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Presence and localization of CCK receptor subtypes in calf pancreas
Regulatory Peptides 111 (2003) 103 – 109 www.elsevier.com/locate/regpep
Presence and localization of CCK receptor subtypes in calf pancreas Jean Morisset a,*, Jean Laine´ a, Judith Bourassa a, Michel Lessard a, Ve´ronique Rome b, Paul Guilloteau b a
Service de gastroente´rologie, De´p. de me´decine et pathologie, Faculte´ de me´decine, Universite´ de Sherbrooke, 3001-12ie`me Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4 b Laboratoire du Jeune Ruminant, Institut National de la Recherche Agronomique, Rennes, France Received 15 July 2002; received in revised form 24 October 2002; accepted 24 October 2002
Abstract This study was undertaken to confirm the presence of CCK receptor subtypes in calf pancreas and establish their cellular localization. Using specific antibodies against CCKA and CCKB receptors, somatostatin, glucagon and insulin, we were able to confirm by Western blot the presence of both CCK receptor protein subtypes in the calf pancreas as a 80 – 85-kDa CCKA receptor and 40 – 45-kDa CCKB receptor. By immunofluorescence, the CCKB receptor colocalizes with the islets’ somatostatin delta cells, confirming what was previously shown in other species, as well as on ductal cells. We could not reproduce in the calf its colocalization with glucagon alpha cells as observed in human and rat. Any specific localization of CCKA receptors with our multiple antibodies failed. Our observation that the CCKB receptor subtype is specifically localized on pancreatic delta cells as well as on ductal cells lets us support the hypothesis that in this species, CCK could be involved in somatostatin metabolism as well as hydrelatic secretion; its effect on enzyme secretion would be indirect. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Calf; Pancreas; CCK receptor subtypes; Somatostatin; Glucagon; Insulin; Ductal cells
1. Introduction Early after birth, periodic activity of the exocrine pancreas exists in calf, and local neural intestinal CCKA receptors could be partly responsible for its control [1]. In milk-fed calves, the short duration of the meal and the long gastric emptying of digesta lead to a longer preprandial phase of pancreatic secretion followed by an intestinal phase associated with a long drop in secretion [2]. It was suggested that the pancreatic secretory response to feeding in the calf would involve the CCKA receptor, but not the CCKB subtype since volume, protein and trypsin outflows were inhibited by the CCKA receptor antagonist, SR27897 but not by the CCKB receptor antagonist, PD135158 [3]. However, when calf pancreatic secretion was stimulated by identical doses of CCK-9 and gastrin 13-S, the secretory response to CCK-9 was inhibited by both CCK receptor antagonists, while gastrin-induced enzyme secretion was reduced by PD1351
* Corresponding author. Tel.: +1-819-820-6813; fax: +1-819-8206826. E-mail address: [email protected] (J. Morisset).
58 and much less by SR27897. It was then concluded that both CCK receptor subtypes are involved in the control of calf pancreatic secretion [4]. Such an involvement of both CCK receptor subtypes in the control of calf pancreatic secretion was strongly supported by the biochemical characterization of both receptors. Indeed, a 78 –96-kDA CCKA receptor protein is predominant in newborn calf, whereas a 40 –52-kDa CCKB receptor expression developed significantly after birth [5]. Furthermore, after molecular cloning of the calf CCKB receptor, its estimated concentration of 4.7 fmol/mg protein at birth increased tremendously to 10, 150 fmol/mg protein at 119 days of age, concomitant with a sharp decrease in its mRNA concentration from 4.2 fg/Ag RNA to 0.5 fg [6]. This figure was later adjusted to 36– 30 fg/Ag RNA at birth, down to 0.05 fg at 150 days of age [7]. These initial studies on the effects of CCK and gastrin on calf pancreatic secretion, on pancreatic CCK receptor subtypes biochemical characterization and on their mRNA estimation left us with great uncertainty as to how two subtypes of the same receptor can control water, electrolytes and enzymes secretion with almost the same efficiency. This study was therefore undertaken to identify by immunoblot-
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ting, and to localize by immunofluorescence both CCK receptors subtypes in calf pancreas and then try to ascribe to each subtype a potential physiological role in the control of pancreatic secretion.
2. Experimental animals Treatments and experiments were conducted according to the European Union regulation concerning the protection of experimental animals. This study was performed on Prim-Holstein-male calves provided by the ‘‘Unite´ Mixte de Recherches sur la Vache Laitie`re’’ (St. Gilles and Le Rheu, Institut National de la Recherche Agronomique). Calves were divided into three groups of four calves each, killed at 14 and 28 days in milk-fed animals, and ruminants killed at 4 months. One calf was sacrificed at birth. Calves in the three groups were given colostrum (25 g kg 1 of live weight per meal, two meals per day) during the first 2 days of life and then received a milk substitute diet based on spray-dried skim milk, whey powder and tallow, containing 25% crude protein, 22% fat, 43% lactose, 3% starch and 7% minerals. Because the calves of the first and second groups were exclusively milk-fed, they were maintained at the preruminant stage (similar to the monogastric stage) until being killed at 14 and 28 days of age. The amount of dry matter in their feed was progressively increased from 250 to 710 g/day between days 7 and 28. From day 29, the ruminants (polygastric stage) calves in the third group were given ad libitum water, hay and commercial concentrate comprising 22% crude protein, 2% fat, 14% cellulose- and nitrogen-free extract, 5.2% starch, and 10% minerals. As the amount of milk substitute was gradually reduced to nothing between days 29 and 56, group 3 calves were functional ruminants from days 57 to 119. All calves in groups 1, 2 and 3 were killed 16 h after the last meal [8]. The newborn calf remained fasted after birth until its sacrifice few hours later.
3.3. Gel electrophoresis and immunoblotting This procedure was performed exactly as described in Ref. [9]. The CCKB receptor antibody (Ab9262) and its corresponding peptide were provided by CURE/Gastroenteric Biology Center, Antibody/RIA Core, NIH grant #DK4 1301. The CCKA receptor antibody AR6 and its corresponding peptide were generous gifts from Dr. Kruse, Kiel, Germany. 3.4. Indirect immunofluorescence Frozen sections of each pancreatic piece from each animal were cut into 3- to 4-Am-thick pieces on a JUNG FRIGOCUT 2800 N cryostat (Leica, Montreal, Quebec), spread on glass slides and air dried for 30 min at room temperature before storage at 80 jC. Fixation and exposition to the different antibodies were performed as described in Ref. [9] with the exception that exposition to the first antibody was
3. Materials and methods 3.1. Tissue preparation At slaughter, the pancreas of each animal from each group was rapidly excised and cooled. Fragments of tissues were quickly embedded in tissue-tek O.C.T. 4583 compound (Sakuta Fine Tek, Torrance, CA) and frozen in liquid nitrogen for indirect immunofluorescence studies or homogenized in a specific buffer for Western blotting analysis. 3.2. Membrane preparation Membranes from each pancreas were collected by centrifugation at 100,000 g for 30 min using a TLS-55 Beckman rotor and they were suspended as described previously [9].
