AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 292: G875-G886, 2007. First published December 7, 2006; doi:10.1152/ajpgi.00558.2005
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Corrigendum
Right arrow All Versions of this Article:
292/3/G875    most recent
00558.2005v1
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 Related articles in AJP - GI
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI 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 ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fischer, L.
Right arrow Articles by Pandol, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fischer, L.
Right arrow Articles by Pandol, S. J.

HORMONES AND SIGNALING

Phosphatidylinositol 3-kinase facilitates bile acid-induced Ca2+ responses in pancreatic acinar cells

L. Fischer,1,2,3 A. S. Gukovskaya,1,2 J. M. Penninger,4 O. A. Mareninova,1,2 H. Friess,3 I. Gukovsky,1,2 and S. J. Pandol1,2

1Veterans Affairs Greater Los Angeles Healthcare System and 2Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; 3Department of Surgery, University of Heidelberg, Germany; and 4Institute for Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria

Submitted 9 December 2005 ; accepted in final form 1 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bile acids are known to induce Ca2+ signals in pancreatic acinar cells. We have recently shown that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca2+ concentration ([Ca2+]i) elicited by CCK by inhibiting sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca2+]i responses. In pancreatic acinar cells, pharmacological inhibition of PI3K with LY-294002 or wortmannin inhibited [Ca2+]i responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxycholate (TCDC). Furthermore, genetic deletion of the PI3K {gamma}-isoform also decreased [Ca2+]i responses to bile acids. Depletion of CCK-sensitive intracellular Ca2+ pools or application of caffeine inhibited bile acid-induced [Ca2+]i signals, indicating that bile acids release Ca2+ from agonist-sensitive endoplasmic reticulum (ER) stores via an inositol (1,4,5)-trisphosphate-dependent mechanism. PI3K inhibitors increased the amount of Ca2+ in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates SERCA-dependent Ca2+ reloading into the ER. Bile acids inhibited Ca2+ reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol (3,4,5)-trisphosphate (PIP3), suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY-294002 completely inhibited trypsinogen activation caused by the bile acid TLC-S. Our results indicate that PI3K and its product, PIP3, facilitate bile acid-induced [Ca2+]i responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca2+ reloading into the ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca2+]i increases and trypsinogen activation mediate key pathological processes in this disorder.

sarco(endo)plasmic reticulum Ca2+-ATPase; taurolithocholic acid 3-sulfate; taurochenodeoxycholate; cholecystokinin; pancreatitis

OVER A CENTURY AGO, Opie (21) proposed that gallstones impacted in the ampulla of Vater cause reflux of bile acids into the pancreatic duct resulting in pancreatitis. Although studies (18, 20) have indicated that bile reflux may not be necessary for gallstone-induced pancreatitis, the results do not exclude the possibility that bile acid reflux mediates some forms of pancreatitis. Bile acids are known to cause severe pancreatitis in experimental animal models (1, 17, 29, 33). However, the mechanisms by which bile acids initiate pancreatitis are not well established.

The fact that bile acids induce free cytosolic Ca2+ concentration ([Ca2+]i) responses in hepatocytes has been known for >10 years (4, 6, 7). Recently, it has been demonstrated (34, 36) that bile acids stimulate both global [Ca2+]i signals and local [Ca2+]i oscillations in pancreatic acinar cells that appear to have the same vectoral pattern as CCK-8 or ACh-induced [Ca2+]i responses, i.e., they start from the apical pole of the acinar cell and then spread to the basolateral region (34). The exact mechanism by which bile acids induce these Ca2+ responses is not known. However, the fact that bile acid-induced Ca2+ signaling patterns are similar to those stimulated by CCK or ACh suggest the possible involvement of second messengers like inositol (1,4,5)-trisphosphate (IP3), cADP-ribose, and nicotinic acid adenine dinucleotide phosphate (34). In fact, Voronina et al. (34) showed that the IP3 receptor inhibitor caffeine is able to block bile acid-induced Ca2+ responses in individual acinar cells, indicating that IP3-dependent pathways are involved in bile acid-induced Ca2+ signals. Another possible mechanism explaining how bile acids are able to induce [Ca2+]i responses in pancreatic acinar cells was introduced by Kim et al. (13). This group demonstrated that bile acids cause the inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), resulting in Ca2+ release from endoplasmic reticulum (ER) stores. The increase in [Ca2+]i, in turn, mediates pathobiological responses such as the activation of the proinflammatory transcription factor NF-{kappa}B, acinar cell death, and trypsinogen activation (13, 14, 27). The latter is considered a key event of acute pancreatitis (16, 30).

Recent studies have demonstrated that phosphatidylinositol 3-kinase (PI3K) plays a significant role in the regulation of [Ca2+]i responses in pancreatic acinar cells as well as the severity of acute pancreatitis in experimental models (10, 11, 19). In particular, we (10) have recently shown that during neurohormonal stimulation with CCK, PI3K regulates [Ca2+]i responses through its inhibitory effect on SERCA. Based on these results and the data obtained by Kim et al. (13), we hypothesized that the [Ca2+]i responses induced by bile acids may also be regulated by PI3K. The present study was designed to determine whether PI3K is involved in bile acid-induced [Ca2+]i responses in the pancreatic acinar cell, in particular, through the inhibition of SERCA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pancreatic acini isolation. Pancreatic acini were isolated from Sprague-Dawley rats (75–100 g), wild-type (WT; p110{gamma}+/+) C57BL/6 mice (30–35 g), and PI3K-{gamma} knockout (KO) C57BL/6 mice deficient in the p110{gamma} catalytic subunit [p110{gamma}–/– (28)] using collagenase digestion methods as previously described (10, 11). Breeding of the p110{gamma}–/– mice and handling of the animals were approved by the Animal Research Committee of the Veterans Affairs Greater Los Angeles Healthcare System in accordance with National Institutes of Health guidelines. Genotyping of PI3K-{gamma} KO mice was done by PCR using specific primers (28). Acini were suspended in solution Q containing (in mM) 120 NaCl, 20 HEPES, 5 KCl, 1 MgCl2, 1 CaCl2, 10 sodium pyruvate, 10 ascorbate, and 10 glucose as well as 0.1% BSA and 0.01% soybean trypsinogen inhibitor. For the "Ca2+-free" medium, CaCl2 was omitted and 1 mM EGTA was added to solution Q. Where indicated, acini were incubated with the PI3K inhibitors LY-294002 and wortmannin at 37°C for 5 min prior to the addition of the agonist.

