- Email: [email protected]
Selective opiate tolerance in the guinea-pig ileum is not associated with selective dependence
Life Sciences, Vol. 33, Sup. I, 1983, pp. 357-360 Printed in the U.S.A.
Pergamon Press
S E L E C T I V E O P I A T E T O L E R A N C E IN THE G U I N E A - P I G ILEUM IS NOT A S S O C I A T E D WITH S E L E C T I V E D E P E N D E N C E Eva Seidl and RHdiger
Schulz
D e p a r t m e n t of N e u r o p h a r m a c o l o g y M a x - P l a n c k - I n s t i t u t fHr P s y c h i a t r i e K r a e p e l i n s t r a s s e 2, D - 8 O O O M 0 n c h e n 40 Federal Republic of Germany (Received in final form June 26, 1983) Summary Opiate d e p e n d e n c e a t t r i b u t a b l e to specific types of opiate receptors was studied in the isolated g u i n e a - p i g ileum made t o l e r a n t / d e p e n d e n t in vivo to a specific narcotic agonist. Apparently, induction of d e p e n d e n c e of b - r e c e p t o r s was a s s o c i a t e d with d e p e n d e n c e of K-receptors and vice versa. These i n v e s t i g a t i o n s involved the use of the i r r e v e r s i b l e ~ - r e c e p t o r a n t a g o n i s t B - f u n a l t r e x a mine to p r o v i d e p r e p a r a t i o n s with functional K-receptors only. The d o c u m e n t e d c r o s s - d e p e n d e n c e b e t w e e n d i f f e r e n t opiate r e c e p t o r types suggests a common system with which these opiate receptors interact. Subtypes of m u l t i p l e opiate receptors may d e v e l o p selective tolerance upon their c h r o n i c a c t i v a t i o n (I ,2). Since tolerance and d e p e n d e n c e have been p o s t u l a t e d to be r e l a t e d to an identical bioc h e m i c a l m e c h a n i s m (3,4,5), a parallel a p p e a r a n c e of both phenom e n a w o u l d be expected. Previous studies from this laboratory with t o l e r a n t / d e p e n d e n t g u i n e a - p i g ileum (GPI) c h a l l e n g e d this p r e s u m p tion. While highly tolerant opiate receptors may d i s p l a y a high degree of dependence, a high degree of d e p e n d e n c e can also be a s s o c i a t e d with a low degree of tolerance (6). These findings c a u s e d us to i n v e s t i g a t e more closely the p h e n o m e n o n of opiate dependence, e m p h a s i z i n g the r e l a t i o n s h i p to specific types of opiate receptors. To a c c o m p l i s h this aim we made use of the irreversible b - o p i a t e r e c e p t o r a n t a g o n i s t B - f u n a l t r e x a m i n e (B-FNA, 7), w h i c h provides G P I - p r e p a r a t i o n s c o n t a i n i n g a single type of functional opiate receptor. Methods Ilea of guinea pigs were r e n d e r e d t o l e r a n t / d e p e n d e n t by subc u t a n e o u s a d m i n i s t r a t i o n of an opioid (6 days) using osmotic m i n i pumps (ALZA, No 2OO1 and 2ML1). The u - a g o n i s t e m p l o y e d was fentanyl (FEN, 10 ug/h) and the K-agonist was e t h y l k e t a z o c i n e (EKC, 50 bg/h). A f t e r set-up of the longitudinal m u s c l e - m y e n t e r i c plexus p r e p a r a t i o n in v i t r o for e l e c t r i c a l s t i m u l a t i o n (8) the tolerancei n d u c i n g opiates were w a s h e d off (60 min). Thereafter, the p r e p a r ations were e x p o s e d to B-FNA (1OO nM, 60 min), w h i c h was followed by a 90 min w a s h procedure. This treatment p r o v i d e s p r e p a r a t i o n s c o n t a i n i n g K-receptors only. Subsequently, the p r e p a r a t i o n s were 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
