AJP - GI Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 286: G784-G790, 2004. First published December 18, 2003; doi:10.1152/ajpgi.00446.2003
0193-1857/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/G784    most recent
00446.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tashiro, M.
Right arrow Articles by Williams, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tashiro, M.
Right arrow Articles by Williams, J. A.

HORMONES AND SIGNALING

Calcineurin mediates pancreatic growth in protease inhibitor-treated mice

Mitsuo Tashiro,1 Linda C. Samuelson,1 Rodger A. Liddle,3 and John A. Williams1,2

Departments of 1Molecular and Integrative Physiology and 2Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622; and 3Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

Submitted 16 October 2003 ; accepted in final form 15 December 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CCK acts on pancreatic acinar cells to increase intracellular Ca2+ leading to secretion of digestive enzymes and, in the long term, pancreatic growth. Calcineurin (CN) is a serine/threonine-specific protein phosphatase activated by Ca2+ and calmodulin that recently has been shown to participate in the growth regulation of cardiac and skeletal myocytes. We therefore tested the effect of two different CN inhibitors, cyclosporine A (CsA) and FK506, on mouse pancreatic growth induced by oral administration of the synthetic protease inhibitor camostat, a known stimulator of endogenous CCK release. Mice were fed a powdered diet with or without 0.1% camostat. Pancreatic wet weight, protein, and DNA were increased in response to camostat in a time-dependent manner over 10 days in ICR mice but not in CCK-deficient mice. Both CsA (15 mg/kg) and FK506 (3 mg/kg) given twice daily blocked the increase in pancreatic wet weight and protein and DNA content induced by camostat. The increase in plasma CCK induced by camostat was not blocked by CsA or FK506. Camostat feeding also increased the relative amount of CN protein, whereas levels of MAPKs, ERKs, and p38 were not altered. In summary, 1) CCK released by chronic camostat feeding induces pancreatic growth in mice; 2) this growth is blocked by treatment with both CsA and FK506, indicating a role for CN; 3) CCK stimulation also increases CN protein. In conclusion, activation and possibly upregulation of CN may participate in regulation of pancreatic growth by CCK in mice.

cholecystokinin; camostat; FK506; cyclosporine A

A VARIETY OF GASTROINTESTINAL peptides have been implicated in the regulation of proliferation and differentiation in the mature gastrointestinal tract and the pancreas (30, 56). It is well known that feeding initiates pancreatic protein synthesis and secretion and that adaptation to a high-protein diet results in pancreatic growth (17, 18). More detailed studies have shown that among gastrointestinal peptides, CCK is released in response to dietary protein leading to pancreatic growth. Injection of CCK increases pancreatic wet weight and protein and DNA content in vivo over 4–10 days (8, 37, 40, 43). Oral administration of soybean trypsin inhibitor (5, 44) or the synthetic protease inhibitor camostat (10, 15, 37, 52, 55), as well as pancreaticobiliary diversion (13, 39), increase the release of endogenous CCK into the blood and induce pancreatic growth. Moreover, CCK-A receptor antagonists that block the action of exogenous and endogenous CCK inhibit pancreatic growth (10, 13, 37, 39, 52). Therefore, most of the studies of pancreatic growth have been focused on CCK as a key regulator.

In pancreatic acinar cells, the intracellular mechanisms of enzyme secretion have been well documented. CCK activates the release of intracellular Ca2+ and the influx of Ca2+ and generates oscillations of intracellular Ca2+ concentration (56). Ca2+ then acts as an intracellular messenger and initiates the secretion of digestive enzymes. CCK has also been reported to activate a variety of other effector pathways, including protein kinase C, the MAPK cascades, phosphatidylinositol 3-kinase, tyrosine kinases, and phosphatases (50, 51, 56). At present, the intracellular mediator(s) regulating pancreatic growth stimulated by CCK is unclear.

Regulation of cellular phosphoproteins involves protein phosphatases, because the state of protein phosphorylation represents a balance between kinase and phosphatase activity. One of the serine/threonine-specific protein phosphatases is calcineurin (CN) (also called protein phosphatase 2B), which is uniquely activated by Ca2+ and calmodulin (6, 24). CN consists of two different subunits, A and B. CN A (a 61-kDa catalytic and calmodulin-binding subunit) is tightly bound to CN B (a 19-kDa calcium-binding subunit homologous to calmodulin). The immunosuppressants cyclosporine A (CsA) and FK506 inhibit CN activity when bound to their intracellular receptors cyclophilin A and FK506-binding protein of 12 kDa (FKBP12), respectively (29, 47). These agents, therefore, can be used as tools to investigate the role of CN in the transduction of Ca2+ signals. Pancreatic cytosol contains Ca2+-activated protein phosphatase activity and immunoreactive CN (4, 54). Groblewski et al. (20, 21) demonstrated that CCK treatment of dissociated pancreatic acini activated CN in that a specific protein substrate, calcium-regulated heat-stable protein of 24 kDa (CRHSP-24), was dephosphorylated after CCK stimulation, and this effect was blocked by CsA or FK506. These inhibitors also had a modest effect in inhibiting pancreatic amylase secretion, leading to the suggestion that CN may play a role in stimulus-secretion coupling in pancreatic acinar cells (20, 48). Recent studies in other tissues, however, have shown that CN plays a critical role in experimental models of cardiac (34, 36, 45) or skeletal muscle (11, 32, 42) hypertrophy. In the present study, we demonstrate that CN mediates the pancreatic growth induced by oral administration of the protease inhibitor camostat in mice and that endogenous CCK is required for camostat-induced pancreatic growth.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Induction of pancreatic growth by camostat feeding. Male ICR mice (Harlan Sprague Dawley, Indianapolis, IN), weighing ~30 g or female CCK-deficient mice on a 129/SV background (26), weighing ~20 g were used in all experiments. The specific pathogen-free animals were housed at 22°C on a 12:12-h light-dark cycle with free access to water and a standard rodent powdered chow (5001 rodent diet, PMI Nutrition International, St. Louis, MO). All studies were approved by the University of Michigan Committee on Use and Care of Animals, and all animals were killed by decapitation under carbon dioxide anesthesia between 8:30 and 10:00 AM.

