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Etomidate stereospecifically stimulates forebrain, but not cerebellar, 3H-diazepam binding
Life Sciences, Vol. 29, pp. 2631-2636 Printed in the U.S.A.
ETOMIDATE
Pergamon Press
STEREOSPECIFICALLY STIMULATES FOREBRAIN, NOT CEREBELLAR, 3H-DIAZEPAM BINDING
D. A s h t o n
, R. G e e r t s ,
C. W a t e r k e y n a n d J . E .
BUT
Leysen
D e p a r t m e n t of P h a r m a c o l o g y ~ a n d B i o c h e m i c a l P h a r m a c o l o g y Janssen Pharmaceutica, B-2340 Beerse, Belgium (Received in final form October 27, 1981)
Summa ry 3 H - D i a z e p a m binding to a total particulate fraction of rat foreb r a i n is e n h a n c e d b y ( + ) - e t o m i d a t e a n d GABA, b u t n o t b y ( - ) - e t o m i d a t e . T h e e n h a n c e m e n t of 3 H - d i a z e p a m b i n d i n g b y ( + ) - e t o m i d a t e w a s due to a t w o - f o l d i n c r e a s e i n b i n d i n g a f f i n i t y , the m a x i m a l n u m b e r of s i t e s r e m a i n e d u n c h a n g e d . T h e d e g r e e of s t i m u l a t i o n with ( + ) - e t o m i d a t e was h i g h e r than that o b t a i n e d with GABA. T H I P d i d n o t s t i m u l a t e 3 H - d i a z e p a m b i n d i n g to f o r e b r a i n , a n d d i d n o t r e v e r s e the e n h a n c e d b i n d i n g s e e n w i t h ( + ) - e t o m i d a t e o r G A B A . In a s y n a p t o s o m a l m e m b r a n e p r e p a r a t i o n of r a t c e r e b e l l u m , u n l i k e ( + ) - e t o m i d a t e , GABA a n d m u s c i m o l p r o d u c e d a m a r k e d s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g . ( + ) - E t o m i d a t e did n o t i n h i b i t 3 H - m u s c i m o l b i n d i n g to GABA r e c e p t o r s , n o r did i t a c t i v a t e o r i n h i b i t o t h e r in v i t r o r e c e p t o r binding a s s a y s . T h e e f f e c t s of ( + ) - e t o m i d a t e o n the b e n z o d i a z e p i n e b i n d i n g a r e d i f f e r e n t f r o m t h o s e of g a b a m i m e t i c d r u g s . It i s p r o p o s e d t h a t l i k e b a r b i t u r a t e s , ( + ) - e t o m i d a t e m a y a f f e c t b e n z o d i a z e p i n e b i n d i n g by i n t e r a c t i o n w i t h the c h l o r i d e i o n o p h o r e w h i c h i s c o u p l e d to the G A B A - r e c e p t o r .
E t o m i d a t e , R - ( + ) e t h y l - 1 - ( 1 - p h e n y l e t h y l ) - 1 H - i m i d a z o l e - c a r b o x y l a t e is a potent, short acting non-barbiturate hypnotic, which lacks analgesic propert i e s ( ] , 2 ) . T h e d r u g h a s two o p t i c a l i s o m e r s , the ( + ) i s o m e r b e i n g r e s p o n s i b l e f o r the i n d u c t i o n of h y p n o s i s . I n a d d i t i o n to i t s h y p n o t i c a c t i v i t y ( + ) - e t o m i d a t e a l s o s h o w s p o t e n t a n t i c o n v u l s a n t a c t i v i t y i n v a r i o u s a n i m a l t e s t s : s u c h a s the maximalmetrazol t e s t (3), the s u b c u t a n e o u s m e t r a z o l t e s t (1), the D - L - a l l y l g l y c i n e t e s t (4), a n d i n a m y g d a l o i d k i n d l e d s e i z u r e s (1, 5). In h u m a n s ( + ) - e t o m i d a t e h a s b e e n s u c c e s s f u l l y u s e d to t e r m i n a t e t h e r a p y - r e s i s t a n t s t a t u s e p i l e p t i c u s (6, 7). R e c e n t e x p e r i m e n t s h a v e d e m o n s t r a t e d a p r o t e c t i v e e f f e c t of the c o m p o u n d i n a n i m a l m o d e l s of h y p o x i a a n d i s c h e m i a (1, 8). In n e u r o p h y s i o l o g i c a l e x p e r i m e n t s b o t h i n v i t r o a n d i n v i v o ( + ) - e t o m i d a t e w a s s h o w n to m i m i c r e s p o n s e s to y - a m i n o b u t y r i c a c i d (GABA) a n d to be s p e c i f i c a l l y a n t a gorized by k n o w n G A B A - a n t a g o n i s t s (9, 10). M a n y e x p e r i m e n t s h a v e s h o w n t h a t GABA is c a p a b l e of a c t i v a t i n g 3 H - b e n z o d i a z e p i n e b i n d i n g i n v i t r o (11). We now r e p o r t the i n t e r a c t i o n s b e t w e e n (+)- a n d ( - ) - e t o m i d a t e i n c o m p a r i s o n to G A B A , m u s c i m o l a n d 4, 5, 6 , 7 - t e t r a h y d r o i s o x a z o l e ( 5 , 4 - C ) p y r i d i n - 3 ( 2 H ) o n e ( T H I P ) on 3 H - d i a z e p a m b i n d i n g i n r a t f o r e b r a i n a n d c e r e b e l l u m .
0024-3205/81/252631-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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M e t h o d s and M a t e r i a l s F e m a l e W i s t a r r a t s w e r e used. F r e s h l y d i s s e c t e d f o r e b r a i n s (whole b r a i n s m i n u s c e r e b e l l u m a n d m e d u l l a o b l o n g a t a ) w e r e h o m o g e n i z e d in 40 v o l u m e s T r i s - H C 1 b u f f e r 50 r a M , p H 7 . 4 , u s i n g a n U l t r a T u r r a x . M e m b r a n e s w e r e c e n t r i f u g e d a t 3 9 , 0 0 0 x g f o r 10 m i n a n d w a s h e d 5 t i m e s by r e s u s p e n s i o n in b u f f e r a n d c e n t r i f u g a t i o n . W a s h e d m e m b r a n e s w e r e s u s p e n d e d in 100 v o l u m e s b u f f e r p e r g r a m o r i g i n a l w e t w e i g h t of t i s s u e a n d f r e s h l y u s e d f o r b i n d i n g
assays. Cerebellar tissue w a s prepared as described for 3 H - m u s c i m o l
binding