Measurement of [Ca2+]i in acinar cell suspensions. Dispersed pancreatic acini were loaded for 30 min at 37°C with 2 µM fura-2 AM in solution Q. After being washed, acini were suspended in the same solution, and fluorescence was measured in a stirred cuvette at 37°C in a Shimadzu RF 1501 spectrofluorimeter with excitation at 340 and 380 nm and emission at 510 nm. [Ca2+]i values were calculated as previously described (10, 11, 23). In addition to measuring the peak and plateau values of the [Ca2+]i response, we also measured the changes in the area under the curve of the [Ca2+]i response (see Fig. 1). This was done by copying the profile of the [Ca2+]i response onto grease-proof paper, cutting out the area under the curve (as illustrated in Fig. 1E), and weighting these areas.


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 1. Phosphatidylinositol 3-kinase (PI3K) inhibitors attenuate free cytosolic Ca2+ concentration ([Ca2+]i) responses to taurolithocholic acid 3-sulfate (TLC-S) in pancreatic acinar cells. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle, 100 µM LY-294002, or 1 µM wortmannin (not shown), and then stimulated with TLC-S at the indicated concentrations (A) or at 450 µM (B–F). A, B, and E: representative tracings from at least 3 independent experiments (i.e., on different preparations of acini). For statistical analyses, the [Ca2+]i response to TLC-S in the absence of PI3K inhibitors (i.e., in acini incubated with vehicle) was considered as 100%. B: representative [Ca2+]i responses to 450 µM TLC-S in the absence (trace a) and presence (trace b) of 100 µM LY294002. C–F: statistical analyses of the effects of 100 µM LY-294002 and 1 µM wortmannin on the magnitude of the peak [Ca2+]i response (C), plateau [Ca2+]i response (D), and area under the curve (E and F) after stimulation with 450 µM TLC-S. E: graphical representation of the area under the curve used to calculate the results shown in F. In this and other figures, the peak [Ca2+]i response was calculated as follows: (peak [Ca2+]i – basal [Ca2+]i); the plateau [Ca2+]i response was calculated as follows: (plateau [Ca2+]i – basal [Ca2+]i). The area under the curve of the [Ca2+]i response was measured as described in MATERIALS AND METHODS. Values are means ± SE from 5–8 tracings for the corresponding [Ca2+]i responses. *P < 0.05 and #P < 0.01 compared with [Ca2+]i responses of acini stimulated by TLC-S in the absence of PI3K inhibitors. Note that in C, D, and F, the scale on the y-axis starts at 50%.

 
Measurement of [Ca2+] in permeabilized acinar cells. Permeabilization was done as previously described (10). Briefly, acini were washed three times in buffer B containing (in mM) 100 KCl, 20 NaCl, 20 HEPES (pH 7.2), and 1 MgCl2 using different EGTA concentrations (0.1, 0.25, and 0.1 mM) each time. After the third wash, acini were transferred to a stirred cuvette at 37°C and resuspended in chelex-treated buffer C (buffer B plus 3 mM ATP, 10 mM creatine phosphate, 10 U/ml creatine phosphokinase, and 1 µM oligomycin). Cells were then permeabilized with 10 µM digitonin. [Ca2+] levels were measured by the fluorescence of fura-2 salt in a stirred cuvette at 37°C with excitation at 340 and 380 nm and emission at 510 nm.

Measurement of LDH release. LDH release was measured using the Cytotoxicity Detection Kit for LDH release according to the manufacturer's protocol (Roche Applied Science). Briefly, acini were incubated for 5 min with or without LY-294002 and then stimulated for 15 min with either 450 µM taurolitocholic acid 3-sulfate (TLC-S) or 1.2 mM sodium taurochenodeoxycholate (TCDC). After that, cells were harvested, and the total amount of LDH and its content in the extracellular medium were determined.

Measurement of trypsin activity. Active trypsin was measured in homogenates of isolated pancreatic acini by a fluorimetric assay as previously described (11). Briefly, isolated pancreatic acini were incubated in medium 199 containing 0.5% BSA and stimulated with 450 µM TLC-S. The reaction was stopped by the addition of ice-cold PBS, and collected pancreatic acini were homogenized in a glass-Teflon homogenizer in homogenization buffer containing 5 mmol/l MES (pH 6.5), 1 mmol/l MgSO4, and 250 mmol/l sucrose. An aliquot of the homogenate was then added to the assay buffer containing 50 mmol/l Tris·HCl (pH 8.0), 150 mmol/l NaCl, 1 mmol/l CaCl2, and 0.1 mg/ml BSA in a stirred cuvette at 37°C. The reaction was started by the addition of a specific substrate, Boc-Gln-Ala-Arg-MCA, which is converted to a fluorescent product by trypsin (12). The product emits fluorescence at 440 nm with excitation at 380 nm. The increase in fluorescence was linear during the 5 min of observation. Trypsin activity in each sample was determined using a standard curve for purified trypsin (Worthington, Freehold, NJ).

Analysis of Akt phosphorylation. Isolated pancreatic acini were incubated for 5 min with vehicle or 100 µM LY-294002 and then exposed to 450 µM TLC-S or 1.2 mM TCDC for 0, 1, 3, 5, and 10 min. Acini incubated with vehicle but not exposed to either bile acid served as controls. Aliquots of pancreatic acini were collected, washed twice with ice-cold PBS, and then resuspended in RIPA phosphorylation buffer [RIPA buffer supplemented with 0.1 M NaH2PO4 + Na2HPO4, 0.1 M NaH2PO4 + 100 mM NaF, 2 mM Na3VO4, and 80 µM beta-glycerolphosphate as well as 1 mM PMSF and protease inhibitor cocktail containing pepstatin, leupeptin, chymostatin, antipain, and aprotinin (5 µg/ml of each)]. Samples were rotated for 20 min at 4°C and centrifuged at 4°C for 15 min at 16,000 g. Supernatants were collected and stored at –80°C. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).

Akt phosphorylation was measured in the supernatants using Western blot analysis and ELISA for phospho-Akt. For Western blot analysis, proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding was blocked by a 1-h incubation of the membranes in 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS; pH 7.5) as suggested by the manufacturer (Cell Signaling). Blots were then incubated for 2 h or overnight with primary antibodies (for both phospho-Akt and Akt) in an antibody buffer containing 1% (wt/vol) nonfat dry milk in Tween 20-TBS [TTBS; 0.05% (vol/vol) Tween 20 in TBS], washed three times with TTBS, and finally incubated for 1 h with a peroxidase-labeled secondary antibody in the antibody buffer. Blots were developed for visualization using an ECL detection kit (Pierce, Rockford, IL). Band intensities in the immunoblots were quantified by densitometry.

The PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit (Cell Signaling) was used for the detection of the levels of Akt1 phosphorylated at Ser473. The quantity of phosphorylated Akt1 protein was measured in a colorimetric assay in accordance with the instructions of the manufacturer by measuring absorbance at 450 nm.

Statistical analysis of data. This was done using a two-tailed Student's t-test. P values of <0.05 were considered statistically significant. The numbers for independent experiments (performed on different acinar cell preparations) as well as the total tracings recorded are given in the figures.

Materials. Thapsigargin (TG), fura-2 AM, and fura-2 pentapotassium salt were purchased from Molecular Probes (Eugene, OR). Wortmannin was from Alexis Biochemicals (Carlsbad, CA). Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) was from Echelon (Salt Lake City, UT). Ionomycin, LY-294002, and creatine phosphate were from Calbiochem (La Jolla, CA). TLC-S, TCDC, IP3, creatine phosphokinase, chelex 100, digitonin, caffeine, and all other chemicals were from Sigma-Aldrich (St. Louis, MO). The Cytotoxicity Detection Kit used to measure LDH release was purchased from Roche Applied Science; the trypsinogen substrate, Boc-Gln-Ala-Arg-MCA, was from Bachem (Torrance, CA); the phospho-Akt (Ser473) antibody was from Cell Signaling; and the Akt antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bile acids exist in both the gallbladder and intestinal lumen in millimolar concentrations (11a). The reason for this is that for their physiological function bile acids need to form micelles. Micelle formation occurs over a narrow concentration range, and the concentration of bile acids necessary to form micelles is called the critical micellar concentration (CMC). Natural primary bile acids all have CMC values in the range of 2–5 mM (11a). In the present study, we used concentrations of bile acids of <1 mM or similar to those used in previously published studies (13, 34).

Both TLC-S (Fig. 1) and TCDC (Fig. 2) elicited an increase in [Ca2+]i in dispersed rat pancreatic acini, but the patterns of the responses were somewhat different. TLC-S induced a peak and plateau response typical for agents that mobilize intracellular [Ca2+]i. The peak response represents mobilization of intracellular stored Ca2+, whereas the plateau results from influx of extracellular Ca2+ into the cytosol. The influx of extracellular Ca2+ is balanced by Ca2+ removal from the cytosol by both the plasma membrane and internal store Ca2+-ATPases. With TLC-S application, the balance of these processes results in maintenance of [Ca2+]i at a constant plateau level (Fig. 1, A and B). TCDC also induced the peak phase of the [Ca2+]i response; however, the second phase of the [Ca2+]i response to TCDC was different. Instead of a constant [Ca2+]i plateau, there was a time-dependent increase in [Ca2+]i (Fig. 2A), suggesting that the Ca2+ influx during TCDC exposure is greater than its extrusion from the cytosol by Ca2+-ATPases. It is unlikely that the Ca2+ influx under the conditions used is due to the ionophoretic properties of TCDC. Indeed, we measured LDH release after the exposure of acini to either 450 µM TLC-S or 1.2 mM TCDC for up to 15 min and did not observe an increase of LDH release above the control level (data not shown), indicating that both bile acids at the concentrations used throughout our experiments did not increase plasma membrane permeability. Similarly, Kim et al. (13) showed that TCDC, in the concentration range used in our experiments, did not increase LDH release from pancreatic acini. These results indicate that the bile acids applied over a short period of time at the concentrations used in this study do not cause a nonspecific increase in the membrane permeability of acinar cells.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 2. PI3K inhibitors attenuate [Ca2+]i responses to taurochenodeoxycholate (TCDC) in pancreatic acinar cells. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (trace c), 100 µM LY-294002 (trace d), or 1 µM wortmannin (not shown), and then stimulated with 1.2 mM TCDC. A: representative tracings from at least 3 independent experiments. B and C: statistical analyses of the effects of 100 µM LY-294002 and 1 µM wortmannin on the magnitude of peak [Ca2+]i responses (B) and area under the curve (C; measured as depicted in Fig. 1E) after stimulation with 1.2 mM TCDC. The [Ca2+]i response to TCDC in the absence of PI3K inhibitors (i.e., in acini incubated with vehicle) was considered as 100%. Values are means ± SE from 6–8 tracings for the corresponding [Ca2+]i responses. *P < 0.05 and #P < 0.01 compared with [Ca2+]i responses of acini stimulated by TCDC in the absence of PI3K inhibitors. Note that in B and C, the scale on the y-axis starts at 50%.

 
Moreover, Voronina et al. (36) reported that bile acids did not induce a leak of fluorescent indicators from the cytosol of acinar cells. Thus, the Ca2+ influx that occurs with TCDC is most likely due to activation of store-operated Ca2+ channels on the plasma membrane, as has been described for other agonists in the pancreatic acinar cell (e.g., Ref. 22).

To elucidate the role of PI3K in bile acid-induced [Ca2+]i responses, we used both pharmacological and genetic approaches. The results shown in Figs. 1 and 2 demonstrate that the specific PI3K inhibitors LY-294002 and wortmannin significantly attenuated both the first and second phases of TLC-S- and TCDC-induced [Ca2+]i responses. These results suggest that PI3K, in part, mediates [Ca2+]i responses induced by TLC-S and TCDC in pancreatic acinar cells. The concentrations of the PI3K inhibitors we used have been applied in pancreatic acinar cells previously (e.g., Refs. 1–5). The inhibitory effects of the PI3K inhibitors on the bile acid-induced [Ca2+]i responses similar to those shown in Figs. 1 and 2 were also observed with 50 µM LY-294002 and 500 nM wortmannin (data not shown).

To show the effect of PI3K inhibition on bile acid-induced [Ca2+]i responses with lower concentrations of both bile acids, we decreased the concentration of TLC-S to 250 µM and TCDC to 800 µM, respectively (data not shown). Even with these lower concentrations of both bile acids, there was a significant inhibition on bile acid-induced [Ca2+]i responses during the exposure to PI3K inhibitors (data not shown). However, the responses to lower doses of bile acids were somewhat variable. For that reason, we did not further analyze the inhibitory effect of PI3K inhibition under these conditions, i.e., with lower concentrations of bile acids. Furthermore, in the experiments in which fura-2-loaded acini were incubated for up to 30 min with vehicle, LY-294002, or wortmannin showed no effect of PI3K inhibition on resting [Ca2+]i levels (10, 11), indicating that both PI3K inhibitors did not significantly change the basal [Ca2+]i level in acinar cells.

