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Morphine-induced desensitization and down-regulation at mu-receptors in 7315c pituitary tumor cells
Life Sciences, Vol. 45, pp. 1937-1942 Printed in the U.S.A.
Pergamon Press
MORPHINE-INDUCED DESENSITIZATION AND DOWN-REGULATION AT MU-RECEPTORS IN 7315C PITUITARY TUMOR CELLS Pamela S. Puttfarcken* and Brian M. Cox Department of Pharmacology Uniformed Services University of the Health Sciences Bethesda, MD 20814, U.S.A. (Received in final form September 8, 1989)
Summary Pituitary 7315c t u m o r cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 BlVl,for periods of five or fortyeight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed m e m b r a n e preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same m e m b r a n e preparations, the n u m b e r and afi~mity of mu-opioid receptors was estimated by m e a s u r e m e n t s of [3H]diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for [3H]diprenorphine. However, after 48 hr of morphine treatment, greater t h a n 25% reductions in receptor n u m b e r were apparent with morphine pretreatment concentrations of 10 BM or higher. These res u l ts suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor n u m b e r occurred after exposure to high concentrations of morphine for periods longer than 5 hr. Most clinically useful opiate drugs and illicitly used opiates act preferentially at the mu-type of opioid receptor (1). Until recently, the mechanisms underlying the development of tolerance to the actions of opioids at mu-receptors were not well understood. Previous studies have utilized NG 108-15 neuroblastoma x glioma hybrid cells to examine the effects of chronic exposure to delta-receptor selective opioid agonists (2,3L At least two different time-dependent changes were reported to occur upon chronic agonist exposure: desensitization, defined as a reduced ability of the agonist to inhibit the enzyme adenylyl cyclase (AC), and receptor downregulation defined as a reduction in the number of delta-receptors in the cell membranes. More recently, morphine has been shown to inhibit AC in cells of the prolactin-secreting rat pituitary tumor, 7315c (4). The inhibitory action of morphine in these cells is mediated by activation of mu-type opioid receptors (4,5,6). Sustained exposure of primary cultures of 7315c cells to m o r p h i n e resulted in a reduced ability of opioids to inhibit AC in m e m b r a n e preparations from the treated cells (5,6). This desensitization was apparent after five hours of exposure to a high concentration of morphine. Receptor number was unchanged at this time. However, after exposure to morphine for periods of 24 hr or more, the number of mu-receptors in the m e m b r a n e s of treated cells was significantly reduced (6). These results suggest that effector desensitization and receptor down-regulation appear to be two distinct consequences of chronic morphine exposure in this mu-receptor regulated system, as in the delta-receptor regulated system in NG 108-15 cells. Present address: Department of Psychiatry, Meyer 4-163, J o h n s Hopkins University, School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21205. 0024-3205/89 $3.00 +.00
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Tolerance at Mu-Opioid Receptors
Vol. 45, No. 20, 1989
Since these two c o n s e q u e n c e s of chronic m o r p h i n e t r e a t m e n t were temporally distinct, it was of interest to d e t e r m i n e if the rates of onset of t h e s e p h e n o m e n a were related to the concentrations of m o r p h i n e to which the cells were exposed. We report here studies in which primary c u l t u r e s of 7315c ceils were treated with a range of c o n c e n t r a t i o n s of m o r p h i n e from 10 nM to 300 IJM for periods of five or forty-eight hours. The extent of desensitization was m e a s u r e d by e s t i m a t i o n of t h e ability of the m u - r e c e p t o r - s e l e c t i v e agonist Tyr-D-Ala-GIy(NMe)Phe-Gly-ol (DAMGO} to inhibit f o r s k o l i n - s t i m u l a t e d AC in m e m b r a n e p r e p a r a t i o n s f r o m t h e t r e a t e d cells. E s t i m a t e s of m u - r e c e p t o r n u m b e r w e r e m a d e by m e a s u r i n g [3H]diprenorphine ([3H]DIP) binding. It is preferable to u s e a radiolabeled antagonist to e s t i m a t e r e c e p t o r density, since t h e binding of a n t a g o n i s t s . u n l i k e t h a t of agonists, is unaffected by t h e p r e s e n c e of g u a n i n e n u c l e o t i d e s or by the c h a n g e s in agonist affinity associated with opiate agonist t r e a t m e n t (5,6). [3H]DIP b i n d s to a h o m o g e n e o u s population of mu-receptors in 7315c cell m e m b r a n e s (5,6). Methods 7315c t u m o r s were propagated in the peritoneal cavities of female Buffalo rats (approx. 250 g; National C a n c e r Institute) a s described by Frey and Kebabian (3). After about three weeks, t u m o r s were r e m o v e d from t h e peritoneal cavities, cells dispersed, a n d grown as primary c u l t u r e s in DMEM m e d i u m containing 2 mM g l u t a m i n e and 10% fetal calf serum. Sources of materials, and p r o c e d u r e s for cell culture, m e m b r a n e preparation, AC assay, and m e a s u r e m e n t s of [3H]DIP binding, are contained in Puttfarcken et al. (6). After pre-incubation with morphine, the cells and m e m b r a n e s u s p e n s i o n s were w a s h e d extensively prior to use in AC or ligand binding assays. No significant a m o u n t s of m o r p h i n e added in the pre-incubation remained in the w a s h e d m e m b r a n e suspensions (6). In the assays of AC activity, estimates of forskolin (10 idVl)-stimulated AC activity were m a d e in triplicate for each concentration of DAMGO tested. For each m o r p h i n e p r e t r e a t m e n t condition, three i n d e p e n d e n t DIP competition c u r v e s against [3H]DIP (1 nM) binding, each employing 18 to 21 c o n c e n t r a t i o n s of unlabeled DIP in triplicate were generated. The reported e s t i m a t e s of KD and B m a x were p r e p a r e d by analysis of the pooled r e s u l t s of the three experiments for each p r e t r e a t m e n t condition using the program LIGAND (7). Results Effects of m o r p h i n e p r e t r e a t m e n t on inhibition of AC activity by DAMGO Cells were grown in m e d i u m without morphine, or in m e d i u m containing m o r p h i n e in concentrations from 10 nM to 300 ~M. Membrane s u s p e n s i o n s were prepared from b a t c h e s of cells exposed to t h e d r u g for periods of 5 h r or 48 hr. In m e m b r a n e s from cells i n c u b a t e d w i t h o u t m o r p h i n e , DAMGO (1 I~M} inhibited f o r s k o l i n - s t i m u l a t e d AC a b o u t 70-75% (Fig. 1A, B). After exposure to m o r p h i n e for 5 h r (Fig. 1A) or 48 h r (Fig 1B) in concentrations of 100 nM or higher the potency of DAMGO in inhibiting AC was reduced. After 5 h r pretreatment, DAMGO inhibition of AC was significantly reduced following m o r p h i n e p r e t r e a t m e n t s at 1 ~M and higher concentrations (ANOVA: 1 IJM m o r p h i n e v. control, FI,4 59.084, P = 0.002; 100 nM morphine v control, FI,4 1.871. P>0.05). After 48 hr of morphine the ability of DAMGO to inhibit AC was significantly reduced by m o r p h i n e p r e t r e a t m e n t concentrations of 100 nM and higher (100 nM morphine v. control, FI,4 106.02, P = 0.001). After 48 h r exposure to the highest m o r p h i n e p r e t r e a t m e n t c o n c e n t r a t i o n a n inhibitory effect of DAMGO was no longer a p p a r e n t at any tested DAMGO concentration. At neither pretreatment time was the inhibitory effect of DAMGO significantly r e d u c e d by p r e t r e a t m e n t with 10 nM m o r p h i n e . P r e t r e a t m e n t with m o r p h i n e did not affect t h e level of f o r s k o l i n - s t i m u l a t e d adenylate cyclase activity in the absence of added opioid (Table 1). After b o t h p r e t r e a t m e n t times, the effects of high DAMGO c o n c e n t r a t i o n s (0.1, 1 ~tM) were r e d u c e d to a g r e a t e r extent t h a n t h o s e of lower DAMGO c o n c e n t r a t i o n s . M a x i m u m inhibitory effects of m u - r e c e p t o r activation were a s s e s s e d by m e a s u r e m e n t of the inhibotory activity of 1 ~tM DAMGO. (Higher c o n c e n t r a t i o n s of DAMGO gave g r e a t e r inhibition of adenylate cyclase, b u t the effects of c o n c e n t r a t i o n s of DAMGO in excess of i ~M were not reliably inhibited by naloxone). The results suggest t h a t the p r e t r e a t m e n t s reduced the m a x i m u m opioid receptor-mediated inhibition that could be obtained with DAMGO wlth little or no increase in the estimated DAMGO IC50 (Fig. I, Table 1).
