Gastrointestinal Research Laboratory, Veterans Affairs Healthcare Connecticut, West Haven, Connecticut
Submitted 27 July 2004 ; accepted in final form 15 September 2004
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ABSTRACT |
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8-bromoadenosine 3',5'-cyclic monophosphate; pancreas; protease
In addition to G protein-coupled receptors linked to Ca2+ signaling, the pancreatic acinar cell has receptors that stimulate adenylate cyclase and cAMP generation. These include secretin, VIP, and pituitary adenylate cyclase-activating peptide (PACAP) receptors. Previous studies have shown that cAMP agonists enhance the effects of secretion elicited by physiological concentrations of CCK (2). Similarly, we have shown that cAMP agonists enhance caerulein-induced zymogen activation (11). These effects were observed with secretin, VIP, and PACAP. Bypassing receptor-mediated events with the cell-permeable cAMP analog, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) also enhances caerulein-induced zymogen activation. Preliminary clinical studies suggest that the cAMP agonist secretin can reduce the severity of pancreatitis. The present study examines the effects of cAMP agonists on carbachol-induced zymogen activation, enzyme secretion, and cellular injury.
Previous studies (5) have suggested that acute acinar cell injury requires two key pathological events: zymogen activation and the retention of active enzymes within the pancreatic acinar cell. We hypothesized that cAMP agonists might reduce acinar cell injury associated with zymogen activation by stimulating secretion of activated enzymes from the acinar cell. In testing this hypothesis, we first demonstrated that cAMP agonists sensitize the acinar cell to carbachol-induced zymogen activation. We also found that cAMP agonists enhance secretion after stimulation with physiological concentrations of carbachol. Surprisingly, the suppression of secretion observed with supraphysiological concentrations of carbachol was overcome when acinar cell cAMP was directly increased. Furthermore, acinar cell injury was reduced by increasing intracellular cAMP. The protective effect of cAMP was associated with enhanced secretion of activated enzymes from the acinar cell. These findings suggest that cAMP agonists may reduce acinar cell injury by enhancing the secretion of active enzymes.
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MATERIALS AND METHODS |
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Acinar experimental protocol. Tissue culture plates containing acini were incubated for 60 min at 37°C under constant O2 with shaking (80 rpm). After exchanging the medium, acini were incubated for an additional 60 min. At this time, acini were treated without changing medium. Treatments included carbachol (1 µM and 1 mM), atropine (1 µM), secretin (0.1-100 nM), 8-Br-cAMP (1 µM1 mM), and forskolin 10 µM. Acini were incubated for 60 min (unless otherwise noted) at which time samples (medium and cells) were collected, placed in 1.5-ml centrifuge tubes (USA Scientific, Waltham, MA) and centrifuged for 1 min at 30 g. Of the resulting cell-free supernatant, 50 µl was removed to a 0.5-ml microcentrifuge tube. All samples were stored at 80°C.
Enzymatic activity assays. Samples containing 450 µl of cells and medium were thawed on ice, homogenized in a conical 1-ml Eppendorf tube with a pestle, and centrifuged at 1,000 g for 1 min. From the resulting postnuclear supernatant, 100 µl was added to wells of a 24-well tissue culture plate containing 350 µl of trypsin assay buffer [50 mM Tris (pH 8.1), 150 mM NaCl, 1 mM CaCl2, 0.01% BSA]. Finally, 50 µl of 400 µM enzyme substrate (trypsin 3135; Peptides International, Louisville, KY and chymotrypsin; Calbiochem, San Diego, CA) diluted in trypsin assay buffer (40 µM final) was added to each well. The plate was read by using a fluorometric microtiter plate reader (model HTS 7000; Perkin-Elmer Analytical Instruments, Shelton, CT) using an 380-nm excitation wavelength and 440-nm emission for 20 reads over 10 min. The slope of the line, which represents enzyme activity of the homogenate, was normalized to total amylase activity and expressed as relative fluorescence units per second per microgram total amylase.
