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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Signal. Author manuscript; available in PMC 2010 April 1.
Published in final edited form as:
PMCID: PMC2677382

Gastrointestinal growth factors and hormones have divergent effects on Akt activation


Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigate in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters[CCK, bombesin,carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations(pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations(nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.

Keywords: Pancreas, Akt, CCK, phosphorylation, kinase, Src, gastrointestinal hormones, PI3K, PK

1. Introduction

The members of the Akt family of protein kinases, including Akt1, Akt2 and Akt 3, have been shown to regulate multiple biological functions including apoptosis, cell survival, protein synthesis and glycogenesis [1-4]. Structurally, Akt contains a N-terminal pleckstrin homology domain (which has been shown to bind phosphoinositides), a catalytic domain related to protein kinases A and C (hence the alternative name of PKB) and a C-terminal regulatory region [2]. Two phosphorylation sites are involved in Akt activation: T308 in the catalytic domain and S473 at the C-terminus. After stimulation by insulin or growth factors, both sites are phosphorylated involving a PI3K-dependant mechanism: T308 is phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase 1) and S473 by a yet unidentified kinase [5]. Akt activation is highly correlated with the S473 phosphorylation level [5-7]. After stimulation by growth factors, generation of 3-phosphoinositides at the plasma membrane cause pleckstrin-domain-mediated translocation of PDK1 and Akt to the membrane, resulting in T308 and S473 phosphorylation and Akt activation [8].

Growth factors almost uniformly activate Akt and the underlying mechanisms have been well studied [9-11]. In contrast, with most G-protein coupled receptors and especially with those mediating the actions of gastrointestinal hormones/neurotransmitters, their effects on Akt activation have been much less well studied and the mechanisms of their effects on Akt activation are largely unknown. Activation of some G-protein coupled receptors has been reported to activate Akt in some studies [1,7,12-17], while others report inhibition of Akt by these stimulants [1,18-20]. In some cases such as with the GI hormone/neurotransmitters, cholecystokinin/gastrin, they are reported to stimulate Akt activation in some tissues, inhibit Akt activation in others and not to alter Akt activity in still others [7,17,21-24]. The cellular mechanisms involved in these differences are unclear.

Pancreatic acinar cells are an excellent model for studying the effect of growth factors and G-protein coupled receptor agonists for gastrointestinal hormones/neurotransmitters, because these cells have been shown to express a number of different G protein-coupled receptors for gastrointestinal hormones/neurotransmitters and growth factors functionally connected to multiple signal transduction pathways [25-32]. Dispersed pancreatic acini can be prepared from intact pancreas that are responsive to a number of stimulants, displaying activation/inhibition of multiple signal transduction pathways and changes in cellular function, which allow their cellular basis of action to be studied in vitro. [25-27,29-31]. Therefore, we choose to study the effect of growth factors and gastrointestinal hormones on Akt activation in pancreatic acini. The examination of their actions in pancreatic acinar cells is also important because PI3K/Akt signaling has been shown to play a central role in pancreatic regeneration [33] and pathological responses of pancreatic acinar cells to CCK [34], but the underlying mechanisms remain unclear. Furthermore, both aberrant signaling through growth factors or gastrointestinal hormones and Akt have been reported to occur in pancreatic pathologies, including cancer and pancreatitis [35-40].

Therefore, the aim of the present study was two-fold. First, to examine the ability of various important gastrointestinal growth factors and gastrointestinal hormones/neurotransmitters to alter Akt activation in a well-established experimental model using primary, non-transfected and non-transformed gastrointestinal cells (i.e. pancreatic acini) and to elucidate the signaling cascades causing the effect on Akt activity by these pancreatic acinar cell stimulants. Furthermore, the signaling cascade for the effects of the GI hormone/neurotransmitter, CCK on Akt activation was studied in detail because of its importance as physiological regulator of pancreatic function as well as its importance in causing experimental pancreatitis, which is a widely used model to study this clinical disorder [21,41].

2. Materials and Methods


Male Sprague-Dawley rats (150-250 g) were obtained from the Small Animals Section, Veterinary Resources Branch, National Institutes of Health (NIH), Bethesda, MD. Rabbit anti-phospho-Akt-pT308, mouse anti-phospho-Akt-pS473, rabbit anti-Akt antibodies, rabbit anti-phospho-Src family (Y416), mouse anti-phospho p44/42 MAPK (T202/Y204) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Bovine anti-goat horseradish peroxidase (HRP)-conjugate and anti-rabbit-HRP-conjugate antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-tyrosine antibody (PY20) was purchased from BD Biosciences (San Jose, CA). Rabbit anti-PI3-kinase p85 was purchased from Upstate Biotechnology (Lake Placid, NY). Tris/HCl pH 8.0 and 7.5 were form Mediatech, Inc. (Herndon, VA). 2-mercaptoethanol, protein assay solution and SDS were from Bio-Rad Laboratories (Hercules, CA). CaCl2 and MgCl2 were from Quality Biological, Inc. (Gaithersburg, MD). Dulbecco's phosphate buffered saline (DPBS), glutamine (200 mM), Tris/glycine/SDS buffer (10×), and Tris/glycine buffer (10×) were from Biosource. Minimal essential media (MEM) vitamin solution, basal medium Eagle (BME) amino acids 100× and Tris-Glycine gels were from Invitrogen (Carlsbad, CA). COOH-terminal octapeptide of cholecystokinin (CCK-8), hepatocyte growth factor (HGF), bombesin, insulin-like growth factor 1 (IGF-I), basic fibroblast growth factor (bFGF), vasoactive intestinal peptide (VIP), endothelin and secretin were from Bachem Bioscience Inc. (King of Prussia, PA). EGF, thapsigargin, PDGF, GF109203X, A23187, LY294002, wortmannin, PD98059, U0126, PP2, PP3 and deoxycholic acid were from Calbiochem (La Jolla, CA). Carbachol, insulin, dimethyl sulfoxide (DMSO), 12-O-tetradecanoylphobol-13-acetate (TPA), L-glutamic acid, fumaric acid, pyruvic acid, trypsin inhibitor, acetic acid, HEPES, Triton X-100, TWEEN® 20, phenylmethanesulfonylfluoride (PMSF), EGTA, NaF, Na4P2O7, phosphatidylinositol, phosphatidylserine, sucrose, sodium-orthovanadate and sodium azide were from Sigma-Aldrich, Inc. (St. Louis, MO). Albumin standard, Protein G, Super Signal West (Pico, Dura) chemiluminescent substrate and stripping buffer were from Pierce (Rockford, IL). Protease inhibitor tablets were from Roche (Basel, Switzerland). Purified collagenase (type CLSPA) was from Worthington Biochemicals (Freehold, NJ). Nitrocellulose membranes were from Schleicher and Schuell BioScience, Inc. (Keene, NH). [γ32P]ATP (3000 Ci/mmol) was purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ). TLC plastic Sheets was purchased from EMD Chemicals (Gibbestown, NJ). 2-propanol and chloroform were purchased from Mallinckrodt Laboratory Chemicals (Phillipsburg, NJ). Alexa 594-conjugated anti-rabbit secondary antibody was from Molecular Probes (Eugene, OR). Poly-L-lysine coated slides and sample chambers were from Wescor (Logan, UT).


Tissue Preparation

Pancreatic acini were obtained by collagenase digestion as previously described [26]. Standard incubation solution contained 25.5 mM HEPES (pH 7.45), 98 mM NaCl, 6 mM KCl, 2.5 mM NaH2PO4, 5 mM sodium pyruvate, 5 mM sodium glutamate, 5 mM sodium fumarate, 11.5 mM glucose, 0.5 mM CaCl2, 1 mM MgCl2, 1 mM glutamine, 1% (w/v) albumin, 0.01% (w/v) trypsin inhibitor, 1% (v/v) vitamin mixture and 1% (v/v) amino acid mixture.

Acini Stimulation

After collagenase digestion, dispersed acini were pre-incubated in standard incubation solution for 2 hrs at 37 °C with or without inhibitors as described previously [26,30]. After pre-incubation 1 ml aliquots of dispersed acini were incubated at 37 °C with or without stimulants. Cells were lysed in lysis buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium azide, 1 mM EGTA, 0.4 mM EDTA, 0.2 mM sodium orthovanadate, 1 mM PMSF, and one protease inhibitor tablet per 10 ml). After sonication, lysates were centrifuged at 10,000× g for 15 min at 4 °C and protein concentration was measured using the Bio-Rad protein assay reagent. Equal amounts of samples were analyzed by SDS-PAGE and Western blotting.

Western Blotting/immunoprecipitation

Western blotting and immunopecipitation were performed as described previously [42]. Whole cell lysates, immunoprecipitates or lysates of subcellular fractions were subjected to SDS-PAGE using 10% and 4-20% Tris-Glycine gels. After electrophoresis, protein was transferred to nitrocellulose membranes. Membranes were blocked in blocking buffer (50 mM Tris/HCl pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.05% Tween® 20, 5% nonfat dry milk) at 4°C overnight or at room temperature for one hour. Membranes were then incubated with primary antibody under constant agitation at antibody dilutions suggested by the antibody supplier. After primary antibody incubation membranes were washed twice in blocking buffer for 4 minutes and then incubated with HRP-conjugated secondary antibody (anti-mouse, anti-rabbit, anti-goat) according to the species of the first antibody for 45 minutes at room temperature under constant agitation. Membranes were then washed again twice in blocking buffer for 4 minutes, twice in washing buffer (50 mM Tris/HCl pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.05% Tween® 20) for 4 minutes, incubated for 4 minutes with chemiluminescense detection reagents and finally exposed to Kodak Biomax film (XAR, MR). The intensity of the protein bands was measured using Kodak ID Image Analysis which were assessed in the linear detection range. When re-probing was necessary membranes were incubated in Stripping buffer (Pierce, Rockford, IL) for 30 minutes at room temperature, washed twice for 10 min in washing buffer, blocked for 1 hour in blocking buffer at room temperature and re-probed as described above.

