Secretin differentially sensitizes rat pancreatic acini to the effects of supramaximal stimulation with caerulein

G. Perides,1 A. Sharma,1 A. Gopal,1 X. Tao,1 K. Dwyer,1 B. Ligon,2 and M. L. Steer1

Departments of 1Surgery and 2Neuroscience, Tufts-New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts

Submitted 18 November 2004 ; accepted in final form 19 May 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Supramaximal stimulation of the rat pancreas with CCK, or its analog caerulein, triggers acute pancreatitis and a number of pancreatitis-associated acinar cell changes including intracellular activation of digestive enzyme zymogens and acinar cell injury. It is generally believed that some of these various acinar cell responses to supramaximal secretagogue stimulation are interrelated and interdependent. In a recent report, Lu et al. (8) showed that secretin, by causing generation of cAMP and activation of PKA, sensitizes acinar cells to secretagogue-induced zymogen activation, and, as a result, submaximally stimulating concentrations of caerulein can, in the presence of secretin, trigger intracellular zymogen activation. We found that secretin also sensitizes acinar cells to secretagogue-induced cell injury and to subapical F-actin redistribution but that it did not alter the caerulein concentration dependence of other pancreatitis-associated changes such as the induction of a peak plateau intracellular [Ca2+] rise, inhibition of secretion, activation of ERK1/2, and activation of NF-{kappa}B. The finding that secretin sensitizes acinar cells to both intracellular zymogen activation and cell injury is consistent with the concept that these two early events in pancreatitis are closely interrelated and, possibly, interdependent. On the other hand, the finding that, in the presence of secretin, caerulein can trigger subapical F-actin redistribution without inhibiting secretion challenges the concept that disruption of the subapical F-actin web is causally related to high-dose secretagogue-induced inhibition of secretion in pancreatic acinar cells.

cAMP; actin; trypsin; zymogen activation; cell injury; pancreatitis; extracellular signal-regulated kinase 1/2; nuclear factor-{kappa}B


THE MOST WIDELY UTILIZED and best-characterized experimental model of acute pancreatitis involves exposing rodents to concentrations of CCK, or its decapeptide analog caerulein, that exceed those that elicit a maximal rate of digestive enzyme secretion from the pancreas (7). In addition to eliciting pancreatic parenchymal injury and inflammation, which are the hallmarks of acute pancreatitis, this supramaximal secretagogue stimulation elicits a number of additional pancreatic responses including redistribution of acinar cell subapical F-actin to the basolateral region of the cell (10), inhibition of digestive enzyme secretion (14), intracellular activation of digestive enzyme zymogens (3), activation of stress-activated kinases such as ERK1/2 (2), and activation of proinflammatory transcription factors such as NF-{kappa}B (4). With the exception of inflammation, each of these responses, including cell injury, can also be elicited by exposing freshly isolated rodent pancreatic acini to supramaximally stimulating concentrations of caerulein under in vitro conditions (15, 16), and, under in vitro conditions, supramaximal stimulation with caerulein is also associated with the induction of a "peak plateau" pattern of increased cytoplasmic [Ca2+] (20).

Previous reports by several groups of investigators have suggested that some of these acinar cell responses to supramaximal secretagogue stimulation may be interrelated and interdependent. For example, the inhibition of digestive enzyme secretion is thought to be the consequence of the dissolution of the subapical F-actin web that is triggered by supramaximal secretagogue stimulation (10) and acinar cell injury is thought to be the consequence of intracellular digestive zymogen activation (13). In addition, each of these responses is thought to be closely related to the changed pattern of intracellular [Ca2+] ([Ca2+]i) rise that follows exposure to a supramaximally stimulating secretagogue concentration (12, 18).

Rodent pancreatic acinar cells display receptors for a variety of secretagogues including CCK and secretin (19). CCK receptor occupancy regulates digestive enzyme secretion via the phospholipase C/inositol trisphosphate/Ca2+/PKC signal transduction pathway. Low concentrations of CCK or caerulein elicit an oscillatory rise and fall in [Ca2+]i, whereas high concentrations elicit a peak plateau [Ca2+]i response. In contrast to CCK or caerulein, occupancy of secretin receptors regulates fluid and electrolyte secretion via activation of adenylate cyclase, generation of cAMP, and activation of PKA. In a recent report, Lu and co-workers (8) noted that secretin, by increasing rat acinar cell cAMP levels, sensitized those cells to caerulein-induced intracellular digestive zymogen activation, i.e., in the presence of secretin, maximally or submaximally stimulating concentrations of caerulein could trigger intracellular activation of trypsinogen and chymotrypsinogen, whereas, in the absence of secretin, higher, supramaximally stimulating caerulein concentrations were required to elicit intracellular zymogen activation.

