Life Sciences, Vol. 50, pp. 1343-1350 Printed in the USA
BIOGENIC
AMINES
Pergamon Press
IN THE GUINEA Yalcin
PIG ENDOCRINE
PANCREAS
cetin
Abt. A n a t o m i e i, M e d i z i n i s c h e H o c h s c h u l e Hannover, W - 3 0 0 0 H a n n o v e r 61, Federal R e p u b l i c of G e r m a n y (Received in final form February 20, 1992) summar7
P a n c r e a t a of g u i n e a - p i g s were i n v e s t i g a t e d for the p r e s e n c e and c e l l u l a r d i s t r i b u t i o n of b i o g e n i c amines. Out of the e s t a b l i s h e d endocrine cell types only insulin (B-) cells c o n t a i n e d i m m u n o r e a c t i v i t y for s e r o t o n i n and noradrenaline. However, the B-cells' content of both amines was quite variable. Serotonin was also c o n f i n e d to e n t e r o c h r o m a f f i n (EC-) cells. No i m m u n o r e a c t i v i t y for d o p a m i n e or h i s t a m i n e was present in any islet cell. T r e a t m e n t of g u i n e a - p i g s with Ro-4-4602 led to a m a r k e d d e c r e a s e of serotonin and n o r a d r e n a l i n e in p a n c r e a t i c e n d o c r i n e cells. The present findings suggest that serotonin and n o r a d r e n a l i n e are involved in the function of the e n d o c r i n e pancreas, p a r t i c u l a r l y of islet B-cells. Biogenic amines, like serotonin, histamine, dopamine or noradr e n a l i n e are involved in the function of the p a n c r e a s (1-7). Their p r e s e n c e in the pancreas has been v e r i f i e d by b i o c h e m i c a l techniques (8-10). Histochemically, b i o g e n i c amines were first d e m o n s t r a t e d by Falck and Hellman (ii) in the p a n c r e a t i c islet u s i n g the f o r m a l d e h y d e - i n d u c e d h i s t o c h e m i c a l f l u o r e s c e n c e technique, and were later localized also in p a n c r e a t a of various species (12-14). Due to the limited s e n s i t i v i t y and s e l e c t i v i t y of this m e t h o d (particularly in case of m i x e d fluorescence colours), the true nature of the fluorescent amines and their c e l l u l a r l o c a l i z a t i o n s in the endocrine p a n c r e a s has remained unclear. R e c e n t investigations on biogenic amines have demonstrated that their d e t e c t i o n by i m m u n o h i s t o c h e m i s t r y is more accurate than by the f l u o r e s c e n c e h i s t o c h e m i c a l t e c h n i q u e (15-18). Using a n t i b o d i e s against serotonin, histamine, d o p a m i n e and noradrenaline we now i n v e s t i g a t e d i m m u n o h i s t o c h e m i c a l l y the g u i n e a - p i g e n d o c r i n e p a n c r e a s for the p r e s e n c e and c e l l u l a r l o c a l i z a t i o n of biogenic amines. Moreover, we studied the effects of the i n h i b i t i o n of the a r o m a t i c - L - a m i n o - a c i d d e c a r b o x y l a s e (an enzyme essential for the formation of serotonin and catecholamines) by Ro-4-4602 (19,20) on the i m m u n o h i s t o c h e m i s t r y of b i o g e n i c amines in the pancreas. Materials Tissue
and methods
preparation
Three groups of adult libitum) were used in the
male g u i n e a - p i g s (food and w a t e r ad present study: (a) normal g u i n e a - p i g s
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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(n=5); (b) g u i n e a - p i g s (n=3) treated with Ro-4-4602 (kindly p r o v i d e d by Dr. U. Fischer, Hoffman-LaRoche, Basle, Switzerland) by intraperitoneal injections (daily 400 mg/kg body weight d i l u t e d in 0.9% NaCl solution) over 3 days; (c) control guineapigs (n=3) treated with 0.9% NaCl solution in the same way for 3 days. A f t e r d e c a p i t a t i o n of the animals u n d e r ether anaesthesia, small specimens from the pancreas were s n a p - f r o z e n in Freon 22 p r e - c o o l e d with liquid nitrogen, f r e e z e - d r i e d and fixed by vaporphase paraformaldehyde (21). For immunohistochemistry of h i s t a m i n e the specimens were fixed by v a p o r - p h a s e d i e t h y l p y r o c a r bonate (16). All specimens were b r i e f l y immersed in p r o p y l e n e oxide and embedded in epoxy resin (Araldite). Specimens of the duodenal mucosa and of the oxyntic mucosa from normal g u i n e a - p i g s and specimens from the bovine adrenal m e d u l l a were p r e p a r e d in the same way and served as reference organs for the immunohistoc h e m i s t r y of the biogenic amines.
Immunohistochemistry Antibodies. Most antibodies used in the p r e s e n t study have been c h a r a c t e r i z e d and employed in previous investigations. Their sources, donor species and w o r k i n g dilutions are listed in Table i. TABLE I Synopsis of the antibodies used in the present study.
Antibody
Donor Species
Source
Working dilution
Ref.
