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Confocal scanning laser microscopy of peptidergic components in the mouse enteric nervous system: Preliminary observations
74 C H E M O T A C T I C A C T I V I T Y OF ~ and ~-CGRPs T O W A R D S M A M M A L I A N E O S I N O P H I L S
L.W. HAYNES, H.C. M A N L E Y and M.J.M. Bristol, U.K.
PERRY, Dept.
Zoology,
U n i v e r s i t y of
The C - t e r m i n u s of rat ~ - 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) contains the a m i n o - a c i d s e q u e n c e of the t e t r a p e p t i d e e o s i n o p h i l c h e m o t a c t i c factor, val-gly-ser-glu. G u i n e a - p i g e o s i n o p h i l p o l y m o r p h o n u c l e a r leukocytes e x h i b i t c o n c e n t r a t i o n - r e l a t e d c h e m o t a x i s t o w a r d s e - C G R P (E.C. 50, 0.5 nM). B-CGRP, w h o s e s e q u e n c e does not c o n t a i n the c h e m o t a c t i c tetrapeptide, is v i r t u a l l y inactive in the c o n c e n t r a t i o n range 0.i nM-0.01 mM. The c h e m o t a c t i c a c t i v i t y of a ~ - C G R P is i n c r e a s e d f o l l o w i n g c o n t r o l l e d tryptic d i g e s t i o n of the peptide. I n c u b a t i o n of e - C G R P with a g u i n e a - p i g lung p a r t i c u l a t e f r a c t i o n (LPC) also results in an i n c r e a s e in its activity. R e v e r s e - p h a s e HPLC e l u t i o n times of some but not all p r o d u c t s of ~-CGRP d i g e s t i o n by t r y p s i n and LPC were similar. The e f f e c t s of C G R P p e p t i d e s on e o s i n o p h i l d e g r a n u l a t i o n and a c t i v a t i o n are being investigated. Our p r e l i m i n a r y findings s u g g e s t that r e c e p t o r s of e o s i n o p h i l s can d i s t i n g u i s h and B forms of CGRP, and that ~-CGRP can be h y d r o l y s e d to one or more smalle~ p e p t i d e s w i t h i n c r e a s e d activity.
C O N F O C A L S C A N N I N G LASER M I C R O S C O P Y OF P E P T I D E R G I C C O M P O N E N T S ENTERIC N E R V O U S SYSTEM: P R E L I M I N A R Y O B S E R V A T I O N S
IN THE MOUSE
1 W1 A 1 1 2 C.F. JOHNSTON , C . SHA ,2E.P. RNSTRONG , J i M . McKILLOP , D . M [ McKAY , I . F A I R ~ E A T H E R , D.W. H A L T O N and K.D. B U C H A N A N , D e p a r t m e n t s of M e d i c i n e and Biology, The Queen's U n i v e r s i t y of Belfast, Belfast, BTI2 6BJ. Samples of mouse stomach, jejunum, ileum and colon were flat fixed between glass slides with p a r a f o r m a l d e h y d e , p e r m e a b i l i s e d in d e t e r g e n t before being s u b j e c t e d to i m m u n o f l u o r e s c e n c e i m m u n o c y t o c h e m i s t r y for CGRP, GRP, NPY, PHI, s u b s t a n c e P and VIP. Tissues were who~e mounted, serosa] surface uppermost, w i t h i n the incident b e a m of a M R C 5 0 0 confoca] scanning laser microscope (CSLM). Optical sections were o b t a i n e d from all levels of the serosa and m u c o s a by m o v i n g the tissue up and down through the n a r r o w focal plane of the instrument. I m m u n o r e a c t i v i t y for CGRP, NPY, PHI, s u b s t a n c e P and VIP was w i d e s p r e a d o c c u r r i n g within nervous elements in the myenteric, deep m u s c l e and submucosal p l e x u s e s and to v a r y i n g degrees w i t h i n the mucosa of all g a s t r o - e n t e r i c regions. G R P - i m m u n o r e a c t i v i t y d i s p l a y e d this d i s t r i b u t i o n only w i t h i n the stomach and was r e s t r i c t e d to the m y e n t e r i c plexus in the intestine and colon. In addition, s u b s t a n c e P - i m m u n o r e a c t i v i t y o c c u r r e d in intestinal mucosal e n d o c r i n e cells. In conclusion, the technique of CSLM c o m b i n e d with i m m u n o f l u o r e s c e n c e g r e a t l y f a c i l i t a t e s e l u c i d a t i o n of the enteric neuronal a r c h i t e c t u r e by o v e r c o m i n g the n e c e s s i t y of image r e c o n s t r u c t i o n from serial routine sections and by e l i m i n a t i n g b a c k g r o u n d flare in whole m o u n t e d sDecimens.
