Advances in Medical Sciences 65 (2020) 46–64
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Review article
Life with the pancreas: A personal experience
T
Jean Morisset (Ph.D.) Division of Gastroenterology, Department of Medicine, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Québec, J1H 5N4, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Pancreatic secretion Enzyme synthesis and growth
This review article has primary objective to summarize pancreatic research which has been done in our laboratory since 1965, the first year of the author's registration in the Ph.D. program at the University of Sherbrooke (Canada). It covers the following major topics of pancreatic physiology: controls of pancreatic adaptation to diet, control of pancreatic enzyme secretion, control of pancreatic enzyme synthesis, control of pancreatic growth, intracellular events stimulated during pancreatic growth, pancreas regeneration after pancreatitis and pancreatectomy, the pancreatic cholecystokinin receptor types 1 and 2, growth control and cell signaling in pancreatic cancer cells and finally, cystic fibrosis.
1. Introduction The present review represents a summary of the research performed on the pancreas by the author as a Ph.D. student at the University of Sherbrooke (Canada) under the supervision of Dr. Jacques Dunnigan, and then at the Medical College of Georgia in Augusta, (Georgia, USA) under the supervision of Dr. Paul D. Webster for two years. His independent research career started in 1970 in the Department of Biology at the Faculty of Sciences of the University of Sherbrooke, and then at this same institution for fifty years. This review article then summarizes the pancreatic research performed by the author and all his research assistants, technicians, M.Sc. and Ph.D. students, as well as his postdoctoral fellows. It covers major topics in pancreatic physiology. Control of pancreatic enzyme secretion, protein synthesis, the muscarinic receptors, the cholecystokinin (CCK) receptors and their implication in pancreas growth are among the subjects that have been investigated in this laboratory. 2. Review 2.1. Pancreas adaptation to diet Many reports on the adaptation of pancreatic enzyme synthesis to the composition of the diet have been published [1] and the general agreement is that a diet rich in carbohydrates causes a preferential increased production of amylolytic enzymes with a concomitant decrease in proteolytic enzyme synthesis. On the contrary, a protein-rich diet will cause the reverse situation in the enzyme production pattern [2]. This process of pancreatic enzyme adaptation to the diet may
involve hormones, products of digestion and the cholinergic system. Our approach to the phenomenon was to study the role of the cholinergic system by looking at the effect of total vagotomy on the adaptation process. We have reported that enzyme adaptation to the carbohydrate and protein-rich diets occurred in the pancreas after vagotomy, as it does in normal rats. These results clearly showed that the cholinergic system is not involved [1]. Among the possible mechanisms proposed to explain this adaptation was a humoral one mediated by products of digestion. In an effort to test this possibility, we determined if parenteral glucose or essential amino acids might totally or partially explain the phenomenon. The data indicated that intraperitoneal (i.p.) glucose administered to rats fed a protein-rich diet significantly increased pancreatic amylase contents; such an increase did not occur with a sugar-rich diet. However, it was also shown that i.p. injection of amino acids had no effect in sugarrich fed rats on neither amylase nor chymotrypsin. Finally, insulin did not seem to be involved in the adaptation process since its administration during the adaptation period to both diets resulted in amylase content reductions with no effect on chymotrypsin contents. It remains unknown how i.p. glucose increases amylase in protein-rich fed rats [3]. Since most of these studies on pancreatic enzyme adaptation to diets were performed on rats, we decided more recently to look at what was happening in large animals, such as horses and roe deer. In horses, the pancreatic tissues were collected within 30 min of euthanasia from 7 adult horses. The data indicated that for each enzyme studied (amylase, lipase, elastase, trypsin and chymotrypsin), the concentration (units/mg DNA) for each enzyme, the cellular concentration in pancreatic tissue was consistent among horses. Interestingly, compared with the other enzymes, lipase was
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[email protected]. https://doi.org/10.1016/j.advms.2019.11.002 Received 18 June 2019; Received in revised form 5 September 2019; Accepted 1 November 2019 1896-1126/ © 2019 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.
Advances in Medical Sciences 65 (2020) 46–64
J. Morisset
treatment, the secretory responses to urecholine were increased but when secretion was expressed as a percentage of enzyme released in the medium with regard to the initial amount present in the gland before the incubation period, data suggested that corticosterone did not modify the maturation of the secretory process itself nor the cholinergic receptors [12]. This response to cholinergic pancreatic stimulation was also studied in cases of rats’ early and delayed weaning [13,14]. Access to solid food as early as 12 days as the unique source of nutrient resulted in increased secretion of amylase and chymotrypsinogen (ChTg) in response to urecholine. If, however, the expression of the enzyme output is calculated as a percentage of enzyme release with regards to its tissue concentration, then we can see that early weaning did not really modify the secretory pattern of enzyme release by their pancreas in response to a cholinergic stimulation [13]. If, however, pups are fed maternal milk only until they reach 27 days of age compared to pups weaned at 21 days with their secretion expressed as percentage of the amount of enzyme released over the total tissue content, the enzyme output in response to urecholine is significantly reduced from day 25 in pups kept on maternal milk only [14]. In higher mammals, pancreatic secretion in conscious suckling piglets was studied by Pierzynowski et al. [15] and by Zabielski et al. [16] in newborn calves. In this species, data indicate that periodic activity of the exocrine pancreas exists in neonatal calves soon after birth and local neural intestinal CCK-A receptors could be partly responsible for the modulation of neonatal calf pancreatic secretion.
predominant (1.090 U/mg DNA), followed by amylase (59 U/mg DNA). The three proteolytic enzymes were detected in small concentrations (1–10 U/mg DNA). The predominance of lipase contrasted to findings in other species, such as pig, rat and calf, particularly with regard to amylase. These findings support use of fats, rather than soluble carbohydrates, as the primary source of extra calories [4]. The physiology of the exocrine pancreas remains quite unknown in wildlife mammals because of either a lack of interest or a relative inaccessibility to the gland. In our study, we compared the pancreatic enzyme contents of bovine milk-fed only ruminant calves and roe deer fed rich easily digestible plant cell contents. In this study, we have compared the cattle and the roe deer which are two species with the closest frame of phylogeny, as shown in their karyotypes. Our data indicate that except for lipase, specific activities of chymotrypsin, elastase I and II, and trypsin were always greater or similar in roe deer than in the calf, and more so for chymotrypsin and elastase II, with 5- to 8-fold increases. In the roe deer, there was no difference between young and old animals for all the enzymes, except for chymotrypsin. It appears from our data that the bovine species could have lost some digestive possibilities when compared with roe deer [5]. The synthesis and availability of the potent CCK receptor antagonist MK-329 greatly helped investigators in their evaluation of the real physiological effects of CCK on the pancreas. In our laboratory, we used this inhibitor to study the role of CCK in the induction and maintenance of dietary protein-stimulated pancreatic growth in rats [6]. The inhibitor was given subcutaneously (s.c.) twice a day at 1.0 mg kg-1 (0800 h) and 1.5 mg kg-1 (1700 h) for 21 days to rats either fed a 5% or 70% protein diet. The data indicated that the switch from the 5% to the 70% protein-rich diet significantly increased total DNA contents, thus inducing hyperplasia, and this effect was not blocked by MK-329. However, the inhibitor prevented pancreatic hypertrophy and suppressed it significantly after one week of feeding 70% casein. The failure to reduce pancreatic hyperplasia may depend on the dose of the inhibitor given or its route of administration. This possibility was later verified [7] when the MK-329 inhibitor was given intravenously (i.v.) at a dose of 0.5 mg-1 h-1. In that study, feeding 70% casein significantly stimulated pancreatic hyperplasia and tissue hypertrophy, effects totally prevented by MK-329. These data indicated that endogenous CCK released by the high protein diet [8] is the major factor responsible for pancreatic growth induced by a high protein diet.
2.2.2. Characterization of the pancreatic muscarinic receptor To explain this maturation process of the pancreatic cholinergic response and its perturbation under certain physiological conditions, we decided to study the pancreatic muscarinic cholinergic receptors to demonstrate possible variations in their affinity constant or their concentration to understand variations previously observed in secretion studies. We first used radiolabeled (3H)-quinuclidinyl benzilate (3H-QNB), an anticholinergic drug, to characterize the pancreatic muscarinic receptor in a binding assay. The pancreatic receptors consist of a single population of saturable sites when studied on pancreas homogenates. The receptor concentration of binding sties was estimated at 100.8 fmol/mg protein (fmol/mg prt) with an apparent KD of 1.81 × 10-10 M. The data also presented for the first time a correlation between ligand binding and physiological secretory responses [17]. In a more elaborate study, we established a great correlation between cholinergic agonists and antagonists in stimulating and inhibiting amylase release and their respective potency in reducing (3H)-QNB binding to pancreatic membranes [18]. In order to better characterize the relationship that exists between muscarinic receptor occupation and enzyme secretion, we switched from pancreatic homogenate to pancreatic acini to evaluate binding studies and enzyme release concomitantly [19]. This study confirmed that the muscarinic receptors found in a pancreas homogenate are also present on freshly dispersed acini, and also that they are involved directly in enzyme secretion. These receptors exhibit and apparent KD value of 3.09 × 10-10 M with binding capacities of 2605 fmol/mg DNA. Using acini, we were able to establish that the receptor population can be divided into two classes of agonist binding sites: a population of high affinity sites and one of low affinity sites. It was also found that in response to carbamylcholine (Cch), maximal stimulation of amylase release occurred with 40% of receptor occupation while occupation of the remaining 60% caused a progressive decrease in stimulation of amylase release. These data suggest that the physiological response of the pancreatic acini to a muscarinic agonist would involve the high affinity site population. Our next effort was to evaluate the development of the pancreatic muscarinic receptor population with age and its relation to the development of the secretory process. We were able to establish that the
2.2. Control of pancreatic enzyme secretion 2.2.1. Development of the pancreatic secretory process In the 70s, our laboratory got interested in studying the development of the secretion of pancreatic enzymes in response to a cholinergic stimulation. Our data indicated that rat pancreata from 18 and 20-day embryos release a very high percentage of their enzyme content and do not respond to cholinergic stimulation. However, 3 days after birth, pancreata showed identical response under basal and stimulated states. The secretory response to urecholine was apparent later after birth and subsequently increased from day 13 until weaning and decreased thereafter when data are expressed as percentage of enzyme release over total content. The absence of response from fetal, newborn and 3day old pancreas could result from nonspecific leakage which could not be influenced by atropine nor dinitrophenol. However, incubation of newborn pancreas at 4 °C reduced the enzyme losses to 5% [9]. These data were later confirmed by Doyle and Jamieson [10]. In order to evaluate if corticosterone given in vivo can modulate the secretory response to a cholinergic agonist in vitro, corticosterone treatment was given in vivo because it had previously been shown that it elicited premature development of the pancreatic enzymes in young rats before weaning [11]. The in vivo corticosterone treatment resulted in immediate increases in pancreatic tissue concentration of amylase, lipase and chymotrypsin in 5 to 21-day old rats. As a consequence of these increased concentrations of enzymes following the steroid 47
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Modification of pancreatic intracellular calcium has been shown to alter subsequent secretory responses to agonists. Furthermore, the ionophore A23187 has been shown to regulate intracellular calcium. We then verified if its addition to the cholinergic agonist Cch in an incubation medium altered its secretory response and the muscarinic receptor characteristics and populations. The data indicated that addition of the ionophore reduced maximal amylase release, increased the maximal effective concentration of agonist and dramatically impaired the agonist's capacity to induced enzyme secretion at low concentrations. It also abolished the inhibition of secretion observed at high agonist concentrations. However, all the modifications in the secretory process brought about by the ionophore did not result from alterations at the level of the muscarinic receptors. Indeed, the ligand binding characteristics of the receptor were not modified; the concentration of receptors 5476 fmol/mg DNA and the apparent KD (2.17 10-10 M) were similar. Furthermore, the agonist binding characteristics of these receptors also remained unaffected in the presence of the ionophore. These data therefore indicated that the decreased responsiveness of the pancreatic acini to carbachol in the presence of the ionophore A23187 did not involve the receptor but should rather be considered as a defect occurring at a site or sites ulterior to the initial recognition step of the agonist by its receptor [26]. To evaluate if the pancreatic secretory response to a cholinergic stimulation and the muscarinic receptor population were affected during acute pancreatitis, this disease was provoked by multiple CAE s.c. injections for 2 days. During the course of pancreatitis induction, Cch showed decreased potency for stimulating amylase release early in the treatment and decreased efficacy appeared later. While these changes occurred on enzyme secretion, progressive reductions occurred in muscarinic receptor concentrations. Moreover, at the end of the 2day treatment, the pancreatic muscarinic receptors left had retained their binding characteristics for the agonist Cch, as the proportion of binding sites for each class remained similar in both groups [27]. Now, what would be the characteristics of the muscarinic receptor in an azaserine-induced cell carcinoma designated CA 20948 in comparison with normal rat acini? We found that the proportion of highand low-affinity agonist binding sites were similar for neoplastic and normal tissues. The density of muscarinic receptors was higher in neoplastic (200 fmol/mg prt) than in normal pancreas homogenates (80 fmol/mg prt). Interestingly, the muscarinic binding sites of the neoplastic and fetal pancreas showed similar KD values which were higher than those found in the normal pancreas [28]. In another type of cancer cell, the AtT-20 mouse pituitary tumor cells, the muscarinic agonist carbachol markedly decreased the stimulatory effect of forskolin, an adenylate cyclase activator, on both cyclic adenosine 3′:5′-monophosphate (cAMP) and adrenocorticotropin hormone (ACTH) formation. On these cells, analysis of QNB binding showed a straight line with a receptor density of 116 fmol/mg prt which was 50% less than that observed on the CA 20948 acinar cell carcinoma [28]. In this system, the muscarinic receptors are associated to the inhibition of cyclic AMP production and ACTH release [29]. Instead of using s.c. injections of a drug over a long period of time, we tried the Alzet mini-pumps for a constant delivery of the muscarinic antagonist N-methyl-scopolamine (NMS) at a dose of 25 mg kg-1 day for 14 days, compared with saline in the mini-pump to study amylase release and muscarinic receptors. Our data indicated that this NMS treatment caused supersensitivity of the acinar cells to Cch stimulation in vitro and an increased number of binding sites with no change in the affinity of high- and low-binding site populations. However, our study showed that the presence of Alzet mini-pumps filled with saline placed in the abdomen of the rats caused by itself desensitization of the secretory response to Cch. Our data also indicated that osmotic minipumps should not be used i.p. to study neural cholinergic control of pancreatic enzyme secretion [30]. Finally, looking at the gut distribution of the muscarinic receptors, we found that the highest concentration (fmol/DNA) of QNB binding
muscarinic receptors are present in rat fetal pancreas; their maximal concentration is attained at the age of 30 days with a significant decrease observed in one-year-old animals. Also, the affinity of the receptor does not change with age. Of interest is the fact that the secretory response to a cholinergic agonist parallels that of the receptor population. It is then obvious that at all ages, from 30 days after birth onwards, the maximal secretory response of the exocrine pancreas to a cholinergic agonist mobilizes the same proportion of the total population of binding sites [20]. Looking at the maturation of the muscarinic agonist receptors, it was observed that the affinity of QNB binding to the muscarinic receptors found in the pancreas of 21-day fetal and adult rats remained the same. It was also demonstrated that the two populations of Cch binding sites were present during all the developmental periods studied with comparable affinities. Moreover, it was established that the ED50 of bethanechol-stimulated amylase secretion did not change either with age. These data confirm our belief that the population of high-affinity site muscarinic receptors is involved in carbamylcholine-induced maximal enzyme secretion [21]. While studying the pancreatic muscarinic receptors, we asked ourselves if their development with age paralleled or not with the enzymes involved in the hydrolysis of acetylcholine (Ach), the agonist of the muscarinic receptor. We were able to identify two different cholinesterase activities, a true acetylcholinesterase and a pseudocholinesterase with their ratios varying with age: the specific activities of both enzymes decreased sharply at weaning. However, no correlation could be established between the development of acetylcholinesterase activity and the muscarinic receptor [22]. 2.2.3. Modulation of the pancreatic muscarinic receptor We have first investigated the effects of early and delayed weaning on the development of the pancreatic muscarinic receptors. It was demonstrated that the affinity of the QNB antagonist for the receptor was not affected by neither early nor delayed weaning. However, early weaning at 12 and 14 days significantly increased the concentrations of the receptors, while delayed weaning caused slight non-significant reductions [23]. In rats treated for 14 days with high doses of bethanechol (12 mg kg1 day-1), this treatment resulted in a 4-fold decrease in sensitivity of the pancreas for amylase release in the presence of Cch with a shift in the EC50 values from 0.69 μM to 2.9 μM, accompanied by a 42% reduction in receptor concentrations from 3360 to 1930 fmol/mg DNA. Also, the shift in the dose-response curve of amylase secretion was accompanied by modifications to the high- and low-affinity forms of receptors. While the maximum number of high-affinity sites remained the same, their affinity was greatly decreased from 0.24 to 6.1 μM. The affinity of the low affinity sites was moderately decreased from 34 to 150 μM, but there was a large drop in their numbers from 2620 to 890 fmol/mg DNA. These results suggest that the shift in the amylase dose-response curve to Cch could be coupled with the observed change in the affinity of the two agonist forms of the receptor [24]. Looking at this modulation process, we also investigated the trophic effects of caerulein (CAE) on the pancreas and if trophism affected the secretory response to a cholinergic agonist and the muscarinic receptor itself. First, CAE treatment for 2 days caused pancreatic hypertrophy while its administration for 4 days caused hyperplasia of the gland. After 2 days of CAE, the concentration of muscarinic receptors per DNA increased by 57% over control without modification of the receptor affinity for the ligand QNB. Such an increase involved mainly the low affinity sites for Cch; their concentration returned to control levels after 4 days of treatment. Interestingly, the functional capacity of the acini was significantly increased after 2 days as amylase release (U/mg DNA) was significantly increased with a substantial reduction in the sensitivity to the secretagogue. After 4 days of CAE, the functional capacity returned towards control values, but the sensitivity remained decreased [25]. 48
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pancreatic secretion desensitization in response to CCK and Cch, the biochemical and pharmacological properties of the InsP3 receptor were not affected with similar Bmax and KD values; the Ca2+ channel property of the InsP3 receptor was not modified, but the quantities of InsP3 formed in response to Cch and CAE were significantly decreased in both desensitized groups; these decreases were not due to increased InsP3 turnover. These modifications observed in the Ca2+ response were related to a decreased formation of InsP3 from PtdInsP2 hydrolysis caused probably by PLC as a target [38]. Dissection of the inositol phosphate produced after acini stimulation indicates that under control conditions, Cch and CAE both stimulated the production of I(1,4,5)P3, I (1,3,4)P3 and I(1,3,4,5)P4. However, following Cch desensitization, the production of the three phosphoinositides could not be stimulated above control values by Cch. However, after CAE desensitization, the CCK analogue kept its potency in increasing the three inositol phosphate releases above control values contrary to what was observed after Cch desensitization. These data point out that desensitization by Cch and CAE affects differently PLC [39]. To close the loop on our experiments studying the desensitization process of the pancreatic secretory responses to Cch and CAE, we investigated if desensitization could reduce phosphoinositide turnover and, above all, PtdIns(4,5)P2 synthesis [40]. Desensitization does not seem to result from a decrease either in total phosphoinositide (PI) or in specific PtdIns(4,5)P2 synthesis, which is needed for inositol triphosphate and DAG production. However, our study revealed a preferential activation of pancreatic phosphotidyl inositol-4P,5 kinase over that of PtdIns3-kinase by Cch while the response to CAE indicates no effect on inositol incorporation into PtdIns(4,5)P2, whereas inositol was preferentially incorporated into PtdIns(3,4)P2 and PtdIns(3,4,5)P3. The CAE-specific activation of PtdIns3-kinase could be related to its trophic effect as previously suggested, since this enzyme was neither activated by Cch nor involved in pancreas growth [41].
