PfliJgers Archiv
Pflfigers Arch. 372, 6 9 - 7 6 (1977)
Etm~peanJournal of Physiology 9 by Springer-Verlag 1977
On the Secretagogue Effect of Dibutyryl Cyclic AMP in the Rat Exocrine Pancreas* H. BAUDUIN 1 **, C. STOCK z, J. F. LAUNAY 2, D. VINCENT 1, P. POTVLIEGE 3, and J. F. GRENIER / 1 Faculty of Medicine, Free University of Brussels, Laboratory of Biochemical Pharmacology, B-1000 Brussels, Belgium z Unit~ de Recherches 61 de I'INSERM de Biophysiopathologie de l'Intestin, F-67200 Strasbourg, France 3 Laboratory of Anatomical Pathology and Fondation Reine Elisabeth, Vrije Universiteit, B-1000 Brussels, Belgium
Summary. DbcAMP > 0.1 mM induces the discharge of exportable enzymes from rat pancreas fragments incubated in vitro. This effect is qualitatively similar to the action of physiological secretagogues acting via hormone receptors: 1) it is accompanied by the appearance of exocytotic images at the acinar cell apex; 2) it is energy dependent but energy supply is low while that required for the carbamylcholine or caerulein response is high and can only be afforded by oxidative phosphorylation; 3) it is calcium dependent, but no alteration of inward or outward calcium movement can be observed; 4) it is altered by agents known to disrupt the microfilamentous microtubular system [41]. However, the secretory response to DbcAMP is quantitatively less than that obtained with hormonal stimuli. A damaging effect of DbcAMP on pancreatic acinar cells is ruled out on histological and biochemical grounds: there is no significant leakage of LDH; protein synthesis, 2-deoxy-D-glucose and L-leucine uptake are unaltered. The secretagogue effect of DbcAMP is reversible, dose-related and specific. It is not mediated by neurotransmitter release or by interaction with their receptors. The evidence presented points to a direct * Part of this work has been presented in abstract from at the VIIIth Symposium of the European Pancreatic Club, Toulouse, France, October, 1975, 2 3 r d - 2 5 t h and at the Biochemical Society of Belgium [40] ** This work was partially carried out under contracts from the Ministbre de la Politique Scientifique within the framework of the Association E u r a t o m - University of Brussels - University of Pisa, and the Institut National de la Sant~ et de la Recherche M6dicale (France) Abbreviations: cAMP : Adenosine-3',5'-monophosphate, cyclic; DbcAMP: N6-2'-O-Dibutyryl-adenosine-3',5'-monophosphate, cyclic; DbcGMP : N2-2'-O-Dibutyryl-guanosine-3',5'-monophosphate, cyclic; Y-AMP: Adenosine-5'-monophosphate; TCA: Trichloracetic acid; ATP: Adenosine triphosphate; NADH: Nicotinamideadenine dinucleotide; Tris: Tris-(hydroxy-methyl) amino-methane; EGTA: Ethylene glycol-bis (/~ aminoethylether)-NN'-tetraacetic acid; LDH: Lactic dehydrogenase
interaction of DbcAMP on the pancreatic acinar cell and suggests the last step of the secretory cycle as the most probable site of action of the nucleotide derivative.
Key words: Dibutyryl cyclic AMP - Pancreas Rat-
Secretion.
INTRODUCTION It is well known that physiological stimuli (pancreozymin, caerulein, acetylcholine) induce the discharge of exportable enzymes in the exocrine pancreas. Dibutyryl cyclic adenosine monophosphate (DbcAMP) or 3',5'-adenosine cyclic monophosphate (cAMP) are generally recognized to have the same effect [2,16,27,29,33,37] though some authors [4,6, 21] have failed to observe any secretagogue action. However, the efficiency of these stimuli is low (3 times the control value) compared to that of hormones and cholinergics ( 5 - 7 times the control value). The intracellular mechanisms involved in pancreatic enzyme secretion are less clear. Ca z + ions have been implicated in stimulus-secretion coupling [8,19, 23, 26, 31,36,42] as well as in the secretagogue effect of DbcAMP [16, 37]. The involvement of cyclic AMP in the mediation of enzymes secretion [15,37] is disputed while it is well established in fluid secretion [6, 7, 25]. The relative unresponsiveness of the adenylate cyclase preparation to pancreozymin [24, 34], the unreliable effect Of the cAMP phosphodiesterase inhibitors on pancreatic enzyme secretion [2, 6, 29] and the failure of pancreozymin or caerulein to increase cAMP levels in isolated acinar cells [13,15] do not support the hypothesis that cAMP is a second messenger in stimulus secretion coupling.
70 However, the description o f a n a d e n y l a t e cyclase [34], a cyclic A M P p h o s p h o d i e s t e r a s e [35] a n d a cyclic A M P activated p r o t e i n kinase [9] in the pancreas, together with the ability of c h o l e c y s t o k i n i n - p a n c r e o zymin, caerulein a n d cholinergics to increase cyclic A M P levels in vitro a n d in vivo [7,11] suggest a role for cyclic A M P in the r e g u l a t i o n o f enzyme secretion. Since the i m p l i c a t i o n of c A M P in the a c t i o n of physiological stimuli is q u e s t i o n a b l e a n d c A M P a n d D b c A M P have dissociated effects in v a r i o u s tissues or cell types [18, 39], it was of great interest to investigate m o r e t h o r o u g h l y the secretagogue p r o p e r t y o f the n u c l e o t i d e derivative o n the exocrine pancreas. This secretagogue effect is c o m p a r e d to t h a t of u s u a l stimuli. T h e d a t a show that D b c A M P acts directly o n the p a n c r e a t i c a c i n a r cell, its m o s t p r o b a b l e site o f a c t i o n being the last step o f the secretory cycle, i.e. exocytosis.
METHODS Procedures for the preparation of the rat pancreatic pieces and their incubationin vitro have been given previously [1]. The incubation medium enriched by g-amino acids and D-glucose (10 raM) was buffered with bicarbonate (25 raM) at pH 7.4. The gas phase was 95 ~ Oz, 5 ~ CO2. Some experiments were carried out in medium buffered with Tris/HC1 (20 raM), pH 7.4. Secretion of digestiveenzymeswas monitored by the determination of the activity of amylase [10], chymotrypsinogen [32] or iipase [28] on aliquots from the incubation medium. The secretion was also checked by estimation of the radioactivity in the 5 ~ TCA precipitable material released in the medium after labelling of the exportable enzymesof the tissue. The exportable proteins were labeled according to the procedure of Jamieson and Palade [20]: eight pancreatic pieces from I single gland were preincubated for 10 min in leucine free medium. They were pulse labeled with L-[4,5-3H]leucine 5 gCi per ml (53 Ci/mmol) for 5 rain. The pieces were further incubated for 30 min in a chase medium containing cold leucine (800 nmol per ml). At the end of this period, considered as time 0 of the final incubation, the pieces were transferred to their respective flasks containing adequate incubation medium (4 ml). Aliquots of the medium (300 gl) were taken every 10 rain for the first 30 min, then every 15 rain for the last 90 rain. They were replaced by new medium of corresponding composition. DbcAMP was added at time 30 min of the final incubation. The activity of the TCA precipitable material was estimated after digestion of the precipitate by soluene 350 (250 gl per sample) overnight. Counting was performed in a Nuclear Chicago liquid scintillation counter (Mark II) using the scintillation:mixture Omn~fluor (New England Nuclear, Frankfurt, Germany) in toluene, 4 g per liter (10 ml per flask). 2-Deoxy-D-glucose uptake as well as calcium exchanges in the pancreatic gland in vitro were analyzed as previously reported [3]. In some experiments, calcium efflux was studied while secretion was concomittantly monitored by the enzymatic assay (amylase) as well as by the isotopic method. Calcium loading 4SCa5 ~tCi/ml (25 Ci/mol) was achieved during the chase in cold leucine medium; calcium concentration was 0.2 raM. The chase was extended to 40 min and follwed by two washes of 7.5 min in calcium free
Pflfigers Arch. 372 (1977) medium containing 0.5 mM EGTA before the final incubation took place. ATP content of incubated gland was estimated by the luciferine luciferase method of Stanley and Williams as previously reported [31. The lactic dehydrogenase (LDH) activity was determined by following the reduction of pyruvate by NADH; the mixture assay contained 50mM phosphate buffer, pH 7.5, 0.18 mM NADH and 0.6 mM sodium pyruvate; it was mixed in a quartz cuvette immediately placed in a Beckman D.B. spectrophotometer. The decrease in absorbance was recorded at 340 nm.
Electron Microscopy. Small pancreatic fragments were fixed in 2 glutaraldehyde buffered with 0A M sodium phosphate or 0.2 M sodium cacodylate, pH 7.3, and postfixed in 1 ~ osmium tetroxide in the same buffer. They were dehydrated in a graded sequence of alcohols and then embedded in araldite. Thin sections were cut with a Reichert OMU2 ultramicrotome, doubly stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron lrficroscope.