Vol. 45, No. 20, 1989
Tolerance at Mu-Opioid Receptors
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Fig. 1. I n h i b i t i o n of f o r s k o l i n - s t i m u l a t e d AC b y DAMGO i n m e m b r a n e s f r o m 7 3 1 5 c cells p r e t r e a t e d w i t h m o r p h i n e for 5 h r (A) or for 48 h r (B). 7 3 1 5 c cells were p r e p a r e d a n d c u l t u r e d a s p r e v i o u s l y d e s c r i b e d (5). After i n c u b a t i o n w i t h m o r p h i n e - c o n t a i n i n g m e d i u m for 5 or 48 hr, t h e cells were h o m o g e n i z e d a n d w a s h e d m e m b r a n e s p r e p a r e d . I n h i b i t i o n of forskolin (10 l~Vl)-sUmulated AC b y c o n c e n t r a t i o n s of DAMGO from 1 n M to 1 ~VI w a s m e a s u r e d . Points r e p r e s e n t m e a n s v a l u e s (+ s.e.m.) of at l e a s t six e s t i m a t e s for e a c h t r e a t m e n t . (In s o m e cases, s t a n d a r d e r r o r b a r s are o m i t t e d for clarity). M o r p h i n e p r e t r e a t m e n t c o n c e n t r a t i o n s : • 10 nM, Z~ 100 nM, • i pM, O I0 I~M, • 300 ~uM; O control medium without added morphine.
T a b l e 1. F o r s k o l i n - s t i m u l a t e d AC activity a n d DAMGO i n h i b i t i o n of f o r s k o l i n - s t l m u l a t e d AC activity a f t e r e x p o s u r e of 7 3 1 5 c cells to m o r p h i n e .
M o r p h i n e pret r e a t m e n t eonc. (nM) 0
D u r a t i o n of p r e t r e a t m e n t 5 hours 48 h o u r s AC activity Approx. AC activity Approx. p m o l / r n g p m t e t n / m i n DAMGO p m o l / m g p m t e i n / m i n DAMGO IC50 (nM) IC50 (nM) 21+3
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AC activity v a l u e s r e p o r t t h e m e a n (+ s.e.m, of a t l e a s t six e s t i m a t e s for e a c h p r e t r e a t m e n t ) of AC a c t i v i t i e s i n t h e p r e s e n c e of 100 BM f o r s k o l i n , m e a s u r e d o v e r a p e r i o d of 10 rain. A p p r o x i m a t e ICs0 v a l u e s for i n h i b i t i o n of e n z y m e activity b y DAMGO w e r e e s t i m a t e d from Fig. 1, a s s u m i n g t h a t m a x i m u m opioid r e c e p t o r - m e d i a t e d i n h i b i t i o n of e n z y m e activity w a s observed a t 1 I~M DAMGO (see text).
1940
Tolerance at Mu-Opioid Receptors
Vol. 45, No. 20, 1989
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Fig. 2. Effects of m o r p h i n e p r e t r e a t m e n t s for 5 h r (open circles) or 48 h r (closed circles) o n the d e n s i t y of [3H]DIP b i n d i n g s i t e s (Bmax) in 7 3 1 5 c cell m e m b r a n e s . W a s h e d cell m e m b r a n e s were i n c u b a t e d w i t h 1 n M [3H]DIP a t 3 7 ° for 2 0 r a i n i n t h e p r e s e n c e or a b s e n c e of a r a n g e of c o n c e n t r a t i o n s of u n l a b e l e d DIP; b i n d i n g w a s t e r m i n a t e d b y f i l t r a t i o n , a n d r e t a i n e d radioactivity e s t i m a t e d b y liquid scintillation c o u n t i n g . E s t i m a t e s of DIP K D a n d Bma x were o b t a i n e d b y a n a l y s i s of t h e pooled d a t a from t h r e e e x p e r i m e n t s , u s i n g t h e p r o g r a m LIGAND (7). D a t a p o i n t s i n d i c a t e t h e c o m p u t e d Bma x e s t i m a t e s ( f m o l / m g m e m b r a n e protein) a n d s t a n d a r d e r r o r s of t h e e s t i m a t e s . P r e t r e a t m e n t c o n c e n t r a t i o n s of m o r p h i n e a r e i n d i c a t e d a s t h e n e g a t i v e l o g a r i t h m of t h e m o r p h i n e c o n c e n t r a t i o n ; C i n d i c a t e s v a l u e s o b t a i n e d from control ceils i n c u b a t e d for t h e i n d i c a t e d t i m e s i n t h e a b s e n c e of a d d e d m o r p h i n e . KD v a l u e s for DIP were n o t a l t e r e d significantly b y a n y t r e a t m e n t ; m e a n K D for all t r e a t m e n t w a s 0 . 7 3 + 0.09 nM.
Effects of m o r p h i n e p r e t r e a t m e n t s o n [3H]DIP binding. P r e t r e a t m e n t of 7 3 1 5 c cells with 100 BlVlm o r p h i n e for 72 h r h a s b e e n s h o w n to r e d u c e t h e n u m b e r o f m u - r e c e p t o r s i n 7 3 1 5 c e e l m e m b r a n e s . However, n o c h a n g e in r e c e p t o r n u m b e r w a s a p p a r e n t after 5 h r (6). The effects o n r e c e p t o r n u m b e r of p r e t r e a t m e n t of 7 3 1 5 c cells with a r a n g e of c o n c e n t r a t i o n s of m o r p h i n e is s h o w n in Fig. 2. No r e d u c t i o n in t h e n u m b e r of r e c e p t o r s w a s o b s e r v e d a f t e r 5 h r of p r e t r e a t m e n t at a n y t e s t e d m o r p h i n e p r e t r e a t m e n t c o n c e n t r a t i o n . After 4 8 h r of p r e t r e a t m e n t t h e r e w a s a s m a l l r e d u c t i o n i n t h e n u m b e r of r e c e p t o r s after e x p o s u r e to 100 n M m o r p h i n e , a n d s u b s t a n t i a l r e d u c t i o n s in r e c e p t o r n u m b e r a f t e r e x p o s u r e to 10 BM a n d 3 0 0 BM m o r p h i n e . None of t h e p r e t r e a t m e n t s p r o d u c e d a n y s i g n i f i c a n t a l t e r a t i o n s in t h e [3H]DIP K D.
