Telenzepine-sensitive muscarinic receptors
on rat pancreatic acinar cells
STEFAN W. SCHMID,1,2 IRVIN M. MODLIN,1 LAURA H. TANG,1
AUBRY STOCH,1 STEVE RHEE,1 MICHAEL H. NATHANSON,1
GEORGE A. SCHEELE,3 AND FRED S. GORELICK1
1Departments 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; 2Department of Visceral and Transplantation Surgery, University
of Bern, 3010 Bern, Switzerland; and 3AlphaGene, Inc., Charlestown, Massachusetts 02129
Schmid, Stefan W., Irvin M. Modlin, Laura H. Tang,
Aubry Stoch, Steve Rhee, Michael H. Nathanson,
George A. Scheele, and Fred S. Gorelick. Telenzepinesensitive muscarinic receptors on rat pancreatic acinar cells.
Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G734–
G741, 1998.—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 (1023 M) than that
required for maximal amylase secretion (1025 M). Although
both secretion and conversion were inhibited by the M3
antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP)
(50% inhibition ,6 3 1027 M and 1 3 1028 M, respectively),
the most potent inhibitor was the M1 antagonist telenzepine
(50% inhibition ,5 3 10210 M and 1 3 10211 M, respectively).
Pirenzepine, another M1 antagonist, and the M2 antagonist
methoctramine did not reduce amylase secretion or zymogen
processing in concentrations up to 1 3 1025 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
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
NEUROHUMORAL STIMULATION
G734
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 (4DAMP), 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.
MATERIALS AND METHODS
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 Ca21, 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
0193-1857/98 $5.00 Copyright r 1998 the American Physiological Society
ACINAR ZYMOGEN PROTEOLYSIS
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 (65% 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 b-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 125Ilabeled 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 (1027 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 [Ca21]i. Intracellular Ca21 concentration
([Ca21]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 Ca21-sensitive
fluorescent dye fluo 3-AM (8.8 mM) for 30 min at room
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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 (1025 M) or 2) pretreatment with telenzepine (1028 M)
followed by serial stimulation with CCh (1025 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, 58-GCACAGGCACCCACCAAGCAG-38, coding for the termini 1073–1093, lower primer, 58AGAGCAGCAGCAGGCGGAACG-38, coding for 1445–1425; m2,
upper primer, 58-CACGAAACCTCTGACCTACCC-38, coding for
826–846, lower primer, 58-TCTGACCCGACGACCCAACTA-38,
coding for 1508–1488; m3, upper primer, 58-GTCTGGCTTGGGTCATCTCCT-38, coding for 1318–1438, lower primer, 58-GCTGCTGCTGTGGTCTTGGTC-38, 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, 58-CCCTGTGGGAGCTGGGC-38, coding for 1335–1351; m2, 58-GGCTCCACGGGACGGCGT-38, coding for 1243–1260; and m3, 58-GCTGGCTGGCCTACAGGC-38, coding for 1541–1558. The 38 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 53 SSC (13 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 23 or 0.53 SSC and 0.1% SDS at room temperature. After 1-h preincubation in a 1% blocking solution, the
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ACINAR ZYMOGEN PROTEOLYSIS
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 3-54-methoxyspiro[1,2-diotexane-3,28-(58-chloro)tricyclo(3.3.1.13,7)decan]-4-yl6phenyl 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 6 SE,
and differences between individual means are assessed by
unpaired Student’s t-test. The significance level was set at
P , 0.05.
RESULTS
Dose and time dependence of CCh-induced zymogen
conversion. Secretion of amylase reached a maximum
level at 1025 M CCh and decreased at higher doses (Fig.
1A), as reported previously (25). The addition of CCh
(1026 to 1023 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).
The conversion of procarboxypeptidase B2 and chymotrypsinogen 2 was also stimulated by high concentrations (1023 M) of CCh and CCK (1027 M) (10). Although
the selective proteolysis of each of these zymogens in
response to neurohumoral stimulation was qualitatively similar, the signal for PCA1 conversion was more
easily quantitated than the signal generated by the
other zymogens. Thus the conversion of PCA1 to CA1
has been used as the marker for selective zymogen
proteolysis.
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 3 1029 M (Fig. 2). Likewise,
atropine in concentrations of 1026 to 1024 M eliminated
the enzyme conversion stimulated by high doses of 1024
M CCh (normalized mean values 6 SE for 1024 M
CCh 5 4.4 6 0.4, and for atropine: 1026 M 5 1.6 6 0.2,
1025 M 5 1.3 6 0.1, and 1024 M 5 1.3 6 0.1; n 5 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
(1028 to 1025 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).
