- Email: [email protected]
Stimulation of dopamine synthesis and release by morphine and D-Ala2-D-Leu5-enkephalin in the mouse striatum in vivo
Life Sclences, Vol. 28, pp. P r l n t e d in the U . S . A
2277-2286
Pergamon
Press
S T I M U L A T I O N OF D O P A M I N E SYNTHESIS AND RELEASE BY M O R P H I N E AND D - A L A 2 - D - L E U 5 - E N K E P H A L I N IN THE M O U S E S T R ~ A T U M IN V I V O Stephan
Urwyler
and Boris
TaDakoff
V.A. W e s t s l d e M e d i c a l C e n t e r and Dept. of P h y s l o l o g y and Biophyslcs, U n l v e r s l t y of Illlnols at the M e d l c a l Center, Chlcago, IL 60680 (Received
in flnal
form F e b r u a r y
27, 1981)
Summar~ The e f f e c t s of o p i a t e s on d o p a m l n e (DA) r e l e a s e and s y n t n e s l s w e r e a s s e s s e d in the mouse strmatum in v l v o by s l m u l t a n e o u s l y measuring 3,4-dihydroxyphenylalanlne (DOPA) and 3 , 4 - d i h y d r o x y p h e n y l a c e t l c acid (DOPAC) levels after i n h i b i t i o n of 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 Thls m e t h o d was d e v e l o p e d to a s s e s s s t l m u l u s - c o u p l e d changes in DA s y n t h e s l s and release. P e r i p h e r a l i n 3 e c t l o ~ s of m ~ r p n l n e and i n t r a v e n t r l c u l a r i n 3 e c t l o n s of D-AIa - D - L e u - - e n k e p h a l l n elevated DOPAC levels, i n d i c a t i n g that "opiates" s t i m u l a t e d DA release. Conc o m l t a n t l y , the rate of DA s y n t h e s i s was increased. The e f f e c t s were d o s e - d e p e n d e n t , s a t u r a b l e and a n t a g o n l z e d by naloxone. W h e n m o r p h i n e and the e n k e p h a l l n a n a l o g were given t o g e t h e r in s a t u r a t i n g doses, the effects of the two a g e n t s w e r e not addmtlve. Thus, the i n v o l v e m e n t of d l f f e r e n t r e c e p t o r s in the m e d i a t i o n of the e f f e c t s of m o r p h i n e and e n k e p h a l l n s could not be d e m o n s t r a t e d .
The p r e s e n c e of o p l a t e b l n d l n g sltes in the strlatum, one thlrd of w h l c h a p p e a r to be l o c a l l z e d on the t e r m i n a l s of d o p a m l n e r g l c n e u r o n s of the n l g r o s t r l a t a l pathway, has been p r e v i o u s l y d e m o n s t r a t e d (i), and a c t i v a t i o n of o p i a t e r e c e p t o r s results in an increase in strlatal DA s y n t h e s i s and t u r n o v e r (2-6). However, the p r e v l o u s l y r e p o r t e d changes in DA m e t a b o l i s m have been p o s t u l a t e d by some a u t h o r s (7) to r e f l e c t m n t r a n e u r o n a l events not a s s o c i a t e d with DA r e l e a s e from neurons, and the a n s w e r to the q u e s t i o n of w h e t h e r the s t i m u l a t i o n of opiate r e c e p t o r s m n n m b l t s or f a c 1 1 1 t a t e s the release of DA r e m a i n s c l o u d e d in controversy. S p e c l e s d e p e n d e n t d i f f e r e n c e s in opiate e f f e c t s may a c c o u n t for some of the d l s p a r a t e flndlngs. For instance, the effect of m o r p h i n e on l o c o m o t o r a c t l v l t y in mlce and rats indicates s l g n l f l c a n t d i f f e r e n c e s in n e u r o p h a r m a c o l o g i c a s p e c t s of opmate a c t i o n s in these two specles (8, 9). However, the c o n f l l c t l n g r e s u l t s in p r e v i o u s studies can not be e x p l a l n e d solely on the basis of specles or g e n e t l c differences. In fact, no effect (10-13), I n c r e a s e s (i4-16) and d e c r e a s e s (17-19) in the r e l e a s e of DA in the s t r l a t u m of the rat In response to the a d m i n i s t r a t i o n of m o r p h l n e or the e n k e p h a l i n s have been reported. C o n f l i c t i n g r e p o r t s also exist wlth r e g a r d to the e f f e c t s of m o r p h l n e on strlatal DA r e l e a s e in the mouse (20-22) but the e f f e c t s of e n k e p n a l i n s or tnelr a n a l o g u e s on the n e u r o n a l r e l e a s e of DA in moase s t r m a t u m nave not been r e p o r t e d 0024-3205/81/202277-I0$02.00/0 C o p y r l g h t (c) 1981 P e r g a m o n Press Ltd.
1278
O p l a t e E f f e c t s on DA N e u r o n s
Vol.
28, No.
20,
1981
S ! n c e two t y p e s of "opiate" r e c e p t o r s have been ShOWn to be p r e s e n t in r o d e n t b r a i n (23) one m a y a l s o ~ o n d e r w h e t h e r the e f f e c t s of m o r p h l n e a n d the e n k e p h a l l n s on s t r l a t a l D A s y n t h e s l s and r e l e a s e are m e d l a t e d by I d e n t l c a l r e c e p t o r systems. In o r d e r to f u r t h e r e l u c i d a t e the a c t i o n s of "oplates" on D A r e l e a s e and m e t a b o l i s m in tne mouse, we have i n v e s t i g a t e d the e f f e c t s of m o r p h l n e a n d D - A l a 2 - D - L e u b - e n k e p h a l l n , a p o t e n t and p e p t l d a s e - r e s l s t a n t e n k e p n a l ~ n a n a l o g (24), on the levels of DA, D O P A and D O P A C an the m o u s e s t r l a t u m ~n v l v o a f t e r i n h i b i t i o n of a r o m a t i c a m i n o acld decarboxylase. The r e p o r t e d m e t h o d s for m o n l t o r l n g DA m e t a b o l i s m w e r e d e v e l o p e d to m o r e c l e a r l y r e l a t e c h a n g e s in D A p r e c u r s o r and m e t a b o l l t e l e v e l s to s t i m u l u s c o u p l e d r e l e a s e of DA from the DA n e u r o n t e r m i n a l s in the strlatum.
M a t e r i a l s and M e t h o d s Animals. M a l e C 5 7 B 1 / 6 m l c e (23-25g) w e r e p u r c h a s e d from ARS/Sprag~leD a w l e y ( ~ d l s o n , WI) a n d w e r e h o u s e d flve per cage an our l a c o r a t o r i e s (22°C, 12hr l i g h t cycle) for at least 7 d a y s w l t h ad l l b l t u m a c c e s s to P u r i n a l a b o r a t o r y c h o w and w a t e r b e f o r e t h e y ~ere u s e d for the e x p e r l ments. M e a s u r e m e n t s of t ~ r o s l n e h y d r o x [ l a s e a c t l v ! t y and r e l e a s e of DA: T y r o s l n e h y d r o x y l a s e a c t i v i t y was m e a s u r e d an v l v o by m o n l t o r l n g the a c c u m u l a t i o n of D O P A a f t e r I n h l b l t l o n of b r a i n a r o m a t i c a m i n o acld d e c a r b o x y l a s e w i t h an l.p. I n ] e c t l o n of N S D - 1 0 2 4 (Sandev Ltd , Harlow, Essex, G.B.). N S D - i 0 2 4 was u s e d ~n our s t u d i e s because, at the d o s e s a d m i n i s t e r e d , at p r o d u c e d a p r o f o u n d i n n l b l t l o n of a r o m a t l c a m i n o a c i d d e c a r b o x y l a s e t25) but had no s i g n i f i c a n t e f f e c t on M A O a c t l v l t i an m o u s e b r a i n (B. T a b a k o f f , u n p u b l i s h e d o b s e r v a t l o n a n d d a t a p r o v i d e d by Sandev, Ltd.). The b l o c k a d e of DA synthesis, w l t h o u t a s l g n ~ f l c a n t e f f e c t on D A c a t a b o l i s m , a l l o w e d for an e s t i m a t i o n of D A r e l e a s e and s u b s e q u e n t r e u p t a k e a n d c a t a b o l i s m by a s s a y of the levels of DA a n d its m e t a b o l l t e , DOPAC. The a n i m a l s ~ere k l l i e d by d e c a p l t a t l o n and t h e ± r b r a i n s w e r e r a p i d l y r e m o v e d ~nd d i s s e c t e d on a c h i l l e d g l a s s plate. All f u r t h e r p r o c e d u r e s w e r e c a r r i e d out on ace or an 4°C. The heads of the c a u d a t e of b o t h h e m i s p h e r e s w e r e p o o l e d and h o m o g e n i z e d !n 5 ml of 0 4 : 4 p e r c n l o r l c a c i d c o n t a i n i n g 0.05% s o d i u m m e t a D l s u l f l t e and 0.2% E D T A u s i n g a Tekmar Tlssuemlzer.