Fig. 1. Pancreatic calf CCKA and CCKB receptor proteins. Thirty micrograms of pancreatic membranes from 14 days, 1- and 4-month-old calves was used for the Western blot analysis with polyclonal antibodies to the Nterminal residues 1 – 20 of the rat CCKA receptor (antibody AR6) at 1:1000 (upper panel) and to the N-terminal residues 42 – 55 of the canine CCKB receptor (antibody 9262) at 1:10,000 (lower panel). Immunoneutralization was performed by pre-incubating each primary antibody for 2 h with 20 Ag ml 1 of their respective peptide antigen. This is a representative picture of what was observed in each pancreas at all ages.
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Fig. 2. Indirect immunofluorescence of the pancreatic CCKB receptor, somatostatin, glucagon and insulin from 4-month ruminant calf. Polyclonal antibodies to CCKB receptor (9262, (A)) at 1:500 and somatostatin (Barbar, (B)) at 10 Ag ml 1 were used; Glucagon (C) and insulin (D) monoclonal antibodies were used at 1:1000 and 2 Ag ml 1, respectively. Incubation proceeded overnight at 4 jC. Secondary antibodies incubated for 1 h at room temperature were anti-rabbit FITC 1:100 for CCKB, anti-goat FITC 1:100 for somatostatin and anti-mouse FITC 1:100 for glucagon and insulin. The residues 42 – 55 of the CCKB receptor (AV), somatostatin-14 (BV), glucagon (CV) and insulin (DV) were all pre-incubated for 2 h at room temperature at the concentration of 40 Ag ml 1 for the immunoneutralization. Magnification 40 . This representation at 4 months reflects what was observed in the three other groups and in each pancreas.
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Fig. 3. Indirect immunofluorescence of CCKB receptor and immunohistochemistry of cytokeratin on calf pancreatic ductal cells. Pancreatic tissues from 4-month ruminant (A – AV), 1-month (B – BV) and newborn (C – D; CV– DV) calves were evaluated for the presence of the CCKB receptor (left panel) and cytokeratin AE1/ AE3 (right panel). The polyclonal CCKB receptor antibody 9262 and its peptide antigen (D) were used as described in Fig. 2. The cytokeratin antibody was used at 1:50. Panel DVrepresents incubation with a pre-immune serum. Magnification 40 .
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performed overnight at 4 jC. The rat polyclonal anti-somatostatin Barbar purified by affinity chromatography against SS-14 was a generous gift from Dr Brazeau, Montreal, Canada; the monoclonal anti-glucagon and anti-insulin antibodies were from Sigma, St.-Louis, MI and Santa Cruz Biotechnology, Santa Cruz, CA, respectively. Somatostatin-14 and glucagon were from Sigma and porcine insulin from Lilly Pharmaceutical, Indianapolis, IN. The following antibodies were also used. The rat CCKA receptor antibody 94159 (N-terminal, 1:200) was from CURE, AR5 (C-terminal 410 –429, 1:200), AR1 (3rd intracellular loop, 1:50) were from Dr. Kruse and the human CCKA receptor antibody R12 (N-terminal 1– 15, 1:50) was from Schweiger and Amselgruber, Stuttgart, Germany. 3.5. Immunohistochemistry Three- to four-micrometer-thick frozen sections of calf pancreas were investigated by immunochemistry for the expression of cytokeratin AE1/AE3 (DAKO M 3515). The antibody was used at a 1:50 dilution to identify ductal cells. Pancreas from the four age groups including the newborn were examined.
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antibody with its corresponding antigen (top to bottom, right panel) prevented recognition of the receptor and each hormone, thus confirming the specificity of each antibody. From this figure, it is quite evident that the CCKB receptor protein colocalizes with somatostatin in the islet’s delta cells; indeed, it is difficult to match the CCKB receptor with glucagon and insulin. Similar colocalizations of the CCKB receptor on somatostatin delta cells were also observed in the pancreas of younger calves including the newborn (data not shown). On a tissue section, 5 to 10 islets can be observed and the colocalization CCKB receptor – somatostatin was similarly observed in each islet. As observed in Fig. 3, the CCKB receptor (A) colocalizes with pancreatic ductal cells (AV) identified by the cytokeratin AE1/AE3 antibody, a specific epithelial cell marker. Such colocalization is also present in the pancreas of 1-month-old calf (B– BV) and of the newborn (C –CV). The presence of the CCKB receptors on ductal cells includes the intra and interlobular cells; our tissue samples did not include the main ducts. Specificity of the CCKB reaction is supported by the observation that immunofluorescence was completely abolished after preabsorption of the antibody with the antigen in the newborn calf (3C vs. 3D). Similar displacements were also observed at the two other ages (data not shown). Fig. 3DVpresents incubation with a preimmune serum.
