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Differentiation of spinal and supraspinal opioid receptors by morphine tolerance
Life Sciences, Vol. 46, pp. 343-350 Printed in the U.S.A.
DIFFERENTIATION
OF S P I N A L AND S U P R A S P I N A L MORPHINE TOLERANCE*
Pergamon Press
OPIOID
RECEPTORS
BY
Byron C. Yoburn, Kabir L u t f y , Saira Azimuddin and Victor Sierra Department of Pharmaceutical Sciences College of Pharmacy and A11ied Health Professions St. John's U n i v e r s i t y Queens, NY 11439 (Received in final form December 4, 1989) Summary
Mice t r e a t e d f o r 72hrs with morphine (subcutaneously implanted p e l l e t s ) were tested with a v a r i e t y of opioid receptor agonists to examine the development of tolerance and cross-tolerance to t h e i r analgesic action. The development of spinal and supraspinal tolerance f o l l o w i n g morphine treatment was evaluated by administering compounds s y s t e m i c a l l y ( s c ) , i n t r a t h e c a l l y ( I T ) and i n t r a c e r e b r o v e n t r i c u l a r l y (ICY). Following morphine treatment, tolerance to morphine analgesia was observed f o l l o w i n g IT, ICV and sc a d m i n i s t r a t i o n . Chronic morphine treatment also produced cross-tolerance to the analgesic e f f e c t s of the s e l e c t i v e 6 opioid receptor agonist [D-Pen2 , D-PenS]enkephalin (DPDPE) f o l l o w i n g IT and ICV a d m i n i s t r a t i o n . However, morphine treatment s e l e c t i v e l y produced cross-tolerance to ICV [DAla 2 , NMePhe4, Gly-olS]enkephalin (DAGO) (p receptor agonist) analgesia, without a l t e r i n g IT DAGO analgesia. These r e s u l t s suggest t h a t brain and spinal cord receptors mediating the e f f e c t s of DAGO d i f f e r in terms of the development of crosstolerance to morphine; and suggest t h a t tolerance to systemic morphine may be due to changes in spinal 6 and brain p and 6 receptor m e c h a n i s m s . Opioid agonists can produce analgesic e f f e c t s when administered both s p i n a l l y and s u p r a s p i n a l l y ( e . g . , 1,2,3,4, 5 , 6 , 7 , 8 , 9 , 1 0 ) . However, studies i n d i c a t e the d i f f e r e n t nature of the o p i o i d receptors t h a t mediate analgesic e f f e c t s at the spinal and supraspina] l e v e l ( e . g . , 2,3,11). For example, supraspina] analgesia f o l l o w i n g the ICV a d m i n i s t r a t i o n of morphine or the p s e l e c t i v e agonist DAGO is blocked by pre-treatment with the longl a s t i n g p receptor antagonist naloxonazine; while spinal analgesia f o l l o w i n g DAGO and morphine is not a l t e r e d (12). Furthermore, using an acute cross-tolerance paradigm, morphine pre-treatment produces cross-tolerance to the analgesic e f f e c t of ICY DAGO and IT DPDPE (6 s e l e c t i v e a g o n i s t ) ; however, cross* Supported in part by National I n s t i t u t e on Drug Abuse Grant DA04185. Address r e p r i n t requests to BCY. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
344
tolerance to
Spinal and Suprasplnal Tolerance
Vol. 46, No. 5, 1990
ICV DPDPE was not observed ( 3 ) .
In an e f f o r t t o determine changes in o p i o i d r e c e p t o r types, we have examined the e x t e n t o f a n a l g e s i c t o l e r a n c e and crosst o l e r a n c e t o morphine and ~ and 6 s e l e c t i v e p e p t i d e s f o l l o w i n g c h r o n i c (72hr) morphine t r e a t m e n t in mice. The development of both s p i n a l and s u p r a s p i n a l t o l e r a n c e has been s t u d i e d in f u l l dose-response experiments.
Procedure. Male, Swiss-Webster mice (22-249, Taconic Farms, Germantown, NY) were employed in a l l experiments. At l e a s t 24hr f o l l o w i n g a r r i v a l , mice were a n e s t h e t i z e d and implanted w i t h a s i n g l e 75mg morphine or placebo p e l l e t at the nape o f the neck. The p e l l e t s were not removed a f t e r i m p l a n t a t i o n . Seventy-two hrs f o l l o w i n g i m p l a n t a t i o n , mice were weighed and a b a s e l i n e n o c i c e p t i v e response ( t a i l f l i c k ) was determined. The t a i l f l i c k apparatus was adjusted so t h a t a focused beam o f l i g h t a p p l i e d t o the d o r s a l surface o f the t a i ] produced b a s e l i n e t a i l f l i c k s t y p i c a l l y between 2-3sec. Animals t h a t did not respond w i t h i n 5sec d u r i n g b a s e l i n e t e s t i n g were discarded from the experiment. F o l l o w i n g b a s e l i n e t e s t i n g , mice were i n j e c t e d sc; or b r i e f l y ( a p p r o x i m a t e l y 2min) a n e s t h e t i z e d and then i n j e c t e d in the s p i n a l lumbar i n t r a t h e c a l ( I T ) space ( I p l ) or in the l a t e r a l v e n t r i c l e (ICV) ( 5 ~ I ) . IT i n j e c t i o n s were made by the method of Hylden and Wilcox (137. ICV drug a d m i n i s t r a t i o n was by a m o d i f i c a t i o n o f a p r e v i o u s l y described method ( 9 ) . All pellet i m p l a n t a t i o n s , IT and ICY drug i n j e c t i o n s were made w h i l e mice were l i g h t l y anesthetized with halothane ( 9 6 : 4 0 x y g e n : H a l o t h a n e ) . Mice were t e s t e d f o r a n a l g e s i a ( t a i l f l i c k ) 30min f o l l o w i n g morphine a d m i n i s t r a t i o n and 15min f o l l o w i n g peptides (see below). I f an animal did not f l i c k by 10sec, the t e s t was t e r m i n a t e d and the mouse was d e f i n e d as a n a l g e s i c . A l l t e s t i n g was conducted by someone who was not informed as t o the dose i n j e c t e d or the pret r e a t m e n t o f the animals. Drugs. Morphine p e l l e t s (T5mg morphine base) and placebo p e l l e t s were o b t a i n e d from the Research Technology Branch of the N a t i o n a l I n s t i t u t e on Drug A b u s e . E a c h p e l l e t was wrapped ir~ nylon mesh p r i o r t o i m p l a n t a t i o n . Morphine s u l f a t e was o b t a i n e d from Penick L a b o r a t o r i e s (Newark, NJ) and was d i s s o l v e d in 0.9% s a l i n e f o r sc, IT and ICV a d m i n i s t r a t i o n . A l l doses are expressed as the base. DPDPE and DAGO were purchased from Penninsula L a b o r a t o r i e s and were d i s s o l v e d in 0.9% s a l i n e .