sites was in the gastric antrum, followed by the fundus, the fore stomach, the colon, the rectum and then the pancreas [31]. 2.2.4. Desensitization of the pancreatic secretory response The phenomenon of diminished response of an organ or gland subsequent to the initial action of an agent has been observed in some cellular system [32] and can be viewed as a protective cellular regulatory mechanism that can modulate the cell's response to wide variations in the concentration of the agonist. We have studied the desensitization process of amylase release by Cch and found that preincubation of pancreatic acini to Cch at 1 μM and greater, showed shifts to the right in the subsequent Cch dose-response curves of amylase release. This desensitization had not recovered after a period of 3 h. Desensitization happened independently of the calcium concentration in the incubation medium. After desensitization, the only changes observed were a significant decrease in the high-affinity and an equivalent increase in that of the low-affinity receptors. In such conditions, the subsequent secretory response to CAE was only slightly modified, whereas amylase release in response to the phorbol ester 12O-tetra-decanoylphorbol-13-acetate and to the ionophore A23187 was not altered. This study indicates that short-term desensitization with a cholinergic agonist is relatively specific to muscarinic agonists, causes changes in the muscarinic receptor high and low affinity concentrations, but does not alter intracellular steps after calcium mobilization or PKC activation known to be involved in the secretion process [33]. So far, we have looked at the desensitization process by Cch to a second stimulation by CAE, two stimuli of the acinar cells associated with the phospholipase C (PLC) activation. In this study, we looked at the subsequent secretory response to secretin, a hormone involved in adenylate cyclase activation. After Cch preexposure, the pancreatic acini exhibited secretin sub-sensitivity only at secretin concentrations above 10-8 M. Recovery was not observed after 3 h. The adenylate cyclase pathway remained unaltered because cholera toxin, forskolin and 8 Bromo Cyclic AMP (8-Br-cAMP) still induced normal amylase release. The cyclic AMP production remained unaffected as well as the inositol phosphates (InsP) InsP1, InsP2 and InsP3. This sub-sensitivity to secretin after Cch desensitization appears to result from modifications at post-second messenger levels [34]. Direct activation of PKC by phorbol 12-myristate-3 acetate (PMA) as pancreatic acini pre-treatment caused desensitization of Cch-induced amylase release in a concentration and time-dependent fashion. PMA also significantly reduced subsequent amylase release induced by CAE or secretin. Such desensitization by PMA did not lead to a decrease in PMA or A23187-stimulated amylase release nor did it cause changes in muscarinic receptor high- and low-affinity populations. The PKC inhibitor H-7 completely prevented the desensitization induced by phorbol-12, 13-dibutyrate (PDBu), another PKC stimulus, but not that induced by carbamylcholine (CBC). It is then suggested that PMA can induce desensitization of muscarinic receptor-stimulated amylase release by a different mechanism than that involved in muscarinic agonist-induced desensitization [35]. This desensitization process by PKC activation was further studied and data suggest that desensitization induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, and CAE used a common pathway involving PKC activation. However, desensitization by Cch is more complex as it is partially prevented by a PKC inhibitor [36]. In order to determine the role of the phosphoinositides phosphate in the desensitization process of the pancreatic secretory enzymes in response to cholinergic agonists, we established a pharmacological characterization of the inositol triphosphate receptor in the rat pancreas. Indeed, inositol 1,4,5-triphosphate (InsP3) hydrolysis, a product of PLC activation and diacylglycerol (DAG), both act as second messengers to mobilize intracellular calcium and activate PLC. The receptor InsP3 obeys to all the criteria for receptor function: high affinity, saturability, reversibility, specificity, and also the ability to trigger the biological response stimulated by CCK and Ach [37]. In the situation of
2.2.5. Role of adenylate cyclase Our group was the first to demonstrate the presence and stimulation of adenylate cyclase in pancreas homogenate by measuring the formation of cyclic AMP. The enzyme activity was very sensitive to the Mg2+/ATP ratio and could be increased by secretin and pancreozymin (PZ) (today CCK) [42]. This adenylate cyclase activation was confirmed later and found to be maximal within 10 s of exposure to CCK-PZ and directly associated to amylase release. Such an amylase release also occurred when pancreatic tissue was exposed in vitro to dibutyryl cyclic AMP (DbcAMP) [43]. It was also reported that DbcAMP given in vivo s.c. was associated with concomitant amylase and protein secretion in pancreas cannulated rats. In such cases, protein synthesis was not modified [44]. 2.2.6. Hormonal regulation of pancreatic enzyme secretion Somatostatin (SS) secreted in gastric and duodenal lumina indicates that both SS-28 (gastric 8%, duodenal 69%) and SS-14 (gastric 92%, duodenal 28%) are secreted from the stomach and the duodenum. These two forms of SS-28 and SS-14 were found in their respective organs and the secreted forms correlated with their relative proportions found in both organs. Inhibition of diversion-stimulated pancreatic enzyme secretion by intraduodenal infusion of SS is dose-dependent with a maximal effective dose of 24 μg kg-1 h-1 for volume and 48 μg kg1 -1 h for protein outputs. Contrary to its infusion into the duodenum, its infusion into the ileum (48 μg kg-1 h-1) did not inhibit but stimulated pancreatic secretion. It is suggested that the action of SS perfused intraduodenally is probably indirect via inhibition of endogenous release of CCK and/or secretin while its stimulatory effect when infused into the ileum could be through the inhibition of a putative ileal inhibitor [45]. Using an SS analog, SMS 201–995, it was shown that dose-inhibition curves of pancreatic response to i.v. infusion of SS indicate it had an IC50 of 0.7 μg kg-1 h-1 for protein secretion and 1.2 μg kg-1 h-1 for fluid secretion. When compared to SS, data showed that SMS is 20 times 49
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2.2.7. Arachidonic acid and pancreatic enzyme secretion The importance of arachidonic acid (AA) as a potential intracellular messenger has been emphasized by numerous studies in different tissues and cell types [54]. From previous observations, the signaling systems involved in AA production remain relatively undefined in pancreatic acini. To answer this question, our first approach was to further attempt to elucidate the signaling pathway(s) involved in AA release in response to CAE. AA can be generated via two major pathways, one of which is phospholipase A2 (PLA2) and the other involves a phosphatidylinositol 4,5-bisphosphate (PIP2) metabolite, DAG, which is catalyzed by DAG lipase to liberate AA [55]. Our initial data on this subject provide strong evidence that, when pancreatic acini are stimulated with CAE, DAG lipase is activated, leading to AA release. This process does not seem to involve the PLA2 or the phospholipase D (PLD) pathways, but rather the combined activation of PLC and DAG lipase [56]. When we studied the secretory response to Cch and the production of AA, we found that the secretory response to Cch involves PLA2 and DAG lipase as they both release AA. Therefore, after Cch stimulation, one pathway involves the sequential action of PLC, PKC and DAG lipase, whereas the other involves PLA2 activation. This effect of Cch on AA production differs from that of CAE which did not implicate PLA2 activation [56,57]. Finally, a comparative study indicates that amylase release from pancreatic acini can be stimulated by exogenous AA at a maximal concentration of 15 μM and was as efficient as Cch at 10 μM with maximal release of 10–12% of total enzyme content. Also, amylase release of about 6% was observed in response to direct activation of PLA2 by mellitin, a known activator of the enzyme, effect inhibited by a known inhibitor mepacrine. This study confirms the roles played by PLA2 and AA in amylase release from pancreatic acini [58]. We also looked at the effect of basic fibroblast growth-factor (bFGF) on AA release and the pathway involved. bFGF activated DAG lipase activity and stimulated intracellular accumulation and extracellular release of AA. Our data do not support any implication of PLA2 nor PLD in these intracellular events leading to AA release. Therefore, it seems that the sequential cellular events leading to AA mobilization include bFGF receptor tyrosine kinase (TRK) leading to PLC activation, generation of DAG which in turn activates PKC, which is responsible for DAG lipase increased activity leading to AA release [59]. bFGF is not known as a secretory agent for the pancreas but a growth factor. It is then proposed that the bFGF-activated AA release could be implicated in its growth effect and this is supported by the selective activation of DAG-lipase by CAE, another growth factor for the pancreas [56].
more potent than SS in inhibiting both secretions when both hormones are given i.v. When pancreatic juice was diverted from the intestine, plasma CCK increased significantly and these increases were inhibited by i.v. infusion of SMS. However, when SMS was infused into the duodenum, plasma CCK increased significantly. In this situation, it is possible that SMS may have strongly inhibited the release of one or many factors that inhibit CCK release, which was not the case when given i.v [46]. In search for such factors, we have demonstrated that i.v. and intraduodenal infusions of SMS significantly reduced plasma peptide YY (PYY) induced by pancreatic juice diversion. Furthermore, neutralization of PYY by a specific antibody significantly increased both fluid and protein secretion induced by pancreatic juice diversion. It is therefore suggested that pancreatic juice diversion stimulates not only hormones involved in stimulating pancreatic fluid and enzyme secretion, but also the release of inhibitory factors such as PYY, as demonstrated in our study. It is also indicated that endogenous PYY can be a physiological inhibitor of pancreatic secretion via a negative feedback regulation and that its release can be controlled by plasma and intestinal luminal SS [47]. We have previously shown that infusion of SS into the duodenum as well as its release into the upper gut resulted in inhibition of stimulated pancreatic enzyme secretion [45]. It was also shown previously that SS is present in the pancreatic juice [48]. Therefore, we have evaluated the presence and secretion of SS in the pancreatic juice during a basal fasting state, following a meal and after s.c. injections of CAE and secretin alone or in combination. Our data confirmed for the first time the presence of SS immunoreactivity in pure rat pancreatic juice. It was also shown that SS released into the pancreatic juice proceeded in the opposite direction of those of pancreatic volume and protein in response to a meal, CAE, secretin and their combination [49]. This decrease in SS presence in the pancreatic juice after stimulation may be through SS extraction by the pancreatic acinar cells [50]. What would happen to SS secretion under constant CAE and secretin stimulation for 10 days? Contrary to the SS secretory pattern after a single stimulation by both hormones indicating inhibition of SS release, constant infusion of the two hormones led to increased release of SS which developed in two waves, the first between days 2 and 7 of treatment, and the second one at days 8–10 with a 3-fold increase compared to day 7, resulting possibly from sensitization of the pancreatic delta cells (δ-cells) to CAE and secretin [51]. In the previous study, we have found relatively high quantities of SS released under basal and stimulated conditions in the range of 351 (basal) to 1887 (stimulated) ng/210 min of collection. We then looked at the origin and forms of this immunoreactive SS present in the pancreatic juice of cannulated non-anesthetized rats. A gel filtration profile of pancreatic extracts showed that SS is present under three different detectable forms in the rat pancreas: SS-14 was the most important (93%) followed by SS-28 (6%) and a pro-SS at 1%. The same procedure performed in pooled unboiled samples of pancreatic juice revealed the presence of a new high molecular weight form of SS of about 23 kDa. None of the forms previously detected in pancreatic extracts were found in the pancreatic juice. After boiling for 15 min under acidic conditions before gel filtration, a procedure known to protect SS from degradation, this procedure of boiling totally eliminated this 23 kDA molecule previously observed in the pancreatic juice. This 23 kDa molecule does not correspond to known forms of SS but could be an unknown thermosensitive compound interfering in the radioimmunoassay (RIA) protocol [52]. In search for this unidentified compound, we started its purification from freshly collected pure pancreatic juice using 125I-labelled Tyr-1-S14 as substrate. At the end, a SS-14 degrading activity was purified; this proteinase was concentrated more than 350-fold and was purified as a single protein with a molecular weight of approximately 29,000. It was finally identified as the rat pancreatic elastase II. The purified elastase II reveals a Km value of 89 μM for SS-14 and we suggest that this enzyme could be specifically involved in the regulation of biological functions of SS-14 in the gastrointestinal tract [53].
2.2.8. Circadian rhythm of exocrine pancreatic secretion Rhythmicity of exocrine pancreatic secretion has been previously reported and has shown circadian rhythms in the secretion of fluid, electrolytes, proteins and specific enzymes [60]. Our own study was undertaken to investigate circadian rhythms in the fasted and fed conscious rat by reducing the sampling period to 30 min and extending the total observation period, and to further describe the relationship among different pancreatic secretory parameters on a feeding state and light-cycle basis. Our results clearly indicate that over the 4- and 2-day period, there was a clear circadian rhythm of 24 h duration: for all measured parameters, secretory rates increased in the dark period and decreased during the light period. This major circadian rhythm was unexpectedly found to be superimposed on by a remarkably constant neurosecretory-like minor cycle of 2-h duration present in both fed and fasted states. The amplitude of the minor cycle was diminished by fasting. The outputs of fluid, total protein and amylase were found to be only modestly correlated with each other, whereas ChTg output was virtually completely independent of the others. The independence of the fluid and individual enzyme output reveals the exocrine pancreas to be a far more flexible and regulated organ than therefore thought [61]. In an attempt to understand how secretion of some enzymes is or is 50
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feeding conditions with equal efficiency [68]. Upon stimulation, the acinar cell appears to respond to the increased demand by enhancing the size of its organelles. Furthermore, the quantities of protein transported by each of the zymogen granules appear also to remain constant [69]. This secretory process initiated by cholinergic and hormone stimulation can be blocked by the intracellular action of specific organelle inhibitors. Tetracaine for example would inhibit the build-up of calcium concentration in the cytosol and thus impair initiation of enzyme secretion by increasing calcium efflux [70]. The final step in the secretory cycle involves the discharge of the zymogen granule content to the duct lumen and may involve microfilaments within the granule as well as in the cytoplasm. It has also been reported that cytochalasin B might affect the microfilament function and integrity. This alkaloid was therefore studied and shown to be a potent inhibitor of urecholine and CCK-induced amylase secretion but did not modify the response to DB-cyclic AMP. It did not change basal secretion. When DBcAMP was added to CCK, it prevented the inhibition of cytochalasin. Furthermore, cytochalasin had no effect on O2 uptake nor protein synthesis. Prevention of the inhibitory effect of cytochalasin B on enzyme secretion by DBcAMP suggests that the cyclic nucleotide would promote somehow a reassembly of the microfilament subunits or the plasma membrane microfilament architecture [71]. The most potent anticholinergic agent to inhibit urecholine-stimulated pancreatic amylase release has been shown to be 3Quinuclidinylbenzilate (QNB); it was shown to be ten times more potent than atropine, its inhibition lasted for at least 90 min after its removal from the incubation medium, and it is also very specific as it does not alter PZ-induced amylase release [72]. Despite the widespread medical and increasing non-medical use of cocaine, there have been non-controlled studies of its effects on the digestive tract. We did report a few years ago on its effect on amylase release in vitro. Cocaine in the incubation medium at 2 or 10 mM did not modify basal release of amylase. When combined to urecholine and CCK, it preferentially inhibited the secretory response to urecholine (-68%) when compared to CCK (-32%) [73].
not correlated, we investigated the effects of SS (SMS-201-995), atropine, and MK-329 (CCK receptor inhibitor) on the role of cholinergic and CCK-related systems and on the secretory relationship between five pancreatic digestive enzymes in rats. We observed that the relationship between paired enzymes significantly varied according to the treatment. The correlation between ChTg and the other enzymes was markedly modulated by MK-329. Our results also suggest that SMS is more of a gate-keeper in the regulation of exocrine pancreatic secretion and that the secretion of each digestive enzyme is individually regulated. It is also suggested that CCK and Ach are essentially initiators of secretory processes of the pancreas [62]. In order to understand the control of the circadian rhythm of the pancreatic enzyme secretion, we studied the behavior of the minor cycle of enzyme release under separated or combined infusion of atropine and MK-329 inhibition. Even though the total mean outputs of fluid, total protein and amylase decreased with antagonist infusion, the overall pattern of pancreatic secretion still exhibited a rise in the dark period and a fall in the light period in a circadian fashion. Also, atropine and MK-329 did not significantly alter the overall pattern of minor cycles of pancreatic secretion, even though total mean outputs markedly changed. The stability of the rhythm of minor cycle peak intervals in antagonist-infused rats suggests that the 1.8-h cycle of pancreatic secretion is independent of both cholinergic and CCK-related mechanisms, even though these antagonists have clear effects on overall secretory outputs [63]. As observed previously [61], in fed and fasted rats, ChTg and amylase secretions were weakly or not at all correlated. To explain this phenomenon, we investigated the influences of cholinergic and CCKrelated systems on the relationship between both enzymes in rats. Our data indicate that atropine did not alter the correlation between amylase and ChTg secretion. However, MK-329 infusion in fed rats induced a strong correlation between the two enzymes and that this MK-329induced switch from non-parallel to parallel secretion is accelerated and amplified by atropine. Our data led to the conclusion that nonparallel secretion of pancreatic digestive enzymes does occur, and it is regulated mainly by CCK-related mechanisms and cholinergic agonists acting essentially as amplifiers of the phenomenon [64]. In a final study, we investigated and defined contributing roles of cholinergic and CCK tones to the specific regulation of rat pancreatic secretion of digestive enzymes. Our results suggest that under fasting conditions, SS and atropine can neutralize basal pancreatic enzyme outputs, leading to a constitutive type of secretion characterized by parallel secretion of the digestive enzymes. Furthermore, it is proposed that under basal secretion conditions, Ach and CCK reaching the pancreatic acinar cells may act to dissociate pancreatic secretion of individual digestive enzymes originating from heterogeneous secretory granules. The data also suggest that SS is a very important regulator of pancreatic secretion, with cholinergic and CCK-related agonists acting to dissociate the secretion of individual pancreatic digestive enzymes [65].