a s s a y s (12). R a t c e r e b e l l u m w a s h o m o g e n i z e d in 10 v o l u m e s 0. 25 M s u c r o s e . N u c l e i a n d c e l l d e b r i s w e r e r e m o v e d by c e n t r i f u g a t i o n a t 1090 g x ] 0 r a i n . F r o m t h e s u p e r n a t a n t , s u b c e l l u l a r p a r t i c l e s w e r e c e n t r i f u g e d a t 39, 100 x 20 r a i n . T h e p e l l e t w a s h o m o g e n i z e d in 10 v o l u m e s H 2 0 u s i n g a n U l t r a T u r r a x f o l l o w e d by c e n t r i f u g a t i o n a t 7720 g x I0 r a i n . T h e s u p e r n a t a n t w a s f u r t h e r c e n t r i f u g e d a t 16, 100 g x 30 r a i n . T h e m e m b r a n e p e l l e t w a s w a s h e d by h o m o g e n i z a t i o n in 20 v o l u m e s T r i s - c i t r a t e b u f f e r , 30 r a M , p H 7. 1 a n d c e n t r i f u g a t i o n a t 39, ]00 g x 20 r a i n . T h e p e l l e t w a s f r o z e n a n d k e p t a t - 2 0 ° C u n t i l u s e . A f t e r t h a w i n g t h e p e l l e t w a s w a s h e d two m o r e t i m e s in T r i s - c i t r a t e b u f f e r a n d f i n a l l y s u s p e n d e d in ]00 v o l u m e s T r i s - H C 1 b u f f e r p e r g r a m o r i g i n a l w e t w e i g h t of t i s s u e . F o r 3 H - d i a z e p a m b i n d i n g , a s s a y m i x t u r e s w e r e c o m p o s e d of 1 m l m e m b r a n e s u s p e n s i o n , 0 . 0 5 m l 3 H - d i a z e p a m ( f i n a l c o n c e n t r a t i o n 1 nM) a n d 0 . 0 5 m l 10 % e t h a n o l o r d r u g d i s s o l v e d in 10 % e t h a n o l . I n c u b a t i o n w a s r u n a t 0 °C for 30rain. L a b e l l e d m e m b r a n e s w e r e h a r v e s t e d by f i l t r a t i o n u n d e r r e d u c e d p r e s s u r e on G F / B g l a s s f i b e r f i l t e r s a n d w a s h e d 3 t i m e s w i t h 5 m l i c e - c o l d b u f f e r . R a d i o a c t i v i t y on t h e f i l t e r s w a s c o u n t e d in a n I n t e r t e c h n i q u e L i q u i d S c i n t i l l a t i o n c o u n t e r e q u i p p e d w i t h a b u i l t - i n c o m p u t e r s y s t e m to e x p r e s s m e a s u r e d r a d i o a c t i v i t y in D P M . Specific 3H-diazepam binding was defined by s u b t r a c t i n g n o n - s p e c i f i c b i n d i n g m e a s u r e d in t h e p r e s e n c e of 100 n M c l o nazepam. 3H-Muscimol b i n d i n g in th e s y n a p t o s o m a l p r e p a r a t i o n of r a t c e r e b e l l u m w a s c a r r i e d o u t a s d e s c r i b e d in r e f . 12. 3 H - D i a z e p a m s a t u r a t i o n c u r v e s in t he f o r e b r a i n w e r e m e a s u r e d in the a b s e n c e a n d t h e p r e s e n c e of 3 x 10 -5 M e t o m i d a t e u s i n g ]0 d i f f e r e n t c o n c e n t r a t i o n s of 3 H - d i a z e p a m in a r a n g e 0 . 2 to 8 riM. S p e c i f i c b i n d i n g w a s t a k e n a s t h e d i f f e r e n c e b e t w e e n t o t a l a n d n o n - s p e c i f i c b i n d i n g m e a s u r e d in t h e p r e s e n c e of 1 0 0 - f o l d e x c e s s of c l o n a z e p a m o v e r t h e c o n c e n t r a t i o n of 3 H - d i a zepam. A l l a s s a y s w e r e r u n in d u p l i c a t e a n d t w o e x p e r i m e n t s w e r e r u n i n dependently. L i n e s of b e s t f i t t h r o u g h S c a t c h a r d p l o t s w e r e c a l c u l a t e d by the m e t h o d of l e a s t s q u a r e s . Materials,
3H-Diazepam
(spec.
radioactivity
76 C i / m m o l e ,
NEN,
Germany), 3 H - m u s c i m o l (spec. radioactivity i0.3 Ci/mmole, N E N , G e r m a n y ~ clonazeparn (Hoffmann-La Roche, Switzerland), T H I P (kindly provided by Dr. J. Scheel-Krtlger and Dr. V. Christensen, Lundbeck, Denmark), (+)and (-)etomidate (Janssen Pharmaceutica, Belgium), m u s c i m o l (Fluka, Switzerland), G A B A (Aldrich, Europe Division).
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Re s u l t s I n w a s h e d t o t a l m e m b r a n e s of r a t f o r e b r a i n , ( + ) - e t o m i d a t e p r o d u c e s a c o n c e n t r a t i o n d e p e n d e n t i n c r e a s e i n 3 H - d i a z e p a m b i n d i n g , to a m a x i m u m of 150 % of s p e c i f i c b i n d i n g a t 10 ~M ( + ) - e t o m i d a t e . G A B A e n h a n c e s the b i n d i n g to m a x i m a l l y 135 % a t 100 ~M. ( - ) - E t o m i d a t e a n d T H I P do n o t c a u s e a n y s t i m u l a t i o n of b i n d i n g , b u t ( - ) - e t o m i d a t e p r o d u c e s a s m a l l i n h i b i t i o n of b i n d i n g a t 10 ~M. M a x i m a l s t i m u l a t i o n of s p e c i f i c 3 H - d i a z e p a m b i n d i n g by 30 ~M (+)e t o m i d a t e o r by 100 ~M GABA i s n o t r e v e r s e d by T H I P up to a c o n c e n t r a t i o n of 100 ~M. G r a p h s a r e s h o w n i n F i g u r e 1. I n a s y n a p t o s o m a l m e m b r a n e f r a c t i o n of r a t c e r e b e l l u m , w h i c h is e n r i c h e d i n GABA r e c e p t o r s (1Z), GABA a n d m u s c i m o l p r o d u c e a m a r k e d s t i m u l a t i o n of s p e c i f i c 3 H - d i a z e p a m b i n d i n g to a m a x i m u m of ZOO % a t ]0 ~M. T h e s t i m u l a t i o n by GABA i n the c e r e b e l l a r m e m b r a n e s i s m u c h m o r e p r o n o u n c e d t h a n i n the f o r e b r a i n m e m b r a n e s a n d o c c u r s a t 10 t i m e s l o w e r c o n c e n t r a t i o n s . In c o n t r a s t ( + ) - e t o m i d a t e i s v i r t u a l l y i n a c t i v e i n the c e r e b e l l a r m e m b r a n e s .
= 1601
°t 120
oi, " ;
~
,&0 ,o60
Drug concentration(pM]
FIG.
o:,
;
,~
,~o ,060
THIP concentratio•(pM)
1
a. E f f e c t of ( + ) - e t o m i d a t e (e) (n = 7), ( - ) - e t o m i d a t e (o) (n = 4), G A B A (•) (n = 4), and T H I P (A) (n = Z) on specific 3H-diazepare (1 n M ) binding to w a s h e d total m e m b r a n e s of rat forebrain. Control specific binding per assay w a s Z 8 , 3 0 0 + Z, 100 D P M , n = 7 and represented 90 ~0 of the total binding, n indicates the n u m b e r of independent experiments of which data are averaged. b. Effect of T H I P on stimulated specific 3 H - d i a z e p a m binding by 3 x 10 -5 M e t o m i d a t e ( ~ ) o r by 1 x l 0 -4 M GABA ( & ) .
A s i g n i f i c a n t s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g is n o t o b s e r v e d up to a c o n c e n t r a t i o n of 1 ~tM a n d a t 10 ~tM o n l y a s m a l l e n h a n c e m e n t of b i n d i n g b y 30 % is n o t e d . T H I P o n l y p r o d u c e s a s m a l l i n c r e a s e i n 3 H - d i a z e p a m b i n d i n g i n the c e r e b e l l a r m e m b r a n e s a t h i g h c o n c e n t r a t i o n . Graphs are shown in Figure Z. I n a d d i t i o n ( + ) - e t o m i d a t e did n o t i n h i b i t 3 H - m u s c i m o l b i n d i n g to GABA r e c e p t o r s i n the c e r e b e l l a r s y n a p t o s o m a l p r e p a r a t i o n a t c o n c e n t r a t i o n s up to 10 ~M.