Data Analysis. Q u a n t a l d o s e - r e s p o n s e d a t a were a n a l y z e d u s i n g a c o m p u t e r i z e d (BLISS 21) P r o b i t A n a l y s i s ( 1 4 ) p r o g r a m , w h i c h e s t i m a t e s EDs0s, r e l a t i v e p o t e n c i e s and 95% c o n f i d e n c e limits. S t a t i s t i c a l s i g n i f i c a n c e was e v a l u a t e d using P r o b i t Analysis r e s u l t s . Dose-response data are p l o t t e d on l o g - p r o b i t axes w i t h the f i t t e d r e g r e s s i o n l i n e . Since doses t h a t produce 0~ or 100% response cannot be p l o t t e d on p r o b i t c o o r d i n a t e s , the response estimated by P r o b i t a n a l y s i s was p l o t t e d . Estimated data p o i n t s are i n d i c a t e d in the f i g u r e s by an a s t e r i s k and the a c t u a l response is d i s c l o s e d in the f i g u r e legend.
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Spinal and Suprasplnal Tolerance
345
Results
In placebo-treated mice, analgesic potency f o l l o w i n g spinal a d m i n i s t r a t i o n was equal to or greater than potency f o l l o w i n g supraspinal a d m i n i s t r a t i o n (Table I ) . Chronic morphine treatment produced tolerance to the analgesic e f f e c t of morphine (Table I ; Figure I ) , regardless of the route of a d m i n i s t r a t i o n . Chronic morphine also produced cross-tolerance to DPDPE administered IT and ICY (Table I ; Figure 2). However, morphine treatment produced s i t e - s e l e c t i v e cross-tolerance to DAGO. The analgesic dose-response function f o r ICV DAGO was s i g n i f i c a n t l y s h i f t e d to the r i g h t by more than 2 . 5 - f o l d , while there was no s i g n i f i c a n t decrease in the potency of IT DAGO (Table I , Figure 3). Discussion
C h r o n i c m o r p h i n e treatment produced t o l e r a n c e to m o r p h i n e a d m i n i s t e r e d by the subcutaneous, IT and ICV route. The d e v e l o p m e n t of t o l e r a n c e to m o r p h i n e following chronic m o r p h i n e
Subcutaneous
(3
°~
o~ 90, o c-
70
< c-
50 30
O I--
10
OMorphine
Q_
1'o
20
Morphine ( m g / k g )
Intrathecal
0 g}
ICV
.m
•Morphine
c~ 90
-• 70 < qJ
OMorphine
I
5O
50
30
3O I
o 10 L
Q~
Q-
90 ¸
0.;
10÷
OPlacebo
o15 llo 21o
i .o
Morphine (p.g/mouse)
0.2
OPlacebo
o15
1.'0
2;o
5.,
Morphine (/~g/mouse)
FIG. 1 The e f f e c t of 72hr morphine treatment on s u b c u t a n e o u s l y (1.5-15.0 mg/kg, N=5-10/dose/group), IT ( 0 . 2 5 - 1 0 . 0 p g / m o u s e , N=514/dose/group) and ICY ( 0 . 5 - 4 . 0 pg/mouse, N=5-11/dose/group) administered morphine-induced analgesia. For data points with asterisks, actual r e s p o n s e was 100% ( s e e m e t h o d s ) . The IT d a t a are from Yoburn et al. (29).
346
Spinal and Supraspinal Tolerance
Vol. 46, No. 5, 1990
Intrathecal
0
ICV
°~
o~ go.
OPlacebo
~
*
o 70. (-
< 50. r- 30"
°
a) o
t..
10.
•Morphine
Q)
n
0.2
90-
705030"
OPlacebo o o
10"
0'.s 1:0 2:0 d.0 10.0 DPDPE (/~g//mouse)
OMorphine 20 DPDPE (/~g//mouse)
FIG. 2 The e f f e c t of 72hr morphine treatment on IT ( 0 . 5 - 4 . 0 pg/mouse, N=6-9/dose/group) and ICV ( 2 . 0 - 1 0 . 0 p g / m o u s e , N=5-12/dose/group) administered DPDPE-induced analgesia. F o r IT and ICV d a t a p o i n t s with asterisks, a c t u a l r e s p o n s e was 100~ and 0 ~ , r e s p e c t i v e l y (see methods).
Intrathecal
0
90
OPlacebo
90.
o 70 t-
o
Q-
10, 1.0
OPlacebo
70. 50. 30-
< 50. t- 30' t..
ICV
IMorphine
21o
s'.0 i~.0 26.0
DAGO (~g/mouse)
10"
1'o
2o
so
DAGO ( ~ g / / m o u s e )
FIG. 3 The e f f e c t of 72hr morphine treatment on IT ( 2 - 2 0 n g / m o u s e , N= 529/dose/group) and ICV ( 5 - 4 0 n g / m o u s e , N=5-8/dose/group) a d m i n i s t e r e d DAGO-induced a n a l g e s i a . Data are p l o t t e d on l o g p r o b i t axes. For ICV d a t a p o i n t s w i t h a s t e r i s k s , t h e a c t u a l r e s p o n s e was 0% (see m e t h o d s ) .
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Spinal and Supraspinal Tolerance
347
was a n t i c i p a t e d , a l t h o u g h o t h e r i n v e s t i g a t o r s have r e p o r t e d t h a t such t r e a t m e n t in the mouse produces t o l e r a n c e o n l y t o s y s t e m i c a l l y a d m i n i s t e r e d morphine and not t o s p i n a l l y and s u p r a s p i n a l l y a d m i n i s t e r e d morphine when the morphine p e l l e t is l e f t i n place d u r i n g t e s t i n g ( 1 5 , 1 6 , 1 7 ) . However, our r e s u l t s i n the mouse are c o n s i s t e n t w i t h those of Porreca et a l . (3) and Vaught and B a r r e t t (18) using an acute t o l e r a n c e p r o t o c o l , as w e l l as demonstrations of t o l e r a n c e to s p i n a l and b r a i n i n j e c t i o n s of morphine f o l l o w i n g c h r o n i c morphine i n the r a t (e.g.,19,20,21). Mice treated chronically with morphine were cross-tolerant to the analgesic effect o f ICV DAGO and ICV DPDPE. Since these peptides are selective f o r p and 6 o p i o i d receptors, respectively, these findings s u g g e s t t h a t b r a i n p and 6 r e c e p t o r
TABLE I for Placebo Treated Mice
Ana}9esic EDse E s t i m a t e s A g o n i s t and Route of Administration Morphine SC (mg/k9)
and 7 2 h r Morphine
EDs0 Placebo
Morphine
2.4 (2.0-2.8)
8.6* (6.5-10.6)
EDso Ratio+ 3.6
Morphine (pg/mouse) ~T#
ICY DPQPE ( p g / m o u s e ) IT ICV DAGO (ng/mouse) IT ICV
0.6
4.8*
8.0
(0.3-0.9)
(3.0-7.4)
0.8 (0.5-1.3)
2.2* (1.1-4.0)
2.8
0.6 (0.1-1.3)
2.6* (1.2-12.7)
4.3
3.3 (1.0-5.6)
8.1' (5.0-36.7)
2.5
4.1 (2.0-6.2)
3.4 (1.2-5.7)
0.8
15.8 (8.7-33.0)
41.8" (25.3-155.2)
2.6
Mice were implanted with a single 75m9 m o r p h i n e or placebo pellet. S e v e n t y - t w o hrs following implantation, mice were injected subcutaneously (SC), intrathecally (IT) or intracerebroventricularly (ICY) and then tested for a n a l g e s i a (tailflick) at the time of peak e f f e c t . EDsos and 95~ confidence l i m i t s were estimated by computerized P r o b i t A n a l y s i s ( B l i s s 21). * s i g n i f i c a n t l y d i f f e r e n t from placebo ( p < . 0 5 ) . # IT morphine data are from Yoburn et a l . (29). • R a t i o is ED~0 f o r morphinet r e a t e d d i v i d e d by EDs0 f o r p l a c e b o - t r e a t e d . See Fig. I , 2 and 3 f o r N's and doses.
348
Spinal and Supraspinal Toleranace
Vol. 46, No. 5, 1990
m e c h a n i s m s u n d e r g o c h a n g e s d u r i n g c h r o n i c m o r p h i n e treatment. The c r o s s - t o l e r a n c e b e t w e e n s y s t e m i c m o r p h i n e and ICV DAGO as well as IT DPDPE is in a g r e e m e n t with the findings of P o r r e c a et al. (3). However, Porreca et al., (3) did not find crosstolerance b e t w e e n m o r p h i n e and ICV DPDPE. A possible explanation of this discrepancy is t h e use o f a c h r o n i c m o r p h i n e t r e a t m e n t in t h e p r e s e n t e x p e r i m e n t as c o m p a r e d t o an a c u t e m o r p h i n e t r e a t m e n t in the previous report. A l t h o u g h m o r p h i n e has g r e a t e s t affinity for the p receptor and has b e e n c o r r e c t l y characterized as principally a p opioid receptor ligand, i t does d i s p l a y affinity and a c t i v i t y at the 6 receptor, (e.g., 22,23,24,25). Chronic, b u t n o t a c u t e , t r e a t m e n t w i t h m o r p h i n e may be s u f f i c i e n t to p r o d u c e t o l e r a n c e at 8 r e c e p t o r s as a result of s u s t a i n e d binding of m o r p h i n e at 6 receptors. U n l i k e DPDPE, c h r o n i c m o r p h i n e p r o d u c e d s e l e c t i v e crosstolerance t o ICY DAGO. Our f i n d i n g of cross-tolerance following c h r o n i c m o r p h i n e f o r ICV DAGO a g r e e s w i t h t h e r e s u l t s of Porreca et al. (3) using the acute cross-tolerance paradigm. Similarly, V a u g h t and B a r r e t t ( 1 8 ) d e m o n s t r a t e d t h a t c h r o n i c and a c u t e morphine treatment produce cross-tolerance t o ICV a d m i n i s t r a t i o n of the p receptor preferring agonists [D-AIa 2 , ProS] e n k e p h a l i n a m i d e and m o r p h i c e p t i n . The l a c k o f c r o s s - t o l e r a n c e to IT DAGO f o l l o w i n g morphine suggests that the receptors mediating analgesia i n t h e c o r d and i n t h e b r a i n a r e q u i t e d i f f e r e n t . This suggestion i s s u p p o r t e d by p r e v i o u s s t u d i e s t h a t h a v e shown t h a t naloxonazine antagonized ICV DAGO a n a l g e s i a , b u t n o t IT DAGO analgesia (12). The n a l o x o n a z i n e results ( 1 2 ) and o u r c u r r e n t differential cross-tolerance results i n t h e c o r d and b r a i n w i t h DAGO a r e i n a g r e e m e n t w i t h r e c e n t s t u d i e s (28 B a s e d upon t h e r e s u l t s in these studies, the possible spinal and s u p r a s p i n a l mechanisms that are involved in morphine a n a l g e s i a may be s u g g e s t e d . S i n c e IT m o r p h i n e and IT DPDPE analgesia display tolerance, i t may be i n f e r r e d that IT m o r p h i n e tolerance i s b a s e d upon t o l e r a n c e a t 5 (DPDPE) r e c e p t o r s . The residual analgesic a c t i o n o f IT m o r p h i n e i n m o r p h i n e t r e a t e d m i c e may be a c c o u n t e d f o r by a c t i o n a t n o n - t o l e r a n t p (DAGO) s i t e s and diminished analgesic activity at tolerant & sites. In t h e b r a i n , h o w e v e r , ICV m o r p h i n e t o l e r a n c e may be b a s e d upon d i m i n i s h e d activity a t b o t h 6 and p r e c e p t o r s w i t h each s u p r a s p i n a ] receptor type contributing some p o r t i o n of the tolerance; o r ICV m o r p h i n e tolerance may be due t o t o l e r a n c e at either t h e DAGO o r DPDPE site independently. Alternatively, tolerance t o ICV m o r p h i n e may be r e l a t e d to a possible single high affinity receptor f o r DAGO and DPDPE; n a m e l y t h e mul s i t e p o s t u l a t e d by P a s t e r n a k and colleagues (26). On t h e b a s i s o f t h e d a t a i n t h e p r e s e n t experiment it is difficult to assign the relative contribution of DAGO and DPDPE s i t e s t o ICV m o r p h i n e t o l e r a n c e since all three a g o n i s t s p r o d u c e c o m p a r a b l e ICV t o l e r a n c e (approximately 2.5-fold reduction i n p o t e n c y , see T a b l e I ) . By a n a l o g y , t o l e r a n c e to systemically administered morphine which acts both spinally and supraspinally (6,7,27), may be due t o t o l e r a n c e at spinal 6 receptors as w e ] ] as t o l e r a n c e at supraspinal 5 and/or p sites. Regardless of the mechanism of morphine tolerance, it remains c l e a r t h a t s p i n a l p (DAGO) r e c e p t o r s are functionally distinct from supraspina] p receptors, since brain sites, but not spinal sites, display cross-tolerance.
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A£.k n_o.w ] e_dgme_n_t_s Our s i n c e r e t h a n k s t o D r . N. T. T u r n o c k f o r and comments and Mr. J o s e p h C a n d i d o and Ms. technical assistance.
he]pful suggestions J a n e t Lee f o r
B ef. er_en.c .e_s 1.
J . S . HEYMAN, J . L . VAUGHT, R.B. RAFFA and F. PORRECA. T r e n d s Pharm. S c i . , 9, 1 3 4 - 1 3 8 ( 1 9 8 8 a ) . 2. G . S . F . LING and G.W. PASTERNAK. B r a i n . R e s . , 271, 1 5 2 - 1 5 6 (1983). 3. F. PORRECA, J . S . HEYMAN, H . I . MOSBERG, J . R . OMNAAS and J . L . VAUGHT. J. P h a r m a c o l . Exp. Ther., 241, 3 9 3 - 4 0 0 ( 1 9 8 7 a ) . 4. F. PORRECA, H . I . MOSBERG, J . R . OMNAAS, T . F . BURKS and A. COWAN. J. P h a r m a c o ] . E x p . T h e r . , 240, 890-894 ( 1 9 8 7 b ) . 5. T . L . YAKSH and T . A . RUDY. S c i e n c e , 192, 1 3 5 7 - 1 3 5 8 ( 1 9 7 6 ) . 6. T . L . YAKSH and T . A . RUDY. J. P h a r m a c o l . E x p . T h e r . , 202, 411412 ( 1 9 7 7 ) . 7. J C. YEUNG and T . A . RUDY. J. P h a r m a c o ] . E x p . T h e r . , 215, 6 2 6 632 ( 1 9 8 0 a ) . 8. J C. YEUNG and T . A . RUDY. J. P h a r m a c o ] . Exp. T h e r . , 215, 633642 ( 1 9 8 0 b ) . 9. B C. YOBURN, M.C. LUKE, G.W. PASTERNAK, and C. E. INTURRISI, L i f e S c i . 43, 1319-1324 ( 1 9 8 8 ) . 10 B C. YOBURN, D. PAUL, S. AZIMUDDIN, K. LUTFY and V. SIERRA, Brain Res., 485, 176-178 (1989). 11 F PORRECA, H . I . MOSBERG, R. HURST, V , J . HRUBY and T . F . BURKS. J. P h a r m a c o l . Exp. T h e r . , 230, 3 4 1 - 3 4 7 ( 1 9 8 4 ) . 12 J S HEYMAN, C . L . WILLIAMS, T . F . BURKS, H . I . MOSBERG and F. PORRECA. J. P h a r m a c o l . E x p . T h e r . , 245, 2 3 8 - 2 4 3 ( 1 9 8 8 b ) . 13 J K L. HYLDEN and G . L . WILCOX, C u r . J. P h a r m a c o l . , 6 7 , 3 1 3 316 ( 1 9 8 0 ) . 14 D J FINNEY. PEobit Anal~sjsj_ ~E~_ ~_, Cambridge University Press, London (1971). 15 D G LANGE, S . C . ROERIG, J . M . FUJIMOXO and R . I . H . WANG. Science, 208, 72-74 (1980). 16 S C ROCRIG and J.M. FUJIMOTO. J. P h a r m a c o l . Exp. T h e r . , 247, 603-608 (1988). 17 S C ROERIG, S . N . O ' B R I E N , J . N . FUJINOTO and G . L . WILCOX. Brain. R e s . , 308, 3 6 0 - 3 6 3 ( 1 9 8 4 ) . 1B J L VAUGH]. and R.W. BARRETT. In: P~j~ ~d H~d~£h~. ~.9_]~ Neurotransmitters and P a i n C o n t r o l , H. A k i l and J.W. Lewis (Eds.), p 1 7 8 - 1 9 3 , S. K r a g e r : B a s e l , 1987. 19. Y . F . JACQUET and A. LAJTHA. B r a i n R e s . , 103, 5 0 1 - 5 1 3 ( 1 9 7 6 ) . 20. T . L . YAKSH, R . L . KOHL and ~ . A . RUDY. E u r . J. P h a r m a c o ] , , 4 2 , 275-285 (1977). 21. C. SCHMAUSS. E u r . J. P h a r m a c o ] . , 137, 1 9 7 - 2 0 5 ( 1 9 8 7 ) . 22. K - J . CHANG and P. CUATRECASAS. J. B i o ] . Chem. 2 5 4 , 2 6 1 0 - 2 6 1 8 (1979). 23. P. L. WOOD, S.C. CHARLESON, D. LANE and R . L . HUDGIN. Neuropharmacol., 20, 1 2 1 5 - t 2 2 0 ( 1 9 8 1 ) . 24. B . L . WOLOZIN and G.W. PASTERNAK. P r o c . N a t ] . A c a d . S c i . , 78, 6181-6185 (1981). 25. A . E . ]AKEMORI and P . S . PORTOGHESE. J. P h a r m a c o l . Exp. Ther., 243, 91-94 ( 1 9 8 7 ) . 26. G.W. PAS]ERNAK and P.L.. WOOD. L i f e S c i . , 38, 1889- t 8 9 8 (1986). 27. S. IRWIN, R.W. HOUDE, D.R. BENNET;, L . C . HENDERSHOT and M.H.
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SEEVERS. J. P h a r m a c o l . E x p . T h e r . , 101, 1 3 2 - 1 4 3 ( 1 9 5 1 ) . D. PAUL, R . J . BODNAR, M.A. GISTRAK and G.W. PASTERNAK. J. P h a r m a c o l . , (in press). B. YOBURN, K. LUTFY, V. SIERRA and F . C . TORTELLA. Eur. Pharmacol., (in press).
Eur. J.
DIFFERENTIATION
OF S P I N A L AND S U P R A S P I N A L MORPHINE TOLERANCE*
Pergamon Press
OPIOID
RECEPTORS
BY
Byron C. Yoburn, Kabir L u t f y , Saira Azimuddin and Victor Sierra Department of Pharmaceutical Sciences College of Pharmacy and A11ied Health Professions St. John's U n i v e r s i t y Queens, NY 11439 (Received in final form December 4, 1989) Summary
Mice t r e a t e d f o r 72hrs with morphine (subcutaneously implanted p e l l e t s ) were tested with a v a r i e t y of opioid receptor agonists to examine the development of tolerance and cross-tolerance to t h e i r analgesic action. The development of spinal and supraspinal tolerance f o l l o w i n g morphine treatment was evaluated by administering compounds s y s t e m i c a l l y ( s c ) , i n t r a t h e c a l l y ( I T ) and i n t r a c e r e b r o v e n t r i c u l a r l y (ICY). Following morphine treatment, tolerance to morphine analgesia was observed f o l l o w i n g IT, ICV and sc a d m i n i s t r a t i o n . Chronic morphine treatment also produced cross-tolerance to the analgesic e f f e c t s of the s e l e c t i v e 6 opioid receptor agonist [D-Pen2 , D-PenS]enkephalin (DPDPE) f o l l o w i n g IT and ICV a d m i n i s t r a t i o n . However, morphine treatment s e l e c t i v e l y produced cross-tolerance to ICV [DAla 2 , NMePhe4, Gly-olS]enkephalin (DAGO) (p receptor agonist) analgesia, without a l t e r i n g IT DAGO analgesia. These r e s u l t s suggest t h a t brain and spinal cord receptors mediating the e f f e c t s of DAGO d i f f e r in terms of the development of crosstolerance to morphine; and suggest t h a t tolerance to systemic morphine may be due to changes in spinal 6 and brain p and 6 receptor m e c h a n i s m s . Opioid agonists can produce analgesic e f f e c t s when administered both s p i n a l l y and s u p r a s p i n a l l y ( e . g . , 1,2,3,4, 5 , 6 , 7 , 8 , 9 , 1 0 ) . However, studies i n d i c a t e the d i f f e r e n t nature of the o p i o i d receptors t h a t mediate analgesic e f f e c t s at the spinal and supraspina] l e v e l ( e . g . , 2,3,11). For example, supraspina] analgesia f o l l o w i n g the ICV a d m i n i s t r a t i o n of morphine or the p s e l e c t i v e agonist DAGO is blocked by pre-treatment with the longl a s t i n g p receptor antagonist naloxonazine; while spinal analgesia f o l l o w i n g DAGO and morphine is not a l t e r e d (12). Furthermore, using an acute cross-tolerance paradigm, morphine pre-treatment produces cross-tolerance to the analgesic e f f e c t of ICY DAGO and IT DPDPE (6 s e l e c t i v e a g o n i s t ) ; however, cross* Supported in part by National I n s t i t u t e on Drug Abuse Grant DA04185. Address r e p r i n t requests to BCY. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
344
tolerance to
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Vol. 46, No. 5, 1990
ICV DPDPE was not observed ( 3 ) .
In an e f f o r t t o determine changes in o p i o i d r e c e p t o r types, we have examined the e x t e n t o f a n a l g e s i c t o l e r a n c e and crosst o l e r a n c e t o morphine and ~ and 6 s e l e c t i v e p e p t i d e s f o l l o w i n g c h r o n i c (72hr) morphine t r e a t m e n t in mice. The development of both s p i n a l and s u p r a s p i n a l t o l e r a n c e has been s t u d i e d in f u l l dose-response experiments.
Procedure. Male, Swiss-Webster mice (22-249, Taconic Farms, Germantown, NY) were employed in a l l experiments. At l e a s t 24hr f o l l o w i n g a r r i v a l , mice were a n e s t h e t i z e d and implanted w i t h a s i n g l e 75mg morphine or placebo p e l l e t at the nape o f the neck. The p e l l e t s were not removed a f t e r i m p l a n t a t i o n . Seventy-two hrs f o l l o w i n g i m p l a n t a t i o n , mice were weighed and a b a s e l i n e n o c i c e p t i v e response ( t a i l f l i c k ) was determined. The t a i l f l i c k apparatus was adjusted so t h a t a focused beam o f l i g h t a p p l i e d t o the d o r s a l surface o f the t a i ] produced b a s e l i n e t a i l f l i c k s t y p i c a l l y between 2-3sec. Animals t h a t did not respond w i t h i n 5sec d u r i n g b a s e l i n e t e s t i n g were discarded from the experiment. F o l l o w i n g b a s e l i n e t e s t i n g , mice were i n j e c t e d sc; or b r i e f l y ( a p p r o x i m a t e l y 2min) a n e s t h e t i z e d and then i n j e c t e d in the s p i n a l lumbar i n t r a t h e c a l ( I T ) space ( I p l ) or in the l a t e r a l v e n t r i c l e (ICV) ( 5 ~ I ) . IT i n j e c t i o n s were made by the method of Hylden and Wilcox (137. ICV drug a d m i n i s t r a t i o n was by a m o d i f i c a t i o n o f a p r e v i o u s l y described method ( 9 ) . All pellet i m p l a n t a t i o n s , IT and ICY drug i n j e c t i o n s were made w h i l e mice were l i g h t l y anesthetized with halothane ( 9 6 : 4 0 x y g e n : H a l o t h a n e ) . Mice were t e s t e d f o r a n a l g e s i a ( t a i l f l i c k ) 30min f o l l o w i n g morphine a d m i n i s t r a t i o n and 15min f o l l o w i n g peptides (see below). I f an animal did not f l i c k by 10sec, the t e s t was t e r m i n a t e d and the mouse was d e f i n e d as a n a l g e s i c . A l l t e s t i n g was conducted by someone who was not informed as t o the dose i n j e c t e d or the pret r e a t m e n t o f the animals. Drugs. Morphine p e l l e t s (T5mg morphine base) and placebo p e l l e t s were o b t a i n e d from the Research Technology Branch of the N a t i o n a l I n s t i t u t e on Drug A b u s e . E a c h p e l l e t was wrapped ir~ nylon mesh p r i o r t o i m p l a n t a t i o n . Morphine s u l f a t e was o b t a i n e d from Penick L a b o r a t o r i e s (Newark, NJ) and was d i s s o l v e d in 0.9% s a l i n e f o r sc, IT and ICV a d m i n i s t r a t i o n . A l l doses are expressed as the base. DPDPE and DAGO were purchased from Penninsula L a b o r a t o r i e s and were d i s s o l v e d in 0.9% s a l i n e .
