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Chronic administration of lithium modulates tryptophan transport by changing the properties of the synaptosomal plasma membrane
Life Sciences, Vol. 41, pp. 643-650 Printed in the U.S.A.
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C H R O N I C A D M I N I S T R A T I O N OF L I T H I U M M O D U L A T E S T R A N S P O R T BY C H A N G I N G T H E P R O P E R T I E S OF T H E MAL PLASMA MEMBRANE E.
Herrero,
C. G i m ~ n e z
and M.C.
TRYPTOPHAN SYNAPTOSO-
Arag6n
D e p a r t a m e n t o de B i o l o g i a M o l e c u l a r . C e n t r o de B i o l o g l a M o l e c u l a r . F a c u l t a d de C i e n c i a s . U n i v e r s i d a d A u t 6 n o m a de M a d r i d . 2 8 0 4 9 - M a d r i d . S p a i n . (Received in final form May 28, 1987) Summary The e f f e c t of c h r o n i c a d m i n i s t r a t i o n of l i t h i u m s a l t s on the s o d i u m d e p e n d e n t h i g h - a f f i n i t y s y s t e m for t r y p tophan uptake w a s e x a m i n e d in p l a s m a m e m b r a n e v e s i c l e s d e r i v e d f r o m rat b r a i n . Tryptophan transport was m e a s u r e d as a f u n c t i o n of the t e m p e r a t u r e , A r r h e n i u s p l o t s of the d a t a w e r e p r e p a r e d , a n d the a p p a r e n t e n e r g i e s of a c t i v a t i o n w e r e c o m p u t e d . B o t h p l o t s w e r e bi ~ p h a s i c , the t r a n s i t i o n t e m p e r a t u r e d e c r e a s i n g f r o m 2 3 ° C in c o n t r o l a n i m a l s to 18°C in t r e a t e d rats. The a v e r a g e a p p a r e n t e n e r g i e s of a c t i v a t i o n for the c a r r i e r a l s o c h a n g e - b o t h b e l o w a n d a b o v e the t r a n s i t i o n t e m p e r a t u r e - in t r e a t e d a n i m a l s w h e n c o m p a r e d to c o n t r o l s . O u r d a t a s u p p o r t the i d e a t h a t c h r o n i c a d m i n i s t r a t i o n of l i t h i u m i n d u c e s a m o r e f l u i d s t a t e of the s y n a p t o s o m a l p l a s m a m e m b r a n e t h a t c o u l d e x p l a i n m a n y of the e f f e c t s of Li + on m e m b r a n e - b o u n d p r o t e i n s . L i t h i u m is u s e d e x t e n s i v e l y in the t h e r a p y of m a n i c - d e p r e s s i v e p s y c h o s e s , b u t its m o d e of a c t i o n is s t i l l u n k n o w n . Although l i t h i u m i o n s h a v e b e e n r e p o r t e d to a c t in s e v e r a l d i f f e r e n t w a y s (1-4), m o s t of the h y p o t h e s e s c o n c e r n i n g the m e c h a n i s m of the t h e r a p e u t i c a c t i o n of l i t h i u m s a l t s are a s s o c i a t e d w i t h a l t e r a t i o n s in a m i n e n e u r o t r a n s m i s s i o n (5-12). Treatment with lithium has b e e n s h o w n to e n h a n c e some b e h a v i o u r a l and n e u r o p h y s i o l o g i c a l e f f e c t s of s e r o t o n i n (13-15 t h r o u g h m e c h a n i s m s as y e t not w e l l known. S e r o t o n i n b i o s y n t h e s i s in the b r a i n d e p e n d s on the i n t r a c e l l u l a r t r y p t o p h a n l e v e l s s i n c e t r y p t o p h a n h y d r o x y l a s e , the e n z y m i c s t e p l i m i t i n g the r a t e of s e r o t o n i n s y n t h e s i s , is not s a t u r a ted w i t h its s u b s t r a t e L - t r y p t o p h a n u n d e r p h y s i o l o g i c a l c o n d i t i o n s (16). As the c o n c e n t r a t i o n of t r y p t o p h a n in b r a i n c e l l s (17,18) is r e l a t i v e l y s m a l l w h e n c o m p a r e d w i t h t h a t of m o s t o t h e r a m i n o a c i d s , it h a s b e e n s u g g e s t e d t h a t its t r a n s p o r t a c r o s s the p l a s m a m e m b r a n e is r a t e l i m i t i n g for its i n t r a c e l l u l a r c o n c e n t r a t i o n (19,20). The o c c u r r e n c e of two of s u c h p r o c e s s e s of u p t a k e in b r a i n p r e p a r a t i o n s , w i t h h i g h a n d low a f f i n i t i e s for t r y p t o p h a n , h a s b e e n d e s c r i b e d ( 6 , 9 , 2 1 , 2 2 ) , and the a c c u m u l a t e d e v i d e n c e a b o u t the ion d e p e n d e n c e of t r a n s p o r t i n d i c a t e s t h a t the e l e c t r o c h e m i c a l p o t e n t i a l c r e a t e d by a Na + g r a d i e n t s e r v e s as a d i r e c t d r i v i n g f o r c e for the p r o c e s s (23-25). It has b e e n r e p o r t e d t h a t in a d u l t 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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rats the t r a n s p o r t of t r y p t o p h a n a c r o s s the neural m e m b r a n e s is d e p e n d e n t on the N a + - g r a d i e n t , and is s t i m u l a t e d by a m e m b r a n e pot e n t i a l (negative inside). We have p r e v i o u s l y d e m o n s t r a t e d that p l a s m a m e m b r a n e v e s i c l e s o b t a i n e d from l o n g - t e r m l i t h i u m - t r e a t e d rats are able to a c c u m u l a t e t r y p t o p h a n to a g r e a t e r e x t e n t than those from n o r m a l rats and m a i n t a i n a m o r e n e g a t i v e m e m b r a n e p o t e ~ tial than c o n t r o l s (22,26). In this r e p o r t we s h o w that t r e a t m e n t w i t h l i t h i u m p r o m o tes d i s c o n t i n u i t i e s in the A r r h e n i u s p l o t s of the m e m b r a n e - b o u n d t r a n s p o r t s y s t e m for t r y p t o p h a n and lowers the t r a n s i t i o n t e m p e r a ture of the system. These facts i n d i c a t e the e x i s t e n c e of p h a s e t r a n s i t i o n s and E a c h a n g e s and s u g g e s t that l i t h i u m t r e a t m e n t ind u c e s a m o r e fluid state of the n e u r o n a l m e m b r a n e . Materials
and M e t h o d s
