1 Departments of Surgery, Medicine, and Cell Biology, Connecticut Health Care Department of Veterans Affairs, West Haven, and Yale University School of Medicine, New Haven, Connecticut 06516; 2 Department of Visceral and Transplantation Surgery, University of Bern, 3010 Bern, Switzerland; and 3 AlphaGene, Inc., Charlestown, Massachusetts 02129
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ABSTRACT |
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To identify the
muscarinic subtype present on the rat pancreatic acinar cell, we
examined the effects of different muscarinic receptor antagonists on
amylase secretion and proteolytic zymogen processing in isolated rat
pancreatic acini. Maximal zymogen processing required a concentration
of carbachol 10- to 100-fold greater (103 M) than that required
for maximal amylase secretion
(10
5 M). Although both
secretion and conversion were inhibited by the M3 antagonist
4-diphenylacetoxy-N-methyl-piperidine
(4-DAMP) (50% inhibition ~6 × 10
7 M and 1 × 10
8 M, respectively), the
most potent inhibitor was the M1 antagonist telenzepine (50%
inhibition ~5 × 10
10 M and 1 × 10
11 M, respectively).
Pirenzepine, another M1 antagonist, and the M2 antagonist methoctramine
did not reduce amylase secretion or zymogen processing in
concentrations up to 1 × 10
5 M. Analysis of acinar
cell muscarinic receptor by PCR revealed expression of both m1 and m3
subtypes. The pancreatic acinar cell has a distinct
pattern of muscarinic antagonist sensitivity (telenzepine
4-DAMP > pirenzepine) with respect to both amylase secretion and zymogen
conversion.
muscarinic antagonist; 4-diphenylacetoxy-N-methyl-piperidine; carboxypeptidase A1; secretion
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INTRODUCTION |
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NEUROHUMORAL STIMULATION of the exocrine pancreas generates a number of physiological responses, including enhanced zymogen secretion. Recent studies suggest that cholinergic pathways may have a more important role in regulating pancreatic secretion than previously appreciated. Thus the primary action of CCK released by the intestinal nutrients may be to stimulate the cholinergic release of acetylcholine (ACh) (1, 16). The neurotransmitter then acts on muscarinic ACh receptors (mAChR) to stimulate acinar cell secretion. Cholinergic pathways may play a role in pathological processes such as the proteolytic processing of zymogens to active forms.
Cholinergic stimulation may contribute to the pathogenesis of pancreatitis such as that induced by the bite of the Tityus serrulatus scorpion (22), alcohol-associated disease, and cholinesterase inhibitors (17). A key step for initiating many forms of pancreatitis appears to be the pathological intracellular activation of digestive zymogens (15). In vivo studies that use supramaximal concentrations (10- to 100-fold greater than that generating a maximal secretory response) of CCK or cholinergic agonists generate acute pancreatitis (9) and zymogen activation (18). Likewise, hyperstimulation of isolated pancreatic acini by CCK leads to the rapid activation of proteases and enhanced zymogen processing (15). The protease activation observed in isolated pancreatic acini may be relevant to the pathogenesis of acute pancreatitis.
The identification of the muscarinic receptors on the pancreatic acinar cell is relevant to pancreatic physiology and pathology. Muscarinic antagonists are most often used to establish the class of muscarinic receptors; based on the relative efficiency of these agents, the acinar cell receptor has been designated an M3 subtype (11, 12, 28), whereas more proximal cholinergic pathways that affect the pancreas appear to be regulated by M1 receptors (26). Multiple subtypes of muscarinic receptor may be present on a single cell (5) and may potentially couple to distinct signaling pathways. Preliminary studies from our laboratory suggest that zymogen secretion and intracellular zymogen processing may be activated by different signaling pathways. Thus different muscarinic receptors on the acinar cell might mediate these distinct cellular responses. In this study we examined the effects of muscarinic antagonists on carbachol (CCh)-induced secretion and zymogen processing. Our results demonstrate that both responses are potently inhibited by the M1 antagonist telenzepine. Similar inhibition is observed with the M3 antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP), but at a concentration about 1,000-fold greater. Thus telenzepine-sensitive muscarinic receptors regulate both secretion and zymogen processing in the pancreatic acinar cell.