Discussion T h e s e r e s u l t s c o n f i r m o u r p r e v i o u s s t u d i e s s h o w i n g t h a t p r e t r e a t m e n t of 7 3 1 5 c p i t u i t a r y t u m o r cells w i t h a h i g h c o n c e n t r a t i o n (100 BM) of m o r p h i n e for a p e r i o d of 5 h r r e s u l t e d i n a loss of t h e ability of a n opioid a g o n i s t to i n h i b i t f o r s k o l i n - s t i m u l a t e d AC at a t i m e w h e n m u - o p i o i d r e c e p t o r n u m b e r w a s u n c h a n g e d (6), Only a f t e r l o n g e r p e r i o d s of m o r p h i n e e x p o s u r e w a s a s i g n i f i c a n t r e d u c t i o n i n t h e n u m b e r of m u - t y p e b i n d i n g s i t e s observed. T h e c u r r e n t r e s u l t s indicate t h a t b o t h d e s e n s i t i z a t i o n of opioid i n h i b i t i o n of AC a n d d o w n - r e g u l a t i o n of m u - t y p e b i n d i n g s i t e s are o b s e r v e d w h e n 7 3 1 5 c cells are exposed to lower c o n c e n t r a t i o n s of m o r p h i n e t h a n u s e d i n t h e initial study. The m a g n i t u d e s of b o t h t h e d e s e n s i t i z a t i o n a n d t h e d o w n - r e g u l a t i o n were related to t h e c o n c e n t r a t i o n of m o r p h i n e to w h i c h t h e ceils were exposed. No s i g n i f i c a n t d e s e n s i t i z a t i o n or
Vol. 45, No. 20, 1989
Tolerance at Mu-Opioid Receptors
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d o w n - r e g u l a t i o n w a s o b s e r v e d w h e n 7 3 1 5 c cells were t r e a t e d w i t h 10 n M m o r p h i n e for 5 or 48 hr, or w i t h 100 n M for 5 h o u r s . After 4 8 h r of e x p o s u r e to 100 n M m o r p h i n e a s l i g h t d e s e n s i t i z a t i o n w a s a p p a r e n t . T h i s c o n c e n t r a t i o n of m o r p h i n e is s i m i l a r to t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t of m o r p h i n e for t h e h i g h e r - a l I l n i t y form of t h e m u - r e c e p t o r m e a s u r e d i n 7 3 1 5 c m e m b r a n e s i n t h e p r e s e n c e of a s t a b l e GTP a n a l o g u e (Werling et al., m a n u s c r i p t i n preparation). M o r e m a r k e d d e s e n s i t i z a t i o n r e q u i r e d t r e a t m e n t of t h e c e l l s w i t h c o n c e n t r a t i o n s of m o r p h i n e of 1 ~M or h i g h e r . S i m i l a r l o s s e s i n opioid i n h i b i t o r y p o t e n c y were o b s e r v e d a f t e r t r e a t m e n t of t h e cells w i t h t h e s e c o n c e n t r a t i o n s of m o r p h i n e for periods of 5 h r or 4 8 hr, s u g g e s t i n g t h a t t h e d e s e n s i t i z a t i o n p r o c e s s is largely c o m p l e t e w i t h i n a period of 5 h r f r o m i n i t i a t i o n of m o r p h i n e e x p o s u r e . No r e c e p t o r d o w n - r e g u l a t i o n w a s o b s e r v e d after 5 h r of m o r p h i n e t r e a t m e n t a t a n y of t h e c o n c e n t r a t i o n s t e s t e d . However, after 48 h r of t r e a t m e n t , a c l e a r loss i n r e c e p t o r n u m b e r w a s a p p a r e n t w h e n t h e cells were e x p o s e d to 10 ~M a n d h i g h e r c o n c e n t r a t i o n s of m o r p h i n e . T r e a t m e n t w i t h 0.1 or 1 lJaVIm o r p h i n e i n d u c e d s m a l l b u t statistically s i g n i f i c a n t r e d u c t i o n s in t h e n u m b e r of receptors. It a p p e a r s to t a k e longer for d o w n - r e g u l a t i o n to occur, a n d to require e x p o s u r e to a m o r p h i n e c o n c e n t r a t i o n of a b o u t 10 ~Vl or m o r e to p r o d u c e m o r e t h a n a 2 5 % loss of r e c e p t o r s . T h i s c o n c e n t r a t i o n of m o r p h i n e is h i g h e r t h a n t h e lowest affinity s t a t e of t h e 7 3 1 5 c m u - b i n d i n g site for m o r p h i n e m e a s u r e d i n t h e p r e s e n c e of a s t a b l e GTP a n a l o g u e (Werling et al., i n p r e p a r a t i o n ) . It a p p e a r s t h a t very h i g h levels of m u - r e c e p t o r o c c u p a n c y b y m o r p h i n e are r e q u i r e d over a period of t i m e i n excess of 5 h r for s u b s t a n t i a l m u - r e c e p t o r downr e g u l a t i o n to b e o b s e r v e d in 7 3 1 5 c cells. T h e m e c h a n i s m s b y w h i c h a g o n i s t p r e t r e a t m e n t i n d u c e s d e s e n s i t i z a t i o n a r e still n o t clear. R e s u l t s p r e s e n t e d h e r e a n d elsewhere (2,6) i n d i c a t e t h a t r e c e p t o r d o w n - r e g u l a t i o n is n o t r e q u i r e d for a loss of a g o n i s t p o t e n c y . A c h a r a c t e r i s t i c f e a t u r e of d e s e n s i t i z a t i o n i n t h i s s y s t e m is a p r o g r e s s i v e r e d u c t i o n i n t h e m a x i m u m opioid r e c e p t o r - m e d i a t e d i n h i b i t i o n of AC activity (as i n d i c a t e d b y t h e i n h i b i t o r y effect of 1 lIM DAMGO; h i g h e r c o n c e n t r a t i o n s of DAMGO s o m e t i m e s p r o d u c e d a g r e a t e r i n h i b i t i o n , b u t t h e i r effects were n o t reliably i n h i b i t e d b y t h e opioid a n t a g o n i s t naloxone). T h e r e w a s very little i n c r e a s e i n t h e e s t i m a t e d IC50 for DAMGO a s a r e s u l t of p r i o r m o r p h i n e exposure. T h u s , t h e effects of m o r p h i n e p r e t r e a t m e n t o n t h e ability of DAMGO to i n h i b i t AC are m o r e a n a l o g o u s to t h e loss of a g o n i s t r e s p o n s e i n d u c e d b y a n irreversible a n t a g o n i s t t h a n to t h e r i g h t w a r d shift i n t h e c o n c e n t r a t i o n - r e s p o n s e c u r v e o b s e r v e d i n t h