- Email: [email protected]
benzodiazepine receptors by benzodiazepine affinity column chromatography and formation of antibody against the purified receptors
S106 THE PURIFICATION OF GABA/BENZODIAZEPINE RECEPTORS BY BENZODIAZEPINE AFFINITY COLUMN CHROMATOGRAPHY AND FORMATION OF ANTIBODY AGAINST THE PURIFIED RECEPTORS JUN-ICHI TAGUCHI*, KINYA KURIYAMA and HIROSHI KIMURA t#, Department of pharmacology, Kyoto Prefectural University of Medicine, Kamikyo-Ku, Kyoto 602, Japan and t#Department of Anatomy, Shiga University of Medical Sciences, Ohtsu, Shiga 520-21, Japan. The purification of solubilized GABA and benzodiazepine (BZP) receptors from the rat brain and bovine cerebral cortex by BZP affinity gel using I012-S, a new BZP exhibiting a high affinity to solubilized BZP receptor, and the formation of antibody against the purified receptors, has been attempted. GABA and BZP receptors were solubilized from synaptic membrane fractions of the rat brain and bovine cerebral cortex with 1% Nonidet P-40, and the solubilized fraction was applied to the affinity column. The bio-specific elution with I012-S (i mM) resu±ted in the elution of GABA receptor protein highly enriched in the specific binding of [3H]muscimol ([3H]Mus). The highest purification fold obtained was 4,576 and 3,789 in the rat and bovine brains, respectively. SDSpolyacrylamide gel electrophoretic profiles showed that the purified receptor protein consisted of two subunits (M.W.: 48,500 and 54,500 in the rat brain, 53,000 and 57,000 in the bovine brain,respectively). Photoaffinity labeling of the purified fraction with [3H]Flunitra~epam ([3H]FIu) in which the [3H]FIu binding was partially detected following anion exchange chromatography using DEAE-Sephacel, showed that BZP recognition sites existed on one band (M.W.: 48,500) and both bands (M.W.: 53,000 and 57,000) of the purified receptor protein from rat and bovine brains, respectively. Irmnunization of mouse with the purified bovine GABA/BZP receptor protein as antigen resulted in the production of the antibody. Furthermore, immunohistochemical studies in the rat cerebellum have suggested that GABAergic neurons reacting on the antibody are localized predominantly in the granular cell layer.
T H E S Y N E R G I S T I C E F F E C T OF C a 2+ I O N O P H O R E RELEASE FROM THE GUINEA PIG ILEUM
AND
PHORBOL
ESTER
IN A C h
KOHTARO TANIYAMA, MASATO KUSUNOKI and CHIKAKO TANAKA, D e p a r t m e n t o f P h a r m a c o l o g y , K o b e U n i v e r s i t y S c h o o l of M e d i c i n e , C h u o - K u , K o b e 650, Japan. B o t h an i n c r e a s e in i n t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n a n d the a c t i v a t i o n of c a l c i u m - p h o s p h o l i p i d dependent protein kinase (protein k i n a s e C) h a v e b e e n d e m o n s t r a t e d to a c t s y n e r g i s t i c a l l y in p r o d u o i n g a f u l l r e s P o n s e in s t i m u l u s - s e c r e t i o n coupling. Therefore, we c a r r i e d o u t e x p e r i m e n t s to d e t e r m i n e w h e t h e r t h e e n z y m e is i n v o l v e d in the A C h r e l e a s e f r o m c h o l i n e r g i c n e r v e e n d i n g s in t h e g u i n e a p i g ileum. T h e i l e a l s t r i p s , p r e l o a d e d w i t h [ 3 H ] c h o l i n e ~ w e r e s u p e r f u s e d w i t h C a 2 + - f r e e m e d i u m a n d s t i m u l a t e d w i t h C a Z+ i o n o p h o r e , A 2 3 1 8 7 , e l e c t r i c a l s t i m u l a t i o n o r h i g h K (20 mM) in t h e p r e s e n c e a n d a b s e n c e of C a 2+ The stimulations provoked ACh r e l e a s e in t h e p r e s e n c e of C a 2+, b u t n o t in the a b s e n c e of Ca 2+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13a c e t a t e (TPA) ( I 0 - 9 - I 0 - 7 M ) w h i c h a c t i v a t e s p r o t e i n k i n a s e C, p o t e n t i a t e d the s t i m u l a t i o n - i n d u c e d A C h r e l e a s e , in a d o s e d e p e n d e n t m a n n e r . T h e e f f e c t of T P A w a s i n h i b i t e d by p o l y m i x i n B, w h i c h r e p o r t e d l y i n h i b i t s the a c t i v a t i o n of p r o t e i n k i n a s e C. T h e e l e c trical stimulation (5 H z - 3 0 Hz) f r e q u e n c y - d e p e n d e n t l y provoked ACh r e l e a s e in t h e p r e s e n c e of C a 2+, a n d t h e m a x i m u m w a s o b t a i n e d at 30 Hz . T P A p o t e n t i a t e d the A C h r e l e a s e i n d u c e d by s t i m u l a t i o n at 20 Hz a n d u n d e r , a n d p o t e n t i a t e d t h o s e at 10 Hz a n d 20 Hz to a maximal response. These results suggest that protein kinase C p l a y s a r o l e in c e l l s u r f a c e s i g n a l t r a n s d u c t i o n , a n d in p r o d u c i n g a f u l l r e s p o n s e r e l a t e d to A C h r e l e a s e f r o m n e r v e e n d i n g s .
