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Adrenergic influence on rat plasma concentrations of tyrosine and tryptophan
Life Sciences, Vol. 30, pp. 1465-1472 Printed in the U.S.A.
ADRENERGIC
Pergamon Press
I N F L U E N C E ON RAT P L A S M A C O N C E N T R A T I O N S OF T Y R O S I N E AND T R Y P T O P H A N
Tomas
Eriksson
and A r v i d
Carlsson
D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of G ~ t e b o r g , P.O. Box 33031, S-400 33 G ~ t e b o r g , Sweden (Received in final form February 16, 1982) Summary I s o p r e n a l i n e g i v e n to rats in doses b e t w e e n 0.08 and 10 m g / k g i n t r a p e r i t o n e a l l y c a u s e d a s i g n i f i c a n t decrease in p l a s m a c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. Low doses of a d r e n a l i n e (0.04 - 0.16 mg/kg, i n t r a p e r i t o n e a l l y ) c a u s e d a 30 per cent d e c r e a s e in p l a s m a c o n c e n t r a t i o n s of t y r o s i n e , while high doses (0.63 - 1.25 mg/kg, i n t r a p e r i t o n e a l l y ) caused an increase in p l a s m a t y r o s i n e to n e a r l y 200 per cent of the controls. High doses of n o r a d r e n a l i n e (0.63 - 2.5 mg/kg, i n t r a p e r i t o n e a l l y ) c a u s e d a s i m i l a r increase in p l a s m a t y r o s i n e c o n c e n t r a t i o n . The d e c r e a s e in plasma amino acids c a u s e d by these c a t e c h o l a m i n e s is inh i b i t e d by p r o p r a n o l o l , s u g g e s t i n g that this effect is m e d i a t e d via a d r e n e r g i c B - r e c e p t o r s , while the increasing effect is i n h i b i t e d by p h e n o x y b e n z a m i n e , w h i c h suggests that this effect is caused by an e - a d r e n e r g i c mechanism. The t r a n s p o r t of amino acids from plasma into the brain is c a r r i e r m e d i a t e d . The large n e u t r a l amino acids are c o n s i d e r e d to be t r a n s p o r t e d via the same c a r r i e r s y s t e m and to c o m p e t e with each o t h e r for the c a r r i e r (1). The r e l a t i o n b e t w e e n the levels of these p l a s m a amino acids r a t h e r than the actual c o n c e n t r a t i o n of a c e r t a i n amino acid seems to d e t e r m i n e h o w m u c h of each of the d i f f e r e n t amino acids that will be t r a n s p o r t e d into the b r a i n (2, 3). A l t e r a t i o n s in the r e l a t i o n b e t w e e n d i f f e r e n t amino acids w h i c h are t r a n s p o r t e d into the b r a i n by the same c a r r i e r might thus be e x p e c t e d to i n f l u e n c e the c o n c e n t r a t i o n s in the b r a i n of the d i f f e r e n t amino acids. In the same way, a l t e r a t i o n s in the t o t a l p l a s m a amino acid pool might be e x p e c t e d to i n f l u e n c e the t r a n s p o r t into the b r a i n of e x o g e n o u s l y a d m i n i s t e r e d amino acids. We have r e c e n t l y found that a c u t e l y a d m i n i s t e r e d ethanol causes a r a p i d d e c r e a s e in almost every p l a s m a amino acid in rat (4) and in man (to be p u b l i s h e d ) . We have also r e p o r t e d that t r e a t m e n t w i t h e t h a n o l i n c r e a s e s the c o n c e n t r a t i o n s in rat b r a i n of a d m i n i s t e r e d L-dopa (5), t y r o s i n e , t r y p t o p h a n , ~ - m e t h y l d o p a and 5 - h y d r o x y t r y p t o p h a n (6). The cause of this i n c r e a s e in b r a i n c o n c e n t r a t i o n s of a d m i n i s t e r e d amino acids is s u g g e s t e d to be, at least partly, the e t h a n o l - i n d u c e d d e c r e a s e in p l a s m a c o n c e n t r a tions of e n d o g e n o u s large n e u t r a l amino acids, thus f a v o u r i n g the t r a n s p o r t of the a d m i n i s t e r e d one into the brain. 0024-3205/82/171465-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
The way in w h i c h e t h a n o l exerts its amino acid d e c r e a s i n g effect is not clear. However, we have found that the B - a d r e n e r g i c a n t a g o n i s t p r o p r a n o l o l , at least partly, can inhibit this effect of ethanol. S h a m o o n et al. (7) has r e c e n t l y r e p o r t e d that the a d m i n i s t r a tion of a d r e n a l i n e to man causes a r e d u c t i o n in most c i r c u l a t i n g amino acids. This effect was not c o r r e l a t e d to c h a n g e d levels of plasma insulin. It could be t o t a l l y p r e v e n t e d by p r o p r a n o l o l . The present study was u n d e r t a k e n to f u r t h e r i n v e s t i g a t e how a d r e n e r g i c m e c h a n i s m s could i n f l u e n c e the c o n c e n t r a t i o n of amino acids in plasma. It deals w i t h the effects of a d r e n e r g i c drugs on rat p l a s m a c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. Methods Male Sprague D a w l e y S o l l e n t u n a , Sweden) were were h o u s e d for at least h l i g h t / d a r k cycle. They
rats w e i g h i n g about 220 g (Anticimex, used. Before the e x p e r i m e n t s the animals one week in a room m a i n t a i n e d on a 14/10 had free access to food and water.
In one e x p e r i m e n t the animals were given i n t r a p e r i t o n e a l injections with various doses of a d r e n a l i n e (ranged b e t w e e n 0.02 and 1.25 mg/kg), n o r a d r e n a l i n e (ranged b e t w e e n 0.02 and 2.50 mg/kg), or i s o p r e n a l i n e (ranged b e t w e e n 0.02 and 10.0 mg/kg). The drugs were d i s s o l v e d in saline in such a c o n c e n t r a t i o n , that the animals r e c e i v e d a c o n s t a n t volume of 10 ml/kg. Control rats r e c e i v e d the same v o l u m e of saline. Forty m i n u t e s a f t e r the i n j e c t i o n s the animals were k i l l e d by d e c a p i t a t i o n . In a second e x p e r i m e n t the rats in one group were i n j e c t e d i n t r a p e r i t o n e a l l y with L - p r o p r a n o l o l , 15 mg/kg, and 20 m i n u t e s later with i s o p r e n a l i n e , 10 m g / k g i.p. With the same time i n t e r v a l s two o t h e r groups of rats r e c e i v e d i n t r a p e r i t o n e a l i n j e c t i o n s with s a l i n e / i s o p r e n a l i n e and s a l i n e / s a l i n e , r e s p e c t i v e l y . A third experiment was c a r r i e d out s i m i l a r to this, except that the administered dosees of i s o p r e n a l i n e and L - p r o p r a n o l o l were 0.1 and 10 mg/kg, r e s p e c t i v e l y . The i n t e r v a l b e t w e e n the last i n j e c t i o n and death was 40 minutes. In a fourth e x p e r i m e n t p h e n o x y b e n z a m i n e , 10 mg/kg, was i n j e c t e d i n t r a p e r i t o n e a l l y to 18 rats. A n o t h e r 18 rats r e c e i v e d injections with e q u i v a l e n t volumes of saline. Fifty m i n u t e s later 6 rats from e i t h e r group were injected w i t h a d r e n a l i n e , 1.25 m g / k g i.p., w h e r e a s 6 rats were i n j e c t e d with n o r a d r e n a l i n e , 1.25 m g / k g i.p., and the last 6 rats from e i t h e r group were i n j e c t e d with saline. Forty m i n u t e s a f t e r the last i n j e c t i o n the rats were k i l l e d by d e c a p i t a t i o n A fifth e x p e r i m e n t was d e s i g n e d in the same way as the fourth one, except that p r o p r a n o l o l , 10 mg/kg, was a d m i n i s t e r e d instead of p h e n o x y b e n z a m i n e and that a d r e n a l i n e and n o r a d r e n a l i n e were given in a dose of 0.1 mg/kg. I m m e d i a t e l y a f t e r death about 5 ml b l o o d was c o l l e c t e d in a tube c o n t a i n i n g 0.5 ml of a 1 % E D T A solution. The p l a s m a samples were p u r i f i e d on a strong c a t i o n e x c h a n g e column (Dowex 50W X-4) (8, 9). S p e c t r o p h o t o f l u o r i m e t r i c a n a l y s e s of t y r o s i n e (10) and t r y p t o p h a n (11) were c a r r i e d out.
Vol. 30, No. 17, 1982
Regulation of Plasma Amino Acids
S t a t i s t i c a l s i g n i f i c a n c e s were of variance followed by t-test.
assessed
1467
by one way analysis
Results Figure 1 shows the effects of various doses of adrenaline, n o r a d r e n a l i n e and i s o p r e n a l i n e on plasma c o n c e n t r a t i o n s of tyro-
* *
Tyrosine in plasma
(n=3_12)
200
,,?.\!
150
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,,m S _- -// . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
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I
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0.31
1.25
I
5.00 Drug, mg.kg"
Tryptophan in plasma (n=3-12) -
~'~~,-~
......
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50
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FIG.
I
1.25
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5.00 Drug, mg.kg"
1
Effects of adrenaline, n o r a d r e n a l i n e and i s o p r e n a l i n e on rat p l a s m a c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. The drugs were i n j e c t e d i n t r a p e r i t o n e a l l y . Control rats r e c e i v e d an e q u i v a l e n t volume of saline. Forty minutes after the injections the animals were k i l l e d by decapitation. Plasma c o n c e n t r a t i o n s of t y r o s i n e and t r y p t o p h a n are shown as per cent of controls. Control values in pmol/l, mean ± s.e.m. (n), were for tyrosine 94 ± 4.7 (32) and for t r y p t o p h a n 86 ± 2.8 (32).
1468
Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
sine and t r y p t o p h a n . Low d o s e s (0.04 - 0.16 m g / k g ) of a d r e n a l i n e c a u s e d a d e c r e a s e by as m u c h as 30 per c e n t in p l a s m a c o n c e n t r a t i o n s of t y r o s i n e . H i g h d o s e s of a d r e n a l i n e (0.63 - 1.25 m g / k g ) on the o t h e r h a n d c a u s e d a s i g n i f i c a n t i n c r e a s e up to n e a r l y 200 per cent of the c o n t r o l . Low d o s e s of n o r a d r e n a l i n e had no e f f e c t on p l a s m a c o n c e n t r a t i o n of t y r o s i n e , w h i l e h i g h d o s e s (0.63 2.50 m g / k g ) c a u s e d an i n c r e a s e s i m i l a r to t h a t of a d r e n a l i n e . N e i t h e r a d r e n a l i n e nor n o r a d r e n a l i n e c a u s e d any s i g n i f i c a n t change: in p l a s m a c o n c e n t r a t i o n s of t r y p t o p h a n t h o u g h t h e r e seems to be a t e n d e n c y in the same d i r e c t i o n as was s e e n in the case of t y r o s i n e All d o s e s of i s o p r e n a l i n e g i v e n (0.04 - 10 m g / k g ) c a u s e d a s i g n i f i c a n t d e c r e a s e by as m u c h as 50 per cent in p l a s m a c o n c e n t r a t i o n s of b o t h t y r s o i n e and t r y p t o p h a n .
I00
Tyrosine I
'~ 6 0
--4--
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6
I00
Tryptophan I
-+-
Saline or L-Propranolol
Saline or Isoprenaline
I
I
0
20
Killed I
minu~es
,'I-
60
-I-. q3
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.~ 2O
0
6
6 FIG.
2
E f f e c t of L - p r o p r a n o l o l on i s o p r e n a l i n e - i n d u c e d decrease in rat p l a s m a c o n c e n t r a t i o n s of t y r o s i n e a n d t r y p t o p h a n . L - p r o p r a n o l o l , 10 or 15 m g / k g , or an e q u i v a l e n t v o l u m e of s a l i n e was i n j e c t e d i.p. and f o l l o w e d 20 m i n u t e s l a t e r by an i.p. i n j e c t i o n of i s o p r e n a l i n e 0.1 or 10 m g / k g , or an e q u i v a l e n t v o l u m e of s a l i n e . F o r t y m i n u t e s l a t e r the a n i m a l s w e r e k i l l e d by d e c a p i t a t i o n . P l a s m a c o n c e n t r a t i o n s are s h o w n as per cent of c o n t r o l s . C o n t o l v a l u e s in p m o l / l , m e a n ± s.e.m. (n), w e r e for t y r o s i n e 96 ± 2.5 (11) a n d for t r y p t o p h a n 104 ± 3.4 (11).
6O
Vol. 30, No. 17, 1982
Regulation of Plasma Amino Acids
1469
Figure 2 illustrates that the decrease in p l a s m a c o n c e n t r a tions of t y r o s i n e and t r y p t o p h a n caused by a low dose of isoprenaline (0.1 mg/kg) could be c o m p l e t e l y b l o c k e d by propranolol, while the decrease caused by a much h i g h e r dose of i s o p r e n a l i n e (10 mg/kg) could not be i n f l u e n c e d by propranolol. Figure 3 shows the changes in plasma c o n c e n t r a t i o n s of tyrosine and t r y p t o p h a n after t r e a t m e n t with p h e n o x y b e n z a m i n e , adrenaline and n o r a d r e n a l i n e and with c o m b i n a t i o n s of p h e n o x y b e n z a m i n e
220
Tyrosine
t •A,
200
-t-
180
~
~r
+
160 140
~o
12o ~o
Saline or Phenoxybenzamine, I0 mg-kg "~ i.p. I 0
Saline or Adrenaline, 1.25 mg-kg "~ i.p. or Noradrenaline, 1.25 mg'kg "~ i.p. Killed I I 5 0 minutes 9O
~oo 8o
!it ,A,
6O
•A,
,A,
,k
4O 2O 6
0 120-
~
I00
u
80
Tryptophan I t
604020O-
6
6
6
FIG.
3
Effects of p h e n o x y b e n z a m i n e , adrenaline, n o r a d r e n a l i n e , and c o m b i n a t i o n s of p h e n o x y b e n z a m i n e with a d r e n a l i n e or n o r a d r e n a l i n e on rat plasma c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. P h e n o x y b e n z a m i n e , 10 mg/kg, or an equivalent volume of saline was injected i.p. Fifty minutes later adrenaline, 1.25 mg/kg, n o r a d r e n a l i n e , 1.25 mg/kg, or an e q u i v a l e n t volume of saline was injected i.p. Forty minutes after the last i n j e c t i o n the rats were killed by decapitation. Plasma c o n c e n t r a t i o n s are shown as per cent of controls. Control values in pmol/l, mean ± s.e.m. (n), were for t y r o s i n e 69 ± 3.3 (6) and for t r y p t o p h a n 75 ± 2.6 (6).
1470
Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
w i t h a d r e n a l i n e or n o r a d r e n a l i n e . P h e n o x y b e n z a m i n e caused a marked decrease in the c o n c e n t r a t i o n s of both p l a s m a t y r o s i n e and t r y p t o p h a n . P h e n o x y b e n z a m i n e also c o m p l e t e l y b l o c k e d the tyros i n e - i n c r e a s i n g effect of both a d r e n a l i n e and n o r a d r e n a l i n e . In figure 4 are shown the effects of L - p r o p p a n o l o l , adrenaline and n o r a d r e n a l i n e and of c o m b i n a t i o n s of L - p r o p r a n o l o l with
120-
Tyrosine
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8O 604o 20"
i
, 6
6
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,
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6
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6
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Saline or /_-Propranolol, IO mg-kg-' i.p. I 0
Saline or Adrenaline, O.mmg.kg ~ i.p. or Noradrenaline, 0.1 mg-kg -t i.p. Killed I I 50 minutes 90 FIG.
4
Effects of L - p r o p r a n o l o l , a d r e n a l i n e , n o r a d r e n a l i n e and c o m b i n a t i o n s of L - p r o p r a n o l o l w i t h a d r e n a l i n e or nora d r e n a l i n e on rat plasma c o n c e n t r a t i o n s of t y r o s i n e and t r y p t o p h a n . L - P r o p r a n o l o l , 10 mg/kg, or an e q u i v a l e n t volume of saline was i n j e c t e d i.p. Fifty m i n u t e s later a d r e n a l i n e , 0.1 mg/kg, n o r a d r e n a l i n e , 0.1 mg/kg, or an e q u i v a l e n t volume of saline was i n j e c t e d i.p. Forty m i n u t e s a f t e r the last i n j e c t i o n the rats were k i l l e d by d e c a p i tation. Plasma c o n c e n t r a t i o n s are shown as per cent of controls. Control values in ~mol/l, m e a n ± s.e.m. (n), were for t y r o s i n e 63 ± 1.9 (6) and for t r y p t o p h a n 72 ± 5.9 (6).
Vol. 30, No. 17, 1982
Regulation of Plasma Amino Acids
1471
a d r e n a l i n e or n o r a d r e n a l i n e on plasma c o n c e n t r a t i o n s of tyrosine and tryptophan. L - P r o p r a n o l o l had by itself no effect on plasma t y r o s i n e or tryptophan, but it caused a complete inhibition of the amino acid d e c r e a s i n g p r o p e r t i e s of a d r e n a l i n e and noradrenaline. Discussion N o r a d r e n a l i n e is c o n s i d e r e d to possess a strong e- (and a weak B-) type activity, while i s o p r e n a l i n e exerts strong B- (and weak ~-) influences. A d r e n a l i n e has both activities, but the Bactions p r e d o m i n a t e (12). As the decrease in plasma amino acids seen after a d m i n i s t r a t i o n of low doses of adrenaline, noradrenaline and i s o p r e n a l i n e could be c o m p l e t e l y inhibited by the Ba d r e n e r g i c a n t a g o n i s t propranolol, we suggest that this effect is m e d i a t e d via a d r e n e r g i c B-receptors. This h y p o t h e s i s is s u p p o r t e d by the fact that the amino acid d e c r e a s i n g effect of i s o p r e n a l i n e is more p r o n o u n c e d than that of adrenaline, which in turn is more p r o n o u n c e d than that of n o r a d r e n a l i n e . Higher doses of a d r e n a l i n e and n o r a d r e n a l i n e , but not of isoprenaline, cause an increase in plasma tyrosine c o n c e n t r a tions. This effect is c o m p l e t e l y i n h i b i t e d by the s - r e c e p t o r b l o c k i n g agent p h e n o x y b e n z a m i n e , s u g g e s t i n g that this effect of a d r e n a l i n e and n o r a d r e n a l i n e is m e d i a t e d via a d r e n e r g i c ~-receptors. P h e n o x y b e n z a m i n e has by itself a d e c r e a s i n g effect on plasma c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. This p h e n o m e n o n could be due to loss of a tonic ~ - a d r e n e r g i c influence on these levels. A l t e r n a t i v e l y , it could be m e d i a t e d via adrenergic 6-receptors s t i m u l a t e d by an i n c r e a s e d s e c r e t i o n of n o r a d r e n a l i n e which is known to occur after t r e a t m e n t with p h e n o x y b e n z a m i n e (13). The m e c h a n i s m s u n d e r l y i n g the a d r e n e r g i c influence on plasma amino acids could not be d i s c l o s e d by the present data. The way in which a d r e n e r g i c m e c h a n i s m s take part in the control of carbohydrate, protein and lipid m e t a b o l i s m is complex (12). Both pancreatic hormones and the h y p o t h a l a m i c - p i t u i t a r y - a d r e n o c o r t i c a l s y s t e m must be c o n s i d e r e d in this context. A n o t h e r p o s s i b i l i t y is that the d e m o n s t r a t e d effects on plasma amino acids are caused by changes in the total blood flow r e c e i v e d by the liver, in which most amino acids are metabolized. This h y p o t h e s i s is s u p p o r t e d by the fact that s t i m u l a t i o n o ~ ~a d r e n e r g i c r e c e p t o r s a c t u a l l y causes v a s o c o n s t r i c t i o n and d e c r e a s e d h e p a t i c blood flow, while B-adrenergic s t i m u l a t i o n gives rise to v a s o d i l a t a t i o n and i n c r e a s e d liver p e r f u s i o n (14). Acknowledgements This work was s u p p o r t e d by the Swedish Medical R e s e a r c h Council (project No. 155), the Faculty of Medicine at the University of G ~ t e b o r g and the G ~ t e b o r g Medical Society. We want to express H o l m g r e n for her expert
our sincere gratitude t e c h n i c a l assistance.
to mrs
Birgitta
1472
Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
References 1.
2 .
3. 4 5 6 7 8 9 10 11 12 13
14.
W.H. O L D E N D O R F , The Nervous System, Vol 1: The Basic Neurosciences, D.B. T o w e r (ed.), pp. 279-289, Raven Press, New York (1975). J.D. F E R N S T R O M and R.J. WURTMAN, Science 178 414-416 (1972). R.J. W U R T M A N and J.D. FERNSTROM, Biochem. Pharmac. 25 16911696 (1976). T. ERIKSSON, A. CARLSSON, S. L I L J E Q U I S T , M. H A G M A N and R. JAGENBURG, J. Pharm. Pharmacol. 32 512-513 (1980). T. ERIKSSON, S. L I L J E Q U I S T and A _ _ C A R L S S O N , J. Pharm. Pharmacol. 31 636-637 (1979). T. E R I K S S O N and A. CARLSSON, N a u n y n - S c h m i e d e b e r g ' s Arch. Pharmacol. 314 47-50 (1980). H. SHAMOON, R. JACOB and R. SHERWIN, Diabetes 29 875-881 (1980). C. ATACK a n d T. MAGNUSSON, A e t a p h a r m , t o x . , Kbh. 42 3 5 - 5 7 (1978). W. KEHR, A. C A R L S S O N and M. LINDQVIST, N a u n y n - S c h m i e d e b e r g ' s Arch. Pharmacol. 274 273-280 (1972). T.P. W A A L K E S and S. U D E N F R I E N D , J. Lab. clin. Med. 50 733736 (1957). P. BEDARD, A. C A R L S S O N and M. LINDQVIST, N a u n y n - S c h m i e d e berg's Arch. Pharmacol. 272 1-15 (1972). C.R. MARTIN, T e x t b o o k of e n d o c r i n e p h y s i o l o g y , W i l l i a m s & Wilkins Company, B a l t i m o r e (1976). J . H A G G E N D A L , F r o n t i e r s in C a t e c h o l a m i n e Research, E. Usdin and S. Snyder (eds.), pp. 531-535, P e r g a m o n Press, New York (1973). C.V. G R E E N W A Y and R.D. STARK, Physiol. Rev. 51 23-68 (1971).
ADRENERGIC
Pergamon Press
I N F L U E N C E ON RAT P L A S M A C O N C E N T R A T I O N S OF T Y R O S I N E AND T R Y P T O P H A N
Tomas
Eriksson
and A r v i d
Carlsson
D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of G ~ t e b o r g , P.O. Box 33031, S-400 33 G ~ t e b o r g , Sweden (Received in final form February 16, 1982) Summary I s o p r e n a l i n e g i v e n to rats in doses b e t w e e n 0.08 and 10 m g / k g i n t r a p e r i t o n e a l l y c a u s e d a s i g n i f i c a n t decrease in p l a s m a c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. Low doses of a d r e n a l i n e (0.04 - 0.16 mg/kg, i n t r a p e r i t o n e a l l y ) c a u s e d a 30 per cent d e c r e a s e in p l a s m a c o n c e n t r a t i o n s of t y r o s i n e , while high doses (0.63 - 1.25 mg/kg, i n t r a p e r i t o n e a l l y ) caused an increase in p l a s m a t y r o s i n e to n e a r l y 200 per cent of the controls. High doses of n o r a d r e n a l i n e (0.63 - 2.5 mg/kg, i n t r a p e r i t o n e a l l y ) c a u s e d a s i m i l a r increase in p l a s m a t y r o s i n e c o n c e n t r a t i o n . The d e c r e a s e in plasma amino acids c a u s e d by these c a t e c h o l a m i n e s is inh i b i t e d by p r o p r a n o l o l , s u g g e s t i n g that this effect is m e d i a t e d via a d r e n e r g i c B - r e c e p t o r s , while the increasing effect is i n h i b i t e d by p h e n o x y b e n z a m i n e , w h i c h suggests that this effect is caused by an e - a d r e n e r g i c mechanism. The t r a n s p o r t of amino acids from plasma into the brain is c a r r i e r m e d i a t e d . The large n e u t r a l amino acids are c o n s i d e r e d to be t r a n s p o r t e d via the same c a r r i e r s y s t e m and to c o m p e t e with each o t h e r for the c a r r i e r (1). The r e l a t i o n b e t w e e n the levels of these p l a s m a amino acids r a t h e r than the actual c o n c e n t r a t i o n of a c e r t a i n amino acid seems to d e t e r m i n e h o w m u c h of each of the d i f f e r e n t amino acids that will be t r a n s p o r t e d into the b r a i n (2, 3). A l t e r a t i o n s in the r e l a t i o n b e t w e e n d i f f e r e n t amino acids w h i c h are t r a n s p o r t e d into the b r a i n by the same c a r r i e r might thus be e x p e c t e d to i n f l u e n c e the c o n c e n t r a t i o n s in the b r a i n of the d i f f e r e n t amino acids. In the same way, a l t e r a t i o n s in the t o t a l p l a s m a amino acid pool might be e x p e c t e d to i n f l u e n c e the t r a n s p o r t into the b r a i n of e x o g e n o u s l y a d m i n i s t e r e d amino acids. We have r e c e n t l y found that a c u t e l y a d m i n i s t e r e d ethanol causes a r a p i d d e c r e a s e in almost every p l a s m a amino acid in rat (4) and in man (to be p u b l i s h e d ) . We have also r e p o r t e d that t r e a t m e n t w i t h e t h a n o l i n c r e a s e s the c o n c e n t r a t i o n s in rat b r a i n of a d m i n i s t e r e d L-dopa (5), t y r o s i n e , t r y p t o p h a n , ~ - m e t h y l d o p a and 5 - h y d r o x y t r y p t o p h a n (6). The cause of this i n c r e a s e in b r a i n c o n c e n t r a t i o n s of a d m i n i s t e r e d amino acids is s u g g e s t e d to be, at least partly, the e t h a n o l - i n d u c e d d e c r e a s e in p l a s m a c o n c e n t r a tions of e n d o g e n o u s large n e u t r a l amino acids, thus f a v o u r i n g the t r a n s p o r t of the a d m i n i s t e r e d one into the brain. 0024-3205/82/171465-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
The way in w h i c h e t h a n o l exerts its amino acid d e c r e a s i n g effect is not clear. However, we have found that the B - a d r e n e r g i c a n t a g o n i s t p r o p r a n o l o l , at least partly, can inhibit this effect of ethanol. S h a m o o n et al. (7) has r e c e n t l y r e p o r t e d that the a d m i n i s t r a tion of a d r e n a l i n e to man causes a r e d u c t i o n in most c i r c u l a t i n g amino acids. This effect was not c o r r e l a t e d to c h a n g e d levels of plasma insulin. It could be t o t a l l y p r e v e n t e d by p r o p r a n o l o l . The present study was u n d e r t a k e n to f u r t h e r i n v e s t i g a t e how a d r e n e r g i c m e c h a n i s m s could i n f l u e n c e the c o n c e n t r a t i o n of amino acids in plasma. It deals w i t h the effects of a d r e n e r g i c drugs on rat p l a s m a c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. Methods Male Sprague D a w l e y S o l l e n t u n a , Sweden) were were h o u s e d for at least h l i g h t / d a r k cycle. They
rats w e i g h i n g about 220 g (Anticimex, used. Before the e x p e r i m e n t s the animals one week in a room m a i n t a i n e d on a 14/10 had free access to food and water.
In one e x p e r i m e n t the animals were given i n t r a p e r i t o n e a l injections with various doses of a d r e n a l i n e (ranged b e t w e e n 0.02 and 1.25 mg/kg), n o r a d r e n a l i n e (ranged b e t w e e n 0.02 and 2.50 mg/kg), or i s o p r e n a l i n e (ranged b e t w e e n 0.02 and 10.0 mg/kg). The drugs were d i s s o l v e d in saline in such a c o n c e n t r a t i o n , that the animals r e c e i v e d a c o n s t a n t volume of 10 ml/kg. Control rats r e c e i v e d the same v o l u m e of saline. Forty m i n u t e s a f t e r the i n j e c t i o n s the animals were k i l l e d by d e c a p i t a t i o n . In a second e x p e r i m e n t the rats in one group were i n j e c t e d i n t r a p e r i t o n e a l l y with L - p r o p r a n o l o l , 15 mg/kg, and 20 m i n u t e s later with i s o p r e n a l i n e , 10 m g / k g i.p. With the same time i n t e r v a l s two o t h e r groups of rats r e c e i v e d i n t r a p e r i t o n e a l i n j e c t i o n s with s a l i n e / i s o p r e n a l i n e and s a l i n e / s a l i n e , r e s p e c t i v e l y . A third experiment was c a r r i e d out s i m i l a r to this, except that the administered dosees of i s o p r e n a l i n e and L - p r o p r a n o l o l were 0.1 and 10 mg/kg, r e s p e c t i v e l y . The i n t e r v a l b e t w e e n the last i n j e c t i o n and death was 40 minutes. In a fourth e x p e r i m e n t p h e n o x y b e n z a m i n e , 10 mg/kg, was i n j e c t e d i n t r a p e r i t o n e a l l y to 18 rats. A n o t h e r 18 rats r e c e i v e d injections with e q u i v a l e n t volumes of saline. Fifty m i n u t e s later 6 rats from e i t h e r group were injected w i t h a d r e n a l i n e , 1.25 m g / k g i.p., w h e r e a s 6 rats were i n j e c t e d with n o r a d r e n a l i n e , 1.25 m g / k g i.p., and the last 6 rats from e i t h e r group were i n j e c t e d with saline. Forty m i n u t e s a f t e r the last i n j e c t i o n the rats were k i l l e d by d e c a p i t a t i o n A fifth e x p e r i m e n t was d e s i g n e d in the same way as the fourth one, except that p r o p r a n o l o l , 10 mg/kg, was a d m i n i s t e r e d instead of p h e n o x y b e n z a m i n e and that a d r e n a l i n e and n o r a d r e n a l i n e were given in a dose of 0.1 mg/kg. I m m e d i a t e l y a f t e r death about 5 ml b l o o d was c o l l e c t e d in a tube c o n t a i n i n g 0.5 ml of a 1 % E D T A solution. The p l a s m a samples were p u r i f i e d on a strong c a t i o n e x c h a n g e column (Dowex 50W X-4) (8, 9). S p e c t r o p h o t o f l u o r i m e t r i c a n a l y s e s of t y r o s i n e (10) and t r y p t o p h a n (11) were c a r r i e d out.
Vol. 30, No. 17, 1982
Regulation of Plasma Amino Acids
S t a t i s t i c a l s i g n i f i c a n c e s were of variance followed by t-test.
assessed
1467
by one way analysis
Results Figure 1 shows the effects of various doses of adrenaline, n o r a d r e n a l i n e and i s o p r e n a l i n e on plasma c o n c e n t r a t i o n s of tyro-
* *
Tyrosine in plasma
(n=3_12)
200
,,?.\!
150
Ad~,/!~/ I00
/,//,.Noradr. /
.o
•
\ ...-"._ •~.,.--~%- . . . . . . . . . _,r,:~r~ •,.,,
".... * * ~ ,
\.
-,.
,
* •
50
I00
/
•*
•
iw
,
•
: . ""0--'"
: + . . . o oo'"
I
I
..-o
Isopren. I
I
0.02
15oi
"a
/
_.,-~.../ .. . * ...... 0. * ** * * ""--..* ....... • ....... 0......
,---.-'@~
0
,,m S _- -// . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
I
I
0.08
|
,,,, I
0.31
1.25
I
5.00 Drug, mg.kg"
Tryptophan in plasma (n=3-12) -
~'~~,-~
......
A
d~
-~. ~
..,-,'~t'-~
;- . . . . . . . . . . . . .
".~ ....... ~'-..~o/' N.oradr . . "--.. , •
50
-0 ..... * •
* "-o
**
....... $... W
,
*
...... • .......
o-
|
I
*
* •
** ...... •
Isopren. 0
J
0.02
I
i
0.08
I
|
I
0.31
FIG.
I
1.25
J
5.00 Drug, mg.kg"
1
Effects of adrenaline, n o r a d r e n a l i n e and i s o p r e n a l i n e on rat p l a s m a c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. The drugs were i n j e c t e d i n t r a p e r i t o n e a l l y . Control rats r e c e i v e d an e q u i v a l e n t volume of saline. Forty minutes after the injections the animals were k i l l e d by decapitation. Plasma c o n c e n t r a t i o n s of t y r o s i n e and t r y p t o p h a n are shown as per cent of controls. Control values in pmol/l, mean ± s.e.m. (n), were for tyrosine 94 ± 4.7 (32) and for t r y p t o p h a n 86 ± 2.8 (32).
1468
Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
sine and t r y p t o p h a n . Low d o s e s (0.04 - 0.16 m g / k g ) of a d r e n a l i n e c a u s e d a d e c r e a s e by as m u c h as 30 per c e n t in p l a s m a c o n c e n t r a t i o n s of t y r o s i n e . H i g h d o s e s of a d r e n a l i n e (0.63 - 1.25 m g / k g ) on the o t h e r h a n d c a u s e d a s i g n i f i c a n t i n c r e a s e up to n e a r l y 200 per cent of the c o n t r o l . Low d o s e s of n o r a d r e n a l i n e had no e f f e c t on p l a s m a c o n c e n t r a t i o n of t y r o s i n e , w h i l e h i g h d o s e s (0.63 2.50 m g / k g ) c a u s e d an i n c r e a s e s i m i l a r to t h a t of a d r e n a l i n e . N e i t h e r a d r e n a l i n e nor n o r a d r e n a l i n e c a u s e d any s i g n i f i c a n t change: in p l a s m a c o n c e n t r a t i o n s of t r y p t o p h a n t h o u g h t h e r e seems to be a t e n d e n c y in the same d i r e c t i o n as was s e e n in the case of t y r o s i n e All d o s e s of i s o p r e n a l i n e g i v e n (0.04 - 10 m g / k g ) c a u s e d a s i g n i f i c a n t d e c r e a s e by as m u c h as 50 per cent in p l a s m a c o n c e n t r a t i o n s of b o t h t y r s o i n e and t r y p t o p h a n .
I00
Tyrosine I
'~ 6 0
--4--
4o 2O 5
6
I00
Tryptophan I
-+-
Saline or L-Propranolol
Saline or Isoprenaline
I
I
0
20
Killed I
minu~es
,'I-
60
-I-. q3
4o
.~ 2O
0
6
6 FIG.
2
E f f e c t of L - p r o p r a n o l o l on i s o p r e n a l i n e - i n d u c e d decrease in rat p l a s m a c o n c e n t r a t i o n s of t y r o s i n e a n d t r y p t o p h a n . L - p r o p r a n o l o l , 10 or 15 m g / k g , or an e q u i v a l e n t v o l u m e of s a l i n e was i n j e c t e d i.p. and f o l l o w e d 20 m i n u t e s l a t e r by an i.p. i n j e c t i o n of i s o p r e n a l i n e 0.1 or 10 m g / k g , or an e q u i v a l e n t v o l u m e of s a l i n e . F o r t y m i n u t e s l a t e r the a n i m a l s w e r e k i l l e d by d e c a p i t a t i o n . P l a s m a c o n c e n t r a t i o n s are s h o w n as per cent of c o n t r o l s . C o n t o l v a l u e s in p m o l / l , m e a n ± s.e.m. (n), w e r e for t y r o s i n e 96 ± 2.5 (11) a n d for t r y p t o p h a n 104 ± 3.4 (11).
6O
Vol. 30, No. 17, 1982
Regulation of Plasma Amino Acids
1469
Figure 2 illustrates that the decrease in p l a s m a c o n c e n t r a tions of t y r o s i n e and t r y p t o p h a n caused by a low dose of isoprenaline (0.1 mg/kg) could be c o m p l e t e l y b l o c k e d by propranolol, while the decrease caused by a much h i g h e r dose of i s o p r e n a l i n e (10 mg/kg) could not be i n f l u e n c e d by propranolol. Figure 3 shows the changes in plasma c o n c e n t r a t i o n s of tyrosine and t r y p t o p h a n after t r e a t m e n t with p h e n o x y b e n z a m i n e , adrenaline and n o r a d r e n a l i n e and with c o m b i n a t i o n s of p h e n o x y b e n z a m i n e
220
Tyrosine
t •A,
200
-t-
180
~
~r
+
160 140
~o
12o ~o
Saline or Phenoxybenzamine, I0 mg-kg "~ i.p. I 0
Saline or Adrenaline, 1.25 mg-kg "~ i.p. or Noradrenaline, 1.25 mg'kg "~ i.p. Killed I I 5 0 minutes 9O
~oo 8o
!it ,A,
6O
•A,
,A,
,k
4O 2O 6
0 120-
~
I00
u
80
Tryptophan I t
604020O-
6
6
6
FIG.
3
Effects of p h e n o x y b e n z a m i n e , adrenaline, n o r a d r e n a l i n e , and c o m b i n a t i o n s of p h e n o x y b e n z a m i n e with a d r e n a l i n e or n o r a d r e n a l i n e on rat plasma c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. P h e n o x y b e n z a m i n e , 10 mg/kg, or an equivalent volume of saline was injected i.p. Fifty minutes later adrenaline, 1.25 mg/kg, n o r a d r e n a l i n e , 1.25 mg/kg, or an e q u i v a l e n t volume of saline was injected i.p. Forty minutes after the last i n j e c t i o n the rats were killed by decapitation. Plasma c o n c e n t r a t i o n s are shown as per cent of controls. Control values in pmol/l, mean ± s.e.m. (n), were for t y r o s i n e 69 ± 3.3 (6) and for t r y p t o p h a n 75 ± 2.6 (6).