Materials. L-[4,5-3H] leucine and 45Ca were obtained from Amersham Bucks (England) and from C.E.A. (Saclay, France). Specific activities were respectively 53 Ci/mmol and 25 Ci/mmol; the label purity was greater than 98.5 ~. Dibutyryl 3',5'-cyclic AMP, monobutyryl 3',5'-cyclic AMP, dibutyryl 3',5'-cyclic GMP and 5'-AMP were purchased from Boehringer Pharma (Mannheim, Germany). Ionophore A 23187 was a generous gift from Eli Lilly (St Cloud, France). Dibenzamine was kindly supplied by R.I.T. (Genval, Belgium). Cyproheptadine was offered by Merck, Sharp and Dohme (Brussels, Belgium) and hexamethonium by A.P.B. (Brussels, Belgium). Haloperidol was fiom Jansen (Beerse, Belgimn). Atropine sulphate was a Federa product. Pancreozymin was a commercialpreparation of Cecekin (Vitrum Pharmaceuticals, Stockholm, Sweden). Carbamylcholine was purchased from K and K. (Plainview, New York, U.S.A.). All other chemicals were Merck products, analytical grade. Tap water was demineralized through a millipore milli-Ro system and milli-Ro-milli-Q system (Millipore); the calcium concentration was lower than 10-s M as checked by atomic absorption.
RESULTS
DbcAMP Stimulates Exportable Enzyme Release and Induces Exocytosis in the Pancreatic Acinar Cell D b c A M P > 0.1 m M stimulates the discharge of enzymes in the exocrine pancreas. T h e m a x i m a l stimulation, r a n g i n g between 2 to 4 times the basal secretory rate, is lower t h a n that o b t a i n e d with p a n c r e o z y m i n or c a r b a m y l c h o l i n e (Fig. 1). T h e release o f exportable enzymes (Fig. 2) as well as t h a t of r a d i o l a b e l e d " p r o teins" (Fig. 3) is steadily increased by D b c A M P 2 m M for at least 90 rain. I n glands treated for 90 m i n with D b c A M P 2 m M , the a c i n a r l u m i n a are enlarged, c o n v o l u t e d ' a n d generally filled with dense s e c r e t o i y m a t e r i a l ; exocytosis are f r e q u e n t l y observed. These u l t r a s t r u c t u r a l changes are similar to those observed in caerulein ]0 n g / m l or c a r b a m y l c h o l i n e 0.1 m M treated tissue
H. Bauduin et al. : DbcAMP and Pancreatic Secretion
71 %
It 150
20.
%~120
, CARBAMYLCHOLINE .................Ii PANCREOZYMIN
15
90 ~6o
10
'~ %
Dbc AMP
30
~o O O
0.01
0.02 0.001 0.1
0.2 0.01 1
2 0.1 10
Dbc AMP(mM) 1 Poncreozymin (U/ml) 100 Corbomylcholine (pM)
Fig. 1. Relationship between the concentration of carbamylcholine, pancreozymin and dibutyryl cyclic AMP and chymotrypsinogen release from the rat pancreas. Results are the means 4- S.E.M. of at least 6 experiments
80
~
Dbc AMP 2mM [ ca++] = 1.8 mM
70 6o
50
Dbc AMP 2mM [Ca ++] = OmM Control [Ca §247=1.8 mM
40 30
/> 0 ~ ' ~
~
15
50
415
i 60
i 75
~0
i 120
TIME
(rain.)
Fig. 3. Influence of DbcAMP on the release of exportable proteins from the rat pancreas. Exportable proteins were prelabeled by a pulse (5 rain) of L-[4,5-3H] leucine 1 gCi/ml followed by a chase of 30 rain in cold leucine medium (0.8 raM). After that, the pancreatic fragments were transferred to a medium of the same composition and incubated for 120 rain in absence (O) or in presence (I) of DbcAMP 2 mM. Addition of DbcAMP to the medium is indicated by the arrow. Release of 3H labeled TCA precipitable proteins is expressed in % of the activity of tissular proteins as calculated from the activity in the medium and in the tissue at the end of the incubation. Each value represents the mean _+ S.E.M. of 8 experiments
Control [;Ca ++] =OmM
20 10 ,
i
,
i
|
15
:50
45
60
75
|
90 time (min.)
Fig. 2. Influence of DbcAMP on amylase release from the rat pancreas. Effect of calcium depletion. Pancreatic fragments were preincubated for 2 x 15 min in EGTA 1 mM containing medium before a 90 rain final incubation without calcium (9 A) or with a calcium concentration of 1.8 mM (I, A). Each value is the mean 4- S.E.M. of 8 experiments
[3]. O n the c o n t r a r y , in n o n - s t i m u l a t e d c o n t r o l glands, the a c i n a r l u m i n a are e m p t y o r c o n t a i n e d a sparse a n d few m a t e r i a l while exocytosis are an e x c e p t i o n a l occurence.
The Secretagogue Properties of DbcAMP do not Result From an Indirect Interaction with the Pancreatic Acinar Cell a) The Acinar Cells were not Damaged by DbcAMP. T h e m o r p h o l o g i c a l integrity o f the g l a n d e x p o s e d to D b c A M P 2 m M for at least 90 rain, the occurence o f
a d o s e - r e s p o n s e r e l a t i o n s h i p (Fig. 1) a n d the reversibility o f the s e c r e t a g o g u e a c t i o n o f D b c A M P (Fig. 4) rule o u t a n y i m p o r t a n t a l t e r a t i o n o f the a c i n a r cell. M e t a b o l i c p a r a m e t e r s such as p r o t e i n synthesis were n o t affected by D b c A M P 2 m M . T h e i n c o r p o r a t i o n o f L-[4,5-3H] leucine into 5 % T C A p r e c i p i t a b l e m a terial was 84.3 + 6 % o f c o n t r o l , n = 12. T h e interm e d i a r y m e t a b o l i s m was u n a l t e r e d as s h o w n b y the oxygen u p t a k e , lactic acid f o r m a t i o n a n d A T P level (see below). T h e unsignificant l e a k a g e o f the cytop l a s m i c m a r k e r , lactic d e h y d r o g e n a s e , was n o t increased (94 __ 19 % o f control, n : 16) while t r a n s p o r t o f 2-deoxy-D-glucose (95 • 7 % o f control, n = 8) o r o f a m i n o acid such as leucine (102.1 __ 7.3 % o f control, n = 18) d i d n o t differ f r o m control. A d d i t i o n a l l y , n e i t h e r b u t y r i c acid (2 r a M ) , 5 ' - A M P (10 r a M ) n o r d i b u t y r y l cyclic G M P (2 m M ) increased the b a s a l release o f enzymes o r i n d u c e d u l t r a s t r u c t u r a l changes in the gland.
b) The Secretory Effect of DbcAMP was not Mediated through the Release of Neurotransmitters. A n t i m u s carinic ( a t r o p i n e sulphate), ~ - a n t i a d r e n e r g i c (dibenzamine), /~ a n t i a d r e n e r g i c ( p r o p r a n o l o l ) , a n t i n i c o t i n i c ( h e x a m e t h o n i u m ) as well as a n t i d o p a m i n e r g i c (halo-
72
Pfliigers Arch. 372 (1977)
UIIO0 mg
8~ 70
/o
9
500
50
~z400
40
500
.~200
20
IO0 0
45
75
120 time
(rnin.) 0
N
OX
N
§
Dbe N
4
OX
+
N+OXCarb. N
.i-
OX AMP Dbc Dbc Dbe AMP AMP AMP
+
Carb.
Fig. 4. Reversibility of DbcAMP induced chymotrypsinogen release from rat pancreas. Pancreatic fragments were incubated with DbcAMP 2 mM for the first 45 min. Then, after 2 brief washes, they were transferred to vials containing medium without DbcAMP; 30 min later, the fragments were reexposed to DbcAMP 2 mM for 45 min. Each graph represents the result from 1 experiment
Fig. 5. Influence of metabolic inhibitors on chymotrypsmogen release from rat pancreas incubated for 2 h in a Krebs-Ringer-Tris/ HC1 buffered medium. Results, expressed in % of control, are mean values • S.E.M. of 8 experiments. Control: 100 % = 16.3 • 2.5 U of chymotrypsinogen/100 mg/2 h. O: control, gas phase oxygen. N: gas phase nitrogen. OX: sodium oxamate 70 raM. Dbc AMP: dibutyryl cyclic AMP 0.4 mM. Carb: carbamylcholine 0. I mM
Table 1. Influence of inhibitors of cholinergic, adrenergic, nicotinic, dopaminergic and serotoninergic systems on the secretagogue effect of DbeAMP. Pancreatic pieces were incubated for 2 h at 37~C in the presence or the absence of DbcAMP 2 mM and pharmacological agents. Release of amylase is expressed in % of control • S.E.M. Number of experiments is given in brackets. Control 100% = 8 • 1 U/lOOmg/h. Statistical significance has been calculated (paired tests) versus the control value. P < 0.05
presence of atropine is the same, regardless of the low rate of basal secretion due to atropine treatment: it strengthens the evidence against an action of D b c A M P on the cholinergic receptor.