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Etomidate and 3H-diazepam Binding
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FIG. 2
;
060, o.61 o'.i
;o
,60
D r u g c o n c e n t rat ion ( pM )
E f f e c t of ( + ) - e t o m i d a t e (e), GABA ( • ) , T H I P ( A ) a n d m u s c i m o l ( Q ) on s p e c i f i c 3 H - d i a z e p a m (1 nM) b i n d i n g to a washed synaptic membrane p r e p a r a t i o n of r a t c e r e b e l l u m . P o i n t s r e p r e s e n t m e a n d a t a of 5 i n d e p e n d e n t e x p e r i m e n t s in duplicate. Control specific b i n d i n g p e r a s s a y w a s 2850 + 680 D P M , n = 5 a n d r e p r e s e n t e d 80 % of t o t a l b i n d i n g .
T h e S c a t c h a r d p l o t ( F i g . 3) i n d i c a t e s a d e c r e a s e in the KD f o r 3 H - d i a z e p a m i n f o r e b r a i n f r o m 5 . 0 7 + 0 . 3 7 n M to 3 . 2 9 + 0 . 2 9 n M i n the p r e s e n c e of 3 x 10-5 M ( + ) - e t o m i d a t e ; h o w e v e r , B m a x did n o t c h a n g e . In a f u r t h e r s e r i e s of e x p e r i m e n t s w i t h f o r e b r a i n t i s s u e i n c u b a t e d a t 0 ° C , p i c r o t o x i n a t d o s e s up to 10 - 4 M d i d n o t i n h i b i t e i t h e r c o n t r o l 3 H - d i a z e p a m b i n d i n g o r 3 H - d i a z e p a m b i n d i n g s t i m u l a t e d by 3 x 1 0 - 5 M ( + ) - e t o m i d a t e . I n the s a m e c o n d i t i o n s b i c u c u l l i n e i n h i b i t e d 5 H - d i a z e p a m b i n d i n g b y 15 % a t 10 - 5 M a n d by 40 % a t 10 -4 M. B i n d i n g of 3 H - d i a z e p a m s t i m u l a t e d to 147 % of c o n t r o l b y 3 x ] 0 - 5 M ( + ) - e t o m i d a t e w a s i n h i b i t e d by 9 % a t 10-5 M a n d by 19 % a t 10-4 M of b i c u c c u l i n e . N e i t h e r i s o m e r of e t o m i d a t e e n h a n c e d o r i n h i b i t e d a t c o n c e n t r a t i o n s up to 10-5 M, the b i n d i n g of 3 H - d e x e t i m i d e to m u s c a r i n i c r e c e p t o r s , 3 H - d i h y d r o a l p r e n o l o l b i n d i n g to ~ - r e c e p t o r s , 3 H - W B 4101 b i n d i n g to ~ l - a d r e n e r g i c r e c e p t o r s , 3 H - c l o n i d i n e b i n d i n g to ~ 2 - r e c e p t o r s , 3 H - s u f e n t a n i l b i n d i n g to o p i a t e r e c e p t o r s , 3 H - h a l o p e r i d o l b i n d i n g to d o p a m i n e r e c e p t o r s a n d 3Hs p i p e r o n e b i n d i n g to s e r o t o n i n 2 r e c e p t o r s . FIG.
0.25
~ 020
~
0
* ETOMIDATE 2x~O-SM
~ 0.1§ u i
0.)0
X
~
0.05
~
0
0.t
02
0.3
0.4
0.5
0.6
0.7
0.0
3H-DIAZEPAM SPECIFICALLYBOUNO(nM)
3
S c a t c h a r d p l o t s of 3 H - d i a z e p a m s p e c i f i c b i n d i n g m e a s u r e d in the p r e s e n c e (A, e) of 3 x 10-5 M e t o m i d a t e a n d w i t h o u t a d d e d d r u g (A, o). D i f f e r e n t s y m b o l s i n d i c a t e m e a s u r e m e n t s in i n d e p e n d e n t e x p e r i m e n t s . T h e f r e e l i g a n d c o n c e n t r a t i o n w a s g i v e n by the d i f f e r e n c e b e t w e e n t o t a l l y a d d e d a n d totally bound radioactivity. The tissue a m o u n t in the a s s a y w a s 9. 09 m g o r i g i n a l w e t w e i g h t p e r m l . C a l c u l a t i o n of regression l i n e s r e v e a l e d the f o l l o w i n g p a r a m e t e r s f o r c o n t r o l s K D = s l o p e - l = 5 . 0 7 + 0. 37 nM, abscissa intercept=0.789 + 0.058 nM or Bmax=86.8 +6.2 fmoles/mg tissue, correlation coefficient rs=-0.955;with 3 x ]0-5M e t o m i d a t e : K D = s l o p e - 1=3.29 + 0 . 2 9 n M , a b s c i s s a i n t e r c e p t = 0 . 824 + 0. 073 n M o r Bmax=90.6 + 8.0 fmoles/mg tissue.
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Discussion T h e p r e s e n t r e s u l t s c l e a r l y show t h a t o v e r t h e s a m e c o n c e n t r a t i o n r a n g e (+), b u t n o t (-), e t o m i d a t e e n h a n c e s i n v i t r o 3 H - d i a z e p a m b i n d i n g i n r a t forebrain. T h e d e g r e e of s t i m u l a t i o n o b t a i n e d w i t h ( + ) - e t o m i d a t e i n f o r e b r a i n (150 %) w a s e v e n g r e a t e r t h a n t h a t o b t a i n e d w i t h GAB/k (130 %) a n d m a x i m a l s t i m u l a t i o n o c c u r r e d a t a 10 t i m e s l o w e r c o n c e n t r a t i o n . T h e e f f e c t of e t o m i d a t e on 3 H - d i a z e p a m b i n d i n g is c a u s e d b y a n i n c r e a s e i n the b i n d i n g a f f i n i t y , i n c r e a s e d b i n d i n g a f f i n i t y h a s a l s o b e e n r e p o r t e d f o r the GAB/k, p e n t o b a r b i t a l a n d e t a z o l a t e s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g (21). T h u s in f o r e b r a i n t i s s u e it a p p e a r s t h a t ( + ) - e t o m i d a t e a n d GAB.& i n c r e a s e b e n z o d i a z e p i n e b i n ding. H o w e v e r , in c e r e b e l l u m u s i n g a s y n a p t i c m e m b r a n e p r e p a r a t i o n which is e n r i c h e d i n G/kBA r e c e p t o r s (12), ( + ) - e t o m i d a t e did n o t a c t i v a t e 3 H - d i a z e p a r e b i n d i n g , w h e r e a s GAB.& (190 %) a n d m u s c i m o l (200 % ) h i g h l y e n h a n c e d the 3 H - d i a z e p a m b i n d i n g . I n t e r e s t i n g l y T H I P s h o w e d n o a c t i v i t y i n f o r e b r a i n , a n d o n l y s l i g h t l y e n h a n c e d c e r e b e l l a r 3 H - d i a z e p a m b i n d i n g (130 % ) a t h i g h concentrations. In a d d i t i o n the s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g i n f o r e b r a i n by ( + ) - e t o m i d a t e a n d G/kB/k w a s n o t r e v e r s e d o r e n h a n c e d by T H I P . H o w e v e r , the e n h a n c e m e n t o r r e v e r s a l of b i n d i n g by T H I P is t e m p e r a t u r e d e p e n d e n t a n d d o e s n o t o c c u r a t 0°C ( ] 9 ) . The f a i l u r e o f p i c r o t o x i n a n d b i c u c c u l i n e to i n h i b i t the s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g by 3 x 10 -5 M ( + ) - e t o m i d a t e a t 0°C is a l s o p r o b a b l y a t e m p e r a t u r e d e p e n d e n t p h e n o m e n o n since these compounds although not significantly affecting