4. Results Using a specific antibody raised against the N-terminal 1 –20 portion of the rat CCKA receptor, this receptor was specifically identified by Western blot in the pancreas of milk fed 14 days, 1- and 4-month-old ruminant calves (Fig. 1, upper panel). This CCKA receptor exhibits a 85– 90-kDa protein at the three ages. Specificity was confirmed by preabsorption of the antiserum with its corresponding antigen, which resulted in complete blocking of the antigenantibody reaction. As observed in Fig. 1, lower panel, at the three ages studied, the calves’ pancreas showed the presence of a 40 –45-kDa CCKB receptor protein identified with the antibody 9262 (amino acids 42– 55) from the N-terminal extracellular part of the canine CCKB receptor. Specificity was again ensured by the observation that the 40 –45-kDa band disappeared after preabsorption of the antibody with its corresponding antigen. These electrophoretic patterns were observed in each pancreas at the three ages studied. By immunofluorescence, using the antibodies 94159, AR5, AR6, AR1 and R1-2 and their specific peptide antigen, we were unable to specifically localize the CCKA receptor to any calf pancreatic cell type because of nonspecific binding. It is important to indicate however that antibody AR5 recognized the CCKA receptor on rat and mouse acinar cells and on rat and pig islets of Langerhans (9). However, serial sections of a 4-month-old ruminant calf pancreas present an islet of Langerhans containing, from top to bottom (Fig. 2A – D, left panel) the CCKB receptor, somatostatin, glucagon and insulin. Preabsorption of each
5. Discussion Our data confirm the presence of both CCKA and CCKB receptor subtypes in the calf pancreas. Furthermore, we present for the first time in calf pancreas the colocalization of the CCKB receptor with somatostatin in the islet of Langerhans delta cells. Finally, this CCKB receptor is also associated with the pancreatic ductal cells, also a first original observation. By photoaffinity labeling, LeMeuth et al. [5] previously identified the calf pancreatic CCKA receptor as a 78- to 96kDa component and the CCKB receptor as a 40- to 47-kDa protein. We confirmed the presence of these two receptor proteins with comparable molecular weight by Western blot with specific CCK receptor subtype antibodies. In their initial pharmacological analysis, LeMeuth et al. [5] indicated a preferential expression of the CCKA receptor at birth with predominance of the CCKB subtype at postnatal ages. The total amount of CCK receptors established by Scatchard analysis of CCK-9 binding at birth, 28 and 119 days after birth, indicated 47, 2857 and 9929 fmol/mg protein. The same group [6] later on cloned the CCKB receptor in calf pancreas and came up with CCKB receptor concentrations of 4.7, 2930 and 10,150 fmol/mg protein at birth, 28 and 119 days after birth. This later study used a similar membrane preparation and the same CCK-9 marker as in their binding studies. Values of both studies are quite comparable and seem to leave few room for a population of CCKA receptor. Although our Western blots are not quantitative, the data
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presented do not support a 200-fold increase estimated by Scatchard analysis [5] in calf pancreatic CCK receptor subtype during development. Indeed, we did not observe a decrease with age in the expression of the CCKA receptor from 14 days old to 4 months as previously indicated [5]. However, expression of the CCKB receptor seems quite stable with age, again, a discrepancy with previously published data [6]. In the rat [10], we previously demonstrated by Western blot that the expression of the CCKB receptor protein did not vary with age. It has to be reminded that our gels were all loaded with the same amount of membrane proteins. The cellular localization of the pancreatic CCKB receptor is still an open debate among investigators in the field. Indeed, Saillan-Barreau et al. [11] claimed that the human pancreatic CCKB receptor was localized on glucagon a cells and responsible for its secretion. Rooman et al. [12] localized the pancreatic CCKB receptor in the periphery of the rat islets and also indicated their presence on the glucagon a cells, as data not shown. However, Schweiger et al. [13] described the location of the CCKA receptor on the glucagon a cells in the pig pancreas, contrary to the two previous studies showing a colocalization of the CCKB receptor with glucagon in human [11] and rat [12] pancreas. Our own initial data [10] colocalized the CCKB receptor with the somatostatin delta cells in the rat, mouse, pig and human pancreas, and now in the calf pancreas. We cannot yet explain all these discrepancies; our past and present results on CCKB receptor and somatostatin colocalization, however, indicate reproducibility among species. Furthermore, we can now indicate that the previously observed CCKB receptor and somatostatin colocalization in the rat pancreas from adjacent sections [10] were recently confirmed in rat purified islets of Langerhans by confocal microscopy [14]. The localization by indirect immunofluorescence of the CCKB receptor on calf pancreatic ductal cells is also an original observation never described before in normal pancreas of any species. Recently, pancreatic ductal complexes expressing the gastrin/CCKB receptor were observed following ligation of the rat pancreatic ducts, a localization never observed in normal pancreas [12]. In the guinea pig interlobular duct segments in culture, CCK stimulated secretion of a bicarbonate-rich fluid; this response was associated with occupation of the CCKA receptor since antagonized by the specific CCKA receptor antagonist devazepide but not by L365,260, the specific CCKB receptor antagonist. However, in this instance, the maximal response to CCK occurred at 10 nM with no inhibition at 100 nM, an atypical CCKA response [15]. The control of pancreatic secretion in the calf is far from being fully understood. In Holstein adult steers equipped with permanent duodenal pouches for collection of exocrine pancreatic secretions, there was no effect of feeding on the pattern of fluid secretion over a 7-h period [16]. In ruminant calves, equipped with a permanent duct catheter for collection of exocrine pancreatic secretions, there was no effect of
feeding on the pattern of fluid and enzyme secretions over an 8-h period [2]. In calf fed milk substitute, plasma concentrations of gastrin and CCK increased significantly after feeding while that of secretin decreased [2,3,8]. The plasma concentrations of CCK (15 pM) and gastrin (30 pM) observed after feeding are in fact too low to stimulate the high affinity CCKB receptor with a KD estimated at 350 pM in calf of that age [5]. However, in response to feeding, juice, protein and trypsin outflows were all significantly increased, increments totally inhibited by the CCK A antagonist SR27897, but not by the CCKB antagonist PD135158 [3]. These data seem indicate that pancreatic fluid and enzyme secretion could be under CCK control. In fasted milk-fed calves, when CCK and gastrin were infused in similar amount to give both hormone plasma concentrations of 80 – 85 pM, a five-fold higher concentration than after feeding, CCK significantly increased juice and enzyme outflows while gastrin selectively increased enzyme output [4]. The CCK response was significantly reduced by both CCKA and CCKB antagonists, SR27897 and PD135158, respectively. On the contrary, the stimulatory effects of gastrin on protein output remained unaffected by both CCK receptor antagonists, whereas trypsin was selectively inhibited by the CCKB antagonist and chymotrypsin reduced by both. A close look at the amount of antagonists infused [4] indicates that SR27897, given at 15 nmol kg 1 min 1 for a final plasma concentration of 9 AM, could interfere with the CCKB receptor. Indeed this high concentration exceeds by 100-fold the plasma concentration of 90 nM PD135158 measured after its infusion at 0.15 nmol kg 1 min 1. It was previously observed that at 10 AM, MK-329, another specific CCKA receptor antagonist, totally inhibited a believed CCKB-induced amylase release by 10 nM caerulein from pig pancreatic acini [17]. From these data, it is quite difficult to ascertain which of the CCK receptor subtype controls calf pancreatic secretion. One may speculate that volume could be under the control of CCK in the calf because it was inhibited by a CCKB receptor antagonist [4] at a time when secretin concentrations were decreased after feeding [3]. Furthermore, we just reported that this CCKB receptor was present on pancreatic ductal cells. New studies are needed to ascertain this possibility. The observations that both CCK receptor subtypes could not be found on calf pancreatic acinar cells under our experimental conditions and that the pancreas secretes enzymes in response to exogenous CCK-8 [18], CCK-9 and gastrin [4], let us support an alternative mode of action for the observed CCK/gastrin responses. A duodeno-pancreatic neural mechanism was previously proposed [18], supported by the finding that CCK can stimulate neural acetylcholine release possibly through the CCKA receptor [19] from the vagus nerve containing CCK58 and CCK8 [20]. This route is further supported by the observation that atropine totally inhibited CCK-induced pancreatic enzyme secretion in the pig [21]. Also, pancreatic response to feeding
J. Morisset et al. / Regulatory Peptides 111 (2003) 103–109
in pigs was neither inhibited by the CCKA receptor antagonist MK329 [22] nor by the CCKB receptor antagonists L365,260 and PD135156 [23]. As for the calf pancreas (this study), we were unable to visualize CCKA and CCKB receptors on pig pancreatic acinar cells [10]. The observation that CCK can release acetylcholine confirms that CCK can act on cholinergic neurons, the potential pathway to mediate pancreatic enzyme release. The experiment in conscious calves with pharmacological block of duodenal mucosal CCKA receptors by a novel selective non-absorbable CCKA receptor antagonist, Tavazepide from Solvey and muscarinic nonselective receptor antagonist, atropine, strongly supports this hypothesis [24]. This study showed that most, but not all, of CCK-related stimulation of pancreatic enzyme is of extrapancreatic neurohormonal origin. Finally, the colocalization of the CCKB receptor with pancreatic somatostatin delta cells in the calf as well as in other species [10] suggests that CCK could control somatostatin metabolism, a possibility presently under investigation.