Immunocytochemistry and immunofluorescence imaging

After treating pancreatic acini with or without stimulants as indicated, cells were fixed in methanol for 15 minutes at -20° C. A thin layer of cells was deposited onto poly-L-lysine coated glass slides by cytocentrifugation (Cytopro cytocentrifuge, Wescor, Logan, UT) at 500 rpm for 5 minutes. Slides were air-dried for 24 hours, blocked in blocking buffer (50 mM Tris/HCl pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.05% Tween® 20, 5% nonfat dry milk) for 60 min and incubated with a rabbit anti-pS916 Akt antibody (Cell Signaling Technology, (Beverly, MA) and a mouse anti-cadherin antibody (BD Transduction laboratories, Lexington, KY) at a dilution of 1:500 overnight at -4° C. Reactivity was demonstrated by incubation with an Alexa 488-conjugated anti-rabbit and an Alexa 555-conjugated anti-mouse secondary antibody at a dilution of 1:500 for 2 hours at room temperature. Negative controls consisted of replacement of primary antibody with an isotype-matched control. Fluorescent images were collected using a Leica CTR5000 microscope with a 63× objective in oil immersion. Images were acquired using a RetigaExi camera (Qimaging, Burnaby, BC, Canada) and the Volocity software (Improvision, Lexington, MA). Post-image collection restoration was executed using the software's fast restoration tool.

PI3K activity assay

After isolation, 1 ml acini were preincubated for 15 minutes with or without 10 μM wortmannin, and then incubated for 10 min in the absence and presence of 100 nM CCK, 1 nM HGF or 10 μM wortmannin. Cells, after been washed in 0.2 mM Na3VO4 Dulbecco's phosphate buffered saline were homogenized and maintained at 4 °C in PI3K lysis buffer (1.25% triton containing 250 mM sucrose, 20 mM Tris/HCl, pH 7.6, 2.5 mM MgCl2, 50 mM 2-mercaptoethanol, 1.2 mM EGTA, 5 mM Na4P2O7, 50 mM NaF, 1 mM Na3VO4, 30 U/ml bacitracin, 2 mM PMSF and one protease inhibitor tablet per 10 ml). After centrifugation at 10 000 g, the supernatant, containing cytosolic and solubilized membranes, was kept at −70 °C until assay. Protein concentration was determined using the Bio-Rad protein assay reagent. PI3K activity was assessed by determining the conversion of phosphatidyl inositol phosphate (PIP2) to PIP3, as described previously [43]. Briefly, PI3K was assessed in p85 immunoprecipitates obtained by treating lysated acini with anti-PI3-kinase p85 and then immunoprecipitating with protein G-agarose. The immunoprecipitates were incubated for 20 min at room temperature with 20 μM [γ32ATP] (5 μCi/nmol) in 6.25 mM HEPES, 5 mM MgCl2 and 0.25 mM EGTA, and in the presence of 0.25 mg/ml phosphatidylinositol/phosphatidylserine as substrate. The reaction was stopped by the addition of 400 μl chloroform/methanol/HCl (1:2:1, v/v), 150 μl chloroform and 150 μl HCl. After centrifugation (10 000 g), the organic phase was treated with an equal volume of methanol/100 mM HCl/2.5 mM EDTA (1:1:1, v/v), and the new organic phase was separated by centrifugation and then dried. The lipid extract, redissolved in chloroform, was spotted on a silicagel TLC plate, and developed in 2-propanol/acetic acid/H2O (66:2:33, v/v). Plates were dried, and radioactive PIP3 was subsequently visualized by autoradiography and analyzed by densitometric scanning. In all experiments, the densitometric measurement of the band corresponding to cells incubated in the absence of hormone or inhibitor was used as the control value.

In vivo experiments

Adult male Wistar rats (200 and 225 g) were fed a regular rodent diet (Panlab, Barcelona, Spain) and divided into 5 groups of 4 rats each. The first group was injected intraperitoneally with sterile saline solution at 37°C and sacrificed 20 min after the injection. The second group was injected intraperitoneally with CCK-8 (10 μg/kg, prepared in sterile saline solution at 37°C) and killed 20 min after the injection. The third group was injected intraperitoneally with 10 μg/kg CCK-8 and sacrificed 60 min after the injection. The fourth and fifth group was injected intraperitoneally with 20 μg/kg and 30 μg/kg CCK-8, respectively, and killed 20 min after the injection. Animals were sacrificed by CO2 asphyxiation followed by cervical dislocation and pancreatic acini were obtained as described above.

Statistical Analysis

All experiments were performed at least 3 times. Data are presented as mean ± SEM and were analyzed using the Student's t-test for paired data using the software StatView (SAS Institute, Casy, NC). P values <0.05 were considered significant.

3. Results

3.1. Growth factors cause Akt phosphorylation in rat pancreatic acini

Various growth factors increase phosphorylation of Akt residues serine 473 (S473) and threonine 308 (T308) in numerous cells and phosphorylation at these sites not only is essential for Akt activation, but the extent of this phosphorylation closes closely with Akt activation, especially for the S473 site [5-7,9-11]. Five of the seven pancreatic growth factor receptors reported to be present on rat pancreatic acini [28,31], when stimulated by concentrations of growth factors known to activate these receptors, caused a significant (p<0.05) increase in Akt S473 (Figure1, left panel) and T308 (Figure 1, right panel) phosphorylation. HGF caused the most pronounced change (>12-fold increase), whereas activation of the VEGF or bFGF receptors did not stimulate Akt phosphorylation. For a given growth factor, S473 and T308 phosphorylation were affected in a similar manner (Figure 1, compare left panel and right panel).

Fig. 1
Ability of growth factors to stimulate Akt S473 and T308 phosphorylation in rat pancreatic acini. Top: rat pancreatic acini were treated with no additions, with 1 nM HGF for 10 min or with EGF, PDGF, bFGF, Insulin, VEGF or IGF1 in the indicated concentrations ...

3.2. Basal and growth factor-stimulated Akt phosphorylation are regulated by PI3K in rat pancreatic acini

Activation of Akt by growth factors in other tissues is generally mediated by PI3K [2,10,44]. To test if this mechanism also controls Akt activity in rat pancreatic acini, we analyzed the effect of PI3K inhibitors wortmannin and LY294002 on basal (Figure 2, left panel), HGF- (Figure 2, middle panel) and PDGF- (Figure 2, right panel) stimulated Akt phosphorylation. Both basal and stimulated phosphorylation of S473 Akt could be inhibited by pre-incubation with PI3K inhibitors, wortmannin and LY204002, at concentrations known to specifically inhibit PI3K in pancreatic acini [45,46] and other experimental systems [47,48]. These results demonstrate that basal and growth factor-stimulated Akt activation for HGF and PDGF is largely dependent on PI3K activation in rat pancreatic acini.

Fig. 2
Basal and growth-factor-stimulated Akt activity is regulated by PI3K in rat pancreatic acini. Rat pancreatic acini were pre-incubated without additions, with wortmannin or LY294002 for 30 min and then treated with no additions (control) or with 1 nM HGF ...

3.3. Gastrointestinal hormones increasing cAMP cause Akt activation in rat pancreatic acini

Some G-protein coupled receptors increasing intracellular cAMP have been reported to stimulate Akt phosphorylation and activation [12-15], however in other cells their activation reduces Akt phosphorylation and activation [18,19,49,50]. In rat pancreatic acini, the gastrointestinal hormones secretin and vasoactive intestinal peptide, which increase intracellular cAMP levels and stimulate PKA by interacting with distinct secretin and VIP preferring receptors on these cells [28,32], when used at maximally effective concentrations for specifically activating these receptors, each caused a significant increase in Akt S473 and T308 phosphorylation (Figure 3). Lower concentrations of each had a lesser stimulatory effect (Figure 3). Both Akt phosphorylation sites were influenced similarly by these stimulants demonstrating in pancreatic acini, receptor mediated activation of adenylate cyclase leads to stimulation of Akt phosphorylation (Figure 3).

Fig. 3
Ability of gastrointestinal hormones that increase cellular cAMP to stimulate Akt S473 and T308 phosphorylation in rat pancreatic acini. Top: rat pancreatic acini were treated with no additions, with 1 nM HGF for 10 min, with Secretin or VIP in the indicated ...

3.4. Gastrointestinal hormones increasing intracellular calcium can decrease Akt activity in rat pancreatic acini (Figure 4)

Fig. 4
Ability of gastrointestinal hormones that increase cellular calcium to alter Akt S473 and T308 phosphorylation in rat pancreatic acini. Top: rat pancreatic acini were treated with no additions, with 1 nM HGF for 10 min, with cholecystokinin (CCK), carbachol ...