Insulin Glucagon Somatostatin BoY. PP Serotonin Serotonin Histamine Dopamine Dopamine Noradrenaline
Guinea-pig Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Rabbit
DAKO (A564), FRG NOVO (K4023), Denmark H. Etzrodt, FRG R.E. Chance, USA Euro Diagn., H o l l a n d Incstar, FRG F. Sundler, Sweden M. Geffard, France M. Geffard, France SFRI, France
1:500 i:i000 1:4000 1:4000 1:4000 i:i0.000 i:i0.000 1:2000 1:4000 1:2000
24 24 23,24 23,24 23,24 -
16,23 17 17 -
Immunohistochemical protocol. Serial semithin (0.5 #m) sections were cut from the pancreas and incubated a l t e r n a t i v e l y with antibodies against biogenic amines and p a n c r e a t i c hormones for 24 h at 4°C. After rinsing in p h o s p h a t e - b u f f e r e d saline (PBS, pH 7.3) they were immunostained by the p e r o x i d a s e antip e r o x i d a s e (PAP) technique (22). The sections were incubated with swine a n t i - r a b b i t IgG (DAKO, Hamburg, FRG) at 1:20 for 30 min, followed by incubation with soluble P A P - c o m p l e x (DAKO) at 1:50 for 30 min. For the monoclonal antibody against dopamine, goat a n t i - m o u s e IgG (1:200 for 30 min) and soluble P A P - c o m p l e x (mice origin; 1:200 for 30 min) was employed. V i s u a l i z a t i o n of immun o r e a c t i v e sites was p e r f o r m e d in DAB-HCI/H202 (0.7 mM diaminob e n z i d i n e - H C l / 0 . 0 0 2 % H202) in TRIS-HCI, pH 7.6, for i0 min.
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Biogenic Amines in Pancreas
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Specificity controls. M e t h o d - d e p e n d e n t n o n - s p e c i f i c i t i e s were excluded by running controls as recommended (22). The antibodyspecificities were checked in sections of the pancreas and/or of the reference organs by p r e a d s o r p t i o n of all antibodies with homologous and heterologous antigens (insulin, glucagon, somatostatin, bov. pancreatic polypeptide, serotonin, histamine, dopamine, noradrenaline) at concentrations of 6.25-100 ~g/ml antibody (working dilution). Moreover, the antibodies used were p r e a d s o r b e d with purified chromogranins (kindly provided by Dr. Gratzl, U n i v e r s i t y Ulm, FRG). All antibodies could be successfully preadsorbed only by their corresponding antigens, at antigen concentrations as low as 6.25 ~g/ml. Results Reference cells: In the duodenal mucosa, serotonin immunoreactivity was demonstrated in enterochromaffin (EC-) cells; likewise, in the oxyntic mucosa histamine immunoreactivity was localized in e n t e r o c h r o m a f f i n - l i k e (ECl-) cells (23). Chromaffin cells exhibited immunoreactivities for noradrenaline and dopamine.
FIG.
1
An enterochromaffin (EC-) cell, located in the exocrine parenchyma of the pancreas of untreated guinea-pigs, contains strong immunoreactivity for serotonin. This cell is u n r e a c t i v e for the pancreatic hormones or for other biogenic amines as tested on consecutive sections (figures not shown). Interferencecontrast. Bar = 20 ~m
In contrast to noradrenaline, of faint to moderate densities of c h r o m a f f i n cells.
immunoreactivity and was confined
for dopamine was to a small number
Pancreas: In normal guinea-pigs, the antibody against histamine and the two antibodies against dopamine showed no immunoreactivities for the corresponding amines in islet cells. The antibody against histamine also exhibited no immunostaining in d i e t h y l p y r o c a r b o n a t e - f i x e d pancreata. On the other hand, various endocrine cells were immunostained by the two antibodies against serotonin and by the noradrenaline antibody. Faint immunostaining for the latter amine was also observed between exocrine cells that in part may represent neuronal elements of the exocrine parenchyma. This immunostaining was completely abolished after p r e a d s o r p t i o n of the antibody with noradrenaline. Serotonin immunoreactivity was regularly found in enterochromaffin (EC-) cells which mostly were distributed in the exocrine p a r e n c h y m a (Figures i, 4) (24). These cells were unreac-
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Biogenic Amines in Pancreas
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tive for t h e pancreatic hormones and for n o r a d r e n a l i n e . The e s t a b l i s h e d p a n c r e a t i c e n d o c r i n e cell types, i.e. g l u c a g o n (A-), somatostatin (D-) or p a n c r e a t i c p o l y p e p t i d e (PP-) c e l l s d i d n o t exhibit immunoreactivities for the b i o g e n i c a m i n e s u n d e r study. O n t h e o t h e r hand, i n s u l i n (B)- c e l l s c o n t a i n e d immunoreactivit i e s for t h e b i o g e n i c amines serotonin and noradrenaline which were partly co-localized in t h e s a m e B - c e l l s . However, t h e Bcells' c o n t e n t of s e r o t o n i n a n d n o r a d r e n a l i n e w a s q u i t e v a r i a b l e . We found B-cells containing strong, moderate or faint immunoreactivities for s e r o t o n i n and noradrenaline (Figures 2, 3); m o r e o v e r , a v a r y i n g n u m b e r of B - c e l l s w e r e c o m p l e t e l y u n r e a c t i v e for t h e b o t h amines. R e m a r k a b l y , t h e d e n s i t y of s e r o t o n i n immunoreactivity d i d not p a r a l l e l t h a t of n o r a d r e n a l i n e in the s a m e B-cells. Thus, t h e r e w a s no c o n s t a n t r e l a t i o n s h i p between serotonin and noradrenaline immunoreactivities in t h e s a m e Bcells. L i k e w i s e , in no c a s e w e found a v i s i b l e i n t e r r e l a t i o n s h i p b e t w e e n t h e B-cells' c o n t e n t of i n s u l i n a n d s e r o t o n i n or n o r a d renaline immunoreactivities. In c o n t r a s t to t h e s e findings, in p a n c r e a t a of R o - 4 - 4 6 0 2 treated guinea-pigs n o n e of the B - c e l l s contained immunoreactivities for serotonin and noradrenaline (Figure 4) or for histamine and dopamine; only the EC-cells showed weak immunoreact i v i t y for s e r o t o n i n (Figure 4). A l s o in t h e s e animals, t h e o t h e r cell t y p e s w e r e u n r e a c t i v e for t h e s e amines. In p a n c r e a t a of saline-treated guinea-pigs serotonin and noradrenaline immunoreactivities could be demonstrated as described for untreated guinea-pigs. For t h e p a n c r e a t i c h o r m o n e i m m u n o r e a c t i v i t i e s no d i f f e r e n c e s were observed between untreated, Ro-4-4602t r e a t e d a n d s a l i n e - t r e a t e d animals.
