- Email: [email protected]
Pathways of secretion in the exocrine pancreas: The status of resting secretion
Life Sciences, Vol. 40, Printed in the U.S.A.
pp.
2453-2460
Pergamon
Journals
PATHWAYS OF SECRETION IN THE EXOCRINE PANCREAS: THE STATUS OF RESTING SECRETION Adrien Centre
de
R.
Beaudoin
and G i l l e s
Crondin
r e c h e r c h e s u r l e s m ~ c a n i s m e s de s ~ c r @ t i o n Facultfi des sciences Universit6 de S h e r b r o o k e S h e r b r o o k e , Q u e b e c , CANADA J1K 2R1
(Received
in
final
form April
13,
1987)
Summary
I n t h e l a s t d e c a d e t h e c o n c e p t of two d i s t i n c t p a t h w a y s of s e cretion in the exocrine pancreas has s lowly e m e rge d. According to this concept, one p a t h w a y i s i n v o l v e d i n s t i m u l a t e d (regulated) conditions and a n o t h e r u n d e r r e s t i n g (constitutive) conditions. This hypothesis was e l a b o r a t e d at first f r o m t h e c o m p a r i s o n o{ the s p e c i f i c radioactivities of s e c r e t o r y proteins r e l e a s e d by the gland under resting and s t i m u l a t e d conditions. A n a l y s i s of t h e protein composition of the iuice relea s e d u n d e r t h e s e two p h y s i o logical conditions further supported that hypothesis. More r e c e n t studies compared the kinetic of a c c u m u l a t i o n of n e w l y s y n t h e s i z e d proteins in zymogen g r a n u l e and t h e i r release in the g l a n d lumen. The l a t t e r results a r e in a g r e e m e n t : w i t h a model in w h i c h s e c r e t o r y proteins are channelled in two separate pathways, one regulated, and one constitutive. Essentially, the constitutive pathway would c o r r e s p o n d to a paragranular route in which the proteins would be immediately secreted instead of being stored in zymogen granules. In addition, some of the proteins released in the juice under "resting" conditions are associated to microvesicles. The term "microvesicular secretion" is used to designate that type of secretion. The pathways of protein secretion in the eukaryot ic cells have been recently reviewed by Kelly (I). As m e n t i o n e d by this author, protein secretion can take several forms: secretion being constitutive if proteins are immediately secreted after their synthesis whereas in regulated secretion the newly synthesized secretory proteins are stored at high c o n c e n t r a t i o n in secretory granules until the cell receives the appropriate secretory stimulus. The pathway of regulated secretion has been extensively studied in the exocrine acinar cell of pancreas by Palade (1975). He proposed a scheme which has been a model for a variety of other secretory endocrine and exocrine cells. According to this classical scheme, the newly synthesized proteins are sequestered in the rough end~plasmic reticulum (RER), then transported via the transitional elements of the RER to the trans-golgi area where they are packed in the secretory granules. The newly formed granules mix with the older granule population. Upon stimulation, secretory granules anchor to the plasma membrane and release their content by exocytosis. (See Grossman for a review on the pan-
creas). During the seventies and early eighties a series of experiments from different laboratories, using radioactive leucine or methionine as precursors, Copyright
0 0 2 4 - 3 2 0 5 / 8 7 $3.00 + .00 (c) 1987 Pergamon Journals
Ltd.
2454
Secretory Pathways in the Pancreas
Vol. 40, No. 25, 1987
demonstrated that the secretory proteins r e l e . a s e d by t h e p a n c r e a s u n d e r "resting" and s t i m u l a t e d conditions were derived from different intracellular p o o l s of s e c r e t o r y proteins (4-7). Distinctions b e t w e e n t h e s e two p r o c e s s e s b e c a m e more e v i d e n t when p a n c r e a t i c juice, harvested in t h e p r e s e n c e o f a secretagogue, was c o m p a r e d w i t h tim j u i c e c o l l e c t e d from unstimulated animals. I n t h e r a t , t h e r a t i o of a m y l a s e t o c h y m o t r y p s i n v a r i e d p r o p o r t i o n a l l y to the a m o u n t of p r o t e i n released by t h e g l a n d ( 8 ) . Specific activity ot a m y l a s e in the pancreatic juice collected from unstimulated p i g s in v i v o was ;~bout t w i c e that from caerulein stimulated animals (9). O t h e r e x a m p l e s ot s u c h an e n r i c h ment in a m y l a s e u n d e r " r e s t i n g " secretion tlave b e e n r e p o r t e d (10). When s p e cific activity of a m y l a s e , c o l l e c t e d from unstimulated p i g s , was c o m p a r e d w i t h t h a t f o u n d in i t s own z y m o g e n g r a n u l e s , i t was a b o u t t w i c e t h e l a t t e r , l'hes(., results indicated that "resting" secretion d i d not d e r i v e f r o m an h o m o g e n e o u s g r a n u l e c o m p a r t m e n t in t h e a c i n a r ( . ' e l l s . Electrophoretic profiles of p a n c r e a tic juice proteins w e r e a l s o c o m p a r e d , i t r e v e a l e d on t h e one h a n d t h e s i m i larity of c o m p o s i t i o n b e t w e e n I he zymogen g r a n u l e c o n t e n t and p a n c r e a t i c . j u i c e collected under caerulein stimulation, and on t h e o L h e r h a n d , c o n f i r m e c t t h e disparities between "resting" and s t i m u l a t e d secretions o r zymogen g r a n u l e content. The h i g h p r o p o r t i o n of the gP-2 glyc oprote in in u n s t i m u l a t e d secret i o n was a l s o of p a r t i c u l a r interest in t h e s e a n a l y s i s (7,1I). The l a t t e r glycoprotein, originally identified in r a t zymogen g r a n u l e m e m b r a n e , had b e e n previously detected in rat pancreatic j u i c e by S c h e f f e r el. a l . ( 1 2 ) . Crucial questions r a i s e d by t h e o b s e r v a t i o n s described above were: Where did resting secretion come f r o m ? Was i t d e r i v e d f r o m t h e zymogen g r a n u l e s o[ a small number of stimulated a(:ini? Or was i t d e r i v e d f r o m m o s t o f t h e ( : e l l s which were slowly releasing their zymogen g r a n u l e c o n t e n t ? Finally, a more fundamental question: Did r e s t i n g secretion d e r i v e from zymogen granuM~s or from another (:ell compartment? ]'he f i r s t two q u e s t i o n s implied the existence of s p e c i a l i z e d granules inv o l v e d in s p e c i f ic conditions. I.et u s c o n s i d e r the q u e s t i o n of h e t e r o g e n e i t y at the c e l l u l a r level: So f a r , t h e r e h a s b e e n no i m m u n o c y t o c h e m i c a l e v i d e n c e supporting such a possibility. On t h e c o n t r a r y , a m y l a s e a p p e a r s co be q u i t { , uniformly distributed i n a l l t h e g r a n u l e s of a g i v e n c e l t o r e v e n a g i v e n acinus (13-16). l m m u n o c y t o c h e m i c a l s t u d i e s a l s o r e v e a l e d t h a t the amount