M a t e r i a l s . L - ( G - 3 H ) T r y p t o p h a n (specific r a d i o a c t i v i t y 3.1 Ci/mmol) was o b t a i n e d from the R a d i o c h e m i c a l Center, A m e r s h a m , Bucks, U.K. F i c o l l was p r o v i d e d by P h a r m a c i a and w a s e x h a u s t i v e l y d i a l y z e d before use. All o t h e r r e a g e n t s used were of the h i g h e s t p u r i t y avai ! able. L i t h i u m a d m i n i s t r a t i o n and l i t h i u m analysis. L i t h i u m was a d m i n i s tered to 300-320 g m a l e W i s t a r rats in the d r i n k i n g w a t e r (I m g / m l LiCI). S e r u m l i t h i u m was m e a s u r e d by flame p h o t o m e t r y (Perkin-Elm e r Mod. 372) in c a r o t i d b l o o d o b t a i n e d at the time of killing. For b r a i n tissue, a l i q u o t s w e r e weighed, h o m o g e n i z e d in 3% p e r c h l o ric acid, and c e n t r i f u g e d ; the l i t h i u m c o n c e n t r a t i o n s w e r e d e t e r m i n e d in the acid extract. M e m b r a n e v e s i c l e s p r e p a r a t i o n . M e m b r a n e v e s i c l e s from a d u l t male W i s t a r rats (300-320 g) w e r e p r e p a r e d e s s e n t i a l l y as d e s c r i b e d p r e v i o u s l y (27). A f t e r o s m o t i c d i s r u p t i o n of s y n a p t o s o m e s , the susp e n s i o n was c e n t r i f u g e d at 27,000 g for 20 min, and the p e l l e t was r e s u s p e n d e d in 10 ml of a 284 m O S M medium, pH 7.4, w i t h the ionic c o m p o s i t i o n d e p e n d i n g on each p a r t i c u l a r e x p e r i m e n t . Finally, the s u s p e n s i o n was c e n t r i f u g e d at 27,000 g for 15 min and the p e l l e t was r e s u s p e n d e d in the former m e d i u m to a p r o t e i n c o n c e n t r a t i o n of 15-25 mg/ml. P o r t i o n s w e r e f r o z e n in liquid n i t r o g e n and stored at -70°C. A l i q u o t s w e r e q u i c k l y t h a w e d at 37°C. Under these conditions, m e m b r a n e v e s i c l e s w e r e f u n c t i o n a l for at least 1 month. T r a n s p o r t assays. P o r t i o n s (20 ~i) of the s u s p e n s i o n of m e m b r a n e v e s i c l e s d e r i v e d from rat b r a i n (about 0.2 mg of p r o t e i n ) , w e r e p r e l o a d e d , u n l e s s o t h e r w i s e stated, w i t h 120 m M KCI-22 m M p o t a s s i u m p h o s p h a t e buffer, pH 7.4 (KCI medium), and p r e i n c u b a t e d for 1 m i n at 25°C. U p t a k e was s t a r t e d by a d d i t i o n of 100 ul of a solution containing L-(G-3H)-tryptophan (5 ~M final c o n c e n t r a t i o n ) and the d e s i r e d ionic c o m p o s i t i o n , and was t e r m i n a t e d by d i l u t i n g w i t h 5 ml of i c e - c o l d 0.8 M NaCI, and i m m e d i a t e l y f i l t e r i n g t h r o u g h a m o i s t e n e d M i l l i p o r e filter RAWP 02500 (1.2 um pore size) a t t a c h e d to a v a c u u m a s s e m b l y . The filters w e r e r i n s e d twice w i t h the i c e - c o l d medium. Dilution, f i l t r a t i o n , and w a s h i n g w e r e per-
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f o r m e d w i t h i n 15 s. The f i l t e r s w e r e d r i e d at 60°C and p l a c e d in m i c r o v i a l s , and t h e i r r a d i o a c t i v i t y was m e a s u r e d in a l i q u i d - s c i n t i l l a t i o n c o u n t e r (Beckman LS-350). R e s u l t s w e r e c o r r e c t e d by m e a n s of a c o n t r o l o b t a i n e d by d i l u t i n g the m e m b r a n e s u s p e n s i o n b e f o r e a d d i n g the r a d i o a c t i v e s u b s t r a t e s o l u t i o n . All s o l u t i o n s u s e d in the p r e p a r a t i o n of the m e m b r a n e v e s i c l e s and in the u p t a k e e x p e r i m e n t s w e r e p r e p a r e d w i t h d i s t i l l e d d e i o n i z e d w a t e r and filt e r e d t h r o u g h M i l l i p o r e f i l t e r s (0.45 ~m) to a v o i d p o s s i b l e b a c t e rial c o n t a m i n a t i o n . The o s m o l a r i t y of all s o l u t i o n s was k e p t c o n s t a n t d u r i n g the u p t a k e e x p e r i m e n t s . The pH of the e x t e r n a l and i n t e r n a l m e d i u m was 7.4 t h r o u g h o u t the e x p e r i m e n t s . The ionic c o m p o s i t i o n of the v e s i c l e s , i n t e r n a l and e x t e r n a l m e d i u m was m o d i fled to i n v e s t i g a t e the e f f e c t s of d i f f e r e n t ions. All i n c u b a t i o n s w e r e c a r r i e d out in t r i p l i c a t e . E a c h exp e r i m e n t was r e p e a t e d at least three times w i t h d i f f e r e n t m e m b r a n e preparations. A g r e e m e n t a m o n g e x p e r i m e n t s was good; thus, t y p i c a l e x p e r i m e n t s are r e p o r t e d . Protein determination. m e t h o d of R e s c h et al.
M e m b r a n e p r o t e i n was d e t e r m i n e d by the (28).
A n a l y s i s of the data. C a l c u l a t i o n of a c t i v a t i o n e n e r g i e s (E a) was a c c o r d i n g to the A r r h e n i u s e q u a t i o n w h i c h m a y be w r i t t e n as log K =-
Ea 2.3RT
1 T
w h e r e K is the rate c o n s t a n t , R the gas c o n s t a n t , and T the absolute t e m p e r a t u r e . In p r a c t i c e the n u m e r i c a l v a l u e of E a is d e t e r m i n e d f r o m the slope of a p l o t of log K v e r s u s 1/T. S t a t i s t i c a l a n a l y s i s was p e r f o r m e d by the S t u d e n t ' s t - t e s t for n o n - p a i r e d s a m p l e s and a c o m p a r i s o n was m a d e w i t h the c o n t r o l group. P v a l u e s of 0.05 or less w e r e taken as s i g n i f i c a n t , and the r e s u l t s are e x p r e s s e d as the m e a n ± S.E.M. R e s u l t s and D i s c u s s i o n Rats g i v e n l i t h i u m c h l o r i d e ad l i b i t u m in t h e i r d r i n k i n g w a t e r for four w e e k s p r e s e n t e d p l a s m a l i t h i u m c o n c e n t r a t i o n s of 0 . 7 5 ± 0 . 2 0 m E q / l i t e r (mean±S.E.M. of ten a n i m a l s ) , e q u i v a l e n t to the t h e r a p e u t i c l e v e l s u s u a l l y found in humans. LiCl-treated a n i m a l s s h o w e d a light p o l y u r i a but did not d i f f e r s i g n i f i c a n t l y f r o m c o n t r o l s in w e i g h t g a i n and e x i b i t e d no signs of l i t h i u m intoxication. The l e v e l s of l i t h i u m in the b r a i n (1.05±0.17 m E q / g wet weight) w e r e a l w a y s h i g h e r than t h o s e in plasma. A l t h o u g h the m e c h a n i s m of a c t i o n of l i t h i u m is not w e l l known, it involves, at l e a s t in part, the a b i l i t y of Li + to m i m i c o t h e r b i o l o g i c a l l y i m p o r t a n t c a t i o n s such as sodium, m a g n e s i u m , or c a l c i u m ions. S i n c e these c a t i o n s are i n t i m a t e l y , r e l a t e d to m e m b r a n e f u n c t i o n s , it seems l i k e l y that l i t h i u m t h e r a p y i n v o l v e s membrane effects. However, the m o l e c u l a r d e t a i l s of the s t r u c t u ral a l t e r a t i o n s u n d e r l y i n g the e f f e c t s of l i t h i u m on b i o m e m b r a n e s
646
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uncharacterized.