2.3. Control of pancreatic enzyme synthesis Protein synthesis at the translational level is influenced by amounts and activities of several constituents, such as amino acids, activating enzymes, t-RNA, initiating and terminating factors, monosomes and polysomes. We then verified whether increases in pancreatic protein synthesis observed after feeding or bethanechol chloride administration were mediated by changes in components of soluble or precipitable fractions of a cell homogenate. Our data indicate that increases in protein synthesis associated with feeding were mediated by factors isolated in both soluble and particulate fractions. However, it seems that bethanechol administration was associated with increases in protein synthesis resulting from changes observed only in cytoplasmic fractions apparent only 15 and 30 min after stimulation. We also showed that microsomes from fed pigeons were about four times more active than those from fasted or fasted urecholine-treated pigeons [74]. As observed above, food deprivation resulted in decreased pancreas rates of synthesis. Also observed with fasting was a decrease in total RNA which can be explained by increases of free and total alkaline RNase activity by 46 and 52% in fasted pigeons for 3 days when compared to fed birds. Furthermore, there was a decrease in amounts of alkaline RNase-inhibitor activity. It is therefore suggested that reductions in pancreatic RNA content following fasting may be due at least in part to enhanced RNA degradation [75]. Still with the pigeon pancreas, we reported that the i.v. injection of CCK-PZ at doses of 5, 10, 20 and 50 IVY units/500 g body weight caused significant increases in amylase release into the duodenum. However, rates of pancreatic protein synthesis remained unaffected in
2.2.9. Negative control of pancreatic enzyme secretion We can assume that postprandial exocrine secretion depends on a balance between stimulation and inhibition initiated by intraluminal and postabsorptive effects of nutrients. Among the physiological inhibitors, there is the negative feedback mechanism, first reported by Green et al. [66], controlled by the pancreatic proteolytic enzymes present in the lumen of the upper gut. This process involves luminal CCK-releasing peptide, secretin-releasing peptide, diazepam binding inhibitor and monitor peptide [67]. This negative feedback also has hormonal support which involves SS, pancreatic polypeptide, PYY, and to a lesser extent, glucagon, ghrelin, leptin and bile and bile salts [67]. The secretory process under fasting and feeding conditions has been inhibited by atropine even in the pigeon pancreas as its administration to pigeons resulted in increases in amylase contents in either fasting and 51
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following feeding or hormonal stimulation is a physiological process and that the pancreas cannot increase its secretion and protein synthesis at the same time [83]. In diabetic rats, the administration of insulin was associated with increases in pancreatic amylase synthesis [84] and content [85]. We have examined and determined how insulin treatment could be responsible for increases in pancreatic enzyme secretion and synthesis. We first established that insulin added to an incubation medium had no direct effects on amylase secretion nor protein synthesis. However, we have confirmed that under hypoglycemia, amylase contents were decreased significantly after 2 or 3 h of insulin administration while protein synthesis was increased at the same time periods. Obviously, these two processes are not directly influenced by insulin because of its neutral effects in vitro. The changes observed in vivo have been abolished by vagotomy and when acid was introduced into the stomach. It is then suggested that these increases in amylase output and protein synthesis in response to insulin hypoglycemia could be due to vagal release of gastrin from the stomach [86]. When we looked at the secretion and synthesis of two individual enzymes, amylase and ChTg following intragastric administration of oleic acid - a CCK releaser - this stimulation evoked a non-parallel secretion of amylase and ChTg. It was also found that part of this phenomenon can be ascribed to a former non-parallelism in the synthesis of the two enzymes [87]. While atropine significantly increased pancreatic amylase content in fasted and in fed pigeons for up to 6 h after its administration [68], it also decreased significantly leucine incorporation into total protein by 36% as well as into amylase also by 35%. These decreases in protein and amylase synthesis were also accompanied by significant decreases in glucose and palmitate oxidation [68]. Furthermore, atropine administration for 2, 4 and 6 h significantly reduced uridine-3H incorporation into soluble RNA but not into nuclear RNA. It was then suggested that the previous decrease in protein synthesis resulted primarily from changes involving translational levels of protein synthesis [88]. When given in vivo, tetracaine significantly reduced protein synthesis by 23%, 30 min after its administration. As previously observed, in vitro CCK was significantly associated with significant decreases of protein synthesis which was further significantly decreased by the addition of tetracaine. A similar effect of tetracaine was observed when combined with urecholine. These data show that both secretagogues cannot overcome the inhibitory effect of tetracaine on protein synthesis and hence add another evidence that pancreatic protein synthesis might be controlled by factors related to the secretory process [89]. Finally, we have also shown that cocaine in vitro at 2 and 10 mM significantly reduced basal pancreatic protein synthesis by 15 and 49%, respectively [73].
fed and fasted birds while they were significantly increased only in fasted birds given 100 units of CCK-PZ. That particular study indicates that secretory and synthetic responses can be dissociated and presents a certain insensitivity of the pigeon pancreas regarding the control of synthesis of protein by CCK-PZ [76]. Moving from the pigeon to the rat model, we have demonstrated that fasting in rats for 24, 48 and 72 h, compared to fed rats, was associated with an 18, 21 and 28% decrease in phenylalanine incorporation into pancreatic proteins. Fasting for 48 h resulted in a 41% decrease in phenylalanine in purified amylase when compared to fed rats. Similar decreases in phenylalanine incorporation into microsomal proteins were noticed in fasted rats when compared to fed rats. These data clearly indicate that fasting was significantly associated with progressive decreases in protein synthesis in microsomal and zymogen proteins when compared to feeding [77]. We tried to understand the relation existing between protein secretion and their synthesis using an in vitro system of measurements. When protein secretion and synthesis were measured simultaneously in vitro, increases in protein synthesis was never observed with urecholine stimulation. However, if we used a protocol consisting of 4 steps, preincubation (10 min), stimulation (30 min), stabilization (40 min) and finally incorporation (30 min), it was possible to observe a 41% increase in protein synthesis only when secretion previously stimulated by urecholine had returned to its basal level. It is then suggested that the energy of the cell is oriented first towards secretion upon stimulation and that synthesis increased in vitro only when secretion had returned to basal level [78]. We then looked at this dissociation phenomenon between secretion and synthesis of protein when the stimulus was CCK-PZ secretin given in vivo and protein synthesis measured in vitro. The results showed that protein synthesis was initially decreased during the first 15 min after a single injection or infusion of both CCK-PZ and secretin during strong release of amylase into the incubation medium. However, 30 min after the hormonal stimulation, protein synthesis was increased while secretion into the incubation medium had returned to control levels. This increase in protein synthesis lasted for at least 1 h. These data confirmed what was previously observed in an entirely in vitro system [78] and suggest that pancreatic enzyme secretion and synthesis are dissociated in the early minutes following hormonal stimulation [79]. When CCK-PZ and secretin were given in vivo and protein synthesis also evaluated in vivo, the incorporation of phenylalanine into pancreatic proteins was significantly decreased after 5 and 11 min, then significantly increased at 17 min by 37% with a return to control at 45 min. This biphasic evolution was not caused by variations in the precursor pool specific radioactivity [80]. A similar decrease in protein synthesis was previously observed 55 min after intragastric intubation of oleic acid, a known releaser of intestinal CCK [81]. Whether protein synthesis is studied entirely in vitro, in vivo-in vitro or totally in vivo, its reduction in the early minutes after stimulation is a reproducible phenomenon. In an effort to demonstrate that the reduced protein synthesis observed after a first hormonal stimulation while enzyme secretion was increased can be reproduced after a second stimulation, we have investigated the effects of two hormonal stimulations of combined CCKPZ and secretin given at 15-min intervals. The results indicate that a second hormonal stimulation can induce a sustained secretion of enzyme and a new decrease in protein synthesis. It is then obvious that secretion and synthesis can be redissociated during the first minutes following the second stimulation [82]. In order to establish that enhanced enzyme secretion and a decrease in total protein synthesis in the early moments following stimulation by CCK-PZ and secretin is physiological, we verified the effects of feeding on pancreatic protein synthesis. Data showed that 45 min after a 15-min meal, protein synthesis is significantly reduced by 18% while significant increases of 18% and 19% are observed at 90 and 105 min. We then concluded that this reduction in protein synthesis observed
2.4. Control of pancreatic growth 2.4.1. Fetal and postnatal pancreatic growth Before we start this section, it is important to define some of the terms used to describe growth. Hypertrophy can refer to tissue and cells. Tissue hypertrophy represents an increase in total tissue mass measured as total tissue weight while atrophy describes a loss of tissue weight, always in comparison to controls. Cellular hypertrophy describes an increase in cell size established as cell mass (gland weight/ DNA) along with concentrations of proteins, specific enzymes and RNA/DNA. Cellular atrophy represents a decrease in these parameters, always in reference to controls. Gland hyperplasia describes an increase in cell numbers and can be estimated by measuring total DNA contents, thymidine incorporation into DNA/DNA, and labeling or mitotic indices. Gland aplasia refers to decreases in all the above parameters, always in comparison with controls [90]. When we study growth of an organ, it is important to establish as to whether or not stimulated rates of DNA synthesis measured in vivo or in 52
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and growth during fetal life until weaning is supported not only by detection of pancreatic gastrin during that time period but, above all, by the observation that gastrin and its CCKB receptor mRNA are coexpressed with equal intensity during the whole pre- and post-natal development process [97]. We had previously shown that gastrin administration to pregnant dams resulted in fetal pancreatic hypertrophy at the dose given [94]. It is now well accepted that the pancreas of suckling rats bears the CCKA receptor type [98]. Our first study on the trophic effects of CCKPZ on the pancreas was performed on nursing rats, right after weaning, in youngsters and in adults. The data indicated that pancreatic weight increased significantly at 26, 47 and adult ages after CCK-PZ, while that of suckling rats did not change. Hyperplasia documented from total DNA was significant at all ages but 11-day old pups and the ChTg concentration was the most affected at 26, 47 and adult ages when compared to those of amylase, lipase and protein. These data seem to indicate that the pancreas of suckling rats was rather insensitive to CCK-PZ, a sensitivity which becomes apparent after weaning [99]. This lack of effect of CCK-PZ on pancreas growth in suckling rats may have resulted from the purity of the hormone used. Indeed, with the availability of synthetic CCK analogues such as CCK-8 and CAE, comparable studies indicate that CAE given at a dose of 1 μg kg-1 every 12 h for 5 days led to pancreatic hyperplasia accompanied, however, with atrophy as cellular mass and concentrations of amylase and protein were decreased. In that same study, secretin given at the dose of 25 μg kg-1 surprisingly caused stronger hyperplasia and hypertrophy. However, when both hormones were given together, CAE reduced both hyperplasia and hypertrophy initiated by secretin when given alone [100]. A more complete description of the hormonal control of pancreatic growth at all ages of the rat appeared in a 2008 study [90]. Besides CCK and secretin involved in pancreas growth, there are some potential growth factors, such as insulin-growth factors I and II, hepatocyte growth factor (HGF) and basic fibroblast factor (bFGF). In the rat, expression of the 1GF-I mRNA was present in 19-day fetal pancreas, reached its maximal value at birth and decreased significantly after weaning. Expression of this factor in adult rat liver exhibits a 40fold higher level than the newborn pancreas. The presence of 1GF-I mRNA in the pancreas from 19-day fetus coincides with the development period in which typical adult-like acini are being formed with intense enzyme production and cytoplasmic zymogen growth accumulation in acinar cells [101]. The expression pattern of 1GF-II is quite similar to that of 1GF-I up until weaning, an expression which almost disappeared after weaning, this growth factor expression in the liver seems to follow that in the pancreas [98]. HGF has several activities in epithelial cells associated to its ligand binding to a dimeric transmembrane TRK receptor (c-met/hgf). We have characterized by Northern blot the HGF and c-met RNA expression in rat pancreas during development. Our findings show that simultaneous expression in both messengers occurs during the early development of the rat pancreas. Pancreatic levels of both mRNAs were higher between late fetal and 11 post-natal days and then decreased to a very low level at 26 days and stayed as such in the adult. These new data support the implication of this growth factor and its receptor in the early days of pancreas development and maturation [102]. Specific proteins are expressed in the pancreas during development and among them, we looked at clusterin and p8. Clusterin mRNA is induced in several tissues in response to most apoptotic stimuli. The protein has an antiapoptotic activity in these tissues. Pancreatic clusterin mRNA level was monitored from late fetal development until adulthood. Northern blot analysis revealed that clusterin mRNA expression was strong in late fetal life and remained high until day 11 postpartum. Levels decreased progressively with a minimum expression at 30–90 days postpartum. Apoptosis was shown to participate in the remodeling of the pancreas in the neonatal period. The increased expression of clusterin mRNA suggests that this protein is involved in the
vitro are comparable and to establish if these increased rates of DNA synthesis observed after stimulation in vivo are uniformly distributed into the various segments of the gland. It is also of interest to determine if the newly formed cells last and for how long, and which cell types are preferentially added to the gland. We have designed experiments to establish whether or not stimulated rates of DNA synthesis measured in vivo or in vitro are comparable and to demonstrate if increased rates of DNA synthesis observed following CAE treatment in vivo are uniformly distributed in the various segments of the pancreas. Our data indicate that the pancreas can incorporate thymidine in vivo and in vitro with the same efficiency under basal control or stimulated conditions by CAE. Also, it was shown that the growth promoting effect of CCK was uniform in all pancreatic segments looked at [91]. In another study, we evaluated whether hypertrophy and hyperplasia of the pancreatic acinar cells induced by CAE remained after termination of the 4-day hormonal treatment for up to 50 days. CAE treatment induced significant increases in pancreatic weight and contents of DNA, RNA, protein, amylase and ChTg, along with an increased number of acinar cells per acinus and zymogen granules per acinar cells. During the post-treatment period, the CAE-treated pancreas reverted to control values for all parameters except for DNA content and acinar cells per acinus. During that same post-treatment period, salinetreated pancreas exhibited constant growth to reach values of the treated pancreas after 50 days. These data indicate that already present and newly formed acinar cells can remain in place once the trophic stimulus is withdrawn [92]. When we looked at the various cell types and their growth after a 4day CAE treatment and a 50-day follow-up, we observed that with age, the proportion of acinar cells decreased significantly whereas those of the ductal and interstitial cells increased. Although CAE induced preferential acinar cell growth, it did not modify the proportion of this cell population with regards to the other cells. The growth-promoting effect of CAE was obvious from the specific increases in total DNA content and DNA synthesis. The labeling indices indicate that all cell populations except the endocrine system were stimulated to grow in response to CAE. Furthermore, all new cells remained for at least 50 days after termination of treatment. Also of interest, CAE induced uniform growth of the pancreatic tissue during intensive treatment. Furthermore, the normal growth rate of these CAE-stimulated cells was halted for the following 50 days while that of the control group cell population proceeded normally [93]. The novelty of our study on fetal pancreas growth and maturation resides in the fact that three important gastrointestinal hormones or their specific agonists were investigated throughout the entire pregnancy: CAE, gastrin releasing peptide (GRP) and pentagastrin, along with a glucocorticoid. In the fetuses, pancreas growth defined as hyperplasia was observed only in response to hydrocortisone, while aplasia occurred in response to CAE. Gastrin and the GRP antagonist were the most effective hypertrophic agents and atrophy was observed only with the CCKB receptor antagonist L-365-260. In summary, hydrocortisone caused proliferation of the fetal rat pancreas, whereas gastrin induced its differentiation and maturation probably through CCKB receptor occupation [94]. In piglets, alterations in digestive enzyme activity are seen as early as 24 h postpartum. Injections of growth hormone releasing factor (GRF) and/or immunization against SS during gestation also have several effects on the development of digestive enzymes in neonatal pigs. Amylase activity was not affected in newborn piglets from GRFinjected dams. However, piglets from SS immunized sows had a higher concentration of amylase per cell at one day of age, in such a case chymotrypsin was decreased. The lack of effect of GRF on pancreatic weight, DNA and protein [95] observed in this trial is in agreement with previous results in the swine [96]. The possible involvement of gastrin in rat pancreas development 53