FIG.
2
Two adjacent semithin (0.5 ~m) sections from the pancreas of untreated guinea-pigs, immunostained for i n s u l i n (A) a n d serotonin (B). In t h e i s l e t of L a n g e r h a n s , serotonin immunoreactiv i t i e s are c o n f i n e d to the B - c e l l p o p u l a t i o n . However, t h e Bcells either contain strong (large arrows) or faint (small arrows) immunoreactivities for serotonin. Some B-cells are completely unreactive for serotonin. A-cells (arrowheads) identified by the glucagon antibody on a c o n s e c u t i v e section (figure n o t shown) are u n r e a c t i v e for s e r o t o n i n . Interferencec o n t r a s t . B a r = 20 ~m
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Biogenic Amines in Pancreas
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Discussion In a series of studies u s i n g the f o r m a l d e h y d e - i n d u c e d fluorescence technique, biogenic amines have been localized in p a n c r e a t i c islet cells of a v a r i e t y of species (13,14). In the g u i n e a - p i g pancreas, most of the islet cells e m i t t e d a light in the g r e e n - y e l l o w to y e l l o w range, w h e r e a s a few of the cells disp l a y e d a clear g r e e n or m u d d y g r e y i s h - y e l l o w f l u o r e s c e n c e or none at all (25,26). F u r t h e r chemical and h i s t o c h e m i c a l studies supp o s e d the p r e s e n c e of either a catechol or a t r y p t a m i n e derivate, or a m i x t u r e of b o t h in g u i n e a - p i g p a n c r e a t i c islets (12,27,28). The p r e s e n t i m m u n o h i s t o c h e m i c a l study shows that b o t h serot o n i n and n o r a d r e n a l i n e are p r e s e n t in the e n d o c r i n e pancreas. Both amines are c o n f i n e d to the B-cells of islets of Langerhans. However, the B-cells' content of s e r o t o n i n and n o r a d r e n a l i n e is quite variable. Although serotonin and n o r a d r e n a l i n e are co-
are u n r e a c t i v e for n o r a d r e n a L line. Interference-contrast. Bar = 30 ~m
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Biogenic Amines in Pancreas
Vol. 50, No. 18, 1992
localized in many B-cells, the densities of their immunoreactivities m o s t l y differ incomparably from cell to cell. This may be a possible explanation for the d i f f e r e n t amine fluorescence colours p r e v i o u s l y observed in the g u i n e a - p i g p a n c r e a s (25,26). On the other hand, immunoreactivities for h i s t a m i n e or dopamine could not be d e m o n s t r a t e d in the pancreas. Both amines either lack in the pancreas, or other than in the r e f e r e n c e organs, their c e l l u l a r quantities are below the d e t e c t i o n level of the m e t h o d used.
rence-contrast.
Bar=
20 ~m
Basically, the amines localized in B-cells might have been taken up via c i r c u l a t i o n by the B-cells. However, the fact that b i o g e n i c amines (8-10) as well as various amine b i o s y n t h e t i c and d e g r a d a t i v e enzymes (29,30) are present in isolated p a n c r e a t i c islets indicates that islet cells are able to p r o d u c e biogenic amines. T h e i r b i o s y n t h e s e s are obviously regulated under various m e t a b o l i c and experimental conditions (8,30,31). Based on the p r e s e n t data, we assume that B-cells are the cellular source of s e r o t o n i n and noradrenaline. The d i f f e r e n c e s b e t w e e n the B-cells
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Biogenic Amines in Pancreas
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r e l a t e d to t h e i r content of s e r o t o n i n and n o r a d r e n a l i n e immunorea c t i v i t i e s m a y also reflect the i n t e r c e l l u l a r d i f f e r e n c e s in enzyme activities. So far, the lack of d o p a m i n e in B-cells m a y also be due to its rapid c o n v e r s i o n during n o r a d r e n a l i n e synthesis. The a p p l i c a t i o n of Ro-4-4602 to g u i n e a - p i g s led to a disappearance of s e r o t o n i n and n o r a d r e n a l i n e in B-cells and to a m a r k e d d e c r e a s e of s e r o t o n i n i m m u n o r e a c t i v i t y in EC-cells. These e f f e c t s may be due to the inhibitory action of Ro-4-4602 on the b i o s y n t h e s i s of s e r o t o n i n and n o r a d r e n a l i n e in the pancreas. This a s s u m p t i o n is in line w i t h findings o b t a i n e d in isolated pancreatic islets (10,30). In any case, the lack of s e r o t o n i n and noradrenaline immunoreactivities in B-cells in these animals underlines the h i g h s p e c i f i c i t y of the c o r r e s p o n d i n g antibodies. A m i n e s have been d e m o n s t r a t e d in a v a r i e t y of e n d o c r i n e cells (13,14,32,33). The c y t o p h y s i o l o g i c a l role of the amines in islet cells or in other e n d o c r i n e cells is p o o r l y understood. They are p o s s i b l y involved in the r e g u l a t i o n of b i o s y n t h e s i s and storage of the c o - s t o r e d p e p t i d e h o r m o n e s (3,14,34) and in the r e g u l a t i o n of p a n c r e a t i c h o r m o n e secretion (1-4,7). So far, s e r o t o n i n and n o r a d r e n a l i n e may play a s i g n i f i c a n t role in the B-cell function. Acknowledqments I am g r a t e f u l to Drs. R.E. Chance, H. Etzrodt, M. G e f f a r d and F. S u n d l e r for t h e i r generous supply of antibodies. For expert t e c h n i c a l a s s i s t a n c e I am indebted to Ms. M. Meyer, Mr. S. Gudat and Mr. W. M~ller. This w o r k was s u p p o r t e d by the Deutsche Forschungsgemeinschaft (SFB 280/B5).