G/kB/k-enhanced b e n z o d i a z e p i n e binding at 0°C, i n h i b i t e d G/kB/k-enhanced b e n z o d i a z e p i n e b i n d i n g a t 37°C i n c h l o r i d e c o n t a i n i n g s o l u t i o n s (20). It w a s a l s o o b s e r v e d t h a t , u n l i k e G/kBA, m u s c i m o l a n d T H I P , ( + ) - e t o m i d a t e w a s n o t c a p a b l e of d i s p l a c i n g 3 H - m u s c i m o l f r o m i t s r e c e p t o r s i t e s i n the c e r e b e l l u m . The ina c t i v i t y of ( + ) - e t o m i d a t e i n c e r e b e l l a r t i s s u e , a s o p p o s e d to the m a r k e d a c t i v i t y of G A B / k - e r g i c d r u g s s u g g e s t s t h a t ( + ) - e t o m i d a t e h a s a d i f f e r e n t m e c h a n i s m of a c t i o n on b e n z o d i a z e p i n e r e c e p t o r s t h a n the G / k B / k - m i m e t i c c o m pounds. It h a s r e c e n t l y b e e n s h o w n t h a t a s e r i e s of b a r b i t u r a t e s a r e c a p a b l e of enhancing 3H-diazeparn binding, and that this correlates strongly with their h y p n o t i c a b i l i t y (13, 14). T h i s e f f e c t is c h l o r i d e d e p e n d e n t a n d i s b l o c k e d b y p i c r o t o x i n ( ] 3 ) . It h a s a l s o b e e n r e p o r t e d t h a t b a r b i t u r a t e e n h a n c e m e n t of benzodiazepine binding varies with brain region such that cortex >hippocamp u s > s t r i a t u r n = t h a l a m u s > c e r e b e l l u m = m e d u l l a p o n s (15). T h e p r e s e n t finding that e t o m i d a t e s t e r e o s p e c i f i c a l l y e n h a n c e s f o r e b r a i n but not c e r e b e l l a r 3 H - d i a z e p a m b i n d i n g p o i n t s i n a s i m i l a r d i r e c t i o n . It i s t h o u g h t t h a t the b a r b i t u r a t e e n h a n c e m e n t of 3 H - d i a z e p a m b i n d i n g o c c u r s v i a a n i n t e r a c t i o n w i t h the c h l o r i d e i o n o p h o r e of the G/kB/k r e c e p t o r , s i n c e a l l h y p n o t i c b a r b i t u r a t e s inhibit 3H-dihydropicrotoxinin binding, a drug which blocks a related posts y n a p t i c s i t e (16, ]7). A l s o in c u l t u r e d m o u s e s p i n a l c o r d n e u r o n e s h y p n o t i c b a r b i t u r a t e s a c t i v a t e c h l o r i d e i o n c h a n n e l s (18). T h e s i m i l a r i t i e s b e t w e e n the p h a r m a c o l o g i c a l p r o p e r t i e s of e t o m i d a t e a n d b a r b i t u r a t e s ( h ~ p n o s i s , a n t i c o n v u l s i v e a n d b r a i n p r o t e c t i v e ) p l u s the c o m p a r a b l e e f f e c t s on J H - d i a z e p a m b i n d i n g m a k e it r e a s o n a b l e to h y p o t h e s i z e t h a t e t o m i d a t e s t i m u l a t e s t h i s b i n d i n g i n the s a m e w a y a s b a r b i t u r a t e s . It i s w o r t h w h i l e to i n v e s t i g a t e w h e t h e r ( + ) - e t o m i d a t e b i n d s to the c h l o r i d e i o n o p h o r e of the G/kB/k r e c e p t o r c o m p l e x . T h e a v a i l a b i l i t y of a p a i r of o p t i c a l i s o m e r s o n l y one of w h i c h s h o w s p h a r m a c o l o g i c a l and b i o c h e m i c a l a c t i v i t y m a y m a k e e t o m i d a t e a useful tool for further research.
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Acknowledgements This s t u d y w a s in p a r t s u p p o r t e d by a g r a n t f r o m the I . W . O . N . L . References
I.
2. 3. 4.
A. WAUQUIER, D. ASHTON, G. CLINCKE, C.J.E. NIEMEGEERSand P.A.J. Janssen, In: Anasthesie bei Zerebralen Krampfanfallen and Intensivtherapie des Status Epilepticus~ edited by A. Opitz and R. Degen, pp. 183-203, Erlangen: Verlagsgesellschaft (1980). P.A.J. JANSSEN, C.J.E. NIEMEGEERS and R.P.H. MARSBOOM, Arch. Int. Pharmacodyn. Ther. 214:92-132 (1975). L.K.C. DESMEDT, C.J.E. NIEMEGEERS, P.J. LEWIand P.A.J. JALNSSEN, A r z n e i m i t t e l - F o r s c h . 2._~6: 1592- 1603 (1976). D. A S H T O N a n d A . W A U Q U I E R , P h a r m a c . B i o c h e m . B e h a v . ] 1 : 2 2 1 - 2 2 6
(]976). 5. 6. 7.
8. 9. 10. ll. ]2. ]3. 14. 15. ]6. 17. 18. 19. 20. 21.
D. A S H T O N a n d A . W A U Q U I E R , P s y c h o p h a r m a c o l o g y 65: 7- 13 (1979). A. WAUQUIER, D. A S H T O N and P. VAN DER S T A R R E , E p i l e p s y I n t e r n a t i o n a l S y m p o s i u m ~ F i r e n z e , I t a l y , A b s t r a c t 135 (1979). P . V A N D E R S T A R R E , In: An~tsthesie b e i Z e r e b r a l e n K r a m p f a n f a l l e n und I n t e n s i v t h e r a p i e d e s Stattts E p i l e p t i c u s , e d i t e d by A. Opitz a n d R. Degen, pp. 205-208, E r l a n g e n : V e r l a g s g e s e l l s c h a f t (1980). A. W A U Q U I E R , D. ASHTON, G. C L I N C K E and C . J . E . N I E M E G E E R S , A r c h . Int. P h a r m a c o d y n . T h e r . 2 4 9 : 3 3 0 - 3 3 4 (]981). R . M . EVANS a n d R . G . H I L L , E x p e r i e n t i a 3 4 : 1 3 2 5 - 1 3 2 6 (1978). R . M . E V A N S a n d R . G . H I L L , B r . J. P h a r m a c . 6.~1:484 (1977). E. COSTA and A. GUIDOTTI, Ann. R e v . P h a r m a c o l . T o x i c o l . ]_~9: 531545 (1979). K. B E A U M O N T , W.S. C H I L T O N , H.I. Y A M A M U R A and S.J. E N N A , Brain Res. 148:153-162 (]976). F. L E E B - L U N D B E R G , A. S N O W M A N and R . W . O L S E N , Proc. natn. Acad. Sci. U S A 77:7468-7472 (1980). T.C. B U T L E R , J. P h a r m a c . 74:I18-128 (1942). R . W . O L S E N a n d F . L E E B - L U N D B E R G , In: N e u r o t r a n s m i t t e r s in E p i l e p s y r e d i t e d by P. M o r s e l l i and K. L l o y d , R a v e n P r e s s , in p r e s s (]981). M . K . TICKN, M. B A N a n d R . W . O L S E N , M o l e c . P h a r m a c . 44: 3 9 ] 402 (1978). M.K. TICKNandR.W. OLSEN, L i f e Sci. 2._~2:]643-1651 (1978). D . A . M A T H E R S and J . L . B A R K E R , S c i e n c e 2 0 9 : 5 0 7 - 5 0 9 (1980). C. B R A E S T R U P and M. N I E L S E N , N e u r o s c i . L e t t . Suppl. 7 : 5 7 (1981). M. K A R O B A T H , G. D R E X L E R a n d P . S U P A V I L A I , Life Sci. 28: 307313 (1981). F . L E E B - L U N D B E R G , A. SNOWMAN a n d R . W . O L S E N , J. N e u r o s c i . 1 ( 5 ) : 47]-477 (1981).
