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The interaction between alpha-adrenoceptors and calcium ion channel blocker binding sites in the rat brain
S148 THE E F F E C T S QF N E U R O P E P T I D E S ON M U S C A R I N I C , R E C E P T O R B I N D I N G IN THE RAT ~ R A I N RIE MIYOSHI , SHOZO KITO, K U M I K O M I Z U N O and HIROAKI MATSUBAYASHI , Third D~partment of i n t e r n a l M e d i c i n e , H i r o s h i m a 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 , I-2-3 Kasumi, M i n a m i k u , H i r o s h i m a 734, J a p a n The a u t h o r s have so far e x a m i n e d the e f f e c t s of s o m a t o s t a t i n on muscarinic acetylcholine receptors (mAChR) in the rat h i p p o c a m p u s f r o m the viewpoint of binding experiments, and it was o b s e r v e d that s o m a t o s t a t i n r e d u c e d the affinity of mAChR agonist binding. In this paper, we r e p o r t on how various other neuropeptides w h i c h w e r e r e l a t e d w i t h the h i p p o c a m p u s i n f l u e n c e d m A C h R binding, and w h i c h s u b t y p e of m A C h R was a f f e c t e d 3 b Y s o m a t o s t a t i n , mAChR agogist binding experiments w e r e p e r f o r m e d w i ~ h use of H-oxotremorine-M-acetate (H-oxo-M) as the r a d i o a c t i v e l i g a n d and 10M acetylcholine as the n o n - r a d i o a c t i v e ligand. A s i n g l e b i n d i n g site was o b t a i n e d w h e n s a t u r a t i o n e x p e r i m e n t s w e r e p e r f o r m e d u s i n g Na-K phosphate b u f f e r solution. One ~ M of v a s o a c t i v e intestinal polypeptide, e n k e p h a l i n , v a s o p r e s s i n and s u b s t a n c e P w e r e a d d e d to the i n c u b a t i o n medium. To prevent the d e g r a d a t i o n of p e p t i d e s , r e a g e n t s such as b o v i n e ser~., albumin and b a c i t r a c i n w e r e added. As a r e s u l t of s a t u r a t i o n e x p e r i m e n t s of H-oxo-M3in the hippocampus, it w a s n o t i c e d that e n k e p h a l i n r e d u c e d the affinity of H-oxo-M b i n d i n g w i t h the B m a x v a l u e u n c h a n g e d , w h i c h w a s the case w i t h s o m a t o s t a t i n , too. Of t~e h i p p o c a m p u s , c e r e b r a l c o r t e x and m e d u l l a - p o n s , the e f f e c t of somatostatin on ~H-oxo-M binding w a s o b s e r v e d o n l y in th~ f o r m e r two, where mAChRs were d o m i n a n t l y of the MI type. From oxotremorine/ H-N-methyl-scopolamine competition experiments, it w a s n o t i c e d that s o m a t o s t a t i n c o n v e r t e d the oxotremorine high a f f i n i t y billding sites to the low a f f i n i t y state in the h i p p o c a m p u s and cerebral cortex. T h e r e f o r e , it seems that s o m a t o s t a t i n a c c e l e r a t e s the receptor-effector coupling mechanisms of the h i g h a f f i n i t y b i n d i n g site of MI receptors. The effect of somatostatin in the h i p p o c a m p u s w e r e not influenced after adding Gpp(NH)p or treating the m e m b r a n e w i t h i s l e t - a c t i v a t i n g protein or N-ethylmaleimide. How somatostatin regulates mAChR binding is under further investigation.
THE I N T E R A C T I O N B E T W E E N A L P H A - A D R E N O C E P T O R S AND C A L C I U M ION C H A N N E L B L O C K E R B I N D I N G S I T E S IN THE RAT B R A I ~ , , HIROAKI MATSUBAYASHI , SHOZO KITO, SADAO KATAYAMA and RIE MIYOSHI , Third Department of i n t e r n a l M e d i c i n e , H i r o s h i m a U n i v e r s i t y S c h o o l of Medicine, I-2-3 Kasumi Minami-ku, Hiroshima, 734, J a p a n •
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In this study, the a u t h o r s i n v e s t i g a t e d H-verapami~ (H-VER) b l n d i n ~ in the rat c e r e b r a l c o r t e x and c o m p a r e d the r e s u l t w i t h that of H-nitrendipine (H-NTD). The effects of Ca ion c h a n n e l b l o c k e r s on b r a i n a d e n y l a t e c y c l a s e a c t i v i t y were also studied. Through these 3 e x p e r i m e n t s , the f o l l o w i n g results were obtained. I) Saturation a n a l y s i s of H - V E R b i n d i n g r e v e a l e d a s i n g l e b i n d i n g site w h o s e Kd and Bmax v a l u e s w e r e 1 3 3 n M and 1 9 . 4 f m o l / m g p r o t e i n r e s p e c t i v e l y . 2) Alpha-adrenergic agents inhibited H-verapamil b i n ~ i n g in the ~ r d e r of prazosin ~ yohimbine > clonidine with IC_ 0 v a l u e s of 10M range. H-nitrendipine binding was also inhibited by ~hes~ a g e n t s in the o r d e r of y o h i m b i n ~ ~ p r a z o s i n . The ~ IC50 values were a g a i n 10M range. 