Although M2 receptors have not been previously
detected on pancreatic acini, the ability of methoctra-
Fig. 1. Concentration dependence of carbachol (CCh)-stimulated
amylase secretion (A) and procarboxypeptidase A1 (PCA1 ) conversion
(B). Acini were incubated for 30 min with various concentrations of
CCh. C: time course of CCh-stimulated PCA1 conversion. After
exposure to CCh (1 3 1024 M) for various times, acini were assayed
for conversion. Values are means 6 SE; n 5 8. * P , 0.01, ** P ,
0.001, *** P , 0.0001 vs. unstimulated control conditions.
ACINAR ZYMOGEN PROTEOLYSIS
G737
Fig. 2. Effect of atropine on CCh-stimulated secretion. After pretreatment (15 min) with atropine, acini were stimulated (30 min) by CCh
(carb; 1025 M) for amylase measurement. Values are means 6 SE;
n 5 4. * P , 0.001, ** P , 0.0001 vs. CCh.
mine, an M2-selective antagonist, to inhibit this conversion was tested. Methoctramine in doses of 1029 to 1026
M failed to inhibit the conversion (normalized mean
values 6 SE for 1023 M CCh 5 1.9 6 0.2, and for
Fig. 4. Effect of 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP)
on CCh-stimulated secretion (A) and PCA1 conversion (B). After
pretreatment (15 min) with 4-DAMP, acini were stimulated (30 min)
by CCh (1025 M) for amylase measurement or hyperstimulated by
CCh (1024 M) for zymogen conversion. Values are means 6 SE; n 5 4
for secretion, n 5 6 for conversion. * P , 0.05, ** P , 0.001, *** P ,
0.0001 vs. CCh.
Fig. 3. Effect of pirenzepine on CCh-stimulated secretion (A) and PCA1
conversion (B). After pretreatment (15 min) with pirenzepine, acini
were stimulated (30 min) by CCh (1025 M) for amylase measurement
or hyperstimulated by CCh (1024 M) for zymogen conversion. Values
are means 6 SE; n 5 4 for secretion, n 5 8 for conversion. * P , 0.01
vs. CCh.
methoctramine: 1026 M 5 1.8 6 0.2, 1025 M 5 1.7 6 0.2,
and 1024 M 5 1.6 6 0.2; n 5 4). However, pretreatment
with 4-DAMP, an M3-selective antagonist, resulted in
concentration-dependent inhibition of CCh-induced secretion (Fig. 4). Similar to pirenzepine, low concentrations of 4-DAMP tended to increase both secretion and
zymogen conversion.
Pretreatment of acini with the M1 antagonist telenzepine unexpectedly resulted in the inhibition of both
secretion and zymogen conversion. Telenzepine was
.1,000 times more potent than 4-DAMP in decreasing
CCh-stimulated amylase secretion (Fig. 5). The estimated telenzepine concentration required to inhibit
50% of the CCh-induced amylase release was 5 3 10210
M. Similarly, 50% zymogen conversion was inhibited by
a concentration of 1 3 10211 M telenzepine. In control
studies telenzepine (10212 to 1028 M) did not affect
basal or CCK (5 3 10210 )-induced secretion.
Telenzepine is thought to affect pancreatic secretion
primarily by acting on neural pathways. To exclude any
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ACINAR ZYMOGEN PROTEOLYSIS
possible effect of CCh on neural elements in the acinar
preparation, two studies were performed. First, before
stimulation, isolated acini were treated with tetrodotoxin, an inhibitor of neural transmission. However,
this compound (10211 to 1025 M) changed neither the
secretory response nor zymogen processing in acini
stimulated by CCh (not shown). Second, a preparation
of mini-acini containing 2–10 cells was generated.
These small groups of acini appear to be devoid of
neural elements when examined by electron microscopy (not shown). Because these preparations exhibit a
poor secretory response compared with whole acini, the
effects of CCh on [Ca21]i in individual acinar cells were
measured (Fig. 6). Every cell (n 5 23) stimulated with
CCh responded with a rapid, sustained increase in
[Ca21]i (Fig. 6A), as has been described previously (19).
In contrast, no cell (n 5 21) pretreated with telenzepine
responded to CCh (Fig. 6B). The telenzepine effect was
specific for CCh; 20 (95%) of the telenzepine-treated
cells displayed an increase in [Ca21]i in response to
Fig. 6. Effect of CCh (1025 M) and telenzepine (1028 M) on cytosolic
Ca21 concentration ([Ca21]i ). A: CCh is added to pancreatic acinar
cells at t 5 10 s. A rapid increase in [Ca21]i occurs soon after exposure
to CCh, denoted by an abrupt increase in fluorescence intensity.