F i g u r e 1 s h o w s the e f f e c t of t e m p e r a t u r e on the u p t a k e r a t e s of L - t r y p t o p h a n in p l a s m a m e m b r a n e v e s i c l e s o b t a i n e d f r o m c o n t r o l s (Fig. IA) a n d l i t h i u m - t r e a t e d r a t s (Fig. IB) . The m i n i m u m t e m p e r a t u r e at w h i c h a n y s u b s t r a t e a c c u m u l a t i o n c o u l d be d e t e c t e d w a s f o u n d to be a b o u t 5°C. As e x p e c t e d , the a c c u m u l a t i o n of t r i p t o p h a n i n c r e a s e s w i t h i n c r e a s i n g t e m p e r a t u r e in b o t h c o n t r o l and l i t h i u m - t r e a t e d rats, a n d the o v e r a l l s h a p e of the c u r v e s is f a i r l y s i m i l a r . H o w e v e r , w h e r e a s the u p t a k e r a t e s u n d e r the n o n - g r a d i e n t c o n d i t i o n s i n c r e a s e l i n e a r l y w i t h the t e m p e r a t u re a n d v e r y s i m i l a r l y in v e s i c l e s f r o m c o n t r o l a n d t r e a t e d rats,
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E f f e c t of t e m p e r a t u r e on the u p t a k e r a t e s of L - t r y p t ~ p h a n in p l a s m a m e m b r a n e v e s i c l e s f r o m rat b r a i n synaptosomes. The m e m b r a n e v e s i c l e s w e r e o b t a i n e d f r o m c o n t r o l r a t s (A) a n d f r o m l o n g - t e r m l i t h i u m - t r e a t e d r a t s (B) and w e r e p r e l o a d e d w i t h KCI m e d i u m and p r e i n c u b a t e d for 2 m i n at the i n d i c a t e d t e m p e r a t u r e s . A f t e r that, the v e s i c l e s w e r e i n c u b a t e d for 15 sec in a m e d i u m c o n t a i n i n g 120 m M NaCl, 2 2 m M p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.4 (NaCl m e d i u m ) (e), or KCl m e d i u m (o) in the p r e s e n c e of 5 u M - L ( G - 3 H ) t r y p t o p h a n at the indicated temperatures. The d i f f e r e n c e s b e t w e e n upt a k e in the N a C l m e d i u m a n d the KCl m e d i u m w a s t a k e n as the s p e c i f i c N a + - d e p e n d e n t u p t a k e (A). The compos i t i o n of the KCl m e d i u m is d e s c r i b e d in the M a t e r i a l s and M e t h o d s S e c t i o n .
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the u p t a k e r a t e s of t r y p t o p h a n t r a n s p o r t are a l w a y s h i g h e r in l i t h i u m - t r e a t e d r a t s t h a n in the c o n t r o l s in the p r e s e n c e of a Na + g r a d i e n t ( o u t s i d e > i n s i d e ) . T h i s f a c t is e v e n c l e a r e r w h e n the u p t a k e r a t e s in the p r e s e n c e of a Na + g r a d i e n t are c o r r e c t e d for the r a t e s u n d e r n o n - g r a d i e n t c o n d i t i o n s (i.e., KCI m e d i u m ) . At the h i g h e s t t e m p e r a t u r e t e s t e d (45°C), the a c c u m u l a t i o n r a t e of t r y p t o p h a n is m o r e t h a n 60% h i g h e r in p l a s m a m e m b r a n e v e s i c l e s f r o m l i t h i u m - t r e a t e d t h a n in t h o s e f r o m c o n t r o l rats. The A r r h e n i u s p l o t s for the t r a n s p o r t s y s t e m t r y p t o p h a n in b o t h s i t u a t i o n s are d e p i c t e d in Fig. 2, a n d the v a l u e s of t r a n s i t i o n t e m p e r a t u r e s and a c t i v a t i o n e n e r g i e s (E a) are s h o w n in Table I. A d i s c o n t i n u i t y in the A r r h e n i u s p l o t w a s o b s e r v e d in p l a s m a m e m b r a n e v e s i c l e s f r o m e i t h e r c o n t r o l s or l i t h i u m - t r e a t e d rats, and the b r e a k - p o i n t t e m p e r a t u r e s are l i s t e d in T a b l e i. The aver a g e a p p a r e n t a c t i v a t i o n e n e r g y for the t r y p t o p h a n t r a n s p o r t syst e m f r o m c o n t r o l r a t s w a s f o u n d to be 3.28 K c a l / m o l a b o v e and 7 . 1 0 K c a l / m o l b e l o w the t r a n s i t i o n t e m p e r a t u r e . The a p p a r e n t a c t i v a t i o n e n e r g y in the c a s e of l i t h i u m - t r e a t e d r a t s w a s f o u n d to be-4.31 K c a l / m o l a b o v e a n d 8.17 K c a l / m o l b e l o w the t r a n s i t i o n t e m p e r a t u r e , b o t h v a l u e s b e i n g s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e of c o n t r o l rats.
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3.6
2
A r r h e n i u s p l o t s of s p e c i f i c N a + - d e p e n d e n t u p t a k e of t r y p t o p h a n in s y n a p t o s o m a l p l a s m a m e m b r a n e v e s i c l e s t e s t e d in c o n t r o l s (A) and in l o n g - t e r m l i t h i u m t r e a t e d r a t s (B) . T h e e x p e r i m e n t a l c o n d i t i o n s are d e s c r i b e d in the l e g e n d to Fig. I.
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A b r u p t c h a n g e s in the slope of A r r h e n i u s p l o t s at a p a r t ~ c u l a r t e m p e r a t u r e have b e e n t a k e n to r e p r e s e n t a p h a s e t r a n s i t i o n in the lipid e n v i r o n m e n t of the m e m b r a n e - b o u n d e n z y m e s and t r a n s p o r t s y s t e m s (29-33). In the p r e s e n t i n v e s t i g a t i o n , the t r y p t o p h a n u p t a k e s y s t e m in s y n a p t o s o m a l p l a s m a m e m b r a n e v e s i c l e s f r o m c o n t r o l a n i m a l s s h o w e d a b r e a k p o i n t at 23°C, w h i c h s h i f t e d to 18°C a f t e r the l i t h i u m t r e a t m e n t , thus r e f l e c t i n g a p e r t u r b a t i o n of the m e m b r a n e l i p i d s in this s i t u a t i o n . The l o w e r i n g of the t r a n s i t i o n t e m p e r a t u r e and the c h a n g e s in the A r r h r e n i u s a c t i v a tion e n e r g y a b o u t the t r a n s i t i o n t e m p e r a t u r e p r o d u c e d by longt e r m l i t h i u m t r e a t m e n t , w o u l d be e x p e c t e d if is a s s u m e d that lit h i u m t r e a t m e n t i n d u c e s a m o r e fluid state of the s y n a p t o s o m a l plasma membrane. It is i n t e r e s t i n g to note that we did not found any a p p a rent e f f e c t of a l o n g - t e r m t r e a t m e n t w i t h l i t h i u m salts on the t r a n s p o r t of t y r o s i n e , a s t r u c t u r a l l y r e l a t e d a m i n o acid and p r e c u r s o r of c a t e c h o l a m i n e s (data not shown). A p o s s i b l e e x p l a n a t i o n for the d i f f e r e n t t h e r m a l b e h a v i o r of the t r y p t o p h a n t r a n s p o r t s y s t e m f o u n d in the l i t h i u m - t r e a t e d a n i m a l s m i g h t be due to d i f f e r e n c e s in the composition of the m e m b r a n e s i n d u c e d by l i t h i u m t r e a t m e n t (i.e. p h o s p h o l i p i d s , fatty acids, or c h o l e s t e r o l ) t h a t c o u l d i n f l u e n c e the l i p i d - p r o t e i n interactions. Such i n t e r a c t i o n c o u l d a l s o a f f e c t the c o n f o r m a tion or a g g r e g a t i o n state of the protein, and the r e s u l t i n g changes in p r o t e i n s t r u c t u r e w i t h t e m p e r a t u r e m i g h t a l s o d e t e r m i n e the b r e a k p o i n t of the A r r h e n i u s plot, as has b e e n e m p h a s i z e d by some a u t h o r s (34-36). A l t h o u g h l i t h i u m is w i d e l y u s e d for the t r e a t m e n t of mania, its e f f e c t s on the c e n t r a l n e r v o u s s y s t e m are p o o r l y u n d e r stood. It is w o r t h n o t i n g that m a n y of the l i t h i u m e f f e c t s des-
TABLE
1
V a l u e s of t r a n s i t i o n t e m p e r a t u r e s and a c t i v a t i o n e n e r g i e s (E a) for the t r y p t o p h a n t r a n s p o r t s y s t e m in p l a s m a m e m b r a n e v e s i c l e s f r o m rat b r a i n s y n a p t o s o m e s . Activation energy (Ea) (Kcal/mol)
Controls Li+-treated animals
Transition temperature ° C
below break ~oint
above break
23. i±i. 0
7. i0_+0.09
3.28+ - 0.05
18.2±i. i***
8.17±0.08"**
4.31±0.06"**
V a l u e s are the m e a n s ± SEM of at least ten i n d e p e n d e n t experiments. S i g n i f i c a n c e of d i f f e r e n c e b e t w e e n t r e a t e d and n o n - t r e a t e d animals: * * * p < 0 ° 0 0 1
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c r i b e d in the l i t e r a t u r e involve a great v a r i e t y of m e m b r a n e - b o u n d a c t i v i t i e s (i.e., c a r r i e r s and r e c e p t o r s ) . Thus it is t e m p t i n g to s p e c u l a t e that the t h e r a p e u t i c a c t i o n s of l i t h i u m take p l a c e t h r o u g h a l t e r a t i o n s in the c o m p o s i t i o n a n d / o r the d y n a m i c p r o p e r ties of the n e u r a l m e m b r a n e s . Acknowledgements This w o r k was m 6 n Areces.