The h o m o g e n a t e
~as c e n t r l f u g e d for 20 man in a
Sorval c e n t r l f e g e at 3 0 , 0 0 0 g and 2.4 ml of 2 M Trls and 50 mg of a l u m l n a w e r e a d d e d to the r e c o v e r e d s u p e r n a t a n t fluld. A f t e r t h o r o u g h mlxlng, the a l u m i n a was w a s h e d twice w i t h d l s t ~ i l e d w a t e r and the b o u n d c a t e c h o l s ~ere e l u t e d f r o m the a l u m i n a w l t h a 2 M a c e t i c a c l d s o l u t i o n w h i c h c o n t a l n e d 0.2% s o d l u m m e t a b l s u l f l t e . The s a m p l e s w e r e then f r o z e n unt~l DA and Its m e t a b o l l t e s w e r e s e p a r a t e d by r e v e r s e p h a s e h l g h p e r f o r m a n c e l l q u l d c h r o m a t o g r a p h y on a C l 8 - ~ B o n d a p a k C o l u m n ~Waters ~ s s o c l a t e s , M i l f o r d , MA) (26) a n d q u a n t l t a t e d by tDe use of an e l e c t r o c h e m l c a l d e t e c t o r ( B l o a n a l y t l c a l Systems, W L a f a y e t t e , IN). A u t n e n t l c s t a n d a r d s w e r e u s e d for e s t a o l l s h l n g c a l l b r a t l o n c u r v e s a n d d ~ h y d r o x y b e n z y l a m l n e was c a r r l e d t h r o u g h the e x t r a c t l o n a n d q u a n t l t a t l o n p r o c e d u r e as an i n t e r n a l standard. T r e a t m e n t of a n l m a ! s w l t h dru~s. NSD-1024 [3-nydroxybenzyloxamlne d ! h y d r o g e n p h o s p n a t e ] was d i s s o l v e d ~n 0.9% NaCI a n d i n 3 e c t e d ±.p. (5C0 mg/kg) 30 m a n b e f o r e the a n i m a l s w e r e kllled, since our p r i o r e x p e r l m e n t s had s h o w n that D © P A a c c u m u ! a t l o n was l i n e a r o v e r that t~me p e r l o d (25) H a l o p e r l d o l was u s e d ±n the form of H a l d o l A i n 3 e c t a b l e s o l u n - o n rMcNe±l L a b o r a t o r i e s , Ft. W a s n l n g t o n , PA) and was d i l u t e d w i t h 0 9% NaC! b e f o r e l,p. i n ] e c ~ i o n (i mg/kg, 9Q mln. b e f o r e N S D - 1 0 2 4 ) . N a l o x o n e (Endo
Vol
28, No.
20,
1981
OPiate
Effects
on DA Neurons
L a o o r a t o r l e s , G a r d e n Cltl, N%) (3 mg/kg) and m o r p h m n e sulfate (Mallanckrodt, St Louis, MC) were also d l s s o l v e d in 0 9% NaCI and ~ n 3 e c t e d at the stated doses (l p.) 20 and 15 m a n u t e s before NSD-1024, respectlvely. C e r t a i n of the sallne (control) and m o r p h l n e in3ected a n l m a l s also were a~3ected (1.9.) w l t n b e n z t r o p l n e m e s y i a t e (Arapahoe, Newport, TN), 25 mg/kg, 20 m i n u t e s before NSD-1024 D - A I a 2 - D - L e u ~e n k e p h a l a n (Bachem, Torrance, CA) was glven i n t r a v e n t r l c u l a r l y (loC v~l under llg>t ether a n e s t h e s i a (27) in 4 ~i of Meri!s' solutaon, i0 mln p r a o r to N S D - 1 0 2 4 A n l m a l s r e c e l v a n g ! p. i n 3 e c t ! o n s of sallne (instead of m o r p h l n e , n a l o p e r l d o l or~. benzt~oplne) or [.c %- in3ectaons of Merlls' s o l u t l o n (instead of D - A l a ~ - D - L e u - - e n k e p n a l a n ) at the a p p r o p r i a t e tlmes p r a o r to N S D - 1 0 2 4 served as controls. In order to obtaln basal levels of DA and DOPAC, o t h e r control m2ce r e c e l v e d only vehlcle, but no N S D - 1 0 2 4 in3ectlons. Results are e x p r e s s e d as means ~ S.EoM. The data were a n a l y z e d by the use of a n a l y s l s of v a r i a n c e and tne Snudent ~-test P values < 0.05 were taken as an indicatlon of s t a t a s t l c a l s ! g n l f l c a n c e of d l ~ f e r e n c e s between mean values
Results M o r p h a n e an doses r a n g l n g from 10-40 mg/kg was found to elevate D O P A and D O P A C levels an the c a u d a t e (Fig. !). DOPA was not d e t e c t a b l e In a n a m a l s r e c e l v l n g no NSD-1024, but the level of D O P A C was s a g n a f l c a n t l y nagher than in a n a m a l s r e c e l v a n g NSD-1024 but no "oplate". The e f f e c t s of m o r p h a n e were d o s e - d e p e n d e n t , saturable and could be b l o c k e d by p r e t r e a t m e n t of the a n a m a l s w a t h n a l o x o n e (Fag. i). Results o b t a a n e d an the p r e s e n c e of b e n z t r o p a n e a n d l c a t e d that the D O P A C levels In the s t r a a t u m of the N S D - 1 0 2 4 t r e a t e d anmmals r e f l e c t e d the stamulus c o u p l e d e x t r a n e u r o n a l r e l e a s e of DA tsee dascusslon) Table I shows that pret r e a t m e n t of the a n l m a l s wlth b e n z t r o p a n e lowered D O P A C levels to a p p r o x i m a t e l y the same e x t e n t an control and m o r p h l n e t r e a t e d anamals, and that D O P A a c c u m u l a t a o n was also l o w e r e d by benztropane. D - A i a 2 - D - L e u 5 - e n k e p h a l a n was d e m o n s t r a t e d to a n c r e a s e DA s y n t h e s a s and relase an the s t r a a ~ u m as i n d l c a t e d by I n c r e a s e s in D O P A and DOPAC l e v e l s over levels of D O P A and D O P A C found in Merlas' solutlon a n 3 e c t e d m l c e (Fag. 2). However, the D O P A levels !n a n l m a l s r e c e ~ v l n g 3ust N S D - 1 0 2 4 and an l.c.v, a n 3 e c t a o n of the Merlas' solutlon were hlgher than the D O P A levels an the a n l m a l s a n 3 e c t e d l.p. wlth sallne. Thas i n d a c a t e d that the l.c.v, a n 3 e c t ~ o n by itself could s t a m u l a t e DA synthesis. After l n n l b l t l o ~ of DA synthesas, an ancrease an DA r e l e a s e w o u l d be e x p e c t e d to be r e f l e c t e d an an a c c e l e r a t e d declane an DA levels. However, d u r l n g the short p e r l o d of s y n t n e s l s a n n l b l t l o n used an the p r e s e n t e x p e r l ments, the r e l e a s e d DA a c c o u n t e d for only 11% of the total DA pool [DA levels an all c o n t r o l mlce r e c e l v ~ n g no NSD-1024 and those r e c e a v ! n g N S D - i C 2 4 were, r e s p e c t l v e l y 100.7+--2.9 (n=22) vs. 8 9 . 7 + 2 . 0 (n=31) nmoles/g] and due to the usual d l f f e r e n c e s an anatlal levels of D A in i n d a v a d u a l m a c e It was an m o s t e x p e r l m e n t s not p o s s l b l e to o b s e r v e s l g n ~ f ~ c a n t d a f f e r ences an D A levels an the p r e s e n c e of "opaates" (Table II).
2279
2280
OplaCe
Effects
on
DA Neurons
Vol.
28,
No.
20,
T 40
w O9
30
,,~
20
o
z
E 4
IO 0
0 d~
i
/Y
o
E c
~-
.r v
4O W OO
30 < UJ n"
20
~ Z
4
i
O_ 0 n
0
[ i
3
i
-b I
I
O
IO
--
i
20
MORPHINE
50
40
SUL,-"ATE(mg/kg ~ p) FIG.
i
E f f e c t of m o r p n l n e s u l f a t e o n D O P A C (upper p a n e l ) a n d D O P A (lower p a n e l ) l e v e l s zn t h e m o u s e n u c l e u s c a u d a t u s . C 5 7 m l c e r e c e z v e d l.p. z n 3 e c t l o n s of s a l l n e or z n c r e a s l n g d o s e s of m o r p h l n e s u l f a t e (-o-) 13 m l n b e f o r e N S D - I 0 2 4 a n d 45 m l n b e f o r e s a c r z f l c e . Certazn anzmals (-o-) recelved 3 m g / k g o f n a l o x o n e l.p. 5 m z n b e f o r e t h e y w e r e z n 3 e c t e d w l t ~ m o r p h l n e or s a l l n e . DOPAC levels were a l s o m e a s u r e d zn c o n t r o l a n l m a l s r e c e z v l n g n o N S D - 1 9 2 4 (~). ~ a ! u ~ s f r c m b e t w e e n 4 a n d 13 a D z m a l s w e r e u s e d to o b t a ± n t h e m e a n + S.E.~4 ~sterlsks indlcate DOPA and DOPAC levels whmcn were slgnzflcantly dzfferent from the respective l e v e l s zn c o n t r o l a n z m a l s r e c e ~ v z n g NSD-1024
but
no m c r p n l n e
sdlfate
(p
. 0.05)
1981
%ol.
28, No.
