Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase
L. Fischer,1,2,3
A. S. Gukovskaya,1,2
S. H. Young,2
I. Gukovsky,1,2
A. Lugea,1,2
P. Buechler,3
J. M. Penninger,4
H. Friess,3 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 90073; 3Department of Surgery, University of Heidelberg, 69120 Heidelberg, Germany; and 4Institute for Molecular Biotechnology, Austrian Academy of Sciences, A-1030 Vienna, Austria
Submitted 9 May 2004
; accepted in final form 20 July 2004
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ABSTRACT
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Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca2+ responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-
, regulates Ca2+ responses and the Ca2+-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca2+ responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca2+ influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca2+ concentration ([Ca2+]i) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production nor Ins(1,4,5)P3-induced Ca2+ mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P3 or Ins(1,4,5)P3 receptors. PI3-kinase inhibition did not affect Ca2+ mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca2+]i signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-
increased the amount of Ca2+ in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca2+ reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca2+ signaling in pancreatic acinar cells through its inhibitory effect on SERCA.
pancreas; cholecystokinin; carbachol
PHOSPHATIDYLINOSITOL 3-KINASE (PI3-kinase) is a key signaling molecule in eukaryotic cells, which plays important roles in the immune response, growth regulation, survival, and metabolism (9, 23, 52). We recently showed (20) that there is a significant role for PI3-kinase in the regulation of Ca2+ responses in pancreatic acinar cells. In particular, pharmacological inhibition of PI3-kinase with LY-294002 and wortmannin and genetic deletion of PI3-kinase-
both resulted in decreased Ca2+ release from intracellular stores and Ca2+ influx, as well as diminished trypsinogen and NF-
B activation induced by supraphysiological concentrations of CCK in pancreatic acinar cells (20). These results suggested that PI3-kinase plays an important role in Ca2+ signaling, which in turn may contribute to Ca2+-dependent pathological responses of pancreatitis (19, 35, 45, 51, 54). In fact, a recent study (49) demonstrated a significant amelioration of the severity of experimental pancreatitis with pharmacological inhibition of PI3-kinase.
Ca2+ is a key messenger mediating pancreatic acinar cell secretory responses (51, 55). Rapid cytosolic Ca2+ signals are mainly generated by liberation of Ca2+ stored in the endoplasmic reticulum (ER) (3, 34, 55), although other compartments of acinar cells (i.e., mitochondria, nucleus, zymogen granules) are also involved in Ca2+ signaling (51, 56). However, the ER contains the vast majority of agonist-sensitive intracellular Ca2+, and it has been shown (24, 51, 55) that the initial rise of cytosolic free Ca2+ concentration ([Ca2+]i) during physiological and supraphysiological neurohormonal stimulation is due to Ca2+ release from the ER into the cytosol. This Ca2+ release results in emptying of the ER of Ca2+, which in turn results in opening of store-operated Ca2+ channels (SOCs) in the plasma membrane, allowing Ca2+ influx into the cell (4, 42). Ca2+ entering the cytosol via SOCs is taken up into the lumen of the ER by SERCA, especially with termination of agonist stimulation (4, 34, 51). During agonist stimulation, there is inhibition of SERCA activity preventing complete reloading of Ca2+ into the ER stores (36, 39). The mechanism of this inhibition has not been determined.
CCK and acetylcholine are two major agonists mediating the secretory response of pancreatic acinar cells with Ca2+ serving as a second messenger for both agonists (8, 36, 55). CCK elicits two patterns of Ca2+ responses in acinar cells (45, 55). Physiological doses of CCK induce [Ca2+]i oscillations, whereas supraphysiological CCK concentrations induce an initial rapid rise in [Ca2+]i followed by a sustained [Ca2+]i plateau. The peak and plateau [Ca2+]i responses are thought to be at least in part responsible for the pathological effect of high-dose CCK on exocrine pancreas (35, 51, 54). Ca2+ responses to CCK are initiated by CCK-induced activation of phospholipase C (PLC) through the G protein-coupled pathway leading to production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (3, 55). Ins(1,4,5)P3 mediates Ca2+ release into the cytosol through its receptors [Ins(1,4,5)P3Rs] on the ER (3, 4, 8, 24, 37). Besides Ins(1,4,5)P3, cADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), and fatty acid ethyl esters are able to release Ca2+ from intracellular stores through effects on Ca2+-regulated ryanodine receptors (RyRs) and NAADP receptors (8, 15, 24, 51, 55). Current evidence (8, 24, 37, 44) suggests that Ins(1,4,5)P3 and its receptors act to trigger Ca2+ release from the ER, whereas the other signals act to amplify the Ca2+ signals.
There are two main pathways operative in removing Ca2+ from the cytosol during and at the termination of neurohormonal stimulation. These pathways are ATP-dependent mechanisms removing Ca2+ either back into the ER through SERCA or to the extracellular space through plasma membrane calcium ATPase (PMCA) (2, 4, 34). SERCA pump activity in pancreatic acinar cells is
14 times greater than non-SERCA pump mechanisms (7), which makes SERCA a key regulator of Ca2+ signaling during neurohormonal stimulation as well as its termination (27).