of zymogen g r a n u l e s in the acini l o c a t e d in c l o s e p r o x i m i t y t o t h e i s l e t s of Langherans (peri-insular) was c o n s i d e r a b l y increased as compared to the content o b s e r v e d in m o r e d i s t a n t acini (tele-insular). There exists a controversy as t o w h e t h e r tile z y m o g e n g r a n u l e s of t h e s e two g r o u p s of a c i n i c o n t a i n d i f f e r e n t r a t i o s of a m y l a s e t o c h y m o t r y p s i n (16). One h a s t o r e m e m b e r t h a t p e r i - i n s u l a r c e l l s do ,lot c o n s t i t u t e a v e r y i m p o r t a n t m a s s ot t i s s u e compared to teleinsular cells and s e c o n d l y , that differences i n t h e r a t i o s o f t h e s e e n z y m e s in the zymogen g r a n u l e s of t h e s e two g r o u p s o t c e l l s a r e s m a l l a s c o m p a r e d t o t h e c h a n g e s o b s e r v e d in t h e j u i c e r e l e a s e d upon s t i m u l a t i o n (8,16). In contrast t o t h e s e i m m u n o c y t o c h e m i c a l ~ b s e r v a t . i o n s t h e r e c e n t r e p o r t of Mroz a n d L e c h ~ n e ( 1 7 ) h a s d e m o n s t r a t , ed a m a r k e d h e t e r o g e n e i t y in t h e a m y l a s e and chymor, r y p s i n c o n t e n t s of individual zymogen g r a n u l e s . The m a j o r v a r i a t i o n s in the r a t i o of these enzymes could well a c c ount for the wide va ria tions in enzyme c o m p o s i t i o n o b s e r v e d b e t w e e n r e s t i n g and s t i m u l a t e d secretions. It is n o t known h o w e v e r i f t h e s e d i f f e r e n t t y p e s of g r a n u l e s w e r e l o c a l i z e d in the same c e l l s . As m e n t i o n e d h,,, t h e s e a u t h o r s s h o r t - t e r m c h a n g e s in t h e c o m p o s i t i o n of p a n c r e a t i c j u i c e a s s e e n in t h e s t u d y by A d e l s o n a n d M i l l e r ( 1 6 ) ~ i t . h different secretagogues could result, from selective secretion of such g r a n u l e s with diIferent composilions. From t h e c o n c e p t u a l p o i n t , o f v i e w i t w o u l d be difficult t o e n v i s a g e how a m y l a s e and c h y m o t r y p s i n e n r i c h e d g r a n u l e s c o u l d be present in a g i v e n c e l l . 11 wot!ld r e q u i r e a s o p h i s t i c a t e d system to fill up a particular condensing vacuol(, with a specitic type of v e s i c l e s e n r i c h e d i n one
Vol. 40, No. 25, 1987
Secretory Pathways
in the Pancreas
2455
particular enzyme. A l t e r n a t i v e l y one could think of different, r,ypes o( ceils bearing granules enriched in one particular enzyme involved in resting or in stimulated conditions respectively. A possible h e t e r o g e n e i t y in enzyme composition at the level of biliary, gasr,ric, duodenal and splenic regions of the pancreas has been examined (19). This study has shown that these various regions contain e s s e n t i a l l y comparable amounts of amylase and chymotrypsin on a DNA or protein basis. Bypass
of :,he zymogen
granule
compartment
(paragranular
pathway)
The concept of a bypass of the zymogen granule compartment by a fraction of the newly synr,hesized secretory molecules was elaborated from experimenr,s with the pig pancreas in rive (9). It was found :,hat the specific radioactivity of amylase secrer,ed in the juice at 50 rain post-pulse and chase, labelin~ wir-h leucine, was I0 to 25 times :,hat present in the zymogen granules. This study was extended at different: times post-pulse and chase labeling, and showed a flagrant a s y n c h r o n i s m between packaging and release of newly synr,hesized amylase. In fact, t h e p e a k of r e l e a s e of n e w l y s y n t h e s i z e d amylase prec ' e e d e d t h e p e a k of i t s a c c u m u l a t i o n in the zymogen granules (20). In parallel with r,hese studies on t h e r e l e a s e el amylase under "resting" conditions, Havinga et al. (21) studied the intracellular transport of t h e major glycoprotein of z y m o g e n g r a n u l e m e m b r a n e s i n rat, p a n c r e a s . "['heir pulse and chase experiments on i s o l a r - o d c e l l s demons:rated that a considerably large:' p a r t of l a b e l e d GP-2 m o l e c u l e s was transported to the plasma membrane than w o u l d be e x p e c t e d ii this transport p r o c e e d e d a t t h e same r a t e a n d v i a t h e same r o u t e a s t h e m a s s of n e w l y s y n t h e s i z e d zymogens. They a l s o e x p l a i n e d their results by a s s u m i n g t h a t a f r a c t i o n of (;P-2 m < ~ I e c u l e s t o l l o w e d a n a l r e r n;it ire ~(I~C r e L () T}# r ( ) ~ [ e . 111 t h e e m b r y o n i c r a t s at o n e d a y b e f o r e b i r t h , SeCTOr_inn o c c u r s solely i n a b a s a l m a n n e r t h a t : i x n o t s" i m u l a t e d by s e c r e t a g o g u e s . By pulse a n d c h a s e experiments on l o b u l e s in vitro, Chang and Aryan (22) haw, recently s how n I h a t the release of l a b e l e d proteins occurred i n two d i s t i n c t phases. A first p h a s e w a s m a x i m a l a t 2 h of c h a s e a n d d e c l i n e d t o a m i n i m u m by 7 h . l)ischar~e ot l a b e l e d p r o r - e i n s b e g a n t o r i s e a g a i n a t 8 h of c h a s e . Analysis of secret i o n by f l u o r o g r a p h y a n d SDS-PAGE r e v e a l e d thaL amylase and the pancreatic zymogens were the major constituents in bolh phases. Pulse-chase E.M. a u r , o radiography showed that Golgi complex, condensing vacuoles a n d immar-ure g r a n t:its ~ere maximally labeled during the first p h a s e of s e c r e t i o n while labeling of m a t u r e g r a n u l e s was m a x i m a l d u r i n g t h e s e c o n d p h a s e . They suggested that the tits:- phase represents constir,utive non g r a n u l e discharge a n d 2nd p h a s e represenr,s consr,itutive granule release. Other kinetic studies by A r y a n a n d Castle (23) on the adult rat pancreas also indicated that newly synthesized zymogens were released i n two k i n e t i c a l l y distinct phases, The f i r s t frc:m 0 to 6.5 h and the second peaked at 9.5 h and declined w i t h a T 1 / 2 e l 50 h . These results a r e i n a g r e e m e n t wir-h t h e a u t o r a d i o g r a p h y studies of Kramer a n d P o o r t ( 2 4 ) who d e s c r i b e d i n 1972 t h e k i n e t i c of r e l e a s e of l a b e l e d proteins from the unstimulated rat exocrine cells. Their data obtained by quantir-alive radioautography and biochemical determinations it: n o n - a n e s t h e t i z e d fasting rats, showed that proteins were r e l e a s e d i n t w o waves, o n e s t , a r t ing a t 20 rain a n d t h e o t h e r at a b o u t 7 h r p o s r - - p u l s e . The r e s u l t s o[ the various studies described above are in agreemenr- with a model in which a fraction of the newly synthesized secretory proteins are routed in a paragranular pathway.