Acknowledgements We wish to thank Mrs. C. Ducharme for her secretarial assistance as well as the Director and the staff of the slaughter house of Rennes for the permission and help to collect samples. This research was supported by grant GP6 369 from the Natural Sciences and Engineering Research Council of Canada.
References [1] Zabielski R, Morisset J, Podgurniak P, Rome´ V, Biernat M, Bernard C, et al. Bovine pancreatic secretion in the first week of life: potential involvement of intestinal CCK receptors. Regul Pept 2002;103: 93 – 104. [2] LeDrean G, LeHuerou-Luron I, Chayvialle JA, Rome´ V, Gestin M, Bernard C, et al. Kinetics of pancreatic exocrine secretion and plasma gut regulatory peptide release in response to feeding in preruminant and ruminant calves. Comp Biochem Physiol 1997;117A:245 – 55. [3] LeDrean G, LeHuerou-Luron I, Gestin M, Rome´ V, Bernard C, Chayvialle JA, et al. Pancreatic secretory response to feeding in the calf: CCK-A receptors, but not CCK-B/gastrin receptors are involved. Can J Physiol Pharmacol 2000;78:813 – 9. [4] LeDrean G, LeHuerou-Luron I, Gestin M, Desbois C, Rome´ V, Bernard C, et al. Exogenous CCK and gastrin stimulate pancreatic exocrine secretion via CCK-A but also via CCK-B/gastrin receptors in the calf. Eur J Physiol 1999;438:86 – 93. [5] LeMeuth V, Philouze-Rome´ V, LeHuerou-Luron I, Formal M, Vaysse N, Gespach C, et al. Differential expression of A- and B-subtypes of cholecystokinin/gastrin receptors in the developing calf pancreas. Endocrinology 1993;133:1182 – 91. [6] Dufresne M, Escrieut C, Clerc P, LeHuerou-Luron I, Prats H, Bertrand V, et al. Molecular cloning, developmental expression and pharmacological characterization of the CCKB/gastrin receptor in the calf pancreas. Eur J Pharmacol 1996;297:165 – 79.
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[7] Desbois C, Clerc P, LeHuerou-Luron I, LeDrean G, Gestin M, Dufresne M, et al. Differential tissular expression of the CCKA and CCKB/gastrin receptor genes during postnatal development in the calf. Life Sci 1998;63:2059 – 70. [8] Le Drean G, LeHuerou-Luron I, Gestin M, Rome´ V, Plodari M, Bernard C, et al. Comparison of the kinetics of pancreatic secretion and gut regulatory peptides in the plasma of preruminant calves fed milk or soybean protein. J Dairy Sci 1998;81:1313 – 21. [9] Bourassa J, Laine´ J, Kruse ML, Gagnon MC, Calvo E, Morisset J. Ontogeny and species differences in the pancreatic expression and localization of the CCKA receptors. Biochem Biophys Res Commun 1999;260:820 – 8. [10] Morisset J, Wong H, Walsh JH, Laine´ J, Bourassa J. Pancreatic CCKB receptors: their potential roles in somatostatin release and y-cell proliferation. Am J Physiol 2000;279:G148 – 56. [11] Saillan-Barreau C, Dufresne M, Clerc P, Sanchez D, Corominola H, Moriscot C, et al. Evidence for a functional role of the cholecystokininB/gastrin receptor in the human fetal and adult pancreas. Diabetes 1999;48:2015 – 21. [12] Rooman I, Lardon J, Flamez D, Schuit F, Bouwens L. Mitogenic effect of gastrin and expression of gastrin receptors in duct-like cells of rat pancreas. Gastroenterology 2001;121:940 – 9. [13] Schweiger M, Erhard MH, Amselgruber WM. Cell specific localization of the cholecystokinin A receptor in the porcine pancreas. Anat Histol Embryol 2000;29:357 – 61. [14] Julien S, Laine´ J, Morisset J. Expectation of pancreatic physiological responses from CCKB receptor occupation in its natural environment. In: Tache´ Y, Ohning G, Lloyd K, Goto Y, editors. Gut – brain peptides in the new millennium: a tribute to John Walsh by his collaborators. Los Angeles, CA, USA: Med Pub; 2002. p. 381 – 91. Chapter 38. [15] Szalmay G, Varga G, Kajiyama F, Yang X-S, Lang TF, Case RM, et al. Bicarbonate and fluid secretion evoked by cholecystokinin, bombesin and acetylcholine in isolated guinea-pig ducts. J Physiol 2001;535: 795 – 807. [16] St-Jean G, Harmon DL, Peters JP, Ames NK. Collection of pancreatic exocrine secretions by formation of a duodenal pouch in cattle. Am J Vet Res 1992;53:2377 – 80. [17] Morisset J, Levenez F, Corring T, Benrezzak O, Pelletier G, Calvo E. Pig pancreatic acinar cells possess predominantly the CCK-B receptor subtype. Am J Physiol 1986;271:E397 – 402. [18] Zabielski R, Onaga T, Mineo H, Pierzynowski SG, Kato S. Local versus peripheral blood administration of cholecystokinin-8 and secretin on pancreatic secretion in calves. Exp Physiol 1994;79:301 – 11. [19] Soudah HC, Lu Y, Hasler WL, Owyang C. Cholecystokinin at physiological levels evokes pancreatic enzyme secretion via a cholinergic pathway. Am J Physiol 1992;263:G102 – 7. [20] Chang T, Thagesen H, Lee KY, Roth FL, Chey WY. Canine vagus nerves stores cholecystokinin-58 and -8 but releases only cholecystokinin-8 upon electrical vagal stimulation. Regul Pept 2000;87:1 – 7. [21] Viau G, Laine´ J, Levenez F, Gueugneau AM, Corring T, Morisset J, et al. Evidence for a secretory pathway originating from the maturation of granules in the pig exocrine pancreas. Am J Physiol 1994;30: G764 – 71. [22] Lhoste EF, Gueugneau AM, Garofano A, Philippe C, Levenez F, Corring T. Role of CCK in the regulation of secretion and adaptation in the pig pancreas. Pancreas 1995;11:86 – 94. [23] Lhoste EF, Fiszlewicz M, Corring T. Administration of two antagonists of the cholecystokininB/gastrin receptor does not totally inhibit the pancreatic response to a meal in the pig. Pancreas 2002;24:47 – 52. [24] Zabielski R, Lenieewska V, Borlak J, Gregory PC, Kiela P, Pierzinowski SG, et al. Effects of intraduodenal administration of Tarazepide on pancreatic secretion and duodenal EMG in neonatal calves. Regul Pept 1998;78:113 – 23.