G-protein coupled receptors increasing intracellular calcium have been reported to increase Akt activity in some [1,16,17], but not all [1,20,51] cell systems. In rat pancreatic acini, the three gastrointestinal hormones/neurotransmitters, which interact with distinct receptors and activate phospholipase C (CCK, carbachol, bombesin) [28,52], when used at a maximally effective dose for activation of PLC [28,52], each significantly decreased basal Akt S473 phosphorylation [by 91± 5.3%, 68 ± 11% and 57±14%, respectively], and similarly inhibited T308 phosphorylation (Figure 4).

3.5. CCK has a biphasic effect on Akt phosphorylation in rat pancreatic acini (Figure 5)

Fig. 5
Dose-response of CCK's effect on Akt phosphorylation in rat pancreatic acini. Top: rat pancreatic acini were treated with no additions, with 1 nM HGF for 10 min or CCK in the indicated concentrations for 2.5 min and then lysed. Western blots were analyzed ...

Because CCK is both important in physiological and pathological processes involving pancreatic acinar cells [21,41,53,54], its effect on Akt phosphorylation was studied in more detail and a detailed dose-response curve was performed. Low CCK concentrations (0.1, 1nM) stimulated Akt S473 (2.8- and 1.9-fold increase over basal) and T308 phosphorylation (Figure 5, lanes 2 and 3), higher CCK doses (>1 nM)) significantly inhibited phosphorylation at each site by 60±4.5-75±9.1%) (Figure 5, lanes 4,5,6). The pancreatic CCKA receptor can exist in two affinity states (i.e. a high affinity state mediating effects of CCK in the pM range and a low affinity state mediating effects of higher CCK doses), which are coupled to different intracellular signaling cascades [45,55-58]. Therefore, we considered the possibility that the stimulation of Akt by low concentrations of CCK (pM) might be mediated by the high-affinity CCKA receptor state and the inhibition at higher concentrations of CCK by the low-affinity receptor state.

3.6. Phosphorylation of Akt by the high-affinity CCKA receptor state is mediated by Src and PI3K in rat pancreatic acini (Figure 6)

Fig. 6
Akt stimulation by the high-affinity CCKA-receptor state is mediated by PI3K stimulation. A: rat pancreatic acini were treated with no additions, with the high affinity CCKA-receptor state agonist JMV (100 nM) or with 1 nM HGF for 2.5 min and then lysed. ...

Because activation of Akt by growth factors is generally mediated by PI3K [2,10,44], we hypothesized that activation of Akt by low doses of CCK might also be mediated by this pathway. Because p85 tyrosine phosphorylation is an important early event in PI3K activation [10,44,59,60], we studied the effect of CCK-JMV180 (referred to as “JMV” in the remaining manuscript), a synthetic CCK analogue that acts as an agonist of only the high-affinity CCKA receptor state [21,53,55,58], on p85 tyrosine and Akt S473 phosphorylation. JMV was used instead of low concentrations of CCK for these experiments because, at the doses used, it can cause maximal stimulation of the high-affinity CCKA receptor without activating the low-affinity receptor, whereas with CCK this stimulation and inhibition at a given dose could not be exactly predicted. Similar to HGF, JMV (100 nM) caused an increase in p85 tyrosine phosphorylation (1.7±2.0-fold) and, increased Akt S473 phosphorylation (Figure 6A, lanes 1-3). Furthermore, pre-incubation with the PI3K inhibitor wortmannin, at a concentration that completely inhibited HGF stimulatory effects (Figure 6B, lanes 3 and 6), also completely blocked Akt activation by JMV, (Figure 6B, lanes 5 and 6), demonstrating that activation of the high affinity CCKA receptor state stimulates Akt phosphorylation and this effect is mediated by PI3K. Moreover, p85 phosphorylation stimulated by CCK (Figure 6C, lanes 1-4) and JMV (Figure 6C, lanes 5-7) could be inhibited by the Src-inhibitor PP2, but not its inactive structural analogue PP3 (Figure 6C, lanes 8-11). These data demonstrate that Akt activation by the high-affinity CCKA receptor is mediated by both Src and PI3K.

3.7. Time-course of Akt inhibition by the low-affinity CCKA receptor (Figure 7)

Fig. 7
Time course of CCK's effect on Akt phosphorylation in rat pancreatic acini. Top: rat pancreatic acini were treated with no additions or with 100 nM CCK for the indicated amount of time. Western blots were analyzed using either anti-pS473 Akt or anti-pT308 ...

To investigate in further detail the ability of activation of the low affinity CCKA receptor state to inhibit Akt phosphorylation, we first studied the time-course of CCK's effect at a concentration activating this state (CCK, 100 nM) (Figure 7). CCK (100 nM) caused a significant and rapid inhibition of both S473 and T308 Akt phosphorylation, which was evident at 2.5 min, maximal at 10-15 min and persisting more than 45 min (Figure 7).

3.8. CCK-induced Akt inhibition is mediated through suppression of p85 tyrosine phosphorylation, leading to inhibition of PI3K (Figure 8)

Fig. 8
Akt inhibition by the low-affinity CCKA-receptor state is mediated by PI3K inhibition. Panel A (top). Rat pancreatic acini were treated with no additions, with CCK (100 nM), with 1 nM HGF or with both CCK and HGF for 2.5 min and then lysed. Lysates were ...

As stimulation of Akt by the high-affinity CCKA receptor state is mediated through activation of PI3K (Figure 6), we wanted to determine if inhibition of Akt by the low-affinity CCKA receptor state is mediated by a decrease in PI3K activity. CCK (100 nM) caused a significant decrease in basal and HGF-stimulated p85 tyrosine phosphorylation (51±13%, p=0.011 and 60±22%, p=0.0061 vs. corresponding controls) (Figure 8 A). Moreover, 100 nM CCK produced a significant decrease in the basal PI3K activity (71.9±6.6% vs. control, p<0.05), as did wortmannin (decrease to 32 ± 12% of control) (Figure 8B). HGF (1 nM) increased basal PIP3 by 11 ± 4%%, and this was markedly inhibited by CCK (100 nM) (47.5±6.1% vs. HGF, p<0.001) and was also inhibited by wortmannin to 39 ± 23% of control (Figure 8B). These results demonstrated that CCK activation of the low affinity CCKA receptor state can inhibit both basal and HGF-stimulated PI3K activity in these cells, in a similar manner to the inhibition caused by the PI3K inhibitor, wortmannin.

3.9. Effect of PKC activation and increase of intracellular calcium on Akt inhibition mediated by the low-affinity CCKA receptor (Figure 9)

Fig. 9
Effect of post-receptor stimulants on Akt S473 and T308 phosphorylation in rat pancreatic acini. Top: rat pancreatic acini were treated with no additions, with the phorbol ester TPA (1 μM), with the calcium ionophore A23187(1 μM), with ...

The low-affinity CCKA receptor state activates phospholipase C (PLC), leading to the formation of diacylglycerol and inositol 1,4,5 triphosphate [53,55,57,61,62], which induces an increase in intracellular calcium levels and activation of PKC's [21,27,28]. Similar to higher concentrations of CCK, the phorbol ester TPA, a potent PKC activator, caused a 67 ± 6.1% decrease in basal Akt S473 and a 41 ± 10% decrease in T308 phosphorylation, whereas increases in intracellular calcium (caused by the calcium ionophore A23187 or short-time incubation with thapsigargin), had no significant effect, either alone or in combination with TPA (Figure 9). These results suggest primarily activation of PKC is mediating the inhibitory effects of CCK on Akt phosphorylation.

3.10. Effect of PKC inhibition and inhibition of calcium signaling either alone or together on Akt inhibition mediated by the low-affinity CCKA receptor (Figure 10)

Fig. 10
Effect of the PKC inhibitor GF109203X and/or depletion of intracellular calcium with thapsigargin on CCK- mediated inhibition of Akt phosphorylation in rat pancreatic acini. Cells were pretreated with no additions, with GF109203X (50 μM) for 2 ...

To investigate the role in Akt inhibition of Ca2+ mobilization and PKC activation by CCK directly, CCK was incubated with acini with or without inhibition of each alone or together using conditions which are known to cause specific inhibition of each [26,27,30,42]. Pre-incubation with the broad-spectrum PKC inhibitor GF109203X (GFX) completely reversed TPA's effect on AKT phosphorylation (data not shown), but had no significant effect on CCK-mediated Akt inhibition (Figure 10, top panel- lanes 5 and 6 and bottom panel). Calcium depletion by long-term pre-incubation with thapsigargin in calcium-free medium alone had no significant effect on Akt inhibition mediated by CCK (Figure 10, top panel lanes 5,7 and bottom panel). To confirm that the thapsigargin or GFX109203X were having the appropriate cellular effect, their effects on CCK stimulated pS916PKD were included as controls and they demonstrate the responses previous reported with each agent [45] showing their lack of effect alone was not do to their lack of inhibition of the appropriate PLC pathway. This conclusion was verified because the simultaneous blockade of both arms of the PLC cascade by pre-incubating acini with GFX109203X and thapsigargin in combination almost completely reversed Akt inhibition by CCK (Figure 10, top panel lanes 5 and 8 and bottom panel). These results demonstrate CCK's inhibitory effect on Akt phosphorylation requires the simultaneous activation of both limbs of the PLC cascade.