_
~t
,
N
i
m
Vol.
40, No.
25,
1987
Secretory
FIGS.
Morphological
aspects
of the apical
Pathways
2457
I A-D
cytoplasm FIG.
in the Pancreas
of the rat pancreatic
acinar
cell
IA
Portion of the apical c y t o p l a s m and lumen (L) showing the zymogen granules coated vesicles (cV), m i n i g r a n u l e s (mG), and several m i c r o v e s i c l e s (mV) dispersed in the lumen. Classical techniques of tissue fixation and staining does not allow a good p r e s e r v a t i o n of microvesicles located in gland lumen (42 7OOX) FIG.
IB
Portion of the apical c y t o p l a s m illustrating the o m e g a - s h a p e d outset of exocytosis. One can also notice some m i c r o v e s i c l e s bourhood of the cell surface (48 2OOX). FIG.
granule at the in the neigh-
IC
Presence of several types of vesicles in the apical cytoplasm. Arrowheads point out several types of vesicles. Some are entirely filled with more or less dense material (minigranules) whereas others contain some aggregates. One cannot exclude that the latter are endocytic vesicles (42 7OOX). FIG.
ID
Immunocytohemical localization of amylase by the protein A gold according to Beaudoin et al. (32). Gold grains can be observed indicating the presence of amylase. Intracellular
structures
involved
in parasranular
technique in minigranules
pathway
Assuming the existence of the paragranular pathway, one can wonder how this transport is effected. Since GP-2 is a g l y c o p r o t e i n with an hydrophobic character, it is reasonable to believe that it is transported by a vesicular structure. Furthermore, as judged by the size of the g l y c o r p o t e i n GP-2 (80 kDa), that is released by the acinar cell, the intracellular vesicle, must derive from the Golgi trans-saccules or the immature granule rather than the endoplasmic reticulum, where its molecular weight was estimated to be about 73 kDa (21). Is there some morphological structures that could potentially operate such a transport in the apical cytoplasm? Yes indeed, one can observe various types of vesicles close to the luminal plasma membrane. The big questions are: where are they coming from or where are they going to? The first category of vesicles have a diameter of the order of 50 nm, some are coated, others are uncoated (Fig. IA-D). The second category of vesicles are larger, with a diameter of about 150 nm. In this latter group, some are filled with dense material, others are practically empty (Fig. IC). Among those that are entirely filled with material some are even as dense as the mature zymogen granules (Fig. IC-D). We termed "minigranules" this last category. Their content ~ills the entire vesicle and is quite opaque to electrons, and for this reason we presume that these are not endocytic vesicles. In addition, as illustrated in Fig. ID, they contain secretory proteins as indicated by amylase immunoreactivity. F o l l o w i n g prolonged stimulation, the zymogen granule size is considerably reduced and these minigranules are more evident (see Fig. 3 in 25). Comparable m i n i g r a n u l e s are numerous in certain pathological situations, such as acinar cell carcinoma (26,27). Indeed among the various cell types found in these more or less differentiated tumors, some contain zymogen granules, others contain e x c l u s i v e l y these minigranules. Presence of amylase in this enzymatic content has been confirmed by i m m u n o c y t o c h e m i s t r y (27). It is
2458
Secretory
Pathways
in the Pancreas
Vo].
40, No.
25,
1987
noteworthy that these tumor cells exhibit a significant spontaneous release of amylase while they show s poor response to secretagogues (unpublished observations). Although m i n i g r a n u ] e s and other vesicles are interesting vehicles, there is still however no definitive proof at this moment, that some of these are responsible for the "paragranu]ar pathway". In this respect, a detailed morphological study of these vesicles in control and carbamylcho]ine stimulated rats has been recently reported by Romagnoli (28). His observations also suggested that some of these vesicles could play a role in enzyme secretion (personal communication). "Resting secretion" soluble proteins.
comprises
two components:
microvesicular
secretion
and
Microvesicles were recently discovered in the lumen of non-stimulated rat pancreas by f r e e z e - s u b s t i t u t i o n and conventional electron microscopy and subsequently isolated from pancreatic juice. SDS-polyacrylamide gel electrophoresis rew~aled that these vesicles conLain only one major protein, identified by an immunoblot technique as GP-2 (29). In the juice collected under resting conditions, this g l y c o p r o t e i n was tota|ly precipitable by high speed centrifugation. A minor g l y c o p r o t e i n was later identified as an ATP diphosphohydrolase (30). This indicated that a very specific domain of the membrane was involved in that shedding process. [mmunocytochemical localization of GP-2 and amylase confirmed that the former was associated with microvesicles whereas amylase was virtually excluded. From these observations, it is clear that "resting" secretion can be separated in two different components: a particulate "microvesicular secretion" and a soluble component comprising amylase and several other minor proteins. The observations raise a key question: Is the intracellular transport of these two secretory proteins related to each other? As mentioned above, pulse and chase experiments of Havinga et al. (2|) on isolated cells of rat pancreas and our experiments with rats and pigs in vivo have indicated that a fraction of both GP-2 and amylase molecules precedes the bulk of labeled secretory proteins. Indeed, these proteins appear at the cell surface at a moment after pulse labeling when most of the labeled proteins are still localized in the Golgi area (30 min) and the zymogen granules are not yet significantly labeled. A second type of observations support the concept of a cotransport of amylase and GP-2 in the acinar cell of rat pancreas. In a recent series of experiments to define the role of steroids in pancreatic function, it was found that adrenalectomy and castration caused a marked reduction in the proportions of amylase in the gland as compared to other secretory proteins (31). We noticed later that this was a c c o m p a n i e d by an important decrease in both amylase and GP-2 o u t p u t u n d e r r e s t i n g conditions. When s u c h a n i m a l s w e r e t r e a t e d with dexamethasone,the proportions of amylase were re s tore d in the t i s s u e w h e r e a s a m y l a s e and GP-2 w e r e i n c r e a s e d a s c o m p a r e d t o o t h e r s e c r e t o r y proteins in t h e pancreatic juice under "resting" conditions (unpublished observations). Although these series of observations do n o t c o n s t i t u t e a proof for the cotransport, per se, ir illustrates a certain d e g r e e of c o u p l i n g in the r e l e a s e o[ t h e s e two p r o t e i n s . A definite proof for the existence of such a p a r a granular pathway of transport w o u l d be t h e i d e n t i f i c a t i o n of a protein typical of " r e s t i n g " secretion t h a t w o u l d be a b s e n t f r o m z y m o g e n g r a n u l e s . Conclusion Although a definite proof [or the existence of the paragranular r o u t e s f o r t h e r e l e a s e o f pancr.eas s e c r e t o r y proteins under resting and s t i m u l a t e d conditions is still to come, b i o c h e m i c a l e v i d e n c e s s t r o n g l y p o i n t o u t in t h a t
Vol.