Amylase assay. Samples were thawed on ice. The cell-free supernatant was assayed for secreted amylase. The remaining sample (450 µl of cells + medium) was homogenized in 1.5-ml centrifuge tubes with a pestle and centrifuged at 1,000 g for 1 min. The resulting postnuclear supernatant was assayed for total amylase. Amylase activity was determined by using a commercial kit (Phaebadas kit; Pharmacia Diagnostic, Rochester, NY). Amylase secretion was calculated as the percent total release (medium/medium + cells).
Lactate dehydrogenase assay. The assay was performed by using the commercially available Cytotox 96 nonradioactive cytotoxicity assay kit according to the manufacturer's instructions (Promega, Madison, WI). In brief, acinar cells were treated with 1 µM or 1 mM carbachol alone or in the presence of either 8-Br-cAMP (100 µM), secretin (100 nM), or atropine (1 µM) for 2 h. Samples were collected and centrifuged at 30 g for 30 s. A 50-µl aliquot of medium was then removed to measure lactate dehydrogenase (LDH) release from the cells. A manufacturer-provided lysis reagent was then added to the remaining 450 µl of cells and medium to determine total LDH. Both cell and medium samples were assayed. The results are expressed as the percent LDH released into the medium.
Trypan blue retention. Acinar cells were treated with 1 µM or 1 mM carbachol alone or in the presence of either 8-Br-cAMP (100 µM) or secretin (100 nM) for 2 h. Samples were then allowed to sediment by gravity. The medium was removed, leaving a cell pellet. The cell pellet was treated with 100 µl of 0.4% trypan blue stain (GIBCO-BRL) for exactly 2 min. The samples were then washed with 1 ml of 1x PBS four times. Samples were frozen at 80°C. After thawing, 100 µl of 1% Triton X-100 was added to the cells, which were then sonicated. The samples were then read on a plate reader at a wavelength of 583. This result was normalized to total nucleic acid content, which was determined by reading the samples at a wavelength of 260. Results were then expressed as fold vs. control.
cAMP assay. cAMP content in acinar homogenates was determined by using a commercial ELISA assay (Amersham Biosciences, Piscataway, NJ). Acini were preincubated with 1 mM IBMX for 15 min to inhibit endogenous cAMP-phosphodiesterases. The cells were then treated with specified agents for 10 min. cAMP content was calculated by using a standard curve and normalized to total amylase content.
Chymotrypsin antibody production and purification.
Trypsin cleaves chymotrypsinogen between arginine 15 and isoleucine 16. The sequence for the first 11 amino acids of human chymotrypsin-B at the amino end of the cleavage site (1626) IVNGEDAVPGS was used to immunize rabbits. Serum was collected, and affinity was purified by using an affinity column with immobilized peptide. With the use of immunoblot analysis, the antibody recognizes a major band at 13 kDa that corresponds to chymotrypsin. Intensity of the band was determined to directly correlate with chymotrypsin activity.
Chymotrypsin immunoblot analysis. Because preliminary data demonstrated that our fluorogenic assays for trypsin and chymotrypsin could not reliably measure secreted enzymes, an immunoblot assay for active enzyme secretion was developed. After treatment, cells and medium (300 µl) were collected. A 50-µl aliquot of cells and medium was collected and assayed for total amylase to ensure equal cell number in each sample. To the remaining 250 µl of cells and medium, 100 mM EGTA and 500 mM benzamidine were added to a final concentration of 1 mM and 5 mM, respectively. Samples were allowed to sediment by gravity and all of the cell-free medium was removed. A sample of 150 µl of 1x Laemmli loading buffer was added to 150 µl cell-free medium. The remaining cell-free medium was discarded. A sample of 300 µl of 1x Laemmli loading buffer was added to the medium-free cell pellet. All samples were heated to 95°C for 5 min and then stored at 80°C. Western blot analysis was performed to detect the presence of precursor or active forms of chymotrypsinogen.