None Dibenzamine 5- 10-s M Propranolol 5- 10-5 M Atropine sulphate 5.10 -s M Hexamethonium 5- 10-5 M Haloperidol 5.10 -5 M Cyproheptadine 10-8 M 10-6 M 5-10 -bM
Control
DbcAMP 2 mM
100
224.1 • 32.0 (6)
86.1 • 22.7 (5) 85.5 • 9.0 (6)
172.5 4- 8.4 (6) 315.7 + 57.8 (6)
52.6 • 11.1 (6)* 230.2 + 39.2(6) 90.8 • 18.9 (5) 142.4 • 17.3 (4)
175.9• 3.6 (6) 280.3 • 77.4 (4)
80.4 + 9.8 (4) 193.1 4- 23.6 (4) 84.8 • 19.7 (4) 170.6• 18.7 (4) 178.1 • 29.0 (4)* 247.8 • 76.2 (4)
peridol) agents completely fail to alter the release of enzyme induced by D b c A M P (Table 1). The antiserotonin agent, cyproheptadine, has by itself a strong releasing effect in the 5 - 10 -5 M range on the exocrine pancreas. Lower concentration neither alter the spontaneous secretion of enzyme nor modify the response t o the nucleotide derivative. The significant depressive effect of atropine sulphate (5 9 10 -2 M) on basal secretion suggests that at least a part of this spontaneous secretion is under cholinergic tone. The magnitude of the maximal response to D b c A M P in
Evaluation of the Characteristics of DbcAMP Secretagogue Properties a) The Secretion Induced by DbcAMP is Energy Dependent. 1. Blockade of both oxidative phosphorylation (by anaerobiosis) and glycolysis (by sodium oxamate 70 m M ) abolishes the secretagogue effect of D b c A M P (Fig. 5). 2. Blockade of oxidative phosphorylation (by anaerobiosis or antimycin A 10 -5 M) or glycolysis (by sodium oxamate 70 raM) does not alter the secretory response to D b c A M P (Fig. 5). Anaerobiosis slightly decreases both control and D b c A M P stimulated enzyme release but the magnitude of the stimulation remains identical to control. 3. D b c A M P 2 m M did not alter the oxygen uptake (97 + 9 % of control, n = 6), lactic acid formation (88 • 10% of control, n = 16) nor the energy balance in the gland as shown by the A T P level (109 • 14 % of control, n = 8). Control values were respectively: oxygen uptake: 6.0 + 0.2 gmole/100 rag/ h, lactic acid formation:0.45 + 0.05 pmole/100 rag/h, A T P level: 0.36 + 0.06 ~tmole/g. 4. The maximal stimulation of enzyme release induced by carbamylcholine 1 0 - b M is 5 to 7 times the control value (Fig, i). Concomittantly, oxygen uptake increases (111.7 _+ 3.6% of control, n = 58) as well as
H. Bauduin et al. : DbcAMP and Pancreatic Secretion
73
45Ca EFFLUX (% of the load) ,oo
/
Ca**
OmM
,_,\
Dbc AMP 2raM
Ca**
,,-A
O.OSmM
~
Ca** 0.05 mM + Dbc AMP 2raM
o AMYLASE RELEASE (U/lOOmg) 100t
Time ~lOmin.
45Ca EFFLUX (% of the load)
100
OmM
50
AMYLASE RELEASE (U/]OOmg) 200
100
Fig. 6 Influence of DbcAMP on calcium efflux and amylase release from "calcium depleted" rat pancreas. Pancreatic fragments were incubated in medium containing calcium 0.2 raM, 45Ca 5 gCi/ml for 45 min, then washed 2 x 7.5 min in calcium free EGTA 0.5 mM supplemented medium. After that, fragments were exposed for 10 rain to ionophore A23187 10 -s M (io), then transferred to a calcium free medium ([Ca2§ ] < 10 -8 M) and incubated for 130 min. The arrow indicates the addition of DbcAMP 2 mM and/or calcium 0.05 raM. eSCa efflux is given in % of the load which was calculated from the residual activity in the tissue at the end of the incubation and the activity released in the medium. The results are representative of one experiment
(Fig. 5), i.e. to the level of the DbcAMP-response in similar condition. Under anaerobiosis the ATP level in the gland is 8 _+ 1.1% of control, n = 8; the lactic acid formation is doubled though not further altered by DbcAMP or carbamylcholine treatment.
b) The Secretion Induced by DbcAMP is Calcium Dependent. The secretory response to DbcAMP 2 mM is reduced in calcium free medium (Fig. 2). A more complete depletion of calcium in the gland, achieved by a wash with ionophore A23 187 in calcium free ( < 10 -8 M) medium, is a perequisite to suppress the secretagogue effect of DbcAMP (Fig. 6). It is restored by increasing the extracellular calcium concentration to 0.05 mM. However, in absence of DbcAMP, such low concentration of calcium has no secretory effect (Fig. 6). DbcAMP 2 mM does not alter the movement of 45Ca in or out of the pancreas (Fig. 7) while carbamylcholine 0.1 mM increased the ~SCa efflux [3].
Time ~10 rain. Fig. 7. Influence of DbcAMP on calcium efflux and amylase release from rat pancreas. Effect of calcium concentration in the incubation medium. Pancreatic fragments were incubated in medium containing calcium 0.2 mM, r 5 gCi/ml for 45 min, then washed 2 x 7.5 rain in calcium free EGTA 0.5 mM supplemented medium. After that, they were transferred to calcium free medimn (0 mM) or medium containing calcium (0.1 mM) and incubated for 130 min. The arrow indicates the addition of DbcAMP 2 mM. eSCa efflux is given in % of the load (see Fig. 6). The results are representative of 1 experiment
the lactic acid formation (125 _+ 18% of control, n = 19); however the energy balance falls to 73 __ 6 % of control, n = 18. Blockade of oxidative phosphorylation by anaerobiosis reduced the stimulation from 5 to 7 times the control to 21/2 times the control
DISCUSSION The secretagogue properties of DbcAMP in the exocrine pancreas have been observed in several animal species including the rat [2,16, 29], rabbit [33], cat [37] and mouse [27]. The guinea pig pancrease apparently failed to respond to the nucleotide derivative [4, 21 ]. The secretagogue action of DbcAMP cannot be explained by a damaging effect on pancreatic acinar cells as suggested by Kempen [25]. Indeed, there is no alteration neither of the intermediary and protein metabolism nor ofhexose and leucine transport under DbcAMP treatment. The morphological integrity, the absence of significant release of lactic dehydrogenase, t h e reversibility of the nucleotide derivative
74 action plead against a damaging effect of DbcAMP on the acinar cell. The increase of L D H leakage noted by Kempen [25] was probably due to the use of slices instead of pieces of pancreatic tissue. The specificity of the action of the nucleotide derivative is further supported by the existence of a dose-response relationship and the failure of DbcGMP, 5'-AMP and butyric acid to induce any enzyme release. The secretory response to DbcAMP could have been mediated through the release of neurotransmitters. Cholinergics are well known stimulators of enzyme release from the pancreas [19]. Epinephrine [27] and isoproterenol [30] are also able to induce secretion of enzymes from the pancreas at least in some species while dopamine has been involved in hydromineral secretion [12]. Pharmacological agents which antagonize the respective transmitters at the level of their receptors fail to depress the DbcAMPresponse. Thus the present investigation affords strong evidence for a direct interaction between the nucleotide derivative and the pancreatic cell. In order to explain why DbcAMP cannot achieve a full secretory response, comparable to that elicited by physiological secretagogues, it was compulsory to investigate, in both cases, the sequence of steps which result in stimulus-secretion coupling in the pancreatic acinar cell. As observed in glands stimulated by physiological secretagogues DbcAMP induces the appearance of numerous exocytotic pictures at the apex of the acinar cell. This process, known to achieve the discharge of zymogen granules is still considered as the main pathway of the release of digestive enzymes from the pancreas. The secretagogue effect of physiological stimuli, as well as that of DbcAMP requires an energy supply. Indeed, the effect is abolished by the simultaneous addition of agents blocking oxidative phosphorylation and glycolysis. Three steps of the secretory cycle, protein synthesis, transport of secretory material through the Golgi area and discharge into the acinar lumen are endergonic processes [20, 21]. DbcAMP has been reported to interfere with protein synthesis, amino acid transport and amino acid activation. However, the secretory effects of DbcAMP in the exocrine pancreas cannot be linked to protein synthesis. Secretion is independent of protein synthesis [21] and the secretagogue activity of DbcAMP does not require any lag time. Moreover, the nucleotide derivative does not alter the incorporation of labeled leucine into 5 ~ TCA precipitable proteins and the amino acid radioactivity in the TCA soluble material remains unchanged.