358
Cross-dependence in the Guinea Pig Ileum
Vol. 33, Sup. I, 1983
e x p o s e d for 30 m i n e i t h e r to a ~- or to a K-agonist. A f t e r e l e c t r i c a l s t i m u l a t i o n was t e r m i n a t e d , p r e c i p i t a t i o n of w i t h d r a w a l was e v o k e d by I pM n a l o x o n e . The w i t h d r a w a l sign was q u a n t i f i e d by c a l c u l a t i n g the p e r c e n t a g e of the t e n s i o n of the c o n t r a c t u r e in r e l a t i o n to that of the e l e c t r i c a l l y e v o k e d t w i t c h e s m e a s u r e d I h a f t e r in v i t r o s e t - u p of the GPI. Results The i n t e n s i t y of w i t h d r a w a l c o n t r a c t i o n s of the GPI c h r o n i c a l l y e x p o s e d in v i v o to the ~ - a g o n i s t FEN a n d t r e a t e d - or n o t t r e a t e d - in v i t r o w i t h B - F N A are g i v e n in f i g u r e I. Panel A ref l e c t s the d a t a of p r e p a r a t i o n s e x p o s e d in v i t r o to d i f f e r e n t c o n c e n t r a t i o n s of FEN. In the a b s e n c e of B - F N A (open c o l u m n s ) , the c o n t r a c t u r e e v o k e d by n a l o x o n e a m o u n t s in all t i s s u e s to a b o u t 50 percent. In c o m p a r i s o n , in the p r e s e n c e of B - F N A (hatched columns) the r e s p o n s e was s t r o n g l y reduced, a l t h o u g h i n c r e a s i n g F E N - c o n c e n t r a t i o n s c a u s e d m o r e i n t e n s e c o n t r a c t i o n s . The fact that p r e p a r a t i o n s r e s p o n d e d to n a l o x o n e in the p r e s e n c e of FEN m o s t l i k e l y r e l a t e s to an i n c o m p l e t e i n a c t i v a t i o n of p - r e c e p t o r s u p o n B - F N A
100 Exposure to FEN
Exposure to EKC
A
t-
B
3 0
J_
o i-
Zo s0 U
/_
J..
'13
JE
10
50
FEN (nM)
1
5
50
EKC (nM)
FIG. I W i t h d r a w a l c o n t r a c t i o n s p r e c i p i t a t e d by n a l o x o n e in p r e p a r a t i o n s r e n d e r e d d e p e n d e n t to f e n t a n y l (FEN) in vivo. O p e n c o l u m n s are d a t a f r o m p r e p a r a t i o n s n o t e x p o s e d to BF N A in vitro; h a t c h e d c o l u m n s i n d i c a t e d a t a f r o m p r e p a r a t i o n s e x p o s e d in v i t r o to B-FNA. The o r d i n a t e r e f l e c t s the t e n s i o n of the w i t h d r a w a l c o n t r a c t u r e e x p r e s s e d in p e r c e n t a g e of e l e c t r i c a l l y - e v o k e d t w i t c h tension. The abs c i s s a r e f l e c t s the c o n c e n t r a t i o n s of F E N and EKC, r e s p e c t i v e l y , e m p l o y e d to e x p o s e the p r e p a r a t i o n s in vitro. D a t a are m e a n s + S.E.M. of at l e a s t 8 p r e p a r a t i o n s .