supported
by a g r a n t
from the F u n d a c i 6 n
Ra-
References i. M.H. SHEARD, T r e n d s N e u r o s c i . 3: 85-86 (1980). 2. B.E. E H R L I C H and J.M. DIAMOND, J. M e m b r a n e Biol. 52: 187-200 (1980). 3. W.R. SHERMAN, L.Y. MUNSELL, G.G. B E B E R L Y and M.P. HONCHAR, J. N e u r o c h e m . 44: 798-807 (1985). 4. M. S C H O U and K. THOMSEN, in J o h n s o n F.N. (ed.) L i t h i u m R e s e a r c h and Therapy, p a g e s 63-64, A c a d e m i c Press, L o n d o n (1975). 5. J. P E R E Z - C R E U E T , P. T A G L I A M O N T E and G.L. GESSA, J. P h a r m a c o l . Exp. Ther. 178: 325-330 (1971). 6. A.J. M A N D E L L and S. KNAPP, Fed. Proc. Fed. Am. Soc. Exp. Biol. 36: 2 1 4 2 - 2 1 4 8 (1977). 7. I.J. WAJDA, M. B A N A Y - S C H W A R T Z , I. M A N I G A U L T and A. LAJTHA, Neurochem. Res. 6: 321:331 (1978). 8. A. PERT, J.E. R O S E N B L A T , C. SIVIT, C.B. P E R T and W.E. BUNNEY, Science. 201: 171-173 (1978). 9. M.L. L A A K S O and S.S. OJA, N e u r o c h e m . Res. 4 : 4 1 1 - 4 2 3 (1979). i0. S. T R E I S E R and K.J. KELLAR, Eur. J. P h a r m a c o l . 58: 85-86 (1980). ii. S. KNAPP, and A.J. MANDELL, S c i e n c e 180: 645-647 (1973). 12. A.J. M A N D E L L and S. KNAPP, in Almgrem, O., C a r l s s o n , A. and Engel, J. (eds.), C h e m i c a l tools in c a t e c h o l a m i n e r e s e a r c h II. Pages 9-16, N o r t h - H o l l a n d , A m s t e r d a m (1975). 13. C. SANGEE, and D.V. F R A N Z (1978) Commun. P s y c h o p h a r m a c o l . 2, 191-198. 14. E. F R I E D M A N , A. D A L L O B and G. LEVINE, Life Sci. 25, 1 2 6 3 - 1 2 6 6 (1979). 15. H.Y. METZER, in Salama, A.I. (ed.) P r e s y n a p t i c m o d u l a t i o n of p o s t s y n a p t i c r e c e p t o r s in m e n t a l diseases. Pages 115-137. Ann, N.Y. Acad. Sci. 430 (1984). 16. J.L. M E E K and S. L O F S T R A N D , Eur. J. P h a r m a c o l . 37: 377-380 (1986) 17. C.M. Mc KEAN, D.E. B O G G S and N.A. PETERSON, J. N e u r o c h e m . 15: 2 3 5 - 2 4 1 (1968). 18. L.A. T Y F I E L D and J. HOLTON, J. N e u r o c h e m . 26: 101-105 (1976). 19. D.G. G R A H A M E - S M I T H and A.G. PARFITT, J. N e u r o c h e m . 17: 13391353 (1970). 20. A. LAJTHA, in A r o m a t i c A m i n o A c i d s in the Brain. Pages 25-49. E l s e v i e r , A m s t e r d a m (1974). 21. E.R. KORPI, N e u r o c h e m . Res. 5 : 4 1 5 - 4 3 1 (1980). 22. E. HERRERO, M.C. ARAGON, C. G I M E N E Z and F. V A L D I V I E S O , J. Neurochem. 40: 332-337 (1983). 23. S.G. SHULTZ and P.F. CURRAN, Physiol. Rev. 50: 637-718.
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24. I.C. WEST, B i o c h i m . B i o p h y s . A c t a 604: 91-126 (1980). 25. B.I. KANNER, Biochim. Biophys. A c t a 726: 239-316 (1983). 26. M.C. A R A G O N , E. HERRERO, C. GIMENEZ, and F. V A L D I V I E S O . B i o c h e m Soc. Trans. ii: 6 9 6 - 6 9 7 (1983). 27. B.I. KANNER, B i o c h e m i s t r y 17: 1 2 0 7 - 1 2 1 1 (1978). 28. K. RESCH, I. IMM, E. FERBER, D.F.H. W A L L A A C H and H. FISHER, N a t u r w i s s e n n s c h a f t e n , 58: 220 (1971). 29. J. K U N A M O T O , J.K. R A I S O N and J.M. LYONS, J. Theor. Biol. 31: 47-51. 30. M. N E M A T - G O R G A N I and E. M E I S A M I , J. N e u r o c h e m . 32: 1 0 2 7 - 1 0 3 2 (1979). 31. T.A. B R A S I T U S and D. SCHATER, B i o c h e m i s t r y 19: 2 7 6 3 - 2 7 6 9 (1980) 32. R.N. M c E L H A N E Y , Curt. T o p i c s in M e m b r a n e and T r a n s p o r t . 17: 3 1 7 - 3 8 0 (1982). 33. C.D. STUBES, E s s a y s in B i o c h e m i s t r y 19: 1-39 (1983). 34. R.N.F. T H O R N E L E Y , R.R. EADY, and M.G. YATES, Biochim. Biophys. A c t a 403: 269-284. 35. J.C. G O M E Z - F E R N A N D E Z , F.M. GONI, D. BACH, C. R E S T A L L and D. CHAPMAN, FEBS Lett. 98: 224-228 (1979). 36. K. ANZAI, Y. K I R I N O and H. SHIMIZU, J. Biochem. 84: 8 1 5 - 8 2 1 (1978).