We examined the possibility that secretin might selectively and differentially sensitize acinar cells to the other responses that are known to follow supramaximal stimulation with caerulein. In addition to confirming the observations reported by Lu et al. (8), we found that secretin also sensitizes acinar cells to caerulein-induced acinar cell injury/death and to caerulein-induced cytoskeletal changes, i.e., redistribution of subapical F-actin to the basolateral region of the cell. On the other hand, secretin stimulation does not sensitize acinar cells to caerulein-induced inhibition of digestive enzyme secretion, to induction of the peak plateau [Ca2+]i response, or to activation of either ERK1/2 or NF-{kappa}B. Our findings led us to conclude that 1) supramaximal secretagogue-induced subapical F-actin redistribution and inhibition of enzyme secretion are not interdependent events and 2) secretin can differentially sensitize acinar cells to some, but not all, of the effects of supramaximal secretagogue stimulation.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Materials. Collagenase was purchased from Worthington (Freehold, NJ). Caerulein was obtained from Bachem Bioscience (King of Prussia, PA). The fluorogenic substrates for trypsin (Boc-Gln-Ala-Arg-MCA) and chymotrypsin (Suc-Ala-Ala-Pro-Phe-MCA) were purchased from Peptides International (Louisville, KY). Phalloidin conjugated to Alexa Fluor 546, DNase I conjugated to Texas red, fura-2 AM, Pluronic F-127, and Live/Dead Reagent were purchased from Molecular Probes (Junction City, OR). Cell-Tak was from BD Pharmigen (San Diego, CA). Secretin, the PKA inhibitors N-(2-guanidinoethyl)-5-isoquinolinesulfonamdie (H89), (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo-(1,2,3-fg:3',2',2'-kl)pyrrolo-(3,4-1)(1,6)benzodiazocine-10-carboxylic acy hexyl ester (KT-5720), and myristoylated protein kinase inhibitor peptide (14–\?\22) (MPKI), the PKA activator dibutyryl-cAMP (db-cAMP), and the PKG activator dibutyryl-cGMP (db-cGMP) were obtained from BioMol Research Laboratories (Plymouth Meeting, PA). The amylase substrate 2-chloro-p-nitrophenyl-{alpha}-D-maltotrioside was obtained from Diagnostics Chemical (Oxford, CT). Antibodies to cAMP-reactive element binding protein (CREB), ERK1/2, and their phosphorylated forms were obtained from Cell Signaling (Beverly, MA). The NF-{kappa}B oligonucleotide consensus binding sequence was purchased from Promega (Madison, WI), and [{gamma}-32P]ATP was purchased from Perkin-Elmer (Boston, MA). All other chemicals were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO).

Preparation of acini. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Tufts-New England Medical Center. Male Sprague-Dawley rats (Charles River Laboratories) weighing 80–100 g were housed in temperature-controlled (20 ± 2°C) rooms with a 12:12-h light-dark cycle and given standard laboratory chow ad libitum. They were fasted overnight but given water ad libitum before each experiment. Pancreatic acini were freshly isolated by collagenase digestion as previously described (11) and used immediately for each experiment. Cell viability, as measured by trypan blue exclusion, was >95% before the start of each experiment. Unless otherwise stated, the acini were suspended in 15 mM HEPES-Ringer buffer (pH 7.4) containing 130 mM NaCl, 5 mM KCl, 10 mM glucose, 9 mM Na pyruvate, 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA and incubated at 37°C.

Zymogen activation. Activation of trypsinogen and chymotrypsinogen was quantitated by measuring trypsin and chymotrypsin activity in acinar homogenates. For this purpose, trypsin and chymotrypsin activity were fluorimetrically measured using their specific substrates Boc-Gln-Ala-Arg-MCA and Suc-Ala-Ala-Pro-Phe-MCA, respectively, as described previously by Lu et al. (8).

Quantitation of acinar cell injury/death. Acinar cell injury/death was quantitated using Live/Dead Reagent (Molecular Probes), to which viable cells are impermeant. This method of quantitating cell injury/death is based on the cytotoxicity assay reported earlier (6). With cell injury/death, the reagent gains entry to the cell interior and can react with membrane proteins within the cell. In our studies, the reagent was added to suspended acini 15 min before completion of the incubation, and excess reagent was subsequently removed by washing the cells twice with PBS. The cells were then lysed either by homogenization in Ringer buffer or by the addition of 0.1% SDS. The amount of Live/Dead Reagent that had permeated the cell was then quantitated by measuring fluorescence emission at 520 nm after excitation at 495 nm. To allow for pooling of data from separate experiments, results were normalized by comparison to absorption at 280 nm as suggested by the manufacturer.