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MATERIALS AND METHODS |
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Preparation of isolated pancreatic acini. Pancreatic acini were prepared in accordance with procedures outlined in The National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Department of Veterans Affairs Animal Studies Subcommittee. Pancreata harvested from fasted male Sprague-Dawley rats (weighing ~100 g) were subjected to collagenase digestion and mechanical dispersion and filtered through Nytex 200 gauze as previously described (15). For the preparation of small groups of acinar cells (mini-acini), acinar preparations were incubated for an additional 10 min in medium with 2 mM EGTA and no Ca2+, washed into standard medium, and filtered through Nytex 20 gauze. Acini were then incubated in plates with 24 wells containing incubation buffer consisting of (in mM) 25 HEPES, 98 NaCl, 4.8 KCl, 2 CaCl2, and 1.2 MgCl2, and 0.2% bovine serum albumin, and 0.1 mg/ml soybean trypsin inhibitor (pH 7.4), under continuous oxygenation at 37°C for 1 h before treatment.
Exposure to secretagogues. After the equilibration period, acini were treated with CCK-8 (Squibb Diagnostics, New Brunswick, NJ) or carbamylcholine chloride (CCh; Sigma, St. Louis, MO), using the doses and duration of treatment indicated. Acini exposed to incubation medium alone served as unstimulated controls.
Receptor specificity. For each individual experiment, acini were pretreated with either atropine (nonselective muscarinic antagonist) (Sigma), pirenzepine (M1-selective receptor antagonist) (Sigma), telenzepine dihydrochloride (telenzepine; M1-selective receptor antagonist; provided by Byk Gulden, Konstanz, Germany), methoctramine (M2-selective receptor antagonist; gift from Dr. G. Makhlouf, Richmond, VA), or 4-DAMP (M3-selective receptor antagonist; gift from Dr. R. Goyal, Boston, MA) for 15 min before stimulation with CCh.
Determination of amylase activity. After pretreatment with antagonists and exposure to secretagogues, amylase activity was measured in the incubation medium with the use of a Phadebas amylase test kit (Pharmacia Diagnostics, Piscataway, NJ), and secretion was expressed as a ratio in comparison to the unstimulated control. Because preliminary experiments had revealed a uniform distribution of acini in the tubes (±5% in total amylase content), the percentage of released amylase in comparison to the total content measured after sonication of the cells was not regularly measured. However, during a 30-min incubation, basal amylase release ranged from 3% to 6% and stimulated release from 12% to 18%. Preparations exhibiting less than a 2.5-fold increase in amylase release over the unstimulated condition were excluded from analysis.
Detection of zymogen proteolysis.
At the conclusion of the incubation period, proteins were solubilized
by boiling in Laemmli sample buffer containing -mercaptoethanol (10%) and subjected to SDS-PAGE (7.5%) as described previously (13).
Separated proteins were electrophoretically transferred to Immobilon-P
transfer membranes (Millipore/Continental Water Systems, Bedford, MA)
and immunoblotted with a rabbit polyclonal antiserum that binds with
comparable affinity to the zymogen procarboxypeptidase A1
(PCA1), and the active form of
PCA1, carboxypeptidase
A1
(CA1), as described
previously (15). Labeled proteins were detected using
125I-labeled goat anti-rabbit IgG
(ICN Biochemicals, Irvine, CA) followed by autoradiography. The
radioactive bands were excised from the membrane, and the amount of
labeling was quantitated by measuring gamma emissions. The amount of
immunoreactivity of the active enzyme form under various treatment
conditions was calculated as a ratio in comparison to unstimulated
control and expressed as relative conversion. The percentage of
PCA1 relative to
CA1 has been estimated in previous
studies; in the basal state 1-4% may be present in the active
form, and the value increases from 6% to 14% in acini stimulated by
CCK (10
7 M) (15). In some
experiments conversion was detected using the enhanced
chemiluminescence kit and horseradish peroxidase-labeled anti-rabbit
IgG (both from Amersham, Buckinghamshire, UK), after brief exposure to
autoradiography film. Computed densitometry was utilized to estimate
the density of labeled bands.