Vol. 33, Sup. I, 1983
Cross-dependence in the Guinea Pig Ileum
359
t r e a t m e n t (7). U n d e r a n a l o g u o u s e x p e r i m e n t a l c o n d i t i o n s , b u t exp o s i n g the p r e p a r a t i o n s in v i t r o to the K - a g o n i s t EKC i n s t e a d of FEN, the w i t h d r a w a l c o n t r a c t i o n s w e r e of s i m i l a r i n t e n s i t y (50 p e r cent) in b o t h B - F N A - e x p o s e d and n o n - e x p o s e d p r e p a r a t i o n s r e g a r d l e s s of the E K C - c o n c e n t r a t i o n e m p l o y e d (panel B). A f u r t h e r set of e x p e r i m e n t s was c a r r i e d out w i t h p r e p a r a tions r e n d e r e d t o l e r a n t / d e p e n d e n t in v i v o to EKC. Those p r e p a r a t i o n s r e c e i v e d in v i t r o the same t r e a t m e n t w i t h B-FNA as d e s c r i b e d for F E N - t o l e r a n t / d e p e n d e n t tissues, thus " e l i m i n a t i n g " u - r e c e p t o r s by a l k y l a t i o n . Again, E K C - r e e x p o s u r e (I, 5 and 50 nM) of E K C - d e p e n d e n t p r e p a r a t i o n s r e s u l t e d in a n a l o x o n e - p r e c i p i t a t e d w i t h d r a w a l c o n t r a c t u r e of a b o u t 50 p e r c e n t of all E K C - c o n c e n t r a t i o n s e m p l o y e d . B - F N A - e x p o s e d p r e p a r a t i o n s did n o t d i f f e r in the d e g r e e of d e p e n d ence d i s p l a y e d as c o m p a r e d to p r e p a r a t i o n s not e x p o s e d to B-FNA. I n t e r e s t i n g l y , e x p o s u r e of in v i v o E K C - t r e a t e d p r e p a r a t i o n s w i t h F E N a l s o r e s u l t e d in the d i s p l a y of a w i t h d r a w a l c o n t r a c t u r e (50 percent). However, the w i t h d r a w a l sign was i n d u c e d only in p r e p a r a tions n o t e x p o s e d to B-FNA. P r e p a r a t i o n s w i t h a l k y l a t e d u - r e c e p t o r s exhibited strongly reduced withdrawal contractions. Discussion' The m a j o r f i n d i n g r e p o r t e d h e r e i n r e l a t e s to the fact that c h r o n i c a c t i v a t i o n of s p e c i f i c o p i a t e r e c e p t o r types in v i v o (e.g. u-receptors) c a u s e s d e p e n d e n c e not only on these s p e c i f i c r e c e p t o r s - b u t a l s o on o t h e r o p i a t e r e c e p t o r types (e.g. K - r e c e p t o r s ) . This o u t c o m e c o n t r a s t s w i t h studies d e m o n s t r a t i n g s e l e c t i v e t o l e r a n c e d e v e l o p m e n t for p a r t i c u l a r o p i a t e r e c e p t o r types (1,2). The d e m o n s t r a t i o n of c o m p l e t e c r o s s - d e p e n d e n c e b e t w e e n different o p i a t e r e c e p t o r s in the same p r e p a r a t i o n d e m a n d s I. the c h r o n i c e x p o s u r e of o p i a t e r e c e p t o r s in v i v o is c o n fined to a single type of o p i a t e r e c e p t o r s l e a v i n g others unaffected; 2. p r e c i p i t a t i o n of w i t h d r a w a l in v i t r o m u s t r e l a t e to a s p e c i f i c type of o p i a t e receptor. T h e s e c o n d i t i o n s a p p e a r to be m e t by the e x p e r i m e n t s p r e s e n t e d here. W i t h r e s p e c t to the s e l e c t i v e a c t i v a t i o n of ~ - r e c e p t o r s in v i v o by FEN, it has b e e n shown that the s e r u m c o n c e n t r a t i o n of FEN d u r i n g i n f u s i o n (10 ug/h) was b e l o w I nM. I nM FEN, however, does n o t a c