Digestive enzyme secretion. Digestive enzyme secretion from acini was quantitated by measuring amylase activity in the sample of acini and amylase activity discharged into the incubation medium. Amylase activity was measured using chloro-p-nitrophenyl-{alpha}-D-maltotrioside as the substrate on a Cobas Fara autoanalyzer. Net stimulated amylase secretion was calculated as the percentage of total amylase content that was discharged into the incubation medium over 30 min at 37°C after subtraction of the basal secretion (i.e., secretion in the absence of a stimulus), which typically ranged from 3% to 5%.

Characterization of F-actin distribution. The distribution of F-actin within individual acinar cells was evaluated by confocal fluorescence microscopy of acini stained with Alexa-Fluor 546-conjugated phalloidin. Briefly, equal volumes of 4% freshly prepared paraformaldehyde in PBS were added to suspensions of acini, and the mixtures were incubated for 5 min at room temperature. The cells were then washed in PBS and permeabilized by incubation in 0.5% Triton X-100 in PBS for 15 min. Fixed acini were treated with 0.1% sodium borohydride in PBS for 10 min, washed in PBS, and then incubated with 1 U/ml Alexa-Fluor 546-phalloidin for 1 h at room temperature. The acini were then washed and viewed under a Leica confocal microscope by an observer unfamiliar with the sample identity. To quantitate F-actin distribution, the intensity of fluorescence was digitally measured at the apical and basolateral regions of cells. The ratio of apical to basolateral F-actin fluorescence intensity was then calculated.

In separate experiments, the ratio of F-actin to G-actin was determined. For this purpose, F-actin and G-actin contents were fluorimetrically determined using acini stained with phalloidin conjugated to Alexa-Fluor 546 and DNAse conjugated to Texas red, respectively.

Evaluation of effect of secretin on caerulein-induced changes in [Ca2+]i. Acini were loaded with fura-2 by incubation with 1 µM fura-2 AM in the presence of 0.005% Pluronic F-127 for 10 min at room temperature followed by extensive washing. They were then placed in a NUNC tissue culture chamber on a Nikon Eclipse TEU 2000 inverted microscope, and single cells within acinar cell clusters were arbitrarily selected for study. Interference filters on a rotational filter wheel allowed selection of alternate excitation wavelengths of 340 and 380 nm from a xenon light source. Images of fluorescence emission at 510 nm were collected and sent, via an intensified charge-coupled device, to a dedicated digital image analysis system (IPLAB 3.6 with ratio plus, Scanalytics; Fairfax, VA). Background subtraction was carried out independently at each of the excitation wavelengths. The ratios of fluorescence emission (510 nm) at the two excitation wavelengths (340 /380 nm) were determined and expressed as the ratio of 340- to 380-nm fluorescence. Two patterns of Ca2+ response were identified and defined as follows. An "oscillatory" response was defined as one characterized by intermittent increases in fluorescence emission, each followed by a rapid decline in fluorescence emission to the resting baseline. A peak plateau response was defined as one characterized by a single large increase in fluorescence emission that, in Ca2+-containing medium, was followed by a slow decline to a lower value that remained above the resting baseline. Under certain conditions, a mixed response pattern was observed in which the response appeared to consist of oscillations superimposed upon a peak plateau pattern.

Activation of CREB, ERK1/2, and NF-{kappa}B. Activation of the PKA-specific substrate CREB and the stress-activated kinase ERK1/2 were quantitated by immunoblot analysis using anti-CREB antibodies, anti-phospho-CREB antibodies, anti-ERK1/2 antibodies, and anti-phospho-ERK1/2 antibodies from Cell Signaling. Activation of NF-{kappa}B was quantitated by electrophoretic mobility shift assay using the NF-{kappa}B consensus sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3' as described previously (5). The resulting gels were dried and exposed to X-ray films. Autoradiograms were analyzed by densitometry using Kodak 1D image-analysis software (Eastman Kodak; Rochester, NY) to quantitate the extent of NF-{kappa}B activation.

Analysis of data. The results reported in this communication reflect the observations made in three or more independent experiments. Figures report mean values ± SE. The significance of changes was evaluated using Student's t-test when two groups were compared or ANOVA when more than two groups were compared. Differences were considered significant when P values were <0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effects of secretin on caerulein-induced intracellular activation of zymogens. Supramaximally stimulating concentrations of caerulein (i.e., 1–10 nM) but not submaximally stimulating caerulein concentrations (i.e., 0.01–0.1 nM) caused intra-acinar cell activation of both trypsinogen (Fig. 1A) and chymotypsinogen (not shown, see Ref. 8). In the presence of 100 nM secretin, the dose-response relationship for caerulein-induced zymogen activation was altered, and digestive enzyme zymogen activation was observed when the caerulein concentration was 0.1 nM (Fig. 1; see Ref. 8). In addition to shifting the dose dependence of caerulein-induced zymogen activation, secretin also increased the magnitude of caerulein-induced zymogen activation (Fig. 1A). The effect of secretin on caerulein-induced trypsinogen activation was reproduced when the cell-permeant form of cAMP (i.e., db-cAMP) but not db-cGMP was used in place of secretin, and the effect of secretin on caerulein-induced zymogen activation was inhibited when the cAMP-dependent PKA inhibitors H89, KT5720, or MPKI were present (Fig. 1B).



