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Am J Physiol Gastrointest Liver Physiol 292: G1594-G1599, 2007. First published March 1, 2007; doi:10.1152/ajpgi.00500.2006
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HORMONES AND SIGNALING

Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin

Sohail Z. Husain,1 Wayne M. Grant,1 Fred S. Gorelick,2 Michael H. Nathanson,2 and Ahsan U. Shah1

Departments of 1Pediatrics and 2Internal Medicine, Yale University School of Medicine and Veterans Affairs Connecticut Health Care, New Haven, Connecticut

Submitted 26 October 2006 ; accepted in final form 22 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Aberrant cytosolic Ca2+ flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca2+-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca2+ changes were recorded in acinar cells loaded with the intermediate Ca2+-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 µM FK506 or 10 µM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca2+ after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca2+ signal.

pancreatitis; protein phosphatase 2B; pancreatic acinar; calcium signaling

ACUTE PANCREATITIS is a necroinflammatory disease of the pancreas that has many inciting factors but few known mechanisms (41). An early, critical disease feature is the pathological activation of digestive proenzymes of proteases (or zymogens) within the pancreatic acinar cell (30, 32). Evidence supporting this conclusion includes observations that 1) the morphological changes of severe pancreatitis resemble those that are typical of digestive necrosis (38); 2) pancreatic and serum levels of activated zymogens increase early in the disease course, before evidence of acinar cell injury (10); 3) pretreatment with serine protease inhibitors blocks both trypsin activation and pancreatitis (27, 32), which has been used successfully in clinical trials to reduce post-endoscopic retrograde cholangiopancreatography-induced pancreatitis (3); and 4) hereditary pancreatitis is the result of genetic mutations in the cationic trypsinogen gene that may lead to enhanced trypsin activation (44).

Pathological intra-acinar zymogen activation is associated with an aberrant rise in acinar cell intracellular (cytosolic) Ca2+ (Cai2+). When this Cai2+ rise is prevented by either chelation of Cai2+ or by depleting Cai2+ stores, CCK-induced trypsinogen activation is blocked (21, 30, 32). Furthermore, acute pancreatic duct obstruction, a potential cause of acute pancreatitis, changes the Cai2+ response of the acinar cell to a signaling pattern that has been associated with intra-acinar zymogen activation (25).

While aberrant Ca2+ signaling has been recognized as central to the pathogenesis of acute pancreatitis and zymogen activation, its downstream effectors have not been identified. An important target of Ca2+ is Ca2+/calmodulin-dependent serine/threonine phosphatase 2B (protein phosphatase 2B or calcineurin). In the exocrine pancreas, the Ca2+-calcineurin pathway is required for acinar cell growth (33, 39) and may have a modest effect on enzyme secretion (5, 11, 43). However, the role of calcineurin in early pancreatitis events is not known. Thus, in this study, we examined whether calcineurin activation mediates pathological pancreatic zymogen activation.


   METHODS
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 METHODS
RESULTS
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Preparation of pancreatic acini. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Groups of pancreatic acinar cells, known as acini, were isolated as previously described with minor modifications (17). Briefly, fasted male Sprague-Dawley rats weighing 50–100 g (Charles River Laboratory, Wilmington, MA) were euthanized using a protocol approved by the Animal Care and Use Committee. Pancreatic tissue from young rats weighing 50–100 g was used because it resulted in more homogeneous acini preparations than tissue from older rats. This might be explained by the fact that the tissue of older rats contains more intrapancreatic fat. Nevertheless, separate experiments performed with cells from older rats weighing >150 g yielded similar data trends. The pancreas was removed and minced for 5 min in buffer containing 40 mM Tris (pH 7.4), 95 mM NaCl, 4.7 mM KCl, 0.6 mM MgCl2, 1.3 mM CaCl2, 1 mM NaH2PO4, 10 mM glucose, and 2 mM glutamine plus 1.0% BSA, 1x MEM nonessential amino acids (GIBCO-BRL), and 50 U/ml type IV collagenase (Worthington, Freehold, NJ). Tissue was incubated for 60 min at 37°C with shaking (120 rpm). The digest was then filtered through a 300-µm mesh (Sefar American, Depew, NY), washed several times in collagenase-free buffer, distributed evenly in 24-well Falcon tissue culture plates (Becton-Dickinson, Franklin Lakes, NJ), and incubated for 60 min at 37°C under constant O2 with shaking (80 rpm). This was followed by an exchange with fresh media. For each day of acinar cell isolation, a single animal was used. Such individual experiments were repeated on at least 3 days.