40, No.
25,
1987
Secretory
Pathways
in the Pancreas
2459
direction. It is not excluded that under "resting" conditions there is some contribution from the regulated pathway by some spontaneous ~usion of zymogen granule. It is also possible that the constitutive "presumably vesicular" pathway would also be functioning under stimulated conditions. In this review we have intentionally left aside the questions of the relative rate of transport of individual zymogens and the parallelism in the transport and release in response to various types of stimuli. Acknowledgement s
cript grant
We wish to express our gratitute to Carolyn Rancourt for typing the manusand to Marielle Martin for the artwork. This work was supported by a from the NSERC and the MRC of Canada. References
I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28.
R.B. KELLY, Science 230 25-31 (1985) G.E. PALADE, ,Science 189 347-35.~ (1975) A. QROSSMAN, Comp. Biochem. Physiol. 78 I-1"3 (1984) S.S. ROTHMAN and L.D. ISENMAN, Amer. J. Physiol. 22(~ 1082-1087 (1974) P. ROBBERECHT, M. CREMER and J. CHR]STOPHE, Gastroenterol. 72 417-420 (1977) A.R. BEAUDOIN, M. FII ION and M. ROBERGE, ]RCS Medical Science 8 774-775 ( 198o ) M. ROBERGE and A.R. BEAUDOIN, B i o c h t m . B i o p h y s . A c t a 716 3 3 1 - 3 3 6 ( 1 9 8 2 ) J . C . DAGORN and A. ESTIVAL, J . P h y s i o l . 290 5 1 - 5 8 ( 1 9 7 9 ) A.R. BEAUDOIN, A. VACHEREAU and P. ST-JEAN, B i o c h i m . B i o p h y s . A c t a 757 302-305 (1983) H. SOMMER, J . SCitREZENMEIR and tt. KASPER, H e p a r o l . ( ; a s t r o e n t e r o l . 32 2 4 6 249 (1985) D. LEBEL and A.R. BEAUDOIN, Biochim. Biophys. Acta 847 132-135 (1985) R.C.T. SCHEFFER, C. POORT and J.W. SLOT, Eur. J. Cell Biol. 23 122-128 ( 1980) J.P. KRAEIIENBUHI , L. RACINE and J.D. JAMIESON, J. Cell Biol. 72 406-423 (1977) M. BENDAYAN, J . ROTH, A. PERRELET a n d [ . ORC1, ,]. H i s t o c h e m . C y t o c h e m . 28 149-160 (1980) M. BENDAYAN, E u r . J . C e l l B i o l . 36 2 6 3 - 2 6 8 ( 1 9 8 5 ) C. POSTHUMA, J . W . SLOT and H.,]. CEIJZE, J . H i s t o c h e m . C y t o h c e m . 34 2 0 3 - 2 0 7 (1986) E.A. MROZ and C. I ECHENE, Scien(e 232 871-873 (1986) J.W. ADEI_SON and P.E. MIILER, Science 228 993-994 (1985) A.R. BEAUDOIN, P. ST-JEAN and (;. GIRARD, Can. .l. Physiol. Pharmacol. 59 8~4-886 (1981) A.R. BEAUDOIN, P. ST-JEAN and A. VACHEREAU, Pancreas I 2-4 (1986a) J.R. HAVIN(;A, G.J. STROUS and C. POOR]', Eur. J. Biochem. 144 177-183 (1984) A. CHANG and P. ARVAN, J. Cell Biol. 103 457a (198(~) P. ARVAN and J.D. CASTLE, J. Cell Biol. 103 458a (1986) M.F. KRAMER and C. POOR]', J. Cell Biol. 52 147-158 (1972) A.R. BEAUDOIN, G. (;RONI)IN, M. FILION and A. [ORD, Can. J. Biochem. Cell Biol. 62 1288-1291 (1984) J.K. REDDY and M.S. RAO, Science 198 78-80 (1977) A.R. BEAUI)OIN, h. BOURGON, (;. GRONDIN, G.G. POIRIER, A. LORD and D.S. EONGNECKER, Experimental Pancreatic Carcinosenosis, In Vitro Carcinosenesis and T r a n s p l a n t a b l e Pancreatic Carcinomas. CRC Press, p. 262-276 (1986) P. ROMAGNOII, The Physiology of Pancreatic Acinar Cells, Questions and perspectives on the secretory process: Bio Essays 2. Raven Press (1985)
2460
29. 30. 31. 32.
Secretory Pathways in the Pancreas
Vol. 40, No. 25, 1987
A.R. BEAUDOIN, G. GRONDIN, A VACHEREAU, P. ST-JEAN and C. CABANA, J. Histochem. Cytochem. 34 1079-1084 (1986b) A.R. BEAUDOIN, A. VACHEREAU, G. GRONDIN, P. ST-JEAN, M.D. ROSENBERG and R. STROBEL, FEBS Letters 203 I-2 (1986e) A.R. BEAUDOIN, G. GRONDIN, P. ST-JEAN, A. VACHEREAU, C. CABANA and A. GROSSMAN, Endocrinology I19 2106-2117 (1986d) A.R. BEAUDOIN, G. GRONDIN, A LORD and M. PELLETIER, J. Histochem. Cytochem. 35 569-575 (1985)
pp.