t i v a t e K - r e c e p t o r s in the m y e n t e r i c plexus, as is d e m o n s t r a t ed by the i n a b i l i t y of this c o n c e n t r a t i o n to i n h i b i t e l e c t r i c a l l y e v o k e d t w i t c h e s in B - F N A e x p o s e d p r e p a r a t i o n s ( u n p u b l i s h e d o b s e r v a tions). The s e r u m c o n c e n t r a t i o n for EKC a l s o a m o u n t e d to < 2 nM. 2 nM EKC does n o t a f f e c t ~ - r e c e p t o r s as has been shown w i t h prep a r a t i o n s e x p o s e d to B - c h l o r n a l t r e x a m i n e (unpublished observation). A s i m i l a r e x p e r i m e n t a l a p p r o a c h p r o v e d the s e l e c t i v e a c t i o n of the c o n c e n t r a t i o n s of the ~- and K - a g o n i s t s e m p l o y e d to a c t i v a t e their r e s p e c t i v e o p i a t e r e c e p t o r types in the p r e p a r a t i o n s set up in vitro. T h e o r e t i c a l l y , these findings in the GPI r e q u i r e a c o m m o n s y s t e m w i t h w h i c h all o p i a t e r e c e p t o r s interact. This s y s t e m is not as y e t identitied, b u t a d e n y l a t e c y c l a s e m a y be a likely cand i d a t e (9,10). The a d a p t a t i o n of such an e n z y m e s y s t e m w o u l d exp l a i n d e p e n d e n c e as w e l l as low level tolerance, since low d e g r e e s of c r o s s - t o l e r a n c e b e t w e e n ~- and K - r e c e p t o r s have b e e n c o n s i s t e n t ly d e m o n s t r a t e d in the GPI (2,11). This f i n d i n g does not c o n f l i c t
360
Cross-dependence
in the Guinea Pig Ileum
Vol. 33, Sup. I, 1983
w i t h the r e p o r t e d s e l e c t i v e d e v e l o p m e n t of t o l e r a n c e (1,2). It m e r e l y r e q u i r e s d i f f e r e n t b i o c h e m i c a l m e c h a n i s m s , e.g. c h a n g e s at the o p i a t e r e c e p t o r level as w e l l as an a d a p t i o n b e y o n d the rec e p t o r site. Thus, an a d a p t a t i o n of the a d e n y l a t e c y c l a s e d u r i n g d e v e l o p m e n t of tolerance~dependence has been shown (9,10) , and " d o w n - r e g u l a t i o n " of o p i a t e r e c e p t o r s has b e e n r e p o r t e d for n e u r o b l a s t o m a h y b r i d cells (Chang et al., 1982; L a w et al.) as well as for the c e n t r a l n e r v o u s s y s t e m (Chang et al., this volume). Acknowled@ements We like to thank Drs. B-funaltrexamine.
Portoghese
and T a k e m o r i
for p r o v i d i n g
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
R. SCHULZ, M. WUSTER, H. KRENSS, and A. HERZ, N a t u r e 285, 242-243 (1980). R. SCHULZ, M. W~STER, P. RUBINI, and A. HERZ, J. P h a r m a c o l . Exp. Ther. 219, 5 4 7 - 5 5 0 (1981). H.O.J. COLLIER, N a t u r e 283, 625-629 (1980). D.B. G O L D S T E I N and A. G O L D S T E I N , Ass. Res. Nerv. Ment. Dis. 46, 265-267 (1968). L. SHUSTER, N a t u r e 189, 314-315 (1961). R. SCHULZ, E. SEIDL, M. WUSTER, and A. HERZ, Europ. J. P h a r m a col. 84, 33-40 (1982). S.J. WARD, P.S. P O R T O G H E S E , and A.E. T A K E M O R I , Europ. J. P h a r m a c o l . 85, 163-170 (1982). R. SCHULZ and A. G O L D S T E I N , J. P h a r m a c o l . Exp. Ther. 183, 4 0 4 - 4 1 0 (1972). H.O.J. C O L L I E R and A.C. ROY, N a t u r e 248, 24-27 (1974). S.K. SHARMA, W.A. KLEE, and M. N I R E N B E R G , Proc. Natl. Acad. Sci. 72, 3 0 9 2 - 3 0 9 6 (1975). M. WUSTER, P. RUBINI, and R. SCHULZ, Life Sci. 29, 1219-1227 (1981).
