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The purification and characterization of amphibian neuromedin U
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CHANGES IN IN-VITRO MOTILITY PATTERN FOLLOWING NEUROPEPTIDE TREATMENT IN MOUSE ILEUM INFECTED WITH THE DIGENEAN ECHINOSTOMA LIEI JOANNA DEKKERS 1 , M~C. THORNDYKE 1 AND P.J. WHITFIELD z
K.A.
BALOGUN 2 ,
J.H.
RIDDELL 1 ,
I. Dept Biology, Wolfson Laboratory, R.H.B.N.C., Egham, Surrey TW20 OEX 2. Parasitology Research Group,Division of Biosphere Science, Kings College, London W.8. The adult stage of the digenean parasite E.liei matures in the ileum of mice to produce a local lesion which includes the previously reported changes in neurohormonal peptide content. We have now studied the motility {amplitude, frequency and tone} of isolated segments of ileum in vitro and monitored the effect of standard applications of acetylcholine (Ach), bombesin and substance P (SP) on the basal pattern. Segments of ileum (7,10,12cm proximal to the ileo-caecal junction) were taken from non-parasitized {control) mice and their responses compared with those from ileal segments of mice infected with cysts of E.liei sacrificed 18-21 days post-infection. Parasitized mouse ileum showed distinct differences in response to application of both SP and bombesin compared to control tissues although the change was not always clear cut and varied according to the site of the ileal segment taken. Thus parasitized gut s h o w e d _ ~ n c r e ~ e s in tone as well as amplitude and frequency in response to SP (10 ~, 10 M) compared to normal ileum. ~his r~sponse was not so marked at the 7cm site. Application of bombesin (19-~, 10--M) produced the expected increases in tone, frequency and amplitude in the normal ileal preparations. In contrast, bombesin in the same dose range induced decreases in amplitude in parasitized ileum, while frequency and tone were unaffected. Whether these changes result from muscle pathology subsequent upon infection, increased intraluminal pressure induced by the parasite burden, alteration in peptide receptor concentration/coupling mechanisms, or by any combination thereof, remains for future investigations to determine.
THE P U R I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF A M P H I B I A N N E U R O M E D I N U J DOMIN, Y YIANGOU, R SPOKES, A AITKEN, K PARMAR, BJ C H R Y S A N T H O U AND SR B L O O M Dept of Medicine, Royal P o s t g r a d u a t e Medical School L o n d o n WI2 Lab of P r o t e i n Structure, National I n s t i t u t e for Medical Research, L o n d o n NW7. N e u r o m e d i n U is a novel b i o a c t i v e p e p t i d e o r i g i n a l l y i s o l a t e d from p o r c i n e spinal cord. Recent studies have d e m o n s t r a t e d high c o n c e n t r a t i o n s of n e u r o m e d i n U i m m u n o r e a c t i v i t y i n both m a m m a l i a n and a m p h i b i a n g a s t r o i n t e s i n a l tract. The aim of this s t u d y was to purify, c h a r a c t e r i z e and e x a m i n e the r e l a t i v e biological a c t i v i t y of the n e u r o m e d i n U like p e p t i d e d e t e c t e d i n the gut of the frog Rana temDoraria. Sequential c o l u m n c h r o m a t o g r a p h y of the entire g a s t r o i n t e s t i n a l tract from 50 animals f o l l o w i n g acid extraction, p r o d u c e d a homologous p e a k of i m m u n o r e a c t i v i t y w h i c h was s u b j e c t e d to a u t o m a t e d Edman degradation. Frog n e u r o m e d i n U was found to be a 25 residue p e p t i d e h a v i n g 60% s e q u e n c e s i m i l a r i t y w i t h the p o r c i n e form and r e t a i n i n g almost total c o n s e r v a t i o n of the b i o l o g i c a l l y active C-terminus. Synthetic Ct e r m i n a l l y a m i d a t e d frog n e u r o m e d i n U c o n s e q u e n t l y retains the a b i l i t y to contract rat uterine s m o o t h muscle in vitro and to exert p r e s s o r effects. The high degree of amino acid sequence c o n s e r v a t i o n is i n d i c a t i v e of s t r o n g e v o l u t i o n a r y p r e s s u r e acting to r e t a i n the p r e s e n c e of this p o s s i b l y p h y s i o l o g i c a l l y i m p o r t a n t p e p t i d e across the v e r t e b r a t e sub-phylum.