Presence and localization of CCK receptor subtypes in calf pancreas Jean Morisset a,*, Jean Laine´ a, Judith Bourassa a, Michel Lessard a, Ve´ronique Rome b, Paul Guilloteau b a
Service de gastroente´rologie, De´p. de me´decine et pathologie, Faculte´ de me´decine, Universite´ de Sherbrooke, 3001-12ie`me Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4 b Laboratoire du Jeune Ruminant, Institut National de la Recherche Agronomique, Rennes, France Received 15 July 2002; received in revised form 24 October 2002; accepted 24 October 2002
Abstract This study was undertaken to confirm the presence of CCK receptor subtypes in calf pancreas and establish their cellular localization. Using specific antibodies against CCKA and CCKB receptors, somatostatin, glucagon and insulin, we were able to confirm by Western blot the presence of both CCK receptor protein subtypes in the calf pancreas as a 80 – 85-kDa CCKA receptor and 40 – 45-kDa CCKB receptor. By immunofluorescence, the CCKB receptor colocalizes with the islets’ somatostatin delta cells, confirming what was previously shown in other species, as well as on ductal cells. We could not reproduce in the calf its colocalization with glucagon alpha cells as observed in human and rat. Any specific localization of CCKA receptors with our multiple antibodies failed. Our observation that the CCKB receptor subtype is specifically localized on pancreatic delta cells as well as on ductal cells lets us support the hypothesis that in this species, CCK could be involved in somatostatin metabolism as well as hydrelatic secretion; its effect on enzyme secretion would be indirect. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Calf; Pancreas; CCK receptor subtypes; Somatostatin; Glucagon; Insulin; Ductal cells
1. Introduction Early after birth, periodic activity of the exocrine pancreas exists in calf, and local neural intestinal CCKA receptors could be partly responsible for its control [1]. In milk-fed calves, the short duration of the meal and the long gastric emptying of digesta lead to a longer preprandial phase of pancreatic secretion followed by an intestinal phase associated with a long drop in secretion [2]. It was suggested that the pancreatic secretory response to feeding in the calf would involve the CCKA receptor, but not the CCKB subtype since volume, protein and trypsin outflows were inhibited by the CCKA receptor antagonist, SR27897 but not by the CCKB receptor antagonist, PD135158 [3]. However, when calf pancreatic secretion was stimulated by identical doses of CCK-9 and gastrin 13-S, the secretory response to CCK-9 was inhibited by both CCK receptor antagonists, while gastrin-induced enzyme secretion was reduced by PD1351
* Corresponding author. Tel.: +1-819-820-6813; fax: +1-819-8206826. E-mail address: [email protected] (J. Morisset).
58 and much less by SR27897. It was then concluded that both CCK receptor subtypes are involved in the control of calf pancreatic secretion [4]. Such an involvement of both CCK receptor subtypes in the control of calf pancreatic secretion was strongly supported by the biochemical characterization of both receptors. Indeed, a 78 –96-kDA CCKA receptor protein is predominant in newborn calf, whereas a 40 –52-kDa CCKB receptor expression developed significantly after birth [5]. Furthermore, after molecular cloning of the calf CCKB receptor, its estimated concentration of 4.7 fmol/mg protein at birth increased tremendously to 10, 150 fmol/mg protein at 119 days of age, concomitant with a sharp decrease in its mRNA concentration from 4.2 fg/Ag RNA to 0.5 fg [6]. This figure was later adjusted to 36– 30 fg/Ag RNA at birth, down to 0.05 fg at 150 days of age [7]. These initial studies on the effects of CCK and gastrin on calf pancreatic secretion, on pancreatic CCK receptor subtypes biochemical characterization and on their mRNA estimation left us with great uncertainty as to how two subtypes of the same receptor can control water, electrolytes and enzymes secretion with almost the same efficiency. This study was therefore undertaken to identify by immunoblot-
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ting, and to localize by immunofluorescence both CCK receptors subtypes in calf pancreas and then try to ascribe to each subtype a potential physiological role in the control of pancreatic secretion.
2. Experimental animals Treatments and experiments were conducted according to the European Union regulation concerning the protection of experimental animals. This study was performed on Prim-Holstein-male calves provided by the ‘‘Unite´ Mixte de Recherches sur la Vache Laitie`re’’ (St. Gilles and Le Rheu, Institut National de la Recherche Agronomique). Calves were divided into three groups of four calves each, killed at 14 and 28 days in milk-fed animals, and ruminants killed at 4 months. One calf was sacrificed at birth. Calves in the three groups were given colostrum (25 g kg 1 of live weight per meal, two meals per day) during the first 2 days of life and then received a milk substitute diet based on spray-dried skim milk, whey powder and tallow, containing 25% crude protein, 22% fat, 43% lactose, 3% starch and 7% minerals. Because the calves of the first and second groups were exclusively milk-fed, they were maintained at the preruminant stage (similar to the monogastric stage) until being killed at 14 and 28 days of age. The amount of dry matter in their feed was progressively increased from 250 to 710 g/day between days 7 and 28. From day 29, the ruminants (polygastric stage) calves in the third group were given ad libitum water, hay and commercial concentrate comprising 22% crude protein, 2% fat, 14% cellulose- and nitrogen-free extract, 5.2% starch, and 10% minerals. As the amount of milk substitute was gradually reduced to nothing between days 29 and 56, group 3 calves were functional ruminants from days 57 to 119. All calves in groups 1, 2 and 3 were killed 16 h after the last meal [8]. The newborn calf remained fasted after birth until its sacrifice few hours later.
3.3. Gel electrophoresis and immunoblotting This procedure was performed exactly as described in Ref. [9]. The CCKB receptor antibody (Ab9262) and its corresponding peptide were provided by CURE/Gastroenteric Biology Center, Antibody/RIA Core, NIH grant #DK4 1301. The CCKA receptor antibody AR6 and its corresponding peptide were generous gifts from Dr. Kruse, Kiel, Germany. 3.4. Indirect immunofluorescence Frozen sections of each pancreatic piece from each animal were cut into 3- to 4-Am-thick pieces on a JUNG FRIGOCUT 2800 N cryostat (Leica, Montreal, Quebec), spread on glass slides and air dried for 30 min at room temperature before storage at 80 jC. Fixation and exposition to the different antibodies were performed as described in Ref. [9] with the exception that exposition to the first antibody was
3. Materials and methods 3.1. Tissue preparation At slaughter, the pancreas of each animal from each group was rapidly excised and cooled. Fragments of tissues were quickly embedded in tissue-tek O.C.T. 4583 compound (Sakuta Fine Tek, Torrance, CA) and frozen in liquid nitrogen for indirect immunofluorescence studies or homogenized in a specific buffer for Western blotting analysis. 3.2. Membrane preparation Membranes from each pancreas were collected by centrifugation at 100,000 g for 30 min using a TLS-55 Beckman rotor and they were suspended as described previously [9].