1470
Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
w i t h a d r e n a l i n e or n o r a d r e n a l i n e . P h e n o x y b e n z a m i n e caused a marked decrease in the c o n c e n t r a t i o n s of both p l a s m a t y r o s i n e and t r y p t o p h a n . P h e n o x y b e n z a m i n e also c o m p l e t e l y b l o c k e d the tyros i n e - i n c r e a s i n g effect of both a d r e n a l i n e and n o r a d r e n a l i n e . In figure 4 are shown the effects of L - p r o p p a n o l o l , adrenaline and n o r a d r e n a l i n e and of c o m b i n a t i o n s of L - p r o p r a n o l o l with
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Saline or Adrenaline, O.mmg.kg ~ i.p. or Noradrenaline, 0.1 mg-kg -t i.p. Killed I I 50 minutes 90 FIG.
4
Effects of L - p r o p r a n o l o l , a d r e n a l i n e , n o r a d r e n a l i n e and c o m b i n a t i o n s of L - p r o p r a n o l o l w i t h a d r e n a l i n e or nora d r e n a l i n e on rat plasma c o n c e n t r a t i o n s of t y r o s i n e and t r y p t o p h a n . L - P r o p r a n o l o l , 10 mg/kg, or an e q u i v a l e n t volume of saline was i n j e c t e d i.p. Fifty m i n u t e s later a d r e n a l i n e , 0.1 mg/kg, n o r a d r e n a l i n e , 0.1 mg/kg, or an e q u i v a l e n t volume of saline was i n j e c t e d i.p. Forty m i n u t e s a f t e r the last i n j e c t i o n the rats were k i l l e d by d e c a p i tation. Plasma c o n c e n t r a t i o n s are shown as per cent of controls. Control values in ~mol/l, m e a n ± s.e.m. (n), were for t y r o s i n e 63 ± 1.9 (6) and for t r y p t o p h a n 72 ± 5.9 (6).
Vol. 30, No. 17, 1982
Regulation of Plasma Amino Acids
1471
a d r e n a l i n e or n o r a d r e n a l i n e on plasma c o n c e n t r a t i o n s of tyrosine and tryptophan. L - P r o p r a n o l o l had by itself no effect on plasma t y r o s i n e or tryptophan, but it caused a complete inhibition of the amino acid d e c r e a s i n g p r o p e r t i e s of a d r e n a l i n e and noradrenaline. Discussion N o r a d r e n a l i n e is c o n s i d e r e d to possess a strong e- (and a weak B-) type activity, while i s o p r e n a l i n e exerts strong B- (and weak ~-) influences. A d r e n a l i n e has both activities, but the Bactions p r e d o m i n a t e (12). As the decrease in plasma amino acids seen after a d m i n i s t r a t i o n of low doses of adrenaline, noradrenaline and i s o p r e n a l i n e could be c o m p l e t e l y inhibited by the Ba d r e n e r g i c a n t a g o n i s t propranolol, we suggest that this effect is m e d i a t e d via a d r e n e r g i c B-receptors. This h y p o t h e s i s is s u p p o r t e d by the fact that the amino acid d e c r e a s i n g effect of i s o p r e n a l i n e is more p r o n o u n c e d than that of adrenaline, which in turn is more p r o n o u n c e d than that of n o r a d r e n a l i n e . Higher doses of a d r e n a l i n e and n o r a d r e n a l i n e , but not of isoprenaline, cause an increase in plasma tyrosine c o n c e n t r a tions. This effect is c o m p l e t e l y i n h i b i t e d by the s - r e c e p t o r b l o c k i n g agent p h e n o x y b e n z a m i n e , s u g g e s t i n g that this effect of a d r e n a l i n e and n o r a d r e n a l i n e is m e d i a t e d via a d r e n e r g i c ~-receptors. P h e n o x y b e n z a m i n e has by itself a d e c r e a s i n g effect on plasma c o n c e n t r a t i o n s of t y r o s i n e and tryptophan. This p h e n o m e n o n could be due to loss of a tonic ~ - a d r e n e r g i c influence on these levels. A l t e r n a t i v e l y , it could be m e d i a t e d via adrenergic 6-receptors s t i m u l a t e d by an i n c r e a s e d s e c r e t i o n of n o r a d r e n a l i n e which is known to occur after t r e a t m e n t with p h e n o x y b e n z a m i n e (13). The m e c h a n i s m s u n d e r l y i n g the a d r e n e r g i c influence on plasma amino acids could not be d i s c l o s e d by the present data. The way in which a d r e n e r g i c m e c h a n i s m s take part in the control of carbohydrate, protein and lipid m e t a b o l i s m is complex (12). Both pancreatic hormones and the h y p o t h a l a m i c - p i t u i t a r y - a d r e n o c o r t i c a l s y s t e m must be c o n s i d e r e d in this context. A n o t h e r p o s s i b i l i t y is that the d e m o n s t r a t e d effects on plasma amino acids are caused by changes in the total blood flow r e c e i v e d by the liver, in which most amino acids are metabolized. This h y p o t h e s i s is s u p p o r t e d by the fact that s t i m u l a t i o n o ~ ~a d r e n e r g i c r e c e p t o r s a c t u a l l y causes v a s o c o n s t r i c t i o n and d e c r e a s e d h e p a t i c blood flow, while B-adrenergic s t i m u l a t i o n gives rise to v a s o d i l a t a t i o n and i n c r e a s e d liver p e r f u s i o n (14). Acknowledgements This work was s u p p o r t e d by the Swedish Medical R e s e a r c h Council (project No. 155), the Faculty of Medicine at the University of G ~ t e b o r g and the G ~ t e b o r g Medical Society. We want to express H o l m g r e n for her expert
our sincere gratitude t e c h n i c a l assistance.
to mrs
Birgitta
1472
Regulation of Plasma Amino Acids
Vol. 30, No. 17, 1982
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