Measurement of
[Ca2+]i.
Intracellular Ca2+ concentration
([Ca2+]i)
was measured in the acinar cells with the use of confocal fluorescence
microscopy (19, 20). Cells were isolated as described above and then
loaded with the Ca2+-sensitive
fluorescent dye fluo 3-AM (8.8 mM) for 30 min at room temperature in
Liebovitz L-15 medium containing 10% fetal calf serum (20). The fluo
3-loaded cells were transferred to a perifusion chamber on the stage of
a Bio-Rad MRC-600 confocal microscope and then continuously perifused
at 37°C with a HEPES-based buffer solution. The fluo 3 was excited
with the 488-nm line of a krypton-argon laser, and emission signals
above 515 nm were collected. Small (2-10 cells) clusters of acini
were identified to ensure that there was no associated nerve tissue,
and then the cells were examined in the line-scanning mode (20) during
either 1) stimulation with CCh
(105 M) or
2) pretreatment with telenzepine
(10
8 M) followed by serial
stimulation with CCh (10
5
M). The spatial resolution was 0.26 mm/pixel so that fluorescence signals from neighboring cells could readily be distinguished, and
signals were collected at a rate of 3-5/s.
Preparation of mRNA and cDNA from isolated pancreatic acini and brain. Rats were killed, and their brains were immediately removed and homogenized in liquid nitrogen. Freshly prepared pancreatic acini or mini-acini from young rats (~100 g) were immediately resuspended in lysis buffer (from FastTrack kit; see below) and subjected to mRNA preparation using the FastTrack mRNA isolation kit (Invitrogen, San Diego, CA). Approximately 2-3 µg of mRNA were obtained from one of the standard acinar preparations and <1 µg was obtained from mini-acini. The first-strand cDNA was then transcribed from 0.4-0.8 µg mRNA, using reverse transcriptase (Superscript kit; Life Technologies, Gaithersburg, MD). To eliminate the contamination of genomic DNA in the mRNA preparation, a reaction without reverse transcriptase was always included in the first-strand cDNA synthesis, which was subsequently evaluated in the gene-specific PCR amplification.
PCR and Southern blotting. Approximately 10% of the resulting cDNA (obtained from 20-40 ng original mRNA) was amplified by gene-specific PCR for 30 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min), using an MJ Research PTC-200 thermal cycler. The following rat mAChR-specific primers were used to direct synthesis of the muscarinic receptor subtypes in PCR reactions (30): m1, upper primer, 5'-GCACAGGCACCCACCAAGCAG-3', coding for the termini 1073-1093, lower primer, 5'-AGAGCAGCAGCAGGCGGAACG-3', coding for 1445-1425; m2, upper primer, 5'-CACGAAACCTCTGACCTACCC-3', coding for 826-846, lower primer, 5'-TCTGACCCGACGACCCAACTA-3', coding for 1508-1488; m3, upper primer, 5'-GTCTGGCTTGGGTCATCTCCT-3', coding for 1318-1438, lower primer, 5'-GCTGCTGCTGTGGTCTTGGTC-3', coding for 1751-1731. PCR products had a size of 373 bp (m1), 683 bp (m2), and 434 bp (m3). They were separated by electrophoresis on 1% agarose gels containing ethidium bromide and photographed under fluorescent illumination. The PCR products were then transferred to nylon membranes (Amersham), and the nucleic acids were fixed by ultraviolet cross-linking. For Southern blot analysis the following internal sequence probes were used for hybridization: m1, 5'-CCCTGTGGGAGCTGGGC-3', coding for 1335-1351; m2, 5'-GGCTCCACGGGACGGCGT-3', coding