Pergamon Press
S E L E C T I V E O P I A T E T O L E R A N C E IN THE G U I N E A - P I G ILEUM IS NOT A S S O C I A T E D WITH S E L E C T I V E D E P E N D E N C E Eva Seidl and RHdiger
Schulz
D e p a r t m e n t of N e u r o p h a r m a c o l o g y M a x - P l a n c k - I n s t i t u t fHr P s y c h i a t r i e K r a e p e l i n s t r a s s e 2, D - 8 O O O M 0 n c h e n 40 Federal Republic of Germany (Received in final form June 26, 1983) Summary Opiate d e p e n d e n c e a t t r i b u t a b l e to specific types of opiate receptors was studied in the isolated g u i n e a - p i g ileum made t o l e r a n t / d e p e n d e n t in vivo to a specific narcotic agonist. Apparently, induction of d e p e n d e n c e of b - r e c e p t o r s was a s s o c i a t e d with d e p e n d e n c e of K-receptors and vice versa. These i n v e s t i g a t i o n s involved the use of the i r r e v e r s i b l e ~ - r e c e p t o r a n t a g o n i s t B - f u n a l t r e x a mine to p r o v i d e p r e p a r a t i o n s with functional K-receptors only. The d o c u m e n t e d c r o s s - d e p e n d e n c e b e t w e e n d i f f e r e n t opiate r e c e p t o r types suggests a common system with which these opiate receptors interact. Subtypes of m u l t i p l e opiate receptors may d e v e l o p selective tolerance upon their c h r o n i c a c t i v a t i o n (I ,2). Since tolerance and d e p e n d e n c e have been p o s t u l a t e d to be r e l a t e d to an identical bioc h e m i c a l m e c h a n i s m (3,4,5), a parallel a p p e a r a n c e of both phenom e n a w o u l d be expected. Previous studies from this laboratory with t o l e r a n t / d e p e n d e n t g u i n e a - p i g ileum (GPI) c h a l l e n g e d this p r e s u m p tion. While highly tolerant opiate receptors may d i s p l a y a high degree of dependence, a high degree of d e p e n d e n c e can also be a s s o c i a t e d with a low degree of tolerance (6). These findings c a u s e d us to i n v e s t i g a t e more closely the p h e n o m e n o n of opiate dependence, e m p h a s i z i n g the r e l a t i o n s h i p to specific types of opiate receptors. To a c c o m p l i s h this aim we made use of the irreversible b - o p i a t e r e c e p t o r a n t a g o n i s t B - f u n a l t r e x a m i n e (B-FNA, 7), w h i c h provides G P I - p r e p a r a t i o n s c o n t a i n i n g a single type of functional opiate receptor. Methods Ilea of guinea pigs were r e n d e r e d t o l e r a n t / d e p e n d e n t by subc u t a n e o u s a d m i n i s t r a t i o n of an opioid (6 days) using osmotic m i n i pumps (ALZA, No 2OO1 and 2ML1). The u - a g o n i s t e m p l o y e d was fentanyl (FEN, 10 ug/h) and the K-agonist was e t h y l k e t a z o c i n e (EKC, 50 bg/h). A f t e r set-up of the longitudinal m u s c l e - m y e n t e r i c plexus p r e p a r a t i o n in v i t r o for e l e c t r i c a l s t i m u l a t i o n (8) the tolerancei n d u c i n g opiates were w a s h e d off (60 min). Thereafter, the p r e p a r ations were e x p o s e d to B-FNA (1OO nM, 60 min), w h i c h was followed by a 90 min w a s h procedure. This treatment p r o v i d e s p r e p a r a t i o n s c o n t a i n i n g K-receptors only. Subsequently, the p r e p a r a t i o n s were 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
