102
Biochimica et Biophysica Acta, 544 (1978) 102--112
© Elsevier/North-Holland Biomedical Press
BBA 28722 A POSSIBLE ROLE FOR GUANOSINE 3',5'-MONOPHOSPHATE IN THE STIMULUS-SECRETION COUPLING IN EXOCRINE PANCREAS
C.L. KAPOOR and G. KRISHNA * Section on Drug-Tissue Interaction, Laboratory o f Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, Md. 20014 (U.S.A.)
(Received April llth, 1978) Summary Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eightfold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 rain and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, C.L. and Krishna, G. (1977) Science 196, 1003-1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium. There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 , M concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated. A variety of digestive enzymes are made in the acinar cells of pancreas, then packaged into zymogen granules, and released into the pancreatic lumen by a * To w h o m r e p r i n t r e q u e s t s s h o u l d be sent.
103 process of exocytosis [1--5]. The release of these enzymes is stimulated by cholinergic agents such as acetylcholine, carbamylcholine and pilocarpine, as well as by polypeptide hormones such as cholecystokinin and caerulein [6--8]. Although it has been suggested that cyclic AMP may mediate the stimulatory effects of these substances in exocrine pancreas, the evidence presented so far has not been very convincing [9--14]. Most of the evidence implicating a mediator role for cyclic AMP was obtained by adding cyclic AMP or its butyryl derivatives to tissue slices and measuring the rate of release of amylase or other enzymes. These studies led to the erroneous conclusion that cyclic AMP mediated the amylase secretions induced by various agents [ 15 ]. It has been shown recently that cyclic GMP, rather than cyclic AMP, may mediate the effects of various secretagogues in isolated pancreatic acinar cells [16,17] and in pancreatic slices and lobules [ 1 8 - 2 1 ] . In this study we have investigated the possible role of cyclic GMP in the pancreatic amylase secretion by correlating the dose response of various secretagogues in the rate of amylase secretion in pancreatic slices with the increase in cyclic GMP accumulation and its secretion. Pancreatic slices and lobules appear to be better models than acinar cells for studying the mechanism of amylase release, since the slices and lobules appear to retain all the functional aspects of pancreas. Even though carbamylcholine and cholecystokinin octapeptide increase cyclic GMP in isolated pancreatic acinar cells, these secretagogues release only &-6% of amylase in 20 min from acinar cells [22] as compared to 15--20% of amylase secretion from pancreatic slices or lobules. Moreover, the concentrations of carbamylcholine required to elicit 50% of the maximal effect (ECs0) were higher in isolated acinar cells than in slices [17,19]. The reason for this is not clear but appears to be due to a loss of structural organization of the lobules in the isolated cell preparation which may be very important for the secretory function. For this reason, pancreatic slices or lobules were employed throughout this study. Materials Synthetic C-terminal octapeptide of porcine cholecystokinin was a gift from Squibb Institute for Medical Research, Princeton, N.J. Caerulein (as diethyl ammonium salt) was a gift from Farmitalia, Milano, Italy. Carbamylcholine chloride was obtained from K & K Laboratories, Plainview, N.Y. Cyclic GMP and bovine albumin Fraction V were obtained from Sigma Chemical Co., Stl Louis, Mo. Cyclic AMP and cyclic GMP antisera were prepared in rabbits according to the m e t h o d of Steiner et al. [23] and iodinated tyrosine methylester 2'-O-succinyl cyclic GMP and cyclic AMP, (specific activity 600 Ci/mmol) were purchased from Collaborative Research, Inc., Waltham, Mass. Partially purified beef heart phosphodiesterase, cyclic GMP and cyclic AMP were purchased from Boehinger Mannheim, New York, N.Y. Amylase azure blue, the substrate for amylase, was obtained from Calbiochem, La Jolla, Calif. Succinic anhydride and triethylamine were from Eastman Kodak, Rochester, N.Y. Methods Guinea pigs (male, N.I.H. Hartley strain, weight 3 0 0 - 3 5 0 g) were fasted overnight and were killed by decapitation. The pancreas was removed and im-
104 mersed in Krebs-Ringer bicarbonate buffer, pH 7.4, containing glucose (1 mg/ ml) and equilibrated with 95% 02/5% CO2. The fat and damaged tissues were trimmed and the tissue was cut out 2 × 2 mm fragments and preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer. At the end of preincubation, slices were collected by decantation of the medium, washed and suspended in a fresh medium. Similarly, pancreatic lobules (2 ~ 1 )! 0.5 mm size, weighing approx. 