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Fig. 1. Effect of caerulein (Caer) and secretin (Secr) on trypsinogen activation. A: freshly prepared rat pancreatic acini were incubated with varying concentrations of caerulein in the absence (solid bars) or presence (open bars) of 100 nM secretin. After 30 min at 37°C, acini were homogenized, and trypsin activity was determined. Note that trypsin activity, in the presence of 100 nM secretin, is increased when the concentration of caerulein is 10–10 nM, whereas increased trypsin activity, in the absence of secretin, is only noted when the caerulein concentration is 10–9 M. Results shown are mean ± SE values from 3 independent experiments, each performed in duplicate. *P < 0.01 when trypsin activity in the presence of caerulein ± secretin is compared with that in the absence of caerulein or secretin. B: freshly prepared rat pancreatic acini were incubated with combinations of 0.1 nM caerulein, 100 nM secretin, 100 µM dibutryl (db)-cAMP, 100 µM db-cGMP, 10 µM H89, 10 µM KT5720, and 10 µM myrisolated protein kinase inhibitor (MPKI). After 30 min at 37°C, acini were homogenized, and trypsin activity was determined. Results shown are mean ± SE values from 3 independent experiments, each performed in duplicate.

 
Effects of secretin and caerulein on acinar cell injury/death. We utilized Live/Dead Reagent to quantitate the effects of secretin on the concentration dependence of caerulein-induced acinar cell injury/death. Although the results obtained roughly paralleled those noted in pilot studies using leakage of lactate dehydrogenase or permeability to trypan blue as indicators of cell injury/death, we noted a greater level of consistency between experiments and closer agreement between replicate samples using the Live/Dead Reagent method. As shown in Fig. 2, secretin concentrations of up to 100 nM did not elicit an increase in acinar cell injury/death. In contrast, caerulein elicited concentration-dependent acinar cell injury/death, and the threshold concentration of caerulein needed for the induction of injury/death in the absence of secretin was 1 nM. In the presence of 100 nM secretin, however, the dose dependence of caerulein-induced cell injury/death was altered, and the threshold concentration of caerulein needed for the induction of injury/death was reduced to 0.1 nM.



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Fig. 2. Effect of caerulein and secretin on cell injury. Acinar cells were incubated with varying concentrations of caerulein in the absence (solid bars) or presence (open bars) of 100 nM secretin. Fifteen minutes before the end of the experiment, Live/Dead Reagent was added, and cell injury/death was measured as described in the text. Results are expressed as the ratio of the sample value to the value noted for samples incubated without stimulants (0). Data shown reflect mean ± SE values obtained from 3 independent experiments, each performed in triplicate. *P < 0.01 compared with samples incubated in the absence of stimulants.

 
Effect of secretin and caerulein on acinar cell secretion of amylase. Both secretin and caerulein stimulated secretion of amylase from freshly isolated rat pancreatic acini (Fig. 3). The dose-response relationship for secretin-stimulated secretion was monophasic, and 100 nM secretin elicited a maximal rate of secretion (not shown). In contrast, the dose-response relationship for caerulein-stimulated secretion was biphasic, and 0.1 nM caerulein elicited a maximal rate of secretion (Fig. 3). Higher concentrations of caerulein led to inhibition of amylase secretion. Maximal secretin-stimulated secretion was smaller in magnitude than maximal caerulein-stimulated secretion but, as noted many years ago by Williams and colleagues (1), the combination of secretin with caerulein elicited additive secretion, i.e., secretion occured at a rate that roughly corresponded to the rate elicited by secretin combined with the rate achieved by the concentration of caerulein being used. As shown in Fig. 3, the dose-response relationship for secretion stimulated by the combination of secretin plus caerulein continued to be biphasic, and secretin failed to prevent the inhibition of secretion noted in the presence of high concentrations of caerulein. Furthermore, the presence of secretin did not alter the concentration dependence of caerulein-induced inhibition of secretion, and, as a result, inhibition of secretion was not observed when the concentration of caerulein was 0.1 nM or lower. Similar results were obtained when acinar cells were incubated with increasing amounts of caerulein in the presence of db-cAMP, i.e., 100 µM db-cAMP had an additive effect on caerulein-stimulated amylase secretion and did not alter either the biphasic character or the concentration dependence of caerulein-regulated amylase secretion (not shown).