2453-2460
Pergamon
Journals
PATHWAYS OF SECRETION IN THE EXOCRINE PANCREAS: THE STATUS OF RESTING SECRETION Adrien Centre
de
R.
Beaudoin
and G i l l e s
Crondin
r e c h e r c h e s u r l e s m ~ c a n i s m e s de s ~ c r @ t i o n Facultfi des sciences Universit6 de S h e r b r o o k e S h e r b r o o k e , Q u e b e c , CANADA J1K 2R1
(Received
in
final
form April
13,
1987)
Summary
I n t h e l a s t d e c a d e t h e c o n c e p t of two d i s t i n c t p a t h w a y s of s e cretion in the exocrine pancreas has s lowly e m e rge d. According to this concept, one p a t h w a y i s i n v o l v e d i n s t i m u l a t e d (regulated) conditions and a n o t h e r u n d e r r e s t i n g (constitutive) conditions. This hypothesis was e l a b o r a t e d at first f r o m t h e c o m p a r i s o n o{ the s p e c i f i c radioactivities of s e c r e t o r y proteins r e l e a s e d by the gland under resting and s t i m u l a t e d conditions. A n a l y s i s of t h e protein composition of the iuice relea s e d u n d e r t h e s e two p h y s i o logical conditions further supported that hypothesis. More r e c e n t studies compared the kinetic of a c c u m u l a t i o n of n e w l y s y n t h e s i z e d proteins in zymogen g r a n u l e and t h e i r release in the g l a n d lumen. The l a t t e r results a r e in a g r e e m e n t : w i t h a model in w h i c h s e c r e t o r y proteins are channelled in two separate pathways, one regulated, and one constitutive. Essentially, the constitutive pathway would c o r r e s p o n d to a paragranular route in which the proteins would be immediately secreted instead of being stored in zymogen granules. In addition, some of the proteins released in the juice under "resting" conditions are associated to microvesicles. The term "microvesicular secretion" is used to designate that type of secretion. The pathways of protein secretion in the eukaryot ic cells have been recently reviewed by Kelly (I). As m e n t i o n e d by this author, protein secretion can take several forms: secretion being constitutive if proteins are immediately secreted after their synthesis whereas in regulated secretion the newly synthesized secretory proteins are stored at high c o n c e n t r a t i o n in secretory granules until the cell receives the appropriate secretory stimulus. The pathway of regulated secretion has been extensively studied in the exocrine acinar cell of pancreas by Palade (1975). He proposed a scheme which has been a model for a variety of other secretory endocrine and exocrine cells. According to this classical scheme, the newly synthesized proteins are sequestered in the rough end~plasmic reticulum (RER), then transported via the transitional elements of the RER to the trans-golgi area where they are packed in the secretory granules. The newly formed granules mix with the older granule population. Upon stimulation, secretory granules anchor to the plasma membrane and release their content by exocytosis. (See Grossman for a review on the pan-
creas). During the seventies and early eighties a series of experiments from different laboratories, using radioactive leucine or methionine as precursors, Copyright
0 0 2 4 - 3 2 0 5 / 8 7 $3.00 + .00 (c) 1987 Pergamon Journals
Ltd.
2454
Secretory Pathways in the Pancreas
Vol. 40, No. 25, 1987
demonstrated that the secretory proteins r e l e . a s e d by t h e p a n c r e a s u n d e r "resting" and s t i m u l a t e d conditions were derived from different intracellular p o o l s of s e c r e t o r y proteins (4-7). Distinctions b e t w e e n t h e s e two p r o c e s s e s b e c a m e more e v i d e n t when p a n c r e a t i c juice, harvested in t h e p r e s e n c e o f a secretagogue, was c o m p a r e d w i t h tim j u i c e c o l l e c t e d from unstimulated animals. I n t h e r a t , t h e r a t i o of a m y l a s e t o c h y m o t r y p s i n v a r i e d p r o p o r t i o n a l l y to the a m o u n t of p r o t e i n released by t h e g l a n d ( 8 ) . Specific activity ot a m y l a s e in the pancreatic juice collected from unstimulated p i g s in v i v o was ;~bout t w i c e that from caerulein stimulated animals (9). O t h e r e x a m p l e s ot s u c h an e n r i c h ment in a m y l a s e u n d e r " r e s t i n g " secretion tlave b e e n r e p o r t e d (10). When s p e cific activity of a m y l a s e , c o l l e c t e d from unstimulated p i g s , was c o m p a r e d w i t h t h a t f o u n d in i t s own z y m o g e n g r a n u l e s , i t was a b o u t t w i c e t h e l a t t e r , l'hes(., results indicated that "resting" secretion d i d not d e r i v e f r o m an h o m o g e n e o u s g r a n u l e c o m p a r t m e n t in t h e a c i n a r ( . ' e l l s . Electrophoretic profiles of p a n c r e a tic juice proteins w e r e a l s o c o m p a r e d , i t r e v e a l e d on t h e one h a n d t h e s i m i larity of c o m p o s i t i o n b e t w e e n I he zymogen g r a n u l e c o n t e n t and p a n c r e a t i c . j u i c e collected under caerulein stimulation, and on t h e o L h e r h a n d , c o n f i r m e c t t h e disparities between "resting" and s t i m u l a t e d secretions o r zymogen g r a n u l e content. The h i g h p r o p o r t i o n of the gP-2 glyc oprote in in u n s t i m u l a t e d secret i o n was a l s o of p a r t i c u l a r interest in t h e s e a n a l y s i s (7,1I). The l a t t e r glycoprotein, originally identified in r a t zymogen g r a n u l e m e m b r a n e , had b e e n previously detected in rat pancreatic j u i c e by S c h e f f e r el. a l . ( 1 2 ) . Crucial questions r a i s e d by t h e o b s e r v a t i o n s described above were: Where did resting secretion come f r o m ? Was i t d e r i v e d f r o m t h e zymogen g r a n u l e s o[ a small number of stimulated a(:ini? Or was i t d e r i v e d f r o m m o s t o f t h e ( : e l l s which were slowly releasing their zymogen g r a n u l e c o n t e n t ? Finally, a more fundamental question: Did r e s t i n g secretion d e r i v e from zymogen granuM~s or from another (:ell compartment? ]'he f i r s t two q u e s t i o n s implied the existence of s p e c i a l i z e d granules inv o l v e d in s p e c i f ic conditions. I.et u s c o n s i d e r the q u e s t i o n of h e t e r o g e n e i t y at the c e l l u l a r level: So f a r , t h e r e h a s b e e n no i m m u n o c y t o c h e m i c a l e v i d e n c e supporting such a possibility. On t h e c o n t r a r y , a m y l a s e a p p e a r s co be q u i t { , uniformly distributed i n a l l t h e g r a n u l e s of a g i v e n c e l t o r e v e n a g i v e n acinus (13-16). l m m u n o c y t o c h e m i c a l s t u d i e s a l s o r e v e a l e d t h a t the amount of zymogen g r a n u l e s in the acini l o c a t e d in c l o s e p r o x