Pfi0.gersArch. 372 (1977) The energy requirements of the 2 other endergonic steps of the secretory cycle are different in the presence of DbcAMP and physiological secretagogues. Glycolysis, which accounts for 15 ~ of the immediately available energy in normal glands [1] can fully support the secretion primed by DbcAMP. Indeed, under anaerobiosis, the maximal secretory response to DbcAMP is maintained while that to carbamylcholine is reduced. These observations suggest that the pancreatic response to physiological secretagogues requires more energy supply than the response to DbcAMP; however, they do not indicate which one of the endergonic steps of the secretory cycle can explain this difference. According to Jamieson and Palade [22] and Singh and Webster [38], carbamylcholine and pancreozymin did not alter the in vitro transport of the secretory material through the Golgi area but stimulate the release of exportable proteins from zymogen granules. This implies 1. a faster migration of these granules from their site of packaging towards the acinar lumen and 2. a more rapid discharge of their content into the acinar lumen. Considering that the same amount of enzyme is discharged under anaerobiosis by DbcAMP and cholinergics, and that ultrastructural examination shows preservation of exocytosis in both cases (unpublished data), the intracellular transport of zymogen granules towards the apical cell membrane would be the step requiring the greater supply of energy while exocytosis can still be stimulated when the energetic supply is low. Thus it is suggested that exocytosis may be the most probable site of action of the nucleotide derivative. In the exocrine pancreas the secretagogue effect of physiological stimuli requires calcium in the environment [19, 23, 31,36, 42]. However, intracellular calcium, probably through redistribution, can trigger this secretory function for some time. The present investigation confirms the calcium dependence of the secretory response to DbcAMP already reported by Heisler et al. [16,17] and Schulz [37]. The importance of the interaction of DbcAMP with calcium has been further emphasized in very severely calcium depleted glands which were permeated to the ion by two EGTA washes followed by ionophore A23 187 treatment. In those conditions, a concentration of calcium too low to trigger any secretion was made effective by DbcAMP. Interaction between calcium and cAMP in the regulation of several cell functions is well known [5]. Also the ratio cAMP to calcium has been reported to be a determining factor in the modulation of zymogen granule discharge through the alteration of the dynamic state of the microtubules [14]. As it has been shown in the secretory response to hormones and
H. Bauduin et al. : DbcAMP and Pancreatic Secretion
cholinergics [3,43], microtubules and microfilaments have been involved in the secretagogue action of DbcAMP [41]. In peculiar, when microtubules have been disrupted by vinblastine, the maximal secretory response to DbcAMP reaches the same level than that obtained normally after stimulation with physiological secretagogues. Considering that this potentiation effect is 1. calcium dependent, 2. inhibited by agents which block the oxidative phosphorylation and 3. inhibited by the cytochalasin B, an agent known to disrupt microfilaments, it seems likely that DbcAMP act on the later step of the secretory cycle in the acinar cell. We thank Dr. C. Koehl for the analysis of calcium and Mrs. E. Schell-Frederick for her critical reading of the manuscript. The devoted collaboration of Mrs. M. Bourguignon is gratefully acknowledged.
REFERENCES 1. Bauduin, H., Colin, M., Dumont, J. E. : Energy sources for protein synthesis and enzymatic secretion in rat pancreas in vitro. Biochim. Biophys. Acta 174, 722-733 (1969) 2. Bauduin, H., Rochus, L., Vincent, D., Dumont, J. E. : Role of cyclic 3',5'-AMP in the action of physiological secretagogues on the metabolism of rat pancreas in vitro. Biochim. Biophys. Acta 252, 171 - 183 (1971) 3. Bauduin, H., Stock, C., Vincent, D., Grenier, J. F. : Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B. J. Cell Biol. 66, 165-181 (1975) 4. Benz, L., Eckstein, B., Matthews, E. K., Williams, J. A. : Control of pancreatic amylase release in vitro: effects of ions, cyclic AMP and colchicine. Br. J. Pharmacol. 46, 6 6 - 7 7 (1972) 5. Berridge, M. J.: The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv. Cyclic Nucleotide Res. 6, 1 - 9 8 (1976) 6. Case, R. M., Scratcherd, T. : The actions of dibutyryl cyclic adenosine 3',5'-monophosphate and methylxanthines on pancreatic exocrine secretion. J. Physiol. (Lond.) 223, 649--667 (1972) 7. Case, R. M., Johnson, M., Scratcherd, T., Sherratt, H. S. A.: Cyclic adenosine 3',5'-monophosphate concentration in the pancreas following stimulation by secretin, cholecystokininpancreozymin and acetylcholine. J. Physiol. (Lond.) 223, 6 6 9 - 684 (1972) 8. Case, R. M., Clausen, T.: The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas. J. Physiol. (Lond.) 235, 75--102 (1973) 9. Cenatiempo, Y., Mangeat, P., Marchis-Mouren, G. : Purification and properties of cyclic AMP dependent and independent protein kinases from rat pancreas. Biochimie 57, 865-873 (1975) 10. Danielsson, A.: Technique for measuring amylase secretion from pieces of mouse pancreas. Anal. Biochem. 59, 220-234 (1974) I 1. Deschodt-Lanckman, M., Robberecht, P., De Neef, P., Labrie, F., Christophe, J.: In vitro interactions of gastrointestinal hormones on cyclic adenosine 3',5'-monophosphate levels and amylase output in the rat pancreas. Gastroent. 68, 3 1 8 325 (1975)
75 12. Fukuta, Y., Iwatsuki, K., Takeuchi, O., Hashimoto, K. : Secretin-like activity of dopamine on canine pancreatic secretion. Tohoku J. Exp. Med. 108, 353-360 (1972) 13. Gardner, J. D., Conlon, T. P., Adams, T. D. : Cyclic AMP in pancreatic acinar cells. Effects of gastrointestinal hormones. Gastroent. 70, 2 9 - 3 5 (1976) 14. Gillespie, E.: Microtubules, cyclic AMP, calcium and secretion. Ann. New York Acad. Sci. 253, 771-779 (1975) 15. Haymovits, A., Scheele, G. A. : Cellular cyclic nucleotides and enzyme secretion in the pancreatic acinar cell. Proc. Natl. Acad. Sci. U.S.A. 73, 156-160 (1976) 16. Heisler, S., Fast, D., Tenenhouse, A. : Role of Ca 2+ and cyclic AMP in protein secretion from rat exocrine pancreas. Biochim. Biophys. Acta 279, 5 6 1 - 572 0972) 17. Heisler, S. : Calcium efflux and secretion of e-amylase from rat pancreas. Br. J. Pharmacol. 52, 387-392 (1974) 18. Hilz, H., Tarnowski, W. : Opposite effects of cyclic AMP and its dibutyryl derivative on glycogen levels in Hela cells. Biochem. Biophys. Res. Commun. 40, 973-98/(1970) 19. Hokin, L. E.: Effects of calcium omission on acetylcholinestimulated amylase secretion and phospholipid synthesis in pigeon pancreas slices. Biochim. Biophys. Acta 115, 219-221 (1966) 20. Jamieson, J. D., Palade, G. E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J. Cell Biol. 39, 589-603 (1968) 21. Jamieson, J. D., Palade, G. E. : Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrine cells. Metabolic studies. J. Cell Biol. 48, 503-522 (1971) 22. Jamieson, J. D., Palade, G. E. : Synthesis, intracellular transport and discharge of secretory proteins in stimulated pancreatic exocrine cells. J. Cell Biol. 50, 135-158 (1971) 23. Kanno, T.: Calcium-dependent amylase release and electrophysiological measurements in cells of the pancreas. J. Physiol. (Lond.) 226, 353-371 (1972) 24. Kempen, H. J. M., De Pont, J. J. H. H. M., Bonting, J. L. : Rat pancreas adenylate cyclase. II. Inactivation and protection of its hormone receptor sites. Biochim. Biophys. Acta 370, 5 7 3 584 (1974) 25. Kempen, H. J. M. : Role of cyclic AMP in exocrine pancreatic secretion. Thesis. Nijmegen, Netherlands 1976 26. Kondo, S., Schulz, I. : Calcium ion uptake in isolated pancreas cells induced by secretagogues. Biochim. Biophys. Acta 419, 7 6 - 9 2 (1976) 27. Kulka, R. G., Sternlicht, E. : Enzyme secretion in mouse pancreas mediated by adenosine 3',5'-cyclic phosphate and inhibited by adenosine 3'-phosphate. Proc. Natl. Acad. Sci. U.S.A. 61, 1123-1128 (1968) 28. Marchis-Mouren, G., Sarda, L., Desnuelle, P. : Purification of hog pancreatic lipase. Arch. Biochem. Biophys. 83, 309-319 (1959) 29. Morisset, J. A., Webster, P. D. : In vitro and in vivo effects of pancreozymin, urecholine and cyclic AMP on rat pancreas. Am. J. Physiol. 230, 202-208 (1971) 30. Pederson, R., Schulz, I. : The effect of isoproterenol on enzyme secretion from the isolated perfused cat pancreas. Biochim. Biophys. Acta 338, 440-446 (1974) 31. Petersen, O. H., Ueda, N. : Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling. J. Physiol. (Lond.) 254, 583-606 (1976) 32. Reboud, J. P., Ben Abdeljlil, A., Desnuelle, P. : Variations de la teneur en enzymes du pancr6as de rat en fonction de la composition des r6gimes. Biochim. Biophys. Acta. 58, 3 2 6 337 (1962) 33. Ridderstapp, A. S., Bonting, S. L. : Cyclic AMP and enzyme secretion by the isolated rabbit pancreas. Pfltigers Arch. 313, 6 2 - 70 (1969)
76 34. Rutten, W. J., De Pont, J. J. H. H. M., Bonting, S. L. : Adenylate cyclase in the rat pancreas; properties and stimulation by hormones. Biochim. Biophys. Acta 274, 201-213 (1972) 35. Rutten, W. J., Schoot, B. M., De Pont, J. J. H. H. M., Bonting, S.L.: Adenosine 3',5'-monophosphate phosphodiesterase in rat pancreas. Biochim. Biophys. Acta 315, 384-393 (1973) 36. Schreurs, V. V. A. M., Swarts, H, G. P., De Pont, J. J. H. H. M., Bonting, S. L. : Role of calcium in exocrine pancreatic secretion. II. Comparison of the effects of carbachol and the ionophore A-23 187 on enzyme secretion and calcium movements in rabbit pancreas. Biochim. Biophys. Acta 419, 320-330 (1976) 37. Schulz, I. : The role of extracellular Ca 2 + and cyclic nucleotides in the mechanism of enzyme secretion from the cat pancreas. Pfl~igers Arch. 360, 165-181 (1975) 38. Singh, M., Webster, P. O. : Effect of hormones on pancreatic macromolecular transport. Gastroent. 68, 1536-1542 (1975) 39. Solomon, S. S., Brush, J. S., Kitabchi, A. E.: Divergent biological effects of adenosine and dibutyryl adenosine 3',5'-
Pfltigers Arch. 372 (1977)
40.