6 mg) were prepared according to the m e t h o d of Scheele and Palade [24] and preincubated at 37°C for 30 rain, washed and suspended in a fresh medium. Unless otherwise stated, all operations were performed at room temperature. Four to eight slices or lobules weighing 20--40 mg were incubated in 1--2 ml of Krebs-Ringer bicarbonate buffer at 37°C for varying periods of time with various agents. As the end of incubation, an aliquot of the medium (25--50 ~l) was removed for the determination of amylase and the rest of the incubation mixture containing the tissue was immersed in boiling water bath for 2 min. (The effective time for termination of reaction was found to be less than 5 s, since the tubes which were held in boiling water for 2 -5 s prior to addition of various hormones did not show any increase in cyclic GMP accumulation. Moreover, the temperature of incubation mixture was increased to 70°C within 5 s after immersion of the tubes in a boiling water bath. After centrifugation of the samples, 50-~1 aliquots of the supernatant fluid were taken for determination of cyclic nucleotides as described below. This procedure effectively extracted the cyclic nucleotides; extraction of cyclic nucleotides by perchloric acid yielded identical values. In some experiments, the tissue slices and the medium were separated after the incubation period and rapidly frozen in liquid nitrogen. The cyclic nucleotides in slices and in the medium were extracted by homogenization of the tissue with 5% trichloroacetic acid. Trichloroacetic acid was removed by repeated ether extraction. Cyclic GMP and cyclic AMP were determined by radioimmunoassay as described by Frandsen and Krishna [25]. Cyclic nucleotides were routinely estimated w i t h o u t purification by Dowex I chromatography, mainly because identical values were obtained with or w i t h o u t purification. The specificity of the m e t h o d has been validated by treating the samples with purified beef heart phosphodiesterase, which resulted in complete loss of cyclic nucleotide-immunoreactivity [25]. Moreover, there was a linear relationship between the a m o u n t of tissue used for assay and the value of cyclic nucleotide obtained in the assay. In 20 separate experiments, the a m o u n t of cyclic GMP in guinea pig pancreatic slices was found to be 571 ~ 58 fmol/mg protein. In order to examine the extent of cell disorganization which could have occurred during the preparation of slices and subsequent incubations, pancreatic slices were incubated as described above, fixed with 3% glutaraldehyde in 20 mM cacodylate buffer (pH 7.4) and treated with OsO4. The samples were dehydrated in a graded series of alcohol and embedded in Epon 812. Sections were cut on LKB microtome and stained with uranyl acetate followed by lead citrate. Electron microscopy was performed by Dr. E.W. Kingsbury on a Hitachi HU-12A at 75 kV at Litton Bionetics Industries, Inc., Rochville, Md. Amylase was assayed according to the m e t h o d of Rinderknecht et al. [26] using amylase azure blue as a substrate and was expressed as a percentage of
105 total enzyme activity present in the tissue. Tissue slices were dissolved in 1 M NaOH and aliquots were taken for the determination of protein using bovine albumin as a standard [27]. Results Ultrastructural characteristics o f pancreatic slices in response to various secretagogues Acinar cells in the pancreatic slices which had been preincubated for 30 min appeared to have a normal cytological appearance. There was a large number of granules which did n o t show any apparent fusion with plasma membrane. Moreover, the plasma membranes of the cells were intact and there was no apparent damage (Fig. la). Pancreatic slices after incubation with 30 pM carbamylcholine for 30 s showed fusion of zymogen granules to the plasmalemma membrane and secretion of contents into the lumen as indicated by the two arrows in Fig. l b . After incubation of the slices with carbamylcholine (30 t~M) for 10 min a larger increase in the secretory materials from zymogens in the granules accumulated in the collecting duct (Fig. lc). A similar fusion and discharge of contents of zymogen granules to the plasmalemma membrane occurred during incubation of pancreatic slices with caerulein or cholecystokinin octapeptide (data not shown). Effect o f various secretagogues on cyclic nucleotide accumulation and amylase secretion from pancreatic slices Incubation of pancreatic slices with 30 ~M carbamylcholine, 3 nM caerulein or 3 nM cholecystokinin octapeptide resulted in a marked increase in cyclic GMP within a few seconds and the amount then declined to a lower level in approx. 1--2 min (Fig. 2). The cyclic GMP level then gradually increased reaching a plateau of approx. 5-fold over the basal level in 10 min and this level was maintained for 30 min (Fig. 2). Cyclic AMP levels did n o t change during this period. These results are in agreement with other studies in which both carbamylcholine and cholecystokinin octapeptide altered cyclic GMP levels in isolated acinar cells [ 17]. Correlation o f cyclic GMP and amylase secretion as a function o f secretagogues concentration In another study [19] we showed that the increase in cyclic GMP at 30 s caused by various agents represent mainly an increase in the tissue level and represent the increase in cyclic GMP at 10 min in the cyclic GMP secreted into the medium. The concentration response studies of the secretagogues were carried out at 30 s and 10 min. 1. Concentration-response o f various secretagogues on cyclic GMP and amylase secretion from pancreatic slices. Results presented in Fig. 3 show the response of carbamylcholine, caerulein and cholecystokinin octapeptide in the accumulation of cyclic GMP in pancreas at 30 s after addition of various secretagogues. Significant increases in cyclic GMP accumulation could be detected with 0.1 nM caerulein or cholecystokinin octapeptide. Concentrations of 3 t~M carbamylcholine, 0.85 nM caerulein and 1 nM cholecystokinin
106
Fig.
