Life Sciences, Vol. 49, pp. 1155-1161
Pergamon P r e s s
P r i n t e d An t h e U . S . A .
PERIPHERAL-TYPE
B E N Z O D I A Z E P I N E RECEPTORS IN H U M A N CEREBRAL CORTEX, KIDNEY, AND C O L O N M. Awad
and M. G a v i s h
R a p p a p o r t Family Institute For R e s e a r c h in the Medical S c i e n c e s and D e p a r t m e n t of Pharmacology, Bruce R a p p a p o r t F a c u l t y of Medicine, T e c h n i o n - I s r a e l Institute of Technology, P.O.B. 9697, 31096 Haifa, Israel (Received in final form August 5, 1991) Summary The specific binding of [3H]PK 11195 and [3H]Ro 5-4864 to h u m a n cerebral cortex, kidney, and colon m e m b r a n e s was studied in order to d e t e r m i n e w h e t h e r p e r i p h e r a l - t y p e b e n z o d i a z e p i n e r e c e p t o r s (PBR) c h a r a c t e r i s t i c s located in human tissues are similar tw those located in calf or rat tissues. W h i l e [vH]PK 11195 (0.05-10 nM, final concentration) b o u n d w i t h high a f f i n i t y (K D about 2 nM) to h u m a n cerebral cortex, kidney, and colon membranes, y i e l d i n g maximal n u m b e r s of b i n d i n g sites of 255 + 23, 1908 + 28, and 1633 Z 98 fm~i/mg protein, respectively, the specific b i n d i n g of [ H]Ro 5-4864 (1.25-40 nM, final concentration), was b a r e l y d e t e c t a b l e (nonspecific b i n d i n g about 90% of the total binding). Furthermore, u n l a b e l e d PK 11195 was two orders of m a g n i t u d e more p ~ t e n t than u n l a b e l e d Ro 5-4864 in d i s p l a c i n g [vH]PK 11195 specific binding from h u m a n cerebral cortex and k i d n e y membranes. These results indicate that PBR b i n d i n g c h a r a c t e r i s t i c s located in h u m a n tissues are similar (but not identical) to those located in calf tissues, but not to those located in rat tissues.
B e n z o d i a z e p i n e (BZ) receptors have been c l a s s i f i e d in two p h a r m a c o l o g i c a l l y distinct types of receptors: "central-type" BZ r e c e p t o r s (CBR) located in the m a m m a l i a n central nervous system (1,2) and "peripheral-type" BZ receptors (PBR) located in rat p e r i p h e r a l tissues and cells such as kidney, lung, liver, heart, mast cells, thymocytes, and platelets, as well as in the b r a i n (2-9).PBR have also been c h a r a c t e r i z e d in some h u m a n tissues such as placenta, irls,myometrium, l y m p h o c y t e s , a n d p l a t e l e t s (10-16). PBR and CBR differ in their d i s t r i b u t i o n w i t h i n the brain, s u b c e l l u l a r localization, and ligand specificity. In rat and guinea pig, Ro 5-4864 (4'-chlorodiazepam) and PK 11195 [l-(2-chloro phenyl)-N-methyl-N-(l-methylpropyl)-3-isoquinoline carboxamide] b i n d w i t h h i g h a f f i n i t y to PBR,but not to C B R ; w h e r e a s clonazepam, * Correspondence
and reprints. 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
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w h i c h b i n d s w i t h h i g h a f f i n i t y to CBR, e x h i b i t s o n l y m i c r o m o l a r b i n d i n g a f f i n i t y to PBR. A d d i t i o n a l l y , C B R are c o u p l e d to y-aminobutyric a c i d r e c e p t o r s a n d a l s o to s p e c i f i c b i n d i n g s i t e s for a n i o n s a n d d i v a l e n t c a t i o n s (17-19), w h e r e a s P B R are not. Recent studies have identified marked species differences b e t w e e n P B R l o c a t e d in c a l f t i s s u e s a n d P B R l o c a t e d in r a t t i s s u e s , w i t h r e f e r e n c e to t h e i r b i n d i n g a f f i n i t y for the c l a s s a c a l p r o t o t y p e P B R h i g h - a f f i n i t y BZ l i g a n d Ro 5-4864. S u c h s p e c i e s d a f f e r e n c e s m a y be a r e s u l t of the e v o l u t i o n a r y p r o c e s s of t h e s e r e c e p t o r s . The m a i n a i m of the ~ r e s e n t s t u d y as ~o compare the binding characteristics of [ ~ H ] P K 11195 a n d [ H ] R o 5 - 4 8 6 4 to h u m a n c e r e b r a l cortex, kidney, a n d c o l o n m e m b r a n e s , an o r d e r to e l u c i d a t e w h e t h e r P B R b i n d i n g c h a r a c t e r i s t a c s l o c a t e d in h u m a n t i s s u e s are s i m i l a r to t h o s e l o c a t e d in c a l f or rat tissues.
Materials
and Methods
Materials. [3H]PK 11195 (85.2 C i / m m o l ) a n d [3H]Ro 5 - 4 8 6 4 (78.9 C i / m m o l ) w e r e p u r c h a s e d f r o m N e w E n g l a n d N u c l e a r (Boston, MA). U n l a b e l e d PK 11195 w a s a g e n e r o u s g i f t f r o m Dr. G. Le Fur (Pharmuka Laboratories,Gennevilliers,France). U n l a b e l e d Ro 5 - 4 8 6 4 a n d c l o n a z e p a m w e r e k i n d l y s u p p l i e d b y Drs. H. G u t m a n a n d E. Kyburz (Hoffmann-La Roche,Basel,Switzerland). All other compounds w e r e p u r c h a s e d f r o m c o m m e r c i a l sources. Membrane preparation. M e n [n= 5, m e a n age 45 + 8 (SD) years] w i t h no h i s t o r y of n e u r o l o g i c a l or p s y c h i a t r i c d i s o r d e r s w e r e d i s s e c t e d a b o u t 5 hr a f t e r death. C e r e b r a l cortex, kidney, and c o l o n w e r e i m m e d i a t e l y r e m o v e d a n d f r o z e n at - 7 0 ° C u n t i l u s e d for b l n d l n g s t u d i e s . S a m p l e s f r o m e a c h t l s s u e w e r e h o m o g e n i z e d in 50 v o l u m e s of 50 m M T r i s - H C l buffer, pH 7.4, at 4 ° C w i t h a B r i n k m a n n p o l y t r o n ( s e t t i n g i0) for i0 sec a n d c e n t r i f u g e d at 4 9 , 0 0 0 x for 30 min. E a c h p e l l e t w a s s u s p e n d e d in 200 v o l u m e s of T r i s b u f f e r a n d u s e d as P B R p r e p a r a t i o n .
B i n d i n g assay. B i n d i n g a s s a y s for [3H]PK 11195 a n d [3H]Ro 5 - 4 8 6 4 w e r e c o n d u c t e d as p r e v i o u s l y d e s c r i b e d (20). B i n d i n g a s s a y c o n t a i n e d 400 Ul m e m b r a n e h o m o g e n a t e (about 200 ug p r o t e i n ) and 25 U1 [ ~ H ] P K 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) in t h e a b s e n c e (total b i n d i n g ) or p r e s e n c e ( n o n s p e c i f i c b i n d i n g ) of i0 ~ M u n l a b e l e d Ro 5 - 4 8 6 4 or PK 11195. A f t e r i n c u b a t i o n for 60 m i n at 4°C, s a m p l e s w e r e f i l t e r e d u n d e r v a c u u m o v e r W h a t m a n G F / C f 1 1 t e r s a n d w a s h e d 3 t i m e s w i t h 4 ml i c e - c o l d T r i s b u f f e r . F i l t e r s w e r e p l a c e d in v i a l s c o n t a i n i n g 5 ml of 1:3 m i x t u r e of L u m a x : x y ~ e n e and c o u n t e d for r a d l o a c t i v l t y a f t e r 12 hr. B i n d i n g a s s a y for [ H ] R o 55 4 8 6 4 ( 1 . 2 5 - 4 0 nM, final c o n c e n t r a t i o n ) w a s s i m i l a r to t h a t of [ H ] P K 1 1 1 9 5 d e s c r i b e d above, e x c e p t t h a t n o n s p e c i f i c b i n d i n g was c a r r i e d o u t u s l n g o n l y i0 uM u n l a b e l e d PK 11195. M a x l m a l n u m b e r s of b i n d i n g s i t e s (B . ) a n d e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t (K~) values were determln~ by S c a t c h a r d a n a l y s l s of s a t u r a t l o n c u r v e s of [ ~ H ] P K 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) . Displacement e x p e r i m e n t s w e r e c o n d u c t e d as r e c e n t l y d e s c r i b e d ( 2 0 ) . I n c u b a t l o n s w e r e p e r f o r m e d for 60 m i n at 4 u C w i t h 1 n M [o H ] P K 11195 in the a b s e n c e or p r e s e n c e of v a r i o u s c o n c e n t r a t i o n s of u n l a b e l e d PK 11195, Ro 5-4864, o r c l o n a z e p a m (i0 --v-10 -M, final c o n c e n t r a t a o n ) as i n h i b i t o r s . T h e ICs0 v a l u e s ( c o n c e n t r a t i o ~ of u n l a b e l e d i n h l b i t o r r e q u a r e d to a n h i b i t 50% s p e c i f i c [ H ] P K 11195 b i n d l n g ) w e r e o b t a i n e d f r o m d i s p l a c e m e n t c u r v e s of t h e s e e x p e r i m e n t s . . m
.
.
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Results T h e s a t u r a t i o n c u r v e s and S c a t c h a r d a n a l y s i s 11195 s p e c i f i c b i n d i n g to h u m a n k i d n e y and c o l o n
of [ 3 H ] P K membranes
are
Colon
1600
1200
800
400 1000 B2000
I
L
]
L
I
I
I
Kidney ~., 160C _u"3
~E © 80(
~',--,~ 4 0 0 3:
1000 B 2000
I
I
I
Cerebral Cortex
240
180
~
40 •
120
60 150 B 3 0 0
I
I
J
L
L
2
4
6
8
10
[3H]PK 11195 (nM)
FIG.
1
S a t u r a t i o n c u r v e s of [3H]PK 11195 b i n d i n g to h u m a n c~lon, kidney, and c e r e b r a l c o r t e x m e m b r a n e s . S p e c i f i c [~H]PK 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) b i n d i n g to h u m a n colon, kidney, and c e r e b r a l c o r t e x m e m b r a n e s w a s d e t e r m i n e d at 4 C for 60 mi~. Insets: S c a t c h a r d p l o t s of s a t u r a t i o n c u r v e s of [ H ] P K 11195 s p e c i f i c b i n d i n g to h u m a n colon, kidney, and c e r e b r a l c o r t e x m e m b r a n e s . D e t a i l s of the b i n d i n g p r o c e d u r e are d e s c r i b e d u n d e r M a t e r i a l s and Methods. R e s u l t s are the m e a n of t h r e e s e p a r a t e e x p e r i m e n t s w i t h less t h a n 15% v a r i a b i l i t y . B, bound; B/F, b o u n d / f r e e .
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p r e s e n t e d in Fig i. [3H]PK 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) b o u n d w i t h h i g h a f f i n i t y to h u m a n k i d n e y m e m b r a n e s , y i e l d i n g B and K n v a l u e s of 1908 + 28 f m o l / m g p r o t e i n a n d 2.3 + 0.i nM, max r e s p e c t i v e l y . N o n s p e c i f i c b i n d i n g of [ H ] P K 11195 t o h u m a n k i d n e y m e m b r a n e s w a s less t h a n 10% of ~he t o t a l b i n d i n g . O n the o t h e r hand, the s p e c i f i c b i n d i n g of [ H ] R o 5 - 4 8 6 4 ( 1 . 2 5 - 4 0 nM, flnal c o n c e n t r a t i o n ) to h u m a n k i d n e y m e m b r a n e s w a s b a r e l y d e t e c t a b l e , s i n c e the n o n s p e c i f i c b i n d i n g of [VH]Ro 5 - 4 8 6 4 to h u m a n k i d n e y m e m b r a n e s w a s a b o u t 90% of t h e ^ t o t a l b i n d i n g . The s p e c l f i c and n o n s p e c i f i c b i n d i n g of 40 n M [JH]Ro 5 - 4 8 6 4 to h u m a n k i d n e y m e m b r a n e s w e r e 4148 + 192 and 3944 + 256 cpm, r e s p e c t i v e l y . No d i f f e r e n c e in n o n s p e c i f i c b i n d i n g w a s o b t a i n e d w h e n the u n l a b e l e d l i g a n d w a s Ro 5 - 4 8 6 4 or PK 11195 (i0 ~M, final c o n c e n t r a t i o n ) (data not shown). T h e r e f o r e , it w a s d i f f i c u l t to c a l c u l a t e the B and K D v a l u e s for [~H]Ro 5-4864. S i m i l a r r e s u l t s w e r e a l s o o b t ~ e d in h u m a n c o l o n m e m b r a n e s ( F i g u r e i). The B and K_ v a l u e s for [~H]PK 11195 in h u m a n c o l o n m e m b r a n e s w e r e m ~ 3 3 Z 9~ f m o l / m g p r o t e i n and 1.6 + 0.I nM, r e s p e c t i v e l y . The s a t u r a t i o n c u r v e and S c a t c h a r d a n a l y s i s of [3H]PK 11195 s p e c i f l c b i n d i n g to h u m a n c ~ r e b r a l c o r t e x m e m b r a n e s are also p r e s e n t e d in Fig i. W h i l e [ H ] P K 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) b o u n d w i t h h i g h a f f i n i t y %o h u m a n c e r e b r a l c o r t e x membranes yielding B and K_ v a l u e s of 255 + 23 f m o l / m g • + aM D -p~oteln and 2.1 0.~ ~M, r e s p e c t i v e l y , the s p e c i f i c b l n d i n g of [ H ] R o 5 - 4 8 6 4 ( 1 7 2 5 - 4 0 nM, final c o n c e n t r a t i o n ) to h u m a n c e r e b r a l c ~ r t e x m e m b r a n e s was b a r e l y d e t e c t a b l e ( n o n s p e c i f i c b i n d i n g of [ H ] R o 5 - 4 8 6 4 w a s a b o u t 90% of the t o t a l b i n d i n g ) . The p o t e n c y e o f u n l a b e l e d PK 11195, Ro 5-4864, and c l o n a z e p a m in i n h i b i t i n g [vH]PK 11195 s p e c i f i c b i n d i n g to rat, human, and c a l f c e r e b r a l c o r t e x and k i d n e y m e m b r a n e s is p r e s e n t e d in T a b l e I. U n l a b e l e d P K 3 1 1 1 9 5 and Ro 5 - 4 8 6 4 e x h i b i t e d s i m i l a r p o t e n c y in d i s p l a c i n g [ H ] P K 11195 s p e c i f i c b i n d i n g f r o m rat c e r e b r a l TABLE
I
I n h i b i t i o n of [3H]PK 11195 S p e c i f i c B i n d i n g V a r i o u s Rat, Human, and C a l f T i s s u e s M e m b r a n e s P K 11195, Ro 5-4864, and C l o n a z e p a m
PK 11195 Cerebral Rat* Human Calf*
Ic- 0 ( ~ ) In~ibitor Ro 5 - 4 8 6 4
to By
Clonazepam
cortex 0.003 0.005 0.004
0.007 0.750 9.850
>i0 >i0 >i0
Kidney Rat* Human Calf*
0.018 0.008 0.003
0.027 0.750 9.860
>i0 >i0 >i0
Colon Human
0.005
0.750
>i0
V a l u e s s h o w n are the m e a n of t h r e e s e p a r a t e w i t h less t h a n 15% v a r i a b i l i t y , * D a t a f r o m A w a d and G a v i s h (20).
experiments
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cortex and k i d n e y membranes; in contrast, u n l a b e l e d PK 11195 was, respectively, two and three orders ~f m a g n i t u d e more potent than u n l a b e l e d Ro 5-4864 in displacing [ H]PK 11195 specific binding from human and calf cerebral cortex and ki~ney m e m b r a n e s . U n l a b e l e d c l o n a z e p a m was ineffective in displacing [ H]PK 11195 specific b i n d i n g from rat, human, and calf cerebral cortex and k i d n e y m e m b r a n e s (ICK0 > i0,000 nM). Similar results were also o b t a i n e d in human color m e m b r a n e s (Table I). Discussion Earlier reports elaborated dramatic q u a n t i t a t i v e species d i f f e r e n c e s and h e t e r o g e n e i t y in PBR,with respect to their binding c h a r a c t e r i s t i c s for the classical prototype PBR h l g h - a f f i n i t y BZ ligand Ro 5-4864 (20-25). These species differences in PBR have been d e m o n s t r a t e d in the binding affinity of [ H]Ro 5-48643to tissues of various s~ecies, in the binding affinity of [ H]Ro 5-4864 as c o m p a r e d to [ H]PK 11195, and in3the rank order of p o t e n c y of u n l a b e l e d Ro 54864 in i n h i b i t i n g [ H]PK 11195 specific ~inding to tissues of various s p e c i e s . T h e binding affinity of [ H]Ro 5-4864 to cow b r a i n m e m b r a n e s has been found to be 200 times lower than it~ affinity to rat brain membranes (21). We d e m o n s t r a t e d that [ H]PK 11195 binds with nanomolar affinity to b~th rat and calf cerebral cortex and peripheral tissues, whereas [ H]Ro 5-4864 binds with n a n o m o l a r affinity to rat cerebral cortex and peripheral tissues but with m i c r o m o l a r afflnity to calf tissues (20). Simila~ d i f f e r e n c e s in the binding affinity of [ H]PK 11195 and [ H]Ro 5-4864 have also been obtained in rat and calf pineal gland (22). A@ditionally, the potency of u n l a b e l e d Ro 5-4864 in inhibiting [~H]PK 11195 specific binding to cat brain sections and membranes has been found to be 140 times lower than that of u n l a b e l e d PK 11195 (23);whereas in rat brain,the potency of u n l a b e l e d Ro 5-4864 is similar to that of unlabeled PK 11195 (24). We found that3the rank order of p o t e n c y of u n l a b e l e d Ro 5-4864 in i n h i b i t i n g [ H]PK 11195 specific binding to cerebral cortex m e m b r a n e s of various species was rat= guinea pig> cat = dog > rabbit > calf. U n l a b e l e d Ro 5-4864 ~as three orders of magnitude more potent in inhibiting specific [ H]PK 11195 binding to rat cerebral cortex m e m b r a n e s than to calf cerebral cortex membranes. On th~ other hand, the p o t e n c y of u n l a b e l e d PK 11195 in inhibiting [ H]PK 11195 specific b i n d i n g to cerebral cortex membranes of various species was almost identical (20). It was speculated that these dramatic species d i f f e r e n c e s in PBR may t h e o r e t i c a l l y be related to v a r i a t i o n s in PBR m o l e c u l a r structure. ~n the present study, we compared the b i n d i n g c h a r a c t e r i s t i c s of [~H]Ro 5-4864 and [ H]PK 11195 to human cerebral cortex, kidney, and colon m e m b r a n e s in order to elaborate w h a t h e r human PBR b i n d i n g c h a r a c t e r i s t i c s resemble that of rat or bovine PBR. The major finding in the present report was that PK 11195 bound with high a f f i n i t y (K n about 2 nM) to human cerebral cortex, kidney, and colon m e m b r a n e s , w h i l e Ro 5-4864 e x h i b i t e d v e r y low binding a f f i n i t y to these tlssues (IC50 = 750 nM). These findings indicate that PBR binding c h a r a c t e r i s t i c s located in human tissues are similar to those located in calf tissues but not to those located in rat tissues. However, the
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f i n d i n g t h a t u n l a b e l e d Ro 5 - 4 8 6 4 w a s t w o a n d t h r e e o ~ d e r s of magnitude, respectively, less p o t e n t in i n h i b i t i n g [ V H ] P K 11195 s p e c i f i c b i n d i n g to h u m a n a n d c a l f t i s s u e s t h a n to rat t i s s u e s (see T a b l e I) i n d i c a t e s t h a t P B R b i n d i n g c h a r a c t e r i s t i c s of h u m a n t i s s u e s are n o t i d e n t i c a l to t h o s e of e i t h e r c a l f or r a t tissues. P B R h a v e a l s o b e e n c h a r a c t e r i z e d in o t h e r h u m a n t i s s u e s s u c h as p l a c e n t a , iris, m y o m e t r i u m , lymphocytes, a n d p l a t e l e t s ~i0-16). A l t h o u g h o u r r e s u l t s c o n c e r n i n g l o w b i n d i n g a f f i n i t y of [ H ] R o 54 8 6 4 to h u m a n c e r e b r a l cortex, kidney, a n d c o l o n m e m b r a n e s are in a c c o r d w i t h p r e v i o u s r e p o r t s (Ii,12,25), o t h e r g r o u p s o b s e r v e d h i g h a f f i n i t y b i n d i n g of [ H ] R O 5 - 4 8 6 4 to h u m a n l y m p h o c y t e s and p l a t e l e t s ( 1 3 , 1 4 , 1 6 ) . The r e a s o n for t h e s e d i s c r e p a n c i e s is not clear. T h e n a t u r e of t h e s e i n t e r - a n d i n t r a s p e c i e s d i f f e r e n c e s in PBR binding characteristics are still u n c l e a r . T h e y m a y r e f l e c t e v o l u t i o n a r y c h a n g e s in the s t r u c t u r e of P B R w h i c h h a v e r e s u l t e d in loss of b i n d i n g a f f i n i t y for Ro 5-4864, b u t h a v e p r e s e r v e d the binding characteristics for PK 11195. F u r t h e r b i o c h e m i c a l s t u d i e s are n e e d e d in o r d e r to c l a r i f y t h i s p h e n o m e n o n .
Acknowledgements T h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m the F u n d for B a s i c R e s e a r c h a d m i n i s t e r e d b y the Israel A c a d e m y of S c i e n c e s a n d H u m a n i t i e s . T h i s p a p e r is in p a r t i a l f u l f i l l m e n t of the r e q u i r e m e n t s for the D.Sc. d e g r e e of M. A w a d at t h e T e c h n i o n I s r a e l I n s t i t u t e of T e c h n o l o g y .
References i. H. M O H L E R a n d T. OKADA, Science, 198 8 4 9 - 8 5 1 (1977). 2. R. S Q U I R E S a n d C. B R A E S T R U P , Nature, 266 7 3 2 - 7 3 4 (1977). 3. L.P. D A V I E S a n d V. HUSTON, Eur. J. P h a r m a c o l . , 73 2 0 9 - 2 1 1 (1981). 4. T. T A N I G U C H I , J.K.T. WANG, a n d S. SPECTOR, B i o c h e m . P h a r m a c o l . , 31 5 8 9 - 5 9 0 (1982). 5. T. T A N I G U C H I , J.K.T. WANG, a n d S. SPECTOR, L i f e Sci., 27 171178 (1980). 6. J.K.T. WANG, T. T A N I G U C H I , M. SUGIURA, a n d S. SPECTOR, Pharmacologist, 23 160 (1981). 7. J.K.T. WANG, T. T A N I G U C H I , a n d S. SPECTOR, L i f e Sci., 27 1 8 8 1 - 1 8 8 8 (1987). 8. J.p. M A R A N G O S , J. PATEL, and R.C. R O S E N B E R G , Mol. P h a r m a c o l . , 22 2 6 - 3 2 (1982). 9. H. S C H O E M A K E R , M. BLISS, and H.I. Y A M A M U R A , Eur. J. P h a r m a c o l . , 71 1 7 3 - 1 7 8 (1981). i0. F. F A R E S a n d M. GAVISH, B i o c h e m . P h a r m a c o l . , 35 2 2 7 - 2 3 0 (1986). ii. D. V A L T I E R , C. M A L G O U R I S , J.-C. G I L B E R T , P. G U I C H E N E Y , A. UZAN, C. G U E R E M Y , G. LE FUR, H. SARAUX, a n d P. MEYER, Neuropharmacology, 26 5 4 9 - 5 5 4 (1987). 12. S. R O N C A - T E S T O N I , P. G A L B A N I , G. MELIS, M. G A M B A C C I A N I , and, P. F I O R E T T I , Int. J. Tiss. React., 4 4 3 7 - 4 4 1 (1984). 13. P. M O I N G E O N , J. BIDART, J.F. A L B E R I C I , a n d C. BOHUON, Eur. J. P h a r m a c o l . , 92 1 4 7 - 1 5 3 (1983).
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14.
J. BENAVIDES, D. QUARTERONET, P.-F. PLOUIN, F. IMBAULT, T. PHAN, A. UZAN, C. RENAULT, M.C. DUBROEUCQ, C. GUEREMY, and G. LE FUR, Biochem. Pharmacol., 33 2467-2472 (1984). 15. M. GAVISH, A. WEIZMAN, L. KARP, S. TYANO, and Z. TANNE, Eur. J. Pharmacol., 121 275-279 (1986). 16. P. MOINGEON, J.J. DESSAUX, R. FELLOUS, J. ALBERICI, J.M. BIDART, P. MOTTE, and C. BOHUON, Life Sci., 35 2003-2009
(1984). 17. M. GAVISH, M. AWAD, and F. FARES, J. Neurochem., 45 760-765 (1985). 18. M.M.S. LO and S.H. SNYDER, J.Neurosci., 3 2270-2279 (1985). 19. R. SQUIRES and E. SAEDERUP, Mol.Pharmacol., 22 327-334 (1982). 20. M. AWAD and M. GAVISH, J. Neurochem., 49 1407-1414 (1987). 21. J. BENAVIDES, N. VAUCHER, M. DANIEL, C. MALGOURIS, A. DOBLE, A. UZAN, C. GUEREMY, and G. LE FUR, Soc. Neurosci. Abstr., ii 278 (1985). 22. A.C. BASSILE, D.C. KLEIN, and P. SKOLNICK, Mol. B r a i n Res., 1 127-135 (1986). 23. J. BENAVIDES, H.E. SAVAKI, C. MALGOURIS, C. LAPLACE, M. DANIEL, F. BEGASSAT, M. DESBAN, A. UZAN, M. DUBROEUCQ, C. RENAULT, C. GUEREMY, and G. LE FUR, Brain Res. Bull., 13 6977 (1984). 24. J. BENAVIDES, D. QUARTERONET, F. IMBAULT, A. MALGOURIS, A. UZAN, C. RENAULT, M.C. DUBROEUCQ, C. GUEREMY, and G. LE FUR, J. Neurochem., 41 1744-1750 (1983). 25. W.C. B R O A D D U S and J.P. BENNETT Jr., B r a i n Res., 518 199-208 (1990).
Pergamon P r e s s
P r i n t e d An t h e U . S . A .
PERIPHERAL-TYPE
B E N Z O D I A Z E P I N E RECEPTORS IN H U M A N CEREBRAL CORTEX, KIDNEY, AND C O L O N M. Awad
and M. G a v i s h
R a p p a p o r t Family Institute For R e s e a r c h in the Medical S c i e n c e s and D e p a r t m e n t of Pharmacology, Bruce R a p p a p o r t F a c u l t y of Medicine, T e c h n i o n - I s r a e l Institute of Technology, P.O.B. 9697, 31096 Haifa, Israel (Received in final form August 5, 1991) Summary The specific binding of [3H]PK 11195 and [3H]Ro 5-4864 to h u m a n cerebral cortex, kidney, and colon m e m b r a n e s was studied in order to d e t e r m i n e w h e t h e r p e r i p h e r a l - t y p e b e n z o d i a z e p i n e r e c e p t o r s (PBR) c h a r a c t e r i s t i c s located in human tissues are similar tw those located in calf or rat tissues. W h i l e [vH]PK 11195 (0.05-10 nM, final concentration) b o u n d w i t h high a f f i n i t y (K D about 2 nM) to h u m a n cerebral cortex, kidney, and colon membranes, y i e l d i n g maximal n u m b e r s of b i n d i n g sites of 255 + 23, 1908 + 28, and 1633 Z 98 fm~i/mg protein, respectively, the specific b i n d i n g of [ H]Ro 5-4864 (1.25-40 nM, final concentration), was b a r e l y d e t e c t a b l e (nonspecific b i n d i n g about 90% of the total binding). Furthermore, u n l a b e l e d PK 11195 was two orders of m a g n i t u d e more p ~ t e n t than u n l a b e l e d Ro 5-4864 in d i s p l a c i n g [vH]PK 11195 specific binding from h u m a n cerebral cortex and k i d n e y membranes. These results indicate that PBR b i n d i n g c h a r a c t e r i s t i c s located in h u m a n tissues are similar (but not identical) to those located in calf tissues, but not to those located in rat tissues.
B e n z o d i a z e p i n e (BZ) receptors have been c l a s s i f i e d in two p h a r m a c o l o g i c a l l y distinct types of receptors: "central-type" BZ r e c e p t o r s (CBR) located in the m a m m a l i a n central nervous system (1,2) and "peripheral-type" BZ receptors (PBR) located in rat p e r i p h e r a l tissues and cells such as kidney, lung, liver, heart, mast cells, thymocytes, and platelets, as well as in the b r a i n (2-9).PBR have also been c h a r a c t e r i z e d in some h u m a n tissues such as placenta, irls,myometrium, l y m p h o c y t e s , a n d p l a t e l e t s (10-16). PBR and CBR differ in their d i s t r i b u t i o n w i t h i n the brain, s u b c e l l u l a r localization, and ligand specificity. In rat and guinea pig, Ro 5-4864 (4'-chlorodiazepam) and PK 11195 [l-(2-chloro phenyl)-N-methyl-N-(l-methylpropyl)-3-isoquinoline carboxamide] b i n d w i t h h i g h a f f i n i t y to PBR,but not to C B R ; w h e r e a s clonazepam, * Correspondence
and reprints. 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
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w h i c h b i n d s w i t h h i g h a f f i n i t y to CBR, e x h i b i t s o n l y m i c r o m o l a r b i n d i n g a f f i n i t y to PBR. A d d i t i o n a l l y , C B R are c o u p l e d to y-aminobutyric a c i d r e c e p t o r s a n d a l s o to s p e c i f i c b i n d i n g s i t e s for a n i o n s a n d d i v a l e n t c a t i o n s (17-19), w h e r e a s P B R are not. Recent studies have identified marked species differences b e t w e e n P B R l o c a t e d in c a l f t i s s u e s a n d P B R l o c a t e d in r a t t i s s u e s , w i t h r e f e r e n c e to t h e i r b i n d i n g a f f i n i t y for the c l a s s a c a l p r o t o t y p e P B R h i g h - a f f i n i t y BZ l i g a n d Ro 5-4864. S u c h s p e c i e s d a f f e r e n c e s m a y be a r e s u l t of the e v o l u t i o n a r y p r o c e s s of t h e s e r e c e p t o r s . The m a i n a i m of the ~ r e s e n t s t u d y as ~o compare the binding characteristics of [ ~ H ] P K 11195 a n d [ H ] R o 5 - 4 8 6 4 to h u m a n c e r e b r a l cortex, kidney, a n d c o l o n m e m b r a n e s , an o r d e r to e l u c i d a t e w h e t h e r P B R b i n d i n g c h a r a c t e r i s t a c s l o c a t e d in h u m a n t i s s u e s are s i m i l a r to t h o s e l o c a t e d in c a l f or rat tissues.
Materials
and Methods
Materials. [3H]PK 11195 (85.2 C i / m m o l ) a n d [3H]Ro 5 - 4 8 6 4 (78.9 C i / m m o l ) w e r e p u r c h a s e d f r o m N e w E n g l a n d N u c l e a r (Boston, MA). U n l a b e l e d PK 11195 w a s a g e n e r o u s g i f t f r o m Dr. G. Le Fur (Pharmuka Laboratories,Gennevilliers,France). U n l a b e l e d Ro 5 - 4 8 6 4 a n d c l o n a z e p a m w e r e k i n d l y s u p p l i e d b y Drs. H. G u t m a n a n d E. Kyburz (Hoffmann-La Roche,Basel,Switzerland). All other compounds w e r e p u r c h a s e d f r o m c o m m e r c i a l sources. Membrane preparation. M e n [n= 5, m e a n age 45 + 8 (SD) years] w i t h no h i s t o r y of n e u r o l o g i c a l or p s y c h i a t r i c d i s o r d e r s w e r e d i s s e c t e d a b o u t 5 hr a f t e r death. C e r e b r a l cortex, kidney, and c o l o n w e r e i m m e d i a t e l y r e m o v e d a n d f r o z e n at - 7 0 ° C u n t i l u s e d for b l n d l n g s t u d i e s . S a m p l e s f r o m e a c h t l s s u e w e r e h o m o g e n i z e d in 50 v o l u m e s of 50 m M T r i s - H C l buffer, pH 7.4, at 4 ° C w i t h a B r i n k m a n n p o l y t r o n ( s e t t i n g i0) for i0 sec a n d c e n t r i f u g e d at 4 9 , 0 0 0 x for 30 min. E a c h p e l l e t w a s s u s p e n d e d in 200 v o l u m e s of T r i s b u f f e r a n d u s e d as P B R p r e p a r a t i o n .
B i n d i n g assay. B i n d i n g a s s a y s for [3H]PK 11195 a n d [3H]Ro 5 - 4 8 6 4 w e r e c o n d u c t e d as p r e v i o u s l y d e s c r i b e d (20). B i n d i n g a s s a y c o n t a i n e d 400 Ul m e m b r a n e h o m o g e n a t e (about 200 ug p r o t e i n ) and 25 U1 [ ~ H ] P K 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) in t h e a b s e n c e (total b i n d i n g ) or p r e s e n c e ( n o n s p e c i f i c b i n d i n g ) of i0 ~ M u n l a b e l e d Ro 5 - 4 8 6 4 or PK 11195. A f t e r i n c u b a t i o n for 60 m i n at 4°C, s a m p l e s w e r e f i l t e r e d u n d e r v a c u u m o v e r W h a t m a n G F / C f 1 1 t e r s a n d w a s h e d 3 t i m e s w i t h 4 ml i c e - c o l d T r i s b u f f e r . F i l t e r s w e r e p l a c e d in v i a l s c o n t a i n i n g 5 ml of 1:3 m i x t u r e of L u m a x : x y ~ e n e and c o u n t e d for r a d l o a c t i v l t y a f t e r 12 hr. B i n d i n g a s s a y for [ H ] R o 55 4 8 6 4 ( 1 . 2 5 - 4 0 nM, final c o n c e n t r a t i o n ) w a s s i m i l a r to t h a t of [ H ] P K 1 1 1 9 5 d e s c r i b e d above, e x c e p t t h a t n o n s p e c i f i c b i n d i n g was c a r r i e d o u t u s l n g o n l y i0 uM u n l a b e l e d PK 11195. M a x l m a l n u m b e r s of b i n d i n g s i t e s (B . ) a n d e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t (K~) values were determln~ by S c a t c h a r d a n a l y s l s of s a t u r a t l o n c u r v e s of [ ~ H ] P K 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) . Displacement e x p e r i m e n t s w e r e c o n d u c t e d as r e c e n t l y d e s c r i b e d ( 2 0 ) . I n c u b a t l o n s w e r e p e r f o r m e d for 60 m i n at 4 u C w i t h 1 n M [o H ] P K 11195 in the a b s e n c e or p r e s e n c e of v a r i o u s c o n c e n t r a t i o n s of u n l a b e l e d PK 11195, Ro 5-4864, o r c l o n a z e p a m (i0 --v-10 -M, final c o n c e n t r a t a o n ) as i n h i b i t o r s . T h e ICs0 v a l u e s ( c o n c e n t r a t i o ~ of u n l a b e l e d i n h l b i t o r r e q u a r e d to a n h i b i t 50% s p e c i f i c [ H ] P K 11195 b i n d l n g ) w e r e o b t a i n e d f r o m d i s p l a c e m e n t c u r v e s of t h e s e e x p e r i m e n t s . . m
.
.
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Peripheral Benzodiazepines in Human Tissues
1157
Results T h e s a t u r a t i o n c u r v e s and S c a t c h a r d a n a l y s i s 11195 s p e c i f i c b i n d i n g to h u m a n k i d n e y and c o l o n
of [ 3 H ] P K membranes
are
Colon
1600
1200
800
400 1000 B2000
I
L
]
L
I
I
I
Kidney ~., 160C _u"3
~E © 80(
~',--,~ 4 0 0 3:
1000 B 2000
I
I
I
Cerebral Cortex
240
180
~
40 •
120
60 150 B 3 0 0
I
I
J
L
L
2
4
6
8
10
[3H]PK 11195 (nM)
FIG.
1
S a t u r a t i o n c u r v e s of [3H]PK 11195 b i n d i n g to h u m a n c~lon, kidney, and c e r e b r a l c o r t e x m e m b r a n e s . S p e c i f i c [~H]PK 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) b i n d i n g to h u m a n colon, kidney, and c e r e b r a l c o r t e x m e m b r a n e s w a s d e t e r m i n e d at 4 C for 60 mi~. Insets: S c a t c h a r d p l o t s of s a t u r a t i o n c u r v e s of [ H ] P K 11195 s p e c i f i c b i n d i n g to h u m a n colon, kidney, and c e r e b r a l c o r t e x m e m b r a n e s . D e t a i l s of the b i n d i n g p r o c e d u r e are d e s c r i b e d u n d e r M a t e r i a l s and Methods. R e s u l t s are the m e a n of t h r e e s e p a r a t e e x p e r i m e n t s w i t h less t h a n 15% v a r i a b i l i t y . B, bound; B/F, b o u n d / f r e e .
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p r e s e n t e d in Fig i. [3H]PK 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) b o u n d w i t h h i g h a f f i n i t y to h u m a n k i d n e y m e m b r a n e s , y i e l d i n g B and K n v a l u e s of 1908 + 28 f m o l / m g p r o t e i n a n d 2.3 + 0.i nM, max r e s p e c t i v e l y . N o n s p e c i f i c b i n d i n g of [ H ] P K 11195 t o h u m a n k i d n e y m e m b r a n e s w a s less t h a n 10% of ~he t o t a l b i n d i n g . O n the o t h e r hand, the s p e c i f i c b i n d i n g of [ H ] R o 5 - 4 8 6 4 ( 1 . 2 5 - 4 0 nM, flnal c o n c e n t r a t i o n ) to h u m a n k i d n e y m e m b r a n e s w a s b a r e l y d e t e c t a b l e , s i n c e the n o n s p e c i f i c b i n d i n g of [VH]Ro 5 - 4 8 6 4 to h u m a n k i d n e y m e m b r a n e s w a s a b o u t 90% of t h e ^ t o t a l b i n d i n g . The s p e c l f i c and n o n s p e c i f i c b i n d i n g of 40 n M [JH]Ro 5 - 4 8 6 4 to h u m a n k i d n e y m e m b r a n e s w e r e 4148 + 192 and 3944 + 256 cpm, r e s p e c t i v e l y . No d i f f e r e n c e in n o n s p e c i f i c b i n d i n g w a s o b t a i n e d w h e n the u n l a b e l e d l i g a n d w a s Ro 5 - 4 8 6 4 or PK 11195 (i0 ~M, final c o n c e n t r a t i o n ) (data not shown). T h e r e f o r e , it w a s d i f f i c u l t to c a l c u l a t e the B and K D v a l u e s for [~H]Ro 5-4864. S i m i l a r r e s u l t s w e r e a l s o o b t ~ e d in h u m a n c o l o n m e m b r a n e s ( F i g u r e i). The B and K_ v a l u e s for [~H]PK 11195 in h u m a n c o l o n m e m b r a n e s w e r e m ~ 3 3 Z 9~ f m o l / m g p r o t e i n and 1.6 + 0.I nM, r e s p e c t i v e l y . The s a t u r a t i o n c u r v e and S c a t c h a r d a n a l y s i s of [3H]PK 11195 s p e c i f l c b i n d i n g to h u m a n c ~ r e b r a l c o r t e x m e m b r a n e s are also p r e s e n t e d in Fig i. W h i l e [ H ] P K 11195 ( 0 . 0 5 - 1 0 nM, final c o n c e n t r a t i o n ) b o u n d w i t h h i g h a f f i n i t y %o h u m a n c e r e b r a l c o r t e x membranes yielding B and K_ v a l u e s of 255 + 23 f m o l / m g • + aM D -p~oteln and 2.1 0.~ ~M, r e s p e c t i v e l y , the s p e c i f i c b l n d i n g of [ H ] R o 5 - 4 8 6 4 ( 1 7 2 5 - 4 0 nM, final c o n c e n t r a t i o n ) to h u m a n c e r e b r a l c ~ r t e x m e m b r a n e s was b a r e l y d e t e c t a b l e ( n o n s p e c i f i c b i n d i n g of [ H ] R o 5 - 4 8 6 4 w a s a b o u t 90% of the t o t a l b i n d i n g ) . The p o t e n c y e o f u n l a b e l e d PK 11195, Ro 5-4864, and c l o n a z e p a m in i n h i b i t i n g [vH]PK 11195 s p e c i f i c b i n d i n g to rat, human, and c a l f c e r e b r a l c o r t e x and k i d n e y m e m b r a n e s is p r e s e n t e d in T a b l e I. U n l a b e l e d P K 3 1 1 1 9 5 and Ro 5 - 4 8 6 4 e x h i b i t e d s i m i l a r p o t e n c y in d i s p l a c i n g [ H ] P K 11195 s p e c i f i c b i n d i n g f r o m rat c e r e b r a l TABLE
I
I n h i b i t i o n of [3H]PK 11195 S p e c i f i c B i n d i n g V a r i o u s Rat, Human, and C a l f T i s s u e s M e m b r a n e s P K 11195, Ro 5-4864, and C l o n a z e p a m
PK 11195 Cerebral Rat* Human Calf*
Ic- 0 ( ~ ) In~ibitor Ro 5 - 4 8 6 4
to By
Clonazepam
cortex 0.003 0.005 0.004
0.007 0.750 9.850
>i0 >i0 >i0
Kidney Rat* Human Calf*
0.018 0.008 0.003
0.027 0.750 9.860
>i0 >i0 >i0
Colon Human
0.005
0.750
>i0
V a l u e s s h o w n are the m e a n of t h r e e s e p a r a t e w i t h less t h a n 15% v a r i a b i l i t y , * D a t a f r o m A w a d and G a v i s h (20).
experiments
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cortex and k i d n e y membranes; in contrast, u n l a b e l e d PK 11195 was, respectively, two and three orders ~f m a g n i t u d e more potent than u n l a b e l e d Ro 5-4864 in displacing [ H]PK 11195 specific binding from human and calf cerebral cortex and ki~ney m e m b r a n e s . U n l a b e l e d c l o n a z e p a m was ineffective in displacing [ H]PK 11195 specific b i n d i n g from rat, human, and calf cerebral cortex and k i d n e y m e m b r a n e s (ICK0 > i0,000 nM). Similar results were also o b t a i n e d in human color m e m b r a n e s (Table I). Discussion Earlier reports elaborated dramatic q u a n t i t a t i v e species d i f f e r e n c e s and h e t e r o g e n e i t y in PBR,with respect to their binding c h a r a c t e r i s t i c s for the classical prototype PBR h l g h - a f f i n i t y BZ ligand Ro 5-4864 (20-25). These species differences in PBR have been d e m o n s t r a t e d in the binding affinity of [ H]Ro 5-48643to tissues of various s~ecies, in the binding affinity of [ H]Ro 5-4864 as c o m p a r e d to [ H]PK 11195, and in3the rank order of p o t e n c y of u n l a b e l e d Ro 54864 in i n h i b i t i n g [ H]PK 11195 specific ~inding to tissues of various s p e c i e s . T h e binding affinity of [ H]Ro 5-4864 to cow b r a i n m e m b r a n e s has been found to be 200 times lower than it~ affinity to rat brain membranes (21). We d e m o n s t r a t e d that [ H]PK 11195 binds with nanomolar affinity to b~th rat and calf cerebral cortex and peripheral tissues, whereas [ H]Ro 5-4864 binds with n a n o m o l a r affinity to rat cerebral cortex and peripheral tissues but with m i c r o m o l a r afflnity to calf tissues (20). Simila~ d i f f e r e n c e s in the binding affinity of [ H]PK 11195 and [ H]Ro 5-4864 have also been obtained in rat and calf pineal gland (22). A@ditionally, the potency of u n l a b e l e d Ro 5-4864 in inhibiting [~H]PK 11195 specific binding to cat brain sections and membranes has been found to be 140 times lower than that of u n l a b e l e d PK 11195 (23);whereas in rat brain,the potency of u n l a b e l e d Ro 5-4864 is similar to that of unlabeled PK 11195 (24). We found that3the rank order of p o t e n c y of u n l a b e l e d Ro 5-4864 in i n h i b i t i n g [ H]PK 11195 specific binding to cerebral cortex m e m b r a n e s of various species was rat= guinea pig> cat = dog > rabbit > calf. U n l a b e l e d Ro 5-4864 ~as three orders of magnitude more potent in inhibiting specific [ H]PK 11195 binding to rat cerebral cortex m e m b r a n e s than to calf cerebral cortex membranes. On th~ other hand, the p o t e n c y of u n l a b e l e d PK 11195 in inhibiting [ H]PK 11195 specific b i n d i n g to cerebral cortex membranes of various species was almost identical (20). It was speculated that these dramatic species d i f f e r e n c e s in PBR may t h e o r e t i c a l l y be related to v a r i a t i o n s in PBR m o l e c u l a r structure. ~n the present study, we compared the b i n d i n g c h a r a c t e r i s t i c s of [~H]Ro 5-4864 and [ H]PK 11195 to human cerebral cortex, kidney, and colon m e m b r a n e s in order to elaborate w h a t h e r human PBR b i n d i n g c h a r a c t e r i s t i c s resemble that of rat or bovine PBR. The major finding in the present report was that PK 11195 bound with high a f f i n i t y (K n about 2 nM) to human cerebral cortex, kidney, and colon m e m b r a n e s , w h i l e Ro 5-4864 e x h i b i t e d v e r y low binding a f f i n i t y to these tlssues (IC50 = 750 nM). These findings indicate that PBR binding c h a r a c t e r i s t i c s located in human tissues are similar to those located in calf tissues but not to those located in rat tissues. However, the
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f i n d i n g t h a t u n l a b e l e d Ro 5 - 4 8 6 4 w a s t w o a n d t h r e e o ~ d e r s of magnitude, respectively, less p o t e n t in i n h i b i t i n g [ V H ] P K 11195 s p e c i f i c b i n d i n g to h u m a n a n d c a l f t i s s u e s t h a n to rat t i s s u e s (see T a b l e I) i n d i c a t e s t h a t P B R b i n d i n g c h a r a c t e r i s t i c s of h u m a n t i s s u e s are n o t i d e n t i c a l to t h o s e of e i t h e r c a l f or r a t tissues. P B R h a v e a l s o b e e n c h a r a c t e r i z e d in o t h e r h u m a n t i s s u e s s u c h as p l a c e n t a , iris, m y o m e t r i u m , lymphocytes, a n d p l a t e l e t s ~i0-16). A l t h o u g h o u r r e s u l t s c o n c e r n i n g l o w b i n d i n g a f f i n i t y of [ H ] R o 54 8 6 4 to h u m a n c e r e b r a l cortex, kidney, a n d c o l o n m e m b r a n e s are in a c c o r d w i t h p r e v i o u s r e p o r t s (Ii,12,25), o t h e r g r o u p s o b s e r v e d h i g h a f f i n i t y b i n d i n g of [ H ] R O 5 - 4 8 6 4 to h u m a n l y m p h o c y t e s and p l a t e l e t s ( 1 3 , 1 4 , 1 6 ) . The r e a s o n for t h e s e d i s c r e p a n c i e s is not clear. T h e n a t u r e of t h e s e i n t e r - a n d i n t r a s p e c i e s d i f f e r e n c e s in PBR binding characteristics are still u n c l e a r . T h e y m a y r e f l e c t e v o l u t i o n a r y c h a n g e s in the s t r u c t u r e of P B R w h i c h h a v e r e s u l t e d in loss of b i n d i n g a f f i n i t y for Ro 5-4864, b u t h a v e p r e s e r v e d the binding characteristics for PK 11195. F u r t h e r b i o c h e m i c a l s t u d i e s are n e e d e d in o r d e r to c l a r i f y t h i s p h e n o m e n o n .
Acknowledgements T h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m the F u n d for B a s i c R e s e a r c h a d m i n i s t e r e d b y the Israel A c a d e m y of S c i e n c e s a n d H u m a n i t i e s . T h i s p a p e r is in p a r t i a l f u l f i l l m e n t of the r e q u i r e m e n t s for the D.Sc. d e g r e e of M. A w a d at t h e T e c h n i o n I s r a e l I n s t i t u t e of T e c h n o l o g y .
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