20,
1981
Opiate
Effects
TABLE
on DA Neurons
228]
I
E f f e c t of P r e t r e a t m e n t wath B e n z t r o p l n e on DOPA and DOPAC Levels an the Strlatum of Control and Morphlne Treated Mace Group
DOPA
% Chan~e
Control (Salane)
3.70+0
Benztropane
3.08~0.24
Morphlne (15 mg/kg)
5.88+0.10
Morphane (15 mg/kg) & Benztropane
3 53+0.38*
Morpnane (30 mg/kg)
6 07+0.68
Morpnlne (30 mg/kg) & Benztropane
3.11+0.18"
39
% Chan~e
DOPAC
3.79+9.29 -17
2.42~0.23"
-36
5.44+0.11
-42
3.29+0.23*
5.70+0
-48
-39
64
2.69+0.15"
-52
A n a m a ! s were an]ected wlth NSD-1024 (500 mg/kg, a.p.) 30 m l n u t e s p r l o r to sacraface. Morphlne sulfate (15 or 30 mg/kg) and b e n z t r o p l n e m e s y l a t e (25 mg/kg, a.p.) were gaven 15 and 20 mlnutes before NSD, respectavely. Control anamals r e c e i v e d salane an3ectaons instead of morphlne and benztropane. Results are glven in nmoles per g of t~ssue. Fave to sax anamals c o m p r l s e d each experam e n t a l group. Per cent change indlcates the extent of d ~ f f e r e n c e s between anamals r e c e a v l n g no b e n z t r o p l n e and those r e c e l v a n g benztropzne. A s t e r a s k s andlcate slgnafacant d a f f e r e n c e s between tnese groups (p < 0.05).
In order to a n v e s t l g a t e w h e t h e r the effects of m o r p h a n e and the e n k p e h a l l n a n a l o g at m a x l m a l doses were addltlve, e x p e r a m e n t s were p e r f o r m ed in w h a c h the two "opaates" were gaven together (Table II). The control (a.p. salane-an3ected) m a c e and anlmals in3ected a.p. wlth m o r p h l n e rec e l v e d an a d d l t a o n a l a.c.v, a n 3 e c t l o n of Merlls' solutlon in these experiments, to control for the effects of l.c.v, zn3ectlons on DA synthesas. Table II shows that the effects of m a x l m a l doses of m o r p h l n e and D - A l a 2 - D - L e u 5 - e n k e p h a l a n were not addltave: no sagnflcant d a f f e r e n c e s were found b e t w e e n m e a n D O P A and D O P A C values an anamals r e c e l v a n g both "oplates" t o g e t h e r and the two groups whlch recelved one of the two groups alone. H a l o p e r l d o l s t l m u l a t e d the synthesls and release of DA to a m u c h nlgher e x t e n t than. dld the m a x l m a l doses of the two "oplates": D O P A and DOPAC levels ~n the caudate of anlmals treated wlth haloperldol and N S D - 1 0 2 4 were 1 2 . 6 8 + 0 . 5 6 n m o l e s / g and 10.84+0.36_ n m o l e s / g (n=6), respectively, c o r r e s p o n d z n g to a 160% and 154% ~ncrease over v a l u e s found in anamals w h a c h r e c e l v e d salzne rather than the haloperadoi. DA levels an tne caudate of a n i m a l s t r e a t e d wlth h a l o p e r l d o l and NSD-1024 were
2282
Oplate
Effects
on D A N e u r o n s
Vol.
28, No.
7 I00
T
75
UJ UD UJ
50
7
~.)
25
r-f<-
o
C3
31: 1
0
i
1
I
~
II
I00
I0 [ i
75
.,t
~
9
E =
8
n 0
~
50
/
7
I
UJ oO <~ td <9 Z
25
2
0 5
L
I
J
J
~
t
1
0
2
4
6
8
I0
D-AI_AZ-D -t_EU~-ENKE~HAI_IN(Ng Icy) FIG.
2
2 5 E f f e c t o f D - A l a - D - L e u - e n k e p h a l l n on D O P A C (upper p a n e l ) a n d D O P A (lower p a n e l ) l e v e l s in t h e m o u s e n u c l e u s caudatus. Anlmals were inDected wlth Merlls' solutlon (ve~icle) o r i n c r e a s i n g a m o u n t s o f t h e e n k e p h a l l n a n a l o g (-e-) I n t r a v e n t r l c u l a r l y under light ether ~nesthesla I 0 m l n b e f o r e t h e y w e r e g i v e n N S D - 1 0 2 4 (500 m g / k g , i.p ) a n d 40 m i n b e f o r e s a c r z f l c e . O t h e r a n i m a l s (-o-) r e c e ! v e d 3 m g / k g o f n a l o x o n e l.p. 5 m ! n p r i o r to the znDectlon of the enkepnalmn analog. DOPAC levels were a l s o m e a s u r e d in c o n t r o l a m ~ m a l s r e c e l v l n g n o N S D - 1 0 2 4 ( r) T h e r e s u l t s s n o w n are the m e a n s ~ S . E . M . of v a l u e s d e r l v e d f r o m b e t w e e n 4 to 18 a n i m a l s . Asterlsks lndlcate DOPA and DOPAC levels whzch were slgnlflcantly d i f f e r e n t f r o m the l e v e l s in c o n t r o l a ~ i m a l s r e c e s v l n g N S D - 1 0 2 4 O u t n o e n k e p h a l l n (p 0 05)
20,
1981
Vol.
28~ No.
20,
1981
Oplate
TABLE
Effects
on D~ Neurons
II
5 Effects of Morphane and D - A I a ' - D - L e u -Enkephalln and the Two "Opiates" in C o m b a n a t z o n on DOPA, DOPAC and DA Levels an the Mouse Strlatum In Vavo Grou~
Control (no NSD)
DOPA DORAC DA (nmoles per g of tissue) not detectable
5.22+0.40* (4)
93.8+1.8 (~)
Control (+ NSD)
5.62+0.33 (i~)
3.50+0.10 (17)
86.6+1.8 (53)
Morphlne
8.61~0.33 *
5.19+0.17"
87.7+1.6
(14)
(l~)
Enkephalan (~ NSD)
8.86+0.56* (I~)
4.92+0.18" (I~)
83.8+1~9 (54)
Morphine + Enkephalln (+ NSD)
9.45+0.54* (I~)
5.41+0.17" (i~)
86.3+1.8 (54)
(+ NSD)
(54)
Anamals were In3ected wath NSD-1024 (500 mg/kg, i.p.) 30 m a n u t e s prlor to sacrlface. D-Ala2-D-Leu5-enkephalln (5 ~g i.c.v.) or Merlas' solutaon (l.c.v.) were admlnlstered i0 mln and morphane sulfate (30 mg/kg, l.p.) or salane (a.p.) were a d m l n a s t e r e d 15 man. prlor to NSD-1024. Anamals an the m o r p h i n e group r e c e a v e d an i.c.v. a n 3 e c t a o n of Merlas' soiutlon and e n k e p h a l ~ n treated anamals r e c e l v e d sallne i.p. Control anlmals recelved both v e h i c l e in3ectlons. DOPA, DOPAC, and DA levels are e x p r e s s e d as means + S.E.M. with the number of animals an parentheses. A s ~ e r l s k s indicate slgnlflcant differences between the respective groups and the control (+ NSD) group (p < 0.01)~
59.4+1.71_ nmoles per g tissue, Indlcatlng that a more p r o f o u n d stimulus for release of DA w o u l d be r e f l e c t e d an s l g n a f l c a n t l y lowered DA levels under our c o n d l t a o n s of inhlblted DA synthesis.
Discussaon The m e a s u r e m e n t of D O P A accu~Lulatlon and DOPAC formation was used an the p r e s e n t study to indicate the synthesis and release of DA and to I n v e s t & g a t e the effects of "oplates" on the n l g r o s t r l a t a l DA pathway. A short d ~ r a t l o n of DA synthesls i n n l b l t l o n was used in our studies such that f e e d b a c k control of tyrosane hydroxylase by DA would not be s e v e r e l y compromlsed. T y r o s l n e nydroxylase as the rate lamltlng enzyme an the b l o s y n t h e s a s of DA and ats p r o d u c t (DOPA) as r a p i d l y d e c a r b o x y l a t e d to DA. Endogenous D O P A levels are e x t r e m e l y low and d a f f a c u l t to detect. However, if aromatac amino acld d e c a r b o x y l a s e Is anhabated, the a c c u m u l a t i o n of D O P A over tame can be used as an andlcator of the rate of DA syntehsas (28). DOPAC has been shown to be formed
2283
2284
O p i a t e E f f e c t s on DA N e u r o n s
Vol.
2~, Ne.