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and CAE-infused (CAE) rats had their pancreatic juice returned to the duodenum. Two other groups had their pancreatic juice either totally diverted outside (DO) or returned into the ileum (DI). Juice was collected every 4 h for 4 days and pancreas collected and evaluated after 4 days. The average volume output per 4 h was 1563 μl in C, while around 5300 μl in DO, DI and CAE groups. Plasma CCK were significantly increased in the DO and DI groups. Pancreatic contents of protein, DNA and pancreatic weight remained comparable to those of the control in the DO group. However, increases in these parameters were significant in the DI and CAE groups when compared to C. These data support the existence of a strong correlation between elevated pancreatic protein secretion and growth of the pancreas and indicate that endogenous CCK can be designated as one of the major mediators. However, for this growth to happen, an adequate supply of essential amino acids is required to support the trophic effect of CCK because no growth happened when the pancreatic juice was not returned to the intestine as observed in the DO group [108]. We also evaluated the importance of the level of protein in the diet on CAE-induced pancreatic growth. The results confirm that pancreatic growth is stimulated by increasing protein concentration of the diet and indicate that a low protein diet, acting through a deficiency of dietary nitrogen and essential amino acids, limits the pancreatic trophic response to CCK or analogues [109]. With all these trophic effects of CCK and secretin on the rat pancreas, it is obvious to ask the question as to whether the human pancreas can grow or regenerate after an aggression. One study indicated that the human pancreas did not regenerate after partial anatomic resection [110], while the pig pancreas exhibited growth in response to bombesin after partial pancreatectomy [111]. Our study was designed to use the pig as a model to study pancreas regeneration in humans. Our results indicate that the pig pancreas can regenerate by itself without any treatment after partial pancreatectomy. In addition, the road leading to remnant regrowth involved cellular processes including inflammation, apoptosis and cell atrophy. Such a chain of events indicates that the pancreatic gland initially responses to the stress of surgery and puts itself at rest before initiating the regeneration process. Signs of regeneration are evident 28 and 30 days after pancreatectomy and regrowth is at this point characterized by acinar cell hypertrophy throughout the remnants. Increases in total DNA content in the remnant 30 days after surgery confirm hyperplasia [112]. Looking at the effects of gastrin on pancreatic growth, our data are consistent with the hypothesis that this hormone also has trophic effects on the gland similar to those of CCK. However, CCK is probably more important because it is more potent than gastrin for both secretory and trophic effects on the pancreas [113]. Besides the gastrointestinal hormones CCK and secretin, there are other hormones involved in the control of pancreatic growth. Our data suggest that pancreatic growth may be partly under control of glucocorticoids only during the suckling period, whereas hypertrophy of the pancreatic tissue can be obtained at all ages [114]. When combined with the glucocorticoid, the GI hormone produced pancreatic hypertrophy after weaning while potentiating the hyperplastic and hypertrophic action of hydrocortisone in suckling rats. Furthermore, hydrocortisone potentiated the hypertrophic effect of CAE after weaning. It is believed that these two hormones may regulate acinar cell proliferation and enzyme synthesis of the exocrine pancreas [115]. Among the other factors which could be involved in the control of pancreatic growth, somatocrinin was tested because it is responsible for growth hormone release and hypophysectomy was associated with reduced pancreatic weight as well as total RNA, protein, and amylase contents [116]. Therefore, we have tested the effect of GRF alone and in association with an SS antibody (ASS) on weaned rats for 14 days. Our data indicate that GRF and ASS interact on total DNA content. GRF alone shows a linear effect and the addition of ASS produced a quadratic effect with the effect of ASS the most significant with the 4 μg kg-1 of GRF. Therefore, GRF has a definite hyperplastic effect of its own on
control of that balance. The levels of clusterin expression followed the pattern of pancreatic cell proliferation and differentiation; this suggests that in the pancreas, the protein is a key factor in these processes as an antiapoptotic molecule [103]. In collaboration with the Iovanna group in Marseille, we have described the cloning, sequence and expression of a new gene, named p8, which is present in rat fetal pancreas and early after birth. High p8 mRNA levels are observed in the fetal pancreas, it is still observed in the newborn with a progressive decrease until day 35. At day 45, p8 mRNA levels drop dramatically and remain constant until day 90. The presence of high levels of p8 mRNA in pancreas of 19-day fetus coincides with the developmental period in which typical adult-like acini are being formed. During the early postnatal period, high levels of p8 mRNA were found, and its expression followed the rate of pancreatic cell proliferation. It is suggested that the p8 protein could play a role in pancreas development. These high levels of p8 mRNA levels were also observed in liver and small intestine [104]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) () is a key glycolysis enzyme present in every tissue. It was shown that this enzyme interacts with tubulin and microtubules with high affinity to the C-terminal region of the alpha subunit of tubulin. The microtubules have been involved in formation and translation of exocrine pancreatic zymogen granules. These observations led us to investigate whether the expression of GAPDH is regulated during pancreatic ontogeny. Its expression revealed its highest level in late fetal life and remained significantly higher than in the adult at birth and during the first 11 days postpartum; it dropped abruptly thereafter and became very low in 26 days and in the adult pancreas. These data indicate that maximal GAPDH mRNA expression is also highly strong, late in fetal life, coinciding with major changes in the developing pancreas [105], as did clusterin [103] and p8 [104]. 2.4.2. Pancreatic growth in adults: stimuli and inhibitors Previous studies indicated that a marked trophic response occurred within days after administration of CAE and secretin to rats; the time course and degree of interaction between these peptides were measured after 1–5 days of treatment. During that period, rats were treated with either CAE and secretin alone or in combination and the pancreatic weight, [3H]-thymidine incorporation into DNA, labeling indices and total DNA content were measured. Incorporation of thymidine into DNA increased 12-fold after 2 days of treatment with the combination of peptides. DNA content increased after 3 days and reached a level 1.8 times higher than control after 5 days. Autoradiography showed that two cell types, acinar and an unidentified type, were the sites of increased DNA synthesis. Acinar cell labeling indices were increased at 1 and 2 days (20-fold) and then fell; non-acinar cells showed an increase only after 2 days and maintained this increase after 5 days. Potentiation was found when CAE and secretin were injected together for all measurements. These data indicate that DNA synthesis in two cell populations is affected by CAE and secretin [106]. Most of the initial observations on the growth-promoting effects of CCK and its analogues were obtained from studies in which the trophic agents were administered exogenously. We decided to determine the role of endogenous CCK in the regulation of pancreatic and intestinal growth. We used the model of bile-pancreatic juice diversion (BPJD) in the rat known to cause hypercholecystokinemia. Our data confirmed the significant increase in plasma CCK after BPJD which was totally blocked by SS but not at all by L-364,718, the specific CCK-A receptor antagonist. Moreover, endogenous CCK released by BPJD caused pancreatic growth as evidenced by significant increases in total pancreatic weight and DNA content. The observation that this trophic effect was completely blocked by SS (reduced plasma CCK) and L-364,718, the potent inhibitor of the CCK-A receptor, strongly indicated that endogenous CCK was involved in pancreatic growth [107]. We then tried to establish if a correlation exists between chronic elevated pancreatic secretion and growth of the pancreas. Control (C) 54
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answer this question, we looked at two potential endogenous inhibitors: PYY - an ileocolonic peptide, and somatostatins - present in all organs of the gut. Our approach to study the role of PYY was to determine if the trophic effect of CCK on the pancreas could be counterbalanced by the release of PYY by CCK itself. Using the model of rats prepared with pancreatic and biliary cannulae, we showed that diversion of the pancreatic juice significantly increased plasma CCK as well as PYY within the first hour of diversion with a maximal effect at 2 h and sustained until 3 h. This effect of juice diversion on PYY release was totally inhibited by the specific and potent CCK receptor antagonist MK-329, indicating a true CCK-mediated effect on PYY release. However, infusion of CAE, bombesin or secretin at concentrations sufficient to increase maximal pancreatic secretion failed to induce PYY release. Our data suggest the involvement of endogenous CCK in PYY release, but this medication seems to require the participation of other yet unknown factors which might also be released by pancreatic juice diversion, and secretin is undoubtedly one of them [123,124]. We then tested PYY as an antitrophic agent for the pancreas. Its infusion in rats for 4 days at a dose of 400 pmol kg-1 h-1 had no effect on pancreatic weight and DNA and RNA contents. However, when combined with CAE at the same dose, it significantly reduced by 39, 35 and 45% increases in pancreatic weight, RNA and DNA contents stimulated by CAE, respectively. In conclusion, PYY can be considered as an antitrophic factor for the pancreas; however, its mode of action remains unexplained and could be in concert with SS [125]. Studies with SS as an antitrophic factor for the pancreas indicated that its administration at a dose of 100 μg kg-1 significantly depressed incorporation of 3H-thymidine into DNA, 3H-uridine into RNA and 14Cphenylalanine into total protein. In long-term studies for 5 days, rats were injected every 8 h with SS at doses of 11, 33 and 100 μg kg-1. Pancreatic contents of DNA, proteins and enzymes were significantly decreased by SS [126]. A follow-up study was designed to substantiate the antitrophic effect of SS on the pancreas under conditions where pancreatic growth is stimulated by CAE. As expected, CAE given for 2 and 4 days increased all parameters indicative of pancreatic hypertrophy and hyperplasia. Interestingly, an SS antibody significantly enhanced the effect of CAE, especially on DNA synthesis and contents after 2 and 4 days. Furthermore, the trophic effect of CAE was significantly reduced by SS with respect to hyperplasia. The study with SS and its specific antibody supports the hypothesis that SS may be considered as an endogenous growth inhibitory factor for the pancreas [127]. We then looked at the effect of SMS 201–995 (SMS), an SS analogue, on normal pancreatic growth as well as food consumption and body weight gain during its constant i.v. infusion for 7 days. We also verified if SMS could affect total pancreatic IGF-1 content and reduce plasma levels of CCK and IGF-1, two known growth factors for the pancreas. SMS was infused at 5 μg kg-1 h-1. After 7 days of treatment, body weight was significantly reduced by 8.8%; this weight loss cannot be ascribed to reduced food intake. SMS caused significant reductions in pancreatic weight accompanied by decreases in total DNA and RNA while total pancreatic enzymes were increased due to inhibition of secretion under constant SMS infusion. At the same time, plasma CCK and IGF-1 were reduced while total IGF-1 pancreatic content was increased. It is suggested that SMS may reduce pancreatic growth by decreasing CCK and IGF-1 release and their specific effects at the pancreatic level [128]. An additional possibility could be that SMS acts directly on the acinar cells which possess the SS receptor [129]. Epidermal growth factor (EGF) given to rats at a dose of 10 μg kg-1 did not alter pancreatic weight or protein content; however, it significantly inhibited DNA synthesis, total DNA content and increased concentrations of amylase, ChTg and proteins. To our surprise, EGF potentiated the action of CAE on pancreatic weight, protein content and ChTg concentrations. CAE, conversely, reversed the inhibitory effect of EGF on thymidine incorporation and total DNA content. This
the pancreas with a potentiation effect of ASS at the intermediate dose of GRF [117]. In order to establish that the trophic effects observed following exogenous hormone treatments are physiological, one must demonstrate that they can be reproduced under normal physiological conditions whereby the endogenous release of these hormones is observed. Hyperphagia observed during pregnancy and lactation would satisfy such a physiological condition. Our data indicate that during pregnancy and lactation, important changes occurred in the pancreas. Indeed, hypertrophy of the pancreatic tissue was first seen during pregnancy and sustained during lactation and was followed by hyperplasia late during the lactation period and the first two weeks after weaning. This study points out that hyperplasia of the pancreatic tissue can occur under physiological conditions whereby food intake is tremendously increased naturally without incitation [118]. Is this phenomenon of increased pancreatic growth during gestation and lactation specific to the rat species? To answer this question, we looked at the pancreas development during pregnancy, lactation and post-lactation in sows. Our study, to our knowledge, is the first report in large animals aimed at determining hypertrophy and hyperplasia of the pancreatic gland during a full reproductive cycle. The data indicate that the pancreas of the gestating and lactating sow is capable of physiological adjustments during the reproductive cycle. The most evident change remains its hyperplasia occurring late during lactation and significantly after weaning. Usually, hyperplasia follows hypertrophy as observed in rats [118], but in this case, it was not evident. The changes observed in the pancreas can be related to increased food intake during lactation and may be mediated by endogenous release of gut hormones still to be identified [119]. What would be the short and long-term effects of 50 or 75% proximal small bowel resection on the function of the exocrine pancreas? After 4 weeks, the pancreatic mass (weight/DNA) was slightly increased, DNA content reduced non-significantly as well as protein and lipase concentrations while amylase and ChTg concentrations were significantly decreased in comparison with the sham-controls. Under these conditions, at 4 weeks after enterectomy, plasma secretin and gastrin concentrations were not modified, the same as total SS contents in the pancreas and the remaining intestinal mucosa. It therefore seems that at least 4 weeks after surgery the pancreas has stabilized its cell population as well as its enzyme contents [120]. We have looked so far at the effects of gut hormones on pancreatic growth. However, since secretion of the pancreatic enzyme is under the control of both the cholinergic and hormonal systems, what would be the effects of a bethanechol treatment for 7 and 14 days on pancreatic growth? Once bethanechol has been given at 2, 6 or 12 mg kg-1 i.p. once daily, after 7 days of treatment, pancreas hypertrophy was observed since protein, amylase and ChTg concentrations were significantly increased only after the highest dose. After 14 days of treatment, the 6 mg dose caused hypertrophy while the 12 mg dose caused both hypertrophy and hyperplasia. It is important to point out that large doses of the cholinergic agent were used in this study and that an acute administration of the 12 mg dose significantly elevated plasma gastrin concentration over the entire 4 h period of observation. It is proposed that gastrin may be the mediator, but we cannot exclude a direct effect of bethanechol [121]. To elaborate the effects of cholinergic stimulation on pancreas growth, we blocked the muscarinic agent effects by infusing for 14 days its antagonist, NMS, at a dose of 25 mg kg-1. This treatment resulted in pancreatic aplasia and hypertrophy. Hypertrophy can be ascribed partially to inhibition of enzyme release elicited either by blocking the acinus muscarinic receptor or the enteropancreatic reflex causing enzyme accumulation in the gland over a long period of time. Aplasia of the pancreatic gland in response to NMS supports nervous implication in the process of pancreatic growth, but a role for gastrin remains [122]. How does the gastrointestinal system control pancreatic growth? To 55
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stimulated by hormones and growth factors are produced by specific enzymes including adenylate cyclase, PLC, PKC and finally PLD involved in the breakdown of phosphatidylcholine (PC) with the production of phosphatidic acid (PA). This PA production would then provide an alternate pathway for the generation of second messengers derived from membrane phospholipids. In view of the potential of PA as a second messenger in the pancreas, we evaluated the involvement of PLD activation in PC hydrolysis as a response to CAE in isolated pancreatic acini. Our data indicated that PC degradation in pancreatic acinar cells in response to CAE is mainly mediated by PLD activation. It is also possible that CAE-induced pancreatic growth can be initiated through its specificity in stimulating PLD activity, then producing PA [138]. Phosphorylation and dephosphorylation of proteins on tyrosyl residues are important reactions involved in cellular activities, namely those associated with growth and differentiation. We have examined if TRK and phosphatase can be modulated by CAE infusion for 48 h, after pancreatic juice diversion and 90% pancreatectomy. Our data suggest that activation of TRK may be an important step in the growth-promoting effect of CAE. Such a proposal is strongly supported by the finding that activation of the particulate and crude TRKs was also observed after pancreatectomy, in a regenerating pancreas in progress and that these enzymes were inhibited by SMS, a potent antigrowth factor to the pancreas. It is also proposed that CAE-activated zymogen granule protein tyrosine phosphatase activity is mostly related to increased enzyme secretion, as corroborated by comparable increases in response to carbachol and pancreatic juice diversion and inhibition of CAE-activated zymogen membrane enzyme by SMS [139]. Binding of CCK to isolated rat pancreatic acini reveals two classes of binding sites: a very high-affinity site and one of lower affinity [140]. We were interested to determine which class of CCK receptor was involved in pancreatic growth with concomitant TRK, PTdIns3-kinase and PLD activation. In order to do so, we compared the effects of CAE and JMV-180, a high-affinity CCK-receptor agonist [141] on pancreatic growth and activation of the three mentioned enzymes. Our data demonstrate clearly that CCK stimulates pancreatic growth via the occupation of the high-affinity CCK receptor and that a concomitant activation of TRK, PtdIns3-kinase and PLD could be among the early biochemical reactions required for initiation and regulation of this growth process [142]. In another study, the activation of TRK, phosphatidylinositol 3-kinase (PI3-kinase) and PLD, was used to delineate the importance of these three enzymes in physiological reactions related to secretion and/ or growth of the pancreatic gland. Our data provide direct evidence for the activation of pancreatic TRKs in response to exogenous and endogenous CCK. Such an activation can be partly associated with secretion of pancreatic enzymes because of the transient activation observed in response to carbachol. However, the sustained TRK activation still observed after 2 h, concomitantly with those of PLD and PTdIns3kinase specifically in response to CAE and not to carbachol, strongly strengthens the hypothesis that these three enzymes may be directly involved in cascade reactions leading to pancreatic growth [143]. We further investigated the effects of three growth factors: EGF, IGF-1 and bFGF on PLD activation. Our data present the description of a new signaling pathway through which EGF, IGF-1 and bFGF may operate to induce some of their specific effects on the pancreas; this pathway involved PLD activation in association with these growth factor receptors’ TRK activity [144]. We then looked at the specificity of PLD activation by CCK and PMA but not by Cch and the ionophore A23187 in rat pancreatic acini. Our data demonstrate that PMA caused PLD activation as evidenced by the release of choline into the incubation medium, the accumulation of PA and the production of phosphatidyl ethanol. This PMA-activated PLD was almost totally inhibited by staurosporine, the PKC inhibitor, but remained unaffected by manoalide, the PLC and PLA2 inhibitor. Under the same conditions, manoalide did not affect Cae-stimulated PLD
antagonistic effect may be due to the inhibitory action of CAE on EGF binding and endocytosis since EGF and/or its receptor must be internalized in the cell in order to trigger inhibition of cell proliferation [130]. 2.5. Intracellular events stimulated during pancreatic growth As seen previously in this review, CCK and its analogue CAE have important trophic effects on the pancreas. However, there is very little information available about the mechanisms by which CCK can regulate DNA synthesis and pancreatic growth. Polyamine metabolism in a variety of mammalian tissues has been activated by growth promoting hormones and factors and, more particularly, ornithine decarboxylase (ODC), responsible for the synthesis of putrescine [131]. Our study on the polyamine metabolism indicated that CCK caused maximal increases in total putrescine (319%), spermidine (63%) and spermine (50%) after 12, 48, and 96 h respectively after the beginning of treatment. This time period coincides with pancreatic hypertrophy and hyperplasia. Rates of pancreatic weight and DNA content increases were significantly correlated with total spermidine and spermine contents. The increases were seen first for putrescine, coincident with observed increases in protein and RNA contents, as well as with initiation of the S phase as evidenced by increase rates of DNA synthesis. These polyamines are needed for pancreatic growth since these growth parameters were reduced following inhibition of ODC by α-difluoromethylornithine (DFMO), a specific inhibitor of the enzyme [132]. The major effect of DFMO was observed on cellularity of the stimulated pancreatic gland as evidenced by its significant reductions in total DNA synthesis and total DNA contents [133]. As we just saw, inhibition of one activity by DFMO resulted in reduced pancreatic growth stimulated by CAE. Interestingly, a putrescine supplement during pancreatic growth initiation in the presence of DFMO totally prevented the loss of pancreatic DNA observed following joint CAE and DFMO treatment. These new data strongly support the suggestion that ODC and polyamines could be actively involved in the stimulated growth process induced by CAE [134]. If ODC is involved in induced pancreatic growth by CAE, are we looking at its activation and/or new synthesis of the enzyme? The enzyme was localized by the immunogold technique in the nucleus and rough endoplasmic reticulum (RER) of cells and the number of gold particles was increased in both organelles by CAE. Peak immunoreactivity was observed in nuclei 4 h after the 2nd CAE injection, whereas in the RER it occurred 8 h after the 3rd injection. These data show that ODC can be induced upon growth stimulation of the pancreas by CAE [135]. ODC and polyamines were also studied during the weaning period and a week after weaning at 21 days. Total DNA was already subnormal after one week of 500 mg kg-1 DFMO with a maximal reduction of 30% after 28 days. These decreases in total DNA contents were accompanied by significant reductions of 36 and 14% after 3 and 4 weeks of treatment, respectively. These data support the view that polyamines play an important role in cell replication and growth of the pancreatic tissue during the neonatal period [136]. Finally, we looked at the circadian variations in pancreatic ODC activity and time-course effects of CAE in fed and fasted rats. Significant circadian variations in ODC activity were observed with the highest values during the dark period and the lowest during the light period. CAE induced hypertrophy and hyperplasia of the pancreas in fed rats; increases in ODC activity preceded the rise in protein and DNA contents, 6 and 12 h after the first injection of CAE. ODC activity was very low in fasted rats and CAE treatment only induced a transient increase in ODC activity 12 h after the first injection. These findings demonstrate that nutritional status is an important factor in the regulation of ODC activity and thereby, in CAE-induced pancreatic growth [137]. Second messengers involved in the intracellular activities 56