We (10, 11) have previously demonstrated a regulatory role for the PI3K {gamma}-isoform in Ca2+ signaling in pancreatic acinar cells. To test the involvement of PI3K-{gamma} in bile acid-induced [Ca2+]i responses, we measured the [Ca2+]i responses to TLC-S and TCDC in pancreatic acinar cells isolated from WT and PI3K-{gamma} KO mice (Fig. 3). Both TLC-S and TCDC elicited [Ca2+]i responses in mouse acini in a fashion similar to those observed in rat acini (Figs. 1 and 2). However, the first (i.e., peak) and second phases of the [Ca2+]i response to TCDC were less resolved in the mouse compared with rat acinar cells. The results shown in Fig. 3 demonstrate that the [Ca2+]i responses induced by TLC-S and TCDC were significantly smaller in PI3K-{gamma}-deficient acinar cells compared with WT acinar cells. Thus, PI3K-{gamma}, in part, mediates bile acid-induced Ca2+ responses in pancreatic acinar cells.


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Genetic deletion of PI3K-{gamma} attenuates [Ca2+]i responses to bile acids. Fura-2-loaded mouse pancreatic acini from wild-type (WT) PI3K-{gamma}-sufficient (trace a) and PI3K-{gamma}-deficient knockout (KO; trace b) mice were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle, and then stimulated with 450 µM TLC-S (A and B) or 1.2 mM TCDC (C–E). A and C: representative tracings from at least 3 independent experiments on both WT and PI3K-{gamma} KO acini. B, D, and E: effects of PI3K-{gamma} genetic deletion on [Ca2+]i responses. [Ca2+]i responses in WT acini were considered as 100%. Values are means ± SE from 5–12 tracings for the corresponding [Ca2+]i responses. *P < 0.05 and #P < 0.01 compared with the [Ca2+]i response in acini isolated from WT mice. Note that in B, D, and E, the scale on the y-axis starts at 50%.

 
We (10, 11) have previously shown that in pancreatic acinar cells, PI3K, and in particular PI3K-{gamma}, mediates [Ca2+]i responses induced by G protein-coupled receptors for the key secretagogues CCK and ACh. The results shown in Figs. 1–3 indicate that PI3K is also involved in the regulation of receptor-independent [Ca2+]i signals that are induced by bile acids. These results further suggest that bile acids and secretagogues may utilize common PI3K-dependent mechanism(s) to stimulate [Ca2+]i increases.

It is known that CCK-induced Ca2+ mobilization is mediated through the IP3 receptor (3, 26). On the other hand, several studies (8, 34) have indicated that bile acids are able to activate IP3-dependent Ca2+-release mechanisms. To test whether bile acids and CCK mobilize Ca2+ from the same intracellular Ca2+ pools, we measured [Ca2+]i signals induced by sequential additions of CCK and bile acids to pancreatic acinar cells. Acini were resuspended in either Ca2+-containing or Ca2+-free medium (to prevent extracellular Ca2+ influx) and stimulated sequentially with CCK and bile acids. In both media (Fig. 4A and data not shown), TLC-S did not elicit a [Ca2+]i response when it was added after the peak [Ca2+]i response to CCK, i.e., in conditions in which CCK-sensitive intracellular Ca2+ pools were depleted (22). TCDC induced a weak [Ca2+]i response when it was added after CCK in the Ca2+-containing medium (Fig. 4B); however, there was no [Ca2+]i response to TCDC in the Ca2+-free medium (Fig. 4C).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Bile acids release [Ca2+]i from the same Ca2+ pool as CCK. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2 (A and B) or in Ca2+-free solution Q, in which CaCl2 was omitted and 1 mM EGTA was added (C). Pancreatic acinar cells were then stimulated with 100 nM CCK-8 followed 100 s later by the addition of 450 µM TLC-S (A) or 1.2 mM TCDC (B and C). Tracings are representative of at least 3 independent experiments.

 
We next applied the IP3 receptor blocker caffeine to further prove that bile acids induce Ca2+ mobilization from IP3-sensitive pools. In intact acinar cells, caffeine significantly inhibited [Ca2+]i responses to TLC-S (Fig. 5) and TCDC (data not shown), suggesting that bile acids induce [Ca2+]i signals through an IP3-dependent mechanism. The combination of maximally effective concentrations of caffeine and LY-294002 attenuated the [Ca2+]i response to bile acids to a greater extent than either agent alone (Fig. 5 and data not shown). This suggests that both IP3-dependent and PI3K-mediated, IP3-independent mechanisms are involved in bile acid-induced [Ca2+]i responses. Furthermore, caffeine significantly inhibited Ca2+ mobilization caused by TLC-S in permeabilized acinar cells (Fig. 6), demonstrating the involvement of the IP3 receptor in the bile acid-induced Ca2+ signal. Of note, the inhibition of the TLC-S-induced Ca2+ signal with caffeine was only partial, again indicating the involvement of IP3-independent mechanism(s). Bile acids are known to depolarize mitochondria (9, 35) and thus modify the energetics of the cell. To eliminate the possible contribution of this effect, all experiments on permeabilized acinar cells were performed in the presence of oligomycin and at a constant ATP level maintained with creatine phosphate/creatine phosphokinase (see MATERIALS AND METHODS).


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 5. Caffeine and PI3K inhibition attenuate bile acid-induced [Ca2+]i responses independent of each other. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (trace a), 100 µM LY-294002 alone (trace b), 20 mM caffeine alone (trace c), or 20 mM caffeine + 100 µM LY-294002 (trace d), and then stimulated with 450 µM TLC-S. Tracings are representative of at least 3 independent experiments. Values in D and E are means ± SE from 4–6 tracings for each condition. The [Ca2+]i response to TLC-S in the absence of caffeine and LY-294002 was considered as 100%. *P < 0.05 and #P < 0.01 compared with the [Ca2+]i response to TLC-S in the absence of caffeine and LY-294002; +P < 0.05 compared with the [Ca2+]i response to TLC-S in the presence of either 20 mM caffeine or 100 µM LY-294002 alone.