Briefly, samples were separated on 15% SDS-PAGE gels (Bio-Rad, Hercules, CA) and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were blocked for 1 h at room temperature with Blotto (PBS, 5% nonfat dry milk, 0.05% Tween-20). Membranes were then probed with primary antibody (1:500) in Blotto for 2 h at room temperature. Membranes were washed and then probed with horseradish peroxidase-labeled goat anti-rabbit IgG (Sigma, St. Louis, MO) for 1 h at room temperature. After treatment with the secondary antibody, membranes were washed in PBS, and autoradiography was performed by using a SuperSignal West Pico chemiluminescence kit (Pierce, Rockford, IL).
Statistical analysis. Data represent means ± SE of at least 3 individual experiments unless otherwise noted, with each experiment being performed in at least duplicate. A Student's t-test analysis was used to determine statistical significance and P values of <0.05 were assigned significance.
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RESULTS |
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When intracellular cAMP was directly increased by the membrane permeable analog of cAMP, 8-Br-cAMP, a different pattern of sensitization was observed. At both 1 µM and 1 mM doses of carbachol, 8-Br-cAMP caused a concentration-dependent, biphasic enhancement of both trypsin and chymotrypsin activation, peaking at 500 µM (Fig. 2, A and B). The addition of 500 µM 8-Br-cAMP increased trypsin and chymotrypsin activation by 2.9- and 5.8-fold, respectively, compared with cells treated with 1 µM carbachol alone. At 1 mM carbachol, 500 µM 8-Br-cAMP caused a 3.2-fold increase in trypsin activation and a 6.8-fold increase in chymotrypsin activation compared with cells treated with carbachol alone.
Note that the magnitude of zymogen activation caused by 8-Br-cAMP is greater than that caused by secretin. Figure 3 compares a near maximal dose of 8-Br-cAMP (100 µM) to the dose of secretin (100 nM) that had the largest effect on zymogen activation. At 1 µM carbachol, secretin caused a 1.8-fold increase in chymotrypsin activation, and 8-Br-cAMP caused a larger 4.4-fold increase in chymotrypsin activation compared with carbachol alone. Whereas 100 nM secretin caused a 1.4-fold increase in chymotrypsin activation compared with 1 mM carbachol, 8-Br-cAMP caused a 5.1-fold enhancement. Neither secretin (100 nM) nor 8-Br-cAMP (100 µM) increased chymotrypsin activation when added to cells not treated with carbachol. These studies demonstrate that cAMP agonists and analogs sensitize the acinar cell to the effects of carbachol on zymogen activation.
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When 8-Br-cAMP was added to cells treated with carbachol, a dose-dependent enhancement in amylase secretion, peaking at 1 mM 8-Br-cAMP was seen at both physiological and supraphysiological concentrations of carbachol (Fig. 4B). When added to 1 µM and 1 mM carbachol, 1 mM 8-Br-cAMP caused an enhancement in amylase secretion to 45 and 30%, respectively. Note that inhibition of secretion produced by supraphysiological carbachol was reversed by 8-Br-cAMP, which increased secretion above levels seen with physiological carbachol alone. The addition of 8-Br-cAMP to control cells had no effect on secretion (data not shown).
Because it is unclear whether activated enzymes will exhibit the same secretory responses as amylase, the release of chymotrypsin in acini and the medium was evaluated by using immunoblot assay (Fig. 5). Whereas supraphysiological carbachol alone caused an increase in the active enzyme form in the cell pellet compared with control, no chymotrypsin was detected in the medium of these samples. The addition of secretin to cells treated with supraphysiological carbachol did not affect these results. However, the addition of 8-Br-cAMP to cells treated with supraphysiological carbachol caused chymotrypsin to appear in the medium. Furthermore, the chymotrypsin in the cell pellet decreased to control levels. These data provide the first evidence that cAMP can overcome the inhibitory effects of carbachol on the secretion of activated enzymes from the acinar cell and suggest that it might protect against acinar cell injury.
