Calcium dependence of proteinase-activated receptor 2 and cholecystokinin- mediated amylase secretion from pancreatic acini
Anupriya Sharma,1
Xiaohong Tao,1
Arun Gopal,1
Brooke Ligon,2
Michael L. Steer,1 and
George Perides1
1Department of Surgery, Tufts-New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts; and 2Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts
Submitted 30 July 2004
; accepted in final form 17 June 2005
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ABSTRACT
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Pancreatic acini secrete digestive enzymes in response to a variety of secretagogues including CCK and agonists acting via proteinase-activated receptor-2 (PAR2). We employed the CCK analog caerulein and the PAR2-activating peptide SLIGRL-NH2 to compare and contrast Ca2+ changes and amylase secretion triggered by CCK receptor and PAR2 stimulation. We found that secretion stimulated by both agonists is dependent on a rise in cytoplasmic Ca2+ concentration ([Ca2+]i) and that this rise in [Ca2+]i reflects both the release of Ca2+ from intracellular stores and accelerated Ca2+ influx. Both agonists, at low concentrations, elicit oscillatory [Ca2+]i changes, and both trigger a peak plateau [Ca2+]i change at high concentrations. Although the two agonists elicit similar rates of amylase secretion, the rise in [Ca2+]i elicited by caerulein is greater than that elicited by SLIGRL-NH2. In Ca2+-free medium, the rise in [Ca2+]i elicited by SLIGRL-NH2 is prevented by the prior addition of a supramaximally stimulating concentration of caerulein, but the reverse is not true; the rise elicited by caerulein is neither prevented nor reduced by prior addition of SLIGRL-NH2. Both the oscillatory and the peak plateau [Ca2+]i changes that follow PAR2 stimulation are prevented by the phospholipase C (PLC) inhibitor U73122
[GenBank]
, but U73122
[GenBank]
prevents only the oscillatory [Ca2+]i changes triggered by caerulein. We conclude that 1) both PAR2 and CCK stimulation trigger amylase secretion that is dependent on a rise in [Ca2+]i and that [Ca2+]i rise reflects release of calcium from intracellular stores as well as accelerated influx of extracellular calcium; 2) PLC mediates both the oscillatory and the peak plateau rise in [Ca2+]i elicited by PAR2 but only the oscillatory rise in [Ca2+]i elicited by CCK stimulation; and 3) the rate of amylase secretion elicited by agonists acting via different types of receptors may not correlate with the magnitude of the [Ca2+]i rise triggered by those different types of secretagogue.
caerulein; phospholipase C
THE PROTEINASE-ACTIVATED FAMILY of receptors (PAR) includes four distinct receptor types (PAR14), each of which is a seven-transmembrane G protein-coupled receptor that is activated by proteolytic cleavage near its extracellular NH2 terminus. Proteolytic cleavage of the receptor releases a tethered ligand, which is part of the receptor molecule itself. That tethered ligand contains an activating peptide sequence, which, following its release, is capable of intramolecular binding to the extracellular, the activating site of the receptor, thus triggering a cascade of signal transduction events and a cellular response that varies depending on both the receptor and the cell type. PAR1, -3, and -4 can be activated by thrombin, and thrombin is generally believed to be the physiological activator of those receptors in vivo (2, 4, 6, 7). PAR2, on the other hand, is not activated by thrombin, but it can be activated by trypsin and other trypsinlike serine proteinases including mast cell tryptase, coagulation factors VIIa and Xa, neutrophil proteinase 3, and membrane tethered serine proteinase-1 (2, 7). The physiologically relevant activator of PAR2 has not been determined, and it may differ among various cell types (4).
In addition to proteolytic cleavage, the PARs can also be directly activated by exposure to soluble synthetic peptides that mimic the receptor-activating sequence of their tethered ligands. Proteolytic cleavage of PAR2 occurs at SKGR-SLIGRL, and the activating peptide sequence of the released PAR2 tethered ligand is SLIGRL (6). Thus PAR2 responses can be experimentally induced using synthetic SLIGRL. On the other hand, the reverse peptide, LRGILS, is inactive (10).
In the gastrointestinal tract, PAR2 has been shown to regulate salivary gland secretion, gastrointestinal smooth muscle motility, intestinal ion transport, and gastric mucous secretion (7). In the pancreas, PAR2 has been found to regulate ion channel function in duct cells (10) and digestive enzyme secretion from acinar cells (8), but the mechanisms responsible for those pancreatic responses to PAR2 stimulation have not been determined. Given the PAR2 local concentrations of trypsin and its zymogen trypsinogen, however, it is likely that PAR2 plays an important role in regulating physiological and/or pathological responses in the exocrine pancreas and that trypsin may be the serine proteinase that regulates these events in vivo. Indeed, in recently completed studies (19), we have found that pharmacological PAR2 activation reduces the severity of secretagogue-induced pancreatitis and that pancreatitis is made worse by genetic deletion of PAR2. These observations have suggested that interventions that lead to PAR2 activation may be beneficial in the treatment or prevention of clinical acute pancreatitis.