The control diet group was fed regular powdered chow. For camostat feeding, 0.1% of camostat (provided by Ono Pharmaceutical, Osaka, Japan) was mixed with regular powdered chow. The mixture was fed ad libitum to mice beginning at 9:00 AM after conditioning by regular powdered diet. ICR or 129/SV mice were fed either regular powdered chow or camostat mixture for 1–10 days. Animals treated with CsA or FK506 were injected subcutaneously twice a day with either 15 mg/kg of CsA (Calbiochem-Novabiochem, La Jolla, CA) suspension in vehicle (5% ethanol in 0.9% saline), 3 mg/kg of FK506 (Fujisawa Healthcare, Deerfield, IL) in vehicle (8% ethanol and 2% polyoxyl 60 hydrogenated castor oil in 0.9% saline), or each vehicle alone. Injection with either CsA, FK506, or vehicle began 2 days before switching to diet containing camostat.

Biochemical determinations. After the pancreas was harvested and weighed, each pancreas was homogenized in 4 ml of buffer containing 5 mM MgCl2 and 0.1% Triton X-100 using a Polytron homogenizer for 20 s and then sonicated for 15 s. Protein concentrations of pancreatic homogenates were determined by protein assay reagent (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as a standard. DNA was measured by Hoechst 33258 dye in the presence of 2.5 M NaCl using calf thymus DNA as a standard. Serum amylase activity was determined by Phadebas Amylase Test (Pharmacia and Upjohn, Uppsala, Sweden).

For determination of plasma CCK, plasma obtained by cardiac puncture was extracted by absorption and elution from C18 Sep-Paks (Waters), and CCK was quantitated by bioassay using amylase secretion from isolated rat pancreatic acini (27, 28). To obtain adequate sensitivity, plasma was pooled from 3 mice fed control diet, whereas plasma from individual mice fed camostat could be determined. Whether CsA and FK506 were extracted is unknown, but the addition of FK506 to assay flasks had no effect on amylase release (unpublished data). Previous inhibitory effects of CsA (20) were reported after preincubation and used higher concentrations than likely to be observed here.

Preparation of pancreatic tissue lysates for electrophoresis. Pancreatic tissue was homogenized with a Polytron homogenizer in ice-cold lysis buffer (pH 7.4) containing (in mM) 50 Tris·HCl, 5 EDTA, 25 NaF, 10 sodium pyrophosphate, 50 {beta}-glycerophosphate, 1 PMSF, 0.2 Na3VO4 and 1 dithiothreitol with 0.2% Triton X-100 (vol/vol), 5% 2-mercaptoethanol (vol/vol), 10 µg/ml aprotinin (Roche), 10 µg/ml leupeptin (Roche), and 10 µg/ml pepstatin (Roche), and then centrifuged at 3,000 rpm at 4°C for 15 min. The supernatant was then prepared for one-dimensional SDS-PAGE and isoelectric focusing (IEF) gel electrophoresis. Samples for SDS-PAGE were adjusted to a final concentration of 62.5 mM Tris·HCl, pH 6.8, 2% SDS (wt/vol), and 10% glycerol followed by boiling for 5 min; samples for IEF gel electrophoresis were adjusted to a final concentration of 4.5 M urea, 2% Nonidet P-40 (vol/vol), and 0.5% 2-mercaptoethanol (vol/vol) without heating. Proteins (30 µg/lane) were then separated by 10% SDS-PAGE or IEF gel electrophoresis using pI 3–10 ampholytes (Bio-Rad Laboratories) and a Mini IEF Cell (model 111; Bio-Rad) (21).

Western blot analysis. After SDS-PAGE or IEF gel electrophoresis, protein was transferred to a nitrocellulose membrane and Western blot analysis was carried out as previously described (19). Membranes were blocked and subsequently incubated overnight at 4°C with mouse monoclonal anti-CN A subunit at 1:1,000 (Transduction Laboratories, Lexington, KY), mouse monoclonal anti-CN B subunit antibody at 1:1,500 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-CRHSP-24 at 1:1,000 (20), rabbit anti-ERK 1, rabbit anti-ERK2, and rabbit anti-p38 MAPK at 1;500 (Santa Cruz Biotechnology, Santa Cruz, CA). After being washed, membranes were incubated with an appropriate second anti-IgG antibody conjugated with horseradish peroxidase. Antibody binding was detected by an enhanced chemiluminescence detection system (Amersham Pharmacia), and the emitted light was recorded on X-ray films. Images were scanned from film with Agfa Arcus II Scanner, and quantification was performed by using Multi-Analyst software (Bio-Rad Laboratories).