20,
p r e s y n a p t l c a l l y e i t h e r f r o m DA w h i c h spAl!s over from s t o r a g e vesicles or from LA w n l c h has b e e n r e l e a s e d into the s y n a p t A c c l e f t and then r e c a p t u r e d Into the p r e s y n a p t l c n e r v e t e r m i n a l (29, 30) In our study, the D O P A C l e v e l s in the c a u d a t e of c o n t r o l a n i m a l s r e c e i v i n g N S D - i C 2 4 were s l g n l f l c a n t l y lower than in c o n t r o l a n i m a l s r e c e i v i n g no d r u g at all. The r e d u c t i o n of D O P A C l e v e l s by a d m l n l s t r a t l o n of N S D - 1 0 2 4 ~ o u l d r e p r e s e n t the e l a m l n a t l o n of s p l l l o v e r f r o m v e s i c l e s due to e x c e s s s y n t n e s l s s i n c e s y n t h e s l s of DA is inhlblted. Thus, the r e m a i n i n g D O P A C l e v e l s p r e s u m a b l y r e f l e c t m a i n l y the d e g r a d a t i o n of D A s u b s e q u e n t to its i m p u l s e - c o u p l e d r e l e a s e into and u p t a k e from the s y n a p t l c cleft. Prot r e a t m e n t of the a n i m a l s w i t h b e n z t r o p l n e , a d r u g w h l c h i n h i b i t s the r e u p t a k e of D A into the p r e s y n a p t l c n e r v e t e r m i n a l (31), a n d / o r t q r o u g h its a n t l - m u s c a r l n l c p r o p e r t i e s , r e d u c e s the f a r i n g rate of n l g r o s t r l a t a l DA n e u r o n s (32, 33) r e s u l t e d in a d e c r e a s e d f o r m a t i o n of D O P A C {Taole i) Thls o b s e r v a t i o n f u r t h e r c o n f i r m s t h a t u n d e r our e x p e r i m e n t a l c o n d i t i o n s c h a n g e s in D O P A C l e v e l s are a g o o d i n d i c a t o r of the e f f e c t s of d r u g s on s t i m u l u s c o u p l e d D A r e l e a s e f r o m the neuron. The l o w e r e d D O P A a c c u m u l a tion in a n i m a l s p r e t r e a t e d w l t h b e n z t r o p l n e c o u l d be a r e s u l t of u p t a k e i n h i b i t i o n w h i c h w o u l d p r o d u c e e l e v a t e d c o n c e n t r a t i o n s of DA in the s y n a p t l c cleft. S u c h e l e v a t e d DA l e v e l s w o u l d i n t e r a c t w l t h 5A r e c e p t o r s c o n t r o l i n g DA s ? n t ~ e s l s (34, 35) Cn the o t ~ e r hand, the l o w e r e d D C P A a c c u m u ! a z ~ o n c o u l d be d u e no nne r e d u c e d firing rate of the LA n e d r c n s p r o d u c e d my a c t i o n s of D e n z t r o p l n e on ~ h o l ~ n e r g ~ c r e c e p t o r s r e s i d i n g on n e u r o n s Df the s t r l a t o - n ~ g r a l f e e d b a c m loop. [orL%ine ant D - h ! a - - u - _ e u ~ - e n k e p n a l i n moth s t i m u l a t e d ~A s / n t ~ e z l s as ~ell as r e l e a s e T h e s e e f f e c t s were m e d i a t e d t n r o u { ~ t-e a c t _ o n of the "o~Aates" on o p i a t e r e c e p t o r s , s~nce the a c t i o n s of m o r ~ q A n e and D - ~ l a - - O - L e u S - e n k e p n a ! A n w e r e a n t a g o n i z e d my n a l o x o n e It aas seen s u g g e s t e d (7, 17) that m o r p h l n e mlgzt i n c r e a s e the l e v e l s of t2e d e a d _ r a z e d d e r i v a t i v e s of D A Dy a n o n f u n c t i o n a l , i n t r a n e u r o n a i r e l e a s e of EA, ~hIle A P r l b l t l n g i m p u l s e - c o u p l e d e x t r a n e u r o n a ! r e l e a s e of 2A. However, .f this w e r e the e a s e the i n c r e a s e in D O P A C f o r m a t i o n an p r e s e n c e of m o r p n l n e ~ould not me e x p e c t e d to De a f f e c t e d my o e n z t r o p l n e B l g g l o et al (2) and Alper et el. (16) n a v e a l s o r e v o r t e d an i n c r e a s e in DOP~ and D C P A C levels an the rat s t r l a t u m a f t e r g i v i n g i.c v i n s e c t l o n s cf a s y n t h e t i c M e t - e n k e p h a l l n a n a l o g or p e r i p h e r a l I n s e c t l o n s of m o r p h i n e . However, trese a u t h o r s d l d not b l o c k DA s y n t n e s l s p r i o r to their m e a s u r e m e n t of D O P A C levels, and t h e r e f o r e w i t n e s s e d c n a n g e s in D O P A C levels p r o m a m l y due in p a r t to an i n c r e a s e d ~ n t e r n e u r o n a ! o v e r f l o w of DA from s t o r a g e sites a f t e r s t i m u l a t i o n of DA s y n t n e s l s by the "o?lates" ir fact, B l g g l o et el. (2) ~ n t e r p r e t e d t h e i r r e s u l t s as b e i n g Ar a g r e e m e n n ~itP the asstunptAon of an I n h i b i t o r , a c t l o n of " o p i a t e s " on DA ~e!ease, bdt our p r e s e n t r e s a l t s an m l c e ~ould c o n t r a d i c t such ~n I n t e r p r e t a t i o n . M o r p h i n e and l e u - e n k e p h a l ! n have bee~ r e p o r t e d to blnd ~o d l f f e r e n n b r a i n r e c e p t o r s ,23) ?.ddltAvlty of the m A o c n e m l c a l e f f e c t s of m o r p h i n e and D - A l a - - D - L e u S - e n k e p h a l i n In v l v o ~ould be s t r o n g e v i d e n c e to =avor i n d e p e n d e n t sites of a c t A c n of the :wo " o p i a t e s " However, % . o n m o r E n l n e a n d the e n < e p h a i l n a n a l o g were g i v e n t o g e t h e r in our e x } e r l m e n t s , their e f f e c t s c l e a r l y w e r e not a d d l t A v e (Tasle I!). The p o s s l b l l t : =~a: DA s ~ n t h e s l s a n d r e l e a s e were s t i m u l a t e d to their fall c a p a c l t y b[ e A t r e r "opAane" a l o n e a n d t h a t these p r o c e s s e s b e c a m e rate l i m i t i n g in our s t u e ± e s car be r a l e d cut, s±nce h a l o p e r A d o l s t i m u l a t e d D~ s : n t ~ e s l s ard r e A e a s e to a m u c h nlgner e x t e n t than the m a x i m a l e f f e c t s o o t a l r e d w r t n the "ofianes" On the o t h e r hand, our r e s u l t s s h o w n zn Figs ! ard 2 are in a g r e e m e n t wlt~ tqe f l n d l n g of C h a n g and C u a t r e c a s e s ~23) that n a ! o < o r e has a n i ~ e r a f f i n i t y for the " m o r ~ n ± n e - r e c e p t o r " than for the " e n k e p n = i ~ n - r e c e p t o r " a n t !s t h e r e f o r e m o r e e f f l c ~ e n t ~n d l s p ~ a c _ ~ j
1981
bol.
28,
No.
20,
1981
Opiate
Effects
on DA N e u r o n s
m o r p n l n e than D - A l a 2 - D - L e u 5 - e n k e p h a ! i n from their r e s p e c t i v e b l n d l n g sltes. It thus a p p e a r s that our r e s u l t s are c o m p a t i b l e w l t h the c o n c e p t that m c r p h l n e and D - A l a - - D - L e u 5 - e n k e p h a l l n may band an a d i f f e r e n t m a n n e r to ~and posslbl> to a d i f f e r e n t portion of) the same receptor.
Acknowled@ements The a u t h o r s wltn to thank Pamela Allen for her e x c e l l e n t t e c h n i c a l assistance This w c r ~ was s d p p o r t e d by the M e d i c a l R e s e a r c h Service of the V e t e r a n s A d m i n i s t r a t i o n , and grants from the Public Health Service (NIAAA 2696, N I D A ~C24, N I D A 1951) and the State of Illinois D e p a r t m e n t of M e n t a l Health and D e v e l o p m e n t a l D l s a b l l l t l e s (8083-13). S Ur~jler ~s the r e c l p l e n t of a Swiss N a t i o n a l Sclence F o u n d a t i o n fellowship.
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14 15. 16. 17. 18. 19.
20. 21.