 

Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 6. Caffeine inhibits TLC-S induced [Ca2+]i responses in permeabilized pancreatic acinar cells. Rat pancreatic acini were resuspended in a permeabilization solution containing 10 µM digitonin. A: permeabilized acini were stimulated with 1 µM inositol (1,4,5)-trisphosphate (IP3). After [Ca2+]i reached a plateau, 20 mM caffeine was added. After 5 min of caffeine exposure (interrupted tracing), permeabilized acini were again stimulated with 1 µM IP3. B: permeabilized acini were incubated for 5 min with vehicle (top trace) or 20 mM caffeine and then stimulated with 180 µM TLC-S. Tracings are representative of 3 independent experiments. C: effect of 20 mM caffeine on the area under the curve of the [Ca2+]i response after stimulation of permeabilized acini with 180 µM TLC-S. The [Ca2+]i response to TLC-S in the absence of caffeine (i.e., in acini incubated with vehicle) was considered as 100%. Values are means ± SE from 3 tracings for the corresponding responses. *P < 0.05 compared with the [Ca2+]i response of acini stimulated by TLC-S in the absence of caffeine.

 
The results shown in Figs. 4–6 indicate that bile acids and CCK mobilize Ca2+ from the same intracellular pools and that bile acids act to cause [Ca2+]i responses through both IP3- and PI3K-dependent mechanisms.

It has been proposed (13) that bile acids inhibit SERCA in pancreatic acinar cells. On the other hand, we (10) have recently shown that PI3K negatively regulates SERCA in these cells and that PI3K inhibits SERCA-dependent Ca2+ reloading of intracellular pools during stimulation with CCK. Thus, we hypothesized that PI3K also regulates bile acid-induced Ca2+ responses through the inhibition of SERCA. To test this hypothesis, we performed the following experiments. The objective of the experiment shown in Fig. 7 was to measure the effect of the PI3K inhibitor LY-294002 on bile acid-induced [Ca2+]i responses under conditions in which SERCA was inhibited. We first treated dispersed rat pancreatic acini with TG to block SERCA followed in 10 s by stimulation with TLC-S or TCDC. Such a short incubation with TG completely inhibits SERCA but does not deplete intracellular Ca2+ pools. Indeed, a previous study (37) has demonstrated that depletion of Ca2+ pools with TG requires >1 min. We (10) have also previously shown that PI3K inhibitors do not affect [Ca2+]i responses to TG. The results shown in Fig. 7 indicate that pretreatment of acini with TG prevented the inhibitory effect of LY-294002 on bile acid-induced [Ca2+]i responses (cf. Figs. 1 and 2). These data suggest that PI3K regulates [Ca2+]i responses to bile acids through the inhibition of SERCA.


Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 7. Thapsigargin (TG) abolishes the effect of LY-294002 on bile acid-induced [Ca2+]i responses. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (traces a and c) or 100 µM LY-294002 (traces b and d), and then stimulated with 2 µM TG and 450 µM TLC-S (A) or with 2 µM TG and 1.2 mM TCDC (B). Tracings are representative of 3 independent experiments. C: statistical analysis of the effect of PI3K inhibition on the area under the curve of the [Ca2+]i response after stimulation of acini with 2 µM TG and the indicated bile acid. Responses were the same in the presence and absence of LY-294002 (i.e., in acini incubated with vehicle); the latter condition was considered as 100%. Values are means ± SE from 3–6 tracings for the corresponding [Ca2+]i responses.

 
To further prove that PI3K regulates Ca2+ reloading to the ER during exposure to bile acids, we next measured the effects of PI3K inhibition on the amount of stored Ca2+ in acinar cells treated with bile acids (Fig. 8). Pancreatic acini were preincubated with or without LY-294002 and stimulated with TLC-S (Fig. 8A) or TCDC (Fig. 8B). EGTA was added 40 s later to block Ca2+ influx, followed by the addition of the Ca2+ ionophore ionomycin to release all stored Ca2+. The results shown in Fig. 8 demonstrate that LY-294002 significantly increased the amount of Ca2+ released by ionomycin, as measured by the area under the curve of the [Ca2+]i response to ionomycin (Fig. 8, C and D). Taken together, the results shown in Figs. 7 and 8 suggest that PI3K facilitates the [Ca2+]i response to bile acids by inhibiting SERCA, which slows down Ca2+ reuptake by the ER resulting in decreased stored Ca2+ and increased [Ca2+]i. Therefore, inhibition of PI3K stimulates Ca2+ reuptake through SERCA, thus attenuating the [Ca2+]i response to bile acids.


Figure 8
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 8. LY-294002 inhibits the depletion of intracellular Ca2+ pools after bile acid-induced [Ca2+]i responses. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (trace a) or 100 µM LY-294002 (trace b), and then stimulated with 450 µM TLC-S or 1.2 mM TCDC. Forty seconds after stimulation with the bile acid, 2 mM EGTA was added to block Ca2+ influx. Another 10 s later, 5 µM ionomycin was applied to release intracellular Ca2+. Tracings are representative of at least 3 independent experiments. C: graphical depiction of the area under the curve after ionomycin administration, which was used to calculate the results shown in D. This area was considered as 100% for acini incubated without LY-294002 (i.e., trace a in A and B). Values are means ± SE from 3–6 tracings for each of the bile acids. #P < 0.01 compared with acini incubated without LY-294002. Note that in D, the scale on the y-axis starts at 50%.

 
The experiments shown in Fig. 9 were performed on permeabilized acinar cells to more directly test the effects of bile acids and PI3K on SERCA-dependent Ca2+ reloading into the ER. TLC-S dose dependently inhibited Ca2+ reloading into the ER after IP3 stimulation (Fig. 9A). A similar dose-dependent effect was observed after stimulation with TCDC (data not shown). These results indicate that in permeabilized cells, i.e., under conditions precluding the direct involvement of PI3K, bile acids inhibit SERCA-dependent Ca2+ reloading into the ER.


Figure 9
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 9. Addition of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) potentiates the inhibitory effect of bile acids on Ca2+ reloading into the endoplasmic reticulum. Rat pancreatic acini were resuspended in a permeabilization medium containing 10 µM digitonin and then stimulated as indicated. A: permeabilized acini were stimulated with 1 µM IP3 either alone (control) or in combination with indicated concentrations of TLC-S. B: permeabilized acini were stimulated with 1 µM IP3, and, after the Ca2+ level had returned back to the basal level, 40 µM PIP3 (traces b and c) or vehicle (trace a) was added. This was followed after 1 min by a second stimulation with 1 µM IP3 either alone (traces a and b) or together with 45 µM TLC-S (trace c). C: mean ± SE values (n = 3–4) for the duration of the [Ca2+] peak after stimulation with 1 µM IP3 either alone (control) or in combination with 40 µM PIP3 or with both 40 µM PIP3 and 45 µM TLC-S, as illustrated in B by traces a–c, respectively. The duration of the [Ca2+] peak was calculated as the duration of time when the [Ca2+] value was greater than half of its maximal increase. #P < 0.01 compared with the [Ca2+] response to IP3 alone (control); +P < 0.05 compared with the [Ca2+] response to IP3 in combination with 40 µM PIP3. D: permeabilized acini were incubated for 1 min with vehicle (traces a and b) or 40 µM PIP3 (traces c and d) and then stimulated with 1 µM IP3 either alone (traces a and c) or in combination with 150 µM TCDC (traces b and d).