Data from several laboratories suggest a role for PI3-kinase in Ca2+ signaling in a number of cell types indicating different mechanisms of the regulation of [Ca2+]i responses by PI3-kinase. In mast cells, PI3-kinase (and in particular, PI3-kinase-
) was shown to regulate [Ca2+]i by activating PLC
and facilitating Ca2+ influx (1, 11, 25). Another mechanism proposed for PI3-kinase regulation of Ca2+ influx in mast cells is through modulating Ins(1,4,5)P3 production (50). In human platelets, PI3-kinase does not regulate the activity of PLC
but likely contributes to a stable interaction of PLC
with the plasma membrane (16). In addition, PI3-kinase is required for activation of store-mediated Ca2+ entry in these cells (47). Coactivation of PLC
and PI3-kinase in Hep G2 cells results in a significant increase of Ins(1,4,5)P3 generation as well as Ca2+ release (43). Several PI3-kinase isoforms were shown to mediate agonist-specific stimulation of Ca2+ channels in vascular myocytes (30). PI3-kinase-
activation plays a critical role in the purinergic regulation of Ca2+ signaling in cardiac cells (6). We recently demonstrated (20) a regulatory role for PI3-kinase (in particular, the PI3-kinase-
isoform) in Ca2+ signaling in pancreatic acinar cells. The goal of the present work was to elucidate the pathway(s) through which PI3-kinase regulates Ca2+ signaling in these cells.
Using pharmacological and genetic means, we found that PI3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of SERCA. In particular, phosphatidylinositol 3,4,5-trisphosphate (PIP3), the main product of PI3-kinase, inhibits Ca2+ reloading into the intracellular pools. Furthermore, our data indicate that other major pathways regulating [Ca2+]i responses in pancreatic acinar cells, i.e., Ins(1,4,5)P3 production, Ins(1,4,5)P3-induced Ca2+ release, and PMCA, are not regulated by PI3-kinase.
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MATERIALS AND METHODS
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Acinar cell isolation.
Pancreatic acini were isolated from Sprague-Dawley rats (75100 g), the wild-type (p110
+/+) C57BL/6 mice (3540g), and homozygous PI3-kinase-
knockout (p110
/) mice (20, 48) using a previously described collagenase digestion method (15, 1720). Breeding of the p110
-deficient 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 the National Institutes of Health guidelines. Genotyping of p110
knockout mice was done by PCR using specific primers (48). Acini were resuspended in solution Q containing (in mM) 120 NaCl, 20 HEPES, 5 KCl, 1 MgCl2, 1 CaCl2, 10 sodium pyruvate, 10 ascorbate, 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 the solution. Where indicated, acini were incubated with the PI3-kinase inhibitors LY-294002 and wortmannin for 5 min at 37°C before the addition of agonist.
Measurement of [Ca2+]i in acinar cell suspension.
Dispersed pancreatic acini were loaded at 37°C with 2 µM fura-2 AM for 30 min in solution Q. After washing, acini were resuspended 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 (15) or presented as the ratio of fluorescence intensities at the two excitation wavelengths [F(340/380)].
Measurement of [Ca2+]i in individual acinar cells.
Dispersed rat acini were loaded with 2 µM fura-2 AM for 30 min at room temperature in solution Q. After loading, acini were placed on a poly-L-lysine-coated coverslip and allowed to attach for 10 min. Where indicated, the PI3-kinase inhibitors were added into the solution. Coverslips were then mounted in a chamber (volume,
0.5 ml) and perfused continuously (
1 ml/min) with solution Q at 37°C. Fluorescence was recorded by using an inverted microscope (model TE 2000-S Nikon Eclipse) and a digital imaging system (MetaFluor; Universal Imaging, Downingtown, PA). Images were obtained every second, and the ratio values [F(340/380)] from small regions (
10 µm2) of each cell were stored for subsequent analysis.
Measurement of Ins(1,4,5)P3 levels.
Pancreatic acini were incubated with or without PI3-kinase inhibitors for 5 min and then stimulated with 100 nM CCK for 0, 5, 10, 30, 60, and 180 s. The reaction was stopped by addition of ice-cold 20% TCA. Samples were centrifuged for 5 min at 10,000 rpm, and the supernatant was washed by adding 2 ml of trioctylamine/1,1,2-trichloro-1,2,2-trifluoroethane mixture (1:3, vol/vol). After vigorous shaking, the supernatant containing the water-soluble Ins(1,4,5)P3 was collected and the amount of Ins(1,4,5)P3 was determined by a radioimmunoassay kit (Amersham Bioscience, Arlington Heights, IL).
Permeabilized acinar cells.
Permeabilization was done as we described previously (15). Rat acini were washed three times in buffer B containing (in mM) 100 KCl, 20 NaCl, 20 HEPES (pH 7.2), and 1 MgCl2, each time using different EGTA concentrations (0.1, 0.25, and 0.1 mM). After the third wash, acini were transferred to a stirred cuvette at 37°C and resuspended in Chelex-treated buffer C (buffer B + 3 mM ATP, 10 mM creatine phosphate, 10 U/ml creatine phosphokinase, and 1 µM oligomycin). Cells were then permeabilized with 10 µM digitonin.
Statistical analysis of data.
Statistical analysis was done by using two-tailed Student's t-test. P values <0.05 were considered statistically significant. The numbers for independent experiments (performed on different acinar cell preparations) and total traces recorded are given in figure legends.
Materials.
Thapsigargin, fura-2 AM, and fura-2 pentapotassium salt were purchased from Molecular Probes (Eugene, OR). Wortmannin was from Alexis Biochemicals (Carlsbad, CA) and Biomol (Butler Pike, PA). Ionomycin, LY-294002, and creatine phosphate were from Calbiochem (La Jolla, CA). CCK-8 was from American Peptide (Sunnyvale, CA). D-myo-inositol 1,4,5-trisphosphate[3H] assay kit was from Amersham Bioscience. D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], PIP3, creatine phosphokinase, oligomycin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), Chelex 100, 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB), dantrolene sodium salt, poly-L-lysine, PMSF, digitonin, and all other chemicals were from Sigma-Aldrich (St. Louis, MO).