3.11. CCK-induced Akt inhibition is abolished after pre-incubation with the tyrosine phosphatase inhibitor pervanadate, but not by the serine phosphatase inhibitor, okadaic acid (Figure 11)

Fig. 11
Effect of protein phosphatase inhibitors on CCK-mediated inhibition of Akt phosphorylation in rat pancreatic acini. Panel A. Cells were pretreated with no additions or with okadaic acid (100 nM) for 30 min. Cells were then stimulated with no additions ...

In some cells various stimulants inhibit Akt phosphorylation by activating the serine phosphatase PP2A [5,63]. To investigate this possibility with CCK, acini were pre-incubation with the PP2A inhibitor okadaic acid (100 nM), which increased basal Akt phosphorylation, demonstrating PP2A can affect basal Akt phosphorylation in pancreatic acini. However, incubation of pancreatic acini with CCK inhibited okadaic acid-stimulated Akt phosphorylation, indicating that the CCK-induced Akt inhibition is independent of PP2A activation (Figure 11A). Because activation of the low-affinity CCKA receptor state causes a marked decrease in p85 tyrosine phosphorylation, we investigated whether this effect could be mediated by activation of a protein tyrosine phosphatase. If so, the protein tyrosine phosphatase inhibitor pervanadate should inhibit CCK-mediated Akt inhibition. Pre-incubation with 50 μM pervanadate, a concentration reported to have specific cellular effects in pancreatic acini [25], completely suppressed CCK-mediated Akt inhibition (Figure 11B).

3.12. Inhibition of Src or p44/42 MAPK does not influence Akt inhibition by CCK (Figure 12)

Fig. 12
CCK-mediated Akt inhibition is not mediated by p44/42 MAPK or Src. Rat pancreatic acini were pretreated with no additions or with the Src inhibitor PP2 (10 μM), the inactive analogue PP3 (10 μM) or the MEK inhibitor U0126 (20 μM) ...

In a number of different cells activation of Src family proteins and/or p44/42 MAPK influence PI3K or Akt phosphorylation and activation [51,64,65]. In pancreatic acini, CCK and HGF both activated p44/42 MAPK and CCK activated Src (Figure 12 lanes 2 and 3) as reported previously [21,45,66]. Inhibition of Src by pre-incubation with PP2 (compare Fig. 12, lanes 2 and 5) or inhibition of p44/42 MAPK by 20 μM U0126 (compare Fig. 12, lanes 2 and 11) had no effect on Akt inhibition by CCK (Figure 12). This lack of effect was not due to insufficient concentrations of PP2 or U0126 because at the concentrations used, they significantly inhibited CCK- or HGF- stimulated Src Y416 and/or p44/42 MAPK T202/Y204 phosphorylation, respectively (Figure 12). These results show that, while Src activation was essential for the stimulation of Akt phosphorylation through activation of the high affinity state of the CCK receptor (Fig. 6), it is not involved in mediating the inhibition of Akt phosphorylation seen with activation of the low affinity state of the CCK receptor.

3.13. CCK has a biphasic effect on Akt activation in vivo (Figure 13)

Fig. 13
Effect of CCK on Akt activation in vivo. Left: rats were injected intraperitoneally with saline or CCK at the indicated dose. After 20 min, rats were sacrificed and acini isolated. Cell lysates were analyzed by Western blotting using anti-pS473 Akt antibody. ...

To test the relevance of our findings in vivo, we treated rats with intraperitoneal injections of different doses of CCK, sacrificed the animals and analyzed Akt phosphorylation in isolated pancreatic acini. Similar to the in vitro results in isolated pancreatic acini, in vivo, low doses of CCK caused an increase in Akt S473 phosphorylation, whereas higher CCK doses inhibited this process (Figure 13, left panel). The stimulatory effects of low doses of CCK were seen after 20 min and were more marked after 1 hour (Figure 13, right panel).

3.13. CCK inhibits Akt activation by HGF in-situ (Figure 14)

Fig. 14
CCK inhibits HGF-mediated Akt activation in situ. Acini were treated with HGF (1 nM) for 5 min or with CCK (100 nM, 5 min) followed by HGF (1 nM, 5 min). Cells were fixed in paraformaldehyde, transferred to poly-L-lysine coated glass slides by cytocentrifugation ...

In some quiescent cells, Akt is reported to be located mainly in the cytoplasm and with stimulation it translocates to the membrane [8] where it is activated by phosphorylation [8]. In rat pancreatic acini incubated with 1 nM HGF, phosphorylated Akt is preferentially located at the cellular plasma membrane, where it co-localizes with membrane marker E-cadherin (Figure 14, middle panels). After pre-incubation with 100 nM CCK followed by incubation with HGF, only very faint staining for phosphorylated Akt was detectable or no co-localization with membrane marker E-cadherin could be found (Figure 14, lower panel). Because translocation of Akt to the membrane is a prerequisite for Akt activation [8], the inhibition of Akt translocation by CCK further supports the conclusion that CCK not only inhibits Akt phosphorylation, but also Akt activation.

3.15. CCK inhibits serum-mediated rescue from apoptosis in Panc-1 cells stably transfected with the CCKA receptor (Figure 15)

Fig. 15
CCK inhibits serum-mediated rescue from apoptosis. Panc-1 cells stably transfected with the CCKA-receptor were cultured in DMEM 10% FCS for 72h (Panel A), in the absence of serum for 72h (Panel B), serum-free for 48h followed by 5% serum for 24h (Panel ...

Activated Akt has been reported to be an important anti-apoptotic factor and inhibition of Akt has been shown to increase apoptosis or to inhibit rescue from apoptosis [2,63]. Because fully intact pancreatic acini cannot be maintained in culture for prolonged periods, to explore this possible effect of CCK, Panc-1 cells stably transfected with the CCKA-receptor were used. Like pancreatic acini, CCKA-receptor Panc-1 cells, when stimulated with CCK showed a rapid and marked inhibition of basal and stimulated Akt phosphorylation (data not shown). In a quiescent culture of CCKA-Panc-1 cells cultured in 10% serum, 4.9±1% of cells were apoptotic as assessed by propidium iodide-annexin staining and FACS analysis (Figure 15A). After 3 days of serum starvation, 19±3% was apoptotic (Figure 15B). The addition of 5% serum for 24h after 48h of serum starvation reduced the number of apoptotic cells to <15% (Figure 15C). However, incubation with CCK with the serum for the 24 hours completely abolished this partial rescue from apoptosis mediated by serum and even increased the number of apoptotic cells (Figure 15D). These results demonstrate that CCK's ability to alter the phosphorylation of Akt altered cell behavior by affecting apoptotic/survival responses.

4. Discussion

The purpose of this study was to examine the ability of gastrointestinal hormones and gastrointestinal growth factors to regulate the activity of Akt and to study the cellular signaling mechanisms involved. This study was performed because of the divergent effects of gastrointestinal hormones/neurotransmitters on Akt activation reported in various tissues [1,7,16,17,17,20-24,51] and the importance of Akt in normal gastrointestinal tissues as well as in gastrointestinal cancers and other cancers for cell survival, response to regeneration, growth, regulation of apoptosis, glycogenesis and protein synthesis [2-4]. Whereas the effects of growth factors on Akt activation have been extensively studied in other tissues [9-11], much less is known of the ability of gastrointestinal hormones/neurotransmitters to affect Akt activation. The present study was carried out in pancreatic acinar cells, because these cells are highly responsive to a number of gastrointestinal growth factors/hormones and each of the gastrointestinal hormones/growth factors studied have been shown to produce changes in acinar cell function [28,31,32,67]. The effects of the hormone/neurotransmitter, cholecystokinin (CCK), which is a physiological regulator of acinar cell function [54], was studied in detail, because numerous studies demonstrate PI3K/Akt activity is not only important in pathological responses of pancreatic acini to CCK [34], but also in growth regulation and resistance to chemotherapy of pancreatic cancer cells [68]. Furthermore, CCK-induced pancreatitis, which is seen with supraphysiological concentrations of CCK (>pM), is one of the main experimental models used to study pancreatitis [34,41]. Studies suggest an important role of alterations in Akt in its pathogenesis [34,41], therefore an understanding of the effects of different concentrations on Akt is particularly important in pancreatic physiology/pathophysiology.

Our results demonstrate that Akt phosphorylation in rat pancreatic acini is altered in response to a wide range of pancreatic secretagogues as well as by a number of the known pancreatic growth factors. Numerous studies in other tissues have demonstrated the Akt T308 and particularly S473 phosphorylation are not only required for Akt activation, but the extent of the phosphorylation correlates closely with Akt activation and thus they are widely used as a measure of Akt activation [5-7,9-11]. Our data support this conclusion because the PI3K inhibitor, wortmannin [47], inhibited both Akt activity as well as Akt T308 and S473 phosphorylation. Our results show that the changes in Akt activity differ with the different secretagogues and growth factors, with some functioning as stimulants and other as inhibitors. Our results support the conclusion that the growth factor-induced activation of Akt in pancreatic acini is mediated by a PI3K-dependent mechanism, because the stimulation of Akt S473 phosphorylation by both HGF and PDGF was inhibited by wortmannin and the specific PI3K inhibitor, LY294002 [48]. That these results are reflective of inhibition of PI3K and not a nonspecific effect of the inhibitors is supported by recent study [34] which show identical results obtained in pancreatic acinar cells using either PI3K knockout mice or inhibition of PI3K using wortmannin or LY294002. Furthermore, HGF stimulated tyrosine phosphorylation of the p85 subunit of PI3K, which has been shown to be correlated with activation of PI3K [10,44,69]. These results have both similarities and differences from other studies in pancreatic acini and from the effects of growth factors in other tissues. They are similar in that insulin, IGF-1, PDGF and EGF [10,69-73] stimulate Akt activation in numerous tissues and this stimulation is mediated by PI3K. Our results are similar to previous studies in pancreatic acini showing that HGF, EGF, PDGF, insulin and IGF-1 cause Akt activation in pancreatic acini [23,31]. These results differ from other studies in that bFGF and VEGF, which are known pancreatic growth factors [31], did not stimulate Akt in the present study, whereas in other tissues such as vascular endothelial cells or intervertebral disc cells, they did [74,75].