Morphological examination. A portion of the pancreas was fixed overnight at 4°C in 3.7% phosphate-buffered formaldehyde (pH 7.4). Tissues were embedded in paraffin, sectioned, and processed for hematoxylin and eosin staining.

Statistical analysis. Results were expressed as means ± SE, and n is the number of mice. Statistical analysis was carried out by unpaired Student's t-test or one-way ANOVA followed by a post hoc test for multiple group comparisons as appropriate. Differences with P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of camostat on pancreatic growth in mice. Chronic feeding of camostat to mice for 10 days has been reported to induce pancreatic growth (37). We therefore first examined the time course of increases in pancreatic wet weight and protein (hypertrophy) and DNA (hyperplasia) in this model. Mice consumed ~4 g/day of diet resulting in an intake of ~130 mg camostat·kg body wt-1·day-1. In response to camostat, pancreatic wet weight, protein, and DNA were increased after 2 days and reached a maximum in 7–10 days (Table 1). There were no significant differences in body weight between control and camostat-fed groups during the experiment. Camostat feeding to mice did not increase the weights of other organs evaluated including liver, spleen, kidney, and heart (data not shown); oral administration of camostat also has been reported to have no effect on the weight of the intestine (14). Histological examination of the pancreas after camostat feeding for 10 days showed intact pancreatic acini of normal size with evidence of hyperstimulation, i.e., increased basolateral basophillia, and some vacuolization compared with control acini. Pancreatic islets were of normal size although decreased in frequency per section as would be expected from distribution in a larger mass of exocrine tissue (data not shown). This selective effect on the exocrine pancreas is consistent with prior reports that total insulin content in the rat pancreas was not changed after camostat administration (16, 35).


View this table:
[in this window]
[in a new window]
  		Table 1. Body weight, pancreatic wet weight, pancreatic protein content, and pancreatic DNA content in camostat fed mice

 

It is believed that oral administration of protease inhibitor or dietary protein induces release of endogenous CCK, resulting in stimulation of pancreatic growth (17, 18). To confirm the role of endogenous CCK in the pancreatic growth induced by camostat, we fed camostat to CCK-deficient mice for 10 days. As observed in the first experiment, in control mice, chronic camostat feeding markedly increased the pancreatic wet weight-to-body weight ratio and protein and DNA content compared with control chow (Fig. 1). In contrast, chronic camostat feeding to CCK-deficient mice did not increase those three parameters compared with control chow. Therefore, this observation confirms that the effect of camostat is mediated by endogenous CCK in mice.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Requirement of CCK for camostat-induced growth of the mouse pancreas. CCK-deficient (CCK-/-) or genetically matched control 129/Sv (CCK+/+) mice were randomly assigned to two different groups and fed either regular powdered chow or camostat mixture for 10 days. Graphs show the means ± SE from 5–6 animals for each condition. *P < 0.05; **P < 0.01 vs. no camostat.

 

Regulation of CN protein and its activity in the pancreas after camostat feeding. We detected both CN A and B subunits by Western blot analysis using specific antibodies. CN A subunit ran at 61 kDa and CN B at 19 kDa in control pancreas (Fig. 2A). In response to camostat feeding, CN A and B subunits were upregulated (5.7 ± 1.5-fold and 2.8 ± 0.3-fold vs. control, n = 6) relative to total protein. In contrast to the pancreas, the amount of both CN A and B subunits were unchanged in the liver, spleen, kidney, or heart (data not shown). We also examined other proteins thought to play a role in cell growth including ERKs and p38 MAPK. In contrast to CN, these signaling proteins were unchanged in amount in mice fed camostat (Fig. 2A).



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. A: effect of camostat on pancreatic signaling protein expression. Both calcineurin (CN) subunits A and B were increased in the camostat-fed mouse pancreas, whereas ERK1/2 and p38 MAPK proteins were unchanged. Representative Western blots of pancreatic protein lysates (30 µg/lane) after SDS-PAGE are shown. B: camostat increased the dephosphorylated forms of pancreatic calcium-regulated heat-stable protein of 24 kDa (CRHSP-24) as revealed by Western blot analysis after isoelectric focusing (IEF) of pancreatic protein lysates (30 µg/lane). Forms: 4P, tetraphosphorylated; 3P, triphosphorylated; 2P, diphosphorylated; 1P, monophosphorylated. Graphs show the means ± SE from 5 animals.