References
I. 2. 3. 4. 5. 6. 7. 8. 9. i0.
11. 12. 13. 14. 15.
J.J. GAGLIARDINO, L.M. ZIEHER, F.C. ITURRIZA, R.E. HERNANDEZ and R.R. RODRIQUEZ, H o r m M e t a b Res ~ 145-150 (1971) I. LUNDQUIST, Acta Physiol Scand (Suppl) 372 1-47 (1971) I. LUNDQUIST, R. E K H O L M and L.E. ERICSON, D i a b e t o l o g i a Z 414422 (1971) K.E. QUICKEL, J.M. F E L D M A N and H.E. LEBOVITZ, E n d o c r i n o l o g y 89 1295-1302 (1971) J.M. F E L D M A N and H.E. LEBOVITZ, Trans A s s o c Am P h y s i c i a n s 35 279-294 (1972) A.E. PONTIROLI, P. MICOSSI and P.P. FOA, H o r m M e t a b Res 1_/2 703-704 (1980) G. SKOGLUND, R.A. GROSS, G.R. BERTRAND, B. A H R E N and M.-M. LOUBATIERES-MARIANI, Diabetes 40 660-665 (1991) S.E. H A N S E N and C.J. HEDESKOV, Acta E n d o c r i n o l 8_66 820-832 (1977) R.T. ZERN, L.B. FOSTER, J.A. B L A L O C K and J.M. FELDMAN, D i a b e t e s 28 185-189 (1979) J.L. BIRD, E.E. W R I G H T and J.M. FELDMAN, Diabetes 29 304-308 (1980) B. FALCK and B. HELI~N, E x p e r i e n t i a 1-9 139-140 (1963) L. CEGRELL, Acta Physiol Scand (Suppl) 314 1-60 (1968) C.H. OWMAN, R. H A K A N S O N and F. SUNDLER, Fed Proc 32 1785-1791 (1973) F. SUNDLER, R. HAKANSON, I. LOREN and I. LUNDQUIST, Invest Cell Pathol 3 87-103 (1980) A. CONSOLAZIONE, C. MILSTEIN, B. W R I G H T and A.C. CUELLO, J
1350
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H i s t o c h e m C y t o c h e m 2 9 1 4 2 5 - 1 4 3 0 (1981) 16. R. HAKANSON, G. BOTTCHER, E. EKBLAD, P. PANULA, M. SIMONSSON, M. DOHLSTEN, T. HALLBERG and F. SUNDLER, H i s t o c h e m i s t r y 8 6 5 17 (1986) 17. M. GEFFARD, O. KAH, B. ONTENIENTE, P. SEGUELA, M. LE M O A L and M. DELAAGE, J N e u r o c h e m 4 2 1 5 9 3 - 1 5 9 9 (1984) 18. A.A.J. VERHOFSTAD, H.W.M. STEINBUSCH, H.W.J. JOOSTEN, O. PENKE, J. V A R G A and M. GOLDSTEIN, Immunocytochemistry: Practical a p p l i c a t i o n s in Pathology and Biology, J.M. Polak and S. Van Noorden (eds), 143-168, John W r i g h t & Sons, Bristol (1983) 19. J. H Y T T E L and B. FJALLAND, Eur J Pharmacol 1-9112-114 (1972) 20. I. KURUMA, G. BARTHOLINI, R. TISSOT and A. PLETSCHER, J Pharm Pharmac 2__44289-294 (1972) 21. B. FALCK, N.-A. HILLARP, G. THIEME and A. TORP, J H i s t o c h e m Cytochem I0348-354 (1962) 22. L.A. STERNBERGER, Immunocytochemistry, 3rd edn, John Wiley, New York (1986) 23. Y. CETIN, L. MULLER-K6PPEL, D. AUNIS, M.F. BADER and D. GRUBE, H i s t o c h e m i s t r y 9-2265-275 (1989) 24. Y. CETIN and D. GRUBE, H i s t o c h e m i s t r y 9__44479-484 (1990) 25. L. CEGRELL, B. FALCK and B. HELLMAN, The structure and m e t a b o l i s m of the p a n c r e a t i c islets, S.E. Brolin, B. Hellman and H. Knutson (eds), 429-435, Pergamon Press, London (1964) 26. L. CEGRELL, B. FALCK and A.-M. ROSENGREN, Life Sci 6 2 4 8 3 2489 (1967) 27. B. FALCK and B. HELLMAN, Acta E n d o c r i n o 1 4 5 1 3 3 - 1 3 8 (1964) 28. L. CEGRELL, Life Sci 6 1 6 4 7 - 1 6 5 2 (1967) 29. H.E. LEBOVITZ and R.W. DOWNS, Frontiers in c a t e c h o l a m i n e research, E. Usdin and S.H. Snyder (eds), 831-833, Pergamon Press, London (1973) 30. J.M. FELDMAN, J.H. H E N D E R S O N and J.A. BLALOCK, D i a b e t o l o g i a 1/7169-174 (1979) 31. I. LUNDQUIST, F. SUNDLER, R. HAKANSON, L.-I. LARSSON and L.G. HEDING, E n d o c r i n o l o g y 9_/7937-947 (1975) 32. B. FALCK and C. OWMAN, Adv Pharmacol 6 2 1 1 - 2 3 1 (1968) 33. Y. CETIN, H i s t o c h e m i s t r y 9__33601-606 (1990) 34. O.M. PULIDO, S.A. BENCOSME, M.L. DE BOLD and A.J. DE BOLD, D i a b e t o l o g i a 1 5 1 9 7 - 2 0 4 (1978)
BIOGENIC
AMINES
Pergamon Press
IN THE GUINEA Yalcin
PIG ENDOCRINE
PANCREAS
cetin
Abt. A n a t o m i e i, M e d i z i n i s c h e H o c h s c h u l e Hannover, W - 3 0 0 0 H a n n o v e r 61, Federal R e p u b l i c of G e r m a n y (Received in final form February 20, 1992) summar7