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RESULTS
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Pharmacological PI3-kinase inhibition attenuates Ca2+ mobilization and influx induced by CCK and carbachol in pancreatic acinar cells.
[Ca2+]i responses to agonists in acinar cell suspension are characterized by an initial peak, representing Ca2+ mobilization from intracellular pools, followed by an elevated [Ca2+]i plateau, which is mediated by extracellular Ca2+ influx (45, 55). We have previously shown (20) that LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-
, inhibit both the initial peak and plateau of the [Ca2+]i response to 100 nM CCK demonstrating that [Ca2+]i responses in acinar cells are regulated by PI3-kinase.
To gain an insight into the mechanism(s) of this regulation, we first measured the effect of the PI3-kinase inhibitors on [Ca2+]i responses to various doses of CCK in isolated rat pancreatic acini. We used concentrations of LY-294002 and wortmannin that have been previously shown to inhibit PI3-kinase-mediated pathways in acinar cells (20, 46, 49, 55). The results in Fig. 1 show that both inhibitors significantly decreased the [Ca2+]i peak and plateau responses in acini stimulated with CCK in the whole concentration range of 10 pM to 100 nM. Inhibition of PI3-kinase also decreased the [Ca2+]i response to carbachol (Fig. 1D). These data indicate that PI3-kinase is involved in the regulation of [Ca2+]i signaling induced by the two major agonists of pancreatic secretion, CCK and acetylcholine. The inhibition of [Ca2+]i responses to carbachol (Fig. 1D) also indicates that the effects of LY-294002 and wortmannin are not mediated through (and are not specific for) the CCK receptor. The decrease of both [Ca2+]i peak and plateau by the PI3-kinase inhibitors indicates inhibition of both Ca2+ influx and mobilization induced by CCK or carbachol. Indeed, in a Ca2+-free medium, we observed similar decreases by the PI3-kinase inhibitors of the [Ca2+]i peak (i.e., Ca2+ mobilization) induced by CCK (Ref. 20 and the present study) and carbachol (data not shown). PI3-kinase inhibitors did not significantly change the basal [Ca2+]i level in acinar cells (Ref. 20 and the present study). Indeed, experiments in which fura-2-loaded acini were incubated for 30 min with vehicle, LY-294002, or wortmannin showed no effect of PI3-kinase inhibition on resting [Ca2+]i levels (data not shown).

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Fig. 1. Phosphatidylinositol 3-kinase (PI3-kinase) inhibitors decrease the peak and plateau of [Ca2+]i response to CCK and carbachol. Fura-2-loaded rat pancreatic acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (a), 100 µM LY-294002 (b), or 1 µM wortmannin (c), and then stimulated with CCK at 1 nM (A, B) or indicated concentrations (C), or with 100 µM carbachol (D). C: inhibitory effect of 100 µM LY-294002 and 1 µM wortmannin on the magnitude of the peak [Ca2+]i response to indicated concentrations of CCK. The [Ca2+]i response to CCK in the absence of the PI3-kinase inhibitors (i.e., in acini incubated with vehicle) was considered as 100%. Values represent means ± SE (n = 35) for the peak [Ca2+]i response and were calculated as (peak [Ca2+]i-basal [Ca2+]i)/basal [Ca2+]i (open bars, vehicle; black bars, LY-294002; gray bars, wortmannin). *P < 0.05 compared with the maximal [Ca2+]i response of acini stimulated with the same dose of CCK in the absence of PI3-kinase inhibitors (vehicle = 100%). [Ca2+]i was measured as described in MATERIALS AND METHODS.
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Both LY-294002 and wortmannin are specific inhibitors of PI3-kinase but also have other targets (13). In particular, LY-294002 inhibits casein kinase 2 (CK2) with a potency similar to that for PI3-kinase (13). Thus we tested the possible involvement of CK2 in the observed effects of LY-294002 on Ca2+ signaling in acinar cells. Incubation of acini with DRB, a specific inhibitor of CK2 (12, 14), had no effect on the CCK-induced [Ca2+]i response (Fig. 2A). Furthermore, the inhibitory effect of LY-294002 on the CCK-induced [Ca2+]i response was preserved in the presence of DRB (Fig. 2B). These results indicate that the observed effect of LY-294002 on Ca2+ signaling is due to PI3-kinase inhibition.

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Fig. 2. Effects of LY-294002 on CCK-induced [Ca2+]i response are not mediated by casein kinase 2. A: fura-2-loaded rat acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 90 min with either vehicle (a) or 60 µM casein kinase inhibitor 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB; b), and then stimulated with 100 nM CCK. B: fura-2-loaded rat acini were resuspended in solution Q containing 1 mM CaCl2 and incubated with DRB for 90 min. Acini were then transferred into a stirred cuvette, further incubated for 5 min with vehicle (c) or 100 µM LY-294002 (d), and stimulated with 100 nM CCK. Tracings are representative of 3 independent experiments on different preparations of acini. In this and subsequent figures, [Ca2+]i is represented by the ratio of fluorescence (F) intensities at 340 and 380 nm.