Fig. 1. Pancreatic calf CCKA and CCKB receptor proteins. Thirty micrograms of pancreatic membranes from 14 days, 1- and 4-month-old calves was used for the Western blot analysis with polyclonal antibodies to the Nterminal residues 1 – 20 of the rat CCKA receptor (antibody AR6) at 1:1000 (upper panel) and to the N-terminal residues 42 – 55 of the canine CCKB receptor (antibody 9262) at 1:10,000 (lower panel). Immunoneutralization was performed by pre-incubating each primary antibody for 2 h with 20 Ag ml 1 of their respective peptide antigen. This is a representative picture of what was observed in each pancreas at all ages.
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Fig. 2. Indirect immunofluorescence of the pancreatic CCKB receptor, somatostatin, glucagon and insulin from 4-month ruminant calf. Polyclonal antibodies to CCKB receptor (9262, (A)) at 1:500 and somatostatin (Barbar, (B)) at 10 Ag ml 1 were used; Glucagon (C) and insulin (D) monoclonal antibodies were used at 1:1000 and 2 Ag ml 1, respectively. Incubation proceeded overnight at 4 jC. Secondary antibodies incubated for 1 h at room temperature were anti-rabbit FITC 1:100 for CCKB, anti-goat FITC 1:100 for somatostatin and anti-mouse FITC 1:100 for glucagon and insulin. The residues 42 – 55 of the CCKB receptor (AV), somatostatin-14 (BV), glucagon (CV) and insulin (DV) were all pre-incubated for 2 h at room temperature at the concentration of 40 Ag ml 1 for the immunoneutralization. Magnification 40 . This representation at 4 months reflects what was observed in the three other groups and in each pancreas.
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Fig. 3. Indirect immunofluorescence of CCKB receptor and immunohistochemistry of cytokeratin on calf pancreatic ductal cells. Pancreatic tissues from 4-month ruminant (A – AV), 1-month (B – BV) and newborn (C – D; CV– DV) calves were evaluated for the presence of the CCKB receptor (left panel) and cytokeratin AE1/ AE3 (right panel). The polyclonal CCKB receptor antibody 9262 and its peptide antigen (D) were used as described in Fig. 2. The cytokeratin antibody was used at 1:50. Panel DVrepresents incubation with a pre-immune serum. Magnification 40 .
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performed overnight at 4 jC. The rat polyclonal anti-somatostatin Barbar purified by affinity chromatography against SS-14 was a generous gift from Dr Brazeau, Montreal, Canada; the monoclonal anti-glucagon and anti-insulin antibodies were from Sigma, St.-Louis, MI and Santa Cruz Biotechnology, Santa Cruz, CA, respectively. Somatostatin-14 and glucagon were from Sigma and porcine insulin from Lilly Pharmaceutical, Indianapolis, IN. The following antibodies were also used. The rat CCKA receptor antibody 94159 (N-terminal, 1:200) was from CURE, AR5 (C-terminal 410 –429, 1:200), AR1 (3rd intracellular loop, 1:50) were from Dr. Kruse and the human CCKA receptor antibody R12 (N-terminal 1– 15, 1:50) was from Schweiger and Amselgruber, Stuttgart, Germany. 3.5. Immunohistochemistry Three- to four-micrometer-thick frozen sections of calf pancreas were investigated by immunochemistry for the expression of cytokeratin AE1/AE3 (DAKO M 3515). The antibody was used at a 1:50 dilution to identify ductal cells. Pancreas from the four age groups including the newborn were examined.
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antibody with its corresponding antigen (top to bottom, right panel) prevented recognition of the receptor and each hormone, thus confirming the specificity of each antibody. From this figure, it is quite evident that the CCKB receptor protein colocalizes with somatostatin in the islet’s delta cells; indeed, it is difficult to match the CCKB receptor with glucagon and insulin. Similar colocalizations of the CCKB receptor on somatostatin delta cells were also observed in the pancreas of younger calves including the newborn (data not shown). On a tissue section, 5 to 10 islets can be observed and the colocalization CCKB receptor – somatostatin was similarly observed in each islet. As observed in Fig. 3, the CCKB receptor (A) colocalizes with pancreatic ductal cells (AV) identified by the cytokeratin AE1/AE3 antibody, a specific epithelial cell marker. Such colocalization is also present in the pancreas of 1-month-old calf (B– BV) and of the newborn (C –CV). The presence of the CCKB receptors on ductal cells includes the intra and interlobular cells; our tissue samples did not include the main ducts. Specificity of the CCKB reaction is supported by the observation that immunofluorescence was completely abolished after preabsorption of the antibody with the antigen in the newborn calf (3C vs. 3D). Similar displacements were also observed at the two other ages (data not shown). Fig. 3DVpresents incubation with a preimmune serum.
4. Results Using a specific antibody raised against the N-terminal 1 –20 portion of the rat CCKA receptor, this receptor was specifically identified by Western blot in the pancreas of milk fed 14 days, 1- and 4-month-old ruminant calves (Fig. 1, upper panel). This CCKA receptor exhibits a 85– 90-kDa protein at the three ages. Specificity was confirmed by preabsorption of the antiserum with its corresponding antigen, which resulted in complete blocking of the antigenantibody reaction. As observed in Fig. 1, lower panel, at the three ages studied, the calves’ pancreas showed the presence of a 40 –45-kDa CCKB receptor protein identified with the antibody 9262 (amino acids 42– 55) from the N-terminal extracellular part of the canine CCKB receptor. Specificity was again ensured by the observation that the 40 –45-kDa band disappeared after preabsorption of the antibody with its corresponding antigen. These electrophoretic patterns were observed in each pancreas at the three ages studied. By immunofluorescence, using the antibodies 94159, AR5, AR6, AR1 and R1-2 and their specific peptide antigen, we were unable to specifically localize the CCKA receptor to any calf pancreatic cell type because of nonspecific binding. It is important to indicate however that antibody AR5 recognized the CCKA receptor on rat and mouse acinar cells and on rat and pig islets of Langerhans (9). However, serial sections of a 4-month-old ruminant calf pancreas present an islet of Langerhans containing, from top to bottom (Fig. 2A – D, left panel) the CCKB receptor, somatostatin, glucagon and insulin. Preabsorption of each
5. Discussion Our data confirm the presence of both CCKA and CCKB receptor subtypes in the calf pancreas. Furthermore, we present for the first time in calf pancreas the colocalization of the CCKB receptor with somatostatin in the islet of Langerhans delta cells. Finally, this CCKB receptor is also associated with the pancreatic ductal cells, also a first original observation. By photoaffinity labeling, LeMeuth et al. [5] previously identified the calf pancreatic CCKA receptor as a 78- to 96kDa component and the CCKB receptor as a 40- to 47-kDa protein. We confirmed the presence of these two receptor proteins with comparable molecular weight by Western blot with specific CCK receptor subtype antibodies. In their initial pharmacological analysis, LeMeuth et al. [5] indicated a preferential expression of the CCKA receptor at birth with predominance of the CCKB subtype at postnatal ages. The total amount of CCK receptors established by Scatchard analysis of CCK-9 binding at birth, 28 and 119 days after birth, indicated 47, 2857 and 9929 fmol/mg protein. The same group [6] later on cloned the CCKB receptor in calf pancreas and came up with CCKB receptor concentrations of 4.7, 2930 and 10,150 fmol/mg protein at birth, 28 and 119 days after birth. This later study used a similar membrane preparation and the same CCK-9 marker as in their binding studies. Values of both studies are quite comparable and seem to leave few room for a population of CCKA receptor. Although our Western blots are not quantitative, the data