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Fig. 3. Effect of caerulein and secretin on amylase secretion by acini. Acinar cells were incubated with varying concentrations of caerulein in the absence or presence of 100 nM secretin. After 30 min, net stimulated amylase secretion was determined as described in the text. Note that secretin increases the rate of amylase secretion at each caerulein concentration but does not change the biphasic response pattern or the concentration of caerulein that elicits the maximal rate of secretion. Results shown are mean ± SE values from 3 independent experiments, each performed in duplicate.

 
Effect of secretin on caerulein-induced subapical F-actin distribution. We found that the F-actin-to-G-actin ratio in acinar cells was not altered by exposure of cells to a supramaximally stimulating concentration of caerulein. That ratio was found to be 0.28 ± 0.01 in the presence of 0.1 nM caerulein and 0.23 ± 0.03 in the presence of 1.0 nM caerulein (P > 0.05). In contrast, we found that the distribution of F-actin changes when acini are exposed to supramaximally stimulating concentrations of caerulein. In unstimulated cells and in cells exposed to submaximally stimulating concentrations of caerulein (0.1 nM), most of the F-actin was located in the subapical region (Figs. 4A and 5). As shown previously by O'Konski et al. (10), supramaximal stimulation with caerulein (1.0 nM) for 30 min led to the loss of subapical F-actin and its redistribution to the basolateral region of the cell (Figs. 4C and 5), but lower concentrations of caerulein did not trigger F-actin redistribution (Fig. 4B). Secretin (100 nM) alone did not alter F-actin distribution, but secretin sensitized acinar cells to caerulein-induced F-actin redistribution, i.e., in the presence of secretin, 0.1 nM caerulein was sufficient to elicit F-actin redistribution from the subapical to basolateral region of the cell (Figs. 4, D–F, and 5). Similarly, db-cAMP (100 µM) alone did not lead to F-actin redistribution but sensitized the acinar cells to caerulein-induced F-actin redistribution (Fig. 4H). As a negative control, 100 µM cGMP was added, which did not alter the F-actin distribution nor have any effect in caerulein-induced F-actin redistribution (Fig. 4G). The F-actin redistribution could be inhibited with 10 µM MPKI (Fig. 4I) and PKA inhibitors H89 (10 µM) or KT5720 (10 µM) (not shown).



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Fig. 4. Effect of caerulein and secretin on F-actin distribution. Pancreatic acini were incubated with 0.01, 0.1, and 1 nM caerulein in the presence or absence of 100 nM secretin for 30 min (A–E) or 5 min (F). They were then fixed, stained for F-actin as described in the text, and visualized using a Leica confocal microscope. Note the redistribution of F-actin from the apical to basolateral region in acini treated, in the absence of secretin, with 1 nM, but not 0.1 nM, caerulein for 30 min. In the presence of secretin, F-actin redistribution is observed in acini exposed to 0.1 nM caerulein for 30 min, and that redistribution is also observed when acini are exposed to secretin plus 0.1 nM caerulein for 5 min (F). db-cAMP at 100 µM had a similar effect as secretin on caerulin-induced F-actin redistribution (H). This effect could not be seen with the addition of db-cGMP (G) and could be inhibited with the addition of 10 µM MPKI (I).

 


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Fig. 5. Quantitation of F-actin redistribution elicited by caerulein in the presence and absence of secretin. Freshly prepared rat acini were incubated with varying concentrations of caerulein in the absence (solid bars) or presence (open bars) of 100 nM secretin. Fixed acini were stained with phalloidin conjugated to Alexa-Fluor 546 and examined using a Leica confocal microscope. Staining intensity at the apical side of cells was digitized, quantitated, and compared with the staining of a similar area at the basal side. Note that a decline in the ratio of apical-to-basal F-actin staining occurs when the caerulein concentration is 10–10 M in the presence of secretin, whereas the decline in the ratio of apical-to-basal F-actin is only observed when the caerulein concentration is 10–9 M in the absence of secretin. Results shown are means ± SE values obtained from examining 10–14 acini in each treatment group from 2 independent experiments. *P < 0.01 when the staining ratio obtained for each group is compared with the ratio found in cells incubated without caerulein or secretin.