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To analyze the effects of PI3-kinase inhibition in more detail, we then measured [Ca2+]i responses in individual acinar cells using fluorescence microscopy. As illustrated in Fig. 3, 100 µM LY-294002 decreased both the magnitude and duration of the [Ca2+]i peak induced by 100 nM CCK, similar to what we observed in acinar cell suspension. LY-294002 also inhibited the [Ca2+]i response to 1 nM CCK in individual acinar cells (data not shown). Inhibition of [Ca2+]i responses to 1 nM and 100 nM CCK in individual acinar cells was also observed with 1 µM wortmannin (data not shown). Application of submaximal doses (<100 pM) of CCK results in [Ca2+]i oscillations, which cannot be seen with dispersed acini because of overlapping [Ca2+]i responses. Figure 4 shows that LY-294002 or wortmannin facilitated the decay of [Ca2+]i oscillations over time after stimulation of acinar cells with 50 pM CCK. This inhibitory effect on the amplitude of [Ca2+]i oscillations was manifest both in Ca2+-containing medium (Fig. 4) and in Ca2+-free medium (data not shown). Of note, we did not observe changes in the frequency of [Ca2+]i oscillations with the PI3-kinase inhibitors.

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Fig. 3. LY-294002 decreases the magnitude and duration (width) of the [Ca2+]i peak after CCK stimulation in individual pancreatic acinar cells. A: fura-2-loaded rat acinar cells were resuspended in solution Q containing 1 mM CaCl2, incubated for 10 min with vehicle (a; n = 47) or 100 µM LY-294002 (b; n = 38), and then stimulated with 1 nM CCK. Shown are representative traces for incubated cells. B: mean ± SE values for the magnitude of the peak [Ca2+]i response calculated as (F340/380 at the [Ca2+]i peak F340/380 at the basal [Ca2+]i) from 6 independent experiments. C: mean ± SE values for the duration of the [Ca2+]i peak (calculated as the duration of time when F340/380 was greater than half its maximal increase) from 6 independent experiments. *P < 0.01 (n = 38) compared with the maximal [Ca2+]i response in acinar cells stimulated with CCK in the absence of LY-294002.
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Fig. 4. PI3-kinase inhibitors facilitate the decay of [Ca2+]i oscillations induced by CCK in individual acinar cells. Fura-2-loaded rat acinar cells were resuspended in solution Q containing 1 mM CaCl2, incubated for 10 min with vehicle (A), 100 µM LY-294002 (B), or 1 µM wortmannin (C), and then stimulated with 50 pM CCK. Shown are representative traces for cells incubated with vehicle (n = 39), LY-294002 (n = 25), or wortmannin (n = 27). D: mean ± SE values for the decrease of [Ca2+]i peak maxima between the first and last peak within a time period of 600 s, as measured in 4 independent experiments. The [Ca2+]i level of the first peak in each trace was considered as 100%. *P < 0.01 compared with the decay of [Ca2+]i peak maxima in the absence of PI3-kinase inhibitors.
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LY-294002 neither decreases Ins(1,4,5)P3 production nor alters Ins(1,4,5)P3-induced Ca2+ release in pancreatic acinar cells.
The observed effect of PI3-kinase inhibitors on [Ca2+]i responses could be due to changes in Ins(1,4,5)P3 production. Therefore, we measured CCK-induced Ins(1,4,5)P3 production in the absence and presence of LY-294002. Figure 5A shows that PI3-kinase inhibition did not decrease Ins(1,4,5)P3 production after stimulation with 100 nM CCK. To the contrary, at the earliest time point measured after CCK stimulation (5 s), Ins(1,4,5)P3 production was significantly increased with LY-294002, compared with control acini.

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Fig. 5. LY-294002 does not decrease CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and does not alter Ins(1,4,5)P3-induced Ca2+ release in permeabilized pancreatic acinar cells. A: rat acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (square) or 100 µM LY-294002 (circle), and then stimulated with 100 nM CCK for indicated times. Values are means ± SE (n = 4) of Ins(1,4,5)P3 concentration per tube. *P < 0.05 compared with Ins(1,4,5)P3 production in the absence of LY-294002. BD: rat acini were resuspended in a permeabilization solution containing 10 µM digitonin and incubated for 5 min with vehicle (a) or 100 µM LY-294002 (b), and then Ins(1,4,5)P3 was added at the indicated concentrations. Traces are representative of 4 independent experiments. E: mean ± SE values (n = 35) for the peak [Ca2+] response to Ins(1,4,5)P3 in digitonin-permeabilized acini incubated with vehicle (square) or LY-294002 (circle). [Ca2+] was measured as described in the MATERIALS AND METHODS.
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To further determine whether the inhibitory effect of LY-294002 on [Ca2+]i responses is mediated through its effect on Ins(1,4,5)P3 receptors, we used permeabilized pancreatic acini stimulated with different concentrations of Ins(1,4,5)P3. PI3-kinase inhibition did not change the Ca2+ release induced by exogenous Ins(1,4,5)P3 (0.1 to 10 µM) in permeabilized acini (Fig. 5, BE). Thus the data in Fig. 5 indicate that the attenuation of [Ca2+]i responses by the PI3-kinase inhibitors is not through an inhibitory effect on Ins(1,4,5)P3 production or Ins(1,4,5)P3Rs in acinar cells.
Pharmacological inhibition of PI3-kinase in pancreatic acinar cells does not affect the plasma membrane Ca2+-ATPase.