In the current study, we report experiments that have employed mouse pancreatic acini to examine the mechanisms responsible for PAR2 regulation of digestive enzyme secretion. We have compared and contrasted PAR2-mediated secretion with secretion mediated via acinar cell CCK receptors. For the PAR2 studies, we have employed the PAR2-activating peptide SLIGRL-NH2, and, for the CCK receptor studies, we have used the CCK analog caerulein. The results of our studies suggest that PAR2-stimulated secretion, similar to CCK-stimulated secretion, is dependent on a rise in cytoplasmic Ca2+ concentration ([Ca2+]i) that reflects both the release of calcium from intracellular stores and an accelerated rate of calcium influx. Like CCK stimulation, PAR2 stimulation elicits an oscillatory change in [Ca2+]i at low agonist concentrations and a peak plateau change at high agonist concentrations, but, whereas only the CCK-stimulated oscillatory change is prevented by inhibition of phospholipase C (PLC), inhibition of PLC prevents both the oscillatory and the peak plateau changes elicited by PAR2 stimulation. Despite the fact that PAR2 and caerulein stimulation elicit comparable rates of amylase secretion from acini, the [Ca2+]i rise elicited by CCK stimulation is greater than that triggered by PAR2 stimulation.
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MATERIALS AND METHODS
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Reagents.
SLIGRL-NH2, TFLLR-NH2, LRGILS-NH2, and GYPGKF-NH2 were synthesized by the Tufts University Core Facility (Boston, MA). Caerulein, the decapeptide analog of the potent pancreatic secretagogue CCK, was obtained from Bachem Bioscience (King of Prussia, PA). Fura-2 AM, BAPTA AM, and Pluronic F-120 were from Molecular Probes (Eugene, OR). The amylase substrate 2-chloro-p-nitrophenyl-
-D-malto-trioside, was obtained from Diagnostics Chemical (Oxford, CT). U73122
[GenBank]
was purchased from BioMol (Plymouth Meeting, PA). All other reagents and chemicals, including collagenase (CLS-4) and thapsigargin, were purchased from Sigma-Aldrich (St. Louis, MO).
Animals.
All experiments were performed using C57BL6 male mice (2530 g) purchased from Charles River Laboratories (Wilmington, MA) or PAR2-deficient (PAR2/) mice bred from breeding pairs kindly provided by Dr. Patricia Andrade-Gordon (Johnson and Johnson Pharmaceutical Research and Development, Spring House, PA) and bred into a C57BL6 background (18). The animals were housed in temperature-controlled (23 ± 2°C) rooms with a 12:12-h light-dark cycle, fed standard laboratory chow, fasted overnight before each experiment, and given water ad libitum. All experiments conformed to protocols approved by the Institutional Animal Care and Use Committee of the Tufts-New England Medical Center.
Preparation of acini.
Dispersed mouse pancreatic acini were freshly prepared for each experiment by collagenase digestion as previously described (13). To reduce the size of acinar cell clusters being harvested and thus facilitate studies monitoring [Ca2+]i in single acinar cells of those clusters, the suspended acini were passed through a 40-µm Nitex mesh (Sefar American, Kansas City, MO) before being loaded with fura-2 AM. Acini were resuspended in HEPES-Ringer buffer, saturated with O2 by bubbling, containing (in mM) 10 HEPES (pH 7.4), 130 NaCl, 5 KCl, 11 glucose, 9 Na-pyruvate, 1 MgCl2, and 1 mM CaCl2, with 0.1% bovine serum albumin. Viability of acinar cells, as assessed by trypan blue exclusion, exceeded 95% before the start of each experiment. Most of the studies evaluating amylase secretion employed acini that were allowed to equilibrate for 5 min at 37°C before the addition of secretagogues. For studies examining the effects on secretion of chelating intracellular Ca2+, the acini were preincubated with 50 µM BAPTA-AM for 30 min at 37°C before the addition of secretagogues. For studies evaluating the effects of Ca2+-free medium on secretion, the freshly prepared acini were extensively washed with nominally Ca2+-free HEPES-Ringer buffer containing 2 mM EGTA and subsequently incubated in that buffer. For studies involving the sequential exposure of acini to two different agonists, the EGTA concentration was reduced to 0.1 mM to minimize cell injury.
Measurement of amylase secretion.