P a n c r e a t a of g u i n e a - p i g s were i n v e s t i g a t e d for the p r e s e n c e and c e l l u l a r d i s t r i b u t i o n of b i o g e n i c amines. Out of the e s t a b l i s h e d endocrine cell types only insulin (B-) cells c o n t a i n e d i m m u n o r e a c t i v i t y for s e r o t o n i n and noradrenaline. However, the B-cells' content of both amines was quite variable. Serotonin was also c o n f i n e d to e n t e r o c h r o m a f f i n (EC-) cells. No i m m u n o r e a c t i v i t y for d o p a m i n e or h i s t a m i n e was present in any islet cell. T r e a t m e n t of g u i n e a - p i g s with Ro-4-4602 led to a m a r k e d d e c r e a s e of serotonin and n o r a d r e n a l i n e in p a n c r e a t i c e n d o c r i n e cells. The present findings suggest that serotonin and n o r a d r e n a l i n e are involved in the function of the e n d o c r i n e pancreas, p a r t i c u l a r l y of islet B-cells. Biogenic amines, like serotonin, histamine, dopamine or noradr e n a l i n e are involved in the function of the p a n c r e a s (1-7). Their p r e s e n c e in the pancreas has been v e r i f i e d by b i o c h e m i c a l techniques (8-10). Histochemically, b i o g e n i c amines were first d e m o n s t r a t e d by Falck and Hellman (ii) in the p a n c r e a t i c islet u s i n g the f o r m a l d e h y d e - i n d u c e d h i s t o c h e m i c a l f l u o r e s c e n c e technique, and were later localized also in p a n c r e a t a of various species (12-14). Due to the limited s e n s i t i v i t y and s e l e c t i v i t y of this m e t h o d (particularly in case of m i x e d fluorescence colours), the true nature of the fluorescent amines and their c e l l u l a r l o c a l i z a t i o n s in the endocrine p a n c r e a s has remained unclear. R e c e n t investigations on biogenic amines have demonstrated that their d e t e c t i o n by i m m u n o h i s t o c h e m i s t r y is more accurate than by the f l u o r e s c e n c e h i s t o c h e m i c a l t e c h n i q u e (15-18). Using a n t i b o d i e s against serotonin, histamine, d o p a m i n e and noradrenaline we now i n v e s t i g a t e d i m m u n o h i s t o c h e m i c a l l y the g u i n e a - p i g e n d o c r i n e p a n c r e a s for the p r e s e n c e and c e l l u l a r l o c a l i z a t i o n of biogenic amines. Moreover, we studied the effects of the i n h i b i t i o n of the a r o m a t i c - L - a m i n o - a c i d d e c a r b o x y l a s e (an enzyme essential for the formation of serotonin and catecholamines) by Ro-4-4602 (19,20) on the i m m u n o h i s t o c h e m i s t r y of b i o g e n i c amines in the pancreas. Materials Tissue
and methods
preparation
Three groups of adult libitum) were used in the
male g u i n e a - p i g s (food and w a t e r ad present study: (a) normal g u i n e a - p i g s
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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(n=5); (b) g u i n e a - p i g s (n=3) treated with Ro-4-4602 (kindly p r o v i d e d by Dr. U. Fischer, Hoffman-LaRoche, Basle, Switzerland) by intraperitoneal injections (daily 400 mg/kg body weight d i l u t e d in 0.9% NaCl solution) over 3 days; (c) control guineapigs (n=3) treated with 0.9% NaCl solution in the same way for 3 days. A f t e r d e c a p i t a t i o n of the animals u n d e r ether anaesthesia, small specimens from the pancreas were s n a p - f r o z e n in Freon 22 p r e - c o o l e d with liquid nitrogen, f r e e z e - d r i e d and fixed by vaporphase paraformaldehyde (21). For immunohistochemistry of h i s t a m i n e the specimens were fixed by v a p o r - p h a s e d i e t h y l p y r o c a r bonate (16). All specimens were b r i e f l y immersed in p r o p y l e n e oxide and embedded in epoxy resin (Araldite). Specimens of the duodenal mucosa and of the oxyntic mucosa from normal g u i n e a - p i g s and specimens from the bovine adrenal m e d u l l a were p r e p a r e d in the same way and served as reference organs for the immunohistoc h e m i s t r y of the biogenic amines.
Immunohistochemistry Antibodies. Most antibodies used in the p r e s e n t study have been c h a r a c t e r i z e d and employed in previous investigations. Their sources, donor species and w o r k i n g dilutions are listed in Table i. TABLE I Synopsis of the antibodies used in the present study.
Antibody
Donor Species
Source
Working dilution
Ref.