Acinar cells remove Ca2+ from the cytosol through two main ATP-dependent pumps, i.e., PMCA and SERCA (2, 41). PMCA extrudes Ca2+ to the extracellular space, whereas SERCA is responsible for reloading Ca2+ into the ER pools (33). To test the involvement of PMCA, fura-2-loaded acini were resuspended in Ca2+-containing medium and stimulated with thapsigargin in the absence and presence of LY-294002 (Fig. 6). Specific and irreversible inhibition of SERCA by thapsigargin causes Ca2+ release into the cytosol and eventually empties the ER stores of Ca2+ (42). The depletion of Ca2+ stores by thapsigargin, in turn, stimulates Ca2+ influx through SOCs (42). Thus similar to hormones and neurotransmitters, thapsigargin causes both Ca2+ mobilization and influx; however, different from the agonists acting through receptors, thapsigargin neither induces Ins(1,4,5)P3 production nor acts through Ins(1,4,5)P3Rs or RyRs. LY-294002 did not affect the [Ca2+]i signal induced by thapsigargin in acinar cells (Fig. 6). The basal and thapsigargin-induced peak [Ca2+]i levels in the presence of 100 µM LY-294002 were 105 ± 6 and 220 ± 15 nM, respectively, compared with 109 ± 5 and 227 ± 18 nM in the absence of LY-294002 (n = 4). Thus pharmacological inhibition of PI3-kinase in acinar cells attenuates the [Ca2+]i responses to CCK and carbachol but not to thapsigargin. The lack of the effect of LY-294002 on thapsigargin-induced [Ca2+]i responses also suggests that PI3-kinase does not directly regulate SOCs in acinar cells.

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Fig. 6. Effects of LY-294002 on [Ca2+]i response are not mediated through the plasma membrane calcium ATPase (PMCA). Fura-2-loaded rat acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (a) or 100 µM LY-294002 (b), and then stimulated with 2 µM thapsigargin (TG). At the maximum of [Ca2+]i response, 2 mM EGTA was added to block Ca2+ influx. Tracings are representative of 3 independent experiments.
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At the maximum increase of [Ca2+]i after stimulation with thapsigargin, we further added EGTA to the acinar cell suspension to prevent Ca2+ influx (Fig. 6). Under these conditions (i.e., complete blockage of SERCA and inhibition of Ca2+ influx), only PMCA regulates [Ca2+]i by extrusion of Ca2+ to the extracellular space. Figure 6 shows that incubation with LY-294002 did not change the rate of [Ca2+]i decrease. This indicates that LY-294002 does not affect PMCA in pancreatic acinar cells.
We applied pharmacological analysis to test the involvement of RyRs and mitochondrial pathways of Ca2+ signaling in the observed effect of PI3-kinase inhibition. To block the RyR-mediated pathway, we used dantrolene, which has been shown to inhibit RyRs (32). Incubation of dispersed acini with 25 µM dantrolene did not change the inhibitory effect of LY-294002 on the CCK-induced [Ca2+]i responses (data not shown), suggesting that RyRs do not mediate this effect.
We then applied the mitochondrial inhibitors oligomycin and CCCP to examine whether mitochondria are involved in the regulation of CCK-induced [Ca2+]i responses by PI3-kinase. Neither oligomycin (2 µM) nor CCCP (2 µM) affected the [Ca2+]i response to 100 nM CCK, nor did they prevent the inhibitory effect of LY-294002 on CCK-induced [Ca2+]i responses (data not shown). Of note, our measurements showed that intracellular ATP level did not significantly decrease during this short incubation with oligomycin or CCCP (data not shown). Acini are incubated in a solution containing 10 mM glucose, and it has been shown in a variety of cell types that in the presence of glucose in the medium, the mitochondrial inhibitors only cause a slow depletion of intracellular ATP (26, 28).
PI3-kinase regulates SERCA during neurohormonal stimulation of pancreatic acinar cells.
The above-described results (Figs. 26) led us to hypothesize that the effect of the PI3-kinase inhibitors on Ca2+ signaling is due to their stimulatory effect on SERCA. To test this hypothesis, we performed the experiments described in Figs. 711. The objective of the experiment shown in Fig. 7 was to measure the effect of LY-294002 on CCK-induced [Ca2+]i responses in conditions in which SERCA is inhibited. We first stimulated dispersed rat pancreatic acini with thapsigargin followed in 10 s by stimulation with CCK. Such short incubation with thapsigargin inhibits SERCA but does not deplete intracellular Ca2+ pools. Indeed, previous studies (57) and our data shown in Fig. 6 demonstrate that to deplete Ca2+ pools with thapsigargin requires >1 min. Thus application of thapsigargin 10 s before CCK stimulation produced a slight but nonsignificant increase in both the magnitude and duration (width) of the CCK-induced [Ca2+]i peak, compared with cells stimulated with CCK alone (Fig. 7A). The [Ca2+]i response to CCK was inhibited by LY-294002. Pretreatment of acini with thapsigargin greatly diminished this inhibitory effect of LY-294002 on CCK-induced [Ca2+]i response in Ca2+-containing medium (Fig. 7, B compared with C) and completely prevented it in Ca2+-free medium (Fig. 7, D compared with E). Thapsigargin abrogated the inhibitory effect of LY-294002 on both the magnitude and duration of the CCK-induced [Ca2+]i peak (Fig. 7F). These data suggest that PI3-kinase inhibition stimulates SERCA, i.e., PI3-kinase regulates [Ca2+]i responses in pancreatic acinar cells through downregulating SERCA.

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Fig. 7. TG abolishes the effects of LY-294002 on CCK-induced [Ca2+]i response. Fura-2-loaded rat acini were resuspended in solution Q either containing 1 mM CaCl2 (AC) or with 1 mM EGTA and no added CaCl2 (DF). A: vehicle (a) or 2 µM TG (b) was added 10 s before stimulation of the acini with 100 nM CCK. BE: acini were incubated for 5 min with vehicle (a) or 100 µM LY-294002 (c), and then stimulated with either 100 nM CCK alone (B and D), or with 2 µM TG followed in 10 s by 100 nM CCK (C and E). Traces are representative of 3 independent experiments. F: mean ± SE values (n = 56) for the magnitude and duration of the [Ca2+]i peak after stimulation with either CCK alone or with TG and CCK, in the absence and presence of LY-294002. The magnitude of the [Ca2+]i peak was calculated as (peak [Ca2+]i basal [Ca2+]i)/basal [Ca2+]i. The duration of the [Ca2+]i peak (width) was measured at maximal [Ca2+]i response. *P < 0.01 compared with the maximal [Ca2+]i response to CCK in the absence of LY-294002 and TG.
