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A specific and sensitive radioimmunoassay for cholecystokinin-39 (CCK-39): Immunological evidence of CCK-39 in brain and gut
S158
mal s u b j e c t s i n c r e a s e d the p l a s m a c o n c e n t r a t i o n f r o m S , 5 p m o l / l ± 2.5 SE to 25.4 ~ 7.2 p m o l / l . C o n c l u s i o n : We r e p o r t the d e v e l o p m e n t of a h i g h l y s i n s i t i v e a n d s p e c i f i c R I A for CCK. D i f f e r e n t m o l e c u l a r f o r m s of CCK are f o u n d in h u m a n t i s s u e e x t r a c t s . A f t e r food t h e r e is a small b u t d e f i n i t e r e l e a s e of CCK in man. S u p p o r t e d by a g r a n t Scha 274/3.
of D e u t s c h e
Forschungsgemeinschaft,
A S P E C I F I C AND S E N S I T I V E R A D I O I M M U N O A S S A Y FOR C H O L E C Y S T O K I N I N - 3 9 (CCK-39): I M M U N O L O G I C A L E V I D E N C E OF CCK-39 IN BRAIN AND GUT. C.B.H.W. Lamers and J.B.M.J. Jansen; D i v i s i o n of G a s t r o e n t e r o l o g y St. R a d b o u d H o s p i t a l , N i j m e g e n , The N e t h e r l a n d s . It has p r e v i o u s l y been shown that in acid e x t r a c t s of p o r c i n e small intestina] m u c o s a c h o l e c y s t o k i n i n (CCK) is p r e s e n t in two m o l e c u l a r forms: C C K - 3 3 and C C K - 3 9 (Mutt, 1976, Clin. E n d o c r i n o l . 5, 1 7 5 s - 1 8 3 s ) . SinCb % p e c i f i c r a d i o i m m u n o a s s a y s for C C K - 3 9 are lacking and C C K - 3 9 c o - e l u t e s w i t h C C K - 3 3 from S e p h a d e x G-50 columns, almost no a t t e n t i o n has been paid to this m o l e c u l a r form of CCK. We have d e v e l o p e d a s p e c i f i c and s e n s i t i v e r a d i o i m m u n o a s s a y for CCK-39. A n t i b o d y 1804 was r a i s e d in a rabbit a g a i n s t 30% pure p o r c i n e CCK c o u p l e d to b o v i n e serum albumen. 1 2 5 1 - C C K - 3 9 was p r e p a r e d by the c h l o r a m i n e T m e t h o d and the label was p u r i f i e d by c h r o m a t o g r a p h y on a C M - S e p h a d e x column. The a n t i b o d y did not c r o s s - r e a c t w i t h CCK-33, CCK-8, CCK 10-20, g a s t r i n s or other polyp e p t i d e h o r m o n e s . The ID-50 of the assay was 1.5 pmol/l incubation m i x t u r e . U s i n g this r a d i o i m m u n o a s s a y C C K - 3 9 was d e m o n s t r a t e d in an a q u e o u s - a c i d e x t r a c t of p o r c i n e small i n t e s t i n e (6.2 i 1.3 pmol/g; m e a n ± SEM, n=3). The c o n c e n t r a t i o n of CCK-39 in aqueousacid e x t r a c t s of f r o n t a l c o r t e x was 6.1 ± 1.5 pmol/g, frontal white 1.5 ± 0.1 pmol/g, t h a l a m u s 0.4 i 0.1 pmol/g, h y p o t h a l a m u s 0.2 ± 0.1 pmol/g, pons 0.1 ± 0.1 pmol/g, and c e r e b e l l u m < 0.1 pmol/g (n=5). S e p h a d e x G-50 SF column c h r o m a t o g r a p h y showed that i m m u n o l o g i c a l l y d e m o n s t r a t e d C C K - 3 9 in brain and in small intestine c o - e l u t e d with the CCK-39 standard. C o n c l u s i o n : C C K - 3 9 is p r e s e n t both in i n t e s t i n e and in b r a i n as shown by a s p e c i f i c r a d i o i m m u n o a s s a y for CCK-39.
COMPARATIVE STUDY OF CHOLECYSTOKININ -LIKE COMPONENTS IN HUMAN AND PORCINE DUODENAL MUCOSAE. A.Condamin,C.Bernard,N.Vaysse,D.Fourmy,L.Pradayrol,and J.A.Chayvialle. INSERM U.45,Hopital E-Herriot,Lyon,and INSERM U.151, CHU Rangueil,Toulouse,France. The molecular heterogeneity of cholecystokinin (CCK)-like immunoreactivity in the mammalian gut has been documented through chromatographic studies using region-specific antisera,but the biological activity of the detected components has been little investigated. ~thods:Samples of human and porcine duodenal mucosae were extracted successively in water and acetic acid,each pair of extracts being processed in parallel as follows : in~nunoadsorption on insolubilized gastrin antibodies crossreacting extensively with CCK, desorption with 15% formic acid and lyophilization, high performance liquid chromatography (HPLC) on NBondapak C18 in conditions allowing for separation of various gastrin and CCK forms, immunoadsorption of the main CCK-like substances recognized at HPLC. The final fractions were tested for I) immunoreactivity in a CCK assay based on Bolton and Hunter
mal s u b j e c t s i n c r e a s e d the p l a s m a c o n c e n t r a t i o n f r o m S , 5 p m o l / l ± 2.5 SE to 25.4 ~ 7.2 p m o l / l . C o n c l u s i o n : We r e p o r t the d e v e l o p m e n t of a h i g h l y s i n s i t i v e a n d s p e c i f i c R I A for CCK. D i f f e r e n t m o l e c u l a r f o r m s of CCK are f o u n d in h u m a n t i s s u e e x t r a c t s . A f t e r food t h e r e is a small b u t d e f i n i t e r e l e a s e of CCK in man. S u p p o r t e d by a g r a n t Scha 274/3.