Freshly prepared acini were incubated for 30 min at 37°C in the presence or absence of secretagogues. Amylase released into the suspending medium and amylase retained within the acini were measured spectrophotometrically using the 2-chloro-p-nitrophenyl-
-D-malto-trioside as a substrate as previously described (13). "Net stimulated amylase secretion," elicited by exposure to secretagogues, was calculated by subtracting the percentage of total amylase content released in the absence of the secretagogue (i.e., basal secretion) from the percentage of total amylase content released in the presence of the secretagogue (i.e., stimulated secretion), and that net stimulated secretion was expressed as a percentage of total amylase content. Basal secretion was consistently 47% of total amylase content over the 30-min period of incubation.
Measurement of [Ca2+]i in single acinar cells.
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. At "time 0," samples were allowed to equilibrate, and, after
3 min, the stimulants (caerulein and/or SLIGRL-NH2) were added. 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, which, in Ca2+-containing medium, was followed by a slow decline to a lower value that remained above the resting baseline. In the absence of extracellular Ca2+, this decline was to the baseline and no plateau indicating that a persisting elevation of [Ca2+]i was observed. Under certain conditions, a mixed response pattern was observed in which some of the cells demonstrated an oscillatory response, whereas others manifested a peak plateau response. The fraction of cells manifesting each type of response was then calculated.
Analysis of data.
The results reported in this communication represent means ± SE values obtained from three or more independent experiments. For each experiment evaluating changes in [Ca2+]i, 1820 separate acinar cells were examined. In all figures, vertical bars denote SE values, and the absence of vertical bars indicates that the SE values are too small to depict. Statistical evaluation of data was accomplished by analysis of variance or Students t-test, and P < 0.05 was considered to denote significant differences.
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RESULTS
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Activation of acinar cell PAR2 accelerates amylase secretion.
As shown in Fig. 1A, the PAR2-activating peptide SLIGRL-NH2 causes concentration-dependent and monophasic secretion of amylase from acini prepared from wild type but not from PAR2/ mice. The reverse PAR2 peptide, LRGILS-NH2, however, does not stimulate amylase secretion even in the acini from wild-type mice. In contrast to SLIGRL-NH2, the CCK analog caerulein elicits biphasic secretion of amylase, i.e., low, submaximally stimulating concentrations of caerulein (
0.1 nM) stimulate secretion, whereas higher, supramaximally stimulating concentrations of caerulein (>0.1 nM) inhibit amylase secretion. This biphasic response to caerulein is noted when acini from either wild-type or PAR2/ mice are examined (Fig. 1B). The peak rate of amylase secretion stimulated by SLIGRL-NH2 is comparable with that elicited by a maximally stimulating concentration of caerulein (Fig. 1 and Table 1). Neither the PAR1- nor the PAR4-activating peptide elicit significant amylase secretion from mouse pancreatic acini (Table 1).

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Fig. 1. Amylase secretion in response to proteinase-activated receptor-2 (PAR2) and caerulein stimulation of acini from wild-type (PAR2+/+) and PAR2/ mice. Freshly prepared pancreatic acini isolated from wild-type or PAR2/ mice were incubated with increasing concentrations of SLIGRL-NH2 or LRGILS-NH2 (01 mM; A) or caerulein (0100 nM; B). Net stimulated amylase secretion was measured and presented as a percentage of total amylase as described in the text.
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PAR2 and caerulein stimulation cause similar patterns of [Ca2+]i change.
Low concentrations of either SLIGRL-NH2 or caerulein elicit an oscillatory change in [Ca2+]i (Fig. 2), although the oscillations elicited by SLIGRL-NH2 are more frequent than those elicited by caerulein. With increasing concentrations of either SLIGRL-NH2 or caerulein, a transition is noted, and progressively more cells demonstrate a peak plateau response, whereas fewer cells show an oscillatory response. The transition between an oscillatory response and the peak plateau response is noted to occur in the presence of a caerulein concentration (0.11 nM) that elicits a maximal rate of digestive enzyme secretion (compare Figs. 1B and 2F). In contrast, the transition between an oscillatory and the peak plateau response to SLIGRL-NH2 occurs at a concentration (300 µM) that elicits a submaximal rate of enzyme secretion (compare Figs. 1A and 2E).

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Fig. 2. Effect of SLIGRL-NH2 and caerulein on acinar cell cytoplasmic Ca2+ concentration ([Ca2+]i). Freshly prepared pancreatic acini were loaded with fura-2 and stimulated with varying concentrations of either SLIGRL-NH2 or caerulein while changes in [Ca2+]i within single acinar cells were monitored as described in the text. Representative tracings obtained using low and high concentrations of the secretagogues are shown in A-D as follows: 30 µM SLIGRL-NH2 (A); 0.01 nM caerulein (B); 1 mM SLIGRL-NH2 (C); and 1 nM caerulein (D). Arrows indicate time of addition of the agonist. E and F: fraction of monitored cells that responded with an oscillatory or a peak plateau [Ca2+]i change in response to SLIGRL-NH2 (E) or caerulein (F).