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Fig. 11. Addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) delays Ca2+ reloading into the endoplasmic reticulum. Rat acini were resuspended in a permeabilization solution containing 10 µM digitonin, incubated for 1 min with vehicle (a), 20 µM PIP3 (b), or 40 µM PIP3 (c), and then stimulated with either 1 µM Ins(1,4,5)P3 alone (A) or with 1 µM Ins(1,4,5)P3 and 2 µM TG (C). B: mean ± SE values (n = 37) for the duration of the [Ca2+] peak after stimulation with Ins(1,4,5)P3 in the absence or presence of PIP3. The duration (width) of the [Ca2+] peak was measured at one half of the maximal [Ca2+] response. *P < 0.01 compared with the maximal [Ca2+] response to Ins(1,4,5)P3 in the absence of PIP3. IP3, Ins(1,4,5)P3.
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We previously reported (20) that genetic deletion of p110
, the catalytic subunit of PI3-kinase
(48), had an inhibitory effect on [Ca2+]i responses in pancreatic acinar cells similar to that of pharmacological PI3-kinase inhibition. Therefore, we asked whether thapsigargin abrogates the inhibition of CCK-induced [Ca2+]i response observed in acini isolated from PI3-kinase-
/ mice. In PI3-kinase-
/ acini stimulated with CCK alone in the Ca2+-free medium, [Ca2+]i rise was significantly less than in the wild-type PI3-kinase-
+/+ acini (Fig. 8A; also Ref. 20). Both the magnitude and width of the [Ca2+]i peak induced by CCK in PI3-kinase-
/ acini were 6570% (n = 8) of those observed in PI3-kinase-
+/+ acini. Thapsigargin almost completely abrogated this decrease in the [Ca2+]i signal; the [Ca2+]i peak induced by the combination of thapsigargin plus CCK did not differ significantly between the PI3-kinase-
+/+ and PI3-kinase-
/ acini (Fig. 8B). Thus inhibition of SERCA eliminates the effect of PI3-kinase-
on CCK-induced [Ca2+]i peak.
SERCA represents a major mechanism in regulating [Ca2+]i during neurohormonal stimulation (7, 27). If PI3-kinase downregulates SERCA, this would prevent Ca2+ reloading into the ER during neurohormonal stimulation and thus augment Ca2+ release from the ER in a manner similar to thapsigargin. In other words, inhibition of PI3-kinase would result in activation of SERCA and more Ca2+ accumulation in the ER during neurohormonal stimulation. To test whether this is the case, we performed the experiment shown in Fig. 9. Rat acini were stimulated with CCK. Forty seconds after addition of CCK, we added EGTA to block Ca2+ influx. Another 10 s later, ionomycin was added to release intracellular Ca2+. Figure 9A shows that there was little ionomycin-releasable Ca2+ in rat acini stimulated with CCK. However, preincubation with LY-294002 (Fig. 9B) or wortmannin (Fig. 9C) significantly increased the amount of releasable Ca2+ in rat acini stimulated with CCK. These data suggest that inhibition of PI3-kinase results in a greater amount of Ca2+ remaining in the ER during neurohormonal stimulation.

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Fig. 9. PI3-kinase inhibitors prevent depletion of intracellular Ca2+ pools. Fura-2-loaded rat acini were resuspended in solution Q containing 1 mM CaCl2, incubated for 5 min with vehicle (A), 100 µM LY-294002 (B), or 1 µM wortmannin (C), and stimulated with 100 nM CCK. At 40 s after CCK stimulation, 2 mM EGTA was added to block Ca2+ influx. Another 10 s later, 5 µM ionomycin was applied to release intracellular Ca2+. Traces are representative of 46 independent experiments.
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We performed similar experiments on acini isolated from PI3-kinase-
/ mice. Figure 10 shows that PI3-kinase-
/ acini have significantly more Ca2+ remaining in intracellular stores (i.e., releasable with ionomycin) than the wild-type, PI3-kinase-
-sufficient acini (Fig. 10, A and B). This augmentation of releasable Ca2+ in PI3-kinase-
/ acini was measured by both the maximal [Ca2+]i increase in response to ionomycin (Fig. 10C) and by the area under the [Ca2+]i curve, which reflects the overall amount of released [Ca2+]i (Fig. 10D).
The results described in Figs. 710 led us to conclude that the changes in [Ca2+]i signaling observed with PI3-kinase inhibition (both pharmacological and genetic) are due to faster Ca2+ reloading into the ER; i.e., Ca2+ reloading is downregulated by PI3-kinase in acinar cells. The experiment in Fig. 11 was designed to test whether the main product of PI3-kinase, PIP3, has an effect on Ca2+ reloading of the ER.
In general, the basal intracellular levels of PIP3 are very low but can rise sharply on neurohormonal stimulation, mediated by receptor-coupled class I PI3-kinase isoforms (52). We found that exogenous PIP3 dose-dependently delayed the decrease of the Ins(1,4,5)P3-induced [Ca2+] signal in permeabilized rat acini (Fig. 11, A and B) demonstrating inhibition of Ca2+ reloading into the intracellular pools. To prove that this effect of PIP3 was not through Ins(1,4,5)P3-induced Ca2+ release, we measured the effect of PIP3 on Ca2+ release induced by the combination of Ins(1,4,5)P3 and thapsigargin. As shown in Fig. 11C, in the presence of thapsigargin (i.e., with SERCA activity blocked) PIP3 did not affect Ins(1,4,5)P3-induced Ca2+ release. The results in Fig. 11 indicate that PIP3 delays SERCA-mediated Ca2+ reloading after Ca2+ release from the ER by Ins(1,4,5)P3.