 

Our laboratory (21) previously identified a novel CN substrate, CRHSP-24, which is dephosphorylated by CN in response to CCK in rat pancreatic acinar cells. Thus an increase in the dephosphorylated states of CRHSP-24 indicates CN activation. From 2 to 10 days after camostat feeding, the percentage of CRHSP-24 in the dephosphorylated bands was increased, indicating CN activation in vivo (Fig. 2B). In mice fed camostat, total CRHSP-24 protein was also modestly upregulated in the pancreas (1.8 ± 0.2-fold vs. control, n = 6) when evaluated on SDS gels, although the example in the IEF gel shown in Fig. 2B shows a greater upregulation as early as 2 days.

Both CsA and FK506 block pancreatic growth induced by camostat. Because CsA and FK506 are CN inhibitors that depend on distinct intracellular receptors (29, 47), we injected either CsA or FK506 to camostat-fed mice to evaluate the role of CN in pancreatic growth. Neither compound affected body weight. We first used CsA, which binds to cyclophilin A, and the complex then inhibits CN. Camostat feeding for 10 days to animals injected with vehicle significantly increased the pancreatic weight-to-body weight ratio compared with normal chow with vehicle injection (Fig. 3, top). CsA injection with camostat feeding significantly reduced this increase, whereas CsA injection with normal chow had only a slight inhibitory action on the ratio (Fig. 3, top). Camostat feeding with vehicle injection also increased protein content and DNA content compared with normal chow with vehicle injection (Fig. 3, middle and bottom). CsA injection with camostat feeding blocked the increase in these parameters, whereas CsA injection with normal chow had no effect compared with vehicle injection with normal chow.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. Effect of cyclosporine A (CsA) on pancreatic growth induced by camostat feeding. Four different groups of ICR mice were fed either regular powdered chow or 0.1% camostat mixture for 10 days. Animals received injections with either 15 mg/kg sc CsA suspensed in vehicle or vehicle alone, twice a day for 10 days. Graphs show the means ± SE from 5–6 animals. *P < 0.01 vs. control group without camostat or CsA.

 

We also studied FK506, because it binds to a distinct receptor, FKBP12, with the complex inhibiting CN. In this study, camostat feeding with vehicle injection also stimulated pancreatic wet weight-to-body weight ratio, protein content, and DNA content compared with normal chow with vehicle injection (Fig. 4). FK506 injection with camostat feeding also greatly reduced the increase in the parameters, resulting in the same levels as vehicle injection with normal chow. As with CsA injection, the effect on pancreatic wet weight-to-body weight ratio was not complete, whereas the effect on protein and DNA content was complete. FK506 injection with normal chow had no effect on the parameters compared with vehicle injection with normal chow.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4. Effect of FK506 on pancreatic growth induced by camostat feeding. Four different groups of ICR mice were fed either regular powdered chow or 0.1% camostat mixture for 10 days. Animals received injections with either 3 mg/kg sc FK506 in vehicle or vehicle alone, twice a day for 10 days. Graphs show the means ± SE from 6 animals. *P < 0.01 vs. control group without camostat or FK506.

 

Histological examination showed no pancreatic damage, edema, cellular infiltration, or fibrosis induced by either CsA or FK506 in the absence or presence of camostat (data not shown). Serum amylase levels also showed no consistent difference compared with vehicle injection (data not shown). Thus the dose of CsA or FK506 used in this study had no effect to induce pancreatitis or pancreatic damage.

Neither CsA nor FK506 blocks releases of endogenous CCK by camostat. We examined the time course of plasma CCK levels during camostat feeding. In response to camostat, plasma CCK was increased after 1 day and remained elevated over the 10-day period (Fig. 5A). To confirm that the immunosuppressants do not block the release of endogenous CCK induced by camostat feeding, we pretreated with either vehicle (CsA or FK506) for 2 days and then fed camostat mixture for 2 days. Both CsA and FK506 slightly increased basal and camostat-induced CCK levels without any indication of inhibition (Fig. 5B).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. Plasma CCK levels after camostat feeding and treatment with immunosuppressants. A: camostat feeding significantly increased plasma CCK measured after 1–10 days. All values are means ± SE of 7–11 measurements. B: mice were treated with 15 mg/kg CsA, 3 mg/kg FK506, or ethanol vehicle twice daily at 7 AM and 7 PM and then switched after 2 days as indicated to camostat-containing diet. Blood was drawn from all mice 2 days later, 2 h after the last dose of inhibitor or vehicle. All values are means ± SE of 3–4 determinations for control diet (each sample was pooled from 3 mice) and 6–11 determinations (individual mice) from animals fed camostat. *P < 0.05 vs. no camostat or inhibitor.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we present evidence that oral administration of the protease inhibitor camostat stimulated pancreatic growth that is mediated by the gastrointestinal hormone CCK. The camostat-induced pancreatic growth requires the Ca2+-activated protein phosphatase CN and is accompanied by selective upregulation of CN A and B subunits.