ETOMIDATE
Pergamon Press
STEREOSPECIFICALLY STIMULATES FOREBRAIN, NOT CEREBELLAR, 3H-DIAZEPAM BINDING
D. A s h t o n
, R. G e e r t s ,
C. W a t e r k e y n a n d J . E .
BUT
Leysen
D e p a r t m e n t of P h a r m a c o l o g y ~ a n d B i o c h e m i c a l P h a r m a c o l o g y Janssen Pharmaceutica, B-2340 Beerse, Belgium (Received in final form October 27, 1981)
Summa ry 3 H - D i a z e p a m binding to a total particulate fraction of rat foreb r a i n is e n h a n c e d b y ( + ) - e t o m i d a t e a n d GABA, b u t n o t b y ( - ) - e t o m i d a t e . T h e e n h a n c e m e n t of 3 H - d i a z e p a m b i n d i n g b y ( + ) - e t o m i d a t e w a s due to a t w o - f o l d i n c r e a s e i n b i n d i n g a f f i n i t y , the m a x i m a l n u m b e r of s i t e s r e m a i n e d u n c h a n g e d . T h e d e g r e e of s t i m u l a t i o n with ( + ) - e t o m i d a t e was h i g h e r than that o b t a i n e d with GABA. T H I P d i d n o t s t i m u l a t e 3 H - d i a z e p a m b i n d i n g to f o r e b r a i n , a n d d i d n o t r e v e r s e the e n h a n c e d b i n d i n g s e e n w i t h ( + ) - e t o m i d a t e o r G A B A . In a s y n a p t o s o m a l m e m b r a n e p r e p a r a t i o n of r a t c e r e b e l l u m , u n l i k e ( + ) - e t o m i d a t e , GABA a n d m u s c i m o l p r o d u c e d a m a r k e d s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g . ( + ) - E t o m i d a t e did n o t i n h i b i t 3 H - m u s c i m o l b i n d i n g to GABA r e c e p t o r s , n o r did i t a c t i v a t e o r i n h i b i t o t h e r in v i t r o r e c e p t o r binding a s s a y s . T h e e f f e c t s of ( + ) - e t o m i d a t e o n the b e n z o d i a z e p i n e b i n d i n g a r e d i f f e r e n t f r o m t h o s e of g a b a m i m e t i c d r u g s . It i s p r o p o s e d t h a t l i k e b a r b i t u r a t e s , ( + ) - e t o m i d a t e m a y a f f e c t b e n z o d i a z e p i n e b i n d i n g by i n t e r a c t i o n w i t h the c h l o r i d e i o n o p h o r e w h i c h i s c o u p l e d to the G A B A - r e c e p t o r .
E t o m i d a t e , R - ( + ) e t h y l - 1 - ( 1 - p h e n y l e t h y l ) - 1 H - i m i d a z o l e - c a r b o x y l a t e is a potent, short acting non-barbiturate hypnotic, which lacks analgesic propert i e s ( ] , 2 ) . T h e d r u g h a s two o p t i c a l i s o m e r s , the ( + ) i s o m e r b e i n g r e s p o n s i b l e f o r the i n d u c t i o n of h y p n o s i s . I n a d d i t i o n to i t s h y p n o t i c a c t i v i t y ( + ) - e t o m i d a t e a l s o s h o w s p o t e n t a n t i c o n v u l s a n t a c t i v i t y i n v a r i o u s a n i m a l t e s t s : s u c h a s the maximalmetrazol t e s t (3), the s u b c u t a n e o u s m e t r a z o l t e s t (1), the D - L - a l l y l g l y c i n e t e s t (4), a n d i n a m y g d a l o i d k i n d l e d s e i z u r e s (1, 5). In h u m a n s ( + ) - e t o m i d a t e h a s b e e n s u c c e s s f u l l y u s e d to t e r m i n a t e t h e r a p y - r e s i s t a n t s t a t u s e p i l e p t i c u s (6, 7). R e c e n t e x p e r i m e n t s h a v e d e m o n s t r a t e d a p r o t e c t i v e e f f e c t of the c o m p o u n d i n a n i m a l m o d e l s of h y p o x i a a n d i s c h e m i a (1, 8). In n e u r o p h y s i o l o g i c a l e x p e r i m e n t s b o t h i n v i t r o a n d i n v i v o ( + ) - e t o m i d a t e w a s s h o w n to m i m i c r e s p o n s e s to y - a m i n o b u t y r i c a c i d (GABA) a n d to be s p e c i f i c a l l y a n t a gorized by k n o w n G A B A - a n t a g o n i s t s (9, 10). M a n y e x p e r i m e n t s h a v e s h o w n t h a t GABA is c a p a b l e of a c t i v a t i n g 3 H - b e n z o d i a z e p i n e b i n d i n g i n v i t r o (11). We now r e p o r t the i n t e r a c t i o n s b e t w e e n (+)- a n d ( - ) - e t o m i d a t e i n c o m p a r i s o n to G A B A , m u s c i m o l a n d 4, 5, 6 , 7 - t e t r a h y d r o i s o x a z o l e ( 5 , 4 - C ) p y r i d i n - 3 ( 2 H ) o n e ( T H I P ) on 3 H - d i a z e p a m b i n d i n g i n r a t f o r e b r a i n a n d c e r e b e l l u m .
0024-3205/81/252631-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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M e t h o d s and M a t e r i a l s F e m a l e W i s t a r r a t s w e r e used. F r e s h l y d i s s e c t e d f o r e b r a i n s (whole b r a i n s m i n u s c e r e b e l l u m a n d m e d u l l a o b l o n g a t a ) w e r e h o m o g e n i z e d in 40 v o l u m e s T r i s - H C 1 b u f f e r 50 r a M , p H 7 . 4 , u s i n g a n U l t r a T u r r a x . M e m b r a n e s w e r e c e n t r i f u g e d a t 3 9 , 0 0 0 x g f o r 10 m i n a n d w a s h e d 5 t i m e s by r e s u s p e n s i o n in b u f f e r a n d c e n t r i f u g a t i o n . W a s h e d m e m b r a n e s w e r e s u s p e n d e d in 100 v o l u m e s b u f f e r p e r g r a m o r i g i n a l w e t w e i g h t of t i s s u e a n d f r e s h l y u s e d f o r b i n d i n g
assays. Cerebellar tissue w a s prepared as described for 3 H - m u s c i m o l
binding