41.
42.
43.
monophosphate on the isolated fat cell. Science 169, 3 8 7 388 (1970) Stock, C , Vincent, D., Bauduin, H.: A p r o p o s de Faction s6cr6tagogue du d6riv6 dibutyryl6 de l'addnosine monophosphate cyclique sur le paner6as exocrine du rat. Arch. Int. Physiol. Biochim. 84, 348-350 (1976) Stock, C., Launay, J. F., Grenier, J. F., Bauduin, H. : Vinblastine potentiates the secretagogue action of dibutyryl cyclic AMP on the exocrine pancreas. Biochem. Biophys. Res. Commun. 76, 217-223 (1977) Williams, J. A., Lee, M. : Pancreatic acinar cells: use of a Ca 2+ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling. Biochem. Biophys. Res. Commun. 60, 542-548 (1974) Williams, J. A., Lee, M. : Microtubules and pancreatic amylase release by mouse pancreas in vitro. J. Cell Biol. 71, 795-806 (1976)
Received May 19, 1977
Pflfigers Arch. 372, 6 9 - 7 6 (1977)
Etm~peanJournal of Physiology 9 by Springer-Verlag 1977
On the Secretagogue Effect of Dibutyryl Cyclic AMP in the Rat Exocrine Pancreas* H. BAUDUIN 1 **, C. STOCK z, J. F. LAUNAY 2, D. VINCENT 1, P. POTVLIEGE 3, and J. F. GRENIER / 1 Faculty of Medicine, Free University of Brussels, Laboratory of Biochemical Pharmacology, B-1000 Brussels, Belgium z Unit~ de Recherches 61 de I'INSERM de Biophysiopathologie de l'Intestin, F-67200 Strasbourg, France 3 Laboratory of Anatomical Pathology and Fondation Reine Elisabeth, Vrije Universiteit, B-1000 Brussels, Belgium
Summary. DbcAMP > 0.1 mM induces the discharge of exportable enzymes from rat pancreas fragments incubated in vitro. This effect is qualitatively similar to the action of physiological secretagogues acting via hormone receptors: 1) it is accompanied by the appearance of exocytotic images at the acinar cell apex; 2) it is energy dependent but energy supply is low while that required for the carbamylcholine or caerulein response is high and can only be afforded by oxidative phosphorylation; 3) it is calcium dependent, but no alteration of inward or outward calcium movement can be observed; 4) it is altered by agents known to disrupt the microfilamentous microtubular system [41]. However, the secretory response to DbcAMP is quantitatively less than that obtained with hormonal stimuli. A damaging effect of DbcAMP on pancreatic acinar cells is ruled out on histological and biochemical grounds: there is no significant leakage of LDH; protein synthesis, 2-deoxy-D-glucose and L-leucine uptake are unaltered. The secretagogue effect of DbcAMP is reversible, dose-related and specific. It is not mediated by neurotransmitter release or by interaction with their receptors. The evidence presented points to a direct * Part of this work has been presented in abstract from at the VIIIth Symposium of the European Pancreatic Club, Toulouse, France, October, 1975, 2 3 r d - 2 5 t h and at the Biochemical Society of Belgium [40] ** This work was partially carried out under contracts from the Ministbre de la Politique Scientifique within the framework of the Association E u r a t o m - University of Brussels - University of Pisa, and the Institut National de la Sant~ et de la Recherche M6dicale (France) Abbreviations: cAMP : Adenosine-3',5'-monophosphate, cyclic; DbcAMP: N6-2'-O-Dibutyryl-adenosine-3',5'-monophosphate, cyclic; DbcGMP : N2-2'-O-Dibutyryl-guanosine-3',5'-monophosphate, cyclic; Y-AMP: Adenosine-5'-monophosphate; TCA: Trichloracetic acid; ATP: Adenosine triphosphate; NADH: Nicotinamideadenine dinucleotide; Tris: Tris-(hydroxy-methyl) amino-methane; EGTA: Ethylene glycol-bis (/~ aminoethylether)-NN'-tetraacetic acid; LDH: Lactic dehydrogenase
interaction of DbcAMP on the pancreatic acinar cell and suggests the last step of the secretory cycle as the most probable site of action of the nucleotide derivative.
Key words: Dibutyryl cyclic AMP - Pancreas Rat-
Secretion.
INTRODUCTION It is well known that physiological stimuli (pancreozymin, caerulein, acetylcholine) induce the discharge of exportable enzymes in the exocrine pancreas. Dibutyryl cyclic adenosine monophosphate (DbcAMP) or 3',5'-adenosine cyclic monophosphate (cAMP) are generally recognized to have the same effect [2,16,27,29,33,37] though some authors [4,6, 21] have failed to observe any secretagogue action. However, the efficiency of these stimuli is low (3 times the control value) compared to that of hormones and cholinergics ( 5 - 7 times the control value). The intracellular mechanisms involved in pancreatic enzyme secretion are less clear. Ca z + ions have been implicated in stimulus-secretion coupling [8,19, 23, 26, 31,36,42] as well as in the secretagogue effect of DbcAMP [16, 37]. The involvement of cyclic AMP in the mediation of enzymes secretion [15,37] is disputed while it is well established in fluid secretion [6, 7, 25]. The relative unresponsiveness of the adenylate cyclase preparation to pancreozymin [24, 34], the unreliable effect Of the cAMP phosphodiesterase inhibitors on pancreatic enzyme secretion [2, 6, 29] and the failure of pancreozymin or caerulein to increase cAMP levels in isolated acinar cells [13,15] do not support the hypothesis that cAMP is a second messenger in stimulus secretion coupling.
70 However, the description o f a n a d e n y l a t e cyclase [34], a cyclic A M P p h o s p h o d i e s t e r a s e [35] a n d a cyclic A M P activated p r o t e i n kinase [9] in the pancreas, together with the ability of c h o l e c y s t o k i n i n - p a n c r e o zymin, caerulein a n d cholinergics to increase cyclic A M P levels in vitro a n d in vivo [7,11] suggest a role for cyclic A M P in the r e g u l a t i o n o f enzyme secretion. Since the i m p l i c a t i o n of c A M P in the a c t i o n of physiological stimuli is q u e s t i o n a b l e a n d c A M P a n d D b c A M P have dissociated effects in v a r i o u s tissues or cell types [18, 39], it was of great interest to investigate m o r e t h o r o u g h l y the secretagogue p r o p e r t y o f the n u c l e o t i d e derivative o n the exocrine pancreas. This secretagogue effect is c o m p a r e d to t h a t of u s u a l stimuli. T h e d a t a show that D b c A M P acts directly o n the p a n c r e a t i c a c i n a r cell, its m o s t p r o b a b l e site o f a c t i o n being the last step o f the secretory cycle, i.e. exocytosis.