Pergamon Journals
C H R O N I C A D M I N I S T R A T I O N OF L I T H I U M M O D U L A T E S T R A N S P O R T BY C H A N G I N G T H E P R O P E R T I E S OF T H E MAL PLASMA MEMBRANE E.
Herrero,
C. G i m ~ n e z
and M.C.
TRYPTOPHAN SYNAPTOSO-
Arag6n
D e p a r t a m e n t o de B i o l o g i a M o l e c u l a r . C e n t r o de B i o l o g l a M o l e c u l a r . F a c u l t a d de C i e n c i a s . U n i v e r s i d a d A u t 6 n o m a de M a d r i d . 2 8 0 4 9 - M a d r i d . S p a i n . (Received in final form May 28, 1987) Summary The e f f e c t of c h r o n i c a d m i n i s t r a t i o n of l i t h i u m s a l t s on the s o d i u m d e p e n d e n t h i g h - a f f i n i t y s y s t e m for t r y p tophan uptake w a s e x a m i n e d in p l a s m a m e m b r a n e v e s i c l e s d e r i v e d f r o m rat b r a i n . Tryptophan transport was m e a s u r e d as a f u n c t i o n of the t e m p e r a t u r e , A r r h e n i u s p l o t s of the d a t a w e r e p r e p a r e d , a n d the a p p a r e n t e n e r g i e s of a c t i v a t i o n w e r e c o m p u t e d . B o t h p l o t s w e r e bi ~ p h a s i c , the t r a n s i t i o n t e m p e r a t u r e d e c r e a s i n g f r o m 2 3 ° C in c o n t r o l a n i m a l s to 18°C in t r e a t e d rats. The a v e r a g e a p p a r e n t e n e r g i e s of a c t i v a t i o n for the c a r r i e r a l s o c h a n g e - b o t h b e l o w a n d a b o v e the t r a n s i t i o n t e m p e r a t u r e - in t r e a t e d a n i m a l s w h e n c o m p a r e d to c o n t r o l s . O u r d a t a s u p p o r t the i d e a t h a t c h r o n i c a d m i n i s t r a t i o n of l i t h i u m i n d u c e s a m o r e f l u i d s t a t e of the s y n a p t o s o m a l p l a s m a m e m b r a n e t h a t c o u l d e x p l a i n m a n y of the e f f e c t s of Li + on m e m b r a n e - b o u n d p r o t e i n s . L i t h i u m is u s e d e x t e n s i v e l y in the t h e r a p y of m a n i c - d e p r e s s i v e p s y c h o s e s , b u t its m o d e of a c t i o n is s t i l l u n k n o w n . Although l i t h i u m i o n s h a v e b e e n r e p o r t e d to a c t in s e v e r a l d i f f e r e n t w a y s (1-4), m o s t of the h y p o t h e s e s c o n c e r n i n g the m e c h a n i s m of the t h e r a p e u t i c a c t i o n of l i t h i u m s a l t s are a s s o c i a t e d w i t h a l t e r a t i o n s in a m i n e n e u r o t r a n s m i s s i o n (5-12). Treatment with lithium has b e e n s h o w n to e n h a n c e some b e h a v i o u r a l and n e u r o p h y s i o l o g i c a l e f f e c t s of s e r o t o n i n (13-15 t h r o u g h m e c h a n i s m s as y e t not w e l l known. S e r o t o n i n b i o s y n t h e s i s in the b r a i n d e p e n d s on the i n t r a c e l l u l a r t r y p t o p h a n l e v e l s s i n c e t r y p t o p h a n h y d r o x y l a s e , the e n z y m i c s t e p l i m i t i n g the r a t e of s e r o t o n i n s y n t h e s i s , is not s a t u r a ted w i t h its s u b s t r a t e L - t r y p t o p h a n u n d e r p h y s i o l o g i c a l c o n d i t i o n s (16). As the c o n c e n t r a t i o n of t r y p t o p h a n in b r a i n c e l l s (17,18) is r e l a t i v e l y s m a l l w h e n c o m p a r e d w i t h t h a t of m o s t o t h e r a m i n o a c i d s , it h a s b e e n s u g g e s t e d t h a t its t r a n s p o r t a c r o s s the p l a s m a m e m b r a n e is r a t e l i m i t i n g for its i n t r a c e l l u l a r c o n c e n t r a t i o n (19,20). The o c c u r r e n c e of two of s u c h p r o c e s s e s of u p t a k e in b r a i n p r e p a r a t i o n s , w i t h h i g h a n d low a f f i n i t i e s for t r y p t o p h a n , h a s b e e n d e s c r i b e d ( 6 , 9 , 2 1 , 2 2 ) , and the a c c u m u l a t e d e v i d e n c e a b o u t the ion d e p e n d e n c e of t r a n s p o r t i n d i c a t e s t h a t the e l e c t r o c h e m i c a l p o t e n t i a l c r e a t e d by a Na + g r a d i e n t s e r v e s as a d i r e c t d r i v i n g f o r c e for the p r o c e s s (23-25). It has b e e n r e p o r t e d t h a t in a d u l t 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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Lithium and Synaptic Plasma Membrane
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rats the t r a n s p o r t of t r y p t o p h a n a c r o s s the neural m e m b r a n e s is d e p e n d e n t on the N a + - g r a d i e n t , and is s t i m u l a t e d by a m e m b r a n e pot e n t i a l (negative inside). We have p r e v i o u s l y d e m o n s t r a t e d that p l a s m a m e m b r a n e v e s i c l e s o b t a i n e d from l o n g - t e r m l i t h i u m - t r e a t e d rats are able to a c c u m u l a t e t r y p t o p h a n to a g r e a t e r e x t e n t than those from n o r m a l rats and m a i n t a i n a m o r e n e g a t i v e m e m b r a n e p o t e ~ tial than c o n t r o l s (22,26). In this r e p o r t we s h o w that t r e a t m e n t w i t h l i t h i u m p r o m o tes d i s c o n t i n u i t i e s in the A r r h e n i u s p l o t s of the m e m b r a n e - b o u n d t r a n s p o r t s y s t e m for t r y p t o p h a n and lowers the t r a n s i t i o n t e m p e r a ture of the system. These facts i n d i c a t e the e x i s t e n c e of p h a s e t r a n s i t i o n s and E a c h a n g e s and s u g g e s t that l i t h i u m t r e a t m e n t ind u c e s a m o r e fluid state of the n e u r o n a l m e m b r a n e . Materials
and M e t h o d s