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To verify that the effects of 8-Br-cAMP on injury were specific, cells were pretreated for 30 min with 10 µM forskolin, a potent stimulator of the catalytic subunit of adenylate cyclase, before the addition of supraphysiological carbachol (1 mM). Effects of forskolin on carbachol-induced activation, secretion, and LDH release were similar to those seen with 8-Br-cAMP. Compared with 1 mM carbachol alone, the addition of forskolin enhanced trypsin and chymotrypsin activation by approximately twofold, increased amylase by 33%, and decreased LDH release by 30% (P value for each comparison <0.05; data not shown). These studies confirm that the effects of 8-Br-cAMP on activation, secretion, and injury are specific to cAMP.
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DISCUSSION |
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Acinar cell secretion was evaluated in two ways in this study: 1) by measuring amylase secretion and 2) by using immunoblot analysis to detect the active form of chymotrypsinogen in the medium. The release of chymotrypsin into the medium was taken as surrogate for the release of other activated enzymes. Because the compartment(s) in which zymogens are converted to active enzymes remains unclear, the relationship between the secretion of amylase and active enzymes is also uncertain. Whereas two studies (6, 14) have suggested that zymogens become activated in a compartment that codistributes with lysosomal markers, another suggested activation occurs in zymogen granules (15). Whether this disparity results from differences in detection methods, the timing of analysis, or other factors awaits further study. In the present study, conditions that enhanced carbachol-induced amylase secretion also caused an increase in activated enzyme secretion. However, increases in activated enzyme secretion caused by 8-Br-cAMP appeared to be more prominent than the increases seen in amylase secretion. Thus it remains unclear whether secretion of amylase and activated enzymes is regulated by a common mechanism and/or is localized to a common compartment.
A novel and surprising result of this study is that 8-Br-cAMP was able to overcome the inhibition of amylase secretion and retention of active enzymes observed with supraphysiological carbachol. A dramatic decrease in enzyme secretion is observed early in the course of experimental pancreatitis and is thought to be required for acinar cell injury (5). The mechanism(s) responsible for the overall reduction in acinar cell secretion and retention of active enzymes observed with supraphysiological concentrations of CCK or carbachol is uncertain. Studies (3, 13a) have noted disruption of the apical acinar cell actin cytoskeleton early in the course of caerulein-induced pancreatitis or after supraphysiological caerulein-treatment in vitro. A study in permeabilized acinar cells concluded that the acinar cell has two actin networks that regulate secretion (12). Whereas one network may act as a regulated barrier to exocytosis, the other may maintain zymogen granules in a distribution required for exocytosis. It has been suggested that a redistribution of zymogen granules away from the apical membrane might account for the suppression of secretion observed with supraphysiological caerulein or carbachol. High doses of cAMP have been shown to partially reverse the effects of caerulein hyperstimulation on the actin cytoskeleton (21). Additional studies are needed to examine whether the effects of cAMP agonists on the apical actin cytoskeleton are responsible for the enhancement of secretion seen with cAMP in this study.
Another interesting finding of this study is the distinct effects of the two cAMP agonists, secretin and 8-Br-cAMP. Secretin and 8-Br-cAMP enhanced amylase secretion and reduced injury when combined with physiological concentrations of carbachol. However, with supraphysiological carbachol stimulation, directly increasing cellular cAMP with 8-Br-cAMP or forskolin was required to enhance secretion and reduce injury. Our data suggest that these differences are likely due to insufficient cAMP generation with secretin when it is combined with supraphysiological carbachol. The blunted production of cAMP under these conditions is likely the result of inhibition of adenylate cyclase by high dose carbachol (1). Because 8-Br-cAMP directly increases intracellular cAMP, it can have similar actions at both physiological and supraphysiological concentrations of carbachol.
