brain of rats injected with pentoxifylline [3,7-dimethyl-l(5-oxo-hexyl)-xanthine]or aminophylline the following was observed: (i) dose-dependent increases of tryptophan and 5-hydroxyindoleacetic acid levels and of 5-hydroxytryptophan and dihydroxyphenylalanine accumulation after treatment with the decarboxylase inhibitor benserazide HCI; (ii) acceleration of norepinephrine turnover following ~-methyl-p-tyrosine; (iii) increase of 3methoxytyramine accumulation following pargyline. On a molar basis pentoxifylline was more potent than aminophylline in affecting brain biogenic amine metabolism. Tryptophan and tyrosine hydroxylation 5-Hydroxyindoleacetic acid

Catecholamine turnover Methylxanthines

1. I n t r o d u c t i o n The m e t h y l x a n t h i n e s are a group of widely used CNS stimulants and have been shown to exert p o t e n t effects on brain m o n o a m i n e metabolism in vivo and in vitro. The turnover o f norepinephrine (NE) (Berkowitz et al., 1970; Waldeck, 1971; Corrodi et al., 1972; Karasawa et al., 1976) and serotonin (5-HT) (Karasawa et al., 1976; Curzon and Fernando, 1976) increases following administration of caffeine or theophylline, whereas dopa m i ne (DA) t u r n o v e r has been r epor t e d to increase (Waldeck, 1971), decrease (Corrodi et al., 1972) or remain u n a f f e c t e d (Karasawa et al., 1976). In vitro, th e ophyl l i ne and pentoxifylline [ 3,7-dimethyl-l(5-oxo-hexyl)-xanthine), Hoechst, West G e r m a ny] inhibited the accumulation in a non-competitive way and augmented th e [K+]-induced release of NE, DA and 5-HT b y synaptosomal fractions o f rat h y p o t h a l a m u s (Cardinali, 1977). On a molar basis, pentoxifylline was m or e p o t e n t

Aminophylline

than theophylline in bringing about changes in transmitter uptake and release. This report describes the effects of pentoxifylline and aminophylline on NE, DA and 5-HT synthesis and turnover in vivo with emphasis on examining the relative effectiveness of bot h drugs to affect brain m o n o a m i n e metabolism.

2. Materials and m et hods Male Wistar rats (180--220 g) were kept in lighted quarters from 0700 to 2100 h daily and given access to food (Purina chow) and water ad libitum. The animals were killed by neck fracture and the brains were quickly removed rostrally to the spino-occipital junction, frozen on solid CO2 and stored at --20 ° C. The following drugs were used: pentoxifylline (Hoechst), benserazide HC1 (Ro44602) (a gift from H offm ann-L a Roche, Switzerland), and aminophylline, pargyline

240

D.P. CARDINALI

HC1 a n d D , L - a - m e t h y l - p - t y r o s i n e m e t h y l ester (AMT) (Sigma C h e m i c a l Co., USA). All drugs were dissolved in 0.9% NaC1 s o l u t i o n a n d i n j e c t e d i.p. in a p p r o x i m a t e l y n e u t r a l solutions. T h e a c c u m u l a t i o n of 3 , 4 - d i h y d r o x y p h e n y l alanine ( D O P A ) a n d 5 - h y d r o x y t r y p t o p h a n (5H T P ) 30 m i n a f t e r t h e i n j e c t i o n o f t h e a r o m a t i c a m i n o acid d e c a r b o x y l a s e i n h i b i t o r Ro 4 - 4 6 0 2 ( 8 0 0 m g / k g ) was t a k e n as an e s t i m a t e o f t h e r a t e - l i m i t i n g steps in t h e synthesis o f c a t e c h o l a m i n e s a n d 5-HT in t h e brain (Carlsson et al., 1972). Brain N E a n d D A t u r n o v e r was e s t i m a t e d f r o m t h e decline o f e n d o g e n o u s a m i n e levels a f t e r synthesis inhibition with AMT (250 mg/kg). The format i o n o f 3 - m e t h o x y t y r a m i n e (3-MT) 60 m i n after injection of the monoamine oxidase (MAO) i n h i b i t o r p a r g y l i n e ( 1 0 0 m g / k g ) was t a k e n as an e s t i m a t e of D A release ( K a r a s a w a et al., 1976). T h e brains w e r e h o m o g e n i z e d in 10 ml 0.4 N p e r c h l o r i c acid c o n t a i n i n g 0.5 g/1 Na2S2Os a n d 2 g / 1 E D T A . T h e h o m o g e n a t e s w e r e c e n t r i f u g e d at 0°C a n d 1 5 0 0 0 g