M a t e r i a l s . L - ( G - 3 H ) T r y p t o p h a n (specific r a d i o a c t i v i t y 3.1 Ci/mmol) was o b t a i n e d from the R a d i o c h e m i c a l Center, A m e r s h a m , Bucks, U.K. F i c o l l was p r o v i d e d by P h a r m a c i a and w a s e x h a u s t i v e l y d i a l y z e d before use. All o t h e r r e a g e n t s used were of the h i g h e s t p u r i t y avai ! able. L i t h i u m a d m i n i s t r a t i o n and l i t h i u m analysis. L i t h i u m was a d m i n i s tered to 300-320 g m a l e W i s t a r rats in the d r i n k i n g w a t e r (I m g / m l LiCI). S e r u m l i t h i u m was m e a s u r e d by flame p h o t o m e t r y (Perkin-Elm e r Mod. 372) in c a r o t i d b l o o d o b t a i n e d at the time of killing. For b r a i n tissue, a l i q u o t s w e r e weighed, h o m o g e n i z e d in 3% p e r c h l o ric acid, and c e n t r i f u g e d ; the l i t h i u m c o n c e n t r a t i o n s w e r e d e t e r m i n e d in the acid extract. M e m b r a n e v e s i c l e s p r e p a r a t i o n . M e m b r a n e v e s i c l e s from a d u l t male W i s t a r rats (300-320 g) w e r e p r e p a r e d e s s e n t i a l l y as d e s c r i b e d p r e v i o u s l y (27). A f t e r o s m o t i c d i s r u p t i o n of s y n a p t o s o m e s , the susp e n s i o n was c e n t r i f u g e d at 27,000 g for 20 min, and the p e l l e t was r e s u s p e n d e d in 10 ml of a 284 m O S M medium, pH 7.4, w i t h the ionic c o m p o s i t i o n d e p e n d i n g on each p a r t i c u l a r e x p e r i m e n t . Finally, the s u s p e n s i o n was c e n t r i f u g e d at 27,000 g for 15 min and the p e l l e t was r e s u s p e n d e d in the former m e d i u m to a p r o t e i n c o n c e n t r a t i o n of 15-25 mg/ml. P o r t i o n s w e r e f r o z e n in liquid n i t r o g e n and stored at -70°C. A l i q u o t s w e r e q u i c k l y t h a w e d at 37°C. Under these conditions, m e m b r a n e v e s i c l e s w e r e f u n c t i o n a l for at least 1 month. T r a n s p o r t assays. P o r t i o n s (20 ~i) of the s u s p e n s i o n of m e m b r a n e v e s i c l e s d e r i v e d from rat b r a i n (about 0.2 mg of p r o t e i n ) , w e r e p r e l o a d e d , u n l e s s o t h e r w i s e stated, w i t h 120 m M KCI-22 m M p o t a s s i u m p h o s p h a t e buffer, pH 7.4 (KCI medium), and p r e i n c u b a t e d for 1 m i n at 25°C. U p t a k e was s t a r t e d by a d d i t i o n of 100 ul of a solution containing L-(G-3H)-tryptophan (5 ~M final c o n c e n t r a t i o n ) and the d e s i r e d ionic c o m p o s i t i o n , and was t e r m i n a t e d by d i l u t i n g w i t h 5 ml of i c e - c o l d 0.8 M NaCI, and i m m e d i a t e l y f i l t e r i n g t h r o u g h a m o i s t e n e d M i l l i p o r e filter RAWP 02500 (1.2 um pore size) a t t a c h e d to a v a c u u m a s s e m b l y . The filters w e r e r i n s e d twice w i t h the i c e - c o l d medium. Dilution, f i l t r a t i o n , and w a s h i n g w e r e per-
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f o r m e d w i t h i n 15 s. The f i l t e r s w e r e d r i e d at 60°C and p l a c e d in m i c r o v i a l s , and t h e i r r a d i o a c t i v i t y was m e a s u r e d in a l i q u i d - s c i n t i l l a t i o n c o u n t e r (Beckman LS-350). R e s u l t s w e r e c o r r e c t e d by m e a n s of a c o n t r o l o b t a i n e d by d i l u t i n g the m e m b r a n e s u s p e n s i o n b e f o r e a d d i n g the r a d i o a c t i v e s u b s t r a t e s o l u t i o n . All s o l u t i o n s u s e d in the p r e p a r a t i o n of the m e m b r a n e v e s i c l e s and in the u p t a k e e x p e r i m e n t s w e r e p r e p a r e d w i t h d i s t i l l e d d e i o n i z e d w a t e r and filt e r e d t h r o u g h M i l l i p o r e f i l t e r s (0.45 ~m) to a v o i d p o s s i b l e b a c t e rial c o n t a m i n a t i o n . The o s m o l a r i t y of all s o l u t i o n s was k e p t c o n s t a n t d u r i n g the u p t a k e e x p e r i m e n t s . The pH of the e x t e r n a l and i n t e r n a l m e d i u m was 7.4 t h r o u g h o u t the e x p e r i m e n t s . The ionic c o m p o s i t i o n of the v e s i c l e s , i n t e r n a l and e x t e r n a l m e d i u m was m o d i fled to i n v e s t i g a t e the e f f e c t s of d i f f e r e n t ions. All i n c u b a t i o n s w e r e c a r r i e d out in t r i p l i c a t e . E a c h exp e r i m e n t was r e p e a t e d at least three times w i t h d i f f e r e n t m e m b r a n e preparations. A g r e e m e n t a m o n g e x p e r i m e n t s was good; thus, t y p i c a l e x p e r i m e n t s are r e p o r t e d . Protein determination. m e t h o d of R e s c h et al.
M e m b r a n e p r o t e i n was d e t e r m i n e d by the (28).
A n a l y s i s of the data. C a l c u l a t i o n of a c t i v a t i o n e n e r g i e s (E a) was a c c o r d i n g to the A r r h e n i u s e q u a t i o n w h i c h m a y be w r i t t e n as log K =-
Ea 2.3RT
1 T
w h e r e K is the rate c o n s t a n t , R the gas c o n s t a n t , and T the absolute t e m p e r a t u r e . In p r a c t i c e the n u m e r i c a l v a l u e of E a is d e t e r m i n e d f r o m the slope of a p l o t of log K v e r s u s 1/T. S t a t i s t i c a l a n a l y s i s was p e r f o r m e d by the S t u d e n t ' s t - t e s t for n o n - p a i r e d s a m p l e s and a c o m p a r i s o n was m a d e w i t h the c o n t r o l group. P v a l u e s of 0.05 or less w e r e taken as s i g n i f i c a n t , and the r e s u l t s are e x p r e s s e d as the m e a n ± S.E.M. R e s u l t s and D i s c u s s i o n Rats g i v e n l i t h i u m c h l o r i d e ad l i b i t u m in t h e i r d r i n k i n g w a t e r for four w e e k s p r e s e n t e d p l a s m a l i t h i u m c o n c e n t r a t i o n s of 0 . 7 5 ± 0 . 2 0 m E q / l i t e r (mean±S.E.M. of ten a n i m a l s ) , e q u i v a l e n t to the t h e r a p e u t i c l e v e l s u s u a l l y found in humans. LiCl-treated a n i m a l s s h o w e d a light p o l y u r i a but did not d i f f e r s i g n i f i c a n t l y f r o m c o n t r o l s in w e i g h t g a i n and e x i b i t e d no signs of l i t h i u m intoxication. The l e v e l s of l i t h i u m in the b r a i n (1.05±0.17 m E q / g wet weight) w e r e a l w a y s h i g h e r than t h o s e in plasma. A l t h o u g h the m e c h a n i s m of a c t i o n of l i t h i u m is not w e l l known, it involves, at l e a s t in part, the a b i l i t y of Li + to m i m i c o t h e r b i o l o g i c a l l y i m p o r t a n t c a t i o n s such as sodium, m a g n e s i u m , or c a l c i u m ions. S i n c e these c a t i o n s are i n t i m a t e l y , r e l a t e d to m e m b r a n e f u n c t i o n s , it seems l i k e l y that l i t h i u m t h e r a p y i n v o l v e s membrane effects. However, the m o l e c u l a r d e t a i l s of the s t r u c t u ral a l t e r a t i o n s u n d e r l y i n g the e f f e c t s of l i t h i u m on b i o m e m b r a n e s
646
remain
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largely
Vol. 41, No. 5, 1987
uncharacterized.