H. POLLARD, C. LLORENS, J.C. SCHWARTZ, C. GROS and F. DRAY, Braln Res. 151: 392-398 (1978). G. BIGGIO, M. CASU, M.G. COKDA, C. DI B E L L O and G.L. GESSA, Science 200 552-554 (1978) D.H. C L O U E T and M. RATNER, Science 1 6 8 : 8 5 4 - 8 5 6 (1970). J.A. G A R C I A - S E V I L L A , L. AHTEE, T. M A G N U S S O N and A. CARLSSON, J. Pharm. Pharmacol. 30. 6 1 3 - 6 2 1 (1978). S.A. PERSSON, Eur. J. Pnarmacol. 5 5 : 1 2 1 - 1 2 8 (1979). J.A. G A R C I A - S E V I L L A , T. M A G N U S S O N and A. CARLSSON, NaunynS c h m l e d e b e r g ' s Arch. Pharmacol. 310. 211-218 (1980). K. K U S C H I N S K Y and O. H O R N K I E W I C Z , Eur. J. Pharmacol. 1 9 : i 1 9 - 1 2 5 (1972). K K U S C H I N S K Y and O. H O R N K I E W I C Z , Eur. J. Pharmacol. 2 6 : 4 1 - 5 0 (1974). P. S L A T E R and C. BLUNDELL, N a u n y n - S c h m l e d e b e r g ' s Arch. Pharmacol. 312: 2 1 9 - 2 2 4 (1980). E. COSTA, D.L. CHENEY, G. R A C A G N I and G. ZSILLA, Life Scl~ 17. 1-18 (1975). S. A R B I L L A and S.Z. LANGER, Nature 2 7 1 : 5 5 9 - 5 6 1 (1978). P. M O L E M A N and J. B R U I N V E L S , Life Scl. 19 1277-1282 (1976). P. M O L E M A N and J. BRUINVELS, J. Neural T r a n s m 46- 225-237 (1979). S. PURI, C. REDDY and H. LAL, Res. Comm. Chem Path. Pharm. 5: 389401 (1973). I. HANBAUEK, J. GIMBLE, K. S N A K A R A N and ~. SHERARD, N e u r o p h a r m a c o l . 18. 8 5 9 - 8 6 4 (1979). R.H. ALPER, K.T. D E M A R E S T and K.E. MOORE, J. Neural Transm. 48: 1 5 7 - 1 6 5 (1980). B. C E L S E N and K. KUSCHINSKY, N a u n y n - S c h m l e d e b e r g ' s Arch. Pharmacol. 284. 1 5 9 - 1 6 5 (1974). H. LOH, D. BRASE, S. S A M P A T H - K A N N A , J. MAR, E. LEONG WAY and C. LI, Nature 264:567-568 (1976). N. SUBRAMA~IAN, P. MITZNEGG, W. SPRUEGEL, W. DOMSCHKE, S. DOMSCHKE, E. W U E N S C H and L. DEMLING, N a u n y n - S c h m l e d e b e r g ' s Arch. Pharmac 299 1 6 3 - 1 6 5 (1977) . S. S A M P A T H KANNA, D.A. BRASE, H.H. LOH and E.L. WAY, P h a r m a c o l o g i s t 18. 545 (1976). S DE LA BAUME, G. PATEY, H. 5tAPCAIS, P. PROTAIS, J. C O N S T E N T I N and J.C. SCHWARTZ, Life Scl. 24. 2333-2342 (1979)
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C. RACAGNI, F. BRUNO, E. I U L I ~ N O and R. PAOLETTI, J pharmacol. Exp. Thero 2 0 9 : 1 1 1 - 1 1 6 (1979). K.J. CHANG and P. CUATRECASAS, J. Biol. Chem. 2 5 4 : 2 6 1 0 - 2 6 1 8 (1979). C.R. BEDDELL, R.B. CLARK, G.W. HARDy, L.A. L O W E , F.B. UBATUBA, J R. VANE, F.R.S. and S. WILKINSON, K.J. CHA~]G, P. C U A T R E C A S A S and R.J. MILLER, Proc. R. Soc. Lond. B. 1 9 8 : 2 4 9 - 2 6 5 (1977). B. T A B A K O F F and P. BLACK, J. Neurochem. 34. 1707-1711 (1980). L.J. FELICE, J.D. FELICE and P.T. KISSINGER, J Neurochem. 31. 1 4 6 1 - 1 4 6 5 (1978) B. TABAKOFF and R. RITZMANN, J. Pharmacol. Exp. Ther. 203. 319-322 (1977). A. CARLSSON, J.N. DAVIS, W. KEHR, M. L I N D Q V I S T and C.V. ATACK, N a u n y n - S c h m l e d e b e r g ' s Arch. Pharmacol. 275 153-168 (1972) R.H. ROTH, L.C. M U R R I N and J.R. WALTERS, Eur. J. Pharmacol. 36: 1 6 3 - 1 7 1 (1976). L.X. CUBEDDU, I.S. HOFFM/LN and G.B. FERRARI, J. pharmaco!. Exp Ther. 2 0 9 : 1 6 5 - 1 7 5 (1979). J.T. COYLE and S.H. SNYDER, Sclence 166 8 9 9 - 9 0 1 (1969) N.E. A N D E N and P.J. BEDARD, J. Pharm. P h a m a c o l . 23 460-462 (1971) E. COSTA, W. FRATTA, J.S. HONG, F. MORONI and H.-f. YANG, in Adv. in Bzochem. p s y c h o p h a r m a c o l . E. Costa and M. Trabucchl, eds., 18 217-226, Raven Press, N.Y. (1978) R.H. RGTH, J.R. WALTERS, L.C. M U ~ R I N and V.H. MORGENROTH, In Pre- and P o s t s y n a p t l c Receptors, E. Usdln and W.E. Bunney, eds , pp. 5-48, Marcel Dekker, New York (1975). A. CARLSSON, In Pre- and P o s t s y n a p t l c Receptors, E Usdln and W.E. Bunney, eds., pp. 49-63, Marcel Dekker, New York (1975)
1981
2277-2286
Pergamon
Press
S T I M U L A T I O N OF D O P A M I N E SYNTHESIS AND RELEASE BY M O R P H I N E AND D - A L A 2 - D - L E U 5 - E N K E P H A L I N IN THE M O U S E S T R ~ A T U M IN V I V O Stephan
Urwyler
and Boris
TaDakoff
V.A. W e s t s l d e M e d i c a l C e n t e r and Dept. of P h y s l o l o g y and Biophyslcs, U n l v e r s l t y of Illlnols at the M e d l c a l Center, Chlcago, IL 60680 (Received
in flnal
form F e b r u a r y
27, 1981)
Summar~ The e f f e c t s of o p i a t e s on d o p a m l n e (DA) r e l e a s e and s y n t n e s l s w e r e a s s e s s e d in the mouse strmatum in v l v o by s l m u l t a n e o u s l y measuring 3,4-dihydroxyphenylalanlne (DOPA) and 3 , 4 - d i h y d r o x y p h e n y l a c e t l c acid (DOPAC) levels after i n h i b i t i o n of 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 Thls m e t h o d was d e v e l o p e d to a s s e s s s t l m u l u s - c o u p l e d changes in DA s y n t h e s l s and release. P e r i p h e r a l i n 3 e c t l o ~ s of m ~ r p n l n e and i n t r a v e n t r l c u l a r i n 3 e c t l o n s of D-AIa - D - L e u - - e n k e p h a l l n elevated DOPAC levels, i n d i c a t i n g that "opiates" s t i m u l a t e d DA release. Conc o m l t a n t l y , the rate of DA s y n t h e s i s was increased. The e f f e c t s were d o s e - d e p e n d e n t , s a t u r a b l e and a n t a g o n l z e d by naloxone. W h e n m o r p h i n e and the e n k e p h a l l n a n a l o g were given t o g e t h e r in s a t u r a t i n g doses, the effects of the two a g e n t s w e r e not addmtlve. Thus, the i n v o l v e m e n t of d l f f e r e n t r e c e p t o r s in the m e d i a t i o n of the e f f e c t s of m o r p h i n e and e n k e p h a l l n s could not be d e m o n s t r a t e d .
The p r e s e n c e of o p l a t e b l n d l n g sltes in the strlatum, one thlrd of w h l c h a p p e a r to be l o c a l l z e d on the t e r m i n a l s of d o p a m l n e r g l c n e u r o n s of the n l g r o s t r l a t a l pathway, has been p r e v i o u s l y d e m o n s t r a t e d (i), and a c t i v a t i o n of o p i a t e r e c e p t o r s results in an increase in strlatal DA s y n t h e s i s and t u r n o v e r (2-6). However, the p r e v l o u s l y r e p o r t e d changes in DA m e t a b o l i s m have been p o s t u l a t e d by some a u t h o r s (7) to r e f l e c t m n t r a n e u r o n a l events not a s s o c i a t e d with DA r e l e a s e from neurons, and the a n s w e r to the q u e s t i o n of w h e t h e r the s t i m u l a t i o n of opiate r e c e p t o r s m n n m b l t s or f a c 1 1 1 t a t e s the release of DA r e m a i n s c l o u d e d in controversy. S p e c l e s d e p e n d e n t d i f f e r e n c e s in opiate e f f e c t s may a c c o u n t for some of the d l s p a r a t e flndlngs. For instance, the effect of m o r p h i n e on l o c o m o t o r a c t l v l t y in mlce and rats indicates s l g n l f l c a n t d i f f e r e n c e s in n e u r o p h a r m a c o l o g i c a s p e c t s of opmate a c t i o n s in these two specles (8, 9). However, the c o n f l l c t l n g r e s u l t s in p r e v i o u s studies can not be e x p l a l n e d solely on the basis of specles or g e n e t l c differences. In fact, no effect (10-13), I n c r e a s e s (i4-16) and d e c r e a s e s (17-19) in the r e l e a s e of DA in the s t r l a t u m of the rat In response to the a d m i n i s t r a t i o n of m o r p h l n e or the e n k e p h a l i n s have been reported. C o n f l i c t i n g r e p o r t s also exist wlth r e g a r d to the e f f e c t s of m o r p h l n e on strlatal DA r e l e a s e in the mouse (20-22) but the e f f e c t s of e n k e p n a l i n s or tnelr a n a l o g u e s on the n e u r o n a l r e l e a s e of DA in moase s t r m a t u m nave not been r e p o r t e d 0024-3205/81/202277-I0$02.00/0 C o p y r l g h t (c) 1981 P e r g a m o n Press Ltd.
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O p l a t e E f f e c t s on DA N e u r o n s
Vol.
28, No.
20,
1981
S ! n c e two t y p e s of "opiate" r e c e p t o r s have been ShOWn to be p r e s e n t in r o d e n t b r a i n (23) one m a y a l s o ~ o n d e r w h e t h e r the e f f e c t s of m o r p h l n e a n d the e n k e p h a l l n s on s t r l a t a l D A s y n t h e s l s and r e l e a s e are m e d l a t e d by I d e n t l c a l r e c e p t o r systems. In o r d e r to f u r t h e r e l u c i d a t e the a c t i o n s of "oplates" on D A r e l e a s e and m e t a b o l i s m in tne mouse, we have i n v e s t i g a t e d the e f f e c t s of m o r p h l n e a n d D - A l a 2 - D - L e u b - e n k e p h a l l n , a p o t e n t and p e p t l d a s e - r e s l s t a n t e n k e p n a l ~ n a n a l o g (24), on the levels of DA, D O P A and D O P A C an the m o u s e s t r l a t u m ~n v l v o a f t e r i n h i b i t i o n of a r o m a t i c a m i n o acld decarboxylase. The r e p o r t e d m e t h o d s for m o n l t o r l n g DA m e t a b o l i s m w e r e d e v e l o p e d to m o r e c l e a r l y r e l a t e c h a n g e s in D A p r e c u r s o r and m e t a b o l l t e l e v e l s to s t i m u l u s c o u p l e d r e l e a s e of DA from the DA n e u r o n t e r m i n a l s in the strlatum.