While it is generally recognized that most growth factors stimulate Akt phosphorylation and activation, the effect of stimulation of G-protein coupled receptors (GPCRs) that increase intracellular cAMP on Akt varies markedly in different cells and nothing is known of their effects on Akt in pancreatic acini. In pancreatic acini activation of either VIP receptors (VPAC1, VPAC2) or secretin receptors, both of which are coupled to adenylate cyclase and their activation stimulates increased cellular cAMP [28,76-78], stimulated Akt activation. These findings are in accordance with studies showing elevations in cAMP, including by other GPCRs can stimulate Akt phosphorylation/activation in fibroblasts [12], thyroid cells [13], 3T3-L1 cells [14] and granulosa cells [15], but differ from studies in PCCL3 thyroid cells [18] and C6 glioma cells [19] which reported no activation of Akt by agents that increase cellular cAMP. These results demonstrate that the effect of adenylate cyclase activation on Akt activity can vary in different cells (i.e. cellular origin, transformed and/or transfected cells). At present the cellular basis for this variation in different cells is unknown.

In contrast to pancreatic growth factors and pancreatic stimulants that activate adenylate cyclase, a number of findings of the present study support the conclusion that gastrointestinal hormones/neurotransmitters that activate phospholipase C can stimulate, as well as inhibit, basal and HGF-stimulated Akt phosphorylation and activity by altering PI3K activity by activating Src. First, low concentrations of CCK (pM range) stimulated Akt activity by stimulating PI3K activity through a Src-dependent mechanism. This conclusion was not due to a nonspecific effect of the Src inhibitor PP2, because it was not seen with the control peptide PP3; this concentration of PP2, but not PP3 has been shown to inhibit CCK stimulated increases in Src activation [66], and the same concentration of PP2 had no effect on Akt inhibition seen with higher concentrations of CCK as discussed below. Second, CCK at higher concentrations (i.e. >pM), carbachol and bombesin significantly inhibited basal Akt S473 and T308 phosphorylation. Third, at higher concentrations CCK caused a significant decrease in basal and HGF-stimulated p85 tyrosine phosphorylation and PI3K activity as well as inhibited HGF-mediated translocation of phosphorylated Akt to the membrane. These results are similar to those reported in other cells with a few other GPCRs, such as the slow inhibition of Akt activation by norepinephrine activating adrenergic receptors in rat adrenal PC12 cells [20], the inhibition of insulin-stimulated Akt activity by angiotensin in vascular smooth muscle cells [1] or ability of dopamine D2/D3 receptor activation to cause inhibition of Akt phosphorylation in striatal tissue[79]. They are also similar to the reported inhibition of IGF-1 mediated Akt activation by carbachol in a number of transfected cells [16] or activation or CCK receptors in the CNS [24]. However, a number of other studies have shown stimulation of Akt by activation of GPCRs coupled to phospholipase C. Angiotensin has been reported to stimulate Akt activation in vascular smooth muscle cells [1], carbachol to stimulate basal Akt activity in transfected cells [16], bombesin-related peptides to stimulate Akt in various tumor cells [80,81] and gastrin to stimulate Akt in AR4-2J cells [17]. These results indicate that not only can the ability of phospholipase C-coupled GPCRs to activate Akt differ markedly in different cells, but also that in some cases (i.e. CCKA receptor) activation of the same receptor can either activate or inhibit Akt activity depending on the receptor's ligand concentration.

Our results, combined with those of others in the literature, provide insights into the cellular signaling mechanisms involved in the biphasic regulation of Akt activity by CCK. Our data support the conclusion that low concentrations of CCK interacting with the high affinity CCKA receptor state stimulates Akt by activating Src and PI3K. These results are consistent with a study reporting low concentrations of CCK [65] in pancreatic acini can stimulate p85 tyrosine phosphorylation, which has been shown to reflect PI3K activation [10,44,59,60]. Furthermore, a number of studies have shown that low concentrations of CCK or activation of the high affinity CCKA receptor state, can activate Src family kinases [25,65,66,82,83] as well as stimulate an association of Src and p85 [65,84]. Taken together, these data support the conclusion that pM concentrations of CCK acting through the high-affinity state of the CCKA receptor activate Src, which in turn associates with and phosphorylates p85, causing an increase in PI3K activity and eventually an increase in Akt activity further downstream. As stimulation of Akt is a proliferative stimulus in many cell types including in pancreatic tissues and pancreatic cancer [35-40], the activation of Akt by low concentrations of CCK might partially account for the stimulation of acinar cell growth by pM-nM doses of CCK [85] or by JMV, an agonist of the high-affinity state of the CCKA receptor [58,86-88]. These conclusions are consistent with studies showing that Akt activation by angiotensin (in mesangial cells) [89], IGF-1 (in oligodendrocytes) [90,91], lysophosphatidic acid (in corneal cells) [92] adenosine (in cardiomyocytes) [93] reactive oxygen species (in fibroblasts) [94] and by estrogen (in endothelial cells) [95], are mediated by Src family kinases. These pathways differ from results of a recent study showing that beta1 integrins can activate Akt through a Src-independent pathway [96].

The inhibition of basal and stimulated Akt activity by higher concentrations of CCK, bombesin and cholinergic agents was a surprising finding. This finding was not an artifact due to dispersed acinar cell preparation because it was seen both in vitro and in vivo. This effect was found in isolated pancreatic acini by Western blotting, by immunocytochemistry in pancreatic acini, in the rat pancreas in vivo and in Panc-1 pancreatic cancer cells transfected with the CCKA receptor. As CCK-mediated Akt inhibition was also present in a cell line, the theoretical possibility that non-acinar cells possibly contaminating our acini preparation could partially mediate this effect can be excluded.

Numerous studies have shown that PKC activation by TPA, LHRH, thrombaxane A2, angiotensin, lysophosphatide C, histamine and thrombin can result in Akt inhibition in different experimental models [1,5,63,73,97-102]. Because CCK, bombesin and cholinergic agents can stimulate phospholipase C in pancreatic acini, resulting in increases in intracellular calcium and generation of diacylglycerol [21,103], which can activate PKC's [21,25,103,104], this mechanism could be responsible for the inhibition of Akt by these GI hormones/neurotransmitters in pancreatic acini. This possibility is partially supported by our finding that with CCK this Akt effect is mediated by the low affinity state of the CCKA receptor, which, in contrast to the high affinity state, has been shown to be coupled to activation and stimulation of phospholipase C and PKCs [45,53,55-57]. A number of our results further support this conclusion. We showed that direct PKC activation by TPA could mimic the effect of high doses of CCK on Akt and that this effect could be inhibited completely by the broad-spectrum PKC inhibitor GF109203X. However, inhibition of PKC by GF109203X did not suppress Akt inhibition by CCK. Furthermore, the inhibition of the CCK-induced increase in intracellular calcium using thapsigargin in calcium-free media did not inhibit CCK's effect on Akt. However, simultaneous inhibition of both CCK-stimulated PKC and calcium increases using GF109203X and thapsigargin in combination suppressed Akt inhibition by CCK. These results demonstrate that the mechanism of inhibition of Akt activation by CCK differs from that of a number of other cell systems with other agents where Akt inhibition was mediated by PKC activation alone.[45,53,55-57]. In contrast with CCK, the Akt inhibition required activation of both limbs of the PLC cascade, i.e. an increase in intracellular calcium and the generation of diacylglycerol acting in synergy.

In general, two different mechanisms of Akt inhibition have been described. First, in some cells, PP2A, a serine phosphatase, is activated by different stimuli and de-phosphorylates Akt, causing a loss of Akt activity [5,63]. Second, tyrosine phosphatases, including members of the Shp family, are activated, causing de-phosphorylation of p85 [44,60] or upstream regulators of p85 [105,106], resulting in decreased PI3K activation and a decrease in Akt phosphorylation. CCK has been reported to stimulate both tyrosine phosphatase activity and serine/threonine phosphatase activity in pancreatic acinar cells as well as other tissues [25,107-110]. A number of findings support the conclusion that CCK-induced Akt inhibition is mediated through an inhibition of p85 phosphorylation by activation of a tyrosine phosphatase. First, concentrations of CCK that caused decreased Akt phosphorylation inhibited basal and HGF-stimulated p85 phosphorylation. Second, the tyrosine phosphatase inhibitor pervanadate, at concentrations shown to have specific cellular effects in pancreatic acini [25], completely reverses CCK-induced Akt inhibition. Third, the serine phosphatase inhibitor okadaic acid, even at concentrations that greatly enhanced basal Akt phosphorylation, thereby showing efficient inhibition of serine phosphatases, did not suppress Akt inhibition mediated by CCK. However, our results do not allow us to identify which specific tyrosine phosphatase is activated by CCK and whether this phosphatase de-phosphorylates p85 directly or acts on an upstream regulator of p85. Furthermore, our data allow the conclusion that p44/42 MAPK and Src, two regulators activated by CCK in pancreatic acini [31,66] and reported in other studies to influence PI3K or Akt phosphorylation [51,64,65] are not involved in CCK-mediated Akt inhibition.