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The reverse PAR2 peptide LRGILS-NH2 does not elicit a change in acinar cell [Ca2+]i (Table 2). As had been noted previously for amylase secretion (Table 1), neither the activating peptide for PAR1 nor the activating peptide for PAR4 elicit a rise in [Ca2+]i in mouse pancreatic acini (Table 2).
The absence of extracellular Ca2+ reduces PAR2- and caerulein-stimulated amylase secretion as well as [Ca2+]i changes elicited by both PAR2 and caerulein stimulation.
As shown in Fig. 3, amylase secretion elicited by exposure to either SLIGRL-NH2 or caerulein is markedly reduced in the presence of a nominally Ca2+-free medium that is supplemented with 2 mM EGTA. Similarly, both SLIGRL-NH2- and caerulein-induced changes in [Ca2+]i are altered under these conditions (Fig. 4). With submaximally stimulating concentrations of SLIGRL-NH2 or caerulein, the oscillations are less frequent and, with time, they disappear. With supramaximally stimulating concentrations, the peak plateau response is altered, and, after an initial peak, [Ca2+]i values return to the baseline value rather than remaining at an elevated plateau level. As shown in Fig. 4, I and J, preincubation of acini in a Ca2+-free medium with the Ca2+-ATPase inhibitor thapsigargin abolishes the rise in [Ca2+]i elicited by either caerulein or SLIGRL-NH2.

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Fig. 3. Effects on amylase secretion of reducing [Ca2+] in the suspending medium. Freshly prepared pancreatic acini, suspended in either standard Ca2+-containing medium or nominally Ca2+-free medium supplemented with 2 mM EGTA, were stimulated either SLIGRL-NH2 (100 µM) or caerulein (1 nM), and net stimulated amylase secretion over 30 min was measured as described in the text. *P < 0.05.
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Fig. 4. Effects on [Ca2+]i changes of reducing [Ca2+] in the suspending medium. Pancreatic acini were loaded with fura-2 and stimulated with either SLIGRL-NH2 (A, C, E, and G) or caerulein (B, D, F, and H) in the presence of either standard Ca2+-containing medium (A, B, E, and F) or nominally Ca2+-free medium supplemented with 0.1 mM EGTA (C, D, G, and H) as described in MATERIALS AND METHODS. Stimulation was accomplished using either a submaximally stimulating concentration of each secretagogue (SLIGRL-NH2, 30 µM; caerulein, 0.01 nM) or a supramaximally stimulating concentration of each secretagogue (SLIGRL-NH2, 1 mM; caerulein, 1 nM). The ratio of fluorescence emitted by single cells at 510 nm after excitation at 340 and 380 nm was monitored as described in MATERIALS AND METHODS. I and J: effects of preincubating acini in nominally Ca2+-free medium with 1 µM thapsigargin on [Ca2+]i changes elicited by SLIGRL-NH2 (I) or caerulein (J). Arrows point to the time point at which SLIGRL-NH2 or caerulein was added.
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Chelating intracellular Ca2+ inhibits both PAR2 and caerulein-stimulated amylase secretion.
Pancreatic acini were preloaded with the cell-permeant calcium chelator BAPTA (50 µM) to determine whether SLIGRL-NH2-induced amylase secretion depends on an increase of [Ca2+]i. Neither SLIGRL-NH2 nor caerulein cause [Ca2+]i to rise in acini preincubated with BAPTA (Fig. 5, A and B) and, as shown in Fig. 5C, the well-described reduction in amylase secretion in response to caerulein under these conditions (12) is also observed when SLIGRL-NH2 is used as the secretagogue.

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Fig. 5. Effects of BAPTA on [Ca2+]i changes and amylase secretion in response to SLIGRL-NH2 and caerulein. Standard acini and BAPTA-loaded acini preloaded with fura-2 were exposed to either 1 mM SLIGRL-NH2 (A) or 1 nM caerulein (B). Arrows indicate time of addition of the agonist. Amylase secretion was also monitored as described in the text using standard acini and acini preloaded with BAPTA after exposure to 1 mM SLIGRL-NH2 or 1 nM caerulein (C). *P < 0.05.
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[Ca2+]i responses following sequential and simultaneous PAR2 and CCK receptor stimulation.