M a t e r i a l s and M e t h o d s Animals. M a l e C 5 7 B 1 / 6 m l c e (23-25g) w e r e p u r c h a s e d from ARS/Sprag~leD a w l e y ( ~ d l s o n , WI) a n d w e r e h o u s e d flve per cage an our l a c o r a t o r i e s (22°C, 12hr l i g h t cycle) for at least 7 d a y s w l t h ad l l b l t u m a c c e s s to P u r i n a l a b o r a t o r y c h o w and w a t e r b e f o r e t h e y ~ere u s e d for the e x p e r l ments. M e a s u r e m e n t s of t ~ r o s l n e h y d r o x [ l a s e a c t l v ! t y and r e l e a s e of DA: T y r o s l n e h y d r o x y l a s e a c t i v i t y was m e a s u r e d an v l v o by m o n l t o r l n g the a c c u m u l a t i o n of D O P A a f t e r I n h l b l t l o n of b r a i n a r o m a t i c a m i n o acld d e c a r b o x y l a s e w i t h an l.p. I n ] e c t l o n of N S D - 1 0 2 4 (Sandev Ltd , Harlow, Essex, G.B.). N S D - i 0 2 4 was u s e d ~n our s t u d i e s because, at the d o s e s a d m i n i s t e r e d , at p r o d u c e d a p r o f o u n d i n n l b l t l o n of a r o m a t l c a m i n o a c i d d e c a r b o x y l a s e t25) but had no s i g n i f i c a n t e f f e c t on M A O a c t l v l t i an m o u s e b r a i n (B. T a b a k o f f , u n p u b l i s h e d o b s e r v a t l o n a n d d a t a p r o v i d e d by Sandev, Ltd.). The b l o c k a d e of DA synthesis, w l t h o u t a s l g n ~ f l c a n t e f f e c t on D A c a t a b o l i s m , a l l o w e d for an e s t i m a t i o n of D A r e l e a s e and s u b s e q u e n t r e u p t a k e a n d c a t a b o l i s m by a s s a y of the levels of DA a n d its m e t a b o l l t e , DOPAC. The a n i m a l s ~ere k l l i e d by d e c a p l t a t l o n and t h e ± r b r a i n s w e r e r a p i d l y r e m o v e d ~nd d i s s e c t e d on a c h i l l e d g l a s s plate. All f u r t h e r p r o c e d u r e s w e r e c a r r i e d out on ace or an 4°C. The heads of the c a u d a t e of b o t h h e m i s p h e r e s w e r e p o o l e d and h o m o g e n i z e d !n 5 ml of 0 4 : 4 p e r c n l o r l c a c i d c o n t a i n i n g 0.05% s o d i u m m e t a D l s u l f l t e and 0.2% E D T A u s i n g a Tekmar Tlssuemlzer.
The h o m o g e n a t e
~as c e n t r l f u g e d for 20 man in a
Sorval c e n t r l f e g e at 3 0 , 0 0 0 g and 2.4 ml of 2 M Trls and 50 mg of a l u m l n a w e r e a d d e d to the r e c o v e r e d s u p e r n a t a n t fluld. A f t e r t h o r o u g h mlxlng, the a l u m i n a was w a s h e d twice w i t h d l s t ~ i l e d w a t e r and the b o u n d c a t e c h o l s ~ere e l u t e d f r o m the a l u m i n a w l t h a 2 M a c e t i c a c l d s o l u t i o n w h i c h c o n t a l n e d 0.2% s o d l u m m e t a b l s u l f l t e . The s a m p l e s w e r e then f r o z e n unt~l DA and Its m e t a b o l l t e s w e r e s e p a r a t e d by r e v e r s e p h a s e h l g h p e r f o r m a n c e l l q u l d c h r o m a t o g r a p h y on a C l 8 - ~ B o n d a p a k C o l u m n ~Waters ~ s s o c l a t e s , M i l f o r d , MA) (26) a n d q u a n t l t a t e d by tDe use of an e l e c t r o c h e m l c a l d e t e c t o r ( B l o a n a l y t l c a l Systems, W L a f a y e t t e , IN). A u t n e n t l c s t a n d a r d s w e r e u s e d for e s t a o l l s h l n g c a l l b r a t l o n c u r v e s a n d d ~ h y d r o x y b e n z y l a m l n e was c a r r l e d t h r o u g h the e x t r a c t l o n a n d q u a n t l t a t l o n p r o c e d u r e as an i n t e r n a l standard. T r e a t m e n t of a n l m a ! s w l t h dru~s. NSD-1024 [3-nydroxybenzyloxamlne d ! h y d r o g e n p h o s p n a t e ] was d i s s o l v e d ~n 0.9% NaCI a n d i n 3 e c t e d ±.p. (5C0 mg/kg) 30 m a n b e f o r e the a n i m a l s w e r e kllled, since our p r i o r e x p e r l m e n t s had s h o w n that D © P A a c c u m u ! a t l o n was l i n e a r o v e r that t~me p e r l o d (25) H a l o p e r l d o l was u s e d ±n the form of H a l d o l A i n 3 e c t a b l e s o l u n - o n rMcNe±l L a b o r a t o r i e s , Ft. W a s n l n g t o n , PA) and was d i l u t e d w i t h 0 9% NaC! b e f o r e l,p. i n ] e c ~ i o n (i mg/kg, 9Q mln. b e f o r e N S D - 1 0 2 4 ) . N a l o x o n e (Endo
Vol
28, No.
20,
1981
OPiate
Effects
on DA Neurons
L a o o r a t o r l e s , G a r d e n Cltl, N%) (3 mg/kg) and m o r p h m n e sulfate (Mallanckrodt, St Louis, MC) were also d l s s o l v e d in 0 9% NaCI and ~ n 3 e c t e d at the stated doses (l p.) 20 and 15 m a n u t e s before NSD-1024, respectlvely. C e r t a i n of the sallne (control) and m o r p h l n e in3ected a n l m a l s also were a~3ected (1.9.) w l t n b e n z t r o p l n e m e s y i a t e (Arapahoe, Newport, TN), 25 mg/kg, 20 m i n u t e s before NSD-1024 D - A I a 2 - D - L e u ~e n k e p h a l a n (Bachem, Torrance, CA) was glven i n t r a v e n t r l c u l a r l y (loC v~l under llg>t ether a n e s t h e s i a (27) in 4 ~i of Meri!s' solutaon, i0 mln p r a o r to N S D - 1 0 2 4 A n l m a l s r e c e l v a n g ! p. i n 3 e c t ! o n s of sallne (instead of m o r p h l n e , n a l o p e r l d o l or~. benzt~oplne) or [.c %- in3ectaons of Merlls' s o l u t l o n (instead of D - A l a ~ - D - L e u - - e n k e p n a l a n ) at the a p p r o p r i a t e tlmes p r a o r to N S D - 1 0 2 4 served as controls. In order to obtaln basal levels of DA and DOPAC, o t h e r control m2ce r e c e l v e d only vehlcle, but no N S D - 1 0 2 4 in3ectlons. Results are e x p r e s s e d as means ~ S.EoM. The data were a n a l y z e d by the use of a n a l y s l s of v a r i a n c e and tne Snudent ~-test P values < 0.05 were taken as an indicatlon of s t a t a s t l c a l s ! g n l f l c a n c e of d l ~ f e r e n c e s between mean values
Results M o r p h a n e an doses r a n g l n g from 10-40 mg/kg was found to elevate D O P A and D O P A C levels an the c a u d a t e (Fig. !). DOPA was not d e t e c t a b l e In a n a m a l s r e c e l v l n g no NSD-1024, but the level of D O P A C was s a g n a f l c a n t l y nagher than in a n a m a l s r e c e l v a n g NSD-1024 but no "oplate". The e f f e c t s of m o r p h a n e were d o s e - d e p e n d e n t , saturable and could be b l o c k e d by p r e t r e a t m e n t of the a n a m a l s w a t h n a l o x o n e (Fag. i). Results o b t a a n e d an the p r e s e n c e of b e n z t r o p a n e a n d l c a t e d that the D O P A C levels In the s t r a a t u m of the N S D - 1 0 2 4 t r e a t e d anmmals r e f l e c t e d the stamulus c o u p l e d e x t r a n e u r o n a l r e l e a s e of DA tsee dascusslon) Table I shows that pret r e a t m e n t of the a n l m a l s wlth b e n z t r o p a n e lowered D O P A C levels to a p p r o x i m a t e l y the same e x t e n t an control and m o r p h l n e t r e a t e d anamals, and that D O P A a c c u m u l a t a o n was also l o w e r e d by benztropane. D - A i a 2 - D - L e u 5 - e n k e p h a l a n was d e m o n s t r a t e d to a n c r e a s e DA s y n t h e s a s and relase an the s t r a a ~ u m as i n d l c a t e d by I n c r e a s e s in D O P A and DOPAC l e v e l s over levels of D O P A and D O P A C found in Merlas' solutlon a n 3 e c t e d m l c e (Fag. 2). However, the D O P A levels !n a n l m a l s r e c e ~ v l n g 3ust N S D - 1 0 2 4 and an l.c.v, a n 3 e c t a o n of the Merlas' solutlon were hlgher than the D O P A levels an the a n l m a l s a n 3 e c t e d l.p. wlth sallne. Thas i n d a c a t e d that the l.c.v, a n 3 e c t ~ o n by itself could s t a m u l a t e DA synthesis. After l n n l b l t l o ~ of DA synthesas, an ancrease an DA r e l e a s e w o u l d be e x p e c t e d to be r e f l e c t e d an an a c c e l e r a t e d declane an DA levels. However, d u r l n g the short p e r l o d of s y n t n e s l s a n n l b l t l o n used an the p r e s e n t e x p e r l ments, the r e l e a s e d DA a c c o u n t e d for only 11% of the total DA pool [DA levels an all c o n t r o l mlce r e c e l v ~ n g no NSD-1024 and those r e c e a v ! n g N S D - i C 2 4 were, r e s p e c t l v e l y 100.7+--2.9 (n=22) vs. 8 9 . 7 + 2 . 0 (n=31) nmoles/g] and due to the usual d l f f e r e n c e s an anatlal levels of D A in i n d a v a d u a l m a c e It was an m o s t e x p e r l m e n t s not p o s s l b l e to o b s e r v e s l g n ~ f ~ c a n t d a f f e r ences an D A levels an the p r e s e n c e of "opaates" (Table II).