358
Cross-dependence in the Guinea Pig Ileum
Vol. 33, Sup. I, 1983
e x p o s e d for 30 m i n e i t h e r to a ~- or to a K-agonist. A f t e r e l e c t r i c a l s t i m u l a t i o n was t e r m i n a t e d , p r e c i p i t a t i o n of w i t h d r a w a l was e v o k e d by I pM n a l o x o n e . The w i t h d r a w a l sign was q u a n t i f i e d by c a l c u l a t i n g the p e r c e n t a g e of the t e n s i o n of the c o n t r a c t u r e in r e l a t i o n to that of the e l e c t r i c a l l y e v o k e d t w i t c h e s m e a s u r e d I h a f t e r in v i t r o s e t - u p of the GPI. Results The i n t e n s i t y of w i t h d r a w a l c o n t r a c t i o n s of the GPI c h r o n i c a l l y e x p o s e d in v i v o to the ~ - a g o n i s t FEN a n d t r e a t e d - or n o t t r e a t e d - in v i t r o w i t h B - F N A are g i v e n in f i g u r e I. Panel A ref l e c t s the d a t a of p r e p a r a t i o n s e x p o s e d in v i t r o to d i f f e r e n t c o n c e n t r a t i o n s of FEN. In the a b s e n c e of B - F N A (open c o l u m n s ) , the c o n t r a c t u r e e v o k e d by n a l o x o n e a m o u n t s in all t i s s u e s to a b o u t 50 percent. In c o m p a r i s o n , in the p r e s e n c e of B - F N A (hatched columns) the r e s p o n s e was s t r o n g l y reduced, a l t h o u g h i n c r e a s i n g F E N - c o n c e n t r a t i o n s c a u s e d m o r e i n t e n s e c o n t r a c t i o n s . The fact that p r e p a r a t i o n s r e s p o n d e d to n a l o x o n e in the p r e s e n c e of FEN m o s t l i k e l y r e l a t e s to an i n c o m p l e t e i n a c t i v a t i o n of p - r e c e p t o r s u p o n B - F N A
100 Exposure to FEN
Exposure to EKC
A
t-
B
3 0
J_
o i-
Zo s0 U
/_
J..
'13
JE
10
50
FEN (nM)
1
5
50
EKC (nM)
FIG. I W i t h d r a w a l c o n t r a c t i o n s p r e c i p i t a t e d by n a l o x o n e in p r e p a r a t i o n s r e n d e r e d d e p e n d e n t to f e n t a n y l (FEN) in vivo. O p e n c o l u m n s are d a t a f r o m p r e p a r a t i o n s n o t e x p o s e d to BF N A in vitro; h a t c h e d c o l u m n s i n d i c a t e d a t a f r o m p r e p a r a t i o n s e x p o s e d in v i t r o to B-FNA. The o r d i n a t e r e f l e c t s the t e n s i o n of the w i t h d r a w a l c o n t r a c t u r e e x p r e s s e d in p e r c e n t a g e of e l e c t r i c a l l y - e v o k e d t w i t c h tension. The abs c i s s a r e f l e c t s the c o n c e n t r a t i o n s of F E N and EKC, r e s p e c t i v e l y , e m p l o y e d to e x p o s e the p r e p a r a t i o n s in vitro. D a t a are m e a n s + S.E.M. of at l e a s t 8 p r e p a r a t i o n s .