F i g u r e 1 s h o w s the e f f e c t of t e m p e r a t u r e on the u p t a k e r a t e s of L - t r y p t o p h a n in p l a s m a m e m b r a n e v e s i c l e s o b t a i n e d f r o m c o n t r o l s (Fig. IA) a n d l i t h i u m - t r e a t e d r a t s (Fig. IB) . The m i n i m u m t e m p e r a t u r e at w h i c h a n y s u b s t r a t e a c c u m u l a t i o n c o u l d be d e t e c t e d w a s f o u n d to be a b o u t 5°C. As e x p e c t e d , the a c c u m u l a t i o n of t r i p t o p h a n i n c r e a s e s w i t h i n c r e a s i n g t e m p e r a t u r e in b o t h c o n t r o l and l i t h i u m - t r e a t e d rats, a n d the o v e r a l l s h a p e of the c u r v e s is f a i r l y s i m i l a r . H o w e v e r , w h e r e a s the u p t a k e r a t e s u n d e r the n o n - g r a d i e n t c o n d i t i o n s i n c r e a s e l i n e a r l y w i t h the t e m p e r a t u re a n d v e r y s i m i l a r l y in v e s i c l e s f r o m c o n t r o l a n d t r e a t e d rats,
un
•~
/
30
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2o
~
/
10
o
10
20
30
~,0
SO
10
Temperature (°C)
Figure
20
30
l,O
SO
Temperature (°C)
1
E f f e c t of t e m p e r a t u r e on the u p t a k e r a t e s of L - t r y p t ~ p h a n in p l a s m a m e m b r a n e v e s i c l e s f r o m rat b r a i n synaptosomes. The m e m b r a n e v e s i c l e s w e r e o b t a i n e d f r o m c o n t r o l r a t s (A) a n d f r o m l o n g - t e r m l i t h i u m - t r e a t e d r a t s (B) and w e r e p r e l o a d e d w i t h KCI m e d i u m and p r e i n c u b a t e d for 2 m i n at the i n d i c a t e d t e m p e r a t u r e s . A f t e r that, the v e s i c l e s w e r e i n c u b a t e d for 15 sec in a m e d i u m c o n t a i n i n g 120 m M NaCl, 2 2 m M p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.4 (NaCl m e d i u m ) (e), or KCl m e d i u m (o) in the p r e s e n c e of 5 u M - L ( G - 3 H ) t r y p t o p h a n at the indicated temperatures. The d i f f e r e n c e s b e t w e e n upt a k e in the N a C l m e d i u m a n d the KCl m e d i u m w a s t a k e n as the s p e c i f i c N a + - d e p e n d e n t u p t a k e (A). The compos i t i o n of the KCl m e d i u m is d e s c r i b e d in the M a t e r i a l s and M e t h o d s S e c t i o n .
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the u p t a k e r a t e s of t r y p t o p h a n t r a n s p o r t are a l w a y s h i g h e r in l i t h i u m - t r e a t e d r a t s t h a n in the c o n t r o l s in the p r e s e n c e of a Na + g r a d i e n t ( o u t s i d e > i n s i d e ) . T h i s f a c t is e v e n c l e a r e r w h e n the u p t a k e r a t e s in the p r e s e n c e of a Na + g r a d i e n t are c o r r e c t e d for the r a t e s u n d e r n o n - g r a d i e n t c o n d i t i o n s (i.e., KCI m e d i u m ) . At the h i g h e s t t e m p e r a t u r e t e s t e d (45°C), the a c c u m u l a t i o n r a t e of t r y p t o p h a n is m o r e t h a n 60% h i g h e r in p l a s m a m e m b r a n e v e s i c l e s f r o m l i t h i u m - t r e a t e d t h a n in t h o s e f r o m c o n t r o l rats. The A r r h e n i u s p l o t s for the t r a n s p o r t s y s t e m t r y p t o p h a n in b o t h s i t u a t i o n s are d e p i c t e d in Fig. 2, a n d the v a l u e s of t r a n s i t i o n t e m p e r a t u r e s and a c t i v a t i o n e n e r g i e s (E a) are s h o w n in Table I. A d i s c o n t i n u i t y in the A r r h e n i u s p l o t w a s o b s e r v e d in p l a s m a m e m b r a n e v e s i c l e s f r o m e i t h e r c o n t r o l s or l i t h i u m - t r e a t e d rats, and the b r e a k - p o i n t t e m p e r a t u r e s are l i s t e d in T a b l e i. The aver a g e a p p a r e n t a c t i v a t i o n e n e r g y for the t r y p t o p h a n t r a n s p o r t syst e m f r o m c o n t r o l r a t s w a s f o u n d to be 3.28 K c a l / m o l a b o v e and 7 . 1 0 K c a l / m o l b e l o w the t r a n s i t i o n t e m p e r a t u r e . The a p p a r e n t a c t i v a t i o n e n e r g y in the c a s e of l i t h i u m - t r e a t e d r a t s w a s f o u n d to be-4.31 K c a l / m o l a b o v e a n d 8.17 K c a l / m o l b e l o w the t r a n s i t i o n t e m p e r a t u r e , b o t h v a l u e s b e i n g s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e of c o n t r o l rats.
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, L 0,5 , , 3,5 3,6 3,1 3,2 I/temperature (K x 10-3)
Figure
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3.3
3.4
3.5
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A r r h e n i u s p l o t s of s p e c i f i c N a + - d e p e n d e n t u p t a k e of t r y p t o p h a n in s y n a p t o s o m a l p l a s m a m e m b r a n e v e s i c l e s t e s t e d in c o n t r o l s (A) and in l o n g - t e r m l i t h i u m t r e a t e d r a t s (B) . T h e e x p e r i m e n t a l c o n d i t i o n s are d e s c r i b e d in the l e g e n d to Fig. I.
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A b r u p t c h a n g e s in the slope of A r r h e n i u s p l o t s at a p a r t ~ c u l a r t e m p e r a t u r e have b e e n t a k e n to r e p r e s e n t a p h a s e t r a n s i t i o n in the lipid e n v i r o n m e n t of the m e m b r a n e - b o u n d e n z y m e s and t r a n s p o r t s y s t e m s (29-33). In the p r e s e n t i n v e s t i g a t i o n , the t r y p t o p h a n u p t a k e s y s t e m in s y n a p t o s o m a l p l a s m a m e m b r a n e v e s i c l e s f r o m c o n t r o l a n i m a l s s h o w e d a b r e a k p o i n t at 23°C, w h i c h s h i f t e d to 18°C a f t e r the l i t h i u m t r e a t m e n t , thus r e f l e c t i n g a p e r t u r b a t i o n of the m e m b r a n e l i p i d s in this s i t u a t i o n . The l o w e r i n g of the t r a n s i t i o n t e m p e r a t u r e and the c h a n g e s in the A r r h r e n i u s a c t i v a tion e n e r g y a b o u t the t r a n s i t i o n t e m p e r a t u r e p r o d u c e d by longt e r m l i t h i u m t r e a t m e n t , w o u l d be e x p e c t e d if is a s s u m e d that lit h i u m t r e a t m e n t i n d u c e s a m o r e fluid state of the s y n a p t o s o m a l plasma membrane. It is i n t e r e s t i n g to note that we did not found any a p p a rent e f f e c t of a l o n g - t e r m t r e a t m e n t w i t h l i t h i u m salts on the t r a n s p o r t of t y r o s i n e , a s t r u c t u r a l l y r e l a t e d a m i n o acid and p r e c u r s o r of c a t e c h o l a m i n e s (data not shown). A p o s s i b l e e x p l a n a t i o n for the d i f f e r e n t t h e r m a l b e h a v i o r of the t r y p t o p h a n t r a n s p o r t s y s t e m f o u n d in the l i t h i u m - t r e a t e d a n i m a l s m i g h t be due to d i f f e r e n c e s in the composition of the m e m b r a n e s i n d u c e d by l i t h i u m t r e a t m e n t (i.e. p h o s p h o l i p i d s , fatty acids, or c h o l e s t e r o l ) t h a t c o u l d i n f l u e n c e the l i p i d - p r o t e i n interactions. Such i n t e r a c t i o n c o u l d a l s o a f f e c t the c o n f o r m a tion or a g g r e g a t i o n state of the protein, and the r e s u l t i n g changes in p r o t e i n s t r u c t u r e w i t h t e m p e r a t u r e m i g h t a l s o d e t e r m i n e the b r e a k p o i n t of the A r r h e n i u s plot, as has b e e n e m p h a s i z e d by some a u t h o r s (34-36). A l t h o u g h l i t h i u m is w i d e l y u s e d for the t r e a t m e n t of mania, its e f f e c t s on the c e n t r a l n e r v o u s s y s t e m are p o o r l y u n d e r stood. It is w o r t h n o t i n g that m a n y of the l i t h i u m e f f e c t s des-
TABLE
1
V a l u e s of t r a n s i t i o n t e m p e r a t u r e s and a c t i v a t i o n e n e r g i e s (E a) for the t r y p t o p h a n t r a n s p o r t s y s t e m in p l a s m a m e m b r a n e v e s i c l e s f r o m rat b r a i n s y n a p t o s o m e s . Activation energy (Ea) (Kcal/mol)
Controls Li+-treated animals
Transition temperature ° C
below break ~oint
above break
23. i±i. 0
7. i0_+0.09
3.28+ - 0.05
18.2±i. i***
8.17±0.08"**
4.31±0.06"**
V a l u e s are the m e a n s ± SEM of at least ten i n d e p e n d e n t experiments. S i g n i f i c a n c e of d i f f e r e n c e b e t w e e n t r e a t e d and n o n - t r e a t e d animals: * * * p < 0 ° 0 0 1
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c r i b e d in the l i t e r a t u r e involve a great v a r i e t y of m e m b r a n e - b o u n d a c t i v i t i e s (i.e., c a r r i e r s and r e c e p t o r s ) . Thus it is t e m p t i n g to s p e c u l a t e that the t h e r a p e u t i c a c t i o n s of l i t h i u m take p l a c e t h r o u g h a l t e r a t i o n s in the c o m p o s i t i o n a n d / o r the d y n a m i c p r o p e r ties of the n e u r a l m e m b r a n e s . Acknowledgements This w o r k was m 6 n Areces.