 
On the other hand, the main product of PI3K, PIP3, also inhibited Ca2+ reuptake upon IP3 stimulation (Fig. 9B) (10). The combination of TLC-S and PIP3 (Fig. 9B) or TCDC and PIP3 (Fig. 9D) slowed the rate of SERCA-dependent Ca2+ reloading more than bile acid or PIP3 alone. These results suggest that bile acids and PIP3 both inhibit SERCA activity, resulting in an augmented inhibition of Ca2+ reloading into the ER. In these experiments, we applied 40 µM PIP3 following our previous experimental protocol (10); a similar but smaller effect was also observed with 20 µM PIP3 (data not shown). The question as to whether these concentrations are physiologically relevant is important but difficult to answer directly. As pointed out previously (15, 32), the concentrations of inositol phospholipids can locally be very high in membranes because of their lipid character.

To further determine the mechanism of PI3K involvement in the effect of bile acids on Ca2+ signaling, we measured whether bile acids activate PI3K in acinar cells by measuring phosphorylation of its key downstream target, Akt/PKB kinase. Using Western blot analysis and a phospho-Akt sandwich ELISA assay for the measurement of phospho-Akt, we found that neither TLC-S or TCDC stimulated Akt phosphorylation in the first 3 min, i.e., in the times of the development of the [Ca2+]i response (Fig. 10). These results suggest that the mechanism of the PI3K effect on bile acid-induced [Ca2+]i responses is not a result of Akt phosphorylation and activation.


Figure 10
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 10. Effects of bile acids on Akt phosphorylation in acinar cells. Pancreatic acinar cells were incubated with 1.2 mM TCDC (A), 450 µM TLC-S (B), or vehicle for the indicated times. Akt phosphorylation was measured in cell lysates with Western blot analysis ({blacksquare}) or with phospho-Akt ELISA ({circ}). A: Western blot analysis was performed with antibodies against Akt phosphorylated at Ser473 and further reprobed with anti-total Akt antibody. The intensities of phospho-Akt bands on the blot were quantified densitometrically and normalized to those of total Akt in the same sample. The mean ratio of phospho-Akt to Akt at a given time point was further normalized to that in control cells treated for the same time with vehicle. B: phospho-Akt levels were measured by a PathScan Phospho-Akt Sandwich ELISA Kit. Values are means ± SE; n = 3. *P < 0.05 compared with the control time point.

 
Finally, we tested whether bile acids induce trypsinogen activation, a key pathological response in pancreatic acinar cells (11, 16, 29), and whether such a response is regulated by PI3K. TLC-S time dependently caused significant trypsinogen activation, which was completely inhibited by LY-294002 (Fig. 11). These results indicate that bile acids induce trypsinogen activation and, furthermore, that it is mediated by PI3K.


Figure 11
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 11. TLC-S causes trypsinogen activation, which is completely blocked by the PI3K inhibitor LY-294002. Rat pancreatic acini were resuspended in medium 199 containing 0.5% BSA, incubated for 5 min with vehicle (open bars) or 100 µM LY-294002 (solid bars), and then exposed to 450 µM TLC-S for the indicated times. Acini incubated with vehicle but not exposed to TLC-S served as controls. Trypsin activity was measured by a fluorogenic assay as described in MATERIALS AND METHODS and normalized to trypsin activity in control acini (i.e., incubated with vehicle and without TLC-S). Values are means ± SE from 3 independent experiments. #P < 0.01 and *P < 0.05 compared with trypsin activity in control acini.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study, using both pharmacological and genetic approaches to inhibit PI3K activity, demonstrates that PI3K, in part, mediates bile acid-induced [Ca2+]i responses in pancreatic acinar cells. Furthermore, the results obtained using mice deficient in the PI3K {gamma}-isoform indicate the involvement of PI3K-{gamma} in bile acid-induced [Ca2+]i responses. We further demonstrated that bile acids release Ca2+ from the same internal pools as CCK. Finally, the experiments on permeabilized acinar cells showed an augmented effect of the combination of bile acids and the PI3K product PIP3 on the inhibition of SERCA activity, suggesting that bile acids and PI3K act independently to inhibit SERCA.

It has been previously shown (5, 7, 34, 36) that bile acids are able to induce specific [Ca2+]i responses in both hepatocytes and pancreatic acinar cells. The exact mechanisms of bile acid-induced pancreatitis in experimental models (1, 17) are not well established and are thought to be through their "blunt" effect as detergents. However, data acquired in different cell types, including the pancreatic acinar cell, have indicated that bile acids regulate specific cellular signals, in particular, [Ca2+]i responses (8, 13, 14, 25, 31, 34). These data also demonstrated a possible role of PI3K in bile acid-induced signaling pathways. We here show for the first time the involvement of PI3K, and in particular PI3K-{gamma}, in the regulation of bile acid-induced [Ca2+]i responses in pancreatic acinar cells.

In terms of Ca2+ signaling, several studies (8, 34) have suggested that bile acids activate IP3-dependent Ca2+-release mechanisms. For example, Devor et al. (8) showed that in the T84 colonic cell line, bile acids induced IP3 accumulation and Ca2+ release from internal stores. In agreement with these studies, our results showed that caffeine, an IP3 receptor antagonist, significantly inhibits [Ca2+]i responses to both TLC-S and TCDC. Our results further showed that in addition to IP3-dependent mobilization of intracellular Ca2+, bile acids employ a second PI3K-dependent mechanism to increase [Ca2+]i. For example, in the presence of the maximally effective dose of caffeine, LY-294002 further decreased bile acid-induced [Ca2+]i responses, suggesting the involvement of both IP3- and PI3K-dependent pathways.