2279
2280
OplaCe
Effects
on
DA Neurons
Vol.
28,
No.
20,
T 40
w O9
30
,,~
20
o
z
E 4
IO 0
0 d~
i
/Y
o
E c
~-
.r v
4O W OO
30 < UJ n"
20
~ Z
4
i
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0
[ i
3
i
-b I
I
O
IO
--
i
20
MORPHINE
50
40
SUL,-"ATE(mg/kg ~ p) FIG.
i
E f f e c t of m o r p n l n e s u l f a t e o n D O P A C (upper p a n e l ) a n d D O P A (lower p a n e l ) l e v e l s zn t h e m o u s e n u c l e u s c a u d a t u s . C 5 7 m l c e r e c e z v e d l.p. z n 3 e c t l o n s of s a l l n e or z n c r e a s l n g d o s e s of m o r p h l n e s u l f a t e (-o-) 13 m l n b e f o r e N S D - I 0 2 4 a n d 45 m l n b e f o r e s a c r z f l c e . Certazn anzmals (-o-) recelved 3 m g / k g o f n a l o x o n e l.p. 5 m z n b e f o r e t h e y w e r e z n 3 e c t e d w l t ~ m o r p h l n e or s a l l n e . DOPAC levels were a l s o m e a s u r e d zn c o n t r o l a n l m a l s r e c e z v l n g n o N S D - 1 9 2 4 (~). ~ a ! u ~ s f r c m b e t w e e n 4 a n d 13 a D z m a l s w e r e u s e d to o b t a ± n t h e m e a n + S.E.~4 ~sterlsks indlcate DOPA and DOPAC levels whmcn were slgnzflcantly dzfferent from the respective l e v e l s zn c o n t r o l a n z m a l s r e c e ~ v z n g NSD-1024
but
no m c r p n l n e
sdlfate
(p
. 0.05)
1981
%ol.
28, No.
20,
1981
Opiate
Effects
TABLE
on DA Neurons
228]
I
E f f e c t of P r e t r e a t m e n t wath B e n z t r o p l n e on DOPA and DOPAC Levels an the Strlatum of Control and Morphlne Treated Mace Group
DOPA
% Chan~e
Control (Salane)
3.70+0
Benztropane
3.08~0.24
Morphlne (15 mg/kg)
5.88+0.10
Morphane (15 mg/kg) & Benztropane
3 53+0.38*
Morpnane (30 mg/kg)
6 07+0.68
Morpnlne (30 mg/kg) & Benztropane
3.11+0.18"
39
% Chan~e
DOPAC
3.79+9.29 -17
2.42~0.23"
-36
5.44+0.11
-42
3.29+0.23*
5.70+0
-48
-39
64
2.69+0.15"
-52
A n a m a ! s were an]ected wlth NSD-1024 (500 mg/kg, a.p.) 30 m l n u t e s p r l o r to sacraface. Morphlne sulfate (15 or 30 mg/kg) and b e n z t r o p l n e m e s y l a t e (25 mg/kg, a.p.) were gaven 15 and 20 mlnutes before NSD, respectavely. Control anamals r e c e i v e d salane an3ectaons instead of morphlne and benztropane. Results are glven in nmoles per g of t~ssue. Fave to sax anamals c o m p r l s e d each experam e n t a l group. Per cent change indlcates the extent of d ~ f f e r e n c e s between anamals r e c e a v l n g no b e n z t r o p l n e and those r e c e l v a n g benztropzne. A s t e r a s k s andlcate slgnafacant d a f f e r e n c e s between tnese groups (p < 0.05).
In order to a n v e s t l g a t e w h e t h e r the effects of m o r p h a n e and the e n k p e h a l l n a n a l o g at m a x l m a l doses were addltlve, e x p e r a m e n t s were p e r f o r m ed in w h a c h the two "opaates" were gaven together (Table II). The control (a.p. salane-an3ected) m a c e and anlmals in3ected a.p. wlth m o r p h l n e rec e l v e d an a d d l t a o n a l a.c.v, a n 3 e c t l o n of Merlls' solutlon in these experiments, to control for the effects of l.c.v, zn3ectlons on DA synthesas. Table II shows that the effects of m a x l m a l doses of m o r p h l n e and D - A l a 2 - D - L e u 5 - e n k e p h a l a n were not addltave: no sagnflcant d a f f e r e n c e s were found b e t w e e n m e a n D O P A and D O P A C values an anamals r e c e l v a n g both "oplates" t o g e t h e r and the two groups whlch recelved one of the two groups alone. H a l o p e r l d o l s t l m u l a t e d the synthesls and release of DA to a m u c h nlgher e x t e n t than. dld the m a x l m a l doses of the two "oplates": D O P A and DOPAC levels ~n the caudate of anlmals treated wlth haloperldol and N S D - 1 0 2 4 were 1 2 . 6 8 + 0 . 5 6 n m o l e s / g and 10.84+0.36_ n m o l e s / g (n=6), respectively, c o r r e s p o n d z n g to a 160% and 154% ~ncrease over v a l u e s found in anamals w h a c h r e c e l v e d salzne rather than the haloperadoi. DA levels an tne caudate of a n i m a l s t r e a t e d wlth h a l o p e r l d o l and NSD-1024 were
2282
Oplate
Effects
on D A N e u r o n s
Vol.
28, No.
7 I00
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D-AI_AZ-D -t_EU~-ENKE~HAI_IN(Ng Icy) FIG.
2
2 5 E f f e c t o f D - A l a - D - L e u - e n k e p h a l l n on D O P A C (upper p a n e l ) a n d D O P A (lower p a n e l ) l e v e l s in t h e m o u s e n u c l e u s caudatus. Anlmals were inDected wlth Merlls' solutlon (ve~icle) o r i n c r e a s i n g a m o u n t s o f t h e e n k e p h a l l n a n a l o g (-e-) I n t r a v e n t r l c u l a r l y under light ether ~nesthesla I 0 m l n b e f o r e t h e y w e r e g i v e n N S D - 1 0 2 4 (500 m g / k g , i.p ) a n d 40 m i n b e f o r e s a c r z f l c e . O t h e r a n i m a l s (-o-) r e c e ! v e d 3 m g / k g o f n a l o x o n e l.p. 5 m ! n p r i o r to the znDectlon of the enkepnalmn analog. DOPAC levels were a l s o m e a s u r e d in c o n t r o l a m ~ m a l s r e c e l v l n g n o N S D - 1 0 2 4 ( r) T h e r e s u l t s s n o w n are the m e a n s ~ S . E . M . of v a l u e s d e r l v e d f r o m b e t w e e n 4 to 18 a n i m a l s . Asterlsks lndlcate DOPA and DOPAC levels whzch were slgnlflcantly d i f f e r e n t f r o m the l e v e l s in c o n t r o l a ~ i m a l s r e c e s v l n g N S D - 1 0 2 4 O u t n o e n k e p h a l l n (p 0 05)
20,
1981
Vol.
28~ No.
20,
1981
Oplate
TABLE
Effects
on D~ Neurons
II
5 Effects of Morphane and D - A I a ' - D - L e u -Enkephalln and the Two "Opiates" in C o m b a n a t z o n on DOPA, DOPAC and DA Levels an the Mouse Strlatum In Vavo Grou~
Control (no NSD)
DOPA DORAC DA (nmoles per g of tissue) not detectable
5.22+0.40* (4)
93.8+1.8 (~)
Control (+ NSD)
5.62+0.33 (i~)
3.50+0.10 (17)
86.6+1.8 (53)
Morphlne
8.61~0.33 *
5.19+0.17"
87.7+1.6
(14)
(l~)
Enkephalan (~ NSD)
8.86+0.56* (I~)
4.92+0.18" (I~)
83.8+1~9 (54)
Morphine + Enkephalln (+ NSD)
9.45+0.54* (I~)
5.41+0.17" (i~)
86.3+1.8 (54)
(+ NSD)
(54)
Anamals were In3ected wath NSD-1024 (500 mg/kg, i.p.) 30 m a n u t e s prlor to sacrlface. D-Ala2-D-Leu5-enkephalln (5 ~g i.c.v.) or Merlas' solutaon (l.c.v.) were admlnlstered i0 mln and morphane sulfate (30 mg/kg, l.p.) or salane (a.p.) were a d m l n a s t e r e d 15 man. prlor to NSD-1024. Anamals an the m o r p h i n e group r e c e a v e d an i.c.v. a n 3 e c t a o n of Merlas' soiutlon and e n k e p h a l ~ n treated anamals r e c e l v e d sallne i.p. Control anlmals recelved both v e h i c l e in3ectlons. DOPA, DOPAC, and DA levels are e x p r e s s e d as means + S.E.M. with the number of animals an parentheses. A s ~ e r l s k s indicate slgnlflcant differences between the respective groups and the control (+ NSD) group (p < 0.01)~
59.4+1.71_ nmoles per g tissue, Indlcatlng that a more p r o f o u n d stimulus for release of DA w o u l d be r e f l e c t e d an s l g n a f l c a n t l y lowered DA levels under our c o n d l t a o n s of inhlblted DA synthesis.