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Fig. 1. Electron microscopy of isolated pancreatic slices. Pancreatic slices (2 X 2 mm) were preincubated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 1 mg glucose/ml for 30 min at 37OC. (a) Control pancreatic slices showing no apparent fusion of zymogen granules with plasmalemma membrane. (b) Pancreatic slices after incubation with 30 PM carbamylcholine for 30 s show fusion of zymogen granules to the plasmalemma membrane in lumen as indicated by the two arrows. (c) Pancreatic slices after 10 min incubation with 30 PM carbamylcholine show discharge of the content of zymogen granules in the collecting duct. The bars in the figures correspond to 1 pm.
octapeptide were required to produce one-half of the maximal effects. 2. Concentration-response of various secretagogues on cyclic GMP and amylase secretion from pancreatic slices. The concentration-dependent increase in cyclic GMP and amylase secretion induced by various secretagogues were closely related (Fig. 4). Significant increases in cyclic GMP and amylase secretion could be detected at 0.1 PM carbamylcholine and the maximal effect occurred at a concentration of 30 PM. Caerulein and cholecystokinin octapeptide also caused significant stimulation of cyclic GMP and amylase secretion at concentrations of 0.1 nM and maximal stimulations occurred at 3 nM. Concentrations of 1.5 PM carbamylcholine, 0.3 nM caerulein and 0.38 nM cholecystokinin octapeptide were required to produce a one-half maximal increase in cyclic GMP as well as amylase secretion. Thus, there was a strict correlation between the concentrations of these agents required to induce cyclic GMP and the amylase secretion.
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b a t e d w i t h s l i c e s . C y c l i c G M P a n d a m y l a s e s e c r e t i o n w e r e d e t e r m i n e d a t 1 0 r a i n o f i n c u b a t i o n . V a l u e s are t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s w h i c h d i d n o t v a r y m o r e t h a n +8%. C o n t r o l (r) o), c a r b a m y l c h o l i n e ( 3 0 /~M) ( o - o), c a e r u l e i n (3 n M ) (A A), c h o l e c y s t o k i n i n o c t a p e p t i d e (3 n M ) , ( i •).
111
tion (Fig. 4). Moreover, the cyclic GMP accumulation and the increase in amylase release by carbamylcholine was inhibited by 50% with the same concentration of atropine. The finding that dibutyryl cyclic GMP ~increases amylase release from pancreatic slices [20], also st~ggests a role for cyclic GMP in the release of amylase even though the mechanism is not clear. These effects may be attributable to inhibition of phosphodiesterase which causes an increase in cyclic GMP. Moreover, sodium butyrate, a hydrolytic product of dibutyryl cyclic GMP, increases amylase release by increasing cyclic GMP in pancreas (Kapoor and Krishna, unpublished findings). The mechanism by which carbamylcholine and cholecystokinin octapeptide increase cyclic GMP is not clear, because these agents do not activate the guanylate cyclase in pancreas homogenates [28]. We have shown earlier that a perfect correlation exists between concentration response of various secretagogues on cyclic GMP increase and calcium efflux in pancreatic acinar cells [16, 17]. Thus it is tempting to speculate that calcium in some way causes the increase in cyclic GMP induced by these agents. Low concentrations of calcium are able to activate the enzyme in cell-free systems [28] but the significance of this activation in an intact cell is not clear. Indeed, it is possible that a mechanism exists by which calcium increases cyclic GMP, other than a direct activation of guanylate cyclase. For example, the increase in calcium efflux observed with these agents in pancreatic acinar cells may trigger other enzyme systems that change the redox potential in cells or increase arachidonic acid release; in turn, these changes may cause an increase in the formation of hydroxyl radicals which directly activate guanytate cyclase in the cell [29]. Whatever the mechanism by which various secretagogues increase cyclic GMP in the pancreatic acinar cells might be, it appears that cyclic GMP is intimately involved in the amylase secretion in pancreas. References 1 Palade, G.E. ( 1 9 5 9 ) F u n c t i o n a l changes in the s t r u c t u r e of cell c o m p o n e n t . In Subcellular particles ( H a y a s h i , T., ed.) p p . 