For these studies, we subjected acini to sequential or simultaneous stimulation with SLIGRL-NH2 and caerulein under conditions of low extracellular [Ca2+] to prevent refilling of intracellular Ca2+ pools via Ca2+ influx. As shown in Fig. 6, initial exposure of acini to a submaximally stimulating concentration of caerulein elicits an oscillatory response, but, with time, the oscillations decrease in amplitude and the time between oscillations increases (Fig. 6B). Subsequent addition of a supramaximally stimulating concentration of caerulein elicits a small peak response (not shown). On the other hand, no rise in [Ca2+]i is observed when the second stimulus is either a submaximally or a supramaximally stimulating concentration of SLIGRL-NH2 (Fig. 6, B and D). Similarly, after the initial stimulation of acini with a supramaximally stimulating concentration of caerulein, the subsequent addition of SLIGRL-NH2 fails to elicit a response (Fig. 6F). In contrast to this pattern of responses that is noted when the sequence of additions is caerulein-SLIGRL-NH2, the pattern of responses is quite different when the sequence is SLIGRL-NH2-caerulein. As had been observed with initial exposure to caerulein, initial exposure to a submaximally stimulating concentration of SLIGRL-NH2 elicits an oscillatory response that, with time, diminishes (Fig. 6A). However, that oscillatory response is restored if the acini are exposed to a submaximally stimulating concentration of caerulein (Fig. 6A), and a peak response pattern is observed if the acini are exposed to a supramaximally stimulating concentration of caerulein (Fig. 6C). When acini are initially exposed to a maximally stimulating concentration of SLIGRL-NH2, an initial peak response is observed. Subsequent exposure to a supramaximally stimulating concentration of caerulein elicits a second peak pattern response (Fig. 6E). As shown in Fig. 7, the simultaneous addition of SLIGRL-NH2 and caerulein to the incubation medium does not result in an increased or additive [Ca2+]i rise.

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Fig. 6. [Ca2+]i responses to the sequential addition of SLIGRL-NH2 and caerulein. Freshly prepared, fura-2-loaded acini were suspended in nominally Ca2+-free medium containing 0.1 mM EGTA as described in the text. [Ca2+]i changes were monitored after sequential addition of SLIGRL-NH2 (30 or 1,000 µM) and caerulein (0.01 or 1 nM). A: low SLIGRL-NH2 followed by low caerulein. B: low caerulein followed by low SLIGRL-NH2. C: low SLIGRL-NH2 followed by high caerulein. D: low caerulein followed by high SLIGRL-NH2. E: high SLIGRL-NH2 followed by high caerulein. F: high caerulein followed by high SLIGRL-NH2. Arrows indicate time of addition of the first and second agonist. Tracings shown are representative of multiple such measurements.
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Fig. 7. Effects of simultaneous exposure to SLIGRL-NH2 and caerulein. Freshly prepared, fura-2-loaded acini were suspended in nominally Ca2+-free medium containing 0.1 mM EGTA as described in the text. [Ca2+]i changes were monitored in cells incubated with 1,000 µM of SLIGRL-NH2 (A), 1 nM caerulein (B), or a combination of 1,000 µM SLIGRL-NH2 and 1 nM caerulein (C). Arrows indicate time of addition of the agonists. Tracings shown are representative of multiple such measurements.
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We performed area under the curve analysis of data derived from experiments evaluating the sequential addition of caerulein-SLIGRL-NH2 or SLIGRL-NH2-caerulein in the absence of extracellular Ca2+. As shown in Fig. 8, initial addition of caerulein releases approximately twice the amount of Ca2+ that is released by initial addition of SLIGRL-NH2. Furthermore, the amount of Ca2+ released by initial exposure to caerulein is not reduced by prior exposure of the acini to SLIGRL-NH2. On the other hand, initial exposure of acini to caerulein abolishes Ca2+ release following subsequent exposure to SLIGRL-NH2.

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Fig. 8. Calcium accumulation after sequential addition of SLIGRL-NH2 and caerulein. Freshly prepared, fura-2-loaded acini were suspended in nominally Ca2+-free medium containing 0.1 mM EGTA as described in the text. [Ca2+]i changes were monitored in cells incubated with 1,000 µM SLIGRL-NH2 followed by 1 nM caerulein (A) or 1 nM caerulein followed by 100 µM SLIGRL-NH2 (B) as in Fig. 6. The relative amounts of intracellular Ca2+ were calculated by determining the area under the curve using Simpsons rule. Bars represent the average from 15 cells from 3 independent experiments. Error bars show the standard deviation.
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Effects of inhibiting PLC on PAR2 and caerulein-stimulated [Ca2+]i changes.
In previously reported studies, Yule et al. (20) have shown that the oscillations induced by submaximally stimulating CCK concentrations are blocked by the PLC inhibitor U73122
[GenBank]
, whereas the peak plateau response to supramaximally stimulating concentrations is reduced by U73122
[GenBank]
. We also find that the PLC inhibitor prevents the oscillatory response elicited by submaximally stimulating concentrations of caerulein, but, in our hands, the peak plateau response to supramaximally stimulating concentrations of caerulein is only partially reduced by U73122
[GenBank]
(Fig. 9). In contrast to these observations with caerulein, we find that the oscillatory response induced by submaximally stimulating concentrations of SLIGRL-NH2, as well as the peak plateau response elicited by higher concentrations of the PAR2-activating peptide, are both completely prevented by U73122
[GenBank]
(Fig. 9).