Response is shown as fluorescence intensity, since fluo 3 cannot be
ratio imaged. Result is typical of that seen in n 5 23 separate cells in
6 experiments. B: telenzepine added to medium at t 5 0 s blocks an
increase of [Ca21]i by CCh added at t 5 15 s. Result is typical of that
seen in n 5 21 separate cells.
Fig. 5. Effect of the M1 antagonist telenzepine on CCh-stimulated
amylase secretion (A) and zymogen conversion (B). After pretreatment (15 min) with telenzepine, acini were stimulated (30 min) by
CCh and either assayed for amylase secretion or processed for
immunoblot analysis using antibodies to carboxypeptidase A1. Values
for amylase secretion and PCA1 processing are means 6 SE; n 5 4 for
each group. * P , 0.05, ** P , 0.01, *** P , 0.001, **** P , 0.0001 vs.
1025 M CCh for secretion and 1023 M CCh for processing.
subsequent stimulation with CCK. These findings confirm that telenzepine is acting directly on acinar cells
and not on nerves.
PCR and Southern blot analysis. cDNA from rat
brain (amplified from 2–4 ng of original mRNA) was
utilized as a positive control in PCR, since it is known to
express all five subtypes of mAChR. As shown in Fig. 7,
a single band of ,370 bp, ,680 bp, or ,430 bp, which
were the predicted sizes corresponding to m1, m2, and
m3, respectively, was amplified in each reaction using
m1, m2, and m3 primer pairs. However, pancreatic
acinar cell cDNA (amplified from 20–40 ng of original
mRNA of either the standard acinar cell preparation or
mini-acini) revealed only the muscarinic subtypes m1
and m3; no m2 DNA was detected. To verify the
specificity of individual PCR products, Southern blot
analysis with specific internal sequences was performed. These results confirmed that PCR products
from brain tissue and pancreatic acinar cells were
specific mAChR subtypes.
ACINAR ZYMOGEN PROTEOLYSIS
Fig. 7. Agarose gel electrophoresis with ethidium bromide staining
for PCR products amplified from rat brain (b) and pancreas (p) with
primer specific for m1, m2, and m3 muscarinic receptor genes.
Typical results are shown for each sample, repeated at least 3 times.
Molecular weight (MW) of bands is labeled in base pairs (bp) at left.
DISCUSSION
Treatment of pancreatic acini with CCh results in the
proteolytic processing of several pancreatic zymogens
to their active forms. Similar to the finding reported for
CCK (15), the effect of CCh on zymogen processing is
concentration dependent. The highest level of processing is elicited by a dose that is approximately 100-fold
greater than that required for maximal secretion. We
have reported that procarboxypeptidase B and chymotrypsinogen 2 exhibit proteolytic responses similar to
that described for PCA1 (10). Saluja et al. (24) found
that hyperstimulation with the CCK analog caerulein
results in the generation of tryptic activity in isolated
acini. Collectively, these findings indicate that this
limited proteolytic cleavage of zymogens is likely to be a
generalized response to supramaximal doses of CCK or
CCh. Thus this assay detects a regulated and limited
proteolytic event that, in addition to enzyme secretion,
may be a physiological response of the acinar cell to
neurohumoral stimulation that becomes pathological
under certain conditions.
The muscarinic receptor(s) that regulates acinar
cells has not been identified at the molecular level.
Although five classes of muscarinic receptors in the
brain have been cloned (3, 4), the classification of these
receptors has to date been largely based on the evaluation of the pharmacological effects of selective receptor
antagonists. Although early reports classified the pancreatic acinar cell muscarinic receptor as the M2 type,
later studies noted that cholinergic secretion was mediated by a 4-DAMP-sensitive M3 acinar cell receptor (11,
12, 28). Our present pharmacological studies are in
agreement with previous reports in regard to the
presence of a 4-DAMP-sensitive muscarinic receptor on
the pancreatic acinar cell. Thus secretion and limited
proteolytic conversion were inhibited by 4-DAMP but
not by equivalent doses of pirenzepine or methoctramine. However, the finding that telenzepine, a putative
M1-selective antagonist, was a potent inhibitor of CChinduced enzyme secretion and zymogen processing in
an acinar cell preparation was unexpected. Telenzepine
was initially described as a selective M1 inhibitor and
evaluated as a therapeutic agent for peptic ulcer dis-
G739
ease. In this respect it proved to be 4–10 times more
potent than the selective M1 antagonist pirenzepine in
the inhibition of gastric acid secretion (8). In addition,
telenzepine was noted to inhibit pancreatic bicarbonate
and protein output in dogs (26, 27) and to completely
block cholinergically stimulated pancreatic secretion in
humans (21). Because the acinar cell was previously
thought to lack an M1 muscarinic receptor, the inhibitory effects of telenzepine on pancreatic secretion have
usually been ascribed to an indirect effect on neural
pathways.
The absence of an effect of tetrodotoxin on CChinduced secretion and the ability of telenzepine to block
a CCh-induced increase in [Ca21]i in small groups of
2–10 acinar cells lacking neural innervation suggest
that it is very unlikely that CCh is acting on neural
pathways in the acinar preparation and confirm that
telenzepine is directly blocking receptors on the acinar
cell.
Similar to other systems (29), it appears that the rat
pancreatic acinar cell may express both the m1 and m3
receptor subtype. Therefore, it was possible that the
two responses to CCh, secretion and zymogen processing, might be regulated by activation of specific receptors. However, the muscarinic antagonists demonstrated a similar pattern for both secretion and zymogen
processing. These findings suggest that the same muscarinic receptor subtype is mediating both of these
responses but that it may couple to distinct signaling
pathways depending on the concentration of agonist.
This conclusion is supported by the observation that
concentration-dependent changes in the pattern of
agonist-induced Ca21 signaling are a recognized property of many G-protein linked receptors (19).
The potent effects of telenzepine on rat pancreatic
acinar cell muscarinic receptor were surprising. For
receptors characterized as the M1 subtype, the usual
rank order of potency is telenzepine . pirenzepine .
4-DAMP; for the M3 subtype the rank order of potency
is 4-DAMP . telenzepine . pirenzepine (14). Comparative studies in the rat parotid gland have demonstrated
that 4-DAMP was 100-fold more potent for displacing
[3H]quinuclidinyl benzilate binding and 500-fold more
potent in blocking the generation of intracellular Ca21
than pirenzepine (6). Similarly, in preliminary studies
we found that the rank order of antagonists in rat
parotid gland, designated as M3 receptor subtype, was
4-DAMP . telenzepine . pirenzepine for inhibiting
CCh-stimulated amylase release (Schmid and Rhee,
unpublished observations). These findings are consistent with previous data obtained from studies of rat
submandibular gland (14) but clearly differ from data
obtained from studies of the pancreas and help to
exclude a trivial explanation for the dramatic effects of
telenzepine on the rat pancreatic acinar cell.
All antagonists (except telenzepine) enhanced the
effect of CCh at low concentrations. The reason for this
phenomenon is unclear. Because secretion is a biphasic
response (decreasing after a peak level is attained), an
enhanced response might have been obtained by competition with the CCh if the agonist had not been used at
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ACINAR ZYMOGEN PROTEOLYSIS
the maximum secretory concentration. However, this
would not explain the similarly enhanced response for
conversion, which is monophasic, nor would it explain
the lack of an effect by telenzepine. It is possible that
the enhanced responses are affected by antagonists: a
recent study has shown that some components of
receptor signaling may be generated by an antagonist
(23).
The reason for the distinct sensitivity of the pancreatic muscarinic receptor to telenzepine is uncertain.
One possibility is that a distinct form of muscarinic
receptor is present in the rat pancreatic acinar cell.
Another explanation is that the sensitivity of a receptor
to an antagonist may be tissue specific. For example,
when muscarinic receptors were transfected into CHO
cells, the M3 antagonist 4-DAMP was found to have a
similar affinity for M1 and M3 receptors (7). Thus the
rank order sensitivity of a receptor to antagonists may
change in a tissue-specific manner.
To provide information on the molecular identity of
acinar cell muscarinic receptors, we performed PCR
analysis and demonstrated that message for both the
m1 and m3 subtypes is present in the acinar cells. An
equal amount of cDNA was used in both reactions, but
amplification of m1 and m3 was carried out with
different PCR conditions. Although the quantity of m1
and m3 mRNA in the pancreatic acini cannot be
directly compared, the message for both receptors
appears to be present in the acinar cell.
These molecular data are consistent with the presence of m1 and m3 muscarinic receptor subtypes on the
pancreatic acinar cell. Additional studies will be required to determine the identity of the muscarinic
receptor on the acinar cell responsible for its sensitivity
to telenzepine.
We acknowledge Dr. Mark Katz and Robert Carangelo for performing some of the assays for zymogen processing and secretion and Dr.
Lisa Matovcik for critical evaluation and commentary on this manuscript.
F. S. Gorelick is a recipient of a Clinical Investigator Career
Development Award and a Merit Award from the Dept. of Veterans
Affairs. I. M. Modlin is a recipient of a Merit Award from the Dept. of
Veterans Affairs. L. H. Tang is a recipient of the FIRST Award from
the National Institute of Diabetes and Digestive and Kidney Diseases
(DK-48820).
Address for reprint requests: F. S. Gorelick, GI Research Laboratory, Bldg. 27, Connecticut Health Care VA, West Haven, CT 06516.
Received 9 June 1997; accepted in final form 23 December 1997.
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