6 4 - - 8 0 , R o n a l d Press, New Y o r k 2 E c k h o l m , R., Zelander, T. a n d Edlung~ Y. ( 1 9 6 2 ) J. U l t r a s t r u c t . Res. 7, 6 1 - - 7 2 3 I c h i k a w a , A. ( 1 9 6 5 ) J. Cell Biol. 24, 3 6 9 - - 3 8 5 4 J a m i s o n , J.D. a n d Palade, G.E. ( 1 9 6 7 ) J. Cell Biol. 3 4 , 5 7 7 - - 5 9 6 5 J a m i s o n , J.D. a n d Palade, G.E. ( 1 9 6 7 ) J. Cell Biol. 34, 5 9 7 - - 6 1 5 6 H a r p e r , A.A. ( 1 9 6 7 ) in H a n d b o o k of Physiol. 2: Sect. 6, (Code, C.F., ed.), pp. 9 6 9 - - 9 9 5 , A m e r i c a n Physiol. S o c i e t y , W a s h i n g t o n 7 S c h r a m m , M. ( 1 9 6 7 ) A n n u . Rev. Biochem. 36, 3 0 7 - - 3 2 0 8 J o r p e , J.E. a n d Mutt, V. ( 1 9 7 3 ) in Secretin, C h o l e c y s t o k i n i n , P a n c r e o z y m i n a n d gasterin ( J o r p e , J.E. a n d Mutt, V., eds.) pp. 1 - - 1 7 7 , Springer Verlag, New Y o r k 9 K u l k a , R.G. a n d S t e r n l i c h , E. ( 1 9 6 8 ) Proc. Natl. A c a d . ScL U.S. 71, 4 0 3 7 - - 4 0 4 1 10 R e d d e r s t a p , A.S. a n d Bonting, S.L. ( 1 9 6 9 ) Pflugens Arch. 3 1 3 , 6 2 - - 7 0 11 Morriset, J.A. a n d Webster, P.D. ( 1 9 7 1 ) Am. J. Physiol. 2 3 0 , 2 0 2 - - 2 0 8 12 Benz, L., Eckstein, B., M a t t h e w s , E.K. a n d William, J.A. ( 1 9 7 2 ) Brit. J. P h a r m a c o l . 46, 6 6 - - 7 7 13 Case, R.M. a n d S c a r t c h e r e d , T. ( 1 9 7 2 ) J. Physiol. 2 2 3 , 6 4 9 - - 6 6 7 14 Heisler, S., Fast~ D. a n d T e e n h o u s e , A. ( 1 9 7 2 ) Biochim. Biophys. A c t a 279, 5 6 1 - - 5 7 2 15 Berridge, M.J. ( 1 9 7 6 ) in A d v a n c e s in Cyclic N u c l e o t i d e R e s e a r c h , Vol. 6 ( G r e e n g a r d , P. a n d R o b i s o n , G.A., eds.), p p . 7 - - 9 8 , R a v e n Press, New Y o r k 16 F r a n d s e n , E.K., C h r i s t o p h e , J., Krishna, G. a n d G a r d n e r , J.D. ( 1 9 7 5 ) P h a r m a c o l o g i s t 17, 269 17 C h r i s t o p h e , J.P., F r a n d s e n , E.K., C o n l o n , T.P., Krishna, G. a n d G a r d n e r , J.D. ( 1 9 7 6 ) J. Biol. Chem. 251, 4640---4645 18 K a P o o r , C.L. a n d Krishna, G. ( 1 9 7 6 ) Fed. Proc. 35, 2 9 5 19 K a p o o r , C.L. a n d Krishna, G. ( 1 9 7 7 ) Science 1 9 6 , 1 0 0 3 - - 1 0 0 5
112
2 0 H a y m o r i t s , A. a n d S c h e e l e , G . A . ( 1 9 7 6 ) P r o c . Natl. A c a d . Sci. U.S. 73, 1 5 6 - - 1 6 0 21 A l b a n o , J., B h o o l a , K . D . a n d H a r v e y , R . F . ( 1 9 7 6 ) N a t u r e 2 6 2 , 4 0 4 - - 4 0 7 2 2 G a r d n e r , J . D . , C o n l o n , T.P., K l a e v e m a n , H., A d a m s , T . D . a n d O m d e t t i , M.A. ( 1 9 7 5 ) J. Clin. Invest. 56,366--375 2 3 S t e i n e r , A . L . , P a r k e r , C.W. a n d K i p n i s , D.M. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 1 1 0 6 - - 1 1 1 3 2 4 S c h e e l e , G . A . a n d P a l a d e , G . E . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 2 6 6 0 - - 2 6 7 0 2 5 F r a n d s e n , E . K . a n d K r i s h n a , G. ( 1 9 7 6 ) Life Sci. 1 3 , 5 2 9 - - 5 4 2 2 6 R i n d e r k n e c h t , H., W i l d i n g , P. a n d H a v e r b a c k , B.J. ( 1 9 6 7 ) E x p e r i e n t i a 2 3 , 8 0 5 - - 8 0 7 2 7 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 6 - - 2 7 5 2 8 K a p o o r , C.L. a n d K r i s h n a , G. ( 1 9 7 8 ) in A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h , Vol. 9, ( a b s t r . ) R a v e n Press, N e w Y o r k , in p r e s s 2 9 M i t t a l , C . K . a n d M u r a d , F. ( 1 9 7 8 ) J. C y c l i c N u c l e o t i d e Res. 3 , 3 8 1 - - 3 9 1
Biochimica et Biophysica Acta, 544 (1978) 102--112
© Elsevier/North-Holland Biomedical Press
BBA 28722 A POSSIBLE ROLE FOR GUANOSINE 3',5'-MONOPHOSPHATE IN THE STIMULUS-SECRETION COUPLING IN EXOCRINE PANCREAS
C.L. KAPOOR and G. KRISHNA * Section on Drug-Tissue Interaction, Laboratory o f Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, Md. 20014 (U.S.A.)