The present study also further elucidates the interaction among zymogen activation, secretion, and injury. Our data indicate that conditions, like supraphysiological carbachol, which enhance zymogen activation and cause retention of active enzymes, lead to increased injury. Whereas it might be predicted that treatments that sensitize to zymogen activation may lead to increased cellular injury, this study confirms that these outcomes are not always generated in parallel. For example, we report that activation induced by carbachol is enhanced by both secretin and 8-Br-cAMP. However, under the same conditions, the addition of 8-Br-cAMP produces a decrease in injury. The dissociation between the effects of cAMP agonists on zymogen activation and injury may be related to enhanced secretion of activated enzymes.
The relationship among activation, secretion, and injury suggested by the present study supports previous results with another secretagogue, bombesin (5). When bombesin binds to the G protein-linked, gastrin-releasing peptide receptor, intracellular calcium and cAMP are increased (19). Like caerulein and carbachol, bombesin causes zymogen activation. However, unlike supraphysiological caerulein and carbachol, active enzymes are secreted from acinar cell after bombesin treatment. Bombesin does not cause cellular injury in vitro or pancreatitis in vivo. That bombesin causes zymogen activation without leading to injury or pancreatitis reinforces the hypothesis that retention of active enzymes is also necessary for damage to the acinar cell. The present study builds on these findings by demonstrating that the injurious effects of a treatment like carbachol, which enhances zymogen activation and causes the retention of active enzymes, may be prevented. This effect presumably occurs through the secretion of active enzymes.
The significance of our finding that physiological doses of carbachol also cause some injury is not clear. Our data confirm previous findings that physiological doses of secretagogue can produce a small level of zymogen activation (10). It is possible that this modest increase in zymogen activation or another event, like secretagogue-induced consumption of ATP, causes stress in our in vitro system that may or may not reflect the in vivo condition. That discrete populations of cells were observed to retain trypan blue after physiological stimulation suggests that a subset of isolated acini is more susceptible to secretagogue-induced injury. Notably, secretin and 8-Br-cAMP, which both enhance carbachol-induced amylase secretion, significantly reduce this injury.
Although we believe this study is the first to demonstrate a direct effect of secretin on acinar cell injury, secretin has been used to treat or prevent acute pancreatitis without an established rationale. In animal studies (7, 13, 16, 17, 22) involving rodents and dogs, there is no consensus about the ability of secretin to prevent or ameliorate pancreatitis. In humans, there has been interest in the ability of secretin to reduce the incidence and severity of post-ERCP-induced pancreatitis. In the few clinical studies (13, 22) evaluating secretin prophylaxis for ERCP-induced pancreatitis, results have been mixed. However, a preliminary randomized, double-blind, placebo-controlled trial suggested that secretin reduces the incidence of post-ERCP pancreatitis when given at the time of the procedure (9). That secretin has a protective effect in our studies when combined with physiological, but not supraphysiological carbachol, might have therapeutic implications. Furthermore, our results showing that secretin can have differing effects may help to explain the divergent results of these animal and human studies. Although the present study provides a rationale for the therapeutic use of secretin to prevent ERCP-induced pancreatitis, it also suggests that an agent with actions more similar to 8-Br-cAMP might be more effective.
In summary, we have shown that increasing intracellular cAMP augments both zymogen activation and secretion in carbachol-treated acinar cells. Notably, cAMP agonists enhance secretion of amylase and markers of activated enzymes. When secretion is increased, the cell injury associated with carbachol treatment is significantly reduced. These results suggest that cAMP-stimulated pathways may reduce cellular injury by causing the secretion of active enzymes from the pancreatic acinar cell. The findings support the hypothesis that zymogen activation and the retention of active enzymes are both required for acinar cell injury.
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GRANTS |
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FOOTNOTES |
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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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REFERENCES |
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