We (10) have previously shown that SERCA inhibition with TG abrogated the effect of PI3K inhibitors on CCK-induced [Ca2+]i responses in the pancreatic acinar cell. In the present study, we similarly found that TG abolished the effect of PI3K inhibition on bile acid-induced [Ca2+]i responses. We further demonstrated that LY-294002 inhibited the depletion of intracellular Ca2+ pools during bile acid exposure. Such results suggest that PI3K negatively regulates SERCA, thus inhibiting Ca2+ reuptake into the ER and augmenting bile acid-induced [Ca2+]i responses. PI3K inhibition reverses this regulation, facilitating SERCA-dependent Ca2+ reloading into the ER.

The experiments performed on permeabilized acinar cells demonstrated that TLC-S and TCDC dose dependently inhibited IP3-induced, SERCA-mediated Ca2+ reuptake. The effect of bile acids to inhibit SERCA was further augmented by PIP3. Our findings from measurements of Akt phosphorylation indicate the role of PI3K to augment bile acid-induced Ca2+ responses is not due to an effect on Akt phosphorylation and activation.

We found that TLC-S time dependently caused trypsinogen activation, a key pathological response of pancreatic acinar cells (11, 16, 29). The extent of activation was less than that caused by hyperstimulating acinar cells with CCK-8, a classical inducer of trypsinogen activation (11, 27), but any premature, intra-acinar cell activation of trypsinogen can have harmful consequences. The TLC-S-induced trypsinogen activation was completely inhibited by LY-294002. These new findings results indicate that bile acids induce a key pathological response that occurs during the course of pancreatitis and, furthermore, that it is mediated by PI3K.

