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Measurement of substance P and calcitonin gene-related peptide fragments in rat CNS
163 M E A S U R E M E N T OF S U B S T A N C E P A N D ~ C A L C I T O N I N G E N E - R E L A T E D P E P T I D E F R A G M E N T S IN RAT CNS T. SAKURADA 1 , 1 Dept. 2 Dept.
P.
LE GREVES 2 a n d
Pharmacology, Pharmacology,
L.
TERENIUS 2
T o h o k u College of Pharmacy, Sendai 983, J a p a n Univ. Uppsala, Uppsala, S w e d e n
C a l c i t o n i n g e n e - r e l a t e d p e p t i d e (CGRP) has been shown to occur in p r i m a r y s e n s o r y neurons, often t o g e t h e r w i t h s u b s t a n c e P (SP). It has been d e m o n s t r a t e d that SP and CGRP can be r e l e a s e d from p r i m a r y s e n s o r y neurons, p r o b a b l y c o - r e l e a s e d from the same neuron. A p o s s i b l e i n t e r a c t i o n b e t w e e n SP and CGRP has b e e n s u g g e s t e d from a b e h a v i o u r a l s t u d y s h o w i n g that CGRP p o t e n t i a t e s and p r o l o n g s the s c r a t c h i n g and b i t i n g r e s p o n s e i n d u c e d by i n t r a t h e c a l a d m i n i s t r a tion of SP (i). Recently, we have shown that CGRP in m i c r o m o l a r c o n c e n t r a t i o n s i n h i b i t s a SP e n d o p e p t i d a s e , o r i g i n a l l y i s o l a t e d from h u m a n c e r e b r o s p i n a l fluid (2). The p r e s e n t study was undertaken to e s t a b l i s h a p r o c e d u r e for m e a s u r e m e n t of SP and CGRP f r a g m e n t s in the b r a i n and spinal cord of rats in order to i n v e s t i gate the d e g r a d a t i o n of both peptides. Male S p r a g u e - D a w l e y rats were u s e d in all experiments. After d e c a p i t a t i o n , the b r a ~ n and spinal c o r d were r a p i d l y removed, d i s s e c t e d and frozen on solid c a r b o n dioxide. The tissue p i e c e s were h o m o g e n i z e d w i t h 1 M acetic acid. The s u p e r n a t a n t s w e r e d i l u t e d w i t h buffer I (0.018 M p y r i d i n e - 0 . 1 M formate). The m i x t u r e was run t h r o u g h a 1 ml s u l p h o p r o p y l S e p h a d e x C-25 i o n - e x c h a n g e r . Buffer II (0.1 M p y r i d i n e - 0 . 1 M formate), b u f f e r III (0.25 M p y r i d i n e - 0.25 M formate) and b u f f e r IV (0.8 M p y r i d i n e - 0 . 8 M formate) w e r e run t h r o u g h the column. SP and SP 1-7 in each f r a c t i o n were a n a l y s e d by r a d i o i m m u n o a s s a y e m p l o y i n g d i f f e r e n t antisera. CGRP and its C - t e r m i n a l f r a g m e n t s were a s s a y e d by u s i n g a CGRP 23-37 d i r e c t e d antiserum. SP and SP 1-7 e l u t e d in good y i e l d s w i t h buffer IV (3). CGRP and CGRP 17-37 e l u t e d almost q u a n t i t a t i v e l y w i t h buffer IV. S y n t h e t i c CGRP 24-37 was e l u t e d by buffer III. R e v e r s e d - p h a s e HPLC a n a l y s i s r e v e a l e d three peaks of i m m u n o r e a c t i v i t y in the b r a i n and spinal cord; a m a j o r p e a k that c o - e l u t e d in the same p o s i t i o n as s y n t h e t i c rat CGRP 1-37, the other peaks e l u t i n g earlier. The highest c o n c e n t r a t i o n s of SP 1-7 and CGRP C - t e r m i n a l f r a g m e n t s were in the d o r s a l part of the spinal cord. The results s u g g e s t that the d o r s a l part of the spinal c o r d m a y have high a c t i v i t y of enzymes d e g r a d i n g SP and CGRP and m a y be a s u i t a b l e tissue to i n v e s t i g a t e the d e g r a d i n g step of b o t h p e p t i d e s in the CNS. The p r o c e d u r e b a s e d on i o n - e x c h a n g e c h r o m a t o g r a p h y is a u s e f u l tool for c h e m i c a l s e p a r a t i o n and r a d i o i m m u n o a s s a y s for q u a n t i t a t i o n of CGRP and its f r a g m e n t s as w e l l as SP and its fragments. i. W i e s e n f e l d - H a l l i n , Z., H~kfel, T., Lundberg, J. M., Forssmann, W. G., Reinecke, M., Tschopp, F. A. and Fischer, J. A. (1984) Neurosci. Lett. 5 2 : 1 9 9 - 2 0 4 2. Le Greves, P., Nyberg, F., Terenius, L. and H6kfelt, T. (1985) Europ. J. Pharmacol. 1 1 5 : 3 0 9 - 3 1 1 3. Sakurada, T., Le Greves, P., Stewart, J. and Terenius, L. (1985) J. Neurochem. 4 4 : 7 1 8 - 7 2 2
P.