Insulin Glucagon Somatostatin BoY. PP Serotonin Serotonin Histamine Dopamine Dopamine Noradrenaline
Guinea-pig Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Rabbit
DAKO (A564), FRG NOVO (K4023), Denmark H. Etzrodt, FRG R.E. Chance, USA Euro Diagn., H o l l a n d Incstar, FRG F. Sundler, Sweden M. Geffard, France M. Geffard, France SFRI, France
1:500 i:i000 1:4000 1:4000 1:4000 i:i0.000 i:i0.000 1:2000 1:4000 1:2000
24 24 23,24 23,24 23,24 -
16,23 17 17 -
Immunohistochemical protocol. Serial semithin (0.5 #m) sections were cut from the pancreas and incubated a l t e r n a t i v e l y with antibodies against biogenic amines and p a n c r e a t i c hormones for 24 h at 4°C. After rinsing in p h o s p h a t e - b u f f e r e d saline (PBS, pH 7.3) they were immunostained by the p e r o x i d a s e antip e r o x i d a s e (PAP) technique (22). The sections were incubated with swine a n t i - r a b b i t IgG (DAKO, Hamburg, FRG) at 1:20 for 30 min, followed by incubation with soluble P A P - c o m p l e x (DAKO) at 1:50 for 30 min. For the monoclonal antibody against dopamine, goat a n t i - m o u s e IgG (1:200 for 30 min) and soluble P A P - c o m p l e x (mice origin; 1:200 for 30 min) was employed. V i s u a l i z a t i o n of immun o r e a c t i v e sites was p e r f o r m e d in DAB-HCI/H202 (0.7 mM diaminob e n z i d i n e - H C l / 0 . 0 0 2 % H202) in TRIS-HCI, pH 7.6, for i0 min.
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Specificity controls. M e t h o d - d e p e n d e n t n o n - s p e c i f i c i t i e s were excluded by running controls as recommended (22). The antibodyspecificities were checked in sections of the pancreas and/or of the reference organs by p r e a d s o r p t i o n of all antibodies with homologous and heterologous antigens (insulin, glucagon, somatostatin, bov. pancreatic polypeptide, serotonin, histamine, dopamine, noradrenaline) at concentrations of 6.25-100 ~g/ml antibody (working dilution). Moreover, the antibodies used were p r e a d s o r b e d with purified chromogranins (kindly provided by Dr. Gratzl, U n i v e r s i t y Ulm, FRG). All antibodies could be successfully preadsorbed only by their corresponding antigens, at antigen concentrations as low as 6.25 ~g/ml. Results Reference cells: In the duodenal mucosa, serotonin immunoreactivity was demonstrated in enterochromaffin (EC-) cells; likewise, in the oxyntic mucosa histamine immunoreactivity was localized in e n t e r o c h r o m a f f i n - l i k e (ECl-) cells (23). Chromaffin cells exhibited immunoreactivities for noradrenaline and dopamine.
FIG.
1
An enterochromaffin (EC-) cell, located in the exocrine parenchyma of the pancreas of untreated guinea-pigs, contains strong immunoreactivity for serotonin. This cell is u n r e a c t i v e for the pancreatic hormones or for other biogenic amines as tested on consecutive sections (figures not shown). Interferencecontrast. Bar = 20 ~m
In contrast to noradrenaline, of faint to moderate densities of c h r o m a f f i n cells.
immunoreactivity and was confined
for dopamine was to a small number
Pancreas: In normal guinea-pigs, the antibody against histamine and the two antibodies against dopamine showed no immunoreactivities for the corresponding amines in islet cells. The antibody against histamine also exhibited no immunostaining in d i e t h y l p y r o c a r b o n a t e - f i x e d pancreata. On the other hand, various endocrine cells were immunostained by the two antibodies against serotonin and by the noradrenaline antibody. Faint immunostaining for the latter amine was also observed between exocrine cells that in part may represent neuronal elements of the exocrine parenchyma. This immunostaining was completely abolished after p r e a d s o r p t i o n of the antibody with noradrenaline. Serotonin immunoreactivity was regularly found in enterochromaffin (EC-) cells which mostly were distributed in the exocrine p a r e n c h y m a (Figures i, 4) (24). These cells were unreac-
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tive for t h e pancreatic hormones and for n o r a d r e n a l i n e . The e s t a b l i s h e d p a n c r e a t i c e n d o c r i n e cell types, i.e. g l u c a g o n (A-), somatostatin (D-) or p a n c r e a t i c p o l y p e p t i d e (PP-) c e l l s d i d n o t exhibit immunoreactivities for the b i o g e n i c a m i n e s u n d e r study. O n t h e o t h e r hand, i n s u l i n (B)- c e l l s c o n t a i n e d immunoreactivit i e s for t h e b i o g e n i c amines serotonin and noradrenaline which were partly co-localized in t h e s a m e B - c e l l s . However, t h e Bcells' c o n t e n t of s e r o t o n i n a n d n o r a d r e n a l i n e w a s q u i t e v a r i a b l e . We found B-cells containing strong, moderate or faint immunoreactivities for s e r o t o n i n and noradrenaline (Figures 2, 3); m o r e o v e r , a v a r y i n g n u m b e r of B - c e l l s w e r e c o m p l e t e l y u n r e a c t i v e for t h e b o t h amines. R e m a r k a b l y , t h e d e n s i t y of s e r o t o n i n immunoreactivity d i d not p a r a l l e l t h a t of n o r a d r e n a l i n e in the s a m e B-cells. Thus, t h e r e w a s no c o n s t a n t r e l a t i o n s h i p between serotonin and noradrenaline immunoreactivities in t h e s a m e Bcells. L i k e w i s e , in no c a s e w e found a v i s i b l e i n t e r r e l a t i o n s h i p b e t w e e n t h e B-cells' c o n t e n t of i n s u l i n a n d s e r o t o n i n or n o r a d renaline immunoreactivities. In c o n t r a s t to t h e s e findings, in p a n c r e a t a of R o - 4 - 4 6 0 2 treated guinea-pigs n o n e of the B - c e l l s contained immunoreactivities for serotonin and noradrenaline (Figure 4) or for histamine and dopamine; only the EC-cells showed weak immunoreact i v i t y for s e r o t o n i n (Figure 4). A l s o in t h e s e animals, t h e o t h e r cell t y p e s w e r e u n r e a c t i v e for t h e s e amines. In p a n c r e a t a of saline-treated guinea-pigs serotonin and noradrenaline immunoreactivities could be demonstrated as described for untreated guinea-pigs. For t h e p a n c r e a t i c h o r m o n e i m m u n o r e a c t i v i t i e s no d i f f e r e n c e s were observed between untreated, Ro-4-4602t r e a t e d a n d s a l i n e - t r e a t e d animals.