Data Analysis. Q u a n t a l d o s e - r e s p o n s e d a t a were a n a l y z e d u s i n g a c o m p u t e r i z e d (BLISS 21) P r o b i t A n a l y s i s ( 1 4 ) p r o g r a m , w h i c h e s t i m a t e s EDs0s, r e l a t i v e p o t e n c i e s and 95% c o n f i d e n c e limits. S t a t i s t i c a l s i g n i f i c a n c e was e v a l u a t e d using P r o b i t Analysis r e s u l t s . Dose-response data are p l o t t e d on l o g - p r o b i t axes w i t h the f i t t e d r e g r e s s i o n l i n e . Since doses t h a t produce 0~ or 100% response cannot be p l o t t e d on p r o b i t c o o r d i n a t e s , the response estimated by P r o b i t a n a l y s i s was p l o t t e d . Estimated data p o i n t s are i n d i c a t e d in the f i g u r e s by an a s t e r i s k and the a c t u a l response is d i s c l o s e d in the f i g u r e legend.
Vol. 46, No. 5, 1990
Spinal and Suprasplnal Tolerance
345
Results
In placebo-treated mice, analgesic potency f o l l o w i n g spinal a d m i n i s t r a t i o n was equal to or greater than potency f o l l o w i n g supraspinal a d m i n i s t r a t i o n (Table I ) . Chronic morphine treatment produced tolerance to the analgesic e f f e c t of morphine (Table I ; Figure I ) , regardless of the route of a d m i n i s t r a t i o n . Chronic morphine also produced cross-tolerance to DPDPE administered IT and ICY (Table I ; Figure 2). However, morphine treatment produced s i t e - s e l e c t i v e cross-tolerance to DAGO. The analgesic dose-response function f o r ICV DAGO was s i g n i f i c a n t l y s h i f t e d to the r i g h t by more than 2 . 5 - f o l d , while there was no s i g n i f i c a n t decrease in the potency of IT DAGO (Table I , Figure 3). Discussion
C h r o n i c m o r p h i n e treatment produced t o l e r a n c e to m o r p h i n e a d m i n i s t e r e d by the subcutaneous, IT and ICV route. The d e v e l o p m e n t of t o l e r a n c e to m o r p h i n e following chronic m o r p h i n e
Subcutaneous
(3
°~
o~ 90, o c-
70
< c-
50 30
O I--
10
OMorphine
Q_
1'o
20
Morphine ( m g / k g )
Intrathecal
0 g}
ICV
.m
•Morphine
c~ 90
-• 70 < qJ
OMorphine
I
5O
50
30
3O I
o 10 L
Q~
Q-
90 ¸
0.;
10÷
OPlacebo
o15 llo 21o
i .o
Morphine (p.g/mouse)
0.2
OPlacebo
o15
1.'0
2;o
5.,
Morphine (/~g/mouse)
FIG. 1 The e f f e c t of 72hr morphine treatment on s u b c u t a n e o u s l y (1.5-15.0 mg/kg, N=5-10/dose/group), IT ( 0 . 2 5 - 1 0 . 0 p g / m o u s e , N=514/dose/group) and ICY ( 0 . 5 - 4 . 0 pg/mouse, N=5-11/dose/group) administered morphine-induced analgesia. For data points with asterisks, actual r e s p o n s e was 100% ( s e e m e t h o d s ) . The IT d a t a are from Yoburn et al. (29).
346
Spinal and Supraspinal Tolerance
Vol. 46, No. 5, 1990
Intrathecal
0
ICV
°~
o~ go.
OPlacebo
~
*
o 70. (-
< 50. r- 30"
°
a) o
t..
10.
•Morphine
Q)
n
0.2
90-
705030"
OPlacebo o o
10"
0'.s 1:0 2:0 d.0 10.0 DPDPE (/~g//mouse)
OMorphine 20 DPDPE (/~g//mouse)
FIG. 2 The e f f e c t of 72hr morphine treatment on IT ( 0 . 5 - 4 . 0 pg/mouse, N=6-9/dose/group) and ICV ( 2 . 0 - 1 0 . 0 p g / m o u s e , N=5-12/dose/group) administered DPDPE-induced analgesia. F o r IT and ICV d a t a p o i n t s with asterisks, a c t u a l r e s p o n s e was 100~ and 0 ~ , r e s p e c t i v e l y (see methods).
Intrathecal
0
90
OPlacebo
90.
o 70 t-
o
Q-
10, 1.0
OPlacebo
70. 50. 30-
< 50. t- 30' t..
ICV
IMorphine
21o
s'.0 i~.0 26.0
DAGO (~g/mouse)
10"
1'o
2o
so
DAGO ( ~ g / / m o u s e )
FIG. 3 The e f f e c t of 72hr morphine treatment on IT ( 2 - 2 0 n g / m o u s e , N= 529/dose/group) and ICV ( 5 - 4 0 n g / m o u s e , N=5-8/dose/group) a d m i n i s t e r e d DAGO-induced a n a l g e s i a . Data are p l o t t e d on l o g p r o b i t axes. For ICV d a t a p o i n t s w i t h a s t e r i s k s , t h e a c t u a l r e s p o n s e was 0% (see m e t h o d s ) .