for 1243-1260; and m3, 5'-GCTGGCTGGCCTACAGGC-3', coding for 1541-1558. The 3' tailing of the oligonucleotides with digoxigenin (DIG)-11-dUTP/ATP was accomplished using the DIG oligonucleotide tailing kit (Boehringer Mannheim, Indianapolis, IN). The membranes were prehybridized for 2 h at 42°C and subsequently hybridized overnight at 42°C with the DIG-labeled probes diluted to 2 pmol/ml with hybridization buffer containing 5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 0.1% N-lauroylsarcosine, 0.02% SDS, and 1% blocking reagent (Boehringer Mannheim). The membranes were then washed four times in a solution of 2× or 0.5× SSC and 0.1% SDS at room temperature. After 1-h preincubation in a 1% blocking solution, the membrane was exposed to anti-DIG Fab fragments conjugated to alkaline phosphatase (working concn 75 mU/ml) for 30 min. The membranes were then washed in 100 mM maleic acid, 150 mM NaCl, and 0.3% Tween 20. Positively hybridized nucleic acids on the membrane were detected with the chemiluminescent alkaline phosphatase substrate disodium 3-{4-methoxyspiro[1,2-diotexane-3,2'-(5'-chloro)tricyclo(3.3.1.13,7)decan]-4-yl}phenyl phosphate (Boehringer Mannheim), using a concentration of 250 mM in 100 mM Tris · HCl and 100 mM NaCl (pH 9.5). After incubation for 15 min at 37°C, DNA bands were revealed by autoradiography. The same membrane was then stripped twice with alkaline probe-stripping buffer (0.2 N NaOH and 0.1% SDS) for 10 min at 37°C before it was reprobed with a second or a third gene-specific probe.
Statistical analysis. Data are expressed as means ± SE, and differences between individual means are assessed by unpaired Student's t-test. The significance level was set at P < 0.05.
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RESULTS |
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Dose and time dependence of CCh-induced zymogen conversion.
Secretion of amylase reached a maximum level at
105 M CCh and decreased at
higher doses (Fig.
1A),
as reported previously (25). The addition of CCh
(10
6 to
10
3 M) resulted in a
concentration-dependent increase of
CA1 (Fig. 1B); zymogen conversion was detected
after 10 min of treatment, reached a maximum level at 30 min, and
decreased after 60 min (Fig. 1C).
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Effects of cholinergic antagonists.
To define the class of cholinergic receptor responsible for mediating
this CCh effect, acini were exposed to atropine, a nonselective muscarinic antagonist. This pretreatment caused a dose-dependent inhibition of amylase secretion, with an approximate
IC50 of 1 × 109 M (Fig.
2). Likewise, atropine in concentrations of
10
6 to
10
4 M eliminated the enzyme
conversion stimulated by high doses of 10
4 M CCh (normalized mean
values ± SE for 10
4 M
CCh = 4.4 ± 0.4, and for atropine:
10
6 M = 1.6 ± 0.2, 10
5 M = 1.3 ± 0.1, and
10
4 M = 1.3 ± 0.1;
n = 7). To examine the subtype of
muscarinic receptors responsible for mediating this conversion, acini
were incubated with selective muscarinic antagonists (2) before the
addition of CCh. Preincubation with pirenzepine
(10
8 to
10
5 M), an M1-selective
antagonist, did not significantly reduce amylase secretion or
PCA1 conversion (Fig.
3). Indeed, there was some increase in both
the secretory response and zymogen conversion in the presence of
pirenzepine (Fig. 3).
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