 
Temporal relationship between F-actin redistribution and inhibition of amylase secretion. The potentially causal relationship between secretagogue-induced F-actin redistribution and the inhibition of amylase secretion was evaluated in time-dependent experiments that tracked both of these responses. The redistribution of subapical F-actin induced by supramaximal stimulation with caerulein (1.0 nM) or by a combination of secretin with a submaximally stimulating concentration of caerulein (0.1 nM) was found to be complete within 5 min of adding the secretagogues to the acini suspension (Fig. 4G). Secretion experiments were then undertaken to examine the effects of F-actin redistribution on amylase secretion. As shown in Fig. 6, left and middle, the percentage of total amylase content that is released per minute, over each 2.5-min interval, in response to either secretin or to secretin plus a submaximally stimulating concentration of caerulein (0.1 nM) rose and declined at a similar rate for at least the first 12.5 min after cells were exposured to the secretagogues despite the fact that, under these conditions, subapical F-actin has been redistributed to the basolateral region of acinar cells (Fig. 4). Similarly, the cumulative secretion of amylase elicited by 0.1 nM caerulein plus secretin continued to exceed that elicited by either secretagogue alone over the initial 12.5 min after cells were exposured to the secretagogues (Fig. 6, right). Taken together, these observations indicate that subapical F-actin can be redistributed without altering the rate of amylase secretion.



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Fig. 6. Time-dependent effects of secretin on caerulein-induced amylase secretion. Freshly isolated acini were preincubated at 37°C for 5 min. Secretin and caerulein were added to the concentrations indicated, and aliquots of the suspending medium were taken for amylase measurement at 2.5-min intervals for 12.5 min. 0, Acinar cells incubated without stimulates; 0S, acinar cells incubated with only 100 µM secretin; 10, acinar cells incubated with 0.1 nM caerulin; 10S, acinar cells incubated with 0.1 nM caerulin and 100 µM secretin. At the completion of the experiment, total amylase content of the acini and suspending medium was measured, and amylase discharge at each time was calculated as a percentage of total content. Left and middle: rates of amylase secretion (i.e., %total content that was discharged/min) during each interval. This rate of secretion was calculated by subtracting the percentage of content discharged at the start of the interval from that discharged at the completion of the interval and then dividing that value by 2.5. Right: cumulative discharge of amylase (i.e., %total content) at each time. Results shown indicate mean ± SD values from 4 independent experiments, each performed in duplicate.

 
cAMP and PKA mediate secretin-induced sensitization of acini to caerulein-induced subapical F-actin redistribution. db-cAMP alone did not cause subapical F-actin redistribution to the basolateral region of the cells, but, in the presence of db-cAMP, a submaximally stimulating concentration of caerulein (0.1 nM) caused subapical F-actin redistribution (Fig. 4 and Table 1). As shown in Fig. 7, exposure of acinar cells to either 100 nM secretin or 100 µM db-cAMP led to phosphorylation of the PKA substrate CREB. Phosphorylation of CREB was prevented by inclusion of the PKA inhibitor H89 (10 µM). As shown in Table 1, the F-actin redistribution that occurs with submaximally stimulating concentration of caerulein (0.1 nM) in the presence of secretin or db-cAMP was prevented by inclusion of 10 µM of the PKA inhibitor H89. Similar results were obtained when the other PKA inhibitors (KT5720 and MPKI) were used (not shown).


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Table 1. Effect of PKA inhibitor H89 on F-actin redistribution

 


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Fig. 7. Effect of PKA inhibitor H89 on activation of cAMP-reactive element binding protein (CREB). Isolated rat pancreatic acini were incubated for 30 min at 37°C with 1 nM caerulein, 100 nM secretin, and 100 µM db-cAMP in the absence or presence of 10 µM of the PKA inhibitor H89. The cells were lysed, and proteins were subjected to immunoblot analysis with anti-phospho-CREB (p-CREB) monoclonal antibody.

 
Effect of secretin on caerulein-induced changes in [Ca2+]i. Secretin (100 nM) alone did not alter [Ca2+]i (Fig. 8). Concentrations of caerulein below 0.1 nM elicited an oscillatory rise and fall in [Ca2+]i, whereas concentrations of 1.0 nM or higher elicited a peak plateau response. An intermediary pattern of response, in which oscillations were superimposed upon a peak plateau response, was observed with 0.1 nM caerulein. Inclusion of secretin along with caerulein did not alter the pattern of response noted with any of the caerulein concentrations. Although small changes in oscillation amplitude and frequency were occasionally observed, the concentration of caerulein required for eliciting an intermediate or peak plateau calcium response were not altered by the inclusion of secretin (Fig. 8).



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Fig. 8. Effects of secretin on caerulein-induced calcium responses. Isolated rat pancreatic acini were incubated with varying concentrations of caerulein in the presence or absence of 100 nM secretin. Note that no significant differences in the intracellular [Ca2+] response were seen, regardless of the presence or absence of secretin in the incubation mixture.