Zymogen activation and amylase secretion. Acini were pretreated with either 10 µM BAPTA-AM (Molecular Probes, Carlsbad, CA), FK506, 11R-calcineurin autoinhibitory peptide (CiP; Calbiochem, San Diego, CA), rapamycin, or okadaic acid (Calbiochem) at specified times. Acini were then stimulated with the CCK ortholog caerulein (100 nM) for 60 min. Protease activity assays were performed by using fluorogenic substrates as previously described (22). Briefly, chymotrypsin substrate (Calbiochem) was added to each homogenized sample, and the accumulation of fluorescence was measured over 10 min using a fluorescent plate reader (HTS 7000, Perkin-Elmer, Shelton, CT) at 380-nm excitation and 440-nm emission wavelengths. Amylase secretion was measured separately from the media and cell homogenates using a Phadebas kit (Amersham Pharmacia, Rochester, NY). Total amylase values were used for the normalization of chymotrypsin results, and the percent amylase secretion into the media was calculated as a measure of enzyme secretion.

Detection of cellular Ca2+ signals. Ca2+ signals were detected as previously described (17). Briefly, acini were incubated for 30 min with the medium-affinity Ca2+-sensing dye fluo-5F-AM (12 µM, KCa = 2.3 µM, Molecular Probes). This dye was used because Cai2+ signals in pancreatic acinar cells can saturate high-affinity dyes such as fluo-4 (KCa = 0.35 µM) (18). A Zeiss LSM510 confocal microscope was used with a x63 (1.4 numerical aperture) objective. The dye was excited at a wavelength of 488 nm, and emission signals of >515 nm were collected at a rate of 2 frames/s with full screen scanning. Fluorescence from individual acinar cells as well as apical and basolateral subcellular regions were recorded. To obviate temperature-dependent pH changes associated with the use of a Tris buffer, in limited experiments in which small temperature changes could not be regulated such as during Ca2+ imaging, 20 mM HEPES was substituted for Tris. In preliminary studies, caerulein-dependent secretion and zymogen activation were the same in Tris and HEPES buffers.

Statistics. Data represent means ± SD or SE, where appropriate, of at least three individual experiments, with each experiment performed in at least duplicate. Statistical significance was determined by a Student's t-test.


   RESULTS
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DISCUSSION
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Chymotrypsin activation is dependent on a rise in Cai2+. Physiological concentrations of the CCK ortholog caerulein (100 pM) induce enzyme secretion. By contrast, a supraphysiological concentration of caerulein (100 nM) induces pathological intra-acinar zymogen activation and pancreatitis (16). Pathological zymogen activation requires a rise in Cai2+. When acinar cells are pretreated with the Cai2+ chelator BAPTA-AM, caerulein-induced trypsin activation is reduced but not eliminated (30, 32). A likely explanation for the lack of total reduction is that BAPTA-AM might be unable to buffer the high micromolar Ca2+ transient that is induced by supraphysiological CCK stimulation. Our study confirmed this finding by demonstrating that another zymogen, chymotrypsinogen, was also activated by caerulein (100 pM) and that this response was decreased with BAPTA (10 µM) by 75% (Fig. 1). Chymotrypsin rather than trypsin activity was used as the standard for zymogen activation because the generation of chymotrypsin activity in acini after supraphysiological stimulation for 60 min more closely follows first-order kinetics; it is also a more sensitive marker of zymogen activation than trypsin (22).


Figure 1
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  		Fig. 1. Caerulein-induced intra-acinar chymotrypsin activation requires a rise in intracellular (cytosolic) Ca2+ (Cai2+). Caerulein (100 nM) induced intra-acinar chymotrypsin activation in isolated pancreatic acinar cells. Pretreatment with the Cai2+ chelator BATPA-AM (10 µM) reduced activation. n = 3. *P < 0.005. Ctl, control.

 
Calcineurin inhibition reduces caerulein-induced chymotrypsin activation. To determine whether calcineurin plays a role in pathological zymogen activation, acinar cells were treated with the calcineurin inhibitor FK506 (Fig. 2). FK506 is an immunosuppressant that indirectly inhibits calcineurin by binding to FK506-binding protein (FKBP) (6). A 50% reduction in chymotrypsin activity was noted after 30 min of pretreatment of acinar cells with 1 µM FK506 (Fig. 2A). This is equivalent to the IC50 for calcineurin phosphatase inhibition by FK506 as shown in pancreatic acinar cells (11). By contrast, FK506 did not affect enzyme secretion (Fig. 2B). Because 10 µM FK506 induced maximal inhibition of chymotrypsin activity and did not affect the secretory response of the acinar cell, this concentration was used in subsequent experiments. The lack of a FK506 effect on secretion was further confirmed by using concentrations of caerulein in ranges that included subphysiological (10 pM) and physiological (100 pM) concentrations, which induced maximal enzyme secretion (100 pM; Fig. 3). This finding is consistent with results from other reports (11, 36) that suggested that FK506 has only a modest effect on pancreatic amylase secretion. The results suggest that FK506 selectively affects zymogen activation.


Figure 2
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  		Fig. 2. Calcineurin inhibition by FK506 reduces caerulein-induced zymogen activation but not enzyme secretion. A: FK506 caused a concentration-dependent decrease in chymotrypsin activity induced by caerulein (100 nM) (B) but did not affect amylase secretion. n = 3. *P < 0.05 with respect to the maximal response.

 

Figure 3
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  		Fig. 3. Amylase secretion induced by a range of caerulein concentrations is not affected by FK506 (10 µM). n = 3–4.

 
Although FK506 is a potent inhibitor of calcineurin, it was shown in nonacinar cells to have calcineurin-independent effects, for example, as an antioxidant or an inducer of heat shock proteins (20). To confirm the specificity of FK506, cell-permeable CiP was used. It was made cell permeable by covalently attaching a poly-arginine-based protein transduction domain (11R) (40). Once inside the cell, CiP inhibits calcineurin by a different mechanism than FK506; it mimics the endogenous autoinhibitory domain of the calcineurin A subunit and blocks its catalytic activity (28). This provides an independent and complementary method for examining the role of calcineurin activity in the acinar cell. As observed with FK506, pretreatment (2 h) of acinar cells with CiP reduced intra-acinar chymotrypsin activation but did not affect enzyme secretion (Fig. 4, A and B). Notably, 50% inhibition of chymotrypsin was noted using 10 µM CiP, which approximates its IC50 (8 µM) in neuronal cells (40). Together, the results using inhibitors FK506 and CiP indicate a role for calcineurin in intra-acinar zymogen activation.


Figure 4
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  		Fig. 4. Calcineurin inhibitory peptide (CiP) reduces caerulein-induced zymogen activation but not enzyme secretion. A: CiP caused a concentration-dependent decrease in chymotrypsin activity induced by caerulein (100 nM; B) but did not affect amylase secretion. n = 3. *P < 0.05 with respect to the maximal response.

 
Calcineurin does not affect the initial Ca2+ signal seen with caerulein stimulation. To determine whether calcineurin plays a role in shaping the Ca2+ signal in acinar cells generated by supraphysiological secretagogue stimulation, isolated acini were loaded with the intermediate affinity Ca2+ dye fluo-5F and Cai2+ was imaged using confocal microscopy. Consistent with a report for carbachol stimulation (17), supraphysiological caerulein (100 nM) induced greater basolateral than apical Ca2+ release (Fig. 5D). However, 30 min of FK506 pretreatment did not affect the intensity of the Cai2+ rise from individual acinar cells or basolateral or apical subregions (Fig. 5E). These results suggest that calcineurin does not play a major role in the upstream regulation of the Ca2+ transient associated with premature zymogen activation. Thus, calcineurin appears to primarily act as a downstream effector of Cai2+ flux (31).


Figure 5
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  		Fig. 5. FK506 does not affect caerulein-induced Cai2+ signals. A: representative phase-contrast images. White box, apical; black box, basolateral. B: resting Ca2+ fluorescence after cells were loaded with fluo-5F-AM; the images were pseudocolored using the Ca2+ scale. C: peak Ca2+. D: representative tracing showing that caerulein (100 nM) induced a higher basolateral than apical Ca2+ rise. E: no differences were observed with FK506 (10 µM) pretreatment. F and G: quantification of peak Ca2+ responses from whole cells (F) and the basolateral/apical regions (G). n = 21–26 cells from each condition.

 
FKBP inhibition by rapamycin or PP1/PP2A inhibition by okadaic acid does not affect chymotrypsin activation. To further determine whether FK506 had a nonselective effect on FKBP isoforms that do not interact with calcineurin, we used rapamycin, a compound that binds FKBP but does not affect calcineurin activation (37). Pretreatment of acinar cells with rapamycin (10 µM) did not affect caerulein-induced chymotrypsin activation or amylase secretion (Table 1). Next, to investigate whether other phosphatases might affect acinar cell responses, the effects of inhibiting the non-Ca2+-dependent serine/threonine phosphatases PP1 and PP2A using okadaic acid were examined (36). In acinar cells, 10 nM okadaic acid has been used to inhibit PP1 and PP2A activity without affecting calcineurin (PP2B) (35). Okadaic acid (10 nM) pretreatment did not inhibit caerulein-induced chymotrypsin activation or amylase secretion but tended to slightly increase zymogen activity (Table 1). Together, the results with rapamycin suggest that the effects of FK506 are specific for calcineurin and those with okadaic acid suggest that calcineurin, but not PP1 or PP2A, mediates zymogen activation but has little effect on secretion.


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  		Table 1. FKBP inhibition by rapamycin or PP1/PP2A inhibition by okadaic acid does not affect caerulain-induced chymotrypsin activation or amylase secretion

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our study shows that calcineurin is an important target of the aberrant Cai2+ rise that causes pathological pancreatic zymogen activation. A rise in Cai2+ in the acinar cell has been linked to several physiological and pathological functions (45). The various effects of Cai2+ are thought to result from distinct spatial and temporal patterns of Ca2+ signals (29). For example, enzyme and fluid secretion from the acinar cell have been linked to Ca2+ spikes from a restricted region of the apical pole where zymogen granules are localized (18). In contrast, a global Ca2+ wave followed by intense basolateral Cai2+ elevations has been associated with stimuli that cause premature zymogen activation and pancreatitis (17). While acinar cell Ca2+ flux has been linked to both normal physiology and disease and much is known about the mechanisms that determine the intensity and duration of the Ca2+ signal, the downstream effectors of the Ca2+ signal remain unclear.

We examined whether the Ca2+-activated phosphatase calcineurin was such a target because it is a major participant in many cellular functions, including the development, trafficking, and regulation of Ca2+ homeostasis(9). In the pancreas, the Ca2+-calcineurin pathway is required for acinar cell growth (33, 39). It has also been linked to disease pathology; for example, in the heart, the activation of calcineurin produces cardiac hypertrophy (15). There have been conflicting reports from both clinical and experiment studies about the role of calcineurin in pancreatitis (7, 19, 24, 26, 34). However, there is some evidence in acinar cells suggesting that NF-{kappa}B activation is dependent on calcineurin (14).

In this study, two calcineurin inhibitors that have different mechanisms of inhibition were found to reduce intra-acinar zymogen activation. FK506, a widely used immunosuppressant, when bound to FKBP, inhibits access of substrates to the catalytic site of calcineurin. CiP is a small peptide sequence that directly inhibits the catalytic site. Cyclosporine A (CsA) is another immunosuppressant and inhibitor of calcineurin, but it was not used because CsA, but not FK506, also affects mitochondrial function by inhibiting the mitochondrial permeability transition pore (8). Because this effect of mitochondria has also been shown to occur in the pancreatic acinar cell (14), we did not use CsA in our experiments.

In this study, calcineurin did not affect enzyme secretion. Previous studies that have reported retention of zymogen granules used chronic FK506 administration (5) or used CsA (11). Our study is consistent with a report (11) suggesting only a modest effect on secretion with acute FK506 use. In some systems, calcineurin is not only a downstream target of Cai2+ but may also provide a feedback mechanism for maintaining cellular Ca2+ homeostasis by regulating intracellular Ca2+ channels (2). We have also shown that calcineurin does not appear to affect the intensity and duration of the initial Ca2+ signal generated by supraphysiological caerulein. This is consistent with a study (5) in which a 2-wk course of FK506 administration to rats did not affect the Cai2+ increase in pooled acini.

Several downstream targets of calcineurin have been identified and are candidates for moderating the responses in zymogen activation that we have observed. Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) was first purified from the pancreatic acinar cell (12). It undergoes rapid sustained calcineurin-dependent dephosphorylation on at least three serine residues in response to Ca2+-mobilizing stimuli such as CCK. While the function of CRHSP-24 is unknown, its acinar cell distribution generally corresponds to regions of initial zymogen activation (12). Another potential target of calcineurin is PKA. A recent study (4) has suggested that PKA can augment pancreatic zymogen activation. Calcineurin dephosphorylates the RII regulatory subunit of PKA, but its effect on PKA activity is not known (1). Thus, it is possible that calcineurin activation mediates zymogen activation via PKA. Other downstream candidates include mitochondrial proteins, such as Bcl-2 family members, that may cause a proapoptotic response (42) and thereby regulate zymogen activation (13). We (23) have examined short-term zymogen activation responses (1-h treatment duration), but long-term responses may be modulated by calcineurin via its actions on transcription factors, such as nuclear factor of activated T cells, NF-{kappa}B, MEF2, or cAMP response element-binding protein (23). Further studies are required to define the calcineurin substrates that mediate pathological zymogen activation within the acinar cell.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants K08 DK-68116 and K12 HD-001401 (to S. Z. Husain), DK-54021 (to F. S. Grant), and DK-45710, TW-01451, and DK-34989 (to M. H. Nathanson); an American Gastroenterological Association/AstraZeneca award (to S. Z. Husain); a Summer undergraduate award (to A. U. Shah); a Veterans Administration merit grant (to F. S. Gorelick); and a grant-in-aid from the American Heart Association (to M. H. Nathanson).


   ACKNOWLEDGMENTS
 
We thank D. Gomes, S. Glaser, E. Thrower, T. Kolodecik, and C. Shugrue for technical advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Husain, Dept. of Pediatrics, 333 Cedar St., FMP408, PO Box 208064, New Haven, CT 06520 (e-mail: sohail.husain{at}yale.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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