f o r 20 min, t h e s u p e r n a t a n t s w e r e d e c a n t e d a n d 0.1 ml o f 2% ascorbic acid was a d d e d b e f o r e t h e s a m p l e s w e r e s u b j e c t e d to c o l u m n c h r o m a t o g r a p h y . F o r synthesis and t u r n o v e r studies, as well as for d e t e r m i n i n g t h e steadys t a t e c o n c e n t r a t i o n o f t h e amines, the samples (at p H 2.0) w e r e p o u r e d o v e r D o w e x c o l u m n s ( D o w e x 50 X 4, 2 0 0 - - 4 0 0 m e s h , H ÷) and e l u t i o n p e r f o r m e d as d e s c r i b e d b y Carlsson et al. ( 1 9 7 2 ) . F o r studies on D A release, DA a n d 3-MT w e r e isolated b y m e a n s o f d o u b l e d columns of aluminum oxide and Amberlite C G - 5 0 as d e s c r i b e d b y K a r a s a w a et al. (1976). The following spectrophotofluorimetric assays w e r e u s e d f o r d e t e r m i n a t i o n s o n the eluates: t y r o s i n e (Waalkes a n d U d e n f r i e n d , 1 9 5 7 ) , NE, DA, D O P A and 3-MT ( L a v e r t y a n d T a y l o r , 1 9 6 8 ) , t r y p t o p h a n (TP), 5-HTP, 5-HT a n d 5 - h y d r o x y i n d o l e a c e t i c acid (5H I A A ) ( H y y p p i i et al., 1973). 3. R e s u l t s Methylxanthines administered 3 h before d e a t h increased t h e r a t e o f t y r o s i n e h y d r o x -

TABLE 1 Effect o f pentoxifylline and aminophylline in combination with the decarboxylase inhibitor Ro 4-4602 on DOPA and 5-HTP formation in rat brain. Treatment 1

Control Pentoxifylline

(~g/g brain)

DOPA (ng/g brain)

TP (pg/g brain)

5-HTP (ng/g brain)

26.4 ± 1.3 (6) 27.2 _+2.0 (7)

157 ± 12 (6) 239 ± 15 (7) 2

4.2 ± 0.3 (6) 4.6 ± 0.2 (7)

174 ± 16 (6) 258 ± 21 (7) 2

25.3 ± 1.9 (6)

316 _+22 (6) 2

5.3 + 0.3 (6) 2

302 ± 25 (6) 2

25.7 ± 1.8 (6)

389 ± 30 (6) 2

6.1 ± 0.3 (6) 2

398 _+31 (5) 2

25.2 ± 1.9 (7)

183 ± 12 (6)

4.2 ± 0.3 (6)

217 _+ 15 (7)

23.8 _+ 1.8 (8)

227 ± 19 (8) 2

4.7 ± 0.3 (8)

243 _+17 (8) 2

24.7 _+ 1.7 (7)

287 + 20 (6) 2

5.1 ± 0.4 (7) 2

271 ± 19 (7) 2

Tyrosine

20 mg/kg Pentoxifylline

40 mg/kg Pentoxifylline

80 mg/kg Aminophylline

16 mg/kg Aminophylline

32 mg/kg Aminophylline

64 mg/kg 1 Pentoxifylline or aminophylline were injected in equimolar amounts 3 h, and Ro 4-4602 (800 mg/kg) 30 rain before death. Controls received only Ro 4-4602. Shown are the means ± S.E.M. Figures in parentheses indicate number o f animals. 2 Significantly different from controls, p < 0.05, analysis of variance.

METHYLXANTHINES AND BRAIN MONOAMINE METABOLISM

ylation by the rat brain in a dose-dependent manner (table 1). On a molar basis pentoxifylline was more potent than aminophylline in increasing DOPA accumulation after Ro 4-4602. The rate of TP h y d r o x y l a t i o n was also augmented by methylxanthines (table 1). The increase apparently resulted from two different phenomena, i.e., the increase of brain TP concentration and an actual increase of the hydroxylating reaction, since the elevation of 5-HTP was greater than that expected from increased precursor levels (table 1). Pentoxifylline was more effective than aminophylline in causing changes of TP hydroxylation. The effect of pentoxifylline on DOPA and 5-HTP accumulation was detected up to 6 h after administration of the drug; at this time, the values for aminophyllineoinjected rats did not differ from the controls (results not shown). Methylxanthine administration to animals subsequently injected with AMT accelerated the rate of decline of NE concentration in brain (table 2). DA was only marginally affected by the highest dose of pentoxifylline

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and remained unchanged following aminophylline. DA release, estimated from the accumulation of its O-methylated metabolite 3-MT 60 min after pargyline injection (3-MT concentration = 34 + 2 ng/g brain) was increased by injecting pentoxifylline, 20 and 80 mg/kg 90 min before death (3-MT = 47 +-2 and 58 + 4 ng/g brain, respectively). Aminophylline augmented DA release only at its highest dose (i.e., 60 mg/kg) (3-MT = 48 -+ 4 ng/g brain). The steady state concentration of brain NE, DA and 5-HT was not affected by methylxanthine treatment (results not shown). 5HIAA levels (379 + 16 ng/g brain) increased 3 h after administering 40 or 80 mg/kg (466 +- 22 and 482 + 19 ng/g brain, respectively) or 64 mg/kg aminophylline (469 ± 27 ng/g brain).

4. Discussion The data indicate that pentoxifylline and aminophylline increase the rate of TP and tyrosine h y d r o x y l a t i o n and of NE turnover

TABLE 2 E f f e c t o f p e n t o x i f y l l i n e a n d a m i n o p h y l t i n e o n t h e decline o f b r a i n NE a n d DA c o n c e n t r a t i o n a f t e r i n j e c t i o n of (~-methyl-p-tyrosine 1 T i m e elapsed a f t e r t~-methyl-p-tyrosine ( m i n ) 0

30

60

120

N E (ng/g b r a i n ) Control Pentoxifylline Aminophylline

375 + 27 (5) 369 + 31 (6) 379 -+ 35 (7)

3 3 9 ± 29 (6) 267 ± 16 (7) 2 289 -+ 19 (7) 2

306 ± 26 (7) 227 ± 15 (6) 2 241 ± 22 (7) 2

284 + 23 (7) 218 _+ 16 (7) 2 237 ± 18 (6) 2

D A (ng/g b r a i n ) Control Pentoxifylline Aminophylline

6 2 4 ± 46 (5) 610 ± 35 (5) 6 5 2 ± 41 (6)

5 1 8 ± 37 (6) 4 4 6 ± 32 (7) 2 4 9 0 ± 4 0 (7)

4 9 9 ± 37 (7) 4 4 2 ± 36 (6) 2 521 ± 45 (6)

4 7 5 ± 39 (7) 4 2 8 _+ 36 (7) 2 4 5 9 ± 47 (6)

I P e n t o x i f y l l i n e (80 m g / k g ) or e q u i m o l a r a m o u n t s o f a m i n o p h y l l i n e (64 m g / k g ) were i n j e c t e d 1 h b e f o r e a - m e t h y l - p - t y r o s i n e ( 2 5 0 mg/kg). T h e a n i m a l s were killed at d i f f e r e n t t i m e s following i n h i b i t o r injection. C o n t r o l s received a - m e t h y l - p - t y r o s i n e o n l y . S h o w n are t h e m e a n s _+ S.E.M. Figures in p a r e n t h e s e s i n d i c a t e n u m b e r o f animals. 2 Significantly d i f f e r e n t f r o m c o n t r o l s , P < 0.05, analysis o f variance.