Inhibition of Akt has been shown to inhibit growth and induce apoptosis [63] or inhibit the ability of growth factors to rescue cells from apoptosis [97], while stimulation of Akt is a potent anti-apoptotic stimulus [2]. CCK has been reported to inhibit growth of human cholangiocarcinoma cell line SLU-132 [111,112] as well as growth of CHO cells, MiaPac2 and Panc-1 human pancreatic cancer cells stably transfected with the CCKA receptor [113,114]. Furthermore, high doses of CCK cause hypoplasia of rat pancreatic acinar tissue with increasing apoptosis [115], stimulate apoptosis in pancreatic acini and acinar tissue [67,116] and induce growth inhibition with increased apoptosis in the rat pancreatic acinar cell line AR4-2J [117]. The exact mechanisms of these anti-growth, pro-apoptotic effects are unknown. The extent of activation of Akt is known to be a potent regulator of apoptotic activity[1-4] and therefore one possible mechanism contributing to these pro-apoptotic effects of CCK could be by regulating Akt activity. Because it is not possible to culture differentiated pancreatic acini for prolonged periods to directly study this possibility in pancreatic acini, we instead studied the effect Panc-1 tumor cells transfected with the CCKA receptor. These cell responded to high doses of CCK in the same way than pancreatic acini, i.e. demonstrating almost complete inhibition of Akt activity. In other cells the inhibition of Akt activity has been reported to have a pro-apoptotic effect manifested by inhibiting growth-factor-mediated rescue from apoptosis [97]. Our finding that CCK inhibits serum-mediated rescue from apoptosis in transfected Panc-1 cells strongly suggests that CCK-mediated Akt inhibition has a functional relevance in these cells and likely contributes to anti-proliferative, pro-apoptotic effects of CCK seen in the above cells.

Based on the results of the present study and published data, we propose a model for the effects of the gastrointestinal hormone CCK on Akt activity in pancreatic acinar cells (Figure 16). In this model, the activation of the high- and low-affinity state of the CCKA receptor have opposing effects on Akt activation. Binding of pM concentrations of CCK to the high-affinity CCKA state induces Src phosphorylation and activation [65,66], binding of Src to p85 [65], p85 phosphorylation with activation of PI3K. This leads to the production of PIP3, the recruitment of PDK1 and Akt to the membrane, followed by the phosphorylation of Akt by PDK1 and the activation of Akt, which has been shown to play a central role in pancreatic regeneration after resection [33]. On the other hand, binding of high doses of CCK (i.e.>pM), which are used to induce pancreatitis, to the low-affinity state of the CCKA receptor induces activation of an unknown tyrosine phosphatase, which directly or indirectly reduces basal and stimulated p85 tyrosine phosphorylation, decreasing basal and stimulated PI3K activity and eventually Akt phosphorylation and activation in a Src independent manner. This model highlights the unique role of Src activation in contributing to the dual effects of CCK on Akt activation, in that its activation is essential for Akt stimulation with low doses of CCK, but is not important for the inhibitory effect of higher concentrations of CCK. In a recent study, these latter concentrations of CCK were reported to stimulate the degradation of c-Met in rat pancreatic acini [118]. In the present study, we report that similar concentrations of CCK inhibit PI3K and Akt activity in these cells, raising the possibility these effects could be caused by an effect on c-Met further downstream in the signal transduction pathway. This possibility can be excluded for a number of reasons. First, c-Met degradation after CCK follows a slow kinetic, starting at 10 minutes and reaching a maximum after 45 minutes [118], while Akt inhibition is maximal after only 2.5 minutes. Second, degradation of growth factor receptors after CCK is limited to c-Met, other growth factor receptors are not degraded [118]. As the latter effects are not regulated by c-Met, they cannot be mediated by CCK-induced c-Met degradation. Therefore, high doses of CCK inhibit growth factor signaling in rat pancreatic acini and in transfected pancreatic tumor cells by at least two different mechanisms: the inhibition of basal and stimulated Akt activation and the degradation of c-Met [118], the most active growth factor receptor in these cells. Numerous recent results suggest the effects seen with higher CCK concentrations on these signaling cascades likely have important functional relevance. Not only could they be important in mediating the anti-proliferative and pro-apoptotic effects of CCK seen in various pancreatic tumors and in normal pancreas [111-115,117], but they could be important in the pathogenesis of acute pancreatitis, the most common clinical inflammatory disorder affecting pancreatic acinar cells. High concentrations of CCK can cause experimental acute pancreatitis that closely resembles that seen in man and is the main experimental model being used to study the signaling mechanisms in pancreatitis. [41,119]. Recent studies show that important mediators of this effect are the ability of CCK to [67] stimulate apoptosis of acinar cells with activation of caspases and that PI3K activity is important in mediating the pancreatitis [41,67]. Furthermore, it has been shown that high doses of growth factors protect from tissue damage in this model [120]. These results coupled with findings in this study on the effects if CCK on Akt activity and in the previous study showing these concentrations of CCK can down-regulate c-Met [118], suggest it will be important to further explore their role in the pathogenesis of acute pancreatitis.

Fig. 16
Differential regulation of the PI3K/Akt pathway by the high- and low-affinity CCKA-receptor in rat pancreatic acini. Arrowheads represent phosphorylation and stimulation while blocked lines represent inhibition. CCK: cholecystokinin; PIP2: phosphatidylinositol-2-phosphate; ...

In summary, we report that gastrointestinal hormones/neurotransmitters can differentially regulate Akt activity with some activating it, with others decreasing activity and in the case of CCK, causing dual regulation depending on the CCK concentration. Pico-molar concentrations of CCK acting through the high-affinity CCKA-receptor, like growth factors, cause an increase in p85 phosphorylation and Akt activation. This effect is mediated by Src and may partially account for CCK's mitogenic /growth effects reported in some normal tissues and various tumors [28,54,121,122]. Higher CCK concentrations (i.e. >pM) signaling through the low-affinity state of the CCKA-receptor cause a decrease in basal and stimulated p85 tyrosine phosphorylation, PI3K activity, Akt activation and Akt translocation to the membrane. This effect requires activation of both limbs of the PLC cascade, i.e. an increase in intracellular calcium and activation of PKC but is not dependent on Src or p44/42 MAPK. This novel effect was seen both in vitro in isolated pancreatic acini, in pancreatic cancer cells and in vivo in the rat pancreas. This CCK-mediated inhibition of Akt signaling may account for CCK's pro-apoptotic and anti-proliferative effects seen in this study and reported by others in previous publications both in vitro and in vivo in pancreatic acini and in other normal tissues and neoplastic tissues [111-115,117]. These observations on the inhibitory effect of CCK on Akt at these concentrations could be particularly important in CCK induction of pancreatitis because the inhibitory effect on Akt occurs at CCK concentrations that are commonly used to induce experimental pancreatitis to study its cellular mechanism and Akt activity has been shown to be important in its pathogenesis [34]. Furthermore, together with a previous report of c-Met degradation [118], with CCK concentrations similar to those causing Akt inhibition in the present study, the present study provides a second mechanism of negative cross-talk between signal transduction pathways initiated by gastrointestinal hormones and growth factors. The understanding of these cross-talk inhibitory mechanisms could greatly enhance our current understanding of pancreatic cell physiology in normal tissue and pancreatic disease, like cancer and pancreatitis, where signaling by growth factors and gastrointestinal hormones is often aberrant.


This work is partially supported by the Intramural Research Program of the NIDDK and NEI, NIH. Marc Berna is supported by a grant from “Action Vaincre le Cancer – Lions Luxembourg”. Jose A. Tapia is supported by the “Programa Ramon y Cajal” and by a research grant (BFU2007-62423) from the “Ministerio de Educación y Ciencia-FEDER” of Spain. Michelle Thill is supported by a “Bourse Formation Recherche (BFR)” grant from the Ministry of Culture, Higher Education and Research of the Grand-Duchy of Luxembourg. Lauro Gonzalez-Fernandez is supported by a “Formacion de personal Investigador (FPI)” grant from the Junta de Extremadura-FEDER, Spain.