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Fig. 9. Effects of U71322
[GenBank]
[Ca2+]i changes elicited by SLIGRL-NH2 and caerulein. Freshly prepared, fura-2-loaded acini were incubated in the presence or absence of 10 µM U73122
[GenBank]
for 10 min. They were then exposed to a submaximally stimulating concentration of either SLIGRL-NH2 (A) or caerulein (B) or a supramaximally stimulating concentration of SLIGRL-NH2 (C) or caerulein (D), and the fluorescence ratio (340/380 nm) was recorded from single cells as described in the text. Arrows indicate time of addition of the agonist. Tracings are representative of multiple such measurements.
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DISCUSSION
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PAR2 is widely distributed in the gastrointestinal tract, and, in a previous report by Kawabata et al. (7), PAR2 stimulation has been shown to accelerate the rate of digestive enzyme secretion by the pancreas. In the current communication, we have confirmed that observation, i.e., the PAR2-activating peptide SLIGRL-NH2 causes concentration-dependent stimulation of amylase secretion (Fig. 1). The magnitude of maximal PAR2-stimulated secretion is similar to that observed when the well-characterized CCK analog caerulein is used at maximally stimulating concentrations, although a much higher concentration of SLIGRL-NH2 is needed to stimulate secretion. As expected, the reverse PAR2 peptide LRGILS-NH2 is inactive as a secretagogue and SLIGRL-NH2-stimulated amylase secretion is not observed when acini from PAR2/ mice are used. In contrast, genetic deletion of PAR2 does not alter caerulein-stimulated amylase secretion (Fig. 1).
Previous studies have shown that caerulein stimulation of pancreatic acini results in a biphasic pattern of amylase secretion, i.e., stimulation of secretion with low concentrations of the secretagogue and inhibition of secretion in the presence of supramaximally stimulating caerulein concentrations (12). This biphasic response reflects interaction of caerulein with two affinity states of the CCK receptor: a high-affinity stimulatory state and a low-affinity inhibitory state (17). We find that the PAR2-activating peptide SLIGRL-NH2 causes only monophasic stimulation of amylase secretion, and no inhibition of secretion by supramaximally stimulating concentrations of SLIGRL-NH2 is observed (Fig. 1). This observation leads us to conclude that acinar cells do not possess low-affinity PAR2 functionally coupled to the inhibition of secretion.
The mechanisms responsible for PAR2-stimulated amylase secretion by acinar cells have not been previously examined. There is, however, substantial evidence indicating that caerulein (or CCK)-stimulated digestive enzyme secretion from pancreatic acinar cells is dependent on a rise in [Ca2+]i and that the observed [Ca2+]i rise is mediated, at least in part, by the PLC/inositol (1,4,5)trisphosphate [Ins(1,4,5,)P3]/Ca2+ signal transduction pathway (11, 15). In studies using other types of cells, PAR2 stimulation has also been reported to cause activation of PLC, generation of Ins(1,4,5)P3, and a rise in [Ca2+]i (3). This led us to wonder whether PAR2-stimulated digestive enzyme secretion by acinar cells might not also be dependent on a PAR2-induced rise in [Ca2+]i, and, to pursue this issue, we embarked on a series of studies comparing and contrasting [Ca2+]i changes elicited by caerulein stimulation with those elicited by PAR2 stimulation.
Submaximally stimulating concentrations of caerulein are known to elicit an oscillatory change in [Ca2+]i, whereas supramaximally stimulating caerulein concentrations elicit a peak plateau response (20). As shown in Fig. 2, PAR2 stimulation also elicits oscillatory calcium changes at low secretagogue concentrations and peak plateau responses when higher SLIGRL-NH2 concentrations are used, but the magnitude of the observed changes in response to SLIGRL-NH2 is lower than that noted to follow stimulation with caerulein. On the other hand, the rate of amylase secretion is comparably increased following either PAR2 or CCK stimulation. These observations indicate that the magnitude of the rise in [Ca2+]i elicited by secretagogues acting via different G protein-coupled receptors does not necessarily correlate with the magnitude of secretion induced by those secretagogues.