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injuries, duct ligation injury and acute inflammation. We have specifically used the model of edematous pancreatitis caused by high doses of CAE (12 μg kg-1) for 2 days to induce pancreatitis. This treatment induced evident depletion of pancreatic protein, digestive enzymes and total DNA which persisted even after 13 days of recovery [149]. Using this model, we evaluated pancreas regeneration starting on day 3 after the 2 days of pancreatitis induction. We have examined 4 different treatments: rat with pancreatitis fed 20% casein without treatment, rat fed 50% casein, rats fed 20% casein supplemented with 1% SBT1 and finally, rats fed 20% casein receiving CAE s.c. at a dose of 1 μg kg-1, 3 times a day. Controls were fed 20% casein with saline s.c. Rats were killed after 5, 10 and 20 days of treatment. Pancreatitis resulted in significant decreases in pancreatic weight and contents of protein, amylase, ChTg, RNA and DNA. During the regenerative process, the CAE treatment increased all parameters to control values within 5 days and induced pancreatic growth thereafter. Soybean trypsin inhibitor (SBTI) restored the pancreas to normal after 10 days with cellular hypertrophy. The 50% casein diet gave a response similar to SBTI without hypertrophy. It can be concluded that exogenous CAE and endogenous release of CCK by SBTI and a high protein diet can accelerate pancreatic regeneration after an attack of acute pancreatitis since without treatment, the pancreas had not returned totally to control after 20 days for some parameters looked at [150]. The recovery of the pancreatic gland after CAE-induced pancreatitis seems to involve endogenous CCK release because recovery was accelerated by feeding SBTI or a high-protein diet [150]. To confirm that CCK release is really involved in the regeneration process, we blocked the CCKA receptor with its specific antagonist, L364,718, given s.c. twice a day at doses of 0.1 mg or 1.0 mg kg-1 for 3–13 days. The low dose of L364,718 did not prevent regeneration. After 13 days, regeneration was still incomplete but the 1.0 mg kg-1 dose of L364,718 strongly inhibited spontaneous regeneration. These data strongly suggest that endogenous CCK is an important and potent trophic factor in the regenerative process of pancreatic tissue following an episode of acute pancreatitis [151]. Insulin-like growth factors are important peptides involved in the regulation of cell growth and differentiation in many tissues. The ontogeny of IGF-1 mRNA in the pancreas indicates the presence of four major transcripts and their levels are much higher in the adult liver than in the adult pancreas. All transcripts of IGF-1 exhibit their highest expression during the first 3 days after pancreatectomy with a peak after the 2nd day. After 6 days, most transcripts were back to control values, apart from the 7.5 kb message. These increases in IGF-1 transcripts paralleled those of pancreatitis-associated protein (PAP) mRNA, a stress protein, during the first 3 days after pancreatectomy. During the course of a 2-day induction of acute pancreatitis, overexpression of IGF1 mRNAs was observed. The same two transcripts, noticed 6 days after pancreatectomy, regressed toward control values. Our observations strongly suggested that IGF-1 was actively involved in pancreatic cell growth and/or differentiation during normal fetal development and after partial pancreatic destruction and removal [152]. We previously observed that exogenous and endogenous CCK were involved in regeneration of the pancreatic gland after induced acute pancreatitis and pancreatectomy. But do these effects of CCK on pancreatic regeneration coincide with changes in the expression of the CCK-1 and CCK-2 receptors? It is interesting to notice that in the course of pancreas destruction during pancreatitis and early during the regeneration period, the gland overexpresses its CCK1 receptor mRNA along with re-expression of the silent CCK2 receptor mRNA whose expression needed the presence of exogenous CAE after the destruction period. These data clearly indicate that CCK is involved in the regeneration process of the rat pancreas and the expression of the silent CCK2 receptor mRNA suggests a partial return to fetal pancreas. It remains to be seen whether gastrin mRNA expression occurs simultaneously with the CCK2 receptor and, most importantly, in which cell
activation but staurosporine decreased PA accumulation in response to CAE only after 15 min. Calcium ionophore and carbachol in concentrations which stimulate enzyme secretion did not stimulate PLD activation in pancreatic acinar cells. On the basis of these results, we can conclude that pancreatic acinar cell PLD can be activated directly by CAE and also indirectly via a PKC dependent pathway. Furthermore, this PLD activity remains insensitive to a muscarinic agonist and to a calcium ionophore [145]. Three distinct forms of PC-PLD have been reported. One is dependent on the presence of oleate for expression of its activity, whereas the others, PLD1 and PLD2, require PIP2 as a cofactor and are inhibited by oleate. After purification, we reported that the pancreas exhibits three types of phosphatidylcholine-specific PLD. An oleate-dependent PLD activity requires the presence of mM concentrations of unsaturated fatty acids. This activity was found predominantly in a microsomal fraction. Another PC-PLD, rPLD1, co-localizes with the oleate-dependent PLD and was purified from the microsomal fraction. In rat pancreas, rPLD1, a protein of 120 kDa, is strongly activated by ARF1 and inhibited by oleate. Finally, rPLD2, a protein of 105 kDa, is associated with a microvesicular fraction. It is distributed throughout the pancreas with a preferential localization in cells in periphery of the islets. We do not know yet which PLD is activated by CCK and associated with pancreatic growth [146]. Finally, we tried to gather information on how CAE activates pancreatic TRKs, PTdIns3-kinase and PLD. The primary roles of CCK on the pancreatic acinar cells are directed towards secretion of the digestive enzymes and growth of the gland. The observation that TRK inhibition resulted in reduced amylase release stimulated by CAE supports the importance of tyrosine phosphorylation in the secretory pathway. The direct implication of PLC and PKC is also supported by the inhibitory effects of manoalide on PLC and of staurosporine on PKC, also resulting in inhibition of CAE-induced enzyme secretion. However, the fact that PLD and PTdIns3-kinase inhibition by wortmannin did not affect CAEinduced amylase secretion, clearly supports the data that these two enzymes are not involved in enzyme secretion since carbachol, a wellknown pancreatic secretagogue, did not activate these two enzymes. Our data suggest an association of PLD and PTdIns3-kinase activations with reactions leading to pancreatic growth [147]. We then looked at some signaling pathways activated in response to CCK in rat pancreatic acini leading to pancreatic growth. Unfortunately, this model of acini was inappropriate to study the biological events preceding mitogenesis and occurrence over hours and days. We thus selected the partial pancreatectomy model in the rat because it gives the opportunity to analyze the cell cycle dynamics of naturally synchronized cells in their normal environment. Our study thus evaluated mitogen-activated protein kinase (MAPK) activation and expression of cell cycle regulatory proteins after pancreatectomy to understand the cellular and molecular mechanisms involved in pancreas regeneration. After partial pancreatectomy, pancreatic remnants exhibited sustained p42/p44 MAPK activation within 8 h until 12 days. Cyclins D1 and E showed maximal expression after 2 and 6 days, coinciding with maximal hyperphosphorylation of pRb and Cdk2 activity. The expression of p15 vanished after 12 h, p27 disappeared gradually, and p21 increased early. The p27 complexed with Cdk2 dissociated after 2 days, whereas p21 associated in a reverse fashion. In conclusion, sustained activation of p42/p44 MAPKS and Cdk2 along with overexpression of cyclin D1 and E and reduction of p15 and p27 cyclin inhibitors occurred early after pancreatectomy and are active factors involved in signaling events which lead to pancreas regeneration [148]. 2.6. Pancreas regeneration after pancreatitis and pancreatectomy Although acinar cells of the adult exocrine pancreas have a relatively low resting mitotic activity, under certain circumstances they can divide rapidly. This is particularly true after partial resection, toxic 57
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protein is localized around the acinar cells and beta cells of the islets of Langerhans. In the adult pig and fetal human pancreas, the CCKA receptor protein was detected by Western blot. By immunofluorescence, its detection was possible only in the islets of Langerhans of the pig pancreas [158]. Because of the relative insensitivity of the pig pancreas to CCK observed either from in vivo studies [159] or from isolated perfused pancreata [160], it was important to confirm these observations using freshly dispersed pig pancreatic acini to determine whether the lack of sensitivity was the result of an occupation of the CCKA low affinity receptor sites, or simply because the pig pancreas possesses a large majority of functional CCKB receptors. The pancreatic acini isolated from pig pancreas are functional as they respond to Cch as do the rat acini; however, the response to very high Cch concentrations (10-5-103 M) did not present the previously observed inhibition of amylase release observed in rat acini. These pig acini remain insensitive to increasing concentrations of secretin as well as to occupation of the high CCKA receptor binding sites by JMV-180. However, the threshold concentration of CAE needed to induce amylase release was 5 × 1010 M with a maximal secretory response at 10-7 M. The secretory response to CAE only at very high concentrations was not blocked by the specific CCKA receptor antagonist MK-329. These data raise the question of whether the pig pancreas possesses any CCKA receptors on its acini. Looking at the expression of CCKA and B receptor mRNA, we found the presence of the CCKA message in adult rat pancreas while the rat brain expresses the B subtype. The A subtype message is also strongly expressed in the gall bladder. The pig brain expresses strongly the B subtype; the rat pancreas does not seem to express the B type, whereas the pig pancreas shows the strongest expression. The B type seems also to be present on the pig gall bladder. The presence of the CCKB receptor on the pig pancreas may explain the secretory response to CAE at very high concentrations [161]. Our data support that CCK could stimulate pancreatic secretion via an indirect effect through the release of Ach in the pancreatic intrinsic innervation [162]. Our next objective was to ascertain the status of the pancreatic CCKB/gastrin receptor regarding its presence and cellular localization in different species. Our data clearly indicate that the CCKB/gastrin receptor mRNA could not be identified in the rat pancreas by Northern blot but was detectable by RT-PCR. This B-type receptor was present in the rat pancreas at all ages in an equal proportion as a 80-kDa protein localized exclusively in the SS δ-cells in the islets of Langerhans. This Btype protein was also identified in mouse, pig and human fetal pancreas with its specific location in the SS δ-cells. The presence of the CCKB/ gastrin receptor in the rat pancreas does not correlate with that of gastrin throughout the development of the gland [163]. Due to their specific location on the islets’ δ-cells, the CCKB/gastrin receptor through CCK and/or gastrin could be important in maintaining the pancreatic δcell population at a normal density. Indeed, in CCKB/gastrin receptordeficient mice, the δ-cell population was reduced by 43% in the gastric antrum [164]. The presence of the CCKB receptor only on the δ-cells of the islets of Langerhans led us to investigate whether this observation was specific to the rat pancreas as identified as a universal phenomenon on this specific type of cell. Our study was initially performed to clarify the controversy in the literature about pancreatic localization of the CCKA and CCKB receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. We performed co-localization assays between the two CCK receptors and the pancreatic hormones - insulin, glucagon and SS. Our data revealed that the CCKA RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig and human pancreas but not in the SS cells. Conversely, the three CCKB RAbs tested - 9262, 9491 and GR4, identified the SS cells. The Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab9262. Finally, the specificity of Ab9262 for the pancreatic CCKB receptor was confirmed
type these expressions occur and for how long [98]. Expression of specific proteins and enzymes during acute pancreatitis has been observed. In the course of necrohaemorrhagic pancreatitis induced by 1% sodium taurocholate into the pancreatic duct, a strong signal of clusterin mRNA expression was observed after 6 h of pancreatitis, it reached a maximum level at 24–48 h, and then decreased progressively to undetectable levels as in healthy pancreas which does not express this mRNA. This expression is of similar intensity 24 h after pancreatitis induction, whether after CAE at a high dose or at different concentrations of sodium taurocholate (0.4, 1.0 or 4%). The protein also appeared in the pancreas and the pancreatic juice of rats with acute pancreatitis but not in normal pancreas. Such an expression of clusterin would be to provide protection against apoptosis [102]. Another protein, p8, is also strongly activated in pancreatic acinar cells during the acute phase of pancreatitis. It is expressed rapidly and specifically in response to cellular pancreatitis-induced injury. Its induction occurred almost similarly in edematous and necrohemorrhagic pancreatitis, indicating that it is maximally expressed even in response to a mild pancreatic injury. This p8 molecule is proposed as a putative transcriptional factor which can regulate pancreatic growth [104]. Expression of GAPDH exhibited a significant increase already 9 h after CAE-induced pancreatitis with its highest level of expression at 18 h. These data strongly suggest that GAPDH might be actively involved in pancreatic cell growth after partial destruction [105]. Parallel increases in TRK and PLD activities were observed from 6 to 48 h during pancreatitis induction by CAE and at the end of the resting period. Activities returned to control values during the regeneration period in the untreated CAE-pancreatitis groups. However, significant increases in both enzyme activities were observed in the pancreatitis rats after the first day of CAE treatment (low dose) during the regeneration period. After 4 days of CAE treatment, TRK activities returned to control values while PLD activity remained significantly elevated. These findings support the involvement of these two enzyme activities with the regenerative processes during the gland's destruction and regeneration [153]. Precautious expression of ODC mRNA, ODC activation and increased DNA synthesis [154] along with the close correlation between activated TRKs and PLD activation implies that these reactions are triggered during pancreas regeneration. Also observed during induced pancreatitis was the rapid induction of the Ki-ras message in parallel with that of ODC in both pancreatitis-saline treated and pancreatitis CAE-treated animals [155]. PAP has been recently found in the rat pancreatic juice during acute pancreatitis. Induced acute pancreatitis disturbed the gross histology and ultra-structure of the acinar cells with the formation of new intra-cellular fibrous material in the cytoplasm which was also found in the acinar lumen. PAP is almost absent from normal acinar cells but after acute pancreatitis it appears in rough endoplasmic reticulum; it is strongly present in normal and abnormal zymogen granules and it remains an important component of the fibrous material. Except for its exclusive presence in fibrous material, PAP is always co-localized with amylase in the other cell compartments. PAP has been suggested as a stress protein or an acute-phase protein induced upon cell insult [156]. 2.7. The pancreatic CCK receptor subtypes 1 and 2 or A and B The characterization of the CCK receptors, their localization in the gut, their biochemical characterization, their interaction with the guanine nucleotide binding proteins, their activation of second messengers, their occupancy and biological functions, as well as their modulation and ontogeny have previously been covered [157]. The presence and localization of the CCKA receptor have been evaluated in rat, mouse, pig and human fetal pancreas by Northern and Western blots and immunofluorescence techniques. In the rat, parallelism exists between development of the CCKA receptor mRNA and intestinal CCK mRNA with maximal peaks of expression during the suckling period. In the rat and mouse pancreas, the CCKA receptor 58
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original SS cell line to study SS secretion, the R1N–14B cells. To our surprise, we have established by Western blot that these R1N cells possess the two CCK receptor subtypes, CCK1 and CCK2. Occupation of the CCK1 receptors by CAE, a CCK analog, led to inhibition of cell proliferation, an effect prevented by L-364718, a specific CCK1 receptor antagonist. Occupation of the CCK2 receptors by gastrin had no effect on cell growth. Proliferation was not affected by SS released from these cells but was inhibited by exogenous SS. It would thus seem that SS released from these cells did not reach the concentration needed to inhibit their growth [175]. Next to the control of growth of these RIN cells in response to occupation of their CCK1 and CCK2 receptors, we studied SS release from them also in response to specific agonists of the two CCK receptor subtypes. These RIN cells contain SS and express both the CCK1 and CCK2 receptors as determined by immunofluorescence. These cells also synthetize CCK and progastrin in an amount estimated at less than 10 pg ml-1. This released amount was not enough to increase basal SS release over a 4 h period and was not significantly reduced by exposure to both CCK receptor antagonists L-364,718 (CCK-1R) and L-365,260 (CCK-2R). We also showed that both CAE and pentagastrin caused a dose-dependent release of SS. However, the response to appeared to be atypical of an occupation of the CCK1 receptor since we did not observe the classical inhibition at high concentrations of the agonist. We believe that here, CAE at higher concentrations occupies the CCK2 receptor. The SS secretory response to concentrations of CAE which stimulate amylase release from rat acini was dose-dependently inhibited by the specific CCK1 receptor antagonist L-364,718, while that to pentagastrin was also inhibited by the specific CCK2 receptor antagonist L-365,260. For a better characterization of the CCK-1R response, we used JMV180, a synthetic peptide which distinguishes high-affinity CCK-1R from its low-affinity receptors. The occupation of the high-affinity CCK-1R by CAE was confirmed by the sustained SS release in response to increasing concentrations of JMV-180. These cells also release more than 60% of their SS content by constitutive secretions confirmed by cycloheximide and brefeldin inhibiting SS synthesis and intracellular trafficking, respectively. These cells, in the end, are not representative of normal SS cells because they possess both CCK R subtypes and release SS through the constitutive instead of the regulated pathways. Finally, we agree that they cannot be used to study the role of CCK-2R occupation on their physiology [176].