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presented do not support a 200-fold increase estimated by Scatchard analysis [5] in calf pancreatic CCK receptor subtype during development. Indeed, we did not observe a decrease with age in the expression of the CCKA receptor from 14 days old to 4 months as previously indicated [5]. However, expression of the CCKB receptor seems quite stable with age, again, a discrepancy with previously published data [6]. In the rat [10], we previously demonstrated by Western blot that the expression of the CCKB receptor protein did not vary with age. It has to be reminded that our gels were all loaded with the same amount of membrane proteins. The cellular localization of the pancreatic CCKB receptor is still an open debate among investigators in the field. Indeed, Saillan-Barreau et al. [11] claimed that the human pancreatic CCKB receptor was localized on glucagon a cells and responsible for its secretion. Rooman et al. [12] localized the pancreatic CCKB receptor in the periphery of the rat islets and also indicated their presence on the glucagon a cells, as data not shown. However, Schweiger et al. [13] described the location of the CCKA receptor on the glucagon a cells in the pig pancreas, contrary to the two previous studies showing a colocalization of the CCKB receptor with glucagon in human [11] and rat [12] pancreas. Our own initial data [10] colocalized the CCKB receptor with the somatostatin delta cells in the rat, mouse, pig and human pancreas, and now in the calf pancreas. We cannot yet explain all these discrepancies; our past and present results on CCKB receptor and somatostatin colocalization, however, indicate reproducibility among species. Furthermore, we can now indicate that the previously observed CCKB receptor and somatostatin colocalization in the rat pancreas from adjacent sections [10] were recently confirmed in rat purified islets of Langerhans by confocal microscopy [14]. The localization by indirect immunofluorescence of the CCKB receptor on calf pancreatic ductal cells is also an original observation never described before in normal pancreas of any species. Recently, pancreatic ductal complexes expressing the gastrin/CCKB receptor were observed following ligation of the rat pancreatic ducts, a localization never observed in normal pancreas [12]. In the guinea pig interlobular duct segments in culture, CCK stimulated secretion of a bicarbonate-rich fluid; this response was associated with occupation of the CCKA receptor since antagonized by the specific CCKA receptor antagonist devazepide but not by L365,260, the specific CCKB receptor antagonist. However, in this instance, the maximal response to CCK occurred at 10 nM with no inhibition at 100 nM, an atypical CCKA response [15]. The control of pancreatic secretion in the calf is far from being fully understood. In Holstein adult steers equipped with permanent duodenal pouches for collection of exocrine pancreatic secretions, there was no effect of feeding on the pattern of fluid secretion over a 7-h period [16]. In ruminant calves, equipped with a permanent duct catheter for collection of exocrine pancreatic secretions, there was no effect of
feeding on the pattern of fluid and enzyme secretions over an 8-h period [2]. In calf fed milk substitute, plasma concentrations of gastrin and CCK increased significantly after feeding while that of secretin decreased [2,3,8]. The plasma concentrations of CCK (15 pM) and gastrin (30 pM) observed after feeding are in fact too low to stimulate the high affinity CCKB receptor with a KD estimated at 350 pM in calf of that age [5]. However, in response to feeding, juice, protein and trypsin outflows were all significantly increased, increments totally inhibited by the CCK A antagonist SR27897, but not by the CCKB antagonist PD135158 [3]. These data seem indicate that pancreatic fluid and enzyme secretion could be under CCK control. In fasted milk-fed calves, when CCK and gastrin were infused in similar amount to give both hormone plasma concentrations of 80 – 85 pM, a five-fold higher concentration than after feeding, CCK significantly increased juice and enzyme outflows while gastrin selectively increased enzyme output [4]. The CCK response was significantly reduced by both CCKA and CCKB antagonists, SR27897 and PD135158, respectively. On the contrary, the stimulatory effects of gastrin on protein output remained unaffected by both CCK receptor antagonists, whereas trypsin was selectively inhibited by the CCKB antagonist and chymotrypsin reduced by both. A close look at the amount of antagonists infused [4] indicates that SR27897, given at 15 nmol kg 1 min 1 for a final plasma concentration of 9 AM, could interfere with the CCKB receptor. Indeed this high concentration exceeds by 100-fold the plasma concentration of 90 nM PD135158 measured after its infusion at 0.15 nmol kg 1 min 1. It was previously observed that at 10 AM, MK-329, another specific CCKA receptor antagonist, totally inhibited a believed CCKB-induced amylase release by 10 nM caerulein from pig pancreatic acini [17]. From these data, it is quite difficult to ascertain which of the CCK receptor subtype controls calf pancreatic secretion. One may speculate that volume could be under the control of CCK in the calf because it was inhibited by a CCKB receptor antagonist [4] at a time when secretin concentrations were decreased after feeding [3]. Furthermore, we just reported that this CCKB receptor was present on pancreatic ductal cells. New studies are needed to ascertain this possibility. The observations that both CCK receptor subtypes could not be found on calf pancreatic acinar cells under our experimental conditions and that the pancreas secretes enzymes in response to exogenous CCK-8 [18], CCK-9 and gastrin [4], let us support an alternative mode of action for the observed CCK/gastrin responses. A duodeno-pancreatic neural mechanism was previously proposed [18], supported by the finding that CCK can stimulate neural acetylcholine release possibly through the CCKA receptor [19] from the vagus nerve containing CCK58 and CCK8 [20]. This route is further supported by the observation that atropine totally inhibited CCK-induced pancreatic enzyme secretion in the pig [21]. Also, pancreatic response to feeding
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in pigs was neither inhibited by the CCKA receptor antagonist MK329 [22] nor by the CCKB receptor antagonists L365,260 and PD135156 [23]. As for the calf pancreas (this study), we were unable to visualize CCKA and CCKB receptors on pig pancreatic acinar cells [10]. The observation that CCK can release acetylcholine confirms that CCK can act on cholinergic neurons, the potential pathway to mediate pancreatic enzyme release. The experiment in conscious calves with pharmacological block of duodenal mucosal CCKA receptors by a novel selective non-absorbable CCKA receptor antagonist, Tavazepide from Solvey and muscarinic nonselective receptor antagonist, atropine, strongly supports this hypothesis [24]. This study showed that most, but not all, of CCK-related stimulation of pancreatic enzyme is of extrapancreatic neurohormonal origin. Finally, the colocalization of the CCKB receptor with pancreatic somatostatin delta cells in the calf as well as in other species [10] suggests that CCK could control somatostatin metabolism, a possibility presently under investigation.