supported
by a g r a n t
from the F u n d a c i 6 n
Ra-
References i. M.H. SHEARD, T r e n d s N e u r o s c i . 3: 85-86 (1980). 2. B.E. E H R L I C H and J.M. DIAMOND, J. M e m b r a n e Biol. 52: 187-200 (1980). 3. W.R. SHERMAN, L.Y. MUNSELL, G.G. B E B E R L Y and M.P. HONCHAR, J. N e u r o c h e m . 44: 798-807 (1985). 4. M. S C H O U and K. THOMSEN, in J o h n s o n F.N. (ed.) L i t h i u m R e s e a r c h and Therapy, p a g e s 63-64, A c a d e m i c Press, L o n d o n (1975). 5. J. P E R E Z - C R E U E T , P. T A G L I A M O N T E and G.L. GESSA, J. P h a r m a c o l . Exp. Ther. 178: 325-330 (1971). 6. A.J. M A N D E L L and S. KNAPP, Fed. Proc. Fed. Am. Soc. Exp. Biol. 36: 2 1 4 2 - 2 1 4 8 (1977). 7. I.J. WAJDA, M. B A N A Y - S C H W A R T Z , I. M A N I G A U L T and A. LAJTHA, Neurochem. Res. 6: 321:331 (1978). 8. A. PERT, J.E. R O S E N B L A T , C. SIVIT, C.B. P E R T and W.E. BUNNEY, Science. 201: 171-173 (1978). 9. M.L. L A A K S O and S.S. OJA, N e u r o c h e m . Res. 4 : 4 1 1 - 4 2 3 (1979). i0. S. T R E I S E R and K.J. KELLAR, Eur. J. P h a r m a c o l . 58: 85-86 (1980). ii. S. KNAPP, and A.J. MANDELL, S c i e n c e 180: 645-647 (1973). 12. A.J. M A N D E L L and S. KNAPP, in Almgrem, O., C a r l s s o n , A. and Engel, J. (eds.), C h e m i c a l tools in c a t e c h o l a m i n e r e s e a r c h II. Pages 9-16, N o r t h - H o l l a n d , A m s t e r d a m (1975). 13. C. SANGEE, and D.V. F R A N Z (1978) Commun. P s y c h o p h a r m a c o l . 2, 191-198. 14. E. F R I E D M A N , A. D A L L O B and G. LEVINE, Life Sci. 25, 1 2 6 3 - 1 2 6 6 (1979). 15. H.Y. METZER, in Salama, A.I. (ed.) P r e s y n a p t i c m o d u l a t i o n of p o s t s y n a p t i c r e c e p t o r s in m e n t a l diseases. Pages 115-137. Ann, N.Y. Acad. Sci. 430 (1984). 16. J.L. M E E K and S. L O F S T R A N D , Eur. J. P h a r m a c o l . 37: 377-380 (1986) 17. C.M. Mc KEAN, D.E. B O G G S and N.A. PETERSON, J. N e u r o c h e m . 15: 2 3 5 - 2 4 1 (1968). 18. L.A. T Y F I E L D and J. HOLTON, J. N e u r o c h e m . 26: 101-105 (1976). 19. D.G. G R A H A M E - S M I T H and A.G. PARFITT, J. N e u r o c h e m . 17: 13391353 (1970). 20. A. LAJTHA, in A r o m a t i c A m i n o A c i d s in the Brain. Pages 25-49. E l s e v i e r , A m s t e r d a m (1974). 21. E.R. KORPI, N e u r o c h e m . Res. 5 : 4 1 5 - 4 3 1 (1980). 22. E. HERRERO, M.C. ARAGON, C. G I M E N E Z and F. V A L D I V I E S O , J. Neurochem. 40: 332-337 (1983). 23. S.G. SHULTZ and P.F. CURRAN, Physiol. Rev. 50: 637-718.
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24. I.C. WEST, B i o c h i m . B i o p h y s . A c t a 604: 91-126 (1980). 25. B.I. KANNER, Biochim. Biophys. A c t a 726: 239-316 (1983). 26. M.C. A R A G O N , E. HERRERO, C. GIMENEZ, and F. V A L D I V I E S O . B i o c h e m Soc. Trans. ii: 6 9 6 - 6 9 7 (1983). 27. B.I. KANNER, B i o c h e m i s t r y 17: 1 2 0 7 - 1 2 1 1 (1978). 28. K. RESCH, I. IMM, E. FERBER, D.F.H. W A L L A A C H and H. FISHER, N a t u r w i s s e n n s c h a f t e n , 58: 220 (1971). 29. J. K U N A M O T O , J.K. R A I S O N and J.M. LYONS, J. Theor. Biol. 31: 47-51. 30. M. N E M A T - G O R G A N I and E. M E I S A M I , J. N e u r o c h e m . 32: 1 0 2 7 - 1 0 3 2 (1979). 31. T.A. B R A S I T U S and D. SCHATER, B i o c h e m i s t r y 19: 2 7 6 3 - 2 7 6 9 (1980) 32. R.N. M c E L H A N E Y , Curt. T o p i c s in M e m b r a n e and T r a n s p o r t . 17: 3 1 7 - 3 8 0 (1982). 33. C.D. STUBES, E s s a y s in B i o c h e m i s t r y 19: 1-39 (1983). 34. R.N.F. T H O R N E L E Y , R.R. EADY, and M.G. YATES, Biochim. Biophys. A c t a 403: 269-284. 35. J.C. G O M E Z - F E R N A N D E Z , F.M. GONI, D. BACH, C. R E S T A L L and D. CHAPMAN, FEBS Lett. 98: 224-228 (1979). 36. K. ANZAI, Y. K I R I N O and H. SHIMIZU, J. Biochem. 84: 8 1 5 - 8 2 1 (1978).