of D e u t s c h e
Forschungsgemeinschaft,
A S P E C I F I C AND S E N S I T I V E R A D I O I M M U N O A S S A Y FOR C H O L E C Y S T O K I N I N - 3 9 (CCK-39): I M M U N O L O G I C A L E V I D E N C E OF CCK-39 IN BRAIN AND GUT. C.B.H.W. Lamers and J.B.M.J. Jansen; D i v i s i o n of G a s t r o e n t e r o l o g y St. R a d b o u d H o s p i t a l , N i j m e g e n , The N e t h e r l a n d s . It has p r e v i o u s l y been shown that in acid e x t r a c t s of p o r c i n e small intestina] m u c o s a c h o l e c y s t o k i n i n (CCK) is p r e s e n t in two m o l e c u l a r forms: C C K - 3 3 and C C K - 3 9 (Mutt, 1976, Clin. E n d o c r i n o l . 5, 1 7 5 s - 1 8 3 s ) . SinCb % p e c i f i c r a d i o i m m u n o a s s a y s for C C K - 3 9 are lacking and C C K - 3 9 c o - e l u t e s w i t h C C K - 3 3 from S e p h a d e x G-50 columns, almost no a t t e n t i o n has been paid to this m o l e c u l a r form of CCK. We have d e v e l o p e d a s p e c i f i c and s e n s i t i v e r a d i o i m m u n o a s s a y for CCK-39. A n t i b o d y 1804 was r a i s e d in a rabbit a g a i n s t 30% pure p o r c i n e CCK c o u p l e d to b o v i n e serum albumen. 1 2 5 1 - C C K - 3 9 was p r e p a r e d by the c h l o r a m i n e T m e t h o d and the label was p u r i f i e d by c h r o m a t o g r a p h y on a C M - S e p h a d e x column. The a n t i b o d y did not c r o s s - r e a c t w i t h CCK-33, CCK-8, CCK 10-20, g a s t r i n s or other polyp e p t i d e h o r m o n e s . The ID-50 of the assay was 1.5 pmol/l incubation m i x t u r e . U s i n g this r a d i o i m m u n o a s s a y C C K - 3 9 was d e m o n s t r a t e d in an a q u e o u s - a c i d e x t r a c t of p o r c i n e small i n t e s t i n e (6.2 i 1.3 pmol/g; m e a n ± SEM, n=3). The c o n c e n t r a t i o n of CCK-39 in aqueousacid e x t r a c t s of f r o n t a l c o r t e x was 6.1 ± 1.5 pmol/g, frontal white 1.5 ± 0.1 pmol/g, t h a l a m u s 0.4 i 0.1 pmol/g, h y p o t h a l a m u s 0.2 ± 0.1 pmol/g, pons 0.1 ± 0.1 pmol/g, and c e r e b e l l u m < 0.1 pmol/g (n=5). S e p h a d e x G-50 SF column c h r o m a t o g r a p h y showed that i m m u n o l o g i c a l l y d e m o n s t r a t e d C C K - 3 9 in brain and in small intestine c o - e l u t e d with the CCK-39 standard. C o n c l u s i o n : C C K - 3 9 is p r e s e n t both in i n t e s t i n e and in b r a i n as shown by a s p e c i f i c r a d i o i m m u n o a s s a y for CCK-39.
COMPARATIVE STUDY OF CHOLECYSTOKININ -LIKE COMPONENTS IN HUMAN AND PORCINE DUODENAL MUCOSAE. A.Condamin,C.Bernard,N.Vaysse,D.Fourmy,L.Pradayrol,and J.A.Chayvialle. INSERM U.45,Hopital E-Herriot,Lyon,and INSERM U.151, CHU Rangueil,Toulouse,France. The molecular heterogeneity of cholecystokinin (CCK)-like immunoreactivity in the mammalian gut has been documented through chromatographic studies using region-specific antisera,but the biological activity of the detected components has been little investigated. ~thods:Samples of human and porcine duodenal mucosae were extracted successively in water and acetic acid,each pair of extracts being processed in parallel as follows : in~nunoadsorption on insolubilized gastrin antibodies crossreacting extensively with CCK, desorption with 15% formic acid and lyophilization, high performance liquid chromatography (HPLC) on NBondapak C18 in conditions allowing for separation of various gastrin and CCK forms, immunoadsorption of the main CCK-like substances recognized at HPLC. The final fractions were tested for I) immunoreactivity in a CCK assay based on Bolton and Hunter