L.W. HAYNES, H.C. M A N L E Y and M.J.M. Bristol, U.K.
PERRY, Dept.
Zoology,
U n i v e r s i t y of
The C - t e r m i n u s of rat ~ - 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) contains the a m i n o - a c i d s e q u e n c e of the t e t r a p e p t i d e e o s i n o p h i l c h e m o t a c t i c factor, val-gly-ser-glu. G u i n e a - p i g e o s i n o p h i l p o l y m o r p h o n u c l e a r leukocytes e x h i b i t c o n c e n t r a t i o n - r e l a t e d c h e m o t a x i s t o w a r d s e - C G R P (E.C. 50, 0.5 nM). B-CGRP, w h o s e s e q u e n c e does not c o n t a i n the c h e m o t a c t i c tetrapeptide, is v i r t u a l l y inactive in the c o n c e n t r a t i o n range 0.i nM-0.01 mM. The c h e m o t a c t i c a c t i v i t y of a ~ - C G R P is i n c r e a s e d f o l l o w i n g c o n t r o l l e d tryptic d i g e s t i o n of the peptide. I n c u b a t i o n of e - C G R P with a g u i n e a - p i g lung p a r t i c u l a t e f r a c t i o n (LPC) also results in an i n c r e a s e in its activity. R e v e r s e - p h a s e HPLC e l u t i o n times of some but not all p r o d u c t s of ~-CGRP d i g e s t i o n by t r y p s i n and LPC were similar. The e f f e c t s of C G R P p e p t i d e s on e o s i n o p h i l d e g r a n u l a t i o n and a c t i v a t i o n are being investigated. Our p r e l i m i n a r y findings s u g g e s t that r e c e p t o r s of e o s i n o p h i l s can d i s t i n g u i s h and B forms of CGRP, and that ~-CGRP can be h y d r o l y s e d to one or more smalle~ p e p t i d e s w i t h i n c r e a s e d activity.
C O N F O C A L S C A N N I N G LASER M I C R O S C O P Y OF P E P T I D E R G I C C O M P O N E N T S ENTERIC N E R V O U S SYSTEM: P R E L I M I N A R Y O B S E R V A T I O N S
IN THE MOUSE
1 W1 A 1 1 2 C.F. JOHNSTON , C . SHA ,2E.P. RNSTRONG , J i M . McKILLOP , D . M [ McKAY , I . F A I R ~ E A T H E R , D.W. H A L T O N and K.D. B U C H A N A N , D e p a r t m e n t s of M e d i c i n e and Biology, The Queen's U n i v e r s i t y of Belfast, Belfast, BTI2 6BJ. Samples of mouse stomach, jejunum, ileum and colon were flat fixed between glass slides with p a r a f o r m a l d e h y d e , p e r m e a b i l i s e d in d e t e r g e n t before being s u b j e c t e d to i m m u n o f l u o r e s c e n c e i m m u n o c y t o c h e m i s t r y for CGRP, GRP, NPY, PHI, s u b s t a n c e P and VIP. Tissues were who~e mounted, serosa] surface uppermost, w i t h i n the incident b e a m of a M R C 5 0 0 confoca] scanning laser microscope (CSLM). Optical sections were o b t a i n e d from all levels of the serosa and m u c o s a by m o v i n g the tissue up and down through the n a r r o w focal plane of the instrument. I m m u n o r e a c t i v i t y for CGRP, NPY, PHI, s u b s t a n c e P and VIP was w i d e s p r e a d o c c u r r i n g within nervous elements in the myenteric, deep m u s c l e and submucosal p l e x u s e s and to v a r y i n g degrees w i t h i n the mucosa of all g a s t r o - e n t e r i c regions. G R P - i m m u n o r e a c t i v i t y d i s p l a y e d this d i s t r i b u t i o n only w i t h i n the stomach and was r e s t r i c t e d to the m y e n t e r i c plexus in the intestine and colon. In addition, s u b s t a n c e P - i m m u n o r e a c t i v i t y o c c u r r e d in intestinal mucosal e n d o c r i n e cells. In conclusion, the technique of CSLM c o m b i n e d with i m m u n o f l u o r e s c e n c e g r e a t l y f a c i l i t a t e s e l u c i d a t i o n of the enteric neuronal a r c h i t e c t u r e by o v e r c o m i n g the n e c e s s i t y of image r e c o n s t r u c t i o n from serial routine sections and by e l i m i n a t i n g b a c k g r o u n d flare in whole m o u n t e d sDecimens.