As seen in Fig. 11A, we observed a small increase in [Ca2+] with the addition of PIP3, due to the fact that commercial preparations of PIP3 contain some calcium. Indeed, experiments with fura-2 acid in a cell-free solution confirmed that PIP3 was slightly contaminated with calcium (data not shown). Addition of 20 µM PIP3 increased [Ca2+] in the medium by 3050 nM and that of 40 µM PIP3 by 70100 nM. Of note, there was no subsequent change in [Ca2+] in response to PIP3 addition to permeabilized acini, indicating that PIP3 does not affect the resting activity of SERCA in unstimulated cells. As shown in Fig. 11A, PIP3 caused a delay of the Ins(1,4,5)P3-induced Ca2+ reloading at the initial Ca2+ concentrations of
100 nM (traces a and b) and
200 nM (trace c). To examine the effect of PIP3 at a higher [Ca2+], we increased the initial [Ca2+] in the medium to 500 nM; under these conditions, 40 µM PIP3 similarly delayed Ca2+ reuptake after Ca2+ release from the ER by Ins(1,4,5)P3 (data not shown).
The exact mechanism through which PIP3 inhibits SERCA-mediated Ca2+ reloading remains to be determined. No specific phosphoinositol lipid-binding domain has been described for SERCA. The observed delaying effect on SERCA-mediated Ca2+ reloading was rather specific for PIP3. We found that incubation of permeabilized acini with phosphatidylinositol 3-monophosphate or phosphatidylinositol 3,4-bisphosphate (at 20 and 40 µM) had no effect on Ins(1,4,5)P3-induced Ca2+ reuptake into the ER (data not shown).
It has been reported (29) that phosphatidylinositols compete with ATP for binding to the COOH terminus of ATP-sensitive K+ channel. To examine the possibility of a similar competitive interaction of PIP3 and ATP with SERCA, we repeated the experiment shown in Fig. 11 in the presence of 1 and 6 mM ATP in the medium (the results in Fig. 11 are with 3 mM ATP in the medium). In the presence of 1 mM ATP, the effect of PIP3 in delaying the Ca2+ reloading was similar to that shown in Fig. 11 (i.e., with ATP concentration of 3 mM). With 6 mM ATP in the medium, the release of Ca2+ by Ins(1,4,5)P3 in permeabilized acini was significantly reduced (data not shown), in agreement with the findings (5, 21) that ATP concentrations above 4 mM cause a progressive reduction in the probability of an open Ins(1,4,5)P3 channel and increase the rate of Ca2+-leakage from the ER. This effect complicates studying of the PIP3 and ATP competitive interactions with SERCA.
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DISCUSSION
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In the present study, we applied pharmacological and genetic inhibition of PI3-kinase to investigate the role of this key signaling molecule in Ca2+ responses to neurohormonal stimulation of the pancreatic acinar cell. The PI3-kinase inhibitors, LY-294002 and wortmannin, are relatively specific and act through different mechanisms (LY-294002 interacts with the ATP binding site, and wortmannin interacts with the catalytic center of PI3-kinase) (13, 52, 53). Thus an effect observed with both inhibitors is a good indication of PI3-kinase involvement. We used mice deficient in the catalytic PI3-kinase-
subunit, p110
(48), for genetic inactivation of PI3-kinase-
, the class IB PI3-kinase isoform mostly implicated in signaling from G protein-coupled receptors (52). The findings in the present and our previous study (20) show that inhibition of PI3-kinase by pharmacological and genetic means results in attenuation of both peak and plateau of the [Ca2+]i responses to CCK as well as carbachol. This indicates that PI3-kinase regulates both agonist-induced Ca2+ mobilization and Ca2+ influx. In the present study, we focused on the mechanism(s) of PI3-kinase regulation of Ca2+ mobilization in pancreatic acinar cells.
We analyzed several mechanisms through which PI3-kinase could possibly regulate Ca2+ signaling in acinar cells. First, we tested whether PI3-kinase inhibition affected Ins(1,4,5)P3 production or Ins(1,4,5)P3 receptors in acinar cells. LY-294002 did not inhibit the Ins(1,4,5)P3-induced Ca2+ release from ER stores (measured in permeabilized acini), which is evidence against the involvement of Ins(1,4,5)P3Rs. Nor did PI3-kinase inhibition decrease Ins(1,4,5)P3 production. To the contrary, we found that LY-294002 stimulated Ins(1,4,5)P3 production in CCK-treated cells. One explanation for this effect could be that PI3-kinase inhibition, by preventing phosphorylation of phosphatidylinositols at the 3' position, increases the availability of phosphatidylinositol 4,5-bisphosphate, the Ins(1,4,5)P3 precursor. Modulation of Ins(1,4,5)P3 production has been suggested as one mechanism of Ca2+ influx regulation by PI3-kinase in mast cells (50). However, in macrophages, wortmannin had no significant effect on Ins(1,4,5)P3 production (31). Our data, using pharmacological analysis, indicated also that RyRs and mitochondria are not involved in the observed effect of PI3-kinase inhibition on Ca2+ signaling in acinar cells.