METHODS Procedures for the preparation of the rat pancreatic pieces and their incubationin vitro have been given previously [1]. The incubation medium enriched by g-amino acids and D-glucose (10 raM) was buffered with bicarbonate (25 raM) at pH 7.4. The gas phase was 95 ~ Oz, 5 ~ CO2. Some experiments were carried out in medium buffered with Tris/HC1 (20 raM), pH 7.4. Secretion of digestiveenzymeswas monitored by the determination of the activity of amylase [10], chymotrypsinogen [32] or iipase [28] on aliquots from the incubation medium. The secretion was also checked by estimation of the radioactivity in the 5 ~ TCA precipitable material released in the medium after labelling of the exportable enzymesof the tissue. The exportable proteins were labeled according to the procedure of Jamieson and Palade [20]: eight pancreatic pieces from I single gland were preincubated for 10 min in leucine free medium. They were pulse labeled with L-[4,5-3H]leucine 5 gCi per ml (53 Ci/mmol) for 5 rain. The pieces were further incubated for 30 min in a chase medium containing cold leucine (800 nmol per ml). At the end of this period, considered as time 0 of the final incubation, the pieces were transferred to their respective flasks containing adequate incubation medium (4 ml). Aliquots of the medium (300 gl) were taken every 10 rain for the first 30 min, then every 15 rain for the last 90 rain. They were replaced by new medium of corresponding composition. DbcAMP was added at time 30 min of the final incubation. The activity of the TCA precipitable material was estimated after digestion of the precipitate by soluene 350 (250 gl per sample) overnight. Counting was performed in a Nuclear Chicago liquid scintillation counter (Mark II) using the scintillation:mixture Omn~fluor (New England Nuclear, Frankfurt, Germany) in toluene, 4 g per liter (10 ml per flask). 2-Deoxy-D-glucose uptake as well as calcium exchanges in the pancreatic gland in vitro were analyzed as previously reported [3]. In some experiments, calcium efflux was studied while secretion was concomittantly monitored by the enzymatic assay (amylase) as well as by the isotopic method. Calcium loading 4SCa5 ~tCi/ml (25 Ci/mol) was achieved during the chase in cold leucine medium; calcium concentration was 0.2 raM. The chase was extended to 40 min and follwed by two washes of 7.5 min in calcium free
Pflfigers Arch. 372 (1977) medium containing 0.5 mM EGTA before the final incubation took place. ATP content of incubated gland was estimated by the luciferine luciferase method of Stanley and Williams as previously reported [31. The lactic dehydrogenase (LDH) activity was determined by following the reduction of pyruvate by NADH; the mixture assay contained 50mM phosphate buffer, pH 7.5, 0.18 mM NADH and 0.6 mM sodium pyruvate; it was mixed in a quartz cuvette immediately placed in a Beckman D.B. spectrophotometer. The decrease in absorbance was recorded at 340 nm.
Electron Microscopy. Small pancreatic fragments were fixed in 2 glutaraldehyde buffered with 0A M sodium phosphate or 0.2 M sodium cacodylate, pH 7.3, and postfixed in 1 ~ osmium tetroxide in the same buffer. They were dehydrated in a graded sequence of alcohols and then embedded in araldite. Thin sections were cut with a Reichert OMU2 ultramicrotome, doubly stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron lrficroscope.
Materials. L-[4,5-3H] leucine and 45Ca were obtained from Amersham Bucks (England) and from C.E.A. (Saclay, France). Specific activities were respectively 53 Ci/mmol and 25 Ci/mmol; the label purity was greater than 98.5 ~. Dibutyryl 3',5'-cyclic AMP, monobutyryl 3',5'-cyclic AMP, dibutyryl 3',5'-cyclic GMP and 5'-AMP were purchased from Boehringer Pharma (Mannheim, Germany). Ionophore A 23187 was a generous gift from Eli Lilly (St Cloud, France). Dibenzamine was kindly supplied by R.I.T. (Genval, Belgium). Cyproheptadine was offered by Merck, Sharp and Dohme (Brussels, Belgium) and hexamethonium by A.P.B. (Brussels, Belgium). Haloperidol was fiom Jansen (Beerse, Belgimn). Atropine sulphate was a Federa product. Pancreozymin was a commercialpreparation of Cecekin (Vitrum Pharmaceuticals, Stockholm, Sweden). Carbamylcholine was purchased from K and K. (Plainview, New York, U.S.A.). All other chemicals were Merck products, analytical grade. Tap water was demineralized through a millipore milli-Ro system and milli-Ro-milli-Q system (Millipore); the calcium concentration was lower than 10-s M as checked by atomic absorption.
RESULTS
DbcAMP Stimulates Exportable Enzyme Release and Induces Exocytosis in the Pancreatic Acinar Cell D b c A M P > 0.1 m M stimulates the discharge of enzymes in the exocrine pancreas. T h e m a x i m a l stimulation, r a n g i n g between 2 to 4 times the basal secretory rate, is lower t h a n that o b t a i n e d with p a n c r e o z y m i n or c a r b a m y l c h o l i n e (Fig. 1). T h e release o f exportable enzymes (Fig. 2) as well as t h a t of r a d i o l a b e l e d " p r o teins" (Fig. 3) is steadily increased by D b c A M P 2 m M for at least 90 rain. I n glands treated for 90 m i n with D b c A M P 2 m M , the a c i n a r l u m i n a are enlarged, c o n v o l u t e d ' a n d generally filled with dense s e c r e t o i y m a t e r i a l ; exocytosis are f r e q u e n t l y observed. These u l t r a s t r u c t u r a l changes are similar to those observed in caerulein ]0 n g / m l or c a r b a m y l c h o l i n e 0.1 m M treated tissue
H. Bauduin et al. : DbcAMP and Pancreatic Secretion
71 %
It 150
20.
%~120
, CARBAMYLCHOLINE .................Ii PANCREOZYMIN
15
90 ~6o
10
'~ %
Dbc AMP
30
~o O O
0.01
0.02 0.001 0.1
0.2 0.01 1
2 0.1 10
Dbc AMP(mM) 1 Poncreozymin (U/ml) 100 Corbomylcholine (pM)
Fig. 1. Relationship between the concentration of carbamylcholine, pancreozymin and dibutyryl cyclic AMP and chymotrypsinogen release from the rat pancreas. Results are the means 4- S.E.M. of at least 6 experiments
80
~
Dbc AMP 2mM [ ca++] = 1.8 mM
70 6o
50
Dbc AMP 2mM [Ca ++] = OmM Control [Ca §247=1.8 mM
40 30
/> 0 ~ ' ~
~
15
50
415
i 60
i 75
~0
i 120
TIME
(rain.)
Fig. 3. Influence of DbcAMP on the release of exportable proteins from the rat pancreas. Exportable proteins were prelabeled by a pulse (5 rain) of L-[4,5-3H] leucine 1 gCi/ml followed by a chase of 30 rain in cold leucine medium (0.8 raM). After that, the pancreatic fragments were transferred to a medium of the same composition and incubated for 120 rain in absence (O) or in presence (I) of DbcAMP 2 mM. Addition of DbcAMP to the medium is indicated by the arrow. Release of 3H labeled TCA precipitable proteins is expressed in % of the activity of tissular proteins as calculated from the activity in the medium and in the tissue at the end of the incubation. Each value represents the mean _+ S.E.M. of 8 experiments
Control [;Ca ++] =OmM
20 10 ,
i
,
i
|
15
:50
45
60
75
|
90 time (min.)
Fig. 2. Influence of DbcAMP on amylase release from the rat pancreas. Effect of calcium depletion. Pancreatic fragments were preincubated for 2 x 15 min in EGTA 1 mM containing medium before a 90 rain final incubation without calcium (9 A) or with a calcium concentration of 1.8 mM (I, A). Each value is the mean 4- S.E.M. of 8 experiments
[3]. O n the c o n t r a r y , in n o n - s t i m u l a t e d c o n t r o l glands, the a c i n a r l u m i n a are e m p t y o r c o n t a i n e d a sparse a n d few m a t e r i a l while exocytosis are an e x c e p t i o n a l occurence.
The Secretagogue Properties of DbcAMP do not Result From an Indirect Interaction with the Pancreatic Acinar Cell a) The Acinar Cells were not Damaged by DbcAMP. T h e m o r p h o l o g i c a l integrity o f the g l a n d e x p o s e d to D b c A M P 2 m M for at least 90 rain, the occurence o f
a d o s e - r e s p o n s e r e l a t i o n s h i p (Fig. 1) a n d the reversibility o f the s e c r e t a g o g u e a c t i o n o f D b c A M P (Fig. 4) rule o u t a n y i m p o r t a n t a l t e r a t i o n o f the a c i n a r cell. M e t a b o l i c p a r a m e t e r s such as p r o t e i n synthesis were n o t affected by D b c A M P 2 m M . T h e i n c o r p o r a t i o n o f L-[4,5-3H] leucine into 5 % T C A p r e c i p i t a b l e m a terial was 84.3 + 6 % o f c o n t r o l , n = 12. T h e interm e d i a r y m e t a b o l i s m was u n a l t e r e d as s h o w n b y the oxygen u p t a k e , lactic acid f o r m a t i o n a n d A T P level (see below). T h e unsignificant l e a k a g e o f the cytop l a s m i c m a r k e r , lactic d e h y d r o g e n a s e , was n o t increased (94 __ 19 % o f control, n : 16) while t r a n s p o r t o f 2-deoxy-D-glucose (95 • 7 % o f control, n = 8) o r o f a m i n o acid such as leucine (102.1 __ 7.3 % o f control, n = 18) d i d n o t differ f r o m control. A d d i t i o n a l l y , n e i t h e r b u t y r i c acid (2 r a M ) , 5 ' - A M P (10 r a M ) n o r d i b u t y r y l cyclic G M P (2 m M ) increased the b a s a l release o f enzymes o r i n d u c e d u l t r a s t r u c t u r a l changes in the gland.
b) The Secretory Effect of DbcAMP was not Mediated through the Release of Neurotransmitters. A n t i m u s carinic ( a t r o p i n e sulphate), ~ - a n t i a d r e n e r g i c (dibenzamine), /~ a n t i a d r e n e r g i c ( p r o p r a n o l o l ) , a n t i n i c o t i n i c ( h e x a m e t h o n i u m ) as well as a n t i d o p a m i n e r g i c (halo-
72
Pfliigers Arch. 372 (1977)
UIIO0 mg
8~ 70
/o
9
500
50
~z400
40
500
.~200
20
IO0 0
45
75
120 time
(rnin.) 0
N
OX
N
§
Dbe N
4
OX
+
N+OXCarb. N
.i-
OX AMP Dbc Dbc Dbe AMP AMP AMP
+
Carb.