FIG.
2
Two adjacent semithin (0.5 ~m) sections from the pancreas of untreated guinea-pigs, immunostained for i n s u l i n (A) a n d serotonin (B). In t h e i s l e t of L a n g e r h a n s , serotonin immunoreactiv i t i e s are c o n f i n e d to the B - c e l l p o p u l a t i o n . However, t h e Bcells either contain strong (large arrows) or faint (small arrows) immunoreactivities for serotonin. Some B-cells are completely unreactive for serotonin. A-cells (arrowheads) identified by the glucagon antibody on a c o n s e c u t i v e section (figure n o t shown) are u n r e a c t i v e for s e r o t o n i n . Interferencec o n t r a s t . B a r = 20 ~m
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Biogenic Amines in Pancreas
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Discussion In a series of studies u s i n g the f o r m a l d e h y d e - i n d u c e d fluorescence technique, biogenic amines have been localized in p a n c r e a t i c islet cells of a v a r i e t y of species (13,14). In the g u i n e a - p i g pancreas, most of the islet cells e m i t t e d a light in the g r e e n - y e l l o w to y e l l o w range, w h e r e a s a few of the cells disp l a y e d a clear g r e e n or m u d d y g r e y i s h - y e l l o w f l u o r e s c e n c e or none at all (25,26). F u r t h e r chemical and h i s t o c h e m i c a l studies supp o s e d the p r e s e n c e of either a catechol or a t r y p t a m i n e derivate, or a m i x t u r e of b o t h in g u i n e a - p i g p a n c r e a t i c islets (12,27,28). The p r e s e n t i m m u n o h i s t o c h e m i c a l study shows that b o t h serot o n i n and n o r a d r e n a l i n e are p r e s e n t in the e n d o c r i n e pancreas. Both amines are c o n f i n e d to the B-cells of islets of Langerhans. However, the B-cells' content of s e r o t o n i n and n o r a d r e n a l i n e is quite variable. Although serotonin and n o r a d r e n a l i n e are co-
are u n r e a c t i v e for n o r a d r e n a L line. Interference-contrast. Bar = 30 ~m
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localized in many B-cells, the densities of their immunoreactivities m o s t l y differ incomparably from cell to cell. This may be a possible explanation for the d i f f e r e n t amine fluorescence colours p r e v i o u s l y observed in the g u i n e a - p i g p a n c r e a s (25,26). On the other hand, immunoreactivities for h i s t a m i n e or dopamine could not be d e m o n s t r a t e d in the pancreas. Both amines either lack in the pancreas, or other than in the r e f e r e n c e organs, their c e l l u l a r quantities are below the d e t e c t i o n level of the m e t h o d used.
rence-contrast.
Bar=
20 ~m
Basically, the amines localized in B-cells might have been taken up via c i r c u l a t i o n by the B-cells. However, the fact that b i o g e n i c amines (8-10) as well as various amine b i o s y n t h e t i c and d e g r a d a t i v e enzymes (29,30) are present in isolated p a n c r e a t i c islets indicates that islet cells are able to p r o d u c e biogenic amines. T h e i r b i o s y n t h e s e s are obviously regulated under various m e t a b o l i c and experimental conditions (8,30,31). Based on the p r e s e n t data, we assume that B-cells are the cellular source of s e r o t o n i n and noradrenaline. The d i f f e r e n c e s b e t w e e n the B-cells
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r e l a t e d to t h e i r content of s e r o t o n i n and n o r a d r e n a l i n e immunorea c t i v i t i e s m a y also reflect the i n t e r c e l l u l a r d i f f e r e n c e s in enzyme activities. So far, the lack of d o p a m i n e in B-cells m a y also be due to its rapid c o n v e r s i o n during n o r a d r e n a l i n e synthesis. The a p p l i c a t i o n of Ro-4-4602 to g u i n e a - p i g s led to a disappearance of s e r o t o n i n and n o r a d r e n a l i n e in B-cells and to a m a r k e d d e c r e a s e of s e r o t o n i n i m m u n o r e a c t i v i t y in EC-cells. These e f f e c t s may be due to the inhibitory action of Ro-4-4602 on the b i o s y n t h e s i s of s e r o t o n i n and n o r a d r e n a l i n e in the pancreas. This a s s u m p t i o n is in line w i t h findings o b t a i n e d in isolated pancreatic islets (10,30). In any case, the lack of s e r o t o n i n and noradrenaline immunoreactivities in B-cells in these animals underlines the h i g h s p e c i f i c i t y of the c o r r e s p o n d i n g antibodies. A m i n e s have been d e m o n s t r a t e d in a v a r i e t y of e n d o c r i n e cells (13,14,32,33). The c y t o p h y s i o l o g i c a l role of the amines in islet cells or in other e n d o c r i n e cells is p o o r l y understood. They are p o s s i b l y involved in the r e g u l a t i o n of b i o s y n t h e s i s and storage of the c o - s t o r e d p e p t i d e h o r m o n e s (3,14,34) and in the r e g u l a t i o n of p a n c r e a t i c h o r m o n e secretion (1-4,7). So far, s e r o t o n i n and n o r a d r e n a l i n e may play a s i g n i f i c a n t role in the B-cell function. Acknowledqments I am g r a t e f u l to Drs. R.E. Chance, H. Etzrodt, M. G e f f a r d and F. S u n d l e r for t h e i r generous supply of antibodies. For expert t e c h n i c a l a s s i s t a n c e I am indebted to Ms. M. Meyer, Mr. S. Gudat and Mr. W. M~ller. This w o r k was s u p p o r t e d by the Deutsche Forschungsgemeinschaft (SFB 280/B5).