LE GREVES 2 a n d
Pharmacology, Pharmacology,
L.
TERENIUS 2
T o h o k u College of Pharmacy, Sendai 983, J a p a n Univ. Uppsala, Uppsala, S w e d e n
C a l c i t o n i n g e n e - r e l a t e d p e p t i d e (CGRP) has been shown to occur in p r i m a r y s e n s o r y neurons, often t o g e t h e r w i t h s u b s t a n c e P (SP). It has been d e m o n s t r a t e d that SP and CGRP can be r e l e a s e d from p r i m a r y s e n s o r y neurons, p r o b a b l y c o - r e l e a s e d from the same neuron. A p o s s i b l e i n t e r a c t i o n b e t w e e n SP and CGRP has b e e n s u g g e s t e d from a b e h a v i o u r a l s t u d y s h o w i n g that CGRP p o t e n t i a t e s and p r o l o n g s the s c r a t c h i n g and b i t i n g r e s p o n s e i n d u c e d by i n t r a t h e c a l a d m i n i s t r a tion of SP (i). Recently, we have shown that CGRP in m i c r o m o l a r c o n c e n t r a t i o n s i n h i b i t s a SP e n d o p e p t i d a s e , o r i g i n a l l y i s o l a t e d from h u m a n c e r e b r o s p i n a l fluid (2). The p r e s e n t study was undertaken to e s t a b l i s h a p r o c e d u r e for m e a s u r e m e n t of SP and CGRP f r a g m e n t s in the b r a i n and spinal cord of rats in order to i n v e s t i gate the d e g r a d a t i o n of both peptides. Male S p r a g u e - D a w l e y rats were u s e d in all experiments. After d e c a p i t a t i o n , the b r a ~ n and spinal c o r d were r a p i d l y removed, d i s s e c t e d and frozen on solid c a r b o n dioxide. The tissue p i e c e s were h o m o g e n i z e d w i t h 1 M acetic acid. The s u p e r n a t a n t s w e r e d i l u t e d w i t h buffer I (0.018 M p y r i d i n e - 0 . 1 M formate). The m i x t u r e was run t h r o u g h a 1 ml s u l p h o p r o p y l S e p h a d e x C-25 i o n - e x c h a n g e r . Buffer II (0.1 M p y r i d i n e - 0 . 1 M formate), b u f f e r III (0.25 M p y r i d i n e - 0.25 M formate) and b u f f e r IV (0.8 M p y r i d i n e - 0 . 8 M formate) w e r e run t h r o u g h the column. SP and SP 1-7 in each f r a c t i o n were a n a l y s e d by r a d i o i m m u n o a s s a y e m p l o y i n g d i f f e r e n t antisera. CGRP and its C - t e r m i n a l f r a g m e n t s were a s s a y e d by u s i n g a CGRP 23-37 d i r e c t e d antiserum. SP and SP 1-7 e l u t e d in good y i e l d s w i t h buffer IV (3). CGRP and CGRP 17-37 e l u t e d almost q u a n t i t a t i v e l y w i t h buffer IV. S y n t h e t i c CGRP 24-37 was e l u t e d by buffer III. R e v e r s e d - p h a s e HPLC a n a l y s i s r e v e a l e d three peaks of i m m u n o r e a c t i v i t y in the b r a i n and spinal cord; a m a j o r p e a k that c o - e l u t e d in the same p o s i t i o n as s y n t h e t i c rat CGRP 1-37, the other peaks e l u t i n g earlier. The highest c o n c e n t r a t i o n s of SP 1-7 and CGRP C - t e r m i n a l f r a g m e n t s were in the d o r s a l part of the spinal cord. The results s u g g e s t that the d o r s a l part of the spinal c o r d m a y have high a c t i v i t y of enzymes d e g r a d i n g SP and CGRP and m a y be a s u i t a b l e tissue to i n v e s t i g a t e the d e g r a d i n g step of b o t h p e p t i d e s in the CNS. The p r o c e d u r e b a s e d on i o n - e x c h a n g e c h r o m a t o g r a p h y is a u s e f u l tool for c h e m i c a l s e p a r a t i o n and r a d i o i m m u n o a s s a y s for q u a n t i t a t i o n of CGRP and its f r a g m e n t s as w e l l as SP and its fragments. i. W i e s e n f e l d - H a l l i n , Z., H~kfel, T., Lundberg, J. M., Forssmann, W. G., Reinecke, M., Tschopp, F. A. and Fischer, J. A. (1984) Neurosci. Lett. 5 2 : 1 9 9 - 2 0 4 2. Le Greves, P., Nyberg, F., Terenius, L. and H6kfelt, T. (1985) Europ. J. Pharmacol. 1 1 5 : 3 0 9 - 3 1 1 3. Sakurada, T., Le Greves, P., Stewart, J. and Terenius, L. (1985) J. Neurochem. 4 4 : 7 1 8 - 7 2 2