Fig. 4. Reversibility of DbcAMP induced chymotrypsinogen release from rat pancreas. Pancreatic fragments were incubated with DbcAMP 2 mM for the first 45 min. Then, after 2 brief washes, they were transferred to vials containing medium without DbcAMP; 30 min later, the fragments were reexposed to DbcAMP 2 mM for 45 min. Each graph represents the result from 1 experiment
Fig. 5. Influence of metabolic inhibitors on chymotrypsmogen release from rat pancreas incubated for 2 h in a Krebs-Ringer-Tris/ HC1 buffered medium. Results, expressed in % of control, are mean values • S.E.M. of 8 experiments. Control: 100 % = 16.3 • 2.5 U of chymotrypsinogen/100 mg/2 h. O: control, gas phase oxygen. N: gas phase nitrogen. OX: sodium oxamate 70 raM. Dbc AMP: dibutyryl cyclic AMP 0.4 mM. Carb: carbamylcholine 0. I mM
Table 1. Influence of inhibitors of cholinergic, adrenergic, nicotinic, dopaminergic and serotoninergic systems on the secretagogue effect of DbeAMP. Pancreatic pieces were incubated for 2 h at 37~C in the presence or the absence of DbcAMP 2 mM and pharmacological agents. Release of amylase is expressed in % of control • S.E.M. Number of experiments is given in brackets. Control 100% = 8 • 1 U/lOOmg/h. Statistical significance has been calculated (paired tests) versus the control value. P < 0.05
presence of atropine is the same, regardless of the low rate of basal secretion due to atropine treatment: it strengthens the evidence against an action of D b c A M P on the cholinergic receptor.
None Dibenzamine 5- 10-s M Propranolol 5- 10-5 M Atropine sulphate 5.10 -s M Hexamethonium 5- 10-5 M Haloperidol 5.10 -5 M Cyproheptadine 10-8 M 10-6 M 5-10 -bM
Control
DbcAMP 2 mM
100
224.1 • 32.0 (6)
86.1 • 22.7 (5) 85.5 • 9.0 (6)
172.5 4- 8.4 (6) 315.7 + 57.8 (6)
52.6 • 11.1 (6)* 230.2 + 39.2(6) 90.8 • 18.9 (5) 142.4 • 17.3 (4)
175.9• 3.6 (6) 280.3 • 77.4 (4)
80.4 + 9.8 (4) 193.1 4- 23.6 (4) 84.8 • 19.7 (4) 170.6• 18.7 (4) 178.1 • 29.0 (4)* 247.8 • 76.2 (4)
peridol) agents completely fail to alter the release of enzyme induced by D b c A M P (Table 1). The antiserotonin agent, cyproheptadine, has by itself a strong releasing effect in the 5 - 10 -5 M range on the exocrine pancreas. Lower concentration neither alter the spontaneous secretion of enzyme nor modify the response t o the nucleotide derivative. The significant depressive effect of atropine sulphate (5 9 10 -2 M) on basal secretion suggests that at least a part of this spontaneous secretion is under cholinergic tone. The magnitude of the maximal response to D b c A M P in
Evaluation of the Characteristics of DbcAMP Secretagogue Properties a) The Secretion Induced by DbcAMP is Energy Dependent. 1. Blockade of both oxidative phosphorylation (by anaerobiosis) and glycolysis (by sodium oxamate 70 m M ) abolishes the secretagogue effect of D b c A M P (Fig. 5). 2. Blockade of oxidative phosphorylation (by anaerobiosis or antimycin A 10 -5 M) or glycolysis (by sodium oxamate 70 raM) does not alter the secretory response to D b c A M P (Fig. 5). Anaerobiosis slightly decreases both control and D b c A M P stimulated enzyme release but the magnitude of the stimulation remains identical to control. 3. D b c A M P 2 m M did not alter the oxygen uptake (97 + 9 % of control, n = 6), lactic acid formation (88 • 10% of control, n = 16) nor the energy balance in the gland as shown by the A T P level (109 • 14 % of control, n = 8). Control values were respectively: oxygen uptake: 6.0 + 0.2 gmole/100 rag/ h, lactic acid formation:0.45 + 0.05 pmole/100 rag/h, A T P level: 0.36 + 0.06 ~tmole/g. 4. The maximal stimulation of enzyme release induced by carbamylcholine 1 0 - b M is 5 to 7 times the control value (Fig, i). Concomittantly, oxygen uptake increases (111.7 _+ 3.6% of control, n = 58) as well as
H. Bauduin et al. : DbcAMP and Pancreatic Secretion
73
45Ca EFFLUX (% of the load) ,oo
/
Ca**
OmM
,_,\
Dbc AMP 2raM
Ca**
,,-A
O.OSmM
~
Ca** 0.05 mM + Dbc AMP 2raM
o AMYLASE RELEASE (U/lOOmg) 100t
Time ~lOmin.
45Ca EFFLUX (% of the load)
100
OmM
50
AMYLASE RELEASE (U/]OOmg) 200
100
Fig. 6 Influence of DbcAMP on calcium efflux and amylase release from "calcium depleted" rat pancreas. Pancreatic fragments were incubated in medium containing calcium 0.2 raM, 45Ca 5 gCi/ml for 45 min, then washed 2 x 7.5 min in calcium free EGTA 0.5 mM supplemented medium. After that, fragments were exposed for 10 rain to ionophore A23187 10 -s M (io), then transferred to a calcium free medium ([Ca2§ ] < 10 -8 M) and incubated for 130 min. The arrow indicates the addition of DbcAMP 2 mM and/or calcium 0.05 raM. eSCa efflux is given in % of the load which was calculated from the residual activity in the tissue at the end of the incubation and the activity released in the medium. The results are representative of one experiment
(Fig. 5), i.e. to the level of the DbcAMP-response in similar condition. Under anaerobiosis the ATP level in the gland is 8 _+ 1.1% of control, n = 8; the lactic acid formation is doubled though not further altered by DbcAMP or carbamylcholine treatment.
b) The Secretion Induced by DbcAMP is Calcium Dependent. The secretory response to DbcAMP 2 mM is reduced in calcium free medium (Fig. 2). A more complete depletion of calcium in the gland, achieved by a wash with ionophore A23 187 in calcium free ( < 10 -8 M) medium, is a perequisite to suppress the secretagogue effect of DbcAMP (Fig. 6). It is restored by increasing the extracellular calcium concentration to 0.05 mM. However, in absence of DbcAMP, such low concentration of calcium has no secretory effect (Fig. 6). DbcAMP 2 mM does not alter the movement of 45Ca in or out of the pancreas (Fig. 7) while carbamylcholine 0.1 mM increased the ~SCa efflux [3].
Time ~10 rain. Fig. 7. Influence of DbcAMP on calcium efflux and amylase release from rat pancreas. Effect of calcium concentration in the incubation medium. Pancreatic fragments were incubated in medium containing calcium 0.2 mM, r 5 gCi/ml for 45 min, then washed 2 x 7.5 rain in calcium free EGTA 0.5 mM supplemented medium. After that, they were transferred to calcium free medimn (0 mM) or medium containing calcium (0.1 mM) and incubated for 130 min. The arrow indicates the addition of DbcAMP 2 mM. eSCa efflux is given in % of the load (see Fig. 6). The results are representative of 1 experiment
the lactic acid formation (125 _+ 18% of control, n = 19); however the energy balance falls to 73 __ 6 % of control, n = 18. Blockade of oxidative phosphorylation by anaerobiosis reduced the stimulation from 5 to 7 times the control to 21/2 times the control
DISCUSSION The secretagogue properties of DbcAMP in the exocrine pancreas have been observed in several animal species including the rat [2,16, 29], rabbit [33], cat [37] and mouse [27]. The guinea pig pancrease apparently failed to respond to the nucleotide derivative [4, 21 ]. The secretagogue action of DbcAMP cannot be explained by a damaging effect on pancreatic acinar cells as suggested by Kempen [25]. Indeed, there is no alteration neither of the intermediary and protein metabolism nor ofhexose and leucine transport under DbcAMP treatment. The morphological integrity, the absence of significant release of lactic dehydrogenase, t h e reversibility of the nucleotide derivative
74 action plead against a damaging effect of DbcAMP on the acinar cell. The increase of L D H leakage noted by Kempen [25] was probably due to the use of slices instead of pieces of pancreatic tissue. The specificity of the action of the nucleotide derivative is further supported by the existence of a dose-response relationship and the failure of DbcGMP, 5'-AMP and butyric acid to induce any enzyme release. The secretory response to DbcAMP could have been mediated through the release of neurotransmitters. Cholinergics are well known stimulators of enzyme release from the pancreas [19]. Epinephrine [27] and isoproterenol [30] are also able to induce secretion of enzymes from the pancreas at least in some species while dopamine has been involved in hydromineral secretion [12]. Pharmacological agents which antagonize the respective transmitters at the level of their receptors fail to depress the DbcAMPresponse. Thus the present investigation affords strong evidence for a direct interaction between the nucleotide derivative and the pancreatic cell. In order to explain why DbcAMP cannot achieve a full secretory response, comparable to that elicited by physiological secretagogues, it was compulsory to investigate, in both cases, the sequence of steps which result in stimulus-secretion coupling in the pancreatic acinar cell. As observed in glands stimulated by physiological secretagogues DbcAMP induces the appearance of numerous exocytotic pictures at the apex of the acinar cell. This process, known to achieve the discharge of zymogen granules is still considered as the main pathway of the release of digestive enzymes from the pancreas. The secretagogue effect of physiological stimuli, as well as that of DbcAMP requires an energy supply. Indeed, the effect is abolished by the simultaneous addition of agents blocking oxidative phosphorylation and glycolysis. Three steps of the secretory cycle, protein synthesis, transport of secretory material through the Golgi area and discharge into the acinar lumen are endergonic processes [20, 21]. DbcAMP has been reported to interfere with protein synthesis, amino acid transport and amino acid activation. However, the secretory effects of DbcAMP in the exocrine pancreas cannot be linked to protein synthesis. Secretion is independent of protein synthesis [21] and the secretagogue activity of DbcAMP does not require any lag time. Moreover, the nucleotide derivative does not alter the incorporation of labeled leucine into 5 ~ TCA precipitable proteins and the amino acid radioactivity in the TCA soluble material remains unchanged.