in six different species: the calf, the pig, the horse, human, the dog and rat, and it always co-localized with SS but never with glucagon in these species. Our data then suggested the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCKA and CCKB receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in our study [165]. In the calf, pancreatic secretory studies indicate that both CCK receptor subtypes are involved [166]. Our study confirms by Western blot the presence of both CCK receptor protein subtypes in the calf pancreas. By immunofluorescence, the CCKB receptor co-localizes with the islets’ SS δ-cells as well as on ductal cells. We were unable to find any specific localization for the CCKA receptors with our multiple antibodies [167]. The absence of CCKA receptors in the calf pancreas, as shown in our study, supports an alternative mechanism to stimulate enzyme secretion in which CCK can stimulate neural Ach release possibly through the CCKA receptor from the vagus nerve containing CCK58 and CCK8 [168]. Such a system could control pancreatic enzyme secretion in higher mammals including man and possibly rodents. We then turned to the islets to determine the role of CCK on somatostain (SS) release via the CCKB receptor. To look at islets of control and diabetic rats, we had to modify the islets’ purification method to improve the yield of islets isolated. This new technique is described in reference [169]. With the clear demonstration that only the CCKB receptors are present on the δ-cells containing SS in the six species studied, we decided to show that the rat pancreatic islets can be used as a reliable tool to study if the presence of the CCKB receptor on the SS cells can be responsible for SS release. This possibility comes from the observation that with an isolated perfused pancreas, SS release was stimulated most efficiently by CCK4 and CCK5 at the high concentrations of 10 nM and 1 μM [170]. Our data clearly indicate that islets purified from fetal and adult human and rat pancreas express identical transcripts of CCKA RmRNAs. The fetal and adult human islets also exhibit a single CCKB mRNA transcript as in the rat islets. The SS mRNAs are identically expressed in islets of both species. The islets from fetal and adult human pancreas synthesize a 50 kDa CCKA R protein different in size from the 80 kDa protein present in rat islets. Both species' islets express a 80 kDa CCKB R protein. In fetal and adult islets from human and rat, the CCKA R colocalized with insulin and glucagon cells and the CCKB R with the SS cells. Finally, the rat purified islets are functional as they can release insulin and SS in response to glucose, forskolin and IBMX. However, to our great surprise, the δ-cells did not respond to two known CCKB receptor agonists, gastrin and pBC264, as both agents failed to induce SS release [171]. To explain this failure of SS release, it is possible that the also secreted islet amyloid polypeptide can impair SS release since it is a known endogenous inhibitor of islets’ hormone release [172]. It has been previously reported that during diabetes development, important modifications occurred including increased secretion, tissue contents and δ-cell population [173]. So, following this observation, we decided to characterize the changes in SS mRNA expression and contents along with those of the CCKB R in normal and diabetic rats and to determine whether insulin treatment can normalize the modifications observed during diabetes development. Our data indicate that diabetes is well established after 7 days; it is controlled by insulin and reappears after insulin cessation. Pancreatic SS mRNA expression and SS content were increased during diabetes, normalized during insulin treatment, and re-increased after insulin cessation. Gland and islet CCKB mRNA and protein almost disappeared during diabetes; CCKB mRNA reappeared in response to insulin, but the protein did not. Confocal microscopy confirmed data obtained on SS and CCKB R as established biochemically in the course of treatment. These results strongly suggest that insulin can negatively control pancreatic SS mRNA and SS content, and positively control CCKB R mRNA with a delayed reappearance of the CCKB receptor protein [174]. Since the islet cells isolated from rat pancreas do not respond to classical CCKB R agonists such as gastrin, we moved to a clone of an
2.8. Growth control and cell signaling in pancreatic cancer cells As a first approach in this field of pancreatic cancer, we used the AR4-2J cells, a rat acinar tumoral cell line, to search for potential cell signaling pathways involved in the growth promoting effect of pituitary adenylate cyclase activating peptide (PACAP). PACAP-38 and -27 caused dose-dependent and parallel activations of TRK and PLD activities which are both inhibited by cell preincubation with genistein and pertussis toxin. The PACAP-induced increase in AR4-2J cell growth was significantly inhibited by increasing concentrations of genistein and wortmannin, inhibitors of TRK, PLD and phosphatidylinositol 3-kinase (PI 3-kinase). PACAP can thus induce concomitant activation of TRK and PLD; this finding and the observation that inhibition of these two enzymes inhibited PACAP-induced AR4-2J cell growth strongly suggests that they are intimately involved in the overall process of PACAPinduced AR4-2J cell proliferation [177]. Using two human pancreatic cell lines of ductal origin, the MIA PaCa-2 and PANC-1 cells, we investigated to identify the growth factors and hormones involved in their proliferation and to establish the intracellular events involved in their growth control. We demonstrated that FGF-2, IGF-1, CAE, and gastrin but not FGF-1, HGF, secretin or PACAP, stimulated proliferation of these two cell lines. Autocrine factors such as gastrin and IGF-1 were also responsible for their proliferation. In response to EGF, FGF-2, IGF-1, CAE, gastrin and bombesin, TRK and tyrosine phosphatase activities were stimulated in both cell 59
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remains possible that cAMP signaling may contribute to phosphorylation of Bad as one way of promoting cell survival; further investigation is needed to unravel the link between apoptosis pathways and other survival pathways activated by cAMP in human pancreatic cancer cells [183]. As to the role of the CCKB receptor and gastrin on development of pancreatic cancer, it remains controversial but some pancreatic cancer cell lines of ductal origin express both the CCKB receptor and gastrin [184].
lines. The close relationship established between cell growth and TRK activation results from the observation that maximal growth stimulation paralleled with maximal enzyme activation and that genistein - the TRK inhibitor - blocked cell growth and enzyme activation. The implication of PLD in this growth process control is doubtful since all growth factors and hormones tested failed to stimulate an already very active PLD activity. A constitutive action of p44 MAPK was observed in both cell lines. However, p38 and p42 were stimulated in both cell lines by all molecules tested [178]. With the same two human pancreatic cell lines, we used SS-14 and its stable analogue SMS 201–995 (SMS) to investigate, identify and clarify the involved intracellular events. In PANC- cells, SS-14 and SMS caused inhibition of both basal- and EGF-1-stimulated growths. To understand this inhibitory mechanism, we tested the effects of both inhibitors on phosphotyrosine phosphatase (PTPase) activities and, more specifically, that of tyrosine phosphatase SHP-1 (PTP1C). Both inhibitors caused significant increases in total cellular PTPase activities and particularly that of PTP1C. Inhibition of membrane TRK and p42 MAP kinase activities was also observed with both inhibitors. In MIA PaCa-2 cells, both SS-14 and SMS were associated with a positive growth response. Total cellular PTPase activity was slightly increased but PTP1C activity could not be detected. Its absence in this cell line was confirmed by Western blot. Membrane TRK activities were significantly increased by both inhibitors p44/p42 which are constitutively active in this cell line, and p38 activities were not affected by SS-14. In conclusion, PTP1C can play a key role in the control of cell proliferation and its cellular presence may determine the therapeutic potential of SS in the control of cancer cell growth [179]. We then concentrated our study on the role of EGF and bombesin, specifically on p44/p42 and p38 MAP kinases in their mitogenic response of these two same cell lines. The two factors stimulated growth of both cell lines. In MIA PaCa-2 cells, both factors stimulated activation of p38 while p44/p42 exhibited high basal activity with no response to stimuli. Growth and p38 activation were inhibited by genistein, wortmannin, PD98059 and SB203580, specific inhibitors of TRK, PI 3-kinase, MEK-1 and p38 kinase, respectively. In PANC-1 cells, EGF and bombesin stimulated p42 activation while p44 remained highly activated and unresponsive to stimuli, and p38 did not respond. Stimulated growth and p42 activation were inhibited by genistein, wortmannin and PD98059. This study indicates that although both cell lines exhibit comparable growth response to EGF and bombesin, the intracellular routes leading to their proliferation show some differences. Isn't this observation a guide towards personalized medicine [180]? To extend our understanding of how pancreatic cancer cells grow and die, we analyzed the role of ERK1/2 pathways in the regulation of cell survival in the MIA PaCa-2 cells. Our data provided several evidences for a direct correlation between elevated ERK1/2 activities and MIA PaCa-2 cell proliferation and survival, whereby inhibition of ERK1/2 activities results in 1) induction of p27kip1 expression which leads to G1 arrest; 2) down-regulation of expression levels of antiapoptotic Bcl-2 homologs (namely, Bcl-2, Bcl-X, Mcl-1) concomitant with an up-regulation (Bad) or steady (Bax, Bak) expression levels of pro-apoptotic Bcl-2 homologs and 3) activation of caspases 3, 6, 8 and 9, along with cleavage of poly (ADP-ribose) polymerase (PARP) and nucleosomal DNA fragmentation. Interestingly, proteasome function appears to be a major component of the apoptotic machinery activated by ERK1/2 inhibition [181]. Recent studies suggest a role for cAMP in cell protection against apoptosis induced by a variety of stimuli including TNFα [182]. In this study, we analyzed the potential role of cAMP in the regulation of pancreatic cancer cell cycle progression and survival and elucidated some of the mechanisms involved. Our data demonstrate that the critical second messenger cAMP protects pancreatic cancer cells against apoptosis induced by the inhibition of ERK. Although one of the major functions of AkT is protection of cells against apoptosis, it does not appear to play a major role in cAMP-mediated antiapoptotic function. It
2.9. Cystic fibrosis Cystic fibrosis (CF) is one of the most common diseases in the Caucasian population. Studies of this disease have been particularly difficult due to the unavailability of pathological human material. Chronically reserpinized animals have been adopted as the CF model because of histologic, physiologic and biochemical similarities of the treated animals with the CF patients [185]. In our study, reserpine was injected i.p. at 0.5 and 1 mg kg-1 for 4 and 7 days. Considering the possible effects of malnutrition, by including appropriate pair-fed animals and measuring parameters that are specifically modified by reserpine (pancreatic weight, lipase and glycoprotein GP-2), this reserpine model can be viewed as a reliable one for CF. The significant decrease in the amount of the zymogen granule GP-2 protein is striking. It suggests that the function of GP-2 could be directly or indirectly linked to some of the manifestations of the pathology in the exocrine pancreas [186]. This follow up article is a thorough illustration at the light and electron microscopic levels of the transformations induced in the rat exocrine pancreas by chronic reserpine treatment. Reserpine induced an accumulation of zymogen granules in 60% of the treated animals and a concomitant decrease of the area occupied by the rough endoplasmic reticulum in the same cells. It was also observed at the light and electron microscopic levels that particles of the pancreatic tissue were strongly affected. Numerous autophagic bodies and lysosomes were observed. Cisternae of the Golgi complex were more distended. Some acinar cells were in the process of lysis. Several vacuolar inclusions were present in some intralobular duct lumina. In these same regions, distended intralobular ducts and acinar lumina were observed. These last two features are important manifestations of the pathology in CF patients where obstruction of ducts was believed to trigger focal destruction of the pancreatic tissue [187]. We then looked at the secretory responses of pancreatic acini in control and reserpinized rats because of the major histological modifications resulting from the reserpine treatment. After 7 days of treatment, both chronic and pair-feeding significantly decreased pancreatic amylase concentration by 75 and 50% of controls. Reserpine caused desensitization of the pancreatic acini secretory response to Cch, without altering maximal amylase release. Secretory responses to CAE and to the phorbol ester TPA were not altered. This observed desensitization of amylase release after reserpine was specific to this muscarinic agonist and may result from increased endogenous Ach release in the course of treatment. Reserpine thus caused a homologous desensitization of the secretory response to Cch muscarinic agonist [188]. In order to substantiate the modified secretory response of the pancreatic acini to Ach after reserpine, this study was undertaken to characterize the secretory alterations caused by reserpine on the secretory responses to secretin and the ionophore A23187 and to substantiate the hypothesis that reserpine alters pancreatic secretory responses to secretagogues through the release of endogenous Ach. Reserpine reduced responses of pancreatic acini to secretin and this reduction was slightly improved by increasing extracellular Ca2+ concentration. In 2.5 mM calcium in the incubation medium, the ionophore response was significantly higher in the reserpine group than in the controls. Our data indicate that reserpine affects the secretory response to secretin as did pre-exposure of pancreatic acini to a high 60
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cycles are not blocked by MK-329 nor atropine. Finally, we have demonstrated with specific CCK receptor antibodies that the pancreases of large mammals, including humans, do not possess the CCK1 receptor and that the CCK2 receptor is present on the pancreatic SS δ-cells in all species studied.
concentration of Cch. The observation that reserpine still caused altered secretin secretory response when the putative Ach release was neutralized by atropine in vivo, at the same time as reserpine, led us to believe that this option could be deleted. Then it remains possible that reserpine may have impaired calcium fluxes [189]. Our next step was to examine if the modified amylase release after reserpine could return to normal values in response to two growth factors to the pancreas, CAE and EGF. Our data indicated that EGF in vivo during the reserpine treatment did not have any major impact on the recovery process or prevention of the alterations caused by malnutrition and reserpine. CAE was the most potent factor capable of promoting recovery or preventing alterations of total pancreatic amylase concentration in the reserpine-treated rats. However, it failed to prevent desensitization of amylase release to Cch or decreases of acinar cell response to secretin. Finally, CAE prevented recovery of the secretory response to Cch and secretin in the absence of any treatment. This study points out that damages caused on the secretory process by reserpine are reversible since recovery of the secretory responses to Cch and secretin as well as amylase concentration were complete after 30 days [190]. As just observed, reserpine treatment caused important modifications in the secretory response to Cch and secretin which could not be reversed by CAE and EGF. Reduced pancreatic weights were also observed. Now, is it possible to reverse pancreatic damages caused by reserpine by two known growth factors to the pancreas, CAE and EGF? Our data indicate that EGF could cause pancreatic hypertrophy without hyperplasia in fed control animals, whereas it had no effect in reserpine-treated rats as a preventive treatment. The most beneficial treatment was that of CAE mostly during the recovery period. This CAE treatment was the most efficient to overcome alterations caused to the pancreatic tissue by malnutrition and reserpine [191].