The abbreviations used are

G protein-coupled receptor
protein kinase B
protein kinase C
protein kinase D
3-phosphoinositide-dependent protein kinase 1
mitogen-activated protein kinase
focal adhesion kinase
luteinizing hormone releasing hormone
phospholipase C
cyclic AMP
hepatocyte growth factor
epidermal growth factor


1. Motley ED, Eguchi K, Gardner C, Hicks AL, Reynolds CM, Frank GD, Mifune M, Ohba M, Eguchi S. Hypertension. 2003;41:775–780. [PubMed]
2. Aikin R, Maysinger D, Rosenberg L. Endocrinology. 2004;145:4522–4531. [PubMed]
3. Yano S, Tokumitsu H, Soderling TR. Nature. 1998;396:584–587. [PubMed]
4. Summers SA, Kao AW, Kohn AD, Backus GS, Roth RA, Pessin JE, Birnbaum MJ. J Biol Chem. 1999;274:17934–17940. [PubMed]
5. Li L, Sampat K, Hu N, Zakari J, Yuspa SH. J Biol Chem. 2006;281:3237–3243. [PubMed]
6. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Science. 2005;307:1098–1101. [PubMed]
7. Rozengurt E. J Cell Physiol. 2007;213:589–602. [PubMed]
8. Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJ, Frech M, Cron P, Cohen P, Lucocq JM, Hemmings BA. J Biol Chem. 1997;272:31515–31524. [PubMed]
9. Liu H, Qiu Y, Xiao L, Dong F. J Immunol. 2006;176:2407–2413. [PubMed]
10. Kwon M, Ling Y, Maile LA, Badley-Clark J, Clemmons DR. Endocrinology. 2006;147:1458–1465. [PubMed]
11. Bellacosa A, Chan TO, Ahmed NN, Datta K, Malstrom S, Stokoe D, McCormick F, Feng J, Tsichlis P. Oncogene. 1998;17:313–325. [PubMed]
12. Cass LA, Summers SA, Prendergast GV, Backer JM, Birnbaum MJ, Meinkoth JL. Mol Cell Biol. 1999;19:5882–5891. [PMC free article] [PubMed]
13. Tsygankova OM, Saavedra A, Rebhun JF, Quilliam LA, Meinkoth JL. Mol Cell Biol. 2001;21:1921–1929. [PMC free article] [PubMed]
14. Song DH, Getty-Kaushik L, Tseng E, Simon J, Corkey BE, Wolfe MM. Gastroenterology. 2007;133:1796–1805. [PMC free article] [PubMed]
15. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL, Richards JS. Mol Endocrinol. 2000;14:1283–1300. [PubMed]
16. Bommakanti RK, Vinayak S, Simonds WF. J Biol Chem. 2000;275:38870–38876. [PubMed]
17. Todisco A, Ramamoorthy S, Witham T, Pausawasdi N, Srinivasan S, Dickinson CJ, Askari FK, Krametter D. Am J Physiol (Gastrointest Liver Physiol) 2001;280:G298–G307. [PubMed]
18. Lou L, Urbani J, Ribeiro-Neto F, Altschuler DL. J Biol Chem. 2002;277:32799–32806. [PubMed]
19. Wang L, Liu F, Adamo ML. J Biol Chem. 2001;276:37242–37249. [PubMed]
20. Mao W, Iwai C, Keng PC, Vulapalli R, Liang CS. Am J Physiol (Cell Physiol) 2006;290:C1373–C1384. [PubMed]
21. Dufresne M, Seva C, Fourmy D. Physiol Rev. 2006;86:805–847. [PubMed]
22. Crozier SJ, Sans MD, Lang CH, D'Alecy LG, Ernst SA, Williams JA. Am J Physiol Gastrointest Liver Physiol. 2008;294:G1148–G1157. [PubMed]
23. Aparicio IM, Garcia-Marin LJ, Andreolotti AG, Bodega G, Jensen RT, Bragado MJ. Biochim Biophys Acta. 2003;1643:37–46. [PubMed]
24. Chhatwal JP, Gutman AR, Maguschak KA, Bowser ME, Yang Y, Davis M, Ressler KJ. Neuropsychopharmacology. 2008
25. Tapia JA, Garcia-Marin LJ, Jensen RT. J Biol Chem. 2003;12:35220–35230. [PubMed]
26. Tapia JA, Ferris HA, Jensen RT, Marin LJ. J Biol Chem. 1999;274:31261–31271. [PubMed]
27. Tapia JA, Bragado MJ, Garcia-Marin LJ, Jensen RT. Biochim Biophys Acta (Mol Cell Res) 2002;1593:99–113. [PubMed]
28. Jensen RT. In: Physiology of the Gastrointestinal Tract. Third. Johnson LR, Jacobson ED, Christensen J, Alpers DH, Walsh JH, editors. Raven Press; New York: 1994. pp. 1377–1446.
29. Tapia JA, Jensen RT, Garcia-Marin LJ. Biochim Biophys Acta. 2006;1763:25–38. [PubMed]
30. Pace A, Garcia-Marin LJ, Tapia JA, Bragado MJ, Jensen RT. J Biol Chem. 2003;278:19008–19016. [PubMed]
31. Hoffmann KM, Tapia JA, Jensen RT. Cell Signal. 2006;18:942–954. [PubMed]
32. Jensen RT, Gardner JD. Fed Proc. 1981;40:2486–2496. [PubMed]
33. Watanabe H, Saito H, Rychahou PG, Uchida T, Evers BM. Gastroenterology. 2005;128:1391–1404. [PubMed]
34. Gukovsky I, Cheng JH, Nam KJ, Lee OT, Lugea A, Fischer L, Penninger JM, Pandol SJ, Gukovskaya AS. Gastroenterology. 2004;126:554–566. [PubMed]
35. Kiehne K, Herzig KH, Folsch UR. Pancreas. 1997;15:35–40. [PubMed]
36. Di Renzo MF, Poulsom R, Olivero M, Comoglio PM, Lemoine NR. Cancer Res. 1995;55:1129–1138. [PubMed]
37. Ebert M, Yokoyama M, Friess H, Büchler MW, Korc M. Cancer Res. 1994;54:5775–5778. [PubMed]
38. Furukawa M, Zhang YQ, Nie L, Shibata H, Kojima I. Diabetologia. 1999;42:450–456. [PubMed]
39. Calvo EL, Boucher C, Pelletier G, Morisset J. Biochem Biophys Res Commun. 1996;229:257–263. [PubMed]
40. Otte JM, Schwenger M, Brunke G, Sparmann G, Emmrich J, Schmitz F, Folsch UR, Herzig KH. Eur J Clin Invest. 2001;31:865–875. [PubMed]
41. Pandol SJ, Saluja AK, Imrie Cw, Banks PA. Gastroenterology. 2007;132:1127–1151. [PubMed]
42. Garcia LJ, Rosado JA, Gonzalez A, Jensen RT. Biochem J. 1997;327:461–472. [PubMed]
43. Sancho V, Trigo MV, Gonzalez N, Valverde I, Malaisse WJ, Villanueva-Penacarrillo ML. J Mol Endocrinol. 2005;35:27–38. [PubMed]
44. Cuevas BD, Lu Y, Mao M, Zhang J, LaPushin R, Siminovitch K, Mills GB. J Biol Chem. 2001;276:27455–27461. [PubMed]
45. Berna MJ, Hoffmann KM, Tapia JA, Thill M, Pace A, Mantey SA, Jensen RT. Biochim Biophys Acta. 2007;1773:483–501. [PMC free article] [PubMed]
46. Tapia JA, Camello C, Jensen RT, Garcia LJ. Biochim Biophys Acta. 1999;1448:486–499. [PubMed]
47. Arcaro A, Wymann MP. Biochem J. 1993;296(Pt 2):297–301. [PubMed]
48. Vlahos CJ, Matter WF, Hui KY, Brown RF. J Biol Chem. 1994;269:5241–5248. [PubMed]
49. Kim S, Jee K, Kim D, Koh H, Chung J. J Biol Chem. 2001;276:12864–12870. [PubMed]
50. Zhou P, Qian L, Chou T, Iadecola C. Neurobiol Dis. 2008;29:543–551. [PMC free article] [PubMed]
51. Andreozzi F, Laratta E, Sciacqua A, Perticone F, Sesti G. Circ Res. 2004;94:1211–1218. [PubMed]
52. Pandol SJ, Jensen RT, Gardner JD. J Biol Chem. 1982;257:12024–12029. [PubMed]
53. Jensen RT, Wank SA, Rowley WH, Sato S, Gardner JD. Trends Pharmacol Sci. 1989;10:418–423. [PubMed]
54. Jensen RT. Pharmacol Toxicol. 2002;91:333–350. [PubMed]
55. Matozaki T, Martinez J, Williams JA. Am J Physiol. 1989;257:G594–600. [PubMed]
56. Lankisch TO, Nozu F, Owyang C, Tsunoda Y. Eur J Cell Biol. 1999;78:632–641. [PubMed]
57. Rowley WH, Sato S, Huang SC, Collado-Escobar DM, Beaven MA, Wang LH, Martinez J, Gardner JD, Jensen RT. Am J Physiol. 1990;259:G655–G665. [PubMed]
58. Stark HA, Sharp CM, Sutliff VE, Martinez J, Jensen RT, Gardner JD. Biochim Biophys Acta. 1989;1010:145–150. [PubMed]
59. Hu ZW, Shi XY, Lin RZ, Hoffman BB. J Biol Chem. 1996;271:8977–8982. [PubMed]
60. Cuevas B, Lu Y, Watt S, Kumar R, Zhang J, Siminovitch KA, Mills GB. J Biol Chem. 1999;274:27583–27589. [PubMed]