(Received April llth, 1978) Summary Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eightfold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 rain and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, C.L. and Krishna, G. (1977) Science 196, 1003-1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium. There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 , M concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated. A variety of digestive enzymes are made in the acinar cells of pancreas, then packaged into zymogen granules, and released into the pancreatic lumen by a * To w h o m r e p r i n t r e q u e s t s s h o u l d be sent.
103 process of exocytosis [1--5]. The release of these enzymes is stimulated by cholinergic agents such as acetylcholine, carbamylcholine and pilocarpine, as well as by polypeptide hormones such as cholecystokinin and caerulein [6--8]. Although it has been suggested that cyclic AMP may mediate the stimulatory effects of these substances in exocrine pancreas, the evidence presented so far has not been very convincing [9--14]. Most of the evidence implicating a mediator role for cyclic AMP was obtained by adding cyclic AMP or its butyryl derivatives to tissue slices and measuring the rate of release of amylase or other enzymes. These studies led to the erroneous conclusion that cyclic AMP mediated the amylase secretions induced by various agents [ 15 ]. It has been shown recently that cyclic GMP, rather than cyclic AMP, may mediate the effects of various secretagogues in isolated pancreatic acinar cells [16,17] and in pancreatic slices and lobules [ 1 8 - 2 1 ] . In this study we have investigated the possible role of cyclic GMP in the pancreatic amylase secretion by correlating the dose response of various secretagogues in the rate of amylase secretion in pancreatic slices with the increase in cyclic GMP accumulation and its secretion. Pancreatic slices and lobules appear to be better models than acinar cells for studying the mechanism of amylase release, since the slices and lobules appear to retain all the functional aspects of pancreas. Even though carbamylcholine and cholecystokinin octapeptide increase cyclic GMP in isolated pancreatic acinar cells, these secretagogues release only &-6% of amylase in 20 min from acinar cells [22] as compared to 15--20% of amylase secretion from pancreatic slices or lobules. Moreover, the concentrations of carbamylcholine required to elicit 50% of the maximal effect (ECs0) were higher in isolated acinar cells than in slices [17,19]. The reason for this is not clear but appears to be due to a loss of structural organization of the lobules in the isolated cell preparation which may be very important for the secretory function. For this reason, pancreatic slices or lobules were employed throughout this study. Materials Synthetic C-terminal octapeptide of porcine cholecystokinin was a gift from Squibb Institute for Medical Research, Princeton, N.J. Caerulein (as diethyl ammonium salt) was a gift from Farmitalia, Milano, Italy. Carbamylcholine chloride was obtained from K & K Laboratories, Plainview, N.Y. Cyclic GMP and bovine albumin Fraction V were obtained from Sigma Chemical Co., Stl Louis, Mo. Cyclic AMP and cyclic GMP antisera were prepared in rabbits according to the m e t h o d of Steiner et al. [23] and iodinated tyrosine methylester 2'-O-succinyl cyclic GMP and cyclic AMP, (specific activity 600 Ci/mmol) were purchased from Collaborative Research, Inc., Waltham, Mass. Partially purified beef heart phosphodiesterase, cyclic GMP and cyclic AMP were purchased from Boehinger Mannheim, New York, N.Y. Amylase azure blue, the substrate for amylase, was obtained from Calbiochem, La Jolla, Calif. Succinic anhydride and triethylamine were from Eastman Kodak, Rochester, N.Y. Methods Guinea pigs (male, N.I.H. Hartley strain, weight 3 0 0 - 3 5 0 g) were fasted overnight and were killed by decapitation. The pancreas was removed and im-
104 mersed in Krebs-Ringer bicarbonate buffer, pH 7.4, containing glucose (1 mg/ ml) and equilibrated with 95% 02/5% CO2. The fat and damaged tissues were trimmed and the tissue was cut out 2 × 2 mm fragments and preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer. At the end of preincubation, slices were collected by decantation of the medium, washed and suspended in a fresh medium. Similarly, pancreatic lobules (2 ~ 1 )! 0.5 mm size, weighing approx. 6 mg) were prepared according to the m e t h o d of Scheele and Palade [24] and preincubated at 37°C for 30 rain, washed and suspended in a fresh medium. Unless otherwise stated, all operations were performed at room temperature. Four to eight slices or lobules weighing 20--40 mg were incubated in 1--2 ml of Krebs-Ringer bicarbonate buffer at 37°C for varying periods of time with various agents. As the end of incubation, an aliquot of the medium (25--50 ~l) was removed for the determination of amylase and the rest of the incubation mixture containing the tissue was immersed in boiling water bath for 2 min. (The effective time for termination of reaction was found to be less than 5 s, since the tubes which were held in boiling water for 2 -5 s prior to addition of various hormones did not show any increase in cyclic GMP accumulation. Moreover, the temperature of incubation mixture was increased to 70°C within 5 s after immersion of the tubes in a boiling water bath. After centrifugation of the samples, 50-~1 aliquots of the supernatant fluid were taken for determination of cyclic nucleotides as described below. This procedure effectively