Financial disclosure The author has no funding to declare. Declaration of competing interest The author declares no conflict of interests. Acknowledgements The author would like to thank Mrs. Helene Morin for her secretarial assistance and Mrs. Inna Furman for her sustained support in writing this review. I would also like to thank both the Departments of Biology and Medicine of the University of Sherbrooke for having given me the opportunity to perform my research over the last 50 years. This review is also a tribute to all my research assistants, technicians, postdoctoral fellows, as well as M.Sc. and Ph.D. students who performed all the research under my supervision. Special thanks also to my family: my wife, Claudine, as well as my son, Louis, and my daughter, Sophie, who missed their father at home while working on all his projects. References [1] Morisset J, Dunnigan J. Exocrine pancreas adaptation to diet in vagotomised rats. Rev Can Biol 1967;26:11–6. [2] Grossman MI, Greengard H, Ivy AC. The effect of dietary composition on pancreatic enzymes. Am J Physiol 1942;138:676–8. [3] Morisset J, Dunnigan J. Effects of glucose, amino acids, and insulin on adaptation of exocrine pancreas to diet. Proc Soc Exp Biol Med 1971;136:231–4. [4] Lorenzo-Figueras M, Morisset S, Morisset J, Lainé J, Merritt AM. Digestive enzyme concentrations and activities in healthy pancreatic tissue of horses. Am J Vet Res 2007;68:1070–2. [5] Guilloteau P, Vitari F, Metzinger-Le Meuth V, LeNormand L, Romé V, Savary G, Delaby L, Domeneghini C, Morisset J. Is there adaptation of the exocrine pancreas in wild animal. The case of the Roe Deer. BMC Vet Res 2012;8:70–8. [6] Green GM, Jurkowska G, Bérubé FL, Rivard N, Guan D, Morisset J. Role of cholecystokinin in induction and maintenance of dietary protein-stimulated pancreatic growth. Am J Physiol 1992;262:G740–6. [7] Morisset J, Guan D, Jurkowska G, Rivard N, Green GM. Endogenous cholecystokinin, the major factor responsible for dietary protein-induced pancreatic growth. Pancreas 1992;7:522–9. [8] Green GM, Levan VH, Liddle RA. Plasma cholecystokinin and pancreatic growth during adaptation to dietary protein. Am J Physiol 1986;251:G70–4. [9] Larose L, Morisset J. Acinar cell responsiveness to urecholine in the rat pancreas during fetal and early postnatal growth. Gastroenterology 1977;73:530–3. [10] Doyle CM, Jamieson JD. Development of secretagogue response in rat pancreatic acini. Dev Biol 1978;65:11–5. [11] Prochazka P. The activity of α-amylase in homogenates of the pancreas of rats during early post-natal development. Physiol Bohemoslov 1964;13:288–91. [12] Morisset J, Larose L. Development of the sensitivity of the rat pancreatic acini to urecholine: effect of corticosterone. In: Brooks FP, Evers PW, editors. Nerves and the gut. Charles B. Slack, Inc.; 1977. p. 142–53. [13] Dumont Y, Larose L, Poirier GG, Morisset J. Effect of early weaning of the neonatal rat on pancreatic acinar cell responsiveness to urecholine. Digestion 1978;17:323–31. [14] Dumont Y, Larose L, Poirier GG, Morisset J. Delayed weaning and denial of solid food nibbling upon pancreatic acinar cell responsiveness to urecholine in neonatal rats. Digestion 1978;18:93–102. [15] Pierzynowski SG, Westrom BR, Svendsen J, Karlsson BW. Development of the exocrine pancreas function in chronically cannulated pigs during 1-13 weeks of postnatal life. J Pediatr Gastroenterol Nutr 1990;10:206–12. [16] Zabielski R, Morisset J, Padgurniak P, Romé V, Bierva TM, Bernard C, Chayvialle JA, Guilloteau P. Bovine pancreatic secretion in the first week of life: potential involvement of intestinal CCK receptors. Regul Pept 2002;103:93–104. [17] Larose L, Lanoe J, Morisset J, Geoffrion L, Dumont Y, Lord A, Poirier GG. Rat pancreatic muscarinic receptors. In: Rosselin G, Fromageot P, Bonfils S, editors. Hormone receptors in digestion and nutrition. Elsevier/North-Holland Biomedical Press; 1979. p. 229–38. [18] Ng KH, Morisset J, Poirier GG. Muscarinic receptors of the pancreas: a correlation
3. Conclusions Over the years, this laboratory has realized important new findings on pancreatic physiology. It has demonstrated that the cholinergic muscarinic system was not involved in the process of pancreatic enzyme adaptation to different diets, at least in rats. Our data have also shown that the sensitivity of the pancreatic acini to a cholinergic stimulation developed early after birth. However, the development of this secretory response after birth is not influenced by corticosterone but was delayed by exclusive milk feeding as the only energy source. This laboratory has been the first to characterize the pancreatic muscarinic receptor and to establish a parallel development of the muscarinic receptor population with maximal enzyme release in response to a cholinergic stimulation. Furthermore, it was established that maximal enzyme release resulted from occupation of the high affinity population of muscarinic receptors. The pancreatic InsP3 receptor was also first characterized in our laboratory. We have also demonstrated, after extensive studies, that there was no SS in the rat pancreatic juice but showed the presence of an elastase II in the juice capable of controlling SS activity in the upper intestine. This laboratory also tried to understand how CCK can control pancreatic growth through specific intracellular enzyme activation when compared to Cch, which is not associated with growth. We found that CCK activates ODC and in successive reactions can sustain activation of TRK, PTd3-kinase and PLD, while Cch cannot sustain activities of the first two enzymes and cannot activate PLD. Furthermore, CCK and bFGF, two growth factors for the pancreas, can specifically activate DAG lipase which from PIP2 can produce arachidonic acid, while Cch does not sustain activation of DAG lipase but that of PLA2. These findings can differentiate pathways leading to pancreatic growth. We were also involved in studying the circadian rhythm of rat pancreatic secretion and established a clear circadian rhythm of secretion accompanied by neurosecretory-like minor cycles of 2-h duration, present in both fed and fasted states. Furthermore, these minor 61
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Pharmacol 1986;64:539–44. [50] Kawai K, Orci L, Unger RH. High somatostatin uptake by the isolated perfused dog pancreas consistent with an “insulo-acinar” axis. Endocrinology 1982;112:303–7. [51] Sarfati P, Green GM, Morisset J. Secretion of protein, fluid and immunoreactive somatostatin in rat pure pancreatic juice: adaptation to chronic caerulein and secretin treatment. Pancreas 1988;3:375–82. [52] Safati P, Morisset J. Origin and characterization of immunoreactive somatostatin in rat pure pancreatic juice. Pancreas 1990;5:158–64. [53] Szilagyi CM, Sarfati P, Pradayrol L, Morisset J. Purification, characterization and substrate specificity of rat pancreatic elastase II. Biochem Biophys Acta 1995;1251:55–65. [54] Naor Z. Is arachidonic acid a second messenger in signal transduction? Mol Cell Endocrinol 1991;80:C181–6. [55] Irvine RF. How is the level of free arachidonic acid controlled in mammalian cells? Biochem J 1982;240:3–16. [56] Hou W, Arita Y, Morisset J. Caerulein-stimulated arachidonic acid release in rat pancreatic acini: a diacylglycerol lipase affair. Am J Physiol 1996;271:C1735–42. [57] Hou W, Arita Y, Morisset J. Dual pathways for carbamylcholine-stimulated arachidonic acid release in rat pancreatic acini. Endocrine 1996;5:67–74. [58] Hou W, Arita Y, Morisset J. Endogenous arachidonic acid release and pancreatic amylase secretion. Pancreas 1997;14:301–8. [59] Hou W, Arita Y, Morisset J. Basic fibroblast growth factor-stimulated arachidonic acid release in rat pancreatic acini: sequential action of tyrosine kinase phospholipase C, protein kinase C and diacylglycerol lipase. Cell Signal 1996;8:487–96. [60] Barrowman J, Brogan D, Fordham J, Mathorn M, Mott A, Rahilly P, Tiptfaft R. A possible diurnal rhythm in rat pancreatic secretion. J Physiol London 1970;208:14P–6P. [61] Maouyo D, Sarfati P, Guan D, Morisset J, Adelson JW. Circadian rhythm of exocrine pancreatic secretion in rats: major and minor cycles. Am J Physiol 1993;264:G792–800. [62] Maouyo D, Morisset J. Amazing pancreas: specific regulation of pancreatic secretion of individual digestive enzymes in rats. Am J Physiol 1995;268:E349–59. [63] Maouyo D, Guan D, Rivard N, Adelson JW, Morisset J. Stability of circadian and minor cycles of exocrine pancreatic secretion in atropine and MK-329-infused rats. Am J Physiol 1995;268:G251–9. [64] Maouyo D, Guan D, Rivard N, Morisset J. Modulation of the relationship between amylase and chymotrypsinogen secretion in atropine and MK329-infused rats. Pancreas 1995;11:330–40. [65] Maouyo D, Morisset J. Modulation of pancreatic secretion of individual digestive enzymes in octreotide (SMS 201-995)-infused rats. Pancreas 1997;14:47–57. [66] Green GM, Lyman RL. Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersecretion in rats. Proc Soc Exp Biol Med 1972;140:6–12. [67] Morisset J. Negative control of human pancreatic secretion. Physiological mechanisms and factors. Pancreas 2008;37:1–12. [68] Morisset J, Webster PD. Effects of atropine on pigeon pancreas. Am J Physiol 1970;219:1286–91. [69] Bendayan M, Bruneau A, Morisset J. Morphometrical and immunochemical studies of rat pancreatic acinar cells under control and experimental conditions. Biol Cell 1985;54:227–34. [70] Morisset J, Beaudoin AR. Biochemical reactions involved in pancreatic enzyme secretion. 2. Inhibitory effects of tetracaine and atropine. Can J Physiol Pharmacol 1977;55:639–43. [71] Morisset J, Beaudoin AR. Biochemical reactions involved in pancreatic enzyme secretion. 4. Effects of cytochalasin B on functions of the exocrine pancreas. Can J Physiol Pharamcol 1977;55:644–51. [72] Morisset J, NG KH, Poirier GG. Comparative inhibitory effects of 3-quinuclidinylbenzylate (QNB) and atropine on amylase release from rat pancreas. Br J Pharmacol 1977;61:97–100. [73] Hamel E, Morisset J. Effects of cocaine on rat pancreatic enzyme secretion and protein synthesis. Am J Dig Dis 1978;23:264–8. [74] Morisset JA, Black O, Webster PD. Effects of fasting, feeding and bethanechol chloride on pancreatic microsomal protein synthesis in vitro. Proc Soc Exp Biol Med 1972;140:1308–14. [75] Morisset JA, Black O, Webster PD. Changes with fasting in pigeon pancreas alkaline and acid ribonuclease. Proc Soc Exp Biol Med 1972;139:562–4. [76] Sahba MM, Morisset JA, Webster PD. Synthetic and secretory effects of cholecystokinin-pancreozymin on the pigeon pancreas. Proc Soc Exp Biol Med 1970;134:728–32. [77] Morisset JA, Webster PD. Effects of fasting and feeding on protein synthesis by rat pancreas. J Clin Investig 1972;51:1–8. [78] Couture Y, Mongeau R, Dunnigan J, Morisset J. Evidence that protein synthesis can be increased in vitro following cholinergic stimulation. Can J Physiol Pharmacol 1972;50:874–82. [79] Mongeau R, Couture Y, Dunnigan J, Morisset J. Early dissociation of protein synthesis and amylase secretion following hormonal stimulation of the pancreas. Can J Physiol Pharmacol 1974;52:198–205. [80] Mongeau R, Dagorn JC, Morisset J. Further evidence that protein synthesis can be decreased in vivo following hormonal stimulation in the rat pancreas. Can J Physiol Pharmacol 1976;54:305–13. [81] Dagorn JC, Michel R. Non parallel courses of intrapancreatic levels of exportable enzymes after a fatty meal. Proc Soc Exp Biol Med 1976;151:608–10. [82] Mongeau R, Morisset J. Modification du profil de la réponse pancréatique chez le rat par une stimulation hormonale répétée. Gastroenterol Clin Biol 1977;1:243–8. [83] Malo C, Morisset J. Time course of pancreatic protein synthesis following feeding. Am J Dig Dis 1978;23:6–8.