We then asked whether PI3-kinase could regulate Ca2+ removal from the cytosol through either PMCA or SERCA. LY-294002 did not affect Ca2+ extrusion after stimulation of acini with thapsigargin (with the Ca2+ influx blocked by EGTA). These results argue against regulation of PMCA activity by PI3-kinase. On the other hand, the finding that LY-294002 and wortmannin significantly decreased the width of the [Ca2+]i peak induced by CCK (as well as carbachol) suggested a faster reloading of Ca2+ into the ER through activation of SERCA. Consistent with faster Ca2+ reloading, we observed that LY-294002 and wortmannin facilitated the decay of CCK-induced [Ca2+]i oscillations in individual acinar cells. Further evidence came from findings that incubation of acinar cells with thapsigargin abolished the inhibitory effects of LY-294002 or PI3-kinase-
genetic deletion on CCK-induced [Ca2+]i responses. These data led us to conclude that PI3-kinase inhibition upregulates SERCA activity during neurohormonal stimulation; in other words, PI3-kinase negatively regulates SERCA in acinar cells.
If PI3-kinase negatively regulates SERCA, the inhibition of PI3-kinase should facilitate Ca2+ reloading and result in a greater amount of Ca2+ in the ER stores during neurohormonal stimulation. Indeed, we found that incubation of acini with LY-294002 or wortmannin resulted in a greater amount of ER-stored (ionomycin-releasable) Ca2+ during CCK stimulation. Moreover, the genetic deletion of PI3-kinase-
had the same effect on ER-stored Ca2+ as the pharmacological PI3-kinase inhibitors. Such an effect of both pharmacological and genetic PI3-kinase inhibition can be explained by abrogation of the negative regulation of SERCA by PI3-kinase, thus allowing SERCA to reload Ca2+ more effectively and increasing the amount of Ca2+ releasable from ER stores. Of note, in permeabilized acini from SERCA2+/ mice, the rate of Ca2+ uptake was
50% slower than in acini isolated from wild-type mice (58).
Finally, we asked whether we could directly observe the reverse effect of attenuating Ca2+ reloading with PIP3, the main product of PI3-kinase. In permeabilized acini, PIP3 significantly inhibited Ca2+ reloading into the ER after Ins(1,4,5)P3 stimulation. No effect of PIP3 was observed in the presence of thapsigargin. These results indicate that PIP3 delayed SERCA-mediated Ca2+ reloading into the ER after Ca2+ release.
As discussed in the introduction, several mechanisms have been proposed for the involvement of PI3-kinase in Ca2+ signaling in other cell types, including activation of PLC
and increase of Ins(1,4,5)P3 production in mast cells (1, 11, 25), activation of store-mediated Ca2+ entry in platelets (47), and regulation of Ca2+ channels in vascular myocytes (30). Obviously, these mechanisms (in particular, the regulatory effect of PI3-kinase on PLC
) are cell-type specific. Our results on CCK-, carbachol-, and thapsigargin-induced Ca2+ signals indicate that in pancreatic acinar cells, PI3-kinase does not interact with the CCK receptor and does not regulate Ca2+ influx through SOCs. By Western blot analysis, we found that LY-294002 had no effect on tyrosine phosphorylation of PLC
in CCK-treated acini (data not shown).
Our results demonstrate the involvement of PI3-kinase in Ca2+ signals elicited by both CCK and carbachol, the two major agonists of digestive enzyme secretion by pancreatic acinar cells. Of note, we found that the regulatory effect of PI3-kinase on CCK-induced [Ca2+]i responses is observed in a wide range of CCK concentrations. At present, there is conflicting evidence on whether CCK (or carbachol) activates PI3-kinase in pancreatic acinar cells (10, 20, 46, 49, 55); alternatively, the role of PI3-kinase in Ca2+ signaling may be permissive in these cells.
In conclusion, our findings indicate that during neurohormonal stimulation, PI3-kinase regulates Ca2+ signals in pancreatic acinar cells through inhibition of SERCA (Fig. 12). Other major pathways regulating [Ca2+]i responses in pancreatic acinar cells, namely, Ins(1,4,5)P3 production, Ins(1,4,5)P3-induced Ca2+ release, and PMCA activity, are not inhibited by PI3-kinase. The functional significance of downregulation of SERCA by PI3-kinase remains to be determined. It may be important for augmenting Ca2+ signals, which is critical for exocytotic secretion (22, 38). Through its effect on Ca2+ signals PI3-kinase may also contribute to other Ca2+-dependent responses in pancreatic acinar cells (20, 49). It remains to be seen whether this novel regulatory mechanism of Ca2+ signaling, i.e., negative regulation of SERCA by PI3-kinase, operates in other cell types. It is also tempting to speculate on possible interrelationships between the two pathways involving the key mediators, Ins(1,4,5)P3 and PIP3, in the regulation of Ca2+ signals (Fig. 12).

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Fig. 12. Schematic depicting the inhibitory effect of PI3-kinase on sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in pancreatic acinar cells. PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PI3K, PI3-kinase; Ins(1,4,5)P3R, Ins(1,4,5)P3 receptor; ER, endoplasmic reticulum.
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GRANTS
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This study was supported by the Department of Veterans Affairs Merit Reviews (for S. J. Pandol and A. S. Gukovskaya) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-59936 and DK-59508. The study was also supported, in part, by the National Institute on Alcohol Abuse and Alcoholism, Research Center for Alcoholic Liver and Pancreatic Diseases Grant P50-AA-11999.
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ACKNOWLEDGMENTS
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The contribution of the Imaging and Morphology Core of the Center for Ulcer Research and Education: Digestive Diseases Research Center (to S. H. Young) is gratefully acknowledged.
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FOOTNOTES
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Address for reprint requests and other correspondence: A. 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.
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