 
Effect of caerulein and secretin on NF-{kappa}B and ERK1/2 activation. NF-{kappa}B activation was noted when acini were exposed to 0.1 nM caerulein, and 0.01 nM caerulein elicited activation of ERK1/2 (Fig. 9). Secretin (100 nM) did not activate either NF-{kappa}B or ERK1/2, and secretin did not alter the caerulein concentrations needed for the activation of either NF-{kappa}B or ERK1/2.



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Fig. 9. Effects of caerulein and secretin on NF-{kappa}B (left) and ERK1/2 (right) activation. Rat pancreatic acini were incubated for 30 min at 37°C with varying concentrations of caerulein in the absence (solid bars) or presence (open bars) of 100 nM secretin. Note that 10–10 and 10–11 M caerulein elicits activation of NF-{kappa}B and ERK1/2, respectively, regardless of the presence or absence of secretin in the incubation mixture. p-ERK1/2, phospho-ERK1/2. Results shown are mean ± SE values from 3 independent experiments. *P < 0.01 when NF-{kappa}B or ERK1/2 activation in the presence of caerulein ± secretin is compared with that in the absence of caerulein or secretin.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Supramaximal stimulation of the rat pancreas with the calcium-mobilizing secretagogue caerulein is associated with the development of acute pancreatitis. Many of the pancreatitis-associated acinar cell changes, including intracellular digestive zymogen activation, acinar cell injury, subapical F-actin redistribution, activation of stress-activated kinases, and activation of the proinflammatory transcription factor NF-{kappa}B, are also observed when freshly isolated rat acini are exposed to supramaximally stimulating concentrations of caerulein under in vitro conditions (15, 16). When studied under in vitro conditions, supramaximal stimulation of acini with caerulein has also been shown to inhibit digestive enzyme secretion and to elicit a peak plateau change in [Ca2+]i (14, 20).

It is generally believed that some of these various acinar cell responses to supramaximal secretagogue stimulation are interrelated and interdependent. For example, redistribution of subapical F-actin is believed to cause inhibition of digestive enzyme secretion (10) and inhibition of secretion and, combined with a pathological peak plateau rise in [Ca2+]i, is believed to cause intracellular activation of digestive enzyme zymogens. Intracellular activation of digestive enzyme zymogens is believed to cause acinar cell injury (13), and, in a recent report, Tando et al. (17) suggested that CCK-induced activation of trypsinogen within acinar cells caused induction of I{kappa}B kinase leading to activation of NF-{kappa}B.

Recently, Lu and co-workers (8) have shown that secretin, an acinar cell secretagogue that acts via cAMP and not via calcium mobilization, can sensitize acinar cells to caerulein-induced digestive zymogen activation. They found that secretin alters the concentration-response relationship between caerulein and zymogen activation such that, in the presence of secretin, low concentrations of caerulein, which would otherwise submaximally stimulate secretion, could trigger activation of trypsinogen and chymotrypsinogen. Furthermore, they noted that this sensitizing effect of secretin is mediated via cAMP.

The observations reported by Lu et al. (8) were confirmed in our preliminary studies, and, in the context of generally accepted concepts regarding the interrelatedness and interdependence of acinar cell events triggered by supramaximal secretagogue stimulation, we hypothesized that secretin would sensitize acinar cells to many, if not all, of the other so-called "pancreatitis-related" effects of supramaximal caerulein stimulation. Thus we expected that secretin would alter the concentration dependence of caerulein-induced changes such that lower concentrations of caerulein would cause acinar cell injury, trigger subapical F-actin redistribution, and activate both ERK1/2 and NF-{kappa}B. Reports by others (1, 9), however, suggested that secretin might not alter the concentration dependence of caerulein-induced effects on digestive enzyme secretion or changes in [Ca2+]i.

As noted in this communication, our expectations were largely but not completely met. Indeed, we found that secretin sensitizes acinar cells to caerulein-induced injury/death, and this observation is certainly compatible with the concept that intracellular zymogen activation and acinar cell injury/death are closely related events. On the other hand, the observation that secretin exerts these sensitizing effects without altering the concentration dependence of caerulein-induced changes in [Ca2+]i is not compatible with the concept that either zymogen activation or cell injury are critically dependent on a peak plateau change in [Ca2+]i. We (13) and others (9) have previously noted that a rise in [Ca2+]i is required but, by itself, insufficient to trigger either zymogen activation or cell injury. These findings combined with the currently reported observations would suggest that an oscillatory, rather than a peak plateau, rise in [Ca2+]i might fulfill this requirement.