a s s a y s (12). R a t c e r e b e l l u m w a s h o m o g e n i z e d in 10 v o l u m e s 0. 25 M s u c r o s e . N u c l e i a n d c e l l d e b r i s w e r e r e m o v e d by c e n t r i f u g a t i o n a t 1090 g x ] 0 r a i n . F r o m t h e s u p e r n a t a n t , s u b c e l l u l a r p a r t i c l e s w e r e c e n t r i f u g e d a t 39, 100 x 20 r a i n . T h e p e l l e t w a s h o m o g e n i z e d in 10 v o l u m e s H 2 0 u s i n g a n U l t r a T u r r a x f o l l o w e d by c e n t r i f u g a t i o n a t 7720 g x I0 r a i n . T h e s u p e r n a t a n t w a s f u r t h e r c e n t r i f u g e d a t 16, 100 g x 30 r a i n . T h e m e m b r a n e p e l l e t w a s w a s h e d by h o m o g e n i z a t i o n in 20 v o l u m e s T r i s - c i t r a t e b u f f e r , 30 r a M , p H 7. 1 a n d c e n t r i f u g a t i o n a t 39, ]00 g x 20 r a i n . T h e p e l l e t w a s f r o z e n a n d k e p t a t - 2 0 ° C u n t i l u s e . A f t e r t h a w i n g t h e p e l l e t w a s w a s h e d two m o r e t i m e s in T r i s - c i t r a t e b u f f e r a n d f i n a l l y s u s p e n d e d in ]00 v o l u m e s T r i s - H C 1 b u f f e r p e r g r a m o r i g i n a l w e t w e i g h t of t i s s u e . F o r 3 H - d i a z e p a m b i n d i n g , a s s a y m i x t u r e s w e r e c o m p o s e d of 1 m l m e m b r a n e s u s p e n s i o n , 0 . 0 5 m l 3 H - d i a z e p a m ( f i n a l c o n c e n t r a t i o n 1 nM) a n d 0 . 0 5 m l 10 % e t h a n o l o r d r u g d i s s o l v e d in 10 % e t h a n o l . I n c u b a t i o n w a s r u n a t 0 °C for 30rain. L a b e l l e d m e m b r a n e s w e r e h a r v e s t e d by f i l t r a t i o n u n d e r r e d u c e d p r e s s u r e on G F / B g l a s s f i b e r f i l t e r s a n d w a s h e d 3 t i m e s w i t h 5 m l i c e - c o l d b u f f e r . R a d i o a c t i v i t y on t h e f i l t e r s w a s c o u n t e d in a n I n t e r t e c h n i q u e L i q u i d S c i n t i l l a t i o n c o u n t e r e q u i p p e d w i t h a b u i l t - i n c o m p u t e r s y s t e m to e x p r e s s m e a s u r e d r a d i o a c t i v i t y in D P M . Specific 3H-diazepam binding was defined by s u b t r a c t i n g n o n - s p e c i f i c b i n d i n g m e a s u r e d in t h e p r e s e n c e of 100 n M c l o nazepam. 3H-Muscimol b i n d i n g in th e s y n a p t o s o m a l p r e p a r a t i o n of r a t c e r e b e l l u m w a s c a r r i e d o u t a s d e s c r i b e d in r e f . 12. 3 H - D i a z e p a m s a t u r a t i o n c u r v e s in t he f o r e b r a i n w e r e m e a s u r e d in the a b s e n c e a n d t h e p r e s e n c e of 3 x 10 -5 M e t o m i d a t e u s i n g ]0 d i f f e r e n t c o n c e n t r a t i o n s of 3 H - d i a z e p a m in a r a n g e 0 . 2 to 8 riM. S p e c i f i c b i n d i n g w a s t a k e n a s t h e d i f f e r e n c e b e t w e e n t o t a l a n d n o n - s p e c i f i c b i n d i n g m e a s u r e d in t h e p r e s e n c e of 1 0 0 - f o l d e x c e s s of c l o n a z e p a m o v e r t h e c o n c e n t r a t i o n of 3 H - d i a zepam. A l l a s s a y s w e r e r u n in d u p l i c a t e a n d t w o e x p e r i m e n t s w e r e r u n i n dependently. L i n e s of b e s t f i t t h r o u g h S c a t c h a r d p l o t s w e r e c a l c u l a t e d by the m e t h o d of l e a s t s q u a r e s . Materials,
3H-Diazepam
(spec.
radioactivity
76 C i / m m o l e ,
NEN,
Germany), 3 H - m u s c i m o l (spec. radioactivity i0.3 Ci/mmole, N E N , G e r m a n y ~ clonazeparn (Hoffmann-La Roche, Switzerland), T H I P (kindly provided by Dr. J. Scheel-Krtlger and Dr. V. Christensen, Lundbeck, Denmark), (+)and (-)etomidate (Janssen Pharmaceutica, Belgium), m u s c i m o l (Fluka, Switzerland), G A B A (Aldrich, Europe Division).
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Re s u l t s I n w a s h e d t o t a l m e m b r a n e s of r a t f o r e b r a i n , ( + ) - e t o m i d a t e p r o d u c e s a c o n c e n t r a t i o n d e p e n d e n t i n c r e a s e i n 3 H - d i a z e p a m b i n d i n g , to a m a x i m u m of 150 % of s p e c i f i c b i n d i n g a t 10 ~M ( + ) - e t o m i d a t e . G A B A e n h a n c e s the b i n d i n g to m a x i m a l l y 135 % a t 100 ~M. ( - ) - E t o m i d a t e a n d T H I P do n o t c a u s e a n y s t i m u l a t i o n of b i n d i n g , b u t ( - ) - e t o m i d a t e p r o d u c e s a s m a l l i n h i b i t i o n of b i n d i n g a t 10 ~M. M a x i m a l s t i m u l a t i o n of s p e c i f i c 3 H - d i a z e p a m b i n d i n g by 30 ~M (+)e t o m i d a t e o r by 100 ~M GABA i s n o t r e v e r s e d by T H I P up to a c o n c e n t r a t i o n of 100 ~M. G r a p h s a r e s h o w n i n F i g u r e 1. I n a s y n a p t o s o m a l m e m b r a n e f r a c t i o n of r a t c e r e b e l l u m , w h i c h is e n r i c h e d i n GABA r e c e p t o r s (1Z), GABA a n d m u s c i m o l p r o d u c e a m a r k e d s t i m u l a t i o n of s p e c i f i c 3 H - d i a z e p a m b i n d i n g to a m a x i m u m of ZOO % a t ]0 ~M. T h e s t i m u l a t i o n by GABA i n the c e r e b e l l a r m e m b r a n e s i s m u c h m o r e p r o n o u n c e d t h a n i n the f o r e b r a i n m e m b r a n e s a n d o c c u r s a t 10 t i m e s l o w e r c o n c e n t r a t i o n s . In c o n t r a s t ( + ) - e t o m i d a t e i s v i r t u a l l y i n a c t i v e i n the c e r e b e l l a r m e m b r a n e s .
= 1601
°t 120
oi, " ;
~
,&0 ,o60
Drug concentration(pM]
FIG.
o:,
;
,~
,~o ,060
THIP concentratio•(pM)
1
a. E f f e c t of ( + ) - e t o m i d a t e (e) (n = 7), ( - ) - e t o m i d a t e (o) (n = 4), G A B A (•) (n = 4), and T H I P (A) (n = Z) on specific 3H-diazepare (1 n M ) binding to w a s h e d total m e m b r a n e s of rat forebrain. Control specific binding per assay w a s Z 8 , 3 0 0 + Z, 100 D P M , n = 7 and represented 90 ~0 of the total binding, n indicates the n u m b e r of independent experiments of which data are averaged. b. Effect of T H I P on stimulated specific 3 H - d i a z e p a m binding by 3 x 10 -5 M e t o m i d a t e ( ~ ) o r by 1 x l 0 -4 M GABA ( & ) .
A s i g n i f i c a n t s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g is n o t o b s e r v e d up to a c o n c e n t r a t i o n of 1 ~tM a n d a t 10 ~tM o n l y a s m a l l e n h a n c e m e n t of b i n d i n g b y 30 % is n o t e d . T H I P o n l y p r o d u c e s a s m a l l i n c r e a s e i n 3 H - d i a z e p a m b i n d i n g i n the c e r e b e l l a r m e m b r a n e s a t h i g h c o n c e n t r a t i o n . Graphs are shown in Figure Z. I n a d d i t i o n ( + ) - e t o m i d a t e did n o t i n h i b i t 3 H - m u s c i m o l b i n d i n g to GABA r e c e p t o r s i n the c e r e b e l l a r s y n a p t o s o m a l p r e p a r a t i o n a t c o n c e n t r a t i o n s up to 10 ~M.
