Immunohistochemical demonstration of gastrin precursor peptides in pig gut

292

IMMUNOHISTOCHEMICAL

D E M O N S T R A T I O N OF G A S T R I N P R E C U R S O R P E P T I D E S

IN PIG GUT.

A N N - C A T H R I N E J O N S S O N and G R A H A M J. DOCKRAY, MRC S e c r e t o r y C o n t r o l Group, Dept. of Physiology, U n i v e r s i t y of Liverpool, B r o w n l o w Hill, P.O. Box 147, Liverpool, L69 3BX. The amino acid sequence of p i g p r e p r o g a s t r i n has r e c e n t l y been d e d u c e d by c D N A sequencing. The p r o p o s e d p r e c u r s o r c o n s i s t s of 104 r e s i d u e s in w h i c h the G-34 s e q u e n c e is e x t e n d e d at b o t h the C- and N-termini. The N - t e r m i n u s i n c l u d e s a signal peptide, an i n t e r v e n i n g sequence d e s i g n a t e d "cryptic A" (probably r e s i d u e s 20-57) and the C - t e r m i n u s c o n s i s t s a sequence d e s i g n a t e d " c r y p t i c B" (residues 96-104). We have e x a m i n e d the l o c a l i z a t i o n of different fragments of p r e p r o g a s t r i n in p i g gut u s i n g r e g i o n specific antibodies. A n t i b o d i e s were r a i s e d in r a b b i t s to fragments of cryptic A (Tyr2720-27) and c r y p t i c B (Tyr9999-104) c o u p l e d to t h y r o g l o b u l i n by t r i a z i n e and g l u t a r a l dehyde, r e s p e c t i v e l y . O t h e r a n t i b o d i e s u s e d were C - t e r m i n a l G-17 and Nt e r m i n a l G-34 specific. The f l u o r e s c e n c e t e c h n i q u e of Coons et al was used t o g e t h e r w i t h the PAP m e t h o d of Sternberger. A n t i b o d i e s to c r y p t i t A and B r e v e a l e d e n d o c r i n e cells t h r o u g h o u t the p o r c i n e antral m u c o s a fixed w i t h p a r a B e n z o q u i n o n e ; a few w e a k l y stained e n d o c r i n e cells could be seen in the d u o d e n a l mucosa, b u t none w e r e found in the m u c o s a of fundus, j e j u n u m or colon. S i m i l a r p o p u l a t i o n s of cells were stained w i t h a n t i b o d i e s to c r y p t i c A and B c o m p a r e d w i t h G-17 and G-34. When the e l u t i o n and r e s t a i n i n g m e t h o d of T r a m u et al was applied the same cells were r e v e a l e d by a n t i s e r a to the cryptic peptides, G-17 and G-34. Absorption c o n t r o l s (i0 and i00 n m o l e s . m l -I) i n d i c a t e d specific l o c a l i z a t i o n in e a c h case. Conclusion: A n t i g e n i c d e t e r m i n a n t s c o r r e s p o n d i n g to the c r y p t i c sequences of p r e p r o g a s t r i n are stored in G - c e l l s of pig antral m u c o s a t o g e t h e r w i t h the G-17 and G-34. R E D U C T I O N OF S T R E S S - I N D U C E D A C T H S E C R E T I O N IN THE RAT BY I M M U N O N E U T R A L I S A T I O N WITH BOTH C R F A N D V A S O P R E S S I N A N T I S E R A IN V I V O L I N T O N E.A. i, TILDERS F.J.H. 2, V E R M E S I. 2, B E R K E N B O S C H F. 2& L O W R Y P.J. 1 iprotein H o r m o n e Unit, St. B a r t h o l o m e w ' s Hospital, London. 2Dept. of P h a r m a c o l ogy, Free University, Amsterdam. S t r e s s - i n d u c e d A C T H release is b e l i e v e d to be c o n t r o l l e d by s e v e r a l i n t e r a c t i n g substances,

which include

catecholamines.

corticotropin-releasing

In these e x p e r i m e n t s ,

sin has been u s e d to i n v e s t i g a t e

attenuated

the p h y s i o l o g i c a l

the A C T H response

58% and 66% r e s p e c t i v e l y . ponse

to both stressors,

in both formalin

a l t h o u g h n o t as m a r k e d l y

abolished,

the p r e s e n c e

regulate ACTH release

and r e s t r a i n t stress by

also r e d u c e d the ACTH res-

as a n t i - C R F

(by 37% in form-

No further depression

c o u l d be o b t a i n e d b y the use of c o m b i n a t i o n s

in c o n t r o l l i n g A C T H secretion.

of CRF and v a s o p r e s -

relevance of the two p e p t i -

P r e t r e a t m e n t w~th a n t i - A V P

s u g g e s t e d that b o t h v a s o p r e s s i n

and

situations.

alin stress and 48% in r e s t r a i n t stress). response

(CRF), v a s o p r e s s i n

immunoneutralisation

des in rats e x p o s e d to d i f f e r e n t stress Anti-CRF

factor

of the stress

of b o t h antibodies.

It is

and an o v i n e - C R F - l i k e p e p t i d e may p l a y a role Since

the stress response

of a n o t h e r factor

is indicated.

could n o t be c o m p l e t e l y

(possibly a catecholamine)

which

can