That camostat-induced pancreatic growth is mediated by CCK is not surprising given the wealth of data that CCK injection can induce pancreatic growth and the fact that most studies have shown that CCK receptor antagonists block the growth response to increasing endogenous CCK by trypsin inhibitor. However, less data exist for mice than rats, and in a recent study, a high-protein diet (containing 78.4% protein) was found to induce pancreatic growth in CCK-deficient mice similar to that seen in control mice compared with control diets (containing 28.4% protein) or high fat or high carbohydrate diets (26), thus indicating the possibility of alternative mediators. In our study, camostat was added to the same control diet in powdered form but had no affect in CCK-deficient mice. Although camostat feeding may also increase the release of endogenous secretin (49), it is clear that it is CCK that is required for camostat-induced growth. The trophic effect of CCK in rats appears primarily to be through high-affinity CCK receptors (7) located on the acinar cells (53). The effect of the high-protein diet may be mediated by another hormone or neural pathway but also may be mediated by amino acids from the dietary protein. Branched-chain amino acids, especially, are known to be effective stimulators of protein synthesis (1). Because oral administrations of amino acids are not significant stimulators of CCK release (26), it is likely that normal pancreatic growth requires both CCK and amino acids and that the amino acid composition of the normal diet was clearly sufficient.

A major finding of this study is that pancreatic growth induced by endogenous CCK can be blocked with two chemically distinct immunosuppressants whose actions are known to be mediated by inhibiting CN. Both CsA and FK506 bind to distinct intracellular proteins, cyclophilin A and FKBP-12. The ligand-binding protein then binds CN, contacting both subunits and preventing access of protein substrates to catalytic residues (23). Although a high concentration of CsA can affect mitochondrial ion permeability, FK506 does not have this action (12). The concentrations of the two agents we used are similar to those used to inhibit cardiac hypertrophy in vivo (34, 45). Moreover, minimal changes in serum amylase level or pancreatic histology were seen after administration of CsA or FK506, making a toxic effect on the pancreas unlikely. Previous reports (9, 48) suggest the potential harmful effect of these agents in rat pancreas. These differences may occur from species, doses, or administration route. Because we are using a pancreatic growth paradigm involving release of endogenous CCK, it was important to confirm that the immunosuppressants are not blocking endogenous CCK release. We therefore measured plasma CCK levels and showed that the increase in CCK induced by camostat was not affected by either CsA or FK506.

The mechanism by which CN mediates growth of the pancreas remains to be established. CN is activated during camostat feeding as a result of the action of CCK to increase intracellular Ca2+ (56). That this was occurring in our study is shown by the dephosphorylation of the CN substrate CRHSP-24. However, the function of CRHSP-24 is unknown. A major known action of CN is to dephosphorylate the nuclear factor of activated T cells (NFAT) (38). NFAT is normally in the cytoplasm, but on dephosphorylation it enters the nucleus and binds to DNA along with other transcription factors and in T cells activates production of interleukin-2. Although NFAT was originally identified in T cells, multiple isoforms are now known to be broadly distributed (22, 38). One mechanism by which CN signaling initiates cardiac hypertrophy is through NFAT3 because transgenic mice expressing constitutively active CN or NFAT3 in cardiac myocytes showed cardiac hypertrophy (34). In this case, NFAT3 was believed to interact with another transcription factor, GATA4. Such a mechanism could also explain the action to induce pancreatic growth, because NFAT isoforms are expressed in the pancreas (38), although whether in acinar cells and which isoform is unknown.

A different possible mechanism could involve a role for CN in protein synthesis. CCK is known to enhance pancreatic protein synthesis in isolated pancreatic acini (25), and both exogenous and endogenous CCK activate various translational mechanisms to enhance protein synthesis (2, 3). In a brief report, Loser et al. (31) reported that CsA inhibited the induction of ornithine decarboxylase (ODC) in camostat-treated rats. Polyamines have long been known to be required for cellular growth, and ODC is known to be under translational control in which its translation is enhanced by ribosomal S6 protein. More directly, we have found that both CsA and FK506 reduce basal and CCK-stimulated protein synthesis in vitro (41). Because protein synthesis is required for cells to exit from G0 and enter the cell cycle, an effect of the immunosupressants could also block mitogenesis. In fact, both CsA and FK506, but not a Ca2+/calmodulin-dependent protein kinase II inhibitor, inhibited DNA synthesis in fibroblasts (46). At present, however, there is no obvious mechanism by which CN might regulate the translation machinery.

Pancreatic hypertrophy and hyperplasia in response to endogenous CCK are also likely to involve activation of other pathways including the ERKs, phosphatidylinositol 3-kinase, and p70 S6 kinase (3, 50, 51). These pathways are likely to be independent of CN signaling. The fact that CN activation appears to be required for pancreatic growth does not by itself mean that CN activation is sufficient to induce growth. Similar conclusions have been reached concerning the regulation of cardiac hypertrophy (33). Further work is clearly needed to establish both the mechanism of CN in the pancreatic growth response and how it interacts with other growth-promoting intracellular pathways.