i m i t y t o t h e i s l e t s of Langherans (peri-insular) was c o n s i d e r a b l y increased as compared to the content o b s e r v e d in m o r e d i s t a n t acini (tele-insular). There exists a controversy as t o w h e t h e r tile z y m o g e n g r a n u l e s of t h e s e two g r o u p s of a c i n i c o n t a i n d i f f e r e n t r a t i o s of a m y l a s e t o c h y m o t r y p s i n (16). One h a s t o r e m e m b e r t h a t p e r i - i n s u l a r c e l l s do ,lot c o n s t i t u t e a v e r y i m p o r t a n t m a s s ot t i s s u e compared to teleinsular cells and s e c o n d l y , that differences i n t h e r a t i o s o f t h e s e e n z y m e s in the zymogen g r a n u l e s of t h e s e two g r o u p s o t c e l l s a r e s m a l l a s c o m p a r e d t o t h e c h a n g e s o b s e r v e d in t h e j u i c e r e l e a s e d upon s t i m u l a t i o n (8,16). In contrast t o t h e s e i m m u n o c y t o c h e m i c a l ~ b s e r v a t . i o n s t h e r e c e n t r e p o r t of Mroz a n d L e c h ~ n e ( 1 7 ) h a s d e m o n s t r a t , ed a m a r k e d h e t e r o g e n e i t y in t h e a m y l a s e and chymor, r y p s i n c o n t e n t s of individual zymogen g r a n u l e s . The m a j o r v a r i a t i o n s in the r a t i o of these enzymes could well a c c ount for the wide va ria tions in enzyme c o m p o s i t i o n o b s e r v e d b e t w e e n r e s t i n g and s t i m u l a t e d secretions. It is n o t known h o w e v e r i f t h e s e d i f f e r e n t t y p e s of g r a n u l e s w e r e l o c a l i z e d in the same c e l l s . As m e n t i o n e d h,,, t h e s e a u t h o r s s h o r t - t e r m c h a n g e s in t h e c o m p o s i t i o n of p a n c r e a t i c j u i c e a s s e e n in t h e s t u d y by A d e l s o n a n d M i l l e r ( 1 6 ) ~ i t . h different secretagogues could result, from selective secretion of such g r a n u l e s with diIferent composilions. From t h e c o n c e p t u a l p o i n t , o f v i e w i t w o u l d be difficult t o e n v i s a g e how a m y l a s e and c h y m o t r y p s i n e n r i c h e d g r a n u l e s c o u l d be present in a g i v e n c e l l . 11 wot!ld r e q u i r e a s o p h i s t i c a t e d system to fill up a particular condensing vacuol(, with a specitic type of v e s i c l e s e n r i c h e d i n one
Vol. 40, No. 25, 1987
Secretory Pathways
in the Pancreas
2455
particular enzyme. A l t e r n a t i v e l y one could think of different, r,ypes o( ceils bearing granules enriched in one particular enzyme involved in resting or in stimulated conditions respectively. A possible h e t e r o g e n e i t y in enzyme composition at the level of biliary, gasr,ric, duodenal and splenic regions of the pancreas has been examined (19). This study has shown that these various regions contain e s s e n t i a l l y comparable amounts of amylase and chymotrypsin on a DNA or protein basis. Bypass
of :,he zymogen
granule
compartment
(paragranular
pathway)
The concept of a bypass of the zymogen granule compartment by a fraction of the newly synr,hesized secretory molecules was elaborated from experimenr,s with the pig pancreas in rive (9). It was found :,hat the specific radioactivity of amylase secrer,ed in the juice at 50 rain post-pulse and chase, labelin~ wir-h leucine, was I0 to 25 times :,hat present in the zymogen granules. This study was extended at different: times post-pulse and chase labeling, and showed a flagrant a s y n c h r o n i s m between packaging and release of newly synr,hesized amylase. In fact, t h e p e a k of r e l e a s e of n e w l y s y n t h e s i z e d amylase prec ' e e d e d t h e p e a k of i t s a c c u m u l a t i o n in the zymogen granules (20). In parallel with r,hese studies on t h e r e l e a s e el amylase under "resting" conditions, Havinga et al. (21) studied the intracellular transport of t h e major glycoprotein of z y m o g e n g r a n u l e m e m b r a n e s i n rat, p a n c r e a s . "['heir pulse and chase experiments on i s o l a r - o d c e l l s demons:rated that a considerably large:' p a r t of l a b e l e d GP-2 m o l e c u l e s was transported to the plasma membrane than w o u l d be e x p e c t e d ii this transport p r o c e e d e d a t t h e same r a t e a n d v i a t h e same r o u t e a s t h e m a s s of n e w l y s y n t h e s i z e d zymogens. They a l s o e x p l a i n e d their results by a s s u m i n g t h a t a f r a c t i o n of (;P-2 m < ~ I e c u l e s t o l l o w e d a n a l r e r n;it ire ~(I~C r e L () T}# r ( ) ~ [ e . 111 t h e e m b r y o n i c r a t s at o n e d a y b e f o r e b i r t h , SeCTOr_inn o c c u r s solely i n a b a s a l m a n n e r t h a t : i x n o t s" i m u l a t e d by s e c r e t a g o g u e s . By pulse a n d c h a s e experiments on l o b u l e s in vitro, Chang and Aryan (22) haw, recently s how n I h a t the release of l a b e l e d proteins occurred i n two d i s t i n c t phases. A first p h a s e w a s m a x i m a l a t 2 h of c h a s e a n d d e c l i n e d t o a m i n i m u m by 7 h . l)ischar~e ot l a b e l e d p r o r - e i n s b e g a n t o r i s e a g a i n a t 8 h of c h a s e . Analysis of secret i o n by f l u o r o g r a p h y a n d SDS-PAGE r e v e a l e d thaL amylase and the pancreatic zymogens were the major constituents in bolh phases. Pulse-chase E.M. a u r , o radiography showed that Golgi complex, condensing vacuoles a n d immar-ure g r a n t:its ~ere maximally labeled during the first p h a s e of s e c r e t i o n while labeling of m a t u r e g r a n u l e s was m a x i m a l d u r i n g t h e s e c o n d p h a s e . They suggested that the tits:- phase represents constir,utive non g r a n u l e discharge a n d 2nd p h a s e represenr,s consr,itutive granule release. Other kinetic studies by A r y a n a n d Castle (23) on the adult rat pancreas also indicated that newly synthesized zymogens were released i n two k i n e t i c a l l y distinct phases, The f i r s t frc:m 0 to 6.5 h and the second peaked at 9.5 h and declined w i t h a T 1 / 2 e l 50 h . These results a r e i n a g r e e m e n t wir-h t h e a u t o r a d i o g r a p h y studies of Kramer a n d P o o r t ( 2 4 ) who d e s c r i b e d i n 1972 t h e k i n e t i c of r e l e a s e of l a b e l e d proteins from the unstimulated rat exocrine cells. Their data obtained by quantir-alive radioautography and biochemical determinations it: n o n - a n e s t h e t i z e d fasting rats, showed that proteins were r e l e a s e d i n t w o waves, o n e s t , a r t ing a t 20 rain a n d t h e o t h e r at a b o u t 7 h r p o s r - - p u l s e . The r e s u l t s o[ the various studies described above are in agreemenr- with a model in which a fraction of the newly synthesized secretory proteins are routed in a paragranular pathway.