We performed a series of experiments using 1) the reverse PAR2-activating peptide, 2) acini prepared from mice after genetic deletion of PAR2, and 3) PAR receptor-specific ligands to be certain that the responses noted using SLIGRL-NH2 were specific to PAR2 stimulation and to determine whether acinar cells might possess other types of PARs linked to digestive enzyme secretion and/or changes in [Ca2+]i. We found that the reverse PAR2-activating peptide did not stimulate amylase secretion nor elicit a rise in [Ca2+]i when acini from wild-type mice were evaluated and that neither the amylase secretion nor the changes in [Ca2+]i elicited by SLIGRL-NH2 in acini prepared from wild-type mice were observed when acini prepared from PAR2-deficient mice were exposed to SLIGRL-NH2 (Tables 1 and 2). These observations indicate that both the secretory changes and the [Ca2+]i changes elicited by SLIGRL-NH2 in acini from wild-type mice are mediated solely via PAR2 receptors. We also found that neither amylase secretion nor a change in [Ca2+]i followed exposure of acini to either the PAR1-activating peptide TFLLR-NH2 or the PAR4-activating peptide GYPGKF-NH2 (Tables 1 and 2). These last observations indicate that acinar cells do not possess either PAR1 or PAR4 receptors that are coupled to either amylase secretion or changes in [Ca2+]i.
It is generally believed that caerulein-stimulated amylase secretion from acini is dependent on a caerulein-induced rise in [Ca2+]i. In the presence of submaximally stimulating concentrations of caerulein, that rise in [Ca2+]i is oscillatory, and it reflects Ins(1,4,5)P3-triggered release of Ca2+ from intracellular storage pools, presumably located within the endoplasmic reticulum and/or zymogen granules, followed by reuptake of Ca2+ into those stores. With supramaximal caerulein stimulation, Ca2+ is released from the intracellular storage pools and, in addition, influx of Ca2+ from the suspending medium is accelerated. Both of these changes result in a sustained rise in [Ca2+]i. To a large extent, the concept that caerulein-induced amylase secretion is a Ca2+-dependent event is based on the observations that 1) caerulein-stimulated amylase secretion is markedly reduced, oscillations fade, and the plateau elevation of [Ca2+]i is lost when acini are suspended in low-[Ca2+] medium (Figs. 3 and 4), and 2) caerulein-stimulated secretion is markedly reduced when the rise in [Ca2+]i is prevented by preloading acini with the Ca2+ chelator BAPTA (Fig. 5). We find that PAR2-stimulated amylase secretion is also reduced when acini are suspended in low-[Ca2+] medium. Under these conditions, the oscillations induced by SLIGRL-NH2 fade and the plateau elevation of [Ca2+]i is lost (Figs. 3 and 4). Furthermore, we note that PAR2-stimulated amylase secretion and the PAR2-stimulated rise in [Ca2+]i are markedly reduced when acini are preloaded with BAPTA (Fig. 5). From these observations, we conclude that PAR2-mediated secretion, similar to caerulein-stimulated secretion, is dependent on the release of Ca2+ from intracellular storage pools and accelerated Ca2+ influx from the suspending medium. When acini are suspended in Ca2+-containing medium, these events facilitate secretion by causing persistence of the [Ca2+]i oscillations and a sustained plateau elevation of [Ca2+]i. With more prolonged PAR2 stimulation, homologous desensitization occurs as PAR2 is phosphorylated, internalized, and degraded (5).
We have performed a series of experiments employing the sequential stimulation of acini with SLIGRL-NH2 and caerulein to further examine the relationship between the calcium changes elicited by PAR2 and CCK stimulation. Our studies were performed in [Ca2+]-free media to prevent refilling of the storage pools following Ca2+ release and Ca2+ efflux from the cell. As shown in Fig. 6, initial stimulation of acini with caerulein prevents the subsequent addition of SLIGRL-NH2 from causing a rise in [Ca2+]i, but the converse is not true, i.e., initial stimulation with SLIGRL-NH2 does not prevent the subsequent addition of caerulein from causing a rise in [Ca2+]i. An area under the curve analysis of these data (Fig. 8) indicates that the initial addition of caerulein causes a [Ca2+]i rise that is approximately twice as great as that observed following the initial addition of SLIGRL-NH2. Furthermore, the [Ca2+]i rise elicited by caerulein is not reduced by prior addition of SLIGRL-NH2, but the addition of caerulein before SLIGRL-NH2 virtually abolishes the SLIGRL-NH2-stimulated [Ca2+]i rise.
These unexpected findings are open to many potentially valid interpretations. For example, the finding that prior addition of caerulein prevents SLIGRL-NH2 from triggering a subsequent rise in [Ca2+]i but that prior exposure to SLIGRL-NH2 does not interfere with caerulein-induced calcium changes could indicate that 1) CCK and PAR2 stimulation trigger release of calcium from separate intracellular storage pools and 2) that CCK stimulation desensitizes acinar cells to subsequent PAR2-stimulated calcium release. On the other hand, our finding that 1) both the caerulein- and the SLIGRL-NH2-induced rises in [Ca2+]i are prevented when acini are preincubated with the Ca2+-ATPase inhibitor thapsigargin (Fig. 4, I and J) and 2) simultaneous exposure of acini to caerulein and SLIGRL-NH2 does not lead to an additive rise in [Ca2+]i (Fig. 7) argue against this "two-calcium pool" explanation and suggest that stimulation with SLIGRL-NH2 leads to only partial emptying of a common intracellular pool that is completely depleted by caerulein stimulation. Further studies exploring the possibilities that CCK- and PAR2-stimulated calcium changes and secretion involve different signal transduction pathways, different calcium storage pools, or even differences in cellular events regulating disposal (i.e., calcium reuptake into storage sites and/or calcium efflux from the cell) will be needed to resolve this issue.