   ACKNOWLEDGMENTS
 
We thank Craig D. Logsdon for critical review of this manuscript, Louis G. D'Alecy for advice on blood collection, and Stephanie Johnston and Bradley Nelson for assistance with the CCK assays.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52860 and DK-59578 (to J. A. Williams), DK-38626 (to R. A. Liddle), DK-48815 (to L. C. Samuelson), and DK-34933 (to The Michigan Digestive Disease Research Core Center).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Williams, Molecular and Integrative Physiology, Univ. of Michigan Medical School, 7744 Medical Science II, Ann Arbor, MI 48109-0622 (E-mail: jawillms{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anthony JC, Anthony TG, Kimball SR, Vary TC, and Jefferson LS. Orally administered leucine stimulates protein synthesis in skeletal muscle of post absorptive rats in association with increased eIF4F formation. J Nutr 130: 139-145, 2000.[Abstract/Free Full Text]
  2. Bragado MJ, Groblewski GE, and Williams JA. Regulation of protein synthesis by cholecystokinin in rat pancreatic acini involves PHAS-1 and the p70 S6 kinase pathway. Gastroenterology 115: 733-742, 1998.[CrossRef][Web of Science][Medline]
  3. Bragado MJ, Tashiro M, and Williams JA. Regulation of the initiation of pancreatic digestive enzyme protein synthesis by cholecystokinin in rat pancreas in vivo. Gastroenterology 119: 1731-1739, 2000.[CrossRef][Web of Science][Medline]
  4. Burnham DB. Characterization of Ca2+-activated protein phosphatase activity in exocrine pancreas. Biochem J 231: 335-341, 1985.[Web of Science][Medline]
  5. Chernick SS, Lepovski S, and Chaikoff IL. A dietary factor regulating the enzyme content of the pancreas: changes induced in size and proteolytic activity of the chick pancreas by the ingestion of raw soy-bean meal. Am J Physiol 155: 33-41, 1948.[Free Full Text]
  6. Crabtree GR. Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96: 611-614, 1999.[CrossRef][Web of Science][Medline]
  7. Dawra R, Saluja A, Lerch MM, Saluja M, Logsdon C, and Steer M. Stimulation of pancreatic growth by cholecystokinin is mediated by high affinity receptors on rat pancreatic acinar cells. Biochem Biophys Res Commun 193: 814-820, 1993.[CrossRef][Web of Science][Medline]
  8. Dembinski AB and Johnson LR. Stimulation of pancreatic growth by secretin, caerulein, and pentagastrin. Endocrinology 106: 323-332, 1980.[Abstract/Free Full Text]
  9. Doi R, Tangoku A, Inoue K, Chowdhury P, and Rayford PL. Effects of FK506 on exocrine pancreas in rats. Pancreas 7: 197-204, 1992.[Web of Science][Medline]
  10. Douglas BR, Woutersen RA, Jansen JB, Rovati LC, and Lamers CB. Comparison of the effect of lorglumide on pancreatic growth stimulated by camostate in rat and hamster. Life Sci 46: 281-286, 1990.[CrossRef][Web of Science][Medline]
  11. Dunn SE, Burns JL, and Michel RN. Calcineurin is required for skeletal muscle hypertrophy. J Biol Chem 274: 21908-21912, 1999.[Abstract/Free Full Text]
  12. Friberg H, Ferrand-Drake M, Bengtsson F, Halestrap AP, and Wieloch T. Cyclosporin A, but not FK506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondria permeability transition in cell death. J Neurosci 18: 5151-5159, 1998.[Abstract/Free Full Text]
  13. Gasslander T, Axelson J, Hakanson R, Ihse I, Lilja I, and Rehfeld JF. Cholecystokinin is responsible for growth of the pancreas after pancreaticobiliary diversion in rats. Scand J Gastroenterol 25: 1060-1065, 1990.[Web of Science][Medline]
  14. Ge YC and Morgan RGH. The effect of trypsin inhibitor on the pancreas and small intestine of mice. Br J Nutr 70: 333-345, 1993.[CrossRef][Web of Science][Medline]
  15. Göke B, Printz H, Koop I, Rausch U, Richter G, Arnold R, and Adler G. Endogenous CCK release and pancreatic growth in rats after feeding a proteinase inhibitor (camostat). Pancreas 1: 509-515, 1986.[Medline]
  16. Göke B, Siegel EG, Stöckmann F, Lankisch PG, and Creutzfeldt W. Effect of a specific serine protease inhibitor on the rat pancreas: influence of camostat on the endocrine pancreas. Digestion 31: 128-135, 1985.[Web of Science][Medline]
  17. Green GM, Jurkowska G, Berube FL, Rivard N, Guan D, and Morisset J. Role of cholecystokinin in induction and maintenance of dietary protein-stimulated pancreatic growth. Am J Physiol Gastrointest Liver Physiol 262: G740-G746, 1992.[Abstract/Free Full Text]
  18. Green GM, Levan VH, and Liddle RA. Plasma cholecystokinin and pancreatic growth during adaptation to dietary protein. Am J Physiol Gastrointest Liver Physiol 251: G70-G74, 1986.[Abstract/Free Full Text]
  19. Groblewski GE, Grady T, Mehta N, Lambert H, Logsdon CD, Landry J, and Williams JA. Cholecystokinin stimulates heat shock protein 27 phosphorylation in rat pancreas both in vivo and in vitro. Gastroenterology 112: 1354-1361, 1997.[CrossRef][Web of Science][Medline]
  20. Groblewski GE, Wagner ACC, and Williams JA. Cyclosporin A inhibits Ca2+/calmodulin-dependent protein phosphatase and secretion in pancreatic acinar cells. J Biol Chem 269: 15111-15117, 1994.[Abstract/Free Full Text]
  21. Groblewski GE, Yoshida M, Bragado MJ, Ernst SA, Leykam J, and Williams JA. Purification and characterization of a novel physiological substrate for calcineurin in mammalian cells. J Biol Chem 273: 22738-22744, 1998.[Abstract/Free Full Text]
  22. Horsley V and Pavlath GK. NFAT: Ubiquitous regulator of cell differentiation and adaptation. J Cell Biol 156: 771-774, 2002.[Abstract/Free Full Text]
  23. Jin L and Harrison SC. Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophillin. Proc Natl Acad Sci USA 99: 13522-13526, 2002.[Abstract/Free Full Text]
  24. Klee CB, Ren H, and Wang X. Regulation of the calmodulin-stimulated protein phosphatase, calcineurin. J Biol Chem 273: 13367-13370, 1998.[Free Full Text]
  25. Korc M, Bailey AC, and Williams JA. Regulation of protein synthesis in normal and diabetic rat pancreas by cholecystokinin. Am J Physiol Gastrointest Liver Physiol 241: G116-G121, 1981.[Abstract/Free Full Text]