Vol. 33, Sup. I, 1983
Cross-dependence in the Guinea Pig Ileum
359
t r e a t m e n t (7). U n d e r a n a l o g u o u s e x p e r i m e n t a l c o n d i t i o n s , b u t exp o s i n g the p r e p a r a t i o n s in v i t r o to the K - a g o n i s t EKC i n s t e a d of FEN, the w i t h d r a w a l c o n t r a c t i o n s w e r e of s i m i l a r i n t e n s i t y (50 p e r cent) in b o t h B - F N A - e x p o s e d and n o n - e x p o s e d p r e p a r a t i o n s r e g a r d l e s s of the E K C - c o n c e n t r a t i o n e m p l o y e d (panel B). A f u r t h e r set of e x p e r i m e n t s was c a r r i e d out w i t h p r e p a r a tions r e n d e r e d t o l e r a n t / d e p e n d e n t in v i v o to EKC. Those p r e p a r a t i o n s r e c e i v e d in v i t r o the same t r e a t m e n t w i t h B-FNA as d e s c r i b e d for F E N - t o l e r a n t / d e p e n d e n t tissues, thus " e l i m i n a t i n g " u - r e c e p t o r s by a l k y l a t i o n . Again, E K C - r e e x p o s u r e (I, 5 and 50 nM) of E K C - d e p e n d e n t p r e p a r a t i o n s r e s u l t e d in a n a l o x o n e - p r e c i p i t a t e d w i t h d r a w a l c o n t r a c t u r e of a b o u t 50 p e r c e n t of all E K C - c o n c e n t r a t i o n s e m p l o y e d . B - F N A - e x p o s e d p r e p a r a t i o n s did n o t d i f f e r in the d e g r e e of d e p e n d ence d i s p l a y e d as c o m p a r e d to p r e p a r a t i o n s not e x p o s e d to B-FNA. I n t e r e s t i n g l y , e x p o s u r e of in v i v o E K C - t r e a t e d p r e p a r a t i o n s w i t h F E N a l s o r e s u l t e d in the d i s p l a y of a w i t h d r a w a l c o n t r a c t u r e (50 percent). However, the w i t h d r a w a l sign was i n d u c e d only in p r e p a r a tions n o t e x p o s e d to B-FNA. P r e p a r a t i o n s w i t h a l k y l a t e d u - r e c e p t o r s exhibited strongly reduced withdrawal contractions. Discussion' The m a j o r f i n d i n g r e p o r t e d h e r e i n r e l a t e s to the fact that c h r o n i c a c t i v a t i o n of s p e c i f i c o p i a t e r e c e p t o r types in v i v o (e.g. u-receptors) c a u s e s d e p e n d e n c e not only on these s p e c i f i c r e c e p t o r s - b u t a l s o on o t h e r o p i a t e r e c e p t o r types (e.g. K - r e c e p t o r s ) . This o u t c o m e c o n t r a s t s w i t h studies d e m o n s t r a t i n g s e l e c t i v e t o l e r a n c e d e v e l o p m e n t for p a r t i c u l a r o p i a t e r e c e p t o r types (1,2). The d e m o n s t r a t i o n of c o m p l e t e c r o s s - d e p e n d e n c e b e t w e e n different o p i a t e r e c e p t o r s in the same p r e p a r a t i o n d e m a n d s I. the c h r o n i c e x p o s u r e of o p i a t e r e c e p t o r s in v i v o is c o n fined to a single type of o p i a t e r e c e p t o r s l e a v i n g others unaffected; 2. p r e c i p i t a t i o n of w i t h d r a w a l in v i t r o m u s t r e l a t e to a s p e c i f i c type of o p i a t e receptor. T h e s e c o n d i t i o n s a p p e a r to be m e t by the e x p e r i m e n t s p r e s e n t e d here. W i t h r e s p e c t to the s e l e c t i v e a c t i v a t i o n of ~ - r e c e p t o r s in v i v o by FEN, it has b e e n shown that the s e r u m c o n c e n t r a t i o n of FEN d u r i n g i n f u s i o n (10 ug/h) was b e l o w I nM. I nM FEN, however, does n o t a c