Pfi0.gersArch. 372 (1977) The energy requirements of the 2 other endergonic steps of the secretory cycle are different in the presence of DbcAMP and physiological secretagogues. Glycolysis, which accounts for 15 ~ of the immediately available energy in normal glands [1] can fully support the secretion primed by DbcAMP. Indeed, under anaerobiosis, the maximal secretory response to DbcAMP is maintained while that to carbamylcholine is reduced. These observations suggest that the pancreatic response to physiological secretagogues requires more energy supply than the response to DbcAMP; however, they do not indicate which one of the endergonic steps of the secretory cycle can explain this difference. According to Jamieson and Palade [22] and Singh and Webster [38], carbamylcholine and pancreozymin did not alter the in vitro transport of the secretory material through the Golgi area but stimulate the release of exportable proteins from zymogen granules. This implies 1. a faster migration of these granules from their site of packaging towards the acinar lumen and 2. a more rapid discharge of their content into the acinar lumen. Considering that the same amount of enzyme is discharged under anaerobiosis by DbcAMP and cholinergics, and that ultrastructural examination shows preservation of exocytosis in both cases (unpublished data), the intracellular transport of zymogen granules towards the apical cell membrane would be the step requiring the greater supply of energy while exocytosis can still be stimulated when the energetic supply is low. Thus it is suggested that exocytosis may be the most probable site of action of the nucleotide derivative. In the exocrine pancreas the secretagogue effect of physiological stimuli requires calcium in the environment [19, 23, 31,36, 42]. However, intracellular calcium, probably through redistribution, can trigger this secretory function for some time. The present investigation confirms the calcium dependence of the secretory response to DbcAMP already reported by Heisler et al. [16,17] and Schulz [37]. The importance of the interaction of DbcAMP with calcium has been further emphasized in very severely calcium depleted glands which were permeated to the ion by two EGTA washes followed by ionophore A23 187 treatment. In those conditions, a concentration of calcium too low to trigger any secretion was made effective by DbcAMP. Interaction between calcium and cAMP in the regulation of several cell functions is well known [5]. Also the ratio cAMP to calcium has been reported to be a determining factor in the modulation of zymogen granule discharge through the alteration of the dynamic state of the microtubules [14]. As it has been shown in the secretory response to hormones and
H. Bauduin et al. : DbcAMP and Pancreatic Secretion
cholinergics [3,43], microtubules and microfilaments have been involved in the secretagogue action of DbcAMP [41]. In peculiar, when microtubules have been disrupted by vinblastine, the maximal secretory response to DbcAMP reaches the same level than that obtained normally after stimulation with physiological secretagogues. Considering that this potentiation effect is 1. calcium dependent, 2. inhibited by agents which block the oxidative phosphorylation and 3. inhibited by the cytochalasin B, an agent known to disrupt microfilaments, it seems likely that DbcAMP act on the later step of the secretory cycle in the acinar cell. We thank Dr. C. Koehl for the analysis of calcium and Mrs. E. Schell-Frederick for her critical reading of the manuscript. The devoted collaboration of Mrs. M. Bourguignon is gratefully acknowledged.
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75 12. Fukuta, Y., Iwatsuki, K., Takeuchi, O., Hashimoto, K. : Secretin-like activity of dopamine on canine pancreatic secretion. Tohoku J. Exp. Med. 108, 353-360 (1972) 13. Gardner, J. D., Conlon, T. P., Adams, T. D. : Cyclic AMP in pancreatic acinar cells. Effects of gastrointestinal hormones. Gastroent. 70, 2 9 - 3 5 (1976) 14. Gillespie, E.: Microtubules, cyclic AMP, calcium and secretion. Ann. New York Acad. Sci. 253, 771-779 (1975) 15. Haymovits, A., Scheele, G. A. : Cellular cyclic nucleotides and enzyme secretion in the pancreatic acinar cell. Proc. Natl. Acad. Sci. U.S.A. 73, 156-160 (1976) 16. Heisler, S., Fast, D., Tenenhouse, A. : Role of Ca 2+ and cyclic AMP in protein secretion from rat exocrine pancreas. Biochim. Biophys. Acta 279, 5 6 1 - 572 0972) 17. Heisler, S. : Calcium efflux and secretion of e-amylase from rat pancreas. Br. J. Pharmacol. 52, 387-392 (1974) 18. Hilz, H., Tarnowski, W. : Opposite effects of cyclic AMP and its dibutyryl derivative on glycogen levels in Hela cells. Biochem. Biophys. Res. Commun. 40, 973-98/(1970) 19. Hokin, L. E.: Effects of calcium omission on acetylcholinestimulated amylase secretion and phospholipid synthesis in pigeon pancreas slices. Biochim. Biophys. Acta 115, 219-221 (1966) 20. Jamieson, J. D., Palade, G. E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J. Cell Biol. 39, 589-603 (1968) 21. Jamieson, J. D., Palade, G. E. : Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrine cells. Metabolic studies. J. Cell Biol. 48, 503-522 (1971) 22. Jamieson, J. D., Palade, G. E. : Synthesis, intracellular transport and discharge of secretory proteins in stimulated pancreatic exocrine cells. J. Cell Biol. 50, 135-158 (1971) 23. Kanno, T.: Calcium-dependent amylase release and electrophysiological measurements in cells of the pancreas. J. Physiol. (Lond.) 226, 353-371 (1972) 24. Kempen, H. J. M., De Pont, J. J. H. H. M., Bonting, J. L. : Rat pancreas adenylate cyclase. II. Inactivation and protection of its hormone receptor sites. Biochim. Biophys. Acta 370, 5 7 3 584 (1974) 25. Kempen, H. J. M. : Role of cyclic AMP in exocrine pancreatic secretion. Thesis. Nijmegen, Netherlands 1976 26. Kondo, S., Schulz, I. : Calcium ion uptake in isolated pancreas cells induced by secretagogues. Biochim. Biophys. Acta 419, 7 6 - 9 2 (1976) 27. Kulka, R. G., Sternlicht, E. : Enzyme secretion in mouse pancreas mediated by adenosine 3',5'-cyclic phosphate and inhibited by adenosine 3'-phosphate. Proc. Natl. Acad. Sci. U.S.A. 61, 1123-1128 (1968) 28. Marchis-Mouren, G., Sarda, L., Desnuelle, P. : Purification of hog pancreatic lipase. Arch. Biochem. Biophys. 83, 309-319 (1959) 29. Morisset, J. A., Webster, P. D. : In vitro and in vivo effects of pancreozymin, urecholine and cyclic AMP on rat pancreas. Am. J. Physiol. 230, 202-208 (1971) 30. Pederson, R., Schulz, I. : The effect of isoproterenol on enzyme secretion from the isolated perfused cat pancreas. Biochim. Biophys. Acta 338, 440-446 (1974) 31. Petersen, O. H., Ueda, N. : Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling. J. Physiol. (Lond.) 254, 583-606 (1976) 32. Reboud, J. P., Ben Abdeljlil, A., Desnuelle, P. : Variations de la teneur en enzymes du pancr6as de rat en fonction de la composition des r6gimes. Biochim. Biophys. Acta. 58, 3 2 6 337 (1962) 33. Ridderstapp, A. S., Bonting, S. L. : Cyclic AMP and enzyme secretion by the isolated rabbit pancreas. Pfltigers Arch. 313, 6 2 - 70 (1969)
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Received May 19, 1977