between displacement of (3H)-quinuclidinyl benzilate binding and amylase secretion. Pharmacology 1979;18:263–70. Larose L, Dumont Y, Asselin J, Morisset J, Poirier GG. Muscarinic receptor of rat pancreatic acini: [3H]QNB binding and amylase secretion. Eur J Pharmacol 1981;76:247–54. Dumont Y, Larose L, Morisset J, Poirier GG. Parallel maturation of the pancreatic secretory response to cholinergic stimulation and the muscarinic receptor population. Br J Pharmacol 1981;73:347–54. Dumont Y, Côté B, Larose L, Poirier GG, Morisset J. Maturation of muscarinic agonist receptors in rat developing pancreas and its relation to maximal enzyme secretion. Life Sci 1981;29:2771–9. Dumont Y, Larose L, Poirier GG, Morisset J. Changes in acetylcholinesterase and cholinesterase activities in rat pancreas during postnatal development. Pharmacology 1984;29:40–6. Dumont Y, Larose L, Poirier GG, Morisset J. Modulation of pancreatic muscarinic receptors by weaning. Life Sci 1982;30:253–7. Larose L, Poirier GG, Dumont Y, Fregeau C, Blanchard L, Morisset J. Modulation of rat pancreatic amylase secretion and muscarinic receptor population by chronic bethanechol treatment. Eur J Pharmacol 1983;95:215–23. Delhaye M, Taton G, Poirier G, Larose L, St-Jacques S, Morisset J. Modulation of rat pancreatic muscarinic cholinergic receptors by caerulein. Biochem Pharmacol 1985;34:1057–63. Larose L, Morisset J. Cholinergic stimulation of pancreatic amylase release and muscarinic receptors. Effect of ionophore A23187. Life Sci 1985;37:255–61. Morisset J, Wood J, Solomon TE, Larose L. Muscarinic receptors and amylase secretion of rat pancreatic acini during caerulein-induced acute pancreatitis. Dig Dis Sci 1987;32:872–7. Taton G, Delhaye M, Swillens S, Morisset J, Larose L, Longnecker DS, Poirier GG. Muscarinic cholinergic receptors in pancreatic acinar carcinoma of rat. Int J Cancer 1985;35:493–7. Heisler S, Larose L, Morisset J. Muscarinic cholinergic inhibition of cyclic AMP formation and adenocorticotropin secretion in mouse pituitary tumor cells. Biochem Biophys Res Commun 1983;114:289–95. Loiselle J, Larose L, Morisset J. Contraindication for osmotic mini-pump in the abdominal cavity to study muscarinic cholinergic control of pancreatic enzyme secretion and muscarinic receptors. Int J Pancreatol 1986;1:249–58. Morisset J, Geoffrion L, Larose L, Lanoe J, Poirier GG. Distribution of muscarinic receptors in the digestive tract organs. Pharmacology 1981;22:189–95. Jun KF, Triggle DJ. Desensitization of ionophore A23187 responses by muscarinic receptor stimulation in intestinal smooth muscle. Biochem Pharmacol 1981;30:95–6. Asselin J, Larose L, Morisset J. Short-term cholinergic desensitization of rat pancreatic secretory response. Am J Physiol 1987;252:G392–7. Larose L, Leclerc L, Asselin J, Ruel S, Morisset J. Muscarinic cholinergic induced secretin subsensitivity in rat isolated pancreatic acini. Effects on amylase release, cyclic adenosine monophosphate and inositol phosphate formation. Pancreas 1989;4:71–8. Blanchard LM, Paquette B, Larose L, Morisset J. Carbamylcholine and phorbol esters desensitize muscarinic receptors by different mechanisms in rat pancreatic acini. Pancreas 1990;5:50–9. Morisset J, Paquette B, Benrezzak O. The ability of staurosporine to modulate pancreatic acinar cell desensitization by TPA, carbamylcholine and caerulein. Cell Signal 1991;3:118–26. Servant M, Guillemette G, Morisset J. Pharmacologic characterization of the inositol trisphosphate receptor in rat pancreas. Pancreas 1994;9:591–8. Servant M, Guillemette G, Morisset J. Pancreatic acinar-cell desensitization alters InsP3 production and Ca2+ mobilisation under conditions where InsP3 receptor remains intact. Biochem J 1995;305:103–10. Lods JS, Nolet B, Morisset J. Selectivity in inositol phosphate production following pancreatic acinar cell desensitization. Biochem Biophys Res Commun 1995;206:870–7. Lods JS, Rossignol B, Dreux C, Morisset J. Phosphoinositide synthesis in desensitized rat pancreatic acinar cells. Am J Physiol 1995;268:G1043–50. Rivard N, Rydzewska G, Boucher C, Lods JS, Calvo E, Morisset J. Cholecystokinin activation of tyrosine kinase, PTdinositol 3-kinase and phospholipase D: a role in pancreas growth induction? Endocr J 1994;2:393–401. Marois C, Morisset J, Dunnigan J. Presence and stimulation of adenylate cyclase in pancreas homogenate. Rev Can Biol 1972;31:253–7. Beaudoin AR, Marois C, Dunnigan J, Morisset J. Biochemical reactions involved in pancreatic enzyme secretion. 1. Activation of the adenylate cyclase complex. Can J Physiol Pharmacol 1974;52:174–82. Morisset J, Webster PD. In vitro and in vivo effects of pancreozymin, urecholine and cyclic AMP on rat pancreas. Am J Physiol 1971;230:202–8. Sarfati P, Morisset J. Regulation of pancreatic enzyme secretion in conscious rats by intraluminal somatostatin: mechanism of action. Endocrinology 1989;124:2406–14. Guan D, Maouyo D, Sarfati P, Morisset J. Effects of SMS 201-995 on basal and stimulated pancreatic secretion in rats. Endocrinology 1990;127:298–304. Guan D, Maouyo D, Taylor IL, Gettys J, Greeley GH, Morisset J. Peptide-YY, a new partner in the negative feedback control of pancreatic secretion. Endocrinology 1991;128:911–6. Conlon JM, Rouiller D, Boden G, Unger RH. Characterization of immunoreactive component of insulin and somatostatin in canine pancreatic juice. FEBS Lett 1979;105:23–6. Sarfati PD, Green GM, Brazeau P, Morisset J. Presence of somatostatin-like immunoreactivity in rat pancreatic juice: a physiological phenomenon. Can J Physiol
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after administration of GH and of TSH. Exp Cell Res 1958;14:201–3. [117] Dubreuil P, Morisset J. Effect of somatocrinin with or without a somatostatin antiserum on pancreatic growth. Pancreas 1986;1:13–9. [118] Jolicoeur L, Asselin J, Morisset J. Trophic effects of gestation and lactation on rat pancreas. Biomed Res 1980;1:482–8. [119] Charbonneau P, Pelletier G, Morisset J. Development of the pancreas during gestation and lactation in swine. Can J Physiol Pharmacol 1982;60:1229–35. [120] Gelinas MD, Morin CL, Morisset J. Exocrine pancreatic function following proximal small bowel resection in rats. J Physiol 1982;322:71–82. [121] Morisset J, Jolicoeur L, Caussignac Y, Solomon TE. Trophic effects of chronic bethanechol on pancreas, stomach, and duodenum in rats. Can J Physiol Pharmacol 1982;60:871–6. [122] Morisset J, Loiselle J. Alteration of the rat pancreas after chronic scopolamine administration. Pharmacology 1985;30:308–13. [123] Chey WY, Shiratori K, Sun G, Jo YH, Ren XS, Lee KY, Chang TM. Role of gut hormones in negative feed-back regulation of exocrine pancreas. Biomed Res 1989;10:87–94. [124] Guan D, Rivard N, Maouyo D, Gettys TW, Morisset J. Importance of cholecystokinin in peptide-YY release in response to pancreatic juice diversion. Regul Pept 1993;43:169–76. [125] Guan D, Rivard N, Morisset J, Greeley GH. Effects of peptide YY on the growth of the pancreas and intestine. Endocrinology 1993;132:219–23. [126] Morisset J, Genick P, Lord A, Solomon TE. Effects of chronic administration of somatostatin on rat exocrine pancreas. Regul Pept 1982;4:49–58. [127] Morisset J. Somatostatin: a potential antigrowth factor for the exocrine pancreas. Regul Pept 1984;10:11–22. [128] Rivard N, Guan D, Turkelson CM, Petitclerc D, Solomon TE, Morisset J. Negative control by sandostatin on pancreatic and duodenal growth: a possible implication of insulin-like growth factor 1. Regul Pept 1991;34:13–23. [129] Esteve JP, Susini C, Vaysse N. Binding of somatostatin to pancreatic acinar cells. Am J Physiol 1984;247:G62–9. [130] Morisset J, Larose L, Korc M. Epidermal growth factor inhibits rat pancreatic cell proliferation, causes acinar cell hypertrophy, and prevents caerulein-induced desensitization of amylase release. Endocrinology 1989;124:2693–8. [131] Pegg AE, McCann PP. Polyamine metabolism and function. Am J Physiol 1982;12:C212–21. [132] Morisset J, Benrezzak O. Polyamines and pancreatic growth induced by caerulein. Life Sci 1984;35:2471–80. [133] Benrezzak O, Morisset J. Effects of α-difluoromethylornithine on pancreatic growth induced by caerulein. Regul Pept 1984;9:143–53. [134] Morisset J, Benrezzak O. Reversal of α-difluoromethylornithine inhibition of caerulein-induced pancreatic growth by putrescine. Regul Pept 1985;11:201–8. [135] Morisset J, Sarfati P, Grondin G. Immunocytochemical demonstration of ornithine decarboxylase in the rat exocrine pancreas using the protein A-gold technique. Can J Physiol Pharmacol 1986;64:444–8. [136] Morisset J, Grondin G. Implication of ornithine decarboxylase and polyamines in pancreatic growth of neonatal rats. Pancreas 1987;2:303–11. [137] Langlois A, Morisset J. Effects of feeding, fasting, and caerulein treatment on ornithine decarboxylase in rat pancreas. Pancreas 1991;6:534–41. [138] Rydzewska G, Rossignol B, Morisset J. Involvement of phospholipase D in caerulein-induced phosphatidylcholine hydrolysis in rat pancreatic acini. Am J Physiol 1993;265:G725–34. [139] Rivard N, LeBel D, Lainé J, Morisset J. Regulation of pancreatic tyrosine kinase and phosphatase activities by cholecystokinin and somatostatin. Am J Physiol 1994;266:G1130–8. [140] Sankaran H, Goldfine ID, Deveney CW, Wong KY, Williams JA. Binding of cholecystokinin to high affinity receptors on isolated rat pancreatic acini. J Biol Chem 1980;255:1849–53. [141] Stark HA, Sharp CM, Sutliff VE, Martinez J, Jensen RT, Gardner JD. CCK-JMV-180: a peptide that distinguishes high-affinity cholecystokinin receptors from low-affinity cholecystokinin receptors. Biochim Biophys Acta 1989;1010:145–50. [142] Rivard N, Rydzewska G, Lods JS, Martinez J, Morisset J. Pancreas growth, tyrosine kinase PTd Ins3-kinase, and PLD involved high affinity CCK-receptor occupation. Am J Physiol 1994;266:G62–70. [143] Rivard N, Rydzewska G, Boucher C, Lods JS, Calvo E, Morisset J. Cholecystokinin activation of tyrosine kinases, PTdinositol 3-kinase and phospholipase D: a role in pancreas growth induction? Endocr J 1994;2:393–401. [144] Rydzewska G, Morisset J. Activation of pancreatic acinar cell phospholipase D by epidermal, insulin-like, and basic fibroblast growth factors involve tyrosine kinase. Pancreas 1995;10:59–65. [145] Rydzewska G, Morisset J. Specificity of phospholipase D activation by cholecystokinin and phorbol myristate acetate but not by carbamylcholine and A23187 in rat pancreatic acini. Digestion 1995;56:127–36. [146] Lainé J, Bourgoin S, Bourassa J, Morisset J. Subcellular distribution and characterization of rat pancreatic phospholipase D isoforms. Pancreas 2000;20:323–36. [147] Rivard N, Rydzewska G, Lods JS, Morisset J. Novel model of integration of signaling pathways in rat pancreatic acinar cells. Am J Physiol 1995;269:G352–62. [148] Morisset J, Aliaga JC, Calvo EL, Bourassa J, Rivard N. Expression and modulation of p42/p44 MAPKs and cell cycle regulatory proteins in rat pancreas regeneration. Am J Physiol 1999;277:G953–9. [149] Wood J, Garcia R, Solomon TE. A simple model for acute pancreatitis: high dose of caerulein injection in rats. Gastroenterology 1982;82:1213. [Abstract]. [150] Jurkowska G, Grondin G, Massé S, Morisset J. 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[84] Palla JC, Ben Abdeljlil A, Desnuelle P. Action de l’insuline sur la biosynthèse de l’amylase et de quelques autres enzymes du pancréas. Biochim Biophys Acta 1968;158:25–35. [85] Ben Abdeljlil A, Palla JC, Desnuelle P. Effect of insulin on pancreatic amylase and chymotrypsinogen. Biochem Biophys Res Commun 1965;18:71–5. [86] Couture Y, Dunnigan J, Morisset J. Stimulation of pancreatic amylase secretion and protein synthesis by insulin. Scand J Gastroenterol 1972;7:257–63. [87] Dagorn JC, Paradis D, Morisset J. Non-parallel response of amylase and chymotrypsinogen biosynthesis following pancreatic simulation: a possible explanation for observed non-parallelism in pancreatic secretion. Digestion 1977;15:110–20. [88] Webster PD, Morisset JA. Effect of atropine on pigeon pancreas. Proc Soc Exp Biol Med 1971;136:245–8. [89] Dragon N, Beaudoin AR, Morisset J. Effect of tetracaine on pancreatic protein synthesis. Proc Soc Exp Biol Med 1975;149:278–81. [90] Morisset J. Hormonal control of pancreatic growth during fetal, neonatal and adult life. Adv Med Sci 2008;53:99–118. [91] Morisset J, Chamberland S, Gilbert L, Lord A, Larose L. Study of pancreatic DNA synthesis in vivo and in vitro following caerulein treatment in vivo and in vitro following caerulein treatment in vivo. Biochem Res 1982;3:151–8. [92] Morisset J, Grondin G. Dynamics of pancreatic tissue cells in the rat exposed to long term caerulein treatment. 1. Biochemical, morphological and morphometrical evaluations. Biol Cell 1989;66:271–8. [93] Morisset J, Grondin G. Dynamic of pancreatic tissue cells in the rat exposed to long term caerulein treatment. 2. Comparative analysis of the various cell types and their growth. Biol Cell 1989;66:279–90. [94] Morisset J, Laine J, Mimeau-Worthington T. Hormonal control of rat fetal pancreas development. Biol Neonate 1999;75:327–36. [95] Farmer C, Brazeau P, Morisset J. Digestive enzyme development in newborn piglets born of sows immunized against somatostatin and/or receiving growth hormone-releasing factor during gestation. Biol Neonate 1993;64:382–91. [96] Hibbard B, Peters JP, Shen RYW, Chester ST. Effect of recombinant porcine somatotropin and dietary protein on pancreatic digestive enzymes in the pig. J Anim Sci 1992;70:2188–94. [97] Morisset J, Wong H, Walsh JH, Lainé J, Bourassa J. Pancreatic CCKB receptors: their potential roles in somatostatin release and δ-cell proliferation. Am J Physiol 2000;279:G148–56. [98] Morisset J, Calvo E. Trophicity of the pancreas: from rodents to man. J Physiol Pharmacol 1998;49(Suppl. 2):25–37. [99] Brants F, Morisset J. Trophic effect of cholecystokinin-pancreozymin on pancreatic acinar cells from rats of different ages. Proc Soc Exp Biol Med 1976;153:523–7. [100] Morisset J. Stimulation of pancreatic growth by secretin and caerulein in suckling rats. Biomed Res 1980;1:405–9. [101] Calvo EL, Bernatchez G, Pelletier G, Iovanna JL, Morisset J. Downregulation of 1GF-I mRNA expression during postnatal pancreatic development and overexpression after subtotal pancreatectomy and acute pancreatitis in rat pancreas. J Mol Endocrinol 1997;18:233–42. [102] Calvo EL, Boucher C, Pelletier G, Morisset J. Ontogeny of hepatocyte growth factor and c-met/hgf receptor in rat pancreas. Biochim Biophys Res Commun 1996;229:257–63. [103] Calvo EL, Mallo GV, Fielder F, Malka D, Vaccaro MI, Keim V, Morisset J, Dagorn JC, Iovanna JI. Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development. Eur J Biochem 1998;254:282–9. [104] Mallo GV, Fielder F, Calvo EL, Ortiz EM, Vasseur S, Keim V, Morisset J, Iovanna JL. Cloning and expression of the rat p8 cDNA, a new gene activated during the acute phase of pancreatitis, pancreatic development and regeneration, and which promote cellular growth. J Biol Chem 1997;272:32360–9. [105] Calvo EL, Boucher C, Coulombe Z, Morisset J. Pancreatic GAPDH gene expression during ontogeny and acute pancreatitis induced by caerulein. Biochem Biophys Res Commun 1997;235:636–40. [106] Solomon TE, Vanier M, Morisset J. Cell site and time course of DNA synthesis in pancreas after caerulein and secretin. Am J Physiol 1983;245:G99–105. [107] Rivard N, Guan D, Maouyo D, Grondin G, Bérubé FL, Morisset J. Endogenous cholecystokinin release responsible for pancreatic growth observed after pancreatic juice diversion. Endocrinology 1991;129:2867–74. [108] Rivard N, Guan D, Maouyo D, Morisset J. Pancreatic protein hypersecretion and elevated plasma CCK: prerequisites for increased pancreatic growth. Pancreas 1993;8:573–80. [109] Green GM, Sarfati PD, Morisset J. Lack of effect of caerulein on pancreatic growth of rats fed a low-protein diet. Pancreas 1991;6:182–9. [110] Tsiotos GG, Barry MK, Johnson CD, Sarr MG. Pancreas regeneration after resection: does it occur in human? 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[171] Julien S, Lainé J, Morisset J. The rat pancratic islets: a reliable tool to study islet responses to cholecystokinin receptor occupation. Regul Pept 2004;121:73–81. [172] Wang F, Adrian TE, Westermark GT, Ding X, Gasslander T, Permet J. Islet amyloid polypeptide totally inhibits β, α and δ-cell secretion in isolated rat pancreatic islets. Am J Physiol 1999;276:E19–24. [173] Patel YC, Weir GC. Increased somatostatin content of islets from streptozotocindiabetic rats. Clin Endocrinol 1976;5:191–4. [174] Julien S, Lainé J, Morisset J. Regulation of rat pancreatic CCKB receptor and somatostatin expression by insulin. Diabetes 2004;53:1526–34. [175] El-Kouhen K, Morisset J. Cholecystokinin and somatostatin negatively affect growth of the somatostatin-RIN-14B cells. Int J Endocrinol 2009:1–6. Article ID 875167. [176] El-Kouhen K, Morisset J. Control of somatostatin (SS) secretion by CCK-1 and CCK2 receptors' occupation in RIN-14B cells, a rat pancreatic islet cell line. Pancreas 2010;39:127–34. [177] Morisset J, Douziech N, Rydzewska G, Buscail L, Rivard N. Cell signalling pathway involved in PACAP-induced AR4-2J cell proliferation. Cell Signal 1995;7:195–205. [178] Douziech N, Lajas A, Coulombe Z, Calvo E, Lainé J, Morisset J. Growth effects of regulatory peptides and intracellular signaling routes in human pancreatic cell lines. Endocrine 1998;9:171–83. [179] Douziech N, Calvo E, Coulombe Z, Muradia G, Bastien J, Aubin RA, Lajas A, Morisset J. Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1. Endocrinology 1999;140:765–77. [180] Douziech N, Calvo E, Lainé J, Morisset J. Activation of MAP kinases in growth responsive pancreatic cancer cells. Cell Signal 1999;11:591–602. [181] Boucher MJ, Morisset J, Vachon PH, Reed JC, Lainé J, Rivard N. MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X1, and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000;79:355–69. [182] Li J, Yang S, Billiar TR. Cyclic nucleotides suppress tumor necrosing factor αmediated apoptosis by inhibiting caspase activation and cytochrome C release in primary hepatocytes via a mechanism independent of Akt activation. J Biol Chem 2000;275:13026–34. [183] Boucher MJ, Duchesne C, Lainé J, Morisset J, Rivard N. cAMP protection of pancreatic cancer cells against apoptosis induced by ERK inhibition. Biochem Biophys Res Commun 2001;285:207–16. [184] Morisset J, Lainé J, Biernat M, Julien S. What are the pancreatic target cells for gastrin and its CCKB receptor? Is this a couple for cancerous cells? Med Sci Monit 2004;10:RA242–6. [185] Martinez JR, Adelstein E, Quissel D, Barbero GJ. The chronically reserpinized rat as a possible model for cystic fibrosis. Pediatr Res 1975;9:463–9. [186] Leblond FA, Morisset J, LeBel D. Alteration of pancreatic growth and of GP-2 content in the reserpinized rat model of cystic fibrosis. Pediatr Res 1989;25:478–81. [187] Grondin G, Leblond FA, Morisset J, LeBel D. Light and electron microscopy of the exocrine pancreas in the chronically reserpinized rat. Pediatr Res 1989;25:482–9. [188] Benrezzak O, Bérubé FL, Morisset J. Alterations of amylase secretion in the chronically reserpinized rat: an acetylcholine-mediated phenomenon. Pancreas 1991;6:679–87. [189] Benrezzak O, Bérubé FL, St-Jean L, Morisset J. Alterations of the pancreatic secretory responses to secretin and to the ionophore A23180 by reserpine: a calciummediated phenomenon? Digestion 1994;55:78–85. [190] Morisset J, Bérubé FL, Vanier M, Benrezzak O. Alterations of pancreatic amylase secretion in the reserpinized rat model of cystic fibrosis. Effects of caerulein and EGF. Int J Pancreatol 1994;16:37–44. [191] Bérubé FL, Benrezzak O, Vanier M, Morisset J. Effects of caerulein and epidermal growth factor on pancreatic growth in the reserpinized rat model. J Pediatr Gastroenterol Nutr 1993;17:39–48.
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