Vol. 46, No. 5, 1990
Spinal and Supraspinal Tolerance
347
was a n t i c i p a t e d , a l t h o u g h o t h e r i n v e s t i g a t o r s have r e p o r t e d t h a t such t r e a t m e n t in the mouse produces t o l e r a n c e o n l y t o s y s t e m i c a l l y a d m i n i s t e r e d morphine and not t o s p i n a l l y and s u p r a s p i n a l l y a d m i n i s t e r e d morphine when the morphine p e l l e t is l e f t i n place d u r i n g t e s t i n g ( 1 5 , 1 6 , 1 7 ) . However, our r e s u l t s i n the mouse are c o n s i s t e n t w i t h those of Porreca et a l . (3) and Vaught and B a r r e t t (18) using an acute t o l e r a n c e p r o t o c o l , as w e l l as demonstrations of t o l e r a n c e to s p i n a l and b r a i n i n j e c t i o n s of morphine f o l l o w i n g c h r o n i c morphine i n the r a t (e.g.,19,20,21). Mice treated chronically with morphine were cross-tolerant to the analgesic effect o f ICV DAGO and ICV DPDPE. Since these peptides are selective f o r p and 6 o p i o i d receptors, respectively, these findings s u g g e s t t h a t b r a i n p and 6 r e c e p t o r
TABLE I for Placebo Treated Mice
Ana}9esic EDse E s t i m a t e s A g o n i s t and Route of Administration Morphine SC (mg/k9)
and 7 2 h r Morphine
EDs0 Placebo
Morphine
2.4 (2.0-2.8)
8.6* (6.5-10.6)
EDso Ratio+ 3.6
Morphine (pg/mouse) ~T#
ICY DPQPE ( p g / m o u s e ) IT ICV DAGO (ng/mouse) IT ICV
0.6
4.8*
8.0
(0.3-0.9)
(3.0-7.4)
0.8 (0.5-1.3)
2.2* (1.1-4.0)
2.8
0.6 (0.1-1.3)
2.6* (1.2-12.7)
4.3
3.3 (1.0-5.6)
8.1' (5.0-36.7)
2.5
4.1 (2.0-6.2)
3.4 (1.2-5.7)
0.8
15.8 (8.7-33.0)
41.8" (25.3-155.2)
2.6
Mice were implanted with a single 75m9 m o r p h i n e or placebo pellet. S e v e n t y - t w o hrs following implantation, mice were injected subcutaneously (SC), intrathecally (IT) or intracerebroventricularly (ICY) and then tested for a n a l g e s i a (tailflick) at the time of peak e f f e c t . EDsos and 95~ confidence l i m i t s were estimated by computerized P r o b i t A n a l y s i s ( B l i s s 21). * s i g n i f i c a n t l y d i f f e r e n t from placebo ( p < . 0 5 ) . # IT morphine data are from Yoburn et a l . (29). • R a t i o is ED~0 f o r morphinet r e a t e d d i v i d e d by EDs0 f o r p l a c e b o - t r e a t e d . See Fig. I , 2 and 3 f o r N's and doses.
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m e c h a n i s m s u n d e r g o c h a n g e s d u r i n g c h r o n i c m o r p h i n e treatment. The c r o s s - t o l e r a n c e b e t w e e n s y s t e m i c m o r p h i n e and ICV DAGO as well as IT DPDPE is in a g r e e m e n t with the findings of P o r r e c a et al. (3). However, Porreca et al., (3) did not find crosstolerance b e t w e e n m o r p h i n e and ICV DPDPE. A possible explanation of this discrepancy is t h e use o f a c h r o n i c m o r p h i n e t r e a t m e n t in t h e p r e s e n t e x p e r i m e n t as c o m p a r e d t o an a c u t e m o r p h i n e t r e a t m e n t in the previous report. A l t h o u g h m o r p h i n e has g r e a t e s t affinity for the p receptor and has b e e n c o r r e c t l y characterized as principally a p opioid receptor ligand, i t does d i s p l a y affinity and a c t i v i t y at the 6 receptor, (e.g., 22,23,24,25). Chronic, b u t n o t a c u t e , t r e a t m e n t w i t h m o r p h i n e may be s u f f i c i e n t to p r o d u c e t o l e r a n c e at 8 r e c e p t o r s as a result of s u s t a i n e d binding of m o r p h i n e at 6 receptors. U n l i k e DPDPE, c h r o n i c m o r p h i n e p r o d u c e d s e l e c t i v e crosstolerance t o ICY DAGO. Our f i n d i n g of cross-tolerance following c h r o n i c m o r p h i n e f o r ICV DAGO a g r e e s w i t h t h e r e s u l t s of Porreca et al. (3) using the acute cross-tolerance paradigm. Similarly, V a u g h t and B a r r e t t ( 1 8 ) d e m o n s t r a t e d t h a t c h r o n i c and a c u t e morphine treatment produce cross-tolerance t o ICV a d m i n i s t r a t i o n of the p receptor preferring agonists [D-AIa 2 , ProS] e n k e p h a l i n a m i d e and m o r p h i c e p t i n . The l a c k o f c r o s s - t o l e r a n c e to IT DAGO f o l l o w i n g morphine suggests that the receptors mediating analgesia i n t h e c o r d and i n t h e b r a i n a r e q u i t e d i f f e r e n t . This suggestion i s s u p p o r t e d by p r e v i o u s s t u d i e s t h a t h a v e shown t h a t naloxonazine antagonized ICV DAGO a n a l g e s i a , b u t n o t IT DAGO analgesia (12). The n a l o x o n a z i n e results ( 1 2 ) and o u r c u r r e n t differential cross-tolerance results i n t h e c o r d and b r a i n w i t h DAGO a r e i n a g r e e m e n t w i t h r e c e n t s t u d i e s (28 B a s e d upon t h e r e s u l t s in these studies, the possible spinal and s u p r a s p i n a l mechanisms that are involved in morphine a n a l g e s i a may be s u g g e s t e d . S i n c e IT m o r p h i n e and IT DPDPE analgesia display tolerance, i t may be i n f e r r e d that IT m o r p h i n e tolerance i s b a s e d upon t o l e r a n c e a t 5 (DPDPE) r e c e p t o r s . The residual analgesic a c t i o n o f IT m o r p h i n e i n m o r p h i n e t r e a t e d m i c e may be a c c o u n t e d f o r by a c t i o n a t n o n - t o l e r a n t p (DAGO) s i t e s and diminished analgesic activity at tolerant & sites. In t h e b r a i n , h o w e v e r , ICV m o r p h i n e t o l e r a n c e may be b a s e d upon d i m i n i s h e d activity a t b o t h 6 and p r e c e p t o r s w i t h each s u p r a s p i n a ] receptor type contributing some p o r t i o n of the tolerance; o r ICV m o r p h i n e tolerance may be due t o t o l e r a n c e at either t h e DAGO o r DPDPE site independently. Alternatively, tolerance t o ICV m o r p h i n e may be r e l a t e d to a possible single high affinity receptor f o r DAGO and DPDPE; n a m e l y t h e mul s i t e p o s t u l a t e d by P a s t e r n a k and colleagues (26). On t h e b a s i s o f t h e d a t a i n t h e p r e s e n t experiment it is difficult to assign the relative contribution of DAGO and DPDPE s i t e s t o ICV m o r p h i n e t o l e r a n c e since all three a g o n i s t s p r o d u c e c o m p a r a b l e ICV t o l e r a n c e (approximately 2.5-fold reduction i n p o t e n c y , see T a b l e I ) . By a n a l o g y , t o l e r a n c e to systemically administered morphine which acts both spinally and supraspinally (6,7,27), may be due t o t o l e r a n c e at spinal 6 receptors as w e ] ] as t o l e r a n c e at supraspinal 5 and/or p sites. Regardless of the mechanism of morphine tolerance, it remains c l e a r t h a t s p i n a l p (DAGO) r e c e p t o r s are functionally distinct from supraspina] p receptors, since brain sites, but not spinal sites, display cross-tolerance.