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Etomidate and 3H-diazepam Binding
Vol. 29, No. 25, 1981
FIG. 2
;
060, o.61 o'.i
;o
,60
D r u g c o n c e n t rat ion ( pM )
E f f e c t of ( + ) - e t o m i d a t e (e), GABA ( • ) , T H I P ( A ) a n d m u s c i m o l ( Q ) on s p e c i f i c 3 H - d i a z e p a m (1 nM) b i n d i n g to a washed synaptic membrane p r e p a r a t i o n of r a t c e r e b e l l u m . P o i n t s r e p r e s e n t m e a n d a t a of 5 i n d e p e n d e n t e x p e r i m e n t s in duplicate. Control specific b i n d i n g p e r a s s a y w a s 2850 + 680 D P M , n = 5 a n d r e p r e s e n t e d 80 % of t o t a l b i n d i n g .
T h e S c a t c h a r d p l o t ( F i g . 3) i n d i c a t e s a d e c r e a s e in the KD f o r 3 H - d i a z e p a m i n f o r e b r a i n f r o m 5 . 0 7 + 0 . 3 7 n M to 3 . 2 9 + 0 . 2 9 n M i n the p r e s e n c e of 3 x 10-5 M ( + ) - e t o m i d a t e ; h o w e v e r , B m a x did n o t c h a n g e . In a f u r t h e r s e r i e s of e x p e r i m e n t s w i t h f o r e b r a i n t i s s u e i n c u b a t e d a t 0 ° C , p i c r o t o x i n a t d o s e s up to 10 - 4 M d i d n o t i n h i b i t e i t h e r c o n t r o l 3 H - d i a z e p a m b i n d i n g o r 3 H - d i a z e p a m b i n d i n g s t i m u l a t e d by 3 x 1 0 - 5 M ( + ) - e t o m i d a t e . I n the s a m e c o n d i t i o n s b i c u c u l l i n e i n h i b i t e d 5 H - d i a z e p a m b i n d i n g b y 15 % a t 10 - 5 M a n d by 40 % a t 10 -4 M. B i n d i n g of 3 H - d i a z e p a m s t i m u l a t e d to 147 % of c o n t r o l b y 3 x ] 0 - 5 M ( + ) - e t o m i d a t e w a s i n h i b i t e d by 9 % a t 10-5 M a n d by 19 % a t 10-4 M of b i c u c c u l i n e . N e i t h e r i s o m e r of e t o m i d a t e e n h a n c e d o r i n h i b i t e d a t c o n c e n t r a t i o n s up to 10-5 M, the b i n d i n g of 3 H - d e x e t i m i d e to m u s c a r i n i c r e c e p t o r s , 3 H - d i h y d r o a l p r e n o l o l b i n d i n g to ~ - r e c e p t o r s , 3 H - W B 4101 b i n d i n g to ~ l - a d r e n e r g i c r e c e p t o r s , 3 H - c l o n i d i n e b i n d i n g to ~ 2 - r e c e p t o r s , 3 H - s u f e n t a n i l b i n d i n g to o p i a t e r e c e p t o r s , 3 H - h a l o p e r i d o l b i n d i n g to d o p a m i n e r e c e p t o r s a n d 3Hs p i p e r o n e b i n d i n g to s e r o t o n i n 2 r e c e p t o r s . FIG.
0.25
~ 020
~
0
* ETOMIDATE 2x~O-SM
~ 0.1§ u i
0.)0
X
~
0.05
~
0
0.t
02
0.3
0.4
0.5
0.6
0.7
0.0
3H-DIAZEPAM SPECIFICALLYBOUNO(nM)
3
S c a t c h a r d p l o t s of 3 H - d i a z e p a m s p e c i f i c b i n d i n g m e a s u r e d in the p r e s e n c e (A, e) of 3 x 10-5 M e t o m i d a t e a n d w i t h o u t a d d e d d r u g (A, o). D i f f e r e n t s y m b o l s i n d i c a t e m e a s u r e m e n t s in i n d e p e n d e n t e x p e r i m e n t s . T h e f r e e l i g a n d c o n c e n t r a t i o n w a s g i v e n by the d i f f e r e n c e b e t w e e n t o t a l l y a d d e d a n d totally bound radioactivity. The tissue a m o u n t in the a s s a y w a s 9. 09 m g o r i g i n a l w e t w e i g h t p e r m l . C a l c u l a t i o n of regression l i n e s r e v e a l e d the f o l l o w i n g p a r a m e t e r s f o r c o n t r o l s K D = s l o p e - l = 5 . 0 7 + 0. 37 nM, abscissa intercept=0.789 + 0.058 nM or Bmax=86.8 +6.2 fmoles/mg tissue, correlation coefficient rs=-0.955;with 3 x ]0-5M e t o m i d a t e : K D = s l o p e - 1=3.29 + 0 . 2 9 n M , a b s c i s s a i n t e r c e p t = 0 . 824 + 0. 073 n M o r Bmax=90.6 + 8.0 fmoles/mg tissue.
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Discussion T h e p r e s e n t r e s u l t s c l e a r l y show t h a t o v e r t h e s a m e c o n c e n t r a t i o n r a n g e (+), b u t n o t (-), e t o m i d a t e e n h a n c e s i n v i t r o 3 H - d i a z e p a m b i n d i n g i n r a t forebrain. T h e d e g r e e of s t i m u l a t i o n o b t a i n e d w i t h ( + ) - e t o m i d a t e i n f o r e b r a i n (150 %) w a s e v e n g r e a t e r t h a n t h a t o b t a i n e d w i t h GAB/k (130 %) a n d m a x i m a l s t i m u l a t i o n o c c u r r e d a t a 10 t i m e s l o w e r c o n c e n t r a t i o n . T h e e f f e c t of e t o m i d a t e on 3 H - d i a z e p a m b i n d i n g is c a u s e d b y a n i n c r e a s e i n the b i n d i n g a f f i n i t y , i n c r e a s e d b i n d i n g a f f i n i t y h a s a l s o b e e n r e p o r t e d f o r the GAB/k, p e n t o b a r b i t a l a n d e t a z o l a t e s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g (21). T h u s in f o r e b r a i n t i s s u e it a p p e a r s t h a t ( + ) - e t o m i d a t e a n d GAB.& i n c r e a s e b e n z o d i a z e p i n e b i n ding. H o w e v e r , in c e r e b e l l u m u s i n g a s y n a p t i c m e m b r a n e p r e p a r a t i o n which is e n r i c h e d i n G/kBA r e c e p t o r s (12), ( + ) - e t o m i d a t e did n o t a c t i v a t e 3 H - d i a z e p a r e b i n d i n g , w h e r e a s GAB.& (190 %) a n d m u s c i m o l (200 % ) h i g h l y e n h a n c e d the 3 H - d i a z e p a m b i n d i n g . I n t e r e s t i n g l y T H I P s h o w e d n o a c t i v i t y i n f o r e b r a i n , a n d o n l y s l i g h t l y e n h a n c e d c e r e b e l l a r 3 H - d i a z e p a m b i n d i n g (130 % ) a t h i g h concentrations. In a d d i t i o n the s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g i n f o r e b r a i n by ( + ) - e t o m i d a t e a n d G/kB/k w a s n o t r e v e r s e d o r e n h a n c e d by T H I P . H o w e v e r , the e n h a n c e m e n t o r r e v e r s a l of b i n d i n g by T H I P is t e m p e r a t u r e d e p e n d e n t a n d d o e s n o t o c c u r a t 0°C ( ] 9 ) . The f a i l u r e o f p i c r o t o x i n a n d b i c u c c u l i n e to i n h i b i t the s t i m u l a t i o n of 3 H - d i a z e p a m b i n d i n g by 3 x 10 -5 M ( + ) - e t o m i d a t e a t 0°C is a l s o p r o b a b l y a t e m p e r a t u r e d e p e n d e n t p h e n o m e n o n since these compounds although not significantly affecting G/kB/k-enhanced