e p r e s e n c e of a competitive antagonist.. M o r p h i n e p r e t r e a t m e n t h a s l e s s effect o n t h e p o t e n c y of DAMGO ( m e a s u r e d b y its IC50) t h a n o n its efficacy (as i n d i c a t e d b y its i n h i b i t o r y effect at 1 gM). T h e s e c h a r a c t e r i s t i c s w o u l d b e expected ff t h e m e c h a n i s m of d e s e n s i t i z a t i o n w a s a r e d u c e d ability of t h e a g o n i s t - o c c u p i e d r e c e p t o r to i n t e r a c t w i t h or to activate t h e g u a n i n e n u c l e o t i d e b i n d i n g p r o t e i n (G-protein) w h i c h m e d i a t e s opioid a c t i o n in t h i s s y s t e m (4,5,8). T h e m a x i m u m i n h i b i t o r y effect is r e d u c e d b e c a u s e a s u b s t a n t i a l f r a c t i o n of t h e r e c e p t o r pool c a n n o l o n g e r a c t i v a t e G - p r o t e i n . T h i s p r o p o s e d m e c h a n i s m is s u p p o r t e d b y o u r p r e v i o u s d e m o n s t r a t i o n t h a t d e s e n s i t i z a t i o n i n d u c e d b y m o r p h i n e is a s s o c i a t e d w i t h a l o s s i n t h e ability of g u a n i n e n u c l e o t i d e s to m o d u l a t e opioid agonist affinity a t m u - r e c e p t o r s i n 7 3 1 5 c cell m e m b r a n e s (6) a n d in c e r e b r a l cortex m e m b r a n e s of g u i n e a pig (9). At t h i s t i m e it is u n c l e a r if t h e i m p a i r m e n t in r e c e p t o r - G p r o t e i n i n t e r a c t i o n is r e l a t e d to c h a n g e s in t h e p r o p e r t i e s of t h e r e c e p t o r or of t h e G protein(s). R e c e p t o r d o w n - r e g u l a t i o n w a s s l o w e r in o n s e t t h a n d e s e n s i t i z a t i o n , a n d w a s o n l y observed after pretreatment with morphine concentrations that induced substantial d e s e n s i t i z a t i o n . It is p o s s i b l e t h a t u n c o u p l i n g of r e c e p t o r from G - p r o t e i n (either physically or functionally) a c c e l e r a t e s t h e r a t e of r e c e p t o r r e m o v a l from t h e m e m b r a n e , or t h a t c h a n g e s i n r e c e p t o r p r o p e r t i e s w h i c h p r e v e n t G - p r o t e i n a c t i v a t i o n also trigger r e c e p t o r d e g r a d a t i o n . It m a y b e r e l e v a n t t h a t a r e d u c t i o n in m u - r e c e p t o r d e n s i t y w a s also o b s e r v e d following p e r t u s s i s t o x i n t r e a t m e n t of 7 3 1 5 c cells to i n d u c e a f u n c t i o n a l u n c o u p l i n g of m u - r e c e p t o r s a n d a s s o c i a t e d G - p r o t e i n s (5). However, it is also possible t h a t s u s t a i n e d r e c e p t o r a c t i v a t i o n r e s u l t s indirectly in a r e d u c e d rate of r e c e p t o r s y n t h e s i s . O u r r e s u l t s s u g g e s t t h a t t h e p r i m a r y m e c h a n i s m for m o r p h i n e t o l e r a n c e a t m u r e c e p t o r s i n 7 3 1 5 c p i t u i t a r y t u m o r cells is a n i m p a i r m e n t of r e c e p t o r - G p r o t e i n i n t e r a c t i o n r e s u l t i n g i n a r e d u c e d ability of a g o n i s t - o c c u p i e d r e c e p t o r s to initiate a r e s p o n s e . At a l a t e r t i m e a n d a f t e r e x p o s u r e to v e r y h i g h c o n c e n t r a t i o n s of m o r p h i n e s o m e r e d u c t i o n i n t h e n u m b e r of m u - r e c e p t o r s also occurs, b u t r e c e p t o r d o w n - r e g u l a t i o n is n o t n e c e s s a r y for a l m o s t c o m p l e t e t o l e r a n c e to b e observed.
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Tolerance at Mu-Opioid Receptors
Vol. 45, No. 20, 1989
Acknowledgements This s t u d y w a s s u p p o r t e d b y a g r a n t from t h e National I n s t i t u t e o n Drug Abuse. We t h a n k Dr. L i n d a Werling a n d Dr. T o m Cote for helpful d i s c u s s i o n i n t h e p r e p a r a t i o n of t h i s m a n u s c r i p t , a n d P a u l M c M a h o n for t e c h n i c a l a s s i s t a n c e . T h e o p i n i o n s a n d a s s e r t i o n s c o n t a i n e d h e r e i n are t h e p r i v a t e o n e s of t h e a u t h o r s , a n d are n o t to b e c o n s t r u e d a s reflecting t h e views of t h e U n i t e d S t a t e s D e p a r t m e n t of D e f e n s e or t h e U n i f o r m e d S e r v i c e s U n i v e r s i t y of t h e H e a l t h Sciences. A n i m a l s u s e d i n t h i s s t u d y were a c q u i r e d a n d c a r e d for i n a c c o r d a n c e w i t h t h e g u i d e l i n e s p u b l i s h e d i n t h e N a t i o n a l I n s t i t u t e s of H e a l t h G u i d e for t h e C a r e a n d Use of L a b o r a t o r y A n i m a l s (National I n s t i t u t e s of H e a l t h P u b l i c a t i o n No. 85-23, revised 1985). References 1. J . MAGNAN, S.J. PATERSON, A, TAVANI, a n d H.W. KOSTERLITZ. N a u n y n - S c h m i e d e b e r g s Arch. Pharmacol. 3 1 9 : 1 9 7 - 2 0 5 (1982). 2. P.Y. LAW, D.S. HOM, a n d H.H.LOH. Mol. Pharmacol. 2 4 : 4 1 3 - 4 2 4 (1983). 3. H.H. LOH, P.-L. TAO andA.P. SMITH. Synapse 2 : 4 5 7 - 4 6 2 (1988). 4. E. F R E Y a n d J . KEBABAIAN,. Endocrinology 1 1 5 : 1 7 9 7 - 1 8 0 4 (1984). 5. L.L.WERLING, P.S. PUTTFARCKEN, a n d B.M. COX. Mol. Pharmacol. 33: 423-431,(1988). 6. P.S. PUTFFARCKEN, L.L.WERLING, a n d B.M. COX. Mol. Pharmacol. 3 3 : 5 2 0 - 5 2 7 (1988). 7. P.J. MUNSON a n d D. RODBARD. Analyt. Biochem. 1 0 7 : 2 2 0 - 2 3 9 (1980). 8. D.L. AUB, E. FREY, R.D. SEKURA, a n d T.E. COTE. J. Biol. Chem. 2 6 1 : 9 3 3 3 - 9 3 4 0 (1986). 9. L.L. WERLING, P.N. McMAHON, a n d B.M. COX. Proc. Natl. Acad. Sci. USA 8 6 : 6 3 9 3 - 6 3 9 7 (1989).
Pergamon Press
MORPHINE-INDUCED DESENSITIZATION AND DOWN-REGULATION AT MU-RECEPTORS IN 7315C PITUITARY TUMOR CELLS Pamela S. Puttfarcken* and Brian M. Cox Department of Pharmacology Uniformed Services University of the Health Sciences Bethesda, MD 20814, U.S.A. (Received in final form September 8, 1989)
Summary Pituitary 7315c t u m o r cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 BlVl,for periods of five or fortyeight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed m e m b r a n e preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same m e m b r a n e preparations, the n u m b e r and afi~mity of mu-opioid receptors was estimated by m e a s u r e m e n t s of [3H]diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for [3H]diprenorphine. However, after 48 hr of morphine treatment, greater t h a n 25% reductions in receptor n u m b e r were apparent with morphine pretreatment concentrations of 10 BM or higher. These res u l ts suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor n u m b e r occurred after exposure to high concentrations of morphine for periods longer than 5 hr. Most clinically useful opiate drugs and illicitly used opiates act preferentially at the mu-type of opioid receptor (1). Until recently, the mechanisms underlying the development of tolerance to the actions of opioids at mu-receptors were not well understood. Previous studies have utilized NG 108-15 neuroblastoma x glioma hybrid cells to examine the effects of chronic exposure to delta-receptor selective opioid agonists (2,3L At least two different time-dependent changes were reported to occur upon chronic agonist exposure: desensitization, defined as a reduced ability of the agonist to inhibit the enzyme adenylyl cyclase (AC), and receptor downregulation defined as a reduction in the number of delta-receptors in the cell membranes. More recently, morphine has been shown to inhibit AC in cells of the prolactin-secreting rat pituitary tumor, 7315c (4). The inhibitory action of morphine in these cells is mediated by activation of mu-type opioid receptors (4,5,6). Sustained exposure of primary cultures of 7315c cells to m o r p h i n e resulted in a reduced ability of opioids to inhibit AC in m e m b r a n e preparations from the treated cells (5,6). This desensitization was apparent after five hours of exposure to a high concentration of morphine. Receptor number was unchanged at this time. However, after exposure to morphine for periods of 24 hr or more, the number of mu-receptors in the m e m b r a n e s of treated cells was significantly reduced (6). These results suggest that effector desensitization and receptor down-regulation appear to be two distinct consequences of chronic morphine exposure in this mu-receptor regulated system, as in the delta-receptor regulated system in NG 108-15 cells. Present address: Department of Psychiatry, Meyer 4-163, J o h n s Hopkins University, School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21205. 0024-3205/89 $3.00 +.00
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Vol. 45, No. 20, 1989
Since these two c o n s e q u e n c e s of chronic m o r p h i n e t r e a t m e n t were temporally distinct, it was of interest to d e t e r m i n e if the rates of onset of t h e s e p h e n o m e n a were related to the concentrations of m o r p h i n e to which the cells were exposed. We report here studies in which primary c u l t u r e s of 7315c ceils were treated with a range of c o n c e n t r a t i o n s of m o r p h i n e from 10 nM to 300 IJM for periods of five or forty-eight hours. The extent of desensitization was m e a s u r e d by e s t i m a t i o n of t h e ability of the m u - r e c e p t o r - s e l e c t i v e agonist Tyr-D-Ala-GIy(NMe)Phe-Gly-ol (DAMGO} to inhibit f o r s k o l i n - s t i m u l a t e d AC in m e m b r a n e p r e p a r a t i o n s f r o m t h e t r e a t e d cells. E s t i m a t e s of m u - r e c e p t o r n u m b e r w e r e m a d e by m e a s u r i n g [3H]diprenorphine ([3H]DIP) binding. It is preferable to u s e a radiolabeled antagonist to e s t i m a t e r e c e p t o r density, since t h e binding of a n t a g o n i s t s . u n l i k e t h a t of agonists, is unaffected by t h e p r e s e n c e of g u a n i n e n u c l e o t i d e s or by the c h a n g e s in agonist affinity associated with opiate agonist t r e a t m e n t (5,6). [3H]DIP b i n d s to a h o m o g e n e o u s population of mu-receptors in 7315c cell m e m b r a n e s (5,6). Methods 7315c t u m o r s were propagated in the peritoneal cavities of female Buffalo rats (approx. 250 g; National C a n c e r Institute) a s described by Frey and Kebabian (3). After about three weeks, t u m o r s were r e m o v e d from t h e peritoneal cavities, cells dispersed, a n d grown as primary c u l t u r e s in DMEM m e d i u m containing 2 mM g l u t a m i n e and 10% fetal calf serum. Sources of materials, and p r o c e d u r e s for cell culture, m e m b r a n e preparation, AC assay, and m e a s u r e m e n t s of [3H]DIP binding, are contained in Puttfarcken et al. (6). After pre-incubation with morphine, the cells and m e m b r a n e s u s p e n s i o n s were w a s h e d extensively prior to use in AC or ligand binding assays. No significant a m o u n t s of m o r p h i n e added in the pre-incubation remained in the w a s h e d m e m b r a n e suspensions (6). In the assays of AC activity, estimates of forskolin (10 idVl)-stimulated AC activity were m a d e in triplicate for each concentration of DAMGO tested. For each m o r p h i n e p r e t r e a t m e n t condition, three i n d e p e n d e n t DIP competition c u r v e s against [3H]DIP (1 nM) binding, each employing 18 to 21 c o n c e n t r a t i o n s of unlabeled DIP in triplicate were generated. The reported e s t i m a t e s of KD and B m a x were p r e p a r e d by analysis of the pooled r e s u l t s of the three experiments for each p r e t r e a t m e n t condition using the program LIGAND (7). Results Effects of m o r p h i n e p r e t r e a t m e n t on inhibition of AC activity by DAMGO Cells were grown in m e d i u m without morphine, or in m e d i u m containing m o r p h i n e in concentrations from 10 nM to 300 ~M. Membrane s u s p e n s i o n s were prepared from b a t c h e s of cells exposed to t h e d r u g for periods of 5 h r or 48 hr. In m e m b r a n e s from cells i n c u b a t e d w i t h o u t m o r p h i n e , DAMGO (1 I~M} inhibited f o r s k o l i n - s t i m u l a t e d AC a b o u t 70-75% (Fig. 1A, B). After exposure to m o r p h i n e for 5 h r (Fig. 1A) or 48 h r (Fig 1B) in concentrations of 100 nM or higher the potency of DAMGO in inhibiting AC was reduced. After 5 h r pretreatment, DAMGO inhibition of AC was significantly reduced following m o r p h i n e p r e t r e a t m e n t s at 1 ~M and higher concentrations (ANOVA: 1 IJM m o r p h i n e v. control, FI,4 59.084, P = 0.002; 100 nM morphine v control, FI,4 1.871. P>0.05). After 48 hr of morphine the ability of DAMGO to inhibit AC was significantly reduced by m o r p h i n e p r e t r e a t m e n t concentrations of 100 nM and higher (100 nM morphine v. control, FI,4 106.02, P = 0.001). After 48 h r exposure to the highest m o r p h i n e p r e t r e a t m e n t c o n c e n t r a t i o n a n inhibitory effect of DAMGO was no longer a p p a r e n t at any tested DAMGO concentration. At neither pretreatment time was the inhibitory effect of DAMGO significantly r e d u c e d by p r e t r e a t m e n t with 10 nM m o r p h i n e . P r e t r e a t m e n t with m o r p h i n e did not affect t h e level of f o r s k o l i n - s t i m u l a t e d adenylate cyclase activity in the absence of added opioid (Table 1). After b o t h p r e t r e a t m e n t times, the effects of high DAMGO c o n c e n t r a t i o n s (0.1, 1 ~tM) were r e d u c e d to a g r e a t e r extent t h a n t h o s e of lower DAMGO c o n c e n t r a t i o n s . M a x i m u m inhibitory effects of m u - r e c e p t o r activation were a s s e s s e d by m e a s u r e m e n t of the inhibotory activity of 1 ~tM DAMGO. (Higher c o n c e n t r a t i o n s of DAMGO gave g r e a t e r inhibition of adenylate cyclase, b u t the effects of c o n c e n t r a t i o n s of DAMGO in excess of i ~M were not reliably inhibited by naloxone). The results suggest t h a t the p r e t r e a t m e n t s reduced the m a x i m u m opioid receptor-mediated inhibition that could be obtained with DAMGO wlth little or no increase in the estimated DAMGO IC50 (Fig. I, Table 1).
Vol. 45, No. 20, 1989
Tolerance at Mu-Opioid Receptors
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Fig. 1. I n h i b i t i o n of f o r s k o l i n - s t i m u l a t e d AC b y DAMGO i n m e m b r a n e s f r o m 7 3 1 5 c cells p r e t r e a t e d w i t h m o r p h i n e for 5 h r (A) or for 48 h r (B). 7 3 1 5 c cells were p r e p a r e d a n d c u l t u r e d a s p r e v i o u s l y d e s c r i b e d (5). After i n c u b a t i o n w i t h m o r p h i n e - c o n t a i n i n g m e d i u m for 5 or 48 hr, t h e cells were h o m o g e n i z e d a n d w a s h e d m e m b r a n e s p r e p a r e d . I n h i b i t i o n of forskolin (10 l~Vl)-sUmulated AC b y c o n c e n t r a t i o n s of DAMGO from 1 n M to 1 ~VI w a s m e a s u r e d . Points r e p r e s e n t m e a n s v a l u e s (+ s.e.m.) of at l e a s t six e s t i m a t e s for e a c h t r e a t m e n t . (In s o m e cases, s t a n d a r d e r r o r b a r s are o m i t t e d for clarity). M o r p h i n e p r e t r e a t m e n t c o n c e n t r a t i o n s : • 10 nM, Z~ 100 nM, • i pM, O I0 I~M, • 300 ~uM; O control medium without added morphine.
T a b l e 1. F o r s k o l i n - s t i m u l a t e d AC activity a n d DAMGO i n h i b i t i o n of f o r s k o l i n - s t l m u l a t e d AC activity a f t e r e x p o s u r e of 7 3 1 5 c cells to m o r p h i n e .
M o r p h i n e pret r e a t m e n t eonc. (nM) 0
D u r a t i o n of p r e t r e a t m e n t 5 hours 48 h o u r s AC activity Approx. AC activity Approx. p m o l / r n g p m t e t n / m i n DAMGO p m o l / m g p m t e i n / m i n DAMGO IC50 (nM) IC50 (nM) 21+3
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AC activity v a l u e s r e p o r t t h e m e a n (+ s.e.m, of a t l e a s t six e s t i m a t e s for e a c h p r e t r e a t m e n t ) of AC a c t i v i t i e s i n t h e p r e s e n c e of 100 BM f o r s k o l i n , m e a s u r e d o v e r a p e r i o d of 10 rain. A p p r o x i m a t e ICs0 v a l u e s for i n h i b i t i o n of e n z y m e activity b y DAMGO w e r e e s t i m a t e d from Fig. 1, a s s u m i n g t h a t m a x i m u m opioid r e c e p t o r - m e d i a t e d i n h i b i t i o n of e n z y m e activity w a s observed a t 1 I~M DAMGO (see text).
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Tolerance at Mu-Opioid Receptors
Vol. 45, No. 20, 1989
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(M)
Fig. 2. Effects of m o r p h i n e p r e t r e a t m e n t s for 5 h r (open circles) or 48 h r (closed circles) o n the d e n s i t y of [3H]DIP b i n d i n g s i t e s (Bmax) in 7 3 1 5 c cell m e m b r a n e s . W a s h e d cell m e m b r a n e s were i n c u b a t e d w i t h 1 n M [3H]DIP a t 3 7 ° for 2 0 r a i n i n t h e p r e s e n c e or a b s e n c e of a r a n g e of c o n c e n t r a t i o n s of u n l a b e l e d DIP; b i n d i n g w a s t e r m i n a t e d b y f i l t r a t i o n , a n d r e t a i n e d radioactivity e s t i m a t e d b y liquid scintillation c o u n t i n g . E s t i m a t e s of DIP K D a n d Bma x were o b t a i n e d b y a n a l y s i s of t h e pooled d a t a from t h r e e e x p e r i m e n t s , u s i n g t h e p r o g r a m LIGAND (7). D a t a p o i n t s i n d i c a t e t h e c o m p u t e d Bma x e s t i m a t e s ( f m o l / m g m e m b r a n e protein) a n d s t a n d a r d e r r o r s of t h e e s t i m a t e s . P r e t r e a t m e n t c o n c e n t r a t i o n s of m o r p h i n e a r e i n d i c a t e d a s t h e n e g a t i v e l o g a r i t h m of t h e m o r p h i n e c o n c e n t r a t i o n ; C i n d i c a t e s v a l u e s o b t a i n e d from control ceils i n c u b a t e d for t h e i n d i c a t e d t i m e s i n t h e a b s e n c e of a d d e d m o r p h i n e . KD v a l u e s for DIP were n o t a l t e r e d significantly b y a n y t r e a t m e n t ; m e a n K D for all t r e a t m e n t w a s 0 . 7 3 + 0.09 nM.
Effects of m o r p h i n e p r e t r e a t m e n t s o n [3H]DIP binding. P r e t r e a t m e n t of 7 3 1 5 c cells with 100 BlVlm o r p h i n e for 72 h r h a s b e e n s h o w n to r e d u c e t h e n u m b e r o f m u - r e c e p t o r s i n 7 3 1 5 c e e l m e m b r a n e s . However, n o c h a n g e in r e c e p t o r n u m b e r w a s a p p a r e n t after 5 h r (6). The effects o n r e c e p t o r n u m b e r of p r e t r e a t m e n t of 7 3 1 5 c cells with a r a n g e of c o n c e n t r a t i o n s of m o r p h i n e is s h o w n in Fig. 2. No r e d u c t i o n in t h e n u m b e r of r e c e p t o r s w a s o b s e r v e d a f t e r 5 h r of p r e t r e a t m e n t at a n y t e s t e d m o r p h i n e p r e t r e a t m e n t c o n c e n t r a t i o n . After 4 8 h r of p r e t r e a t m e n t t h e r e w a s a s m a l l r e d u c t i o n i n t h e n u m b e r of r e c e p t o r s after e x p o s u r e to 100 n M m o r p h i n e , a n d s u b s t a n t i a l r e d u c t i o n s in r e c e p t o r n u m b e r a f t e r e x p o s u r e to 10 BM a n d 3 0 0 BM m o r p h i n e . None of t h e p r e t r e a t m e n t s p r o d u c e d a n y s i g n i f i c a n t a l t e r a t i o n s in t h e [3H]DIP K D.
Discussion T h e s e r e s u l t s c o n f i r m o u r p r e v i o u s s t u d i e s s h o w i n g t h a t p r e t r e a t m e n t of 7 3 1 5 c p i t u i t a r y t u m o r cells w i t h a h i g h c o n c e n t r a t i o n (100 BM) of m o r p h i n e for a p e r i o d of 5 h r r e s u l t e d i n a loss of t h e ability of a n opioid a g o n i s t to i n h i b i t f o r s k o l i n - s t i m u l a t e d AC at a t i m e w h e n m u - o p i o i d r e c e p t o r n u m b e r w a s u n c h a n g e d (6), Only a f t e r l o n g e r p e r i o d s of m o r p h i n e e x p o s u r e w a s a s i g n i f i c a n t r e d u c t i o n i n t h e n u m b e r of m u - t y p e b i n d i n g s i t e s observed. T h e c u r r e n t r e s u l t s indicate t h a t b o t h d e s e n s i t i z a t i o n of opioid i n h i b i t i o n of AC a n d d o w n - r e g u l a t i o n of m u - t y p e b i n d i n g s i t e s are o b s e r v e d w h e n 7 3 1 5 c cells are exposed to lower c o n c e n t r a t i o n s of m o r p h i n e t h a n u s e d i n t h e initial study. The m a g n i t u d e s of b o t h t h e d e s e n s i t i z a t i o n a n d t h e d o w n - r e g u l a t i o n were related to t h e c o n c e n t r a t i o n of m o r p h i n e to w h i c h t h e ceils were exposed. No s i g n i f i c a n t d e s e n s i t i z a t i o n or
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d o w n - r e g u l a t i o n w a s o b s e r v e d w h e n 7 3 1 5 c cells were t r e a t e d w i t h 10 n M m o r p h i n e for 5 or 48 hr, or w i t h 100 n M for 5 h o u r s . After 4 8 h r of e x p o s u r e to 100 n M m o r p h i n e a s l i g h t d e s e n s i t i z a t i o n w a s a p p a r e n t . T h i s c o n c e n t r a t i o n of m o r p h i n e is s i m i l a r to t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t of m o r p h i n e for t h e h i g h e r - a l I l n i t y form of t h e m u - r e c e p t o r m e a s u r e d i n 7 3 1 5 c m e m b r a n e s i n t h e p r e s e n c e of a s t a b l e GTP a n a l o g u e (Werling et al., m a n u s c r i p t i n preparation). M o r e m a r k e d d e s e n s i t i z a t i o n r e q u i r e d t r e a t m e n t of t h e c e l l s w i t h c o n c e n t r a t i o n s of m o r p h i n e of 1 ~M or h i g h e r . S i m i l a r l o s s e s i n opioid i n h i b i t o r y p o t e n c y were o b s e r v e d a f t e r t r e a t m e n t of t h e cells w i t h t h e s e c o n c e n t r a t i o n s of m o r p h i n e for periods of 5 h r or 4 8 hr, s u g g e s t i n g t h a t t h e d e s e n s i t i z a t i o n p r o c e s s is largely c o m p l e t e w i t h i n a period of 5 h r f r o m i n i t i a t i o n of m o r p h i n e e x p o s u r e . No r e c e p t o r d o w n - r e g u l a t i o n w a s o b s e r v e d after 5 h r of m o r p h i n e t r e a t m e n t a t a n y of t h e c o n c e n t r a t i o n s t e s t e d . However, after 48 h r of t r e a t m e n t , a c l e a r loss i n r e c e p t o r n u m b e r w a s a p p a r e n t w h e n t h e cells were e x p o s e d to 10 ~M a n d h i g h e r c o n c e n t r a t i o n s of m o r p h i n e . T r e a t m e n t w i t h 0.1 or 1 lJaVIm o r p h i n e i n d u c e d s m a l l b u t statistically s i g n i f i c a n t r e d u c t i o n s in t h e n u m b e r of receptors. It a p p e a r s to t a k e longer for d o w n - r e g u l a t i o n to occur, a n d to require e x p o s u r e to a m o r p h i n e c o n c e n t r a t i o n of a b o u t 10 ~Vl or m o r e to p r o d u c e m o r e t h a n a 2 5 % loss of r e c e p t o r s . T h i s c o n c e n t r a t i o n of m o r p h i n e is h i g h e r t h a n t h e lowest affinity s t a t e of t h e 7 3 1 5 c m u - b i n d i n g site for m o r p h i n e m e a s u r e d i n t h e p r e s e n c e of a s t a b l e GTP a n a l o g u e (Werling et al., i n p r e p a r a t i o n ) . It a p p e a r s t h a t very h i g h levels of m u - r e c e p t o r o c c u p a n c y b y m o r p h i n e are r e q u i r e d over a period of t i m e i n excess of 5 h r for s u b s t a n t i a l m u - r e c e p t o r downr e g u l a t i o n to b e o b s e r v e d in 7 3 1 5 c cells. T h e m e c h a n i s m s b y w h i c h a g o n i s t p r e t r e a t m e n t i n d u c e s d e s e n s i t i z a t i o n a r e still n o t clear. R e s u l t s p r e s e n t e d h e r e a n d elsewhere (2,6) i n d i c a t e t h a t r e c e p t o r d o w n - r e g u l a t i o n is n o t r e q u i r e d for a loss of a g o n i s t p o t e n c y . A c h a r a c t e r i s t i c f e a t u r e of d e s e n s i t i z a t i o n i n t h i s s y s t e m is a p r o g r e s s i v e r e d u c t i o n i n t h e m a x i m u m opioid r e c e p t o r - m e d i a t e d i n h i b i t i o n of AC activity (as i n d i c a t e d b y t h e i n h i b i t o r y effect of 1 lIM DAMGO; h i g h e r c o n c e n t r a t i o n s of DAMGO s o m e t i m e s p r o d u c e d a g r e a t e r i n h i b i t i o n , b u t t h e i r effects were n o t reliably i n h i b i t e d b y t h e opioid a n t a g o n i s t naloxone). T h e r e w a s very little i n c r e a s e i n t h e e s t i m a t e d IC50 for DAMGO a s a r e s u l t of p r i o r m o r p h i n e exposure. T h u s , t h e effects of m o r p h i n e p r e t r e a t m e n t o n t h e ability of DAMGO to i n h i b i t AC are m o r e a n a l o g o u s to t h e loss of a g o n i s t r e s p o n s e i n d u c e d b y a n irreversible a n t a g o n i s t t h a n to t h e r i g h t w a r d shift i n t h e c o n c e n t r a t i o n - r e s p o n s e c u r v e o b s e r v e d i n t h