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A£.k n_o.w ] e_dgme_n_t_s Our s i n c e r e t h a n k s t o D r . N. T. T u r n o c k f o r and comments and Mr. J o s e p h C a n d i d o and Ms. technical assistance.
he]pful suggestions J a n e t Lee f o r
B ef. er_en.c .e_s 1.
J . S . HEYMAN, J . L . VAUGHT, R.B. RAFFA and F. PORRECA. T r e n d s Pharm. S c i . , 9, 1 3 4 - 1 3 8 ( 1 9 8 8 a ) . 2. G . S . F . LING and G.W. PASTERNAK. B r a i n . R e s . , 271, 1 5 2 - 1 5 6 (1983). 3. F. PORRECA, J . S . HEYMAN, H . I . MOSBERG, J . R . OMNAAS and J . L . VAUGHT. J. P h a r m a c o l . Exp. Ther., 241, 3 9 3 - 4 0 0 ( 1 9 8 7 a ) . 4. F. PORRECA, H . I . MOSBERG, J . R . OMNAAS, T . F . BURKS and A. COWAN. J. P h a r m a c o ] . E x p . T h e r . , 240, 890-894 ( 1 9 8 7 b ) . 5. T . L . YAKSH and T . A . RUDY. S c i e n c e , 192, 1 3 5 7 - 1 3 5 8 ( 1 9 7 6 ) . 6. T . L . YAKSH and T . A . RUDY. J. P h a r m a c o l . E x p . T h e r . , 202, 411412 ( 1 9 7 7 ) . 7. J C. YEUNG and T . A . RUDY. J. P h a r m a c o ] . E x p . T h e r . , 215, 6 2 6 632 ( 1 9 8 0 a ) . 8. J C. YEUNG and T . A . RUDY. J. P h a r m a c o ] . Exp. T h e r . , 215, 633642 ( 1 9 8 0 b ) . 9. B C. YOBURN, M.C. LUKE, G.W. PASTERNAK, and C. E. INTURRISI, L i f e S c i . 43, 1319-1324 ( 1 9 8 8 ) . 10 B C. YOBURN, D. PAUL, S. AZIMUDDIN, K. LUTFY and V. SIERRA, Brain Res., 485, 176-178 (1989). 11 F PORRECA, H . I . MOSBERG, R. HURST, V , J . HRUBY and T . F . BURKS. J. P h a r m a c o l . Exp. T h e r . , 230, 3 4 1 - 3 4 7 ( 1 9 8 4 ) . 12 J S HEYMAN, C . L . WILLIAMS, T . F . BURKS, H . I . MOSBERG and F. PORRECA. J. P h a r m a c o l . E x p . T h e r . , 245, 2 3 8 - 2 4 3 ( 1 9 8 8 b ) . 13 J K L. HYLDEN and G . L . WILCOX, C u r . J. P h a r m a c o l . , 6 7 , 3 1 3 316 ( 1 9 8 0 ) . 14 D J FINNEY. PEobit Anal~sjsj_ ~E~_ ~_, Cambridge University Press, London (1971). 15 D G LANGE, S . C . ROERIG, J . M . FUJIMOXO and R . I . H . WANG. Science, 208, 72-74 (1980). 16 S C ROCRIG and J.M. FUJIMOTO. J. P h a r m a c o l . Exp. T h e r . , 247, 603-608 (1988). 17 S C ROERIG, S . N . O ' B R I E N , J . N . FUJINOTO and G . L . WILCOX. Brain. R e s . , 308, 3 6 0 - 3 6 3 ( 1 9 8 4 ) . 1B J L VAUGH]. and R.W. BARRETT. In: P~j~ ~d H~d~£h~. ~.9_]~ Neurotransmitters and P a i n C o n t r o l , H. A k i l and J.W. Lewis (Eds.), p 1 7 8 - 1 9 3 , S. K r a g e r : B a s e l , 1987. 19. Y . F . JACQUET and A. LAJTHA. B r a i n R e s . , 103, 5 0 1 - 5 1 3 ( 1 9 7 6 ) . 20. T . L . YAKSH, R . L . KOHL and ~ . A . RUDY. E u r . J. P h a r m a c o ] , , 4 2 , 275-285 (1977). 21. C. SCHMAUSS. E u r . J. P h a r m a c o ] . , 137, 1 9 7 - 2 0 5 ( 1 9 8 7 ) . 22. K - J . CHANG and P. CUATRECASAS. J. B i o ] . Chem. 2 5 4 , 2 6 1 0 - 2 6 1 8 (1979). 23. P. L. WOOD, S.C. CHARLESON, D. LANE and R . L . HUDGIN. Neuropharmacol., 20, 1 2 1 5 - t 2 2 0 ( 1 9 8 1 ) . 24. B . L . WOLOZIN and G.W. PASTERNAK. P r o c . N a t ] . A c a d . S c i . , 78, 6181-6185 (1981). 25. A . E . ]AKEMORI and P . S . PORTOGHESE. J. P h a r m a c o l . Exp. Ther., 243, 91-94 ( 1 9 8 7 ) . 26. G.W. PAS]ERNAK and P.L.. WOOD. L i f e S c i . , 38, 1889- t 8 9 8 (1986). 27. S. IRWIN, R.W. HOUDE, D.R. BENNET;, L . C . HENDERSHOT and M.H.
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28. 29.
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SEEVERS. J. P h a r m a c o l . E x p . T h e r . , 101, 1 3 2 - 1 4 3 ( 1 9 5 1 ) . D. PAUL, R . J . BODNAR, M.A. GISTRAK and G.W. PASTERNAK. J. P h a r m a c o l . , (in press). B. YOBURN, K. LUTFY, V. SIERRA and F . C . TORTELLA. Eur. Pharmacol., (in press).
Eur. J.