61. Tsunoda Y, Yoshida H, Owyang C. Am J Physiol. 1996;271:G8–19. [PubMed]
62. Sato S, Stark HA, Martinez J, Beaven MA, Jensen RT, Gardner JD. Am J Physiol. 1989;257:G202–G209. [PubMed]
63. Tanaka Y, Gavrielides MV, Mitsuuchi Y, Fujii T, Kazanietz MG. J Biol Chem. 2003;278:33753–33762. [PubMed]
64. Daulhac L, Kowalski-Chauvel A, Pradayrol L, Vaysse N, Seva C. J Biol Chem. 1999;274:20657–20663. [PubMed]
65. Nozu F, Owyang C, Tsunoda Y. Eur J Cell Biol. 2000;79:803–809. [PubMed]
66. Pace A, Tapia JA, Garcia-Marin LJ, Jensen RT. Biochim Biophys Acta. 2006;1763:356–365. [PubMed]
67. Gukovskaya AS, Gukovsky I, Jung Y, Mouria M, Pandol SJ. J Biol Chem. 2002;277:22595–22604. [PubMed]
68. Fahy BN, Schlieman MG, Virudachalam S, Bold RJ. J Am Coll Surg. 2004;198:591–599. [PubMed]
69. Hayashi H, Nishioka Y, Kamohara S, Kanai F, Ishii K, Fukui Y, Shibasaki F, Takenawa T, Kido H, Katsunuma N. J Biol Chem. 1993;268:7107–7117. [PubMed]
70. Anderson KE, Coadwell J, Stephens LR, Hawkins PT. Curr Biol. 1998;8:684–691. [PubMed]
71. Yu CF, Liu ZX, Cantley LG. J Biol Chem. 2002;277:19382–19388. [PubMed]
72. Chow JY, Barrett KE. Am J Physiol (Cell Physiol) 2007;292:C452–C459. [PubMed]
73. Barthel A, Nakatani K, Dandekar AA, Roth RA. Biochem Biophys Res Commun. 1998;243:509–513. [PubMed]
74. Nakashio A, Fujita N, Tsuruo T. Int J Cancer. 2002;98:36–41. [PubMed]
75. Pratsinis H, Kletsas D. Eur Spine J. 2007;16:1858–1866. [PMC free article] [PubMed]
76. Ito T, Hou W, Katsuno T, Igarashi H, Pradhan TK, Mantey SA, Coy DH, Jensen RT. Am J Physiol (Gastrointest Liver Physiol) 2000;278:G64–G74. [PubMed]
77. Bissonnette BM, Collen MJ, Adachi H, Jensen RT, Gardner JD. Am J Physiol. 1984;246:G710–G717. [PubMed]
78. Jensen RT, Charlton CG, Adachi H, Jones SW, O'Donohue TL, Gardner JD. Am J Physiol. 1983;245:G186–G195. [PubMed]
79. Beaulieu JM, Tirotta E, Sotnikova TD, Masri B, Salahpour A, Gainetdinov RR, Borrelli E, Caron MG. J Neurosci. 2007;27:881–885. [PubMed]
80. Ishola TA, Kang J, Qiao J, Evers BM, Chung DH. Biochim Biophys Acta. 2007;1770:927–932. [PMC free article] [PubMed]
81. Liu X, Carlisle DL, Swick MC, Gaither-Davis A, Grandis JR, Siegfried JM. Exp Cell Res. 2007;313:1361–1372. [PubMed]
82. Piiper A, Elez R, You SJ, Kronenberger B, Loitsch S, Roche S, Zeuzem S. J Biol Chem. 2003;278:7065–7072. [PubMed]
83. Freedman SD, Katz MH, Parker EM, Gelrud A. Am J Physiol. 1999;276:C306–C311. [PubMed]
84. Kim M, Nozu F, Kusama K, Imawari M. Biochem Biophys Res Commun. 2006;339:271–276. [PubMed]
85. Logsdon CD. Am J Physiol. 1986;251:G487–G494. [PubMed]
86. Dawra R, Saluja A, Lerch MM, Saluja M, Logsdon C, Steer M. Biochem Biophys Res Commun. 1993;193:814–820. [PubMed]
87. Spanarkel MB, Martinez J, Briet C, Jensen RT, Gardner JD. J Biol Chem. 1983;258:6746–6749. [PubMed]
88. Matozaki T, Goke B, Tsunoda Y, Rodriguez M, Martinez J, Williams JA. J Biol Chem. 1990;265:6247–6254. [PubMed]
89. Yano N, Suzuki D, Endoh M, Zhao TC, Padbury JF, Tseng YT. J Biol Chem. 2007;282:18819–18830. [PubMed]
90. Cui QL, Almazan G. J Neurochem. 2007;100:1480–1493. [PubMed]
91. Cui QL, Zheng WH, Quirion R, Almazan G. J Biol Chem. 2005;280:8918–8928. [PubMed]
92. Xu KP, Yin J, Yu FS. Invest Ophthalmol Vis Sci. 2007;48:636–643. [PMC free article] [PubMed]
93. Germack R, Griffin M, Dickenson JM. J Mol Cell Cardiol. 2004;37:989–999. [PubMed]
94. Esposito F, Chirico G, Montesano GN, Posadas I, Ammendola R, Russo T, Cirino G, Cimino F. J Biol Chem. 2003;278:20828–20834. [PubMed]
95. Haynes MP, Li L, Sinha D, Russell KS, Hisamoto K, Baron R, Collinge M, Sessa WC, Bender JR. J Biol Chem. 2003;278:2118–2123. [PubMed]
96. Velling T, Stefansson A, Johansson S. Exp Cell Res. 2008;314:309–316. [PubMed]
97. Rose A, Froment P, Perrot V, Quon MJ, LeRoith D, Dupont J. J Biol Chem. 2004;279:52500–52516. [PubMed]
98. Mao M, Fang X, Lu Y, LaPushin R, Bast RC, Jr, Mills GB. Biochem J. 2000;352(Pt 2):475–482. [PubMed]
99. Shizukuda Y, Buttrick PM. Am J Physiol Heart Circ Physiol. 2002;282:H320–H327. [PubMed]
100. Motley ED, Kabir SM, Gardner CD, Eguchi K, Frank GD, Kuroki T, Ohba M, Yamakawa T, Eguchi S. Hypertension. 2002;39:508–512. [PubMed]
101. Wen HC, Huang WC, Ali A, Woodgett JR, Lin WW. Cell Signal. 2003;15:37–45. [PubMed]
102. Thors B, Halldorsson H, Clarke GD, Thorgeirsson G. Atherosclerosis. 2003;168:245–253. [PubMed]
103. Pollo DA, Baldassare JJ, Honda T, Henderson PA, Talkad VD, Gardner JD. Biochim Biophys Acta. 1994;1224:127–138. [PubMed]
104. Satoh A, Gukovskaya AS, Nieto JM, Cheng JH, Gukovsky I, Reeve JR, Jr, Shimosegawa T, Pandol SJ. Am J Physiol (Gastrointest Liver Physiol) 2004;287:G582–G591. [PubMed]
105. Haider UG, Roos TU, Kontaridis MI, Neel BG, Sorescu D, Griendling KK, Vollmar AM, Dirsch VM. Mol Pharmacol. 2005;68:41–48. [PubMed]
106. Zhang SQ, Tsiaras WG, Araki T, Wen G, Minichiello L, Klein R, Neel BG. Mol Cell Biol. 2002;22:4062–4072. [PMC free article] [PubMed]
107. Rivard N, Lebel D, Laine J, Morisset J. Am J Physiol. 1994;266:G1130–G1138. [PubMed]
108. Meyer-Alber A, Hocker M, Fetz I, Fornefeld H, Waschulewski IH, Folsch UR, Schmidt WE. Scand J Gastroenterol. 1995;30:384–391. [PubMed]
109. Siegel EG, Meyer-Alber A, Seebeck J, Folsch UR, Schmidt WE. Scand J Gastroenterol. 1999;34:208–214. [PubMed]
110. Wagner AC, Wishart MJ, Yule DI, Williams JA. Am J Physiol. 1992;263:C1172–C1180. [PubMed]
111. Evers BM, Gomez G, Townsend CM, Jr, Rajaraman S, Thompson JC. Ann Surg. 1989;210:317–322. [PubMed]
112. Hudd C, Euhus DM, LaRegina MC, Herbold DR, Palmer DC, Johnson FE. Cancer Res. 1985;45:1372–1377. [PubMed]
113. Detjen K, Fenrich MC, Logsdon CD. Gastroenterology. 1997;112:952–959. [PubMed]
114. Detjen K, Tseng MJ, Logsdon CD. Biochem Biophys Res Commun. 1995;213:44–51. [PubMed]
115. Trulsson LM, Gasslander T, Svanvik J. Basic Clin Pharmacol Toxicol. 2004;95:183–190. [PubMed]
116. Kaiser AM, Saluja AK, Sengupta A, Saluja M, Steer ML. Am J Physiol. 1995;269:C1295–C1304. [PubMed]
117. Sata N, Klonowski-Stumpe H, Han B, Luthen R, Haussinger D, Niederau C. Pancreas. 1999;19:76–82. [PubMed]
118. Hoffmann KM, Tapia JA, Berna MJ, Thill M, Braunschweig T, Mantey S, Moody T, Jensen RT. J Biol Chem. 2006;281:37705–37719. [PubMed]
119. Steer ML. Baillieres Best Pract Res Clin Gastroenterol. 1999;13:213–225. [PubMed]
120. Warzecha Z, Dembinski A, Konturek PC, Ceranowicz P, Konturek SJ, Tomaszewska R, Schuppan D, Stachura J, Nakamura T. Eur J Pharmacol. 2001;430:113–121. [PubMed]
121. Baldwin GS. J Gastroenterol Hepatol. 1995;10:215–232. [PubMed]
122. Morisset J. J Physiol Pharmacol. 2003;54 4:127–141. [PubMed]