Discussaon The m e a s u r e m e n t of D O P A accu~Lulatlon and DOPAC formation was used an the p r e s e n t study to indicate the synthesis and release of DA and to I n v e s t & g a t e the effects of "oplates" on the n l g r o s t r l a t a l DA pathway. A short d ~ r a t l o n of DA synthesls i n n l b l t l o n was used in our studies such that f e e d b a c k control of tyrosane hydroxylase by DA would not be s e v e r e l y compromlsed. T y r o s l n e nydroxylase as the rate lamltlng enzyme an the b l o s y n t h e s a s of DA and ats p r o d u c t (DOPA) as r a p i d l y d e c a r b o x y l a t e d to DA. Endogenous D O P A levels are e x t r e m e l y low and d a f f a c u l t to detect. However, if aromatac amino acld d e c a r b o x y l a s e Is anhabated, the a c c u m u l a t i o n of D O P A over tame can be used as an andlcator of the rate of DA syntehsas (28). DOPAC has been shown to be formed
2283
2284
O p i a t e E f f e c t s on DA N e u r o n s
Vol.
2~, Ne.
20,
p r e s y n a p t l c a l l y e i t h e r f r o m DA w h i c h spAl!s over from s t o r a g e vesicles or from LA w n l c h has b e e n r e l e a s e d into the s y n a p t A c c l e f t and then r e c a p t u r e d Into the p r e s y n a p t l c n e r v e t e r m i n a l (29, 30) In our study, the D O P A C l e v e l s in the c a u d a t e of c o n t r o l a n i m a l s r e c e i v i n g N S D - i C 2 4 were s l g n l f l c a n t l y lower than in c o n t r o l a n i m a l s r e c e i v i n g no d r u g at all. The r e d u c t i o n of D O P A C l e v e l s by a d m l n l s t r a t l o n of N S D - 1 0 2 4 ~ o u l d r e p r e s e n t the e l a m l n a t l o n of s p l l l o v e r f r o m v e s i c l e s due to e x c e s s s y n t n e s l s s i n c e s y n t h e s l s of DA is inhlblted. Thus, the r e m a i n i n g D O P A C l e v e l s p r e s u m a b l y r e f l e c t m a i n l y the d e g r a d a t i o n of D A s u b s e q u e n t to its i m p u l s e - c o u p l e d r e l e a s e into and u p t a k e from the s y n a p t l c cleft. Prot r e a t m e n t of the a n i m a l s w i t h b e n z t r o p l n e , a d r u g w h l c h i n h i b i t s the r e u p t a k e of D A into the p r e s y n a p t l c n e r v e t e r m i n a l (31), a n d / o r t q r o u g h its a n t l - m u s c a r l n l c p r o p e r t i e s , r e d u c e s the f a r i n g rate of n l g r o s t r l a t a l DA n e u r o n s (32, 33) r e s u l t e d in a d e c r e a s e d f o r m a t i o n of D O P A C {Taole i) Thls o b s e r v a t i o n f u r t h e r c o n f i r m s t h a t u n d e r our e x p e r i m e n t a l c o n d i t i o n s c h a n g e s in D O P A C l e v e l s are a g o o d i n d i c a t o r of the e f f e c t s of d r u g s on s t i m u l u s c o u p l e d D A r e l e a s e f r o m the neuron. The l o w e r e d D O P A a c c u m u l a tion in a n i m a l s p r e t r e a t e d w l t h b e n z t r o p l n e c o u l d be a r e s u l t of u p t a k e i n h i b i t i o n w h i c h w o u l d p r o d u c e e l e v a t e d c o n c e n t r a t i o n s of DA in the s y n a p t l c cleft. S u c h e l e v a t e d DA l e v e l s w o u l d i n t e r a c t w l t h 5A r e c e p t o r s c o n t r o l i n g DA s ? n t ~ e s l s (34, 35) Cn the o t ~ e r hand, the l o w e r e d D C P A a c c u m u ! a z ~ o n c o u l d be d u e no nne r e d u c e d firing rate of the LA n e d r c n s p r o d u c e d my a c t i o n s of D e n z t r o p l n e on ~ h o l ~ n e r g ~ c r e c e p t o r s r e s i d i n g on n e u r o n s Df the s t r l a t o - n ~ g r a l f e e d b a c m loop. [orL%ine ant D - h ! a - - u - _ e u ~ - e n k e p n a l i n moth s t i m u l a t e d ~A s / n t ~ e z l s as ~ell as r e l e a s e T h e s e e f f e c t s were m e d i a t e d t n r o u { ~ t-e a c t _ o n of the "o~Aates" on o p i a t e r e c e p t o r s , s~nce the a c t i o n s of m o r ~ q A n e and D - ~ l a - - O - L e u S - e n k e p n a ! A n w e r e a n t a g o n i z e d my n a l o x o n e It aas seen s u g g e s t e d (7, 17) that m o r p h l n e mlgzt i n c r e a s e the l e v e l s of t2e d e a d _ r a z e d d e r i v a t i v e s of D A Dy a n o n f u n c t i o n a l , i n t r a n e u r o n a i r e l e a s e of EA, ~hIle A P r l b l t l n g i m p u l s e - c o u p l e d e x t r a n e u r o n a ! r e l e a s e of 2A. However, .f this w e r e the e a s e the i n c r e a s e in D O P A C f o r m a t i o n an p r e s e n c e of m o r p n l n e ~ould not me e x p e c t e d to De a f f e c t e d my o e n z t r o p l n e B l g g l o et al (2) and Alper et el. (16) n a v e a l s o r e v o r t e d an i n c r e a s e in DOP~ and D C P A C levels an the rat s t r l a t u m a f t e r g i v i n g i.c v i n s e c t l o n s cf a s y n t h e t i c M e t - e n k e p h a l l n a n a l o g or p e r i p h e r a l I n s e c t l o n s of m o r p h i n e . However, trese a u t h o r s d l d not b l o c k DA s y n t n e s l s p r i o r to their m e a s u r e m e n t of D O P A C levels, and t h e r e f o r e w i t n e s s e d c n a n g e s in D O P A C levels p r o m a m l y due in p a r t to an i n c r e a s e d ~ n t e r n e u r o n a ! o v e r f l o w of DA from s t o r a g e sites a f t e r s t i m u l a t i o n of DA s y n t n e s l s by the "o?lates" ir fact, B l g g l o et el. (2) ~ n t e r p r e t e d t h e i r r e s u l t s as b e i n g Ar a g r e e m e n n ~itP the asstunptAon of an I n h i b i t o r , a c t l o n of " o p i a t e s " on DA ~e!ease, bdt our p r e s e n t r e s a l t s an m l c e ~ould c o n t r a d i c t such ~n I n t e r p r e t a t i o n . M o r p h i n e and l e u - e n k e p h a l ! n have bee~ r e p o r t e d to blnd ~o d l f f e r e n n b r a i n r e c e p t o r s ,23) ?.ddltAvlty of the m A o c n e m l c a l e f f e c t s of m o r p h i n e and D - A l a - - D - L e u S - e n k e p h a l i n In v l v o ~ould be s t r o n g e v i d e n c e to =avor i n d e p e n d e n t sites of a c t A c n of the :wo " o p i a t e s " However, % . o n m o r E n l n e a n d the e n < e p h a i l n a n a l o g were g i v e n t o g e t h e r in our e x } e r l m e n t s , their e f f e c t s c l e a r l y w e r e not a d d l t A v e (Tasle I!). The p o s s l b l l t : =~a: DA s ~ n t h e s l s a n d r e l e a s e were s t i m u l a t e d to their fall c a p a c l t y b[ e A t r e r "opAane" a l o n e a n d t h a t these p r o c e s s e s b e c a m e rate l i m i t i n g in our s t u e ± e s car be r a l e d cut, s±nce h a l o p e r A d o l s t i m u l a t e d D~ s : n t ~ e s l s ard r e A e a s e to a m u c h nlgner e x t e n t than the m a x i m a l e f f e c t s o o t a l r e d w r t n the "ofianes" On the o t h e r hand, our r e s u l t s s h o w n zn Figs ! ard 2 are in a g r e e m e n t wlt~ tqe f l n d l n g of C h a n g and C u a t r e c a s e s ~23) that n a ! o < o r e has a n i ~ e r a f f i n i t y for the " m o r ~ n ± n e - r e c e p t o r " than for the " e n k e p n = i ~ n - r e c e p t o r " a n t !s t h e r e f o r e m o r e e f f l c ~ e n t ~n d l s p ~ a c _ ~ j
1981
bol.
28,
No.
20,
1981
Opiate
Effects
on DA N e u r o n s
m o r p n l n e than D - A l a 2 - D - L e u 5 - e n k e p h a ! i n from their r e s p e c t i v e b l n d l n g sltes. It thus a p p e a r s that our r e s u l t s are c o m p a t i b l e w l t h the c o n c e p t that m c r p h l n e and D - A l a - - D - L e u 5 - e n k e p h a l l n may band an a d i f f e r e n t m a n n e r to ~and posslbl> to a d i f f e r e n t portion of) the same receptor.
Acknowled@ements The a u t h o r s wltn to thank Pamela Allen for her e x c e l l e n t t e c h n i c a l assistance This w c r ~ was s d p p o r t e d by the M e d i c a l R e s e a r c h Service of the V e t e r a n s A d m i n i s t r a t i o n , and grants from the Public Health Service (NIAAA 2696, N I D A ~C24, N I D A 1951) and the State of Illinois D e p a r t m e n t of M e n t a l Health and D e v e l o p m e n t a l D l s a b l l l t l e s (8083-13). S Ur~jler ~s the r e c l p l e n t of a Swiss N a t i o n a l Sclence F o u n d a t i o n fellowship.
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1981