  26. Lacourse KA, Swanberg LJ, Gillespie PJ, Rehfeld JF, Saunders TL, and Samuelson LC. Pancreatic function in CCK-deficient mice: adaptation to dietary protein does not require CCK. Am J Physiol Gastrointest Liver Physiol 276: G1302-G1309, 1999.[Abstract/Free Full Text]
  27. Liddle RA, Goldfine ID, and Williams JA. Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor and alcohol. Gastroenterology 87: 542-549, 1984.[Web of Science][Medline]
  28. Liddle RA, Green GM, Conrad CK, and Williams JA. Proteins but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am J Physiol Gastrointest Liver Physiol 251: G243-G248, 1986.[Abstract/Free Full Text]
  29. Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, and Schreiber SL. Calcineurin is a common target of cyclophillin-cyclosporine A and FKBP-FK506 complexes. Cell 66: 807-815, 1991.[CrossRef][Web of Science][Medline]
  30. Logsdon CD. Role of cholecystokinin in physiologic, and pathophysiologic growth of the pancreas. In: Gastrointestinal Endocrinology, edited by Greeley GH. Totowa, NJ: Humana, 1999, p. 393-422.
  31. Loser C, Stockmann F, Folsch UR, and Creutzfeldt W. Dose-dependent inhibition by cyclosporine A of the induction of pancreatic ornithine decarboxylase (ODC) in rats. Int J Pancreatol 5: 179-189, 1989.[Web of Science][Medline]
  32. Mitchell PO, Mills ST, and Pavlath GK. Calcineurin differentially regulates maintenance and growth of phenotypically distinct muscles. Am J Physiol Cell Physiol 282: C984-C992, 2002.[Abstract/Free Full Text]
  33. Molkentin JD and Dorn GW. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol 63: 391-426, 2001.[CrossRef][Web of Science][Medline]
  34. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215-228, 1998.[CrossRef][Web of Science][Medline]
  35. Müller MK, Goebell H, Alfen R, Ehlers J, Jäger M, and Plümpe H. Effects of camostat, a synthetic protease inhibitor, on endocrine and exocrine pancreas of the rat. J Nutr 118: 645-650, 1988.[Abstract/Free Full Text]
  36. Musarò A, McCullagh KJ, Naya FJ, Olson EN, and Rosenthal N. IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-Atc1. Nature 400: 581-585, 1999.[CrossRef][Medline]
  37. Niederau C, Liddle RA, Williams JA, and Grendell JH. Pancreatic growth: interaction of exogenous cholecystokinin, a protease inhibitor, and a cholecystokinin receptor antagonist in mice. Gut 28, Suppl 1: 63-69, 1987.[Abstract/Free Full Text]
  38. Rao A, Luo C, and Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15: 707-747, 1997.[CrossRef][Web of Science][Medline]
  39. Rivard N, Guan D, Maouyo D, Grondin G, Berube FL, and Morisset J. Endogenous cholecystokinin release responsible for pancreatic growth observed after pancreatic juice diversion. Endocrinology 129: 2867-2874, 1991.[Abstract/Free Full Text]
  40. Rothman SS and Wells H. Enhancement of pancreatic enzyme synthesis by pancreozymin. Am J Physiol 213: 215-218, 1967.[Free Full Text]
  41. Sans MD and Williams JA. Calcineurin is required for protein synthesis by normal rat pancreatic acini (Abstract). Gastroenterology 118, Suppl 2: A158, 2000.
  42. Semsarian C, Wu MJ, Ju YK, Marciniec T, Yeoh T, Allen DG, Harvey RP, and Graham RM. Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signaling pathway. Nature 400: 576-581, 1999.[CrossRef][Medline]
  43. Solomon TE, Petersen H, Elashoff J, and Grossman MI. Interaction of caerulein and secretin on pancreatic size and composition in rat. Am J Physiol Endocrinol Metab Gastrointest Physiol 235: E714-E719, 1978.[Abstract/Free Full Text]
  44. Struthers BJ, MacDonald JR, Dahlgren RR, and Hopkins DT. Effects on the monkey, pig and rat pancreas of soy products with varying levels of trypsin inhibitor and comparison with the administration of cholecystokinin. J Nutr 113: 86-97, 1983.[Abstract/Free Full Text]
  45. Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, Colbert MC, Gualberto A, Wieczorek DF, and Molkentin JD. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 281: 1690-1693, 1998.[Abstract/Free Full Text]
  46. Tomono M, Toyoshima K, Ito M, and Amano H. Calcineurin is essential for DNA synthesis in Swiss 3T3 fibroblasts. Biochem J 317: 675-680, 1996.[Medline]
  47. Walsh CT, Zydowsky LD, and McKeon FD. Cyclosporin A, the cyclophillin class of peptidylprolyl isomerases and blockade of T cell signal transduction. J Biol Chem 267: 13115-13118, 1992.[Abstract/Free Full Text]
  48. Waschulewski IH, Hall DV, Kern HF, and Edwardson JM. Effects of the immunosuppressants cyclosporin A and FK506 on exocytosis in the rat exocrine pancreas in vivo. Br J Pharmacol 108: 892-900, 1993.[Web of Science][Medline]
  49. Watanabe S, Takeuchi T, and Chey WY. Mediation of trypsin inhibitor-induced pancreatic hypersecretion by secretin and cholecystokinin in rats. Gastroenterology 102: 621-628, 1992.[Web of Science][Medline]
  50. Williams JA. Intracellular signaling mechanisms activated by cholecystokinin regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 63: 77-79, 2001.[CrossRef][Web of Science][Medline]
  51. Williams JA. Receptor biology and intracellular regulatory mechanisms in pancreatic acinar cells. Curr Opin Gastroenterol 18: 529-535, 2002.[Medline]
  52. Wisner JR Jr, McLaughlin RE, Rich KA, Ozawa S, and Renner IG. Effects of L-364,718, a new cholecystokinin receptor antagonist, on camostat-induced growth of the rat pancreas. Gastroenterology 94: 109-113, 1988.[Web of Science][Medline]
  53. Yamamoto M, Otani M, Jia DM, Fukumitsu KI, Yoshkawa H, Akiyama T, and Otsuki M. Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats. Am J Physiol Gastrointest Liver Physiol 285: G681-G687, 2003.[Abstract/Free Full Text]
  54. Yokoyama N, Furuyama S, and Wang JH. Demonstration of calmodulin-stimulated phosphatase isoenzymes by monoclonal antibodies. J Biol Chem 265: 8170-8175, 1990.[Abstract/Free Full Text]
  55. Yonezawa H. Secretory responses of hypertrophied rat pancreas induced by repeated oral administration of a synthetic protease inhibitor. Jpn J Physiol 33: 183-195, 1983.[Web of Science][Medline]
  56. Yule DI and Williams JA. Stimulus-secretion coupling in the pancreatic acinus. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994, p. 1447-1472.



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
A. U. Shah, A. Sarwar, A. I. Orabi, S. Gautam, W. M. Grant, A. J. Park, A. U. Shah, J. Liu, P. K. Mistry, D. Jain, et al.
Protease activation during in vivo pancreatitis is dependent on calcineurin activation
Am J Physiol Gastrointest Liver Physiol, November 1, 2009; 297(5): G967 - G973.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. J. Crozier, M. D. Sans, C. H. Lang, L. G. D'Alecy, S. A. Ernst, and J. A. Williams
CCK-induced pancreatic growth is not limited by mitogenic capacity in mice
Am J Physiol Gastrointest Liver Physiol, May 1, 2008; 294(5): G1148 - G1157.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
G. T. Gurda, L. Guo, S.-H. Lee, J. D. Molkentin, and J. A. Williams
Cholecystokinin Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo
Mol. Biol. Cell, January 1, 2008; 19(1): 198 - 206.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Z. Husain, W. M. Grant, F. S. Gorelick, M. H. Nathanson, and A. U. Shah
Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin
Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1594 - G1599.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
L. Guo, M. D. Sans, G. T. Gurda, S.-H. Lee, S. A. Ernst, and J. A. Williams
Induction of early response genes in trypsin inhibitor-induced pancreatic growth
Am J Physiol Gastrointest Liver Physiol, February 1, 2007; 292(2): G667 - G677.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
S. J. Crozier, M. D. Sans, L. Guo, L. G. D'Alecy, and J. A. Williams
Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice
J. Physiol., June 15, 2006; 573(3): 775 - 786.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
N. Kloting, N. Follak, and I. Kloting
Is there an autoimmune process in bone? Gene expression studies in diabetic and nondiabetic BB rats as well as BB rat-related and -unrelated rat strains
Physiol Genomics, December 14, 2005; 24(1): 59 - 64.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. D. Sans and J. A. Williams
Calcineurin is required for translational control of protein synthesis in rat pancreatic acini
Am J Physiol Cell Physiol, August 1, 2004; 287(2): C310 - C319.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/G784    most recent
00446.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tashiro, M.
Right arrow Articles by Williams, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tashiro, M.
Right arrow Articles by Williams, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.