In conclusion, the results of the present study indicate that bile acid-induced increases in [Ca2+]i are mediated by both the IP3-dependent mobilization of intracellular Ca2+ and the inhibition of SERCA-dependent Ca2+ reloading into intracellular pools. Our findings further indicate that PI3K facilitates bile acid-induced Ca2+ responses in pancreatic acinar cells through the inhibition of SERCA-dependent Ca2+ reloading into the ER. Accordingly, pharmacological or genetic inhibition of PI3K results in the activation of SERCA, leading to an attenuation of bile acid-induced [Ca2+]i responses by more rapid Ca2+ reuptake into internal stores. The results of the present study, together with our previous findings (10, 11), indicate an important role for PI3K in facilitating [Ca2+]i responses and trypsinogen activation induced by both neurohormonal stimulation and bile acids. In turn, abnormal [Ca2+]i increases may mediate key pathological responses of pancreatitis, including the activation of inflammatory signals, premature trypsinogen activation, and acinar cell necrosis (14, 24). Bile acids are known to induce acute pancreatitis responses in experimental models (1, 17, 29, 33). Based on our findings, inhibition of PI3K to attenuate cellular [Ca2+]i responses and trypsinogen activation represents a potential therapeutic strategy for the treatment of biliary pancreatitis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a Department of Veterans Affairs (Veterans Affairs Merit Review to S. J. Pandol) and by National Institutes of Health (NIH) Grants DK-59936 and DK-59508. This study was also supported in part by NIH Grant P50-AA-11999.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. S. Gukovskaya, Veterans Affairs Greater Los Angeles Healthcare System, West Los Angeles Veterans Affairs Healthcare Center, 11301 Wilshire Blvd., Bldg. 258, Rm. 340, Los Angeles, CA 90073 (e-mail: agukovsk{at}ucla.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aho HJ, Koskensalo SM, Nevalainen TJ. Experimental pancreatitis in the rat. Sodium taurocholate-induced acute haemorrhagic pancreatitis. Scand J Gastroenterol 15: 411–416, 1980.[ISI][Medline]
  2. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529, 2003.[CrossRef][ISI][Medline]
  3. Beuers U, Nathanson MH, Boyer JL. Effects of tauroursodeoxycholic acid on cytosolic Ca2+ signals in isolated rat hepatocytes. Gastroenterology 104: 604–612, 1993.[ISI][Medline]
  4. Combettes L, Berthon B, Doucet E, Erlinger S, Claret M. Bile acids mobilise internal Ca2+ independently of external Ca2+ in rat hepatocytes. Eur J Biochem 190: 619–623, 1990.[ISI][Medline]
  5. Combettes L, Dumont M, Berthon B, Erlinger S, Claret M. Effect of the bile acid taurolithocholate on cell calcium in saponin-treated rat hepatocytes. FEBS Lett 227: 161–166, 1988.[CrossRef][ISI][Medline]
  6. Combettes L, Dumont M, Berthon B, Erlinger S, Claret M. Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells. J Biol Chem 263: 2299–2303, 1988.[Abstract/Free Full Text]
  7. Devor DC, Sekar MC, Frizzell RA, Duffey ME. Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84). J Clin Invest 92: 2173–2181, 1993.[ISI][Medline]
  8. Ferreira M, Coxito PM, Sardao VA, Palmeira CM, Oliveira PJ. Bile acids are toxic for isolated cardiac mitochondria: a possible cause for hepatic-derived cardiomyopathies? Cardiovasc Toxicol 5: 63–73, 2005.[CrossRef][ISI][Medline]
  9. Fischer L, Gukovskaya AS, Young SH, Gukovsky I, Lugea A, Buechler P, Penninger JM, Friess H, Pandol SJ. Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase. Am J Physiol Gastrointest Liver Physiol 287: G1200–G1212, 2004.[Abstract/Free Full Text]
  10. Gukovsky I, Cheng JH, Nam KJ, Lee OT, Lugea A, Fischer L, Penninger JM, Pandol SJ, Gukovskaya AS. Phosphatidylinositide 3-kinase gamma regulates key pathologic responses to cholecystokinin in pancreatic acinar cells. Gastroenterology 126: 554–566, 2004.[CrossRef][ISI]
  11. Hofmann AF. Biliary secretion and excretion. In: Physiological Basis of Medical Practice (11th ed.), edited by West JB. Baltimore, MD: Williams & Wilkins, 1990, p. 675–691.
  12. Kawabata S, Miura T, Morita T, Kato H, Fujikawa K, Iwanaga S, Takada K, Kimura T, Sakakibara S. Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur J Biochem 172: 17–25, 1988.[ISI][Medline]
  13. Kim JY, Kim KH, Lee JA, Namkung W, Sun AQ, Ananthanarayanan M, Suchy FJ, Shin DM, Muallem S, Lee MG. Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells. Gastroenterology 122: 1941–1953, 2002.[CrossRef][ISI]
  14. Kruger B, Albrecht E, Lerch MM. The role of intracellular calcium signaling in premature protease activation and the onset of pancreatitis. Am J Pathol 157: 43–50, 2000.[Abstract/Free Full Text]
  15. Leevers SJ, Vanhaesebroeck B, Waterfield MD. Signalling through phosphoinositide 3-kinases: the lipids take centre stage. Curr Opin Cell Biol 11: 219–225, 1999.[CrossRef][ISI][Medline]
  16. Lerch MM, Gorelick FS. Early trypsinogen activation in acute pancreatitis. Med Clin North Am 84: 549–563, viii, 2000.[CrossRef][ISI][Medline]
  17. Lerch MM, Saluja AK, Runzi M, Dawra R, Saluja M, Steer ML. Pancreatic duct obstruction triggers acute necrotizing pancreatitis in the opossum. Gastroenterology 104: 853–861, 1993.[ISI][Medline]
  18. Lerch MM, Weidenbach H, Hernandez CA, Preclik G, Adler G. Pancreatic outflow obstruction as the critical event for human gall stone induced pancreatitis. Gut 35: 1501–1503, 1994.[Abstract/Free Full Text]
  19. Lupia E, Goffi A, De Giuli P, Azzolino O, Bosco O, Patrucco E, Vivaldo MC, Ricca M, Wymann MP, Hirsch E, Montrucchio G, Emanuelli G. Ablation of phosphoinositide 3-kinase-gamma reduces the severity of acute pancreatitis. Am J Pathol 165: 2003–2011, 2004.[Abstract/Free Full Text]
  20. Moody FG, Senninger N, Runkel N. Another challenge to the Opie myth. Gastroenterology 104: 927–931, 1993.[ISI][Medline]
  21. Opie EL. The etiology of acute hermorrhagic pancreatitis. Johns Hopkins Hosp Bull 12: 182–188, 1901.
  22. Pandol SJ, Schoeffield MS, Fimmel CJ, Muallem S. The agonist-sensitive calcium pool in the pancreatic acinar cell. Activation of plasma membrane Ca2+ influx mechanism. J Biol Chem 262: 16963–16968, 1987.[Abstract/Free Full Text]
  23. Pandol SJ, Schoeffield-Payne MS, Gukovskaya AS, Rutherford RE. Sphingosine regulates Ca2+-ATPase and reloading of intracellular Ca2+ stores in the pancreatic acinar cell. Biochim Biophys Acta 1195: 45–50, 1994.[Medline]
  24. Raraty M, Ward J, Erdemli G, Vaillant C, Neoptolemos JP, Sutton R, Petersen OH. Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells. Proc Natl Acad Sci USA 97: 13126–13131, 2000.[Abstract/Free Full Text]
  25. Rust C, Bauchmuller K, Fickert P, Fuchsbichler A, Beuers U. Phosphatidylinositol 3-kinase-dependent signaling modulates taurochenodeoxycholic acid-induced liver injury and cholestasis in perfused rat livers. Am J Physiol Gastrointest Liver Physiol 289: G88–G94, 2005.[Abstract/Free Full Text]
  26. Rutherford RE, Schoeffield-Payne M, Pandol SJ. Cellular mechanisms and physiological implications of quantal Ca2+ release in pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 267: G1058–G1066, 1994.[Abstract/Free Full Text]
  27. Saluja AK, Bhagat L, Lee HS, Bhatia M, Frossard JL, Steer ML. Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini. Am J Physiol Gastrointest Liver Physiol 276: G835–G842, 1999.[Abstract/Free Full Text]
  28. Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzki I, Joza N, Mak TW, Ohashi PS, Suzuki A, Penninger JM. Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science 287: 1040–1046, 2000.[Abstract/Free Full Text]
  29. Singh VP, Saluja AK, Bhagat L, van Acker GJ, Song AM, Soltoff SP, Cantley LC, Steer ML. Phosphatidylinositol 3-kinase-dependent activation of trypsinogen modulates the severity of acute pancreatitis. J Clin Invest 108: 1387–1395, 2001.[CrossRef][ISI][Medline]
  30. Steer ML. Early events in acute pancreatitis. Baillieres Best Pract Res Clin Gastroenterol 13: 213–225, 1999.[CrossRef][Medline]
  31. Takikawa Y, Miyoshi H, Rust C, Roberts P, Siegel R, Mandal PK, Millikan RE, Gores GJ. The bile acid-activated phosphatidylinositol 3-kinase pathway inhibits Fas apoptosis upstream of bid in rodent hepatocytes. Gastroenterology 120: 1810–1817, 2001.[CrossRef][ISI][Medline]
  32. Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC, Woscholski R, Parker PJ, Waterfield MD. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem 70: 535–602, 2001.[CrossRef][ISI][Medline]
  33. Vaquero E, Gukovsky I, Zaninovic V, Gukovskaya AS, Pandol SJ. Localized pancreatic NF-{kappa}B activation and inflammatory response in taurocholate-induced pancreatitis. Am J Physiol Gastrointest Liver Physiol 280: G1197–G1208, 2001.[Abstract/Free Full Text]
  34. Voronina S, Longbottom R, Sutton R, Petersen OH, Tepikin A. Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology. J Physiol 540: 49–55, 2002.[Abstract/Free Full Text]
  35. Voronina SG, Barrow SL, Gerasimenko OV, Petersen OH, Tepikin AV. Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating DeltaPsim. J Biol Chem 279: 27327–27338, 2004.[Abstract/Free Full Text]
  36. Voronina SG, Gryshchenko OV, Gerasimenko OV, Green AK, Petersen OH, Tepikin AV. Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration. J Biol Chem 280: 1764–1770, 2005.[Abstract/Free Full Text]
  37. Zhang BX, Zhao H, Loessberg P, Muallem S. Activation of the plasma membrane Ca2+ pump during agonist stimulation of pancreatic acini. J Biol Chem 267: 15419–15425, 1992.[Abstract/Free Full Text]

Related articles in AJP - GI:

Corrigendum

AJP - GI 2007 292: G1815. [Full Text]  



This article has been cited by other articles:


Home page
 GutHome page
V Venglovecz, Z Rakonczay Jr, B Ozsvari, T Takacs, J Lonovics, A Varro, M A Gray, B E Argent, and P Hegyi
Effects of bile acids on pancreatic ductal bicarbonate secretion in guinea pig
Gut, August 1, 2008; 57(8): 1102 - 1112.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
J. M Laukkarinen, G. J D Van Acker, E. R Weiss, M. L Steer, and G. Perides
A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate
Gut, November 1, 2007; 56(11): 1590 - 1598.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Corrigendum
Right arrow All Versions of this Article:
292/3/G875    most recent
00558.2005v1
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 Related articles in AJP - GI
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed