Correlation of inhibitory potencies of putative antagonists for σ receptors in brain and spleen

European Journal of Pharmacology, 148 (1988) 467-470

467

Elsevier EJP 20105 Short communication

Correlation of inhibitory potencies of putative antagonists for o receptors in brain and spleen T s u n g - P i n g Su, S u z a n n e E. Schell, Felicia Y. F o r d - R i c e a n d E d y t h e D. L o n d o n * Neuropharmacology Laboratory, Addiction Research Center, National Institute on Drug Abuse, P. 0. Box 5180, Baltimore, Maryland 21224, U.S.A.

Received 24 December 1987, accepted 2 February 1988 The putative a receptor antagonists, haloperidol, HR 375, BMY 14802 and BW 234U potently inhibited both [3H]d-N-allylnormetazocine binding to o receptors in brain homogenates and [3H]haloperidol binding to a receptors in spleen homogenates. An excellent correlation of inhibitory potencies in the two assay systems was obtained. The results support the view that [3H]d-N-allylnormetazocine and [3H]haloperidol both label the same receptor populations, and suggest that o antagonists may be useful in elucidating physiological role(s) of o receptors in the nervous and immune systems. o Receptors; N-Allylnormetazocine; SK & F 10047; Spleen; Brain; Haloperidol; HR 375; BMY 14802; BW 234U 1. Introduction The existence of o receptors has been postulated by Martin et al. (1976) to explain canine delirium and human psychosis induced by N-allylnormetazocine ( N A N M , SKF-10047). Subsequently, two distinct populations of binding sites have been implicated as mediators of the psychotomimetic effects induced by N A N M . One of these has been identified using [3H]phencyclidine as the ligand (Zukin and Zukin, 1979). The other site is haloperidol-sensitive and displays a higher affinity for [3H]NANM than phencyclidine (Su, 1982). Several ligands possess affinities for both sites, implying a structural similarity between the sites; however, other drugs exhibit selective binding to only one of the sites. In keeping with the findings of a committee on nomenclature, the haloperidol-insensitive receptor labelled with [3H]phencyclidine has been termed the 'phencyclidine receptor' and the other site, the 'o receptor' (Quirion et al., 1987).

* To whom all correspondence should be addressed.

Although it is still not clear whether either of the two receptors mediates the psychotomimetic effects of N A N M , potential antagonists for the o receptor have been synthesized. In preclinical trials, these drugs, BW 234U (Ferris et al., 1986), H R 375 (Su, 1986) and BMY 14802 (Taylor and Dekleva, in press) were characterized as 'atypical' antipsychotics that do not exert their effects through dopaminergic antagonism. Haloperidol, which was the most potent inhibitor in the o receptor binding assay (Su, 1982) also antagonized the effects of o agonists in potentiating the electrically induced contractions of the guinea-pig vas deferens (Vaupal and Su, 1987). Recent studies have demonstrated the existence of o receptors in spleen homogenates as well as on peripheral blood lymphocytes (Wolfe et al., 1987). Those results suggest therefore that o receptors may also play an important role in immune functions. If so, the potential therapeutic application of o antagonists in pathophysiological states involving the lymphoid o receptors should not be overlooked. In this study we examined the inhibitory potencies of the four putative o receptor antago-

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

468 nists (i.e., haloperidol, H R 375, B M Y 14802 a n d BW 234U) in o r e c e p t o r b i n d i n g assays using c r u d e m e m b r a n e p r e p a r a t i o n s of the g u i n e a - p i g b r a i n and spleen, a n d c o r r e l a t e d their p o t e n c i e s in the respective assays.

ml c o n t a i n e d 500 /*1 of h o m o g e n a t e , 1 n M [ 3 H ] h a l o p e r i o d o l , 50 n M s p i p e r o n e to block radiol i g a n d b i n d i n g to D 2 d o p a m i n e receptors, and inhibitor. Specific b i n d i n g was d e f i n e d in the presence of 0.1 m M u n l a b e l l e d d - N A N M . T h e i n c u b a t i o n was carried out at 2 5 ° C for 2 h, a n d t e r m i n a t e d by r e d u c e d p r e s s u r e filtration using a Brandel cell harvester. IC50 values ( c o n c e n t r a t i o n required to inhibit 50% of specific b i n d i n g of r a d i o l i g a n d to r e c e p t o r sites) were o b t a i n e d from d o s e - i n h i b i t i o n studies p e r f o r m e d with five to ten i n h i b i t o r concentrations, of which at least three c o n c e n t r a t i o n s red u c e d specific b i n d i n g by 20 to 80%. K i values of the i n h i b i t o r s in the o r e c e p t o r b i n d i n g assay were c a l c u l a t e d f r o m the IC5o values using the ChengPrusoff e q u a t i o n , with e s t i m a t e d K d values for [ 3 H ] d - N A N M a n d [ 3 H ] h a l o p e r i d o l being 90 nM + 8 ( n = 3 ) a n d 0.3 n M _ + 0 . 0 4 ( n = 4 ) , respectively. BW 234U, B M Y 14802 a n d H R 375 were generous gifts from Drs. R.M. Ferris ( W e l l c o m e R e s e a r c h L a b o r a t o r i e s , Research Triangle Park, N C ) , D.P. T a y l o r (Bristol-Myers, W a l l i n g f o r d , CT) a n d F.J. H o c k (Hoechst, F . R . G . ) respectively.

2. Materials and methods Tissue was o b t a i n e d f r o m m a l e H a r t l e y guinea-pigs weighing 300 to 400 g. Brains a n d spleens were excised after c a r b o n d i o x i d e a s p h y x i a t i o n of the animals. Brains were frozen a n d stored at - 7 0 ° C until they were used. Brain h o m o genates were p r e p a r e d as d e s c r i b e d in detail elsewhere (Su, 1982). A final c o n c e n t r a t i o n of 2 n M of [ 3 H ] d - N A N M (23 C i / m m o l ; N e w E n g l a n d N u c l e a r ) was used to label o receptors in the b r a i n h o m o g e n a t e s . Specific b i n d i n g was det e r m i n e d in the presence of 0.1 m M of u n l a b e l e d d - N A N M . The rest of the p r o c e d u r e s were the same as d e s c r i b e d elsewhere (Su, 1986). Spleens were kept on ice a n d assayed imm e d i a t e l y on the same day. T h e y were h o m o g e nized in 40 volumes ( w / v ) of ice-cold 0.05 M T r i s - H C l ( p H 8.0) using a B r i n k m a n n p o l y t r o n (setting 5, twice for 5 s each). A f t e r c e n t r i f u g a t i o n at 3 5 0 0 0 × g at 4 ° C for 10 min, the resultant pellets were r e h o m o g e n i z e d . T h e c e n t r i f u g a t i o n a n d r e h o m o g e n i z a t i o n steps were r e p e a t e d once more. The final h o m o g e n a t e s r e p r e s e n t e d an original weight of a p p r o x i m a t e l y 7 mg wet t i s s u e / m l buffer. [ 3 H ] H a l o p e r i d o l (8 C i / m m o l ; N e w Eng l a n d N u c l e a r ) was used to label o r e c e p t o r s in the spleen h o m o g e n a t e s . A final assay v o l u m e of 3

3. Results T h e r a n k o r d e r of p o t e n c i e s of the four inhibitors in o r e c e p t o r b i n d i n g was the same in b o t h b r a i n a n d spleen h o m o g e n a t e s (table 1). Haloperidol, a b u t e r o p h e n o n e D 2 d o p a m i n e receptor a n t a g o n i s t , was the most p o t e n t of the four drugs

TABLE 1 K i values and Hill slopes of the dose-inhibition curves of the putative o antagonists in guinea-pig brain and spleen o receptor binding assays. Specific binding to o receptors in brain was determined using 2 nM [3H]d-NANM; specific binding in the spleen was determined using 1 nM [~H]haloperidol in the presence of 50 nM spiperone. Values represent means+ S.E.M. for the number of experiments indicated in parentheses. Brain assays were performed in quadruplicates and spleen assays in triplicates.

Drug Haloperidol HR375 BMY 14802 BW 234U

Brain [3H]d-NANM

Spleen [ 3H]Haloperidol

K i (nM)

nH

(n)

K i (nM)

nH

(n)

1.46 + 0.3 30.6 + 1.1 223 + 28 689 +117

1.09 + 0.07 1.30_+0.05 0.59_+0.10 0.97_+0.14

(4) (3) (2) (4)

0.24 + 0.02 7.33_+ 0 . 5 6 150 +_ 7 403 +184

1.07 _+0.02 1.23+0.16 1.62_+0.14 1.07+0.16

(3) (3) (3) (3)

469 Z

The existence of o receptors in the spleen and lymphoid tissues suggests that o receptors may

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-I 400

0 40

4

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200

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~oo

-~ re

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0 i

0

i

100

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200

i

300

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400

i

500

:

600

i

700

i

800

i

900

Ki VALUES (nM) IN GUINEA-PIG B R A I N <~ RECEPTOR BINDING ASSAY

Fig. 1. Correlation of K i values of haloperidol (1), HR 375 (2), BMY 14802 (3) and BW 234U (4) from o receptor binding assays in the brain and in the spleen. The K i values were taken from table 1. The line was drawn from all points by best-fit linear regression analysis.

as a n i n h i b i t o r of o receptor b i n d i n g in b r a i n a n d spleen homogenates. H R 375, which also b i n d s to b o t h D 2 d o p a m i n e receptors a n d o receptors in the b r a i n (Su, 1986), was the next p o t e n t i n h i b i t o r in both o receptor assay systems. H R 375 was a b o u t 30-fold less p o t e n t than haloperidol, a n d racemic B M Y 14802 was slightly more p o t e n t t h a n BW 234U. BW 234U was the least p o t e n t of the drugs tested. W h e n the K i values o b t a i n e d for the four putative o antagonists in the two different assay systems were compared, a correlation coefficient of 0.99 was o b t a i n e d (fig. 1).

4. Discussion T h e r a n k orders of potencies a n d the excellent correlation of the K i values of the four putative o antagonists in the b r a i n a n d spleen e receptor b i n d i n g assays indicate that the four drugs were i n h i b i t i n g [ 3 H ] d - N A N M a n d [3H]haloperidol b i n d i n g to a c o m m o n site. The b i n d i n g site would most likely be o receptors as the c o n d i t i o n s used in both assay systems optimized the b i n d i n g of the labelled ligands to o receptors. O u r results corroborate the previous f i n d i n g that o receptors exist in the spleen (Wolfe et al., 1987).

represent a cellular substrate i n f l u e n c i n g certain i m m u n e functions. The speculation is in keeping with the previous f i n d i n g that p h e n c y c l i d i n e depressed cellular a n d h u m o r a l i m m u n e responses of i m m u n o c y t e s in vitro ( K h a n s a r i et al., 1984). Although p h e n c y c l i d i n e b i n d s preferentially to p h e n c y c l i d i n e receptors, it shows m o d e r a t e affinity for o receptors (Su, 1982). Thus, the i m m u n o suppressive effects of p h e n c y c l i d i n e could reflect a n action at o receptors. The d e m o n s t r a t i o n of o receptors in the spleen raises the possibility that specific o antagonists m a y have clinical applications in disorders of imm u n e function. T h e present findings d e m o n s t r a t e that several p u t a t i v e o antagonists b i n d to o receptors in l y m p h o i d tissue. These drugs m a y be helpful in elucidating the i n v o l v e m e n t of o receptors in i m m u n e function.

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470 Vaupal, D.B. and T.-P. Su, 1987, Guinea-pig vas deferens preparation may contain both o receptors and phencyclidine receptors, European J. Pharmacol. 139, 125. Wolfe, S.A. Jr., C. Kulsakdinun and E.B. De Souza, 1987, Sigma receptors in human peripheral blood leukocytes

(HPBL) and rat spleen: identification, characterization and autoradiographic localization, Soc. Neurosci. 13, 1437. Zukin, S.R. and R.S. Zukin, 1979, Specific [3H]phencyclidine binding in rat central nervous systems, Proc. Natl. Acad. Sci. 76~ 5372.