T H E S Y N E R G I S T I C E F F E C T OF C a 2+ I O N O P H O R E RELEASE FROM THE GUINEA PIG ILEUM
AND
PHORBOL
ESTER
IN A C h
KOHTARO TANIYAMA, MASATO KUSUNOKI and CHIKAKO TANAKA, D e p a r t m e n t o f P h a r m a c o l o g y , K o b e U n i v e r s i t y S c h o o l of M e d i c i n e , C h u o - K u , K o b e 650, Japan. B o t h an i n c r e a s e in i n t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n a n d the a c t i v a t i o n of c a l c i u m - p h o s p h o l i p i d dependent protein kinase (protein k i n a s e C) h a v e b e e n d e m o n s t r a t e d to a c t s y n e r g i s t i c a l l y in p r o d u o i n g a f u l l r e s P o n s e in s t i m u l u s - s e c r e t i o n coupling. Therefore, we c a r r i e d o u t e x p e r i m e n t s to d e t e r m i n e w h e t h e r t h e e n z y m e is i n v o l v e d in the A C h r e l e a s e f r o m c h o l i n e r g i c n e r v e e n d i n g s in t h e g u i n e a p i g ileum. T h e i l e a l s t r i p s , p r e l o a d e d w i t h [ 3 H ] c h o l i n e ~ w e r e s u p e r f u s e d w i t h C a 2 + - f r e e m e d i u m a n d s t i m u l a t e d w i t h C a Z+ i o n o p h o r e , A 2 3 1 8 7 , e l e c t r i c a l s t i m u l a t i o n o r h i g h K (20 mM) in t h e p r e s e n c e a n d a b s e n c e of C a 2+ The stimulations provoked ACh r e l e a s e in t h e p r e s e n c e of C a 2+, b u t n o t in the a b s e n c e of Ca 2+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13a c e t a t e (TPA) ( I 0 - 9 - I 0 - 7 M ) w h i c h a c t i v a t e s p r o t e i n k i n a s e C, p o t e n t i a t e d the s t i m u l a t i o n - i n d u c e d A C h r e l e a s e , in a d o s e d e p e n d e n t m a n n e r . T h e e f f e c t of T P A w a s i n h i b i t e d by p o l y m i x i n B, w h i c h r e p o r t e d l y i n h i b i t s the a c t i v a t i o n of p r o t e i n k i n a s e C. T h e e l e c trical stimulation (5 H z - 3 0 Hz) f r e q u e n c y - d e p e n d e n t l y provoked ACh r e l e a s e in t h e p r e s e n c e of C a 2+, a n d t h e m a x i m u m w a s o b t a i n e d at 30 Hz . T P A p o t e n t i a t e d the A C h r e l e a s e i n d u c e d by s t i m u l a t i o n at 20 Hz a n d u n d e r , a n d p o t e n t i a t e d t h o s e at 10 Hz a n d 20 Hz to a maximal response. These results suggest that protein kinase C p l a y s a r o l e in c e l l s u r f a c e s i g n a l t r a n s d u c t i o n , a n d in p r o d u c i n g a f u l l r e s p o n s e r e l a t e d to A C h r e l e a s e f r o m n e r v e e n d i n g s .