CHANGES IN IN-VITRO MOTILITY PATTERN FOLLOWING NEUROPEPTIDE TREATMENT IN MOUSE ILEUM INFECTED WITH THE DIGENEAN ECHINOSTOMA LIEI JOANNA DEKKERS 1 , M~C. THORNDYKE 1 AND P.J. WHITFIELD z
K.A.
BALOGUN 2 ,
J.H.
RIDDELL 1 ,
I. Dept Biology, Wolfson Laboratory, R.H.B.N.C., Egham, Surrey TW20 OEX 2. Parasitology Research Group,Division of Biosphere Science, Kings College, London W.8. The adult stage of the digenean parasite E.liei matures in the ileum of mice to produce a local lesion which includes the previously reported changes in neurohormonal peptide content. We have now studied the motility {amplitude, frequency and tone} of isolated segments of ileum in vitro and monitored the effect of standard applications of acetylcholine (Ach), bombesin and substance P (SP) on the basal pattern. Segments of ileum (7,10,12cm proximal to the ileo-caecal junction) were taken from non-parasitized {control) mice and their responses compared with those from ileal segments of mice infected with cysts of E.liei sacrificed 18-21 days post-infection. Parasitized mouse ileum showed distinct differences in response to application of both SP and bombesin compared to control tissues although the change was not always clear cut and varied according to the site of the ileal segment taken. Thus parasitized gut s h o w e d _ ~ n c r e ~ e s in tone as well as amplitude and frequency in response to SP (10 ~, 10 M) compared to normal ileum. ~his r~sponse was not so marked at the 7cm site. Application of bombesin (19-~, 10--M) produced the expected increases in tone, frequency and amplitude in the normal ileal preparations. In contrast, bombesin in the same dose range induced decreases in amplitude in parasitized ileum, while frequency and tone were unaffected. Whether these changes result from muscle pathology subsequent upon infection, increased intraluminal pressure induced by the parasite burden, alteration in peptide receptor concentration/coupling mechanisms, or by any combination thereof, remains for future investigations to determine.
THE P U R I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF A M P H I B I A N N E U R O M E D I N U J DOMIN, Y YIANGOU, R SPOKES, A AITKEN, K PARMAR, BJ C H R Y S A N T H O U AND SR B L O O M Dept of Medicine, Royal P o s t g r a d u a t e Medical School L o n d o n WI2 Lab of P r o t e i n Structure, National I n s t i t u t e for Medical Research, L o n d o n NW7. N e u r o m e d i n U is a novel b i o a c t i v e p e p t i d e o r i g i n a l l y i s o l a t e d from p o r c i n e spinal cord. Recent studies have d e m o n s t r a t e d high c o n c e n t r a t i o n s of n e u r o m e d i n U i m m u n o r e a c t i v i t y i n both m a m m a l i a n and a m p h i b i a n g a s t r o i n t e s i n a l tract. The aim of this s t u d y was to purify, c h a r a c t e r i z e and e x a m i n e the r e l a t i v e biological a c t i v i t y of the n e u r o m e d i n U like p e p t i d e d e t e c t e d i n the gut of the frog Rana temDoraria. Sequential c o l u m n c h r o m a t o g r a p h y of the entire g a s t r o i n t e s t i n a l tract from 50 animals f o l l o w i n g acid extraction, p r o d u c e d a homologous p e a k of i m m u n o r e a c t i v i t y w h i c h was s u b j e c t e d to a u t o m a t e d Edman degradation. Frog n e u r o m e d i n U was found to be a 25 residue p e p t i d e h a v i n g 60% s e q u e n c e s i m i l a r i t y w i t h the p o r c i n e form and r e t a i n i n g almost total c o n s e r v a t i o n of the b i o l o g i c a l l y active C-terminus. Synthetic Ct e r m i n a l l y a m i d a t e d frog n e u r o m e d i n U c o n s e q u e n t l y retains the a b i l i t y to contract rat uterine s m o o t h muscle in vitro and to exert p r e s s o r effects. The high degree of amino acid sequence c o n s e r v a t i o n is i n d i c a t i v e of s t r o n g e v o l u t i o n a r y p r e s s u r e acting to r e t a i n the p r e s e n c e of this p o s s i b l y p h y s i o l o g i c a l l y i m p o r t a n t p e p t i d e across the v e r t e b r a t e sub-phylum.