We found that secretin sensitizes acinar cells to caerulein-induced redistribution of subapical F-actin to the basolateral region of the cell and that, like secretin sensitization of intracellular zymogen activation, the sensitizing effect of secretin on caerulein-induced cytoskeletal changes is 1) mediated via cAMP and 2) involves activation of PKA, i.e., it is a phenomenon that can be induced by exposure to a cell-permeant form of cAMP and it can be aborted by inhibitors of PKA. Taken together, these observations suggest that F-actin redistribution is closely related to both intracellular zymogen activation and acinar cell injury, but whether the cytoskeletal change is the result or cause of zymogen activation/cell injury cannot be determined from our studies. In any case, however, F-actin redistribution is clearly not dependent on a peak plateau change in [Ca2+]i.

Redistribution of subapical F-actin and inhibition of digestive enzyme secretion are observed when acini are exposed to 1.0 nM or higher concentrations of caerulein. This phenomenon has been generally interpreted as indicating that secretagogue-induced inhibition of secretion is, in fact, caused by secretagogue-induced disruption of the subapical F-actin web and redistribution of apical F-actin to the basolateral region of the cell (10). We were surprised, therefore, to find that, in experiments spanning 30 min, secretin could sensitize acinar cells to caerulein-induced F-actin redistribution without sensitizing those same cells to caerulein-induced inhibition of secretion. Although these observations might suggest that F-actin redistribution and inhibition of secretion may be independent events, they are also compatible with the conclusion that the secretin-induced sensitization of acinar cells to caerulein-induced F-actin redistribution occurs slowly and that, therefore, most of the secretion measured over 30 min is complete before F-actin redistribution. In that case, a causal relationship between F-actin redistribution and 30-min secretion of amylase might still be possible. To examine this possibility, time-dependent experiments evaluating the effects of secretin on caerulein-induced F-actin redistribution and amylase secretion over short intervals were performed. We found that secretin-induced sensitization of F-actin redistribution could be clearly observed within 5 min of exposing acini to the combination of secretin and a submaximally stimulating concentration of caerulein but that no inhibition of amylase secretion could be detected during the initial 12.5 min of incubation. Taken together, these findings clearly indicate that F-actin redistribution can occur without leading to a reduction in the rate of amylase secretion, and they cast considerable doubt on the widely held belief that subapical F-actin redistribution is the proximate cause of secretagogue-induced inhibition of secretion.

Finally, our study has that secretin does not sensitize acinar cells to caerulein-induced activation of either ERK1/2 or NF-{kappa}B. We found that caerulein-induced activation of both ERK1/2 and NF-{kappa}B was observed in the presence of caerulein concentrations that are below those required to elicit intracellular activation of trypsinogen and that the dose dependence of caerulein-induced NF-{kappa}B and ERK1/2 activation was not altered by the presence of secretin. Our observations, particularly those regarding caerulein-induced activation of NF-{kappa}B, are at variance with those recently reported by Tando et al. (17), which suggested that CCK induction of I{kappa}B kinase and activation of NF-{kappa}B are mediated by intracellular activation of trypsinogen. Although NF-{kappa}B and ERK1/2 activation in response to caerulein may play important roles in the evolution of secretagogue-induced pancreatitis, and they may ultimately regulate the severity of that pathological process, the intracellular events leading to ERK1/2 and NF-{kappa}B activation clearly differ from those that lead to zymogen activation, F-actin redistribution, and acinar cell injury.

In summary, we have shown that, in addition to sensitizing acinar cells to caerulein-induced intracellular zymogen activation, secretin, acting via cAMP and PKA, also sensitizes acinar cells to caerulein-induced F-actin redistribution and acinar cell injury but does not alter the concentration dependence of caerulein-induced inhibition of enzyme secretion, peak plateau change in [Ca2+]i levels, ERK1/2 activation, or NF-{kappa}B activation. These observations led us to the following conclusions: 1) secretagogue-induced zymogen activation, cell injury, and F-actin redistribution are closely related and potentially interdependent events, but they are not dependent on inhibition of secretion or on a peak plateau change in [Ca2+]i; 2) secretagogue-induced zymogen activation, cell injury, and F-actin redistribution can all occur in the absence of an inhibition of digestive enzyme secretion from acinar cells; and 3) redistribution of subapical F-actin to the basolateral region of the cell is not, by itself, sufficient to inhibit digestive enzyme secretion.


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This study was supported by National Institutes of Health Grants RO1-AM31396-20 (to M. L. Steer) and P30-DK-34928-20 (to the Center for Gastroenterology Research on Absorptive and Secretory Processes of Tufts-New England Medical Center).


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Perides, Dept. of Surgery, Tufts-New England Medical Center, 860 Washington St., Boston, MA 02111 (e-mail: gperides{at}tufts-nemc.org)

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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