b e n z o d i a z e p i n e binding at 0°C, i n h i b i t e d G/kB/k-enhanced b e n z o d i a z e p i n e b i n d i n g a t 37°C i n c h l o r i d e c o n t a i n i n g s o l u t i o n s (20). It w a s a l s o o b s e r v e d t h a t , u n l i k e G/kBA, m u s c i m o l a n d T H I P , ( + ) - e t o m i d a t e w a s n o t c a p a b l e of d i s p l a c i n g 3 H - m u s c i m o l f r o m i t s r e c e p t o r s i t e s i n the c e r e b e l l u m . The ina c t i v i t y of ( + ) - e t o m i d a t e i n c e r e b e l l a r t i s s u e , a s o p p o s e d to the m a r k e d a c t i v i t y of G A B / k - e r g i c d r u g s s u g g e s t s t h a t ( + ) - e t o m i d a t e h a s a d i f f e r e n t m e c h a n i s m of a c t i o n on b e n z o d i a z e p i n e r e c e p t o r s t h a n the G / k B / k - m i m e t i c c o m pounds. It h a s r e c e n t l y b e e n s h o w n t h a t a s e r i e s of b a r b i t u r a t e s a r e c a p a b l e of enhancing 3H-diazeparn binding, and that this correlates strongly with their h y p n o t i c a b i l i t y (13, 14). T h i s e f f e c t is c h l o r i d e d e p e n d e n t a n d i s b l o c k e d b y p i c r o t o x i n ( ] 3 ) . It h a s a l s o b e e n r e p o r t e d t h a t b a r b i t u r a t e e n h a n c e m e n t of benzodiazepine binding varies with brain region such that cortex >hippocamp u s > s t r i a t u r n = t h a l a m u s > c e r e b e l l u m = m e d u l l a p o n s (15). T h e p r e s e n t finding that e t o m i d a t e s t e r e o s p e c i f i c a l l y e n h a n c e s f o r e b r a i n but not c e r e b e l l a r 3 H - d i a z e p a m b i n d i n g p o i n t s i n a s i m i l a r d i r e c t i o n . It i s t h o u g h t t h a t the b a r b i t u r a t e e n h a n c e m e n t of 3 H - d i a z e p a m b i n d i n g o c c u r s v i a a n i n t e r a c t i o n w i t h the c h l o r i d e i o n o p h o r e of the G/kB/k r e c e p t o r , s i n c e a l l h y p n o t i c b a r b i t u r a t e s inhibit 3H-dihydropicrotoxinin binding, a drug which blocks a related posts y n a p t i c s i t e (16, ]7). A l s o in c u l t u r e d m o u s e s p i n a l c o r d n e u r o n e s h y p n o t i c b a r b i t u r a t e s a c t i v a t e c h l o r i d e i o n c h a n n e l s (18). T h e s i m i l a r i t i e s b e t w e e n the p h a r m a c o l o g i c a l p r o p e r t i e s of e t o m i d a t e a n d b a r b i t u r a t e s ( h ~ p n o s i s , a n t i c o n v u l s i v e a n d b r a i n p r o t e c t i v e ) p l u s the c o m p a r a b l e e f f e c t s on J H - d i a z e p a m b i n d i n g m a k e it r e a s o n a b l e to h y p o t h e s i z e t h a t e t o m i d a t e s t i m u l a t e s t h i s b i n d i n g i n the s a m e w a y a s b a r b i t u r a t e s . It i s w o r t h w h i l e to i n v e s t i g a t e w h e t h e r ( + ) - e t o m i d a t e b i n d s to the c h l o r i d e i o n o p h o r e of the G/kB/k r e c e p t o r c o m p l e x . T h e a v a i l a b i l i t y of a p a i r of o p t i c a l i s o m e r s o n l y one of w h i c h s h o w s p h a r m a c o l o g i c a l and b i o c h e m i c a l a c t i v i t y m a y m a k e e t o m i d a t e a useful tool for further research.
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Acknowledgements This s t u d y w a s in p a r t s u p p o r t e d by a g r a n t f r o m the I . W . O . N . L . References
I.
2. 3. 4.
A. WAUQUIER, D. ASHTON, G. CLINCKE, C.J.E. NIEMEGEERSand P.A.J. Janssen, In: Anasthesie bei Zerebralen Krampfanfallen and Intensivtherapie des Status Epilepticus~ edited by A. Opitz and R. Degen, pp. 183-203, Erlangen: Verlagsgesellschaft (1980). P.A.J. JANSSEN, C.J.E. NIEMEGEERS and R.P.H. MARSBOOM, Arch. Int. Pharmacodyn. Ther. 214:92-132 (1975). L.K.C. DESMEDT, C.J.E. NIEMEGEERS, P.J. LEWIand P.A.J. JALNSSEN, A r z n e i m i t t e l - F o r s c h . 2._~6: 1592- 1603 (1976). D. A S H T O N a n d A . W A U Q U I E R , P h a r m a c . B i o c h e m . B e h a v . ] 1 : 2 2 1 - 2 2 6
(]976). 5. 6. 7.
8. 9. 10. ll. ]2. ]3. 14. 15. ]6. 17. 18. 19. 20. 21.
D. A S H T O N a n d A . W A U Q U I E R , P s y c h o p h a r m a c o l o g y 65: 7- 13 (1979). A. WAUQUIER, D. A S H T O N and P. VAN DER S T A R R E , E p i l e p s y I n t e r n a t i o n a l S y m p o s i u m ~ F i r e n z e , I t a l y , A b s t r a c t 135 (1979). P . V A N D E R S T A R R E , In: An~tsthesie b e i Z e r e b r a l e n K r a m p f a n f a l l e n und I n t e n s i v t h e r a p i e d e s Stattts E p i l e p t i c u s , e d i t e d by A. Opitz a n d R. Degen, pp. 205-208, E r l a n g e n : V e r l a g s g e s e l l s c h a f t (1980). A. W A U Q U I E R , D. ASHTON, G. C L I N C K E and C . J . E . N I E M E G E E R S , A r c h . Int. P h a r m a c o d y n . T h e r . 2 4 9 : 3 3 0 - 3 3 4 (]981). R . M . EVANS a n d R . G . H I L L , E x p e r i e n t i a 3 4 : 1 3 2 5 - 1 3 2 6 (1978). R . M . E V A N S a n d R . G . H I L L , B r . J. P h a r m a c . 6.~1:484 (1977). E. COSTA and A. GUIDOTTI, Ann. R e v . P h a r m a c o l . T o x i c o l . ]_~9: 531545 (1979). K. B E A U M O N T , W.S. C H I L T O N , H.I. Y A M A M U R A and S.J. E N N A , Brain Res. 148:153-162 (]976). F. L E E B - L U N D B E R G , A. S N O W M A N and R . W . O L S E N , Proc. natn. Acad. Sci. U S A 77:7468-7472 (1980). T.C. B U T L E R , J. P h a r m a c . 74:I18-128 (1942). R . W . O L S E N a n d F . L E E B - L U N D B E R G , In: N e u r o t r a n s m i t t e r s in E p i l e p s y r e d i t e d by P. M o r s e l l i and K. L l o y d , R a v e n P r e s s , in p r e s s (]981). M . K . TICKN, M. B A N a n d R . W . O L S E N , M o l e c . P h a r m a c . 44: 3 9 ] 402 (1978). M.K. TICKNandR.W. OLSEN, L i f e Sci. 2._~2:]643-1651 (1978). D . A . M A T H E R S and J . L . B A R K E R , S c i e n c e 2 0 9 : 5 0 7 - 5 0 9 (1980). C. B R A E S T R U P and M. N I E L S E N , N e u r o s c i . L e t t . Suppl. 7 : 5 7 (1981). M. K A R O B A T H , G. D R E X L E R a n d P . S U P A V I L A I , Life Sci. 28: 307313 (1981). F . L E E B - L U N D B E R G , A. SNOWMAN a n d R . W . O L S E N , J. N e u r o s c i . 1 ( 5 ) : 47]-477 (1981).