t i v a t e K - r e c e p t o r s in the m y e n t e r i c plexus, as is d e m o n s t r a t ed by the i n a b i l i t y of this c o n c e n t r a t i o n to i n h i b i t e l e c t r i c a l l y e v o k e d t w i t c h e s in B - F N A e x p o s e d p r e p a r a t i o n s ( u n p u b l i s h e d o b s e r v a tions). The s e r u m c o n c e n t r a t i o n for EKC a l s o a m o u n t e d to < 2 nM. 2 nM EKC does n o t a f f e c t ~ - r e c e p t o r s as has been shown w i t h prep a r a t i o n s e x p o s e d to B - c h l o r n a l t r e x a m i n e (unpublished observation). A s i m i l a r e x p e r i m e n t a l a p p r o a c h p r o v e d the s e l e c t i v e a c t i o n of the c o n c e n t r a t i o n s of the ~- and K - a g o n i s t s e m p l o y e d to a c t i v a t e their r e s p e c t i v e o p i a t e r e c e p t o r types in the p r e p a r a t i o n s set up in vitro. T h e o r e t i c a l l y , these findings in the GPI r e q u i r e a c o m m o n s y s t e m w i t h w h i c h all o p i a t e r e c e p t o r s interact. This s y s t e m is not as y e t identitied, b u t a d e n y l a t e c y c l a s e m a y be a likely cand i d a t e (9,10). The a d a p t a t i o n of such an e n z y m e s y s t e m w o u l d exp l a i n d e p e n d e n c e as w e l l as low level tolerance, since low d e g r e e s of c r o s s - t o l e r a n c e b e t w e e n ~- and K - r e c e p t o r s have b e e n c o n s i s t e n t ly d e m o n s t r a t e d in the GPI (2,11). This f i n d i n g does not c o n f l i c t
360
Cross-dependence
in the Guinea Pig Ileum
Vol. 33, Sup. I, 1983
w i t h the r e p o r t e d s e l e c t i v e d e v e l o p m e n t of t o l e r a n c e (1,2). It m e r e l y r e q u i r e s d i f f e r e n t b i o c h e m i c a l m e c h a n i s m s , e.g. c h a n g e s at the o p i a t e r e c e p t o r level as w e l l as an a d a p t i o n b e y o n d the rec e p t o r site. Thus, an a d a p t a t i o n of the a d e n y l a t e c y c l a s e d u r i n g d e v e l o p m e n t of tolerance~dependence has been shown (9,10) , and " d o w n - r e g u l a t i o n " of o p i a t e r e c e p t o r s has b e e n r e p o r t e d for n e u r o b l a s t o m a h y b r i d cells (Chang et al., 1982; L a w et al.) as well as for the c e n t r a l n e r v o u s s y s t e m (Chang et al., this volume). Acknowled@ements We like to thank Drs. B-funaltrexamine.
Portoghese
and T a k e m o r i
for p r o v i d i n g
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
R. SCHULZ, M. WUSTER, H. KRENSS, and A. HERZ, N a t u r e 285, 242-243 (1980). R. SCHULZ, M. W~STER, P. RUBINI, and A. HERZ, J. P h a r m a c o l . Exp. Ther. 219, 5 4 7 - 5 5 0 (1981). H.O.J. COLLIER, N a t u r e 283, 625-629 (1980). D.B. G O L D S T E I N and A. G O L D S T E I N , Ass. Res. Nerv. Ment. Dis. 46, 265-267 (1968). L. SHUSTER, N a t u r e 189, 314-315 (1961). R. SCHULZ, E. SEIDL, M. WUSTER, and A. HERZ, Europ. J. P h a r m a col. 84, 33-40 (1982). S.J. WARD, P.S. P O R T O G H E S E , and A.E. T A K E M O R I , Europ. J. P h a r m a c o l . 85, 163-170 (1982). R. SCHULZ and A. G O L D S T E I N , J. P h a r m a c o l . Exp. Ther. 183, 4 0 4 - 4 1 0 (1972). H.O.J. C O L L I E R and A.C. ROY, N a t u r e 248, 24-27 (1974). S.K. SHARMA, W.A. KLEE, and M. N I R E N B E R G , Proc. Natl. Acad. Sci. 72, 3 0 9 2 - 3 0 9 6 (1975). M. WUSTER, P. RUBINI, and R. SCHULZ, Life Sci. 29, 1219-1227 (1981).