242 and DA release b y t h e rat brain in vivo. In a g r e e m e n t with previous results o n NE, DA and 5-HT u p t a k e and release by h y p o t h a l a m i c s y n a p t o s o m e s in vitro (Cardinali, 1977) p e n t o x i f y l l i n e was f o u n d t o be m o r e p o t e n t t h a n a m i n o p h y l l i n e in affecting brain m o n o a m i n e metabolism. T h e present results also agree with previous r e p o r t s o n m e t h y l x a n t h i n e effects on NE and 5-HT m e t a b o l i s m in the brains o f rodents. T h e o p h y l l i n e a n d / o r caffeine have b e e n s h o w n to e n h a n c e b o t h t h e disappearance o f e n d o g e n o u s NE a f t e r inhibition o f its biosynthesis ( B e r k o w i t z et al., 1970; Waldeck, 1971; C o r r o d i et al., 1972) and the conversion o f radioactive t y r o s i n e into catecholamines (Waldeck, 1 9 7 1 ; B e r k o w i t z et al., 1 9 7 0 ) as well as t o increase the c o n c e n t r a t i o n o f the brain NE and 5-HT m e t a b o l i t e s 3m e t h o x y - 4 - h y d r o x y p h e n y l e t h y l e n e glycol sulp h a t e and 5-HIAA (Karasawa et al., 1976; C u r z o n and F e r n a n d o , 1976). H o w e v e r o u r d a t a o n a u g m e n t a t i o n o f brain DA release b y p e n t o x i f y l l i n e (and in higher doses b y a m i n o p h y l l i n e ) c o n t r a s t with t h e r e p o r t e d lack o f effects o f t h e o p h y l l i n e o n 3-MT levels a f t e r MAO inhibition even in the presence o f i m i p r a m i n e (Karasawa et al., 1976). F u r t h e r e x p e r i m e n t s are n e e d e d to explain this discrepancy. B o t h p e n t o x i f y l l i n e and a m i n o p h y l l i n e augm e n t brain T P levels (table 1). Changes in brain TP u p o n a m i n o p h y l l i n e injection have been r e p o r t e d t o d e p e n d o n increased availability o f plasma TP d u e t o increased lipolysis and o n increased effectiveness o f u p t a k e o f TP b y the brain d u e to e n h a n c e d insulin secret i o n ( C u r z o n and F e r n a n d o , 1976). H o w e v e r t h e increase o f 5-HTP a c c u m u l a t i o n a f t e r R o 4 - 4 6 0 2 b y m e t h y l x a n t h i n e s c o u l d n o t be explained o n l y in terms o f increased p r e c u r s o r a m i n o acid levels since the 5-HTP rise was greater t h a n t h a t e x p e c t e d f r o m the elevation o f brain TP c o n c e n t r a t i o n . D a t a o n the actual c o n c e n t r a t i o n o f i n t r a n e u r o n a l TP available for h y d r o x y l a t i o n are n e e d e d b e f o r e a d e f i n i t e p i c t u r e o f this p h e n o m e n o n is obtained.

D.P. CARDINALI In conclusion, t h e present results o n the relative effectiveness o f p e n t o x i f y l l i n e and a m i n o p h y l l i n e to a f f e c t brain m o n o a m i n e m e t a b o l i s m in vivo agree with previous d a t a f r o m s y n a p t o s o m a l p r e p a r a t i o n s in vitro. It seems possible that, t h r o u g h e n h a n c e m e n t o f NE, DA and 5-HT release and i n h i b i t i o n o f r e u p t a k e (Cardinali, 1 9 7 7 ) , m e t h y l x a n t h i n e s m e d i a t e t h e r e p o r t e d changes in synthesis and t u r n o v e r o f biogenic amines in the rat brain.

Acknowledgements

The author is an established investigator from the Comisi6n Nacional de Estudios Geo-Heliof/sicos, Argentina. The financial support from Hoechst Aktiengesellschaft is gratefully acknowledged.

References

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