extracted the cyclic nucleotides; extraction of cyclic nucleotides by perchloric acid yielded identical values. In some experiments, the tissue slices and the medium were separated after the incubation period and rapidly frozen in liquid nitrogen. The cyclic nucleotides in slices and in the medium were extracted by homogenization of the tissue with 5% trichloroacetic acid. Trichloroacetic acid was removed by repeated ether extraction. Cyclic GMP and cyclic AMP were determined by radioimmunoassay as described by Frandsen and Krishna [25]. Cyclic nucleotides were routinely estimated w i t h o u t purification by Dowex I chromatography, mainly because identical values were obtained with or w i t h o u t purification. The specificity of the m e t h o d has been validated by treating the samples with purified beef heart phosphodiesterase, which resulted in complete loss of cyclic nucleotide-immunoreactivity [25]. Moreover, there was a linear relationship between the a m o u n t of tissue used for assay and the value of cyclic nucleotide obtained in the assay. In 20 separate experiments, the a m o u n t of cyclic GMP in guinea pig pancreatic slices was found to be 571 ~ 58 fmol/mg protein. In order to examine the extent of cell disorganization which could have occurred during the preparation of slices and subsequent incubations, pancreatic slices were incubated as described above, fixed with 3% glutaraldehyde in 20 mM cacodylate buffer (pH 7.4) and treated with OsO4. The samples were dehydrated in a graded series of alcohol and embedded in Epon 812. Sections were cut on LKB microtome and stained with uranyl acetate followed by lead citrate. Electron microscopy was performed by Dr. E.W. Kingsbury on a Hitachi HU-12A at 75 kV at Litton Bionetics Industries, Inc., Rochville, Md. Amylase was assayed according to the m e t h o d of Rinderknecht et al. [26] using amylase azure blue as a substrate and was expressed as a percentage of
105 total enzyme activity present in the tissue. Tissue slices were dissolved in 1 M NaOH and aliquots were taken for the determination of protein using bovine albumin as a standard [27]. Results Ultrastructural characteristics o f pancreatic slices in response to various secretagogues Acinar cells in the pancreatic slices which had been preincubated for 30 min appeared to have a normal cytological appearance. There was a large number of granules which did n o t show any apparent fusion with plasma membrane. Moreover, the plasma membranes of the cells were intact and there was no apparent damage (Fig. la). Pancreatic slices after incubation with 30 pM carbamylcholine for 30 s showed fusion of zymogen granules to the plasmalemma membrane and secretion of contents into the lumen as indicated by the two arrows in Fig. l b . After incubation of the slices with carbamylcholine (30 t~M) for 10 min a larger increase in the secretory materials from zymogens in the granules accumulated in the collecting duct (Fig. lc). A similar fusion and discharge of contents of zymogen granules to the plasmalemma membrane occurred during incubation of pancreatic slices with caerulein or cholecystokinin octapeptide (data not shown). Effect o f various secretagogues on cyclic nucleotide accumulation and amylase secretion from pancreatic slices Incubation of pancreatic slices with 30 ~M carbamylcholine, 3 nM caerulein or 3 nM cholecystokinin octapeptide resulted in a marked increase in cyclic GMP within a few seconds and the amount then declined to a lower level in approx. 1--2 min (Fig. 2). The cyclic GMP level then gradually increased reaching a plateau of approx. 5-fold over the basal level in 10 min and this level was maintained for 30 min (Fig. 2). Cyclic AMP levels did n o t change during this period. These results are in agreement with other studies in which both carbamylcholine and cholecystokinin octapeptide altered cyclic GMP levels in isolated acinar cells [ 17]. Correlation o f cyclic GMP and amylase secretion as a function o f secretagogues concentration In another study [19] we showed that the increase in cyclic GMP at 30 s caused by various agents represent mainly an increase in the tissue level and represent the increase in cyclic GMP at 10 min in the cyclic GMP secreted into the medium. The concentration response studies of the secretagogues were carried out at 30 s and 10 min. 1. Concentration-response o f various secretagogues on cyclic GMP and amylase secretion from pancreatic slices. Results presented in Fig. 3 show the response of carbamylcholine, caerulein and cholecystokinin octapeptide in the accumulation of cyclic GMP in pancreas at 30 s after addition of various secretagogues. Significant increases in cyclic GMP accumulation could be detected with 0.1 nM caerulein or cholecystokinin octapeptide. Concentrations of 3 t~M carbamylcholine, 0.85 nM caerulein and 1 nM cholecystokinin
106
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Fig. 1. Electron microscopy of isolated pancreatic slices. Pancreatic slices (2 X 2 mm) were preincubated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 1 mg glucose/ml for 30 min at 37OC. (a) Control pancreatic slices showing no apparent fusion of zymogen granules with plasmalemma membrane. (b) Pancreatic slices after incubation with 30 PM carbamylcholine for 30 s show fusion of zymogen granules to the plasmalemma membrane in lumen as indicated by the two arrows. (c) Pancreatic slices after 10 min incubation with 30 PM carbamylcholine show discharge of the content of zymogen granules in the collecting duct. The bars in the figures correspond to 1 pm.