It is certainly possible that the caerulein- and SLIGRL-NH2-sensitive intracellular storage pools release Ca2+ in response to differing signals (i.e., messengers). In preliminary studies, we have examined the role of PLC in these processes. In earlier studies, Yule et al. (20) showed that the oscillations induced by submaximal CCK receptor stimulation were blocked by the PLC inhibitor U73122
[GenBank]
, and, on the basis of this observation, they concluded that those oscillations reflected Ins(1,4,5)P3-mediated release of Ca2+ from intracellular stores, although intracellular Ins(1,4,5)P3 levels were not measurably elevated by submaximally stimulating caerulein concentrations. In addition, they found that U73122
[GenBank]
partially, but incompletely, reduced the peak plateau response elicited by supramaximal CCK receptor stimulation. On the basis of the latter finding, one might conclude that the peak plateau response is, at best, only partially mediated by PLC-generated Ins(1,4,5)P3 and that the peak plateau response also reflects Ca2+ released by another, as yet unidentified, intracellular second messenger. In our studies using caerulein, we have confirmed those earlier findings (Fig. 9), although, in our studies, U73122
[GenBank]
reduces the peak plateau response elicited by a supramaximaly stimulating concentration of caerulein to an even lesser extent than had been observed by Yule et al. (20). In contrast to these findings with caerulein, we have found that both the oscillatory and the peak plateau responses elicited by PAR2 stimulation are completely prevented by U73122
[GenBank]
.
Taken together, these latter findings lead us to suggest that the oscillatory and peak plateau [Ca2+]i rises and the subsequent secretory responses to both low and high concentrations of SLIGRL-NH2 are mediated by a second messenger system that is dependent on PLC. On the other hand, the second messenger systems involved in caerulein-stimulated changes appear to depend on the concentration of caerulein that is used, i.e., submaximally stimulating caerulein concentrations elicit PLC-dependent oscillatory [Ca2+]i changes and amylase secretion, whereas supramaximally stimulating caerulein concentrations elicit PLC-independent peak plateau [Ca2+]i changes and inhibition of amylase. These observations demonstrate the independence of the secretory response from the pattern of calcium change, i.e., the peak plateau change in [Ca2+]i elicited by PAR2 stimulation is associated with stimulation of secretion, whereas that associated with high concentrations of caerulein is associated with inhibition of secretion.
In summary, our studies have shown that mouse pancreatic acini express PAR2 and that activation of acinar cell PAR2 leads to monophasic acceleration of amylase secretion. That accelerated secretion is dependent on a PAR2-induced rise in [Ca2+]i, which reflects release of Ca2+ from intracellular pools and accelerated influx of Ca2+ from the extracellular medium. Preventing the rise in [Ca2+]i elicited by PAR2 stimulation, or removing Ca2+ from the suspending medium, inhibits PAR2-stimulated enzyme secretion. In accord with previous studies, we find that caerulein stimulation of acinar cells also accelerates amylase secretion and that secretion is also dependent on a caerulein-induced rise in [Ca2+]i that reflects release of Ca2+ from intracellular pools and accelerated Ca2+ influx. Finally, activation of PLC and, presumably, generation of Ins(1,4,5)P3 plays a critical role in mediating both the oscillatory and the peak plateau rise in [Ca2+]i elicited by PAR2 stimulation and in mediating the oscillatory rise in [Ca2+]i elicited by submaximally stimulating concentrations of caerulein, but another PLC-independent intracellular second messenger mediates the Ca2+ release and inhibition of secretion that is elicited by supramaximally stimulating concentrations of caerulein.
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GRANTS
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These studies were supported by National Institutes of Health Grants RO1-AM-31396-20 (to M. L. Steer) and P30-DK-34928-20 (to Tufts-New England Medical Center, Center for Gastroenterology Research on Absorptive and Secretory Processes).
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ACKNOWLEDGMENTS
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The authors thank Drs. P. Andrade-Gordon (Johnson and Johnson, Spring Hill, PA) for generously providing breeding pairs of PAR2/ mice and T. Ducibella (Tufts University School of Medicine, Boston, MA) for kindly providing us access to a single cell Ca2+-sensing device.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. Perides, Tupper 205, Dept. of Surgery #37, Tufts-New England Medical Center, 750 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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Copyright © 2005 by the American Physiological Society.