References
I. 2. 3. 4. 5. 6. 7. 8. 9. i0.
11. 12. 13. 14. 15.
J.J. GAGLIARDINO, L.M. ZIEHER, F.C. ITURRIZA, R.E. HERNANDEZ and R.R. RODRIQUEZ, H o r m M e t a b Res ~ 145-150 (1971) I. LUNDQUIST, Acta Physiol Scand (Suppl) 372 1-47 (1971) I. LUNDQUIST, R. E K H O L M and L.E. ERICSON, D i a b e t o l o g i a Z 414422 (1971) K.E. QUICKEL, J.M. F E L D M A N and H.E. LEBOVITZ, E n d o c r i n o l o g y 89 1295-1302 (1971) J.M. F E L D M A N and H.E. LEBOVITZ, Trans A s s o c Am P h y s i c i a n s 35 279-294 (1972) A.E. PONTIROLI, P. MICOSSI and P.P. FOA, H o r m M e t a b Res 1_/2 703-704 (1980) G. SKOGLUND, R.A. GROSS, G.R. BERTRAND, B. A H R E N and M.-M. LOUBATIERES-MARIANI, Diabetes 40 660-665 (1991) S.E. H A N S E N and C.J. HEDESKOV, Acta E n d o c r i n o l 8_66 820-832 (1977) R.T. ZERN, L.B. FOSTER, J.A. B L A L O C K and J.M. FELDMAN, D i a b e t e s 28 185-189 (1979) J.L. BIRD, E.E. W R I G H T and J.M. FELDMAN, Diabetes 29 304-308 (1980) B. FALCK and B. HELI~N, E x p e r i e n t i a 1-9 139-140 (1963) L. CEGRELL, Acta Physiol Scand (Suppl) 314 1-60 (1968) C.H. OWMAN, R. H A K A N S O N and F. SUNDLER, Fed Proc 32 1785-1791 (1973) F. SUNDLER, R. HAKANSON, I. LOREN and I. LUNDQUIST, Invest Cell Pathol 3 87-103 (1980) A. CONSOLAZIONE, C. MILSTEIN, B. W R I G H T and A.C. CUELLO, J
1350
Biogenic Amines in Pancreas
Vol. 50, No. 18, 1992
H i s t o c h e m C y t o c h e m 2 9 1 4 2 5 - 1 4 3 0 (1981) 16. R. HAKANSON, G. BOTTCHER, E. EKBLAD, P. PANULA, M. SIMONSSON, M. DOHLSTEN, T. HALLBERG and F. SUNDLER, H i s t o c h e m i s t r y 8 6 5 17 (1986) 17. M. GEFFARD, O. KAH, B. ONTENIENTE, P. SEGUELA, M. LE M O A L and M. DELAAGE, J N e u r o c h e m 4 2 1 5 9 3 - 1 5 9 9 (1984) 18. A.A.J. VERHOFSTAD, H.W.M. STEINBUSCH, H.W.J. JOOSTEN, O. PENKE, J. V A R G A and M. GOLDSTEIN, Immunocytochemistry: Practical a p p l i c a t i o n s in Pathology and Biology, J.M. Polak and S. Van Noorden (eds), 143-168, John W r i g h t & Sons, Bristol (1983) 19. J. H Y T T E L and B. FJALLAND, Eur J Pharmacol 1-9112-114 (1972) 20. I. KURUMA, G. BARTHOLINI, R. TISSOT and A. PLETSCHER, J Pharm Pharmac 2__44289-294 (1972) 21. B. FALCK, N.-A. HILLARP, G. THIEME and A. TORP, J H i s t o c h e m Cytochem I0348-354 (1962) 22. L.A. STERNBERGER, Immunocytochemistry, 3rd edn, John Wiley, New York (1986) 23. Y. CETIN, L. MULLER-K6PPEL, D. AUNIS, M.F. BADER and D. GRUBE, H i s t o c h e m i s t r y 9-2265-275 (1989) 24. Y. CETIN and D. GRUBE, H i s t o c h e m i s t r y 9__44479-484 (1990) 25. L. CEGRELL, B. FALCK and B. HELLMAN, The structure and m e t a b o l i s m of the p a n c r e a t i c islets, S.E. Brolin, B. Hellman and H. Knutson (eds), 429-435, Pergamon Press, London (1964) 26. L. CEGRELL, B. FALCK and A.-M. ROSENGREN, Life Sci 6 2 4 8 3 2489 (1967) 27. B. FALCK and B. HELLMAN, Acta E n d o c r i n o 1 4 5 1 3 3 - 1 3 8 (1964) 28. L. CEGRELL, Life Sci 6 1 6 4 7 - 1 6 5 2 (1967) 29. H.E. LEBOVITZ and R.W. DOWNS, Frontiers in c a t e c h o l a m i n e research, E. Usdin and S.H. Snyder (eds), 831-833, Pergamon Press, London (1973) 30. J.M. FELDMAN, J.H. H E N D E R S O N and J.A. BLALOCK, D i a b e t o l o g i a 1/7169-174 (1979) 31. I. LUNDQUIST, F. SUNDLER, R. HAKANSON, L.-I. LARSSON and L.G. HEDING, E n d o c r i n o l o g y 9_/7937-947 (1975) 32. B. FALCK and C. OWMAN, Adv Pharmacol 6 2 1 1 - 2 3 1 (1968) 33. Y. CETIN, H i s t o c h e m i s t r y 9__33601-606 (1990) 34. O.M. PULIDO, S.A. BENCOSME, M.L. DE BOLD and A.J. DE BOLD, D i a b e t o l o g i a 1 5 1 9 7 - 2 0 4 (1978)