3) S a t u r a t i o n a n a l y s i s of H-yohimbin~ binding revealed that the i n h i b i t o r y e i f e c t s of b o t h v e r a p a m i l a n d d i l t i a z e m on H-yohimbine binding were to reduce the B m a x v a l u e w i t h the Kd v a l u e unchanged. 4) Thez i n h i b i t o r y effects of v e r a p a m i l on s u c h a l p h a - a d r e n e r g i c a g e n t s as ~H-WB-4101, ~H-clonidine and H-yohimbine bindings w e r e i n d e p e n d e n t of Ca ion. 5 ) 3 B a y k 8644, a Ca ion channel agonist of d i h y d ~ o p y r i d i n e derivative inhibited H-nitrendipine b~nding w h i l e it h a r d l y i n h i b i t e d ~ H - v e ~ a p a m i l binding. 6) G p p ( N H ) p had no e f f e c t on - H - V E R binding whereas it i n h i b i t e d H - N T D binding. 7) Nifedipine inhibit=d ade~ylate cyclase activity, which w a s n o t the case w i t h verapamil. 8) Decreased -H-NTD binding after EDTA-treatment was r e c o v e r e d by t~e a d d i t i o n of a Ca ion. On the other hand, EDTA- t r e a t m e n t c a u s e d no c h a n g e of H - V E R binding. N e v e r t h e l e s s , the a d d i t i o n of a Ca ion d e c r e a s e d H - V E R binding. All these r e s u l t s s h o w e d that there was heterogenecity of Ca a n t a g o n i s t b i n d i n g sites in the brain. F u r t h e r m o r e , it w a s also a s s u m e d that n i f e d i p i n e and n i t r e n d i p i n e b i n d i n g sites w e r e c o u p l i n g w i t h Gi protein.
THE I N T E R A C T I O N B E T W E E N A L P H A - A D R E N O C E P T O R S AND C A L C I U M ION C H A N N E L B L O C K E R B I N D I N G S I T E S IN THE RAT B R A I ~ , , HIROAKI MATSUBAYASHI , SHOZO KITO, SADAO KATAYAMA and RIE MIYOSHI , Third Department of i n t e r n a l M e d i c i n e , H i r o s h i m a U n i v e r s i t y S c h o o l of Medicine, I-2-3 Kasumi Minami-ku, Hiroshima, 734, J a p a n •
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In this study, the a u t h o r s i n v e s t i g a t e d H-verapami~ (H-VER) b l n d i n ~ in the rat c e r e b r a l c o r t e x and c o m p a r e d the r e s u l t w i t h that of H-nitrendipine (H-NTD). The effects of Ca ion c h a n n e l b l o c k e r s on b r a i n a d e n y l a t e c y c l a s e a c t i v i t y were also studied. Through these 3 e x p e r i m e n t s , the f o l l o w i n g results were obtained. I) Saturation a n a l y s i s of H - V E R b i n d i n g r e v e a l e d a s i n g l e b i n d i n g site w h o s e Kd and Bmax v a l u e s w e r e 1 3 3 n M and 1 9 . 4 f m o l / m g p r o t e i n r e s p e c t i v e l y . 2) Alpha-adrenergic agents inhibited H-verapamil b i n ~ i n g in the ~ r d e r of prazosin ~ yohimbine > clonidine with IC_ 0 v a l u e s of 10M range. H-nitrendipine binding was also inhibited by ~hes~ a g e n t s in the o r d e r of y o h i m b i n ~ ~ p r a z o s i n . The ~ IC50 values were a g a i n 10M range. 3) S a t u r a t i o n a n a l y s i s of H-yohimbin~ binding revealed that the i n h i b i t o r y e i f e c t s of b o t h v e r a p a m i l a n d d i l t i a z e m on H-yohimbine binding were to reduce the B m a x v a l u e w i t h the Kd v a l u e unchanged. 4) Thez i n h i b i t o r y effects of v e r a p a m i l on s u c h a l p h a - a d r e n e r g i c a g e n t s as ~H-WB-4101, ~H-clonidine and H-yohimbine bindings w e r e i n d e p e n d e n t of Ca ion. 5 ) 3 B a y k 8644, a Ca ion channel agonist of d i h y d ~ o p y r i d i n e derivative inhibited H-nitrendipine b~nding w h i l e it h a r d l y i n h i b i t e d ~ H - v e ~ a p a m i l binding. 6) G p p ( N H ) p had no e f f e c t on - H - V E R binding whereas it i n h i b i t e d H - N T D binding. 7) Nifedipine inhibit=d ade~ylate cyclase activity, which w a s n o t the case w i t h verapamil. 8) Decreased -H-NTD binding after EDTA-treatment was r e c o v e r e d by t~e a d d i t i o n of a Ca ion. On the other hand, EDTA- t r e a t m e n t c a u s e d no c h a n g e of H - V E R binding. N e v e r t h e l e s s , the a d d i t i o n of a Ca ion d e c r e a s e d H - V E R binding. All these r e s u l t s s h o w e d that there was heterogenecity of Ca a n t a g o n i s t b i n d i n g sites in the brain. F u r t h e r m o r e , it w a s also a s s u m e d that n i f e d i p i n e and n i t r e n d i p i n e b i n d i n g sites w e r e c o u p l i n g w i t h Gi protein.