_
~t
,
N
i
m
Vol.
40, No.
25,
1987
Secretory
FIGS.
Morphological
aspects
of the apical
Pathways
2457
I A-D
cytoplasm FIG.
in the Pancreas
of the rat pancreatic
acinar
cell
IA
Portion of the apical c y t o p l a s m and lumen (L) showing the zymogen granules coated vesicles (cV), m i n i g r a n u l e s (mG), and several m i c r o v e s i c l e s (mV) dispersed in the lumen. Classical techniques of tissue fixation and staining does not allow a good p r e s e r v a t i o n of microvesicles located in gland lumen (42 7OOX) FIG.
IB
Portion of the apical c y t o p l a s m illustrating the o m e g a - s h a p e d outset of exocytosis. One can also notice some m i c r o v e s i c l e s bourhood of the cell surface (48 2OOX). FIG.
granule at the in the neigh-
IC
Presence of several types of vesicles in the apical cytoplasm. Arrowheads point out several types of vesicles. Some are entirely filled with more or less dense material (minigranules) whereas others contain some aggregates. One cannot exclude that the latter are endocytic vesicles (42 7OOX). FIG.
ID
Immunocytohemical localization of amylase by the protein A gold according to Beaudoin et al. (32). Gold grains can be observed indicating the presence of amylase. Intracellular
structures
involved
in parasranular
technique in minigranules
pathway
Assuming the existence of the paragranular pathway, one can wonder how this transport is effected. Since GP-2 is a g l y c o p r o t e i n with an hydrophobic character, it is reasonable to believe that it is transported by a vesicular structure. Furthermore, as judged by the size of the g l y c o r p o t e i n GP-2 (80 kDa), that is released by the acinar cell, the intracellular vesicle, must derive from the Golgi trans-saccules or the immature granule rather than the endoplasmic reticulum, where its molecular weight was estimated to be about 73 kDa (21). Is there some morphological structures that could potentially operate such a transport in the apical cytoplasm? Yes indeed, one can observe various types of vesicles close to the luminal plasma membrane. The big questions are: where are they coming from or where are they going to? The first category of vesicles have a diameter of the order of 50 nm, some are coated, others are uncoated (Fig. IA-D). The second category of vesicles are larger, with a diameter of about 150 nm. In this latter group, some are filled with dense material, others are practically empty (Fig. IC). Among those that are entirely filled with material some are even as dense as the mature zymogen granules (Fig. IC-D). We termed "minigranules" this last category. Their content ~ills the entire vesicle and is quite opaque to electrons, and for this reason we presume that these are not endocytic vesicles. In addition, as illustrated in Fig. ID, they contain secretory proteins as indicated by amylase immunoreactivity. F o l l o w i n g prolonged stimulation, the zymogen granule size is considerably reduced and these minigranules are more evident (see Fig. 3 in 25). Comparable m i n i g r a n u l e s are numerous in certain pathological situations, such as acinar cell carcinoma (26,27). Indeed among the various cell types found in these more or less differentiated tumors, some contain zymogen granules, others contain e x c l u s i v e l y these minigranules. Presence of amylase in this enzymatic content has been confirmed by i m m u n o c y t o c h e m i s t r y (27). It is
2458
Secretory
Pathways
in the Pancreas
Vo].
40, No.
25,
1987
noteworthy that these tumor cells exhibit a significant spontaneous release of amylase while they show s poor response to secretagogues (unpublished observations). Although m i n i g r a n u ] e s and other vesicles are interesting vehicles, there is still however no definitive proof at this moment, that some of these are responsible for the "paragranu]ar pathway". In this respect, a detailed morphological study of these vesicles in control and carbamylcho]ine stimulated rats has been recently reported by Romagnoli (28). His observations also suggested that some of these vesicles could play a role in enzyme secretion (personal communication). "Resting secretion" soluble proteins.
comprises
two components:
microvesicular
secretion
and
Microvesicles were recently discovered in the lumen of non-stimulated rat pancreas by f r e e z e - s u b s t i t u t i o n and conventional electron microscopy and subsequently isolated from pancreatic juice. SDS-polyacrylamide gel electrophoresis rew~aled that these vesicles conLain only one major protein, identified by an immunoblot technique as GP-2 (29). In the juice collected under resting conditions, this g l y c o p r o t e i n was tota|ly precipitable by high speed centrifugation. A minor g l y c o p r o t e i n was later identified as an ATP diphosphohydrolase (30). This indicated that a very specific domain of the membrane was involved in that shedding process. [mmunocytochemical localization of GP-2 and amylase confirmed that the former was associated with microvesicles whereas amylase was virtually excluded. From these observations, it is clear that "resting" secretion can be separated in two different components: a particulate "microvesicular secretion" and a soluble component comprising amylase and several other minor proteins. The observations raise a key question: Is the intracellular transport of these two secretory proteins related to each other? As mentioned above, pulse and chase experiments of Havinga et al. (2|) on isolated cells of rat pancreas and our experiments with rats and pigs in vivo have indicated that a fraction of both GP-2 and amylase molecules precedes the bulk of labeled secretory proteins. Indeed, these proteins appear at the cell surface at a moment after pulse labeling when most of the labeled proteins are still localized in the Golgi area (30 min) and the zymogen granules are not yet significantly labeled. A second type of observations support the concept of a cotransport of amylase and GP-2 in the acinar cell of rat pancreas. In a recent series of experiments to define the role of steroids in pancreatic function, it was found that adrenalectomy and castration caused a marked reduction in the proportions of amylase in the gland as compared to other secretory proteins (31). We noticed later that this was a c c o m p a n i e d by an important decrease in both amylase and GP-2 o u t p u t u n d e r r e s t i n g conditions. When s u c h a n i m a l s w e r e t r e a t e d with dexamethasone,the proportions of amylase were re s tore d in the t i s s u e w h e r e a s a m y l a s e and GP-2 w e r e i n c r e a s e d a s c o m p a r e d t o o t h e r s e c r e t o r y proteins in t h e pancreatic juice under "resting" conditions (unpublished observations). Although these series of observations do n o t c o n s t i t u t e a proof for the cotransport, per se, ir illustrates a certain d e g r e e of c o u p l i n g in the r e l e a s e o[ t h e s e two p r o t e i n s . A definite proof for the existence of such a p a r a granular pathway of transport w o u l d be t h e i d e n t i f i c a t i o n of a protein typical of " r e s t i n g " secretion t h a t w o u l d be a b s e n t f r o m z y m o g e n g r a n u l e s . Conclusion Although a definite proof [or the existence of the paragranular r o u t e s f o r t h e r e l e a s e o f pancr.eas s e c r e t o r y proteins under resting and s t i m u l a t e d conditions is still to come, b i o c h e m i c a l e v i d e n c e s s t r o n g l y p o i n t o u t in t h a t
Vol.