octapeptide were required to produce one-half of the maximal effects. 2. Concentration-response of various secretagogues on cyclic GMP and amylase secretion from pancreatic slices. The concentration-dependent increase in cyclic GMP and amylase secretion induced by various secretagogues were closely related (Fig. 4). Significant increases in cyclic GMP and amylase secretion could be detected at 0.1 PM carbamylcholine and the maximal effect occurred at a concentration of 30 PM. Caerulein and cholecystokinin octapeptide also caused significant stimulation of cyclic GMP and amylase secretion at concentrations of 0.1 nM and maximal stimulations occurred at 3 nM. Concentrations of 1.5 PM carbamylcholine, 0.3 nM caerulein and 0.38 nM cholecystokinin octapeptide were required to produce a one-half maximal increase in cyclic GMP as well as amylase secretion. Thus, there was a strict correlation between the concentrations of these agents required to induce cyclic GMP and the amylase secretion.
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b a t e d w i t h s l i c e s . C y c l i c G M P a n d a m y l a s e s e c r e t i o n w e r e d e t e r m i n e d a t 1 0 r a i n o f i n c u b a t i o n . V a l u e s are t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s w h i c h d i d n o t v a r y m o r e t h a n +8%. C o n t r o l (r) o), c a r b a m y l c h o l i n e ( 3 0 /~M) ( o - o), c a e r u l e i n (3 n M ) (A A), c h o l e c y s t o k i n i n o c t a p e p t i d e (3 n M ) , ( i •).
111
tion (Fig. 4). Moreover, the cyclic GMP accumulation and the increase in amylase release by carbamylcholine was inhibited by 50% with the same concentration of atropine. The finding that dibutyryl cyclic GMP ~increases amylase release from pancreatic slices [20], also st~ggests a role for cyclic GMP in the release of amylase even though the mechanism is not clear. These effects may be attributable to inhibition of phosphodiesterase which causes an increase in cyclic GMP. Moreover, sodium butyrate, a hydrolytic product of dibutyryl cyclic GMP, increases amylase release by increasing cyclic GMP in pancreas (Kapoor and Krishna, unpublished findings). The mechanism by which carbamylcholine and cholecystokinin octapeptide increase cyclic GMP is not clear, because these agents do not activate the guanylate cyclase in pancreas homogenates [28]. We have shown earlier that a perfect correlation exists between concentration response of various secretagogues on cyclic GMP increase and calcium efflux in pancreatic acinar cells [16, 17]. Thus it is tempting to speculate that calcium in some way causes the increase in cyclic GMP induced by these agents. Low concentrations of calcium are able to activate the enzyme in cell-free systems [28] but the significance of this activation in an intact cell is not clear. Indeed, it is possible that a mechanism exists by which calcium increases cyclic GMP, other than a direct activation of guanylate cyclase. For example, the increase in calcium efflux observed with these agents in pancreatic acinar cells may trigger other enzyme systems that change the redox potential in cells or increase arachidonic acid release; in turn, these changes may cause an increase in the formation of hydroxyl radicals which directly activate guanytate cyclase in the cell [29]. Whatever the mechanism by which various secretagogues increase cyclic GMP in the pancreatic acinar cells might be, it appears that cyclic GMP is intimately involved in the amylase secretion in pancreas. References 1 Palade, G.E. ( 1 9 5 9 ) F u n c t i o n a l changes in the s t r u c t u r e of cell c o m p o n e n t . In Subcellular particles ( H a y a s h i , T., ed.) p p . 