e p r e s e n c e of a competitive antagonist.. M o r p h i n e p r e t r e a t m e n t h a s l e s s effect o n t h e p o t e n c y of DAMGO ( m e a s u r e d b y its IC50) t h a n o n its efficacy (as i n d i c a t e d b y its i n h i b i t o r y effect at 1 gM). T h e s e c h a r a c t e r i s t i c s w o u l d b e expected ff t h e m e c h a n i s m of d e s e n s i t i z a t i o n w a s a r e d u c e d ability of t h e a g o n i s t - o c c u p i e d r e c e p t o r to i n t e r a c t w i t h or to activate t h e g u a n i n e n u c l e o t i d e b i n d i n g p r o t e i n (G-protein) w h i c h m e d i a t e s opioid a c t i o n in t h i s s y s t e m (4,5,8). T h e m a x i m u m i n h i b i t o r y effect is r e d u c e d b e c a u s e a s u b s t a n t i a l f r a c t i o n of t h e r e c e p t o r pool c a n n o l o n g e r a c t i v a t e G - p r o t e i n . T h i s p r o p o s e d m e c h a n i s m is s u p p o r t e d b y o u r p r e v i o u s d e m o n s t r a t i o n t h a t d e s e n s i t i z a t i o n i n d u c e d b y m o r p h i n e is a s s o c i a t e d w i t h a l o s s i n t h e ability of g u a n i n e n u c l e o t i d e s to m o d u l a t e opioid agonist affinity a t m u - r e c e p t o r s i n 7 3 1 5 c cell m e m b r a n e s (6) a n d in c e r e b r a l cortex m e m b r a n e s of g u i n e a pig (9). At t h i s t i m e it is u n c l e a r if t h e i m p a i r m e n t in r e c e p t o r - G p r o t e i n i n t e r a c t i o n is r e l a t e d to c h a n g e s in t h e p r o p e r t i e s of t h e r e c e p t o r or of t h e G protein(s). R e c e p t o r d o w n - r e g u l a t i o n w a s s l o w e r in o n s e t t h a n d e s e n s i t i z a t i o n , a n d w a s o n l y observed after pretreatment with morphine concentrations that induced substantial d e s e n s i t i z a t i o n . It is p o s s i b l e t h a t u n c o u p l i n g of r e c e p t o r from G - p r o t e i n (either physically or functionally) a c c e l e r a t e s t h e r a t e of r e c e p t o r r e m o v a l from t h e m e m b r a n e , or t h a t c h a n g e s i n r e c e p t o r p r o p e r t i e s w h i c h p r e v e n t G - p r o t e i n a c t i v a t i o n also trigger r e c e p t o r d e g r a d a t i o n . It m a y b e r e l e v a n t t h a t a r e d u c t i o n in m u - r e c e p t o r d e n s i t y w a s also o b s e r v e d following p e r t u s s i s t o x i n t r e a t m e n t of 7 3 1 5 c cells to i n d u c e a f u n c t i o n a l u n c o u p l i n g of m u - r e c e p t o r s a n d a s s o c i a t e d G - p r o t e i n s (5). However, it is also possible t h a t s u s t a i n e d r e c e p t o r a c t i v a t i o n r e s u l t s indirectly in a r e d u c e d rate of r e c e p t o r s y n t h e s i s . O u r r e s u l t s s u g g e s t t h a t t h e p r i m a r y m e c h a n i s m for m o r p h i n e t o l e r a n c e a t m u r e c e p t o r s i n 7 3 1 5 c p i t u i t a r y t u m o r cells is a n i m p a i r m e n t of r e c e p t o r - G p r o t e i n i n t e r a c t i o n r e s u l t i n g i n a r e d u c e d ability of a g o n i s t - o c c u p i e d r e c e p t o r s to initiate a r e s p o n s e . At a l a t e r t i m e a n d a f t e r e x p o s u r e to v e r y h i g h c o n c e n t r a t i o n s of m o r p h i n e s o m e r e d u c t i o n i n t h e n u m b e r of m u - r e c e p t o r s also occurs, b u t r e c e p t o r d o w n - r e g u l a t i o n is n o t n e c e s s a r y for a l m o s t c o m p l e t e t o l e r a n c e to b e observed.
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Acknowledgements This s t u d y w a s s u p p o r t e d b y a g r a n t from t h e National I n s t i t u t e o n Drug Abuse. We t h a n k Dr. L i n d a Werling a n d Dr. T o m Cote for helpful d i s c u s s i o n i n t h e p r e p a r a t i o n of t h i s m a n u s c r i p t , a n d P a u l M c M a h o n for t e c h n i c a l a s s i s t a n c e . T h e o p i n i o n s a n d a s s e r t i o n s c o n t a i n e d h e r e i n are t h e p r i v a t e o n e s of t h e a u t h o r s , a n d are n o t to b e c o n s t r u e d a s reflecting t h e views of t h e U n i t e d S t a t e s D e p a r t m e n t of D e f e n s e or t h e U n i f o r m e d S e r v i c e s U n i v e r s i t y of t h e H e a l t h Sciences. A n i m a l s u s e d i n t h i s s t u d y were a c q u i r e d a n d c a r e d for i n a c c o r d a n c e w i t h t h e g u i d e l i n e s p u b l i s h e d i n t h e N a t i o n a l I n s t i t u t e s of H e a l t h G u i d e for t h e C a r e a n d Use of L a b o r a t o r y A n i m a l s (National I n s t i t u t e s of H e a l t h P u b l i c a t i o n No. 85-23, revised 1985). References 1. J . MAGNAN, S.J. PATERSON, A, TAVANI, a n d H.W. KOSTERLITZ. N a u n y n - S c h m i e d e b e r g s Arch. Pharmacol. 3 1 9 : 1 9 7 - 2 0 5 (1982). 2. P.Y. LAW, D.S. HOM, a n d H.H.LOH. Mol. Pharmacol. 2 4 : 4 1 3 - 4 2 4 (1983). 3. H.H. LOH, P.-L. TAO andA.P. SMITH. Synapse 2 : 4 5 7 - 4 6 2 (1988). 4. E. F R E Y a n d J . KEBABAIAN,. Endocrinology 1 1 5 : 1 7 9 7 - 1 8 0 4 (1984). 5. L.L.WERLING, P.S. PUTTFARCKEN, a n d B.M. COX. Mol. Pharmacol. 33: 423-431,(1988). 6. P.S. PUTFFARCKEN, L.L.WERLING, a n d B.M. COX. Mol. Pharmacol. 3 3 : 5 2 0 - 5 2 7 (1988). 7. P.J. MUNSON a n d D. RODBARD. Analyt. Biochem. 1 0 7 : 2 2 0 - 2 3 9 (1980). 8. D.L. AUB, E. FREY, R.D. SEKURA, a n d T.E. COTE. J. Biol. Chem. 2 6 1 : 9 3 3 3 - 9 3 4 0 (1986). 9. L.L. WERLING, P.N. McMAHON, a n d B.M. COX. Proc. Natl. Acad. Sci. USA 8 6 : 6 3 9 3 - 6 3 9 7 (1989).