40, No.
25,
1987
Secretory
Pathways
in the Pancreas
2459
direction. It is not excluded that under "resting" conditions there is some contribution from the regulated pathway by some spontaneous ~usion of zymogen granule. It is also possible that the constitutive "presumably vesicular" pathway would also be functioning under stimulated conditions. In this review we have intentionally left aside the questions of the relative rate of transport of individual zymogens and the parallelism in the transport and release in response to various types of stimuli. Acknowledgement s
cript grant
We wish to express our gratitute to Carolyn Rancourt for typing the manusand to Marielle Martin for the artwork. This work was supported by a from the NSERC and the MRC of Canada. References
I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28.
R.B. KELLY, Science 230 25-31 (1985) G.E. PALADE, ,Science 189 347-35.~ (1975) A. QROSSMAN, Comp. Biochem. Physiol. 78 I-1"3 (1984) S.S. ROTHMAN and L.D. ISENMAN, Amer. J. Physiol. 22(~ 1082-1087 (1974) P. ROBBERECHT, M. CREMER and J. CHR]STOPHE, Gastroenterol. 72 417-420 (1977) A.R. BEAUDOIN, M. FII ION and M. ROBERGE, ]RCS Medical Science 8 774-775 ( 198o ) M. ROBERGE and A.R. BEAUDOIN, B i o c h t m . B i o p h y s . A c t a 716 3 3 1 - 3 3 6 ( 1 9 8 2 ) J . C . DAGORN and A. ESTIVAL, J . P h y s i o l . 290 5 1 - 5 8 ( 1 9 7 9 ) A.R. BEAUDOIN, A. VACHEREAU and P. ST-JEAN, B i o c h i m . B i o p h y s . A c t a 757 302-305 (1983) H. SOMMER, J . SCitREZENMEIR and tt. KASPER, H e p a r o l . ( ; a s t r o e n t e r o l . 32 2 4 6 249 (1985) D. LEBEL and A.R. BEAUDOIN, Biochim. Biophys. Acta 847 132-135 (1985) R.C.T. SCHEFFER, C. POORT and J.W. SLOT, Eur. J. Cell Biol. 23 122-128 ( 1980) J.P. KRAEIIENBUHI , L. RACINE and J.D. JAMIESON, J. Cell Biol. 72 406-423 (1977) M. BENDAYAN, J . ROTH, A. PERRELET a n d [ . ORC1, ,]. H i s t o c h e m . C y t o c h e m . 28 149-160 (1980) M. BENDAYAN, E u r . J . C e l l B i o l . 36 2 6 3 - 2 6 8 ( 1 9 8 5 ) C. POSTHUMA, J . W . SLOT and H.,]. CEIJZE, J . H i s t o c h e m . C y t o h c e m . 34 2 0 3 - 2 0 7 (1986) E.A. MROZ and C. I ECHENE, Scien(e 232 871-873 (1986) J.W. ADEI_SON and P.E. MIILER, Science 228 993-994 (1985) A.R. BEAUDOIN, P. ST-JEAN and (;. GIRARD, Can. .l. Physiol. Pharmacol. 59 8~4-886 (1981) A.R. BEAUDOIN, P. ST-JEAN and A. VACHEREAU, Pancreas I 2-4 (1986a) J.R. HAVIN(;A, G.J. STROUS and C. POOR]', Eur. J. Biochem. 144 177-183 (1984) A. CHANG and P. ARVAN, J. Cell Biol. 103 457a (198(~) P. ARVAN and J.D. CASTLE, J. Cell Biol. 103 458a (1986) M.F. KRAMER and C. POOR]', J. Cell Biol. 52 147-158 (1972) A.R. BEAUDOIN, G. (;RONI)IN, M. FILION and A. [ORD, Can. J. Biochem. Cell Biol. 62 1288-1291 (1984) J.K. REDDY and M.S. RAO, Science 198 78-80 (1977) A.R. BEAUI)OIN, h. BOURGON, (;. GRONDIN, G.G. POIRIER, A. LORD and D.S. EONGNECKER, Experimental Pancreatic Carcinosenosis, In Vitro Carcinosenesis and T r a n s p l a n t a b l e Pancreatic Carcinomas. CRC Press, p. 262-276 (1986) P. ROMAGNOII, The Physiology of Pancreatic Acinar Cells, Questions and perspectives on the secretory process: Bio Essays 2. Raven Press (1985)
2460
29. 30. 31. 32.
Secretory Pathways in the Pancreas
Vol. 40, No. 25, 1987
A.R. BEAUDOIN, G. GRONDIN, A VACHEREAU, P. ST-JEAN and C. CABANA, J. Histochem. Cytochem. 34 1079-1084 (1986b) A.R. BEAUDOIN, A. VACHEREAU, G. GRONDIN, P. ST-JEAN, M.D. ROSENBERG and R. STROBEL, FEBS Letters 203 I-2 (1986e) A.R. BEAUDOIN, G. GRONDIN, P. ST-JEAN, A. VACHEREAU, C. CABANA and A. GROSSMAN, Endocrinology I19 2106-2117 (1986d) A.R. BEAUDOIN, G. GRONDIN, A LORD and M. PELLETIER, J. Histochem. Cytochem. 35 569-575 (1985)