6 4 - - 8 0 , R o n a l d Press, New Y o r k 2 E c k h o l m , R., Zelander, T. a n d Edlung~ Y. ( 1 9 6 2 ) J. U l t r a s t r u c t . Res. 7, 6 1 - - 7 2 3 I c h i k a w a , A. ( 1 9 6 5 ) J. Cell Biol. 24, 3 6 9 - - 3 8 5 4 J a m i s o n , J.D. a n d Palade, G.E. ( 1 9 6 7 ) J. Cell Biol. 3 4 , 5 7 7 - - 5 9 6 5 J a m i s o n , J.D. a n d Palade, G.E. ( 1 9 6 7 ) J. Cell Biol. 34, 5 9 7 - - 6 1 5 6 H a r p e r , A.A. ( 1 9 6 7 ) in H a n d b o o k of Physiol. 2: Sect. 6, (Code, C.F., ed.), pp. 9 6 9 - - 9 9 5 , A m e r i c a n Physiol. S o c i e t y , W a s h i n g t o n 7 S c h r a m m , M. ( 1 9 6 7 ) A n n u . Rev. Biochem. 36, 3 0 7 - - 3 2 0 8 J o r p e , J.E. a n d Mutt, V. ( 1 9 7 3 ) in Secretin, C h o l e c y s t o k i n i n , P a n c r e o z y m i n a n d gasterin ( J o r p e , J.E. a n d Mutt, V., eds.) pp. 1 - - 1 7 7 , Springer Verlag, New Y o r k 9 K u l k a , R.G. a n d S t e r n l i c h , E. ( 1 9 6 8 ) Proc. Natl. A c a d . ScL U.S. 71, 4 0 3 7 - - 4 0 4 1 10 R e d d e r s t a p , A.S. a n d Bonting, S.L. ( 1 9 6 9 ) Pflugens Arch. 3 1 3 , 6 2 - - 7 0 11 Morriset, J.A. a n d Webster, P.D. ( 1 9 7 1 ) Am. J. Physiol. 2 3 0 , 2 0 2 - - 2 0 8 12 Benz, L., Eckstein, B., M a t t h e w s , E.K. a n d William, J.A. ( 1 9 7 2 ) Brit. J. P h a r m a c o l . 46, 6 6 - - 7 7 13 Case, R.M. a n d S c a r t c h e r e d , T. ( 1 9 7 2 ) J. Physiol. 2 2 3 , 6 4 9 - - 6 6 7 14 Heisler, S., Fast~ D. a n d T e e n h o u s e , A. ( 1 9 7 2 ) Biochim. Biophys. A c t a 279, 5 6 1 - - 5 7 2 15 Berridge, M.J. ( 1 9 7 6 ) in A d v a n c e s in Cyclic N u c l e o t i d e R e s e a r c h , Vol. 6 ( G r e e n g a r d , P. a n d R o b i s o n , G.A., eds.), p p . 7 - - 9 8 , R a v e n Press, New Y o r k 16 F r a n d s e n , E.K., C h r i s t o p h e , J., Krishna, G. a n d G a r d n e r , J.D. ( 1 9 7 5 ) P h a r m a c o l o g i s t 17, 269 17 C h r i s t o p h e , J.P., F r a n d s e n , E.K., C o n l o n , T.P., Krishna, G. a n d G a r d n e r , J.D. ( 1 9 7 6 ) J. Biol. Chem. 251, 4640---4645 18 K a P o o r , C.L. a n d Krishna, G. ( 1 9 7 6 ) Fed. Proc. 35, 2 9 5 19 K a p o o r , C.L. a n d Krishna, G. ( 1 9 7 7 ) Science 1 9 6 , 1 0 0 3 - - 1 0 0 5
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2 0 H a y m o r i t s , A. a n d S c h e e l e , G . A . ( 1 9 7 6 ) P r o c . Natl. A c a d . Sci. U.S. 73, 1 5 6 - - 1 6 0 21 A l b a n o , J., B h o o l a , K . D . a n d H a r v e y , R . F . ( 1 9 7 6 ) N a t u r e 2 6 2 , 4 0 4 - - 4 0 7 2 2 G a r d n e r , J . D . , C o n l o n , T.P., K l a e v e m a n , H., A d a m s , T . D . a n d O m d e t t i , M.A. ( 1 9 7 5 ) J. Clin. Invest. 56,366--375 2 3 S t e i n e r , A . L . , P a r k e r , C.W. a n d K i p n i s , D.M. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 1 1 0 6 - - 1 1 1 3 2 4 S c h e e l e , G . A . a n d P a l a d e , G . E . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 2 6 6 0 - - 2 6 7 0 2 5 F r a n d s e n , E . K . a n d K r i s h n a , G. ( 1 9 7 6 ) Life Sci. 1 3 , 5 2 9 - - 5 4 2 2 6 R i n d e r k n e c h t , H., W i l d i n g , P. a n d H a v e r b a c k , B.J. ( 1 9 6 7 ) E x p e r i e n t i a 2 3 , 8 0 5 - - 8 0 7 2 7 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 6 - - 2 7 5 2 8 K a p o o r , C.L. a n d K r i s h n a , G. ( 1 9 7 8 ) in A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h , Vol. 9, ( a b s t r . ) R a v e n Press, N e w Y o r k , in p r e s s 2 9 M i t t a l , C . K . a n d M u r a d , F. ( 1 9 7 8 ) J. C y c l i c N u c l e o t i d e Res. 3 , 3 8 1 - - 3 9 1