Acknowledgements We wish to thank Mrs. C. Ducharme for her secretarial assistance as well as the Director and the staff of the slaughter house of Rennes for the permission and help to collect samples. This research was supported by grant GP6 369 from the Natural Sciences and Engineering Research Council of Canada.
References [1] Zabielski R, Morisset J, Podgurniak P, Rome´ V, Biernat M, Bernard C, et al. Bovine pancreatic secretion in the first week of life: potential involvement of intestinal CCK receptors. Regul Pept 2002;103: 93 – 104. [2] LeDrean G, LeHuerou-Luron I, Chayvialle JA, Rome´ V, Gestin M, Bernard C, et al. Kinetics of pancreatic exocrine secretion and plasma gut regulatory peptide release in response to feeding in preruminant and ruminant calves. Comp Biochem Physiol 1997;117A:245 – 55. [3] LeDrean G, LeHuerou-Luron I, Gestin M, Rome´ V, Bernard C, Chayvialle JA, et al. Pancreatic secretory response to feeding in the calf: CCK-A receptors, but not CCK-B/gastrin receptors are involved. Can J Physiol Pharmacol 2000;78:813 – 9. [4] LeDrean G, LeHuerou-Luron I, Gestin M, Desbois C, Rome´ V, Bernard C, et al. Exogenous CCK and gastrin stimulate pancreatic exocrine secretion via CCK-A but also via CCK-B/gastrin receptors in the calf. Eur J Physiol 1999;438:86 – 93. [5] LeMeuth V, Philouze-Rome´ V, LeHuerou-Luron I, Formal M, Vaysse N, Gespach C, et al. Differential expression of A- and B-subtypes of cholecystokinin/gastrin receptors in the developing calf pancreas. Endocrinology 1993;133:1182 – 91. [6] Dufresne M, Escrieut C, Clerc P, LeHuerou-Luron I, Prats H, Bertrand V, et al. Molecular cloning, developmental expression and pharmacological characterization of the CCKB/gastrin receptor in the calf pancreas. Eur J Pharmacol 1996;297:165 – 79.
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[7] Desbois C, Clerc P, LeHuerou-Luron I, LeDrean G, Gestin M, Dufresne M, et al. Differential tissular expression of the CCKA and CCKB/gastrin receptor genes during postnatal development in the calf. Life Sci 1998;63:2059 – 70. [8] Le Drean G, LeHuerou-Luron I, Gestin M, Rome´ V, Plodari M, Bernard C, et al. Comparison of the kinetics of pancreatic secretion and gut regulatory peptides in the plasma of preruminant calves fed milk or soybean protein. J Dairy Sci 1998;81:1313 – 21. [9] Bourassa J, Laine´ J, Kruse ML, Gagnon MC, Calvo E, Morisset J. Ontogeny and species differences in the pancreatic expression and localization of the CCKA receptors. Biochem Biophys Res Commun 1999;260:820 – 8. [10] Morisset J, Wong H, Walsh JH, Laine´ J, Bourassa J. Pancreatic CCKB receptors: their potential roles in somatostatin release and y-cell proliferation. Am J Physiol 2000;279:G148 – 56. [11] Saillan-Barreau C, Dufresne M, Clerc P, Sanchez D, Corominola H, Moriscot C, et al. Evidence for a functional role of the cholecystokininB/gastrin receptor in the human fetal and adult pancreas. Diabetes 1999;48:2015 – 21. [12] Rooman I, Lardon J, Flamez D, Schuit F, Bouwens L. Mitogenic effect of gastrin and expression of gastrin receptors in duct-like cells of rat pancreas. Gastroenterology 2001;121:940 – 9. [13] Schweiger M, Erhard MH, Amselgruber WM. Cell specific localization of the cholecystokinin A receptor in the porcine pancreas. Anat Histol Embryol 2000;29:357 – 61. [14] Julien S, Laine´ J, Morisset J. Expectation of pancreatic physiological responses from CCKB receptor occupation in its natural environment. In: Tache´ Y, Ohning G, Lloyd K, Goto Y, editors. Gut – brain peptides in the new millennium: a tribute to John Walsh by his collaborators. Los Angeles, CA, USA: Med Pub; 2002. p. 381 – 91. Chapter 38. [15] Szalmay G, Varga G, Kajiyama F, Yang X-S, Lang TF, Case RM, et al. Bicarbonate and fluid secretion evoked by cholecystokinin, bombesin and acetylcholine in isolated guinea-pig ducts. J Physiol 2001;535: 795 – 807. [16] St-Jean G, Harmon DL, Peters JP, Ames NK. Collection of pancreatic exocrine secretions by formation of a duodenal pouch in cattle. Am J Vet Res 1992;53:2377 – 80. [17] Morisset J, Levenez F, Corring T, Benrezzak O, Pelletier G, Calvo E. Pig pancreatic acinar cells possess predominantly the CCK-B receptor subtype. Am J Physiol 1986;271:E397 – 402. [18] Zabielski R, Onaga T, Mineo H, Pierzynowski SG, Kato S. Local versus peripheral blood administration of cholecystokinin-8 and secretin on pancreatic secretion in calves. Exp Physiol 1994;79:301 – 11. [19] Soudah HC, Lu Y, Hasler WL, Owyang C. Cholecystokinin at physiological levels evokes pancreatic enzyme secretion via a cholinergic pathway. Am J Physiol 1992;263:G102 – 7. [20] Chang T, Thagesen H, Lee KY, Roth FL, Chey WY. Canine vagus nerves stores cholecystokinin-58 and -8 but releases only cholecystokinin-8 upon electrical vagal stimulation. Regul Pept 2000;87:1 – 7. [21] Viau G, Laine´ J, Levenez F, Gueugneau AM, Corring T, Morisset J, et al. Evidence for a secretory pathway originating from the maturation of granules in the pig exocrine pancreas. Am J Physiol 1994;30: G764 – 71. [22] Lhoste EF, Gueugneau AM, Garofano A, Philippe C, Levenez F, Corring T. Role of CCK in the regulation of secretion and adaptation in the pig pancreas. Pancreas 1995;11:86 – 94. [23] Lhoste EF, Fiszlewicz M, Corring T. Administration of two antagonists of the cholecystokininB/gastrin receptor does not totally inhibit the pancreatic response to a meal in the pig. Pancreas 2002;24:47 – 52. [24] Zabielski R, Lenieewska V, Borlak J, Gregory PC, Kiela P, Pierzinowski SG, et al. Effects of intraduodenal administration of Tarazepide on pancreatic secretion and duodenal EMG in neonatal calves. Regul Pept 1998;78:113 – 23.