Epidermal growth
factor (EGF) has acute inhibitory and chronic stimulatory effects on
gastric acid secretion. Because a cascade of intracellular events
culminating in the activation of a family of serine-threonine protein
kinases called extracellular signal-regulated protein kinases (ERKs) is
known to mediate the actions of EGF, we undertook studies to explore
the functional role of the ERKs in gastric acid secretion. ERK2 was
immunoprecipitated from cell lysates of highly purified (>95%)
gastric canine parietal cells, and its activity was quantified using
in-gel kinase assays. Of the primary gastric secretagogues, carbachol
was the most potent inducer of ERK2 activity. Gastrin and EGF had
weaker stimulatory effects, whereas no induction was noted in response
to histamine. The effect of carbachol appeared to be independent of
Ca2+ signaling. PD-98059, a
selective inhibitor of the upstream ERK activator mitogen-activated
protein kinase/ERK kinase, dose-dependently inhibited both carbachol-
and EGF-stimulated ERK2 activity, with a maximal effect observed
between 50 and 100 µM. ERKs activation is required for induction of
the early gene c-fos via
phosphorylation of the transcription factor Elk-1 which binds to the
c-fos serum response element (SRE).
Carbachol stimulated a two- to threefold induction of luciferase
activity in cultured parietal cells transfected with either a
SRE-luciferase reporter plasmid or with a chimeric GAL4-ElkC expression
vector and the 5×GAL-luciferase reporter plasmid. To examine the
significance of ERK activation in gastric acid secretion, we tested the
effect of PD-98059 on carbachol-stimulated uptake of
14C-labeled aminopyrine (AP).
Acute inhibition of the ERKs by PD-98059 led to a small increase in AP
uptake and a complete reversal of the acute inhibitory effect of EGF on
AP uptake induced by either carbachol or histamine. In contrast,
exposure of the cells to PD-98059 for 16 h led to a reversal of the
chronic stimulatory effect of EGF on AP uptake induced by carbachol.
Our data led us to conclude that carbachol induces a cascade of events
in parietal cells that results in ERK activation. Although the acute
effect of the ERKs on gastric acid secretion appears to be inhibitory, the activation of transcription factors and of early gene expression could be responsible for its chronic stimulatory effects.
mitogen-activated protein kinase; gastric acid secretion; early
response genes; transcriptional regulation; c-fos
 |
INTRODUCTION |
GASTRIC ACID SECRETION is a complex physiological
process that is regulated by numerous hormones and neurotransmitters.
Carbachol, gastrin, and histamine are among the major gastric acid
secretagogues, and these agents are known to activate multiple signal
transduction pathways upon interaction with their specific parietal
cell receptors. Although carbachol and gastrin increase
Ca2+ mobilization and protein
kinase C activation, histamine is known to induce adenylate cyclase
activation and adenosine 3',5'-cyclic monophosphate (cAMP)
generation (12).
In addition to these well-established signal transduction pathways,
recent studies have indicated that mammalian parietal cells contain
multiple isoforms of a recently discovered family of protein kinases
known as mitogen-activated protein kinases (MAPKs) or extracellular
signal-regulated protein kinases (ERKs) (7, 26). The ERKs are important
elements in a cascade of phosphorylation reactions that is known to
involve upstream protein kinases such as Raf and MAPK/ERK kinase (MEK)
(8, 11). Activation of the ERKs is known to target numerous cellular
proteins, including downstream protein kinases such as 90-kDa S6 kinase
(8, 11) and transcription factors such as Elk-1 that regulate the
activity of the promoter of the early response gene
c-fos through the serum response
element (SRE) (22, 36). Thus the ERKs, via activation of immediate
early gene function and of downstream protein kinases, appear to play a
crucial role in the process of amplification, integration, and
transmission of the extracellular signals from the cell surface to the
nucleus leading to induction of cellular growth and proliferation and,
in some systems, cellular differentiation (8, 11, 17, 23). Our
understanding of the function and physiological role of the ERK pathway
has been improved significantly by the recent development of PD-98059,
a highly specific inhibitor of the ERK activator MEK (1). With the use
of this compound, it was possible to establish, for example, that
activation of the ERK pathway is necessary for the induction of nitric
oxide in cardiac myocytes, whereas it is not needed to mediate insulin stimulation of glucose uptake, lipogenesis, and glycogen synthesis (20,
32).
Epidermal growth factor (EGF) is the prototypic member of a large
family of peptide growth factors that have biological actions on the
function of many organs. In addition to its well-known growth-promoting
properties, EGF has been shown to modulate pituitary hormone
production, pancreatic amylase secretion, insulin synthesis, and
intestinal electrolyte transport (18). One of the best-characterized signal transduction pathways activated in response to EGF stimulation involves the induction of the ERKs and other signaling molecules such
as the early response gene c-fos (11,
17, 23).
In the stomach, EGF affects acid secretion in a divergent fashion.
Under acute conditions, EGF has a long-recognized inhibitory effect on
acid secretion, whereas prolonged administration of EGF increases both
basal and maximal acid secretion in vivo and acid production in
isolated parietal cells in vitro (7, 18, 37). The mechanisms
responsible for this phenomenon are currently only partially
understood. Some studies have suggested that these effects of EGF could
be mediated by the activation of protein tyrosine kinases, since they
are fully reversed by the addition of protein tyrosine kinase
inhibitors (37). In addition, Chew et al. (7) observed that inhibition
of the chronic stimulatory effect of EGF by these agents was associated
with a decrease in phosphorylation of a 44-kDa protein identified as an
ERK isoform. Taken together, these results suggest that the ERKs are
likely to play an important role in the regulation of numerous
physiological functions of the gastric parietal cells.
In this study, we sought to investigate the regulation and the
functional relevance of the ERKs in the process of gastric acid
secretion. Using highly purified canine gastric parietal cells in
primary culture, we were able to demonstrate that although the acute
effect of the ERKs on gastric acid secretion appears to be inhibitory,
the activation of transcription factors and of early gene expression
could be responsible for its chronic stimulatory effects.
 |
MATERIALS AND METHODS |
Plasmids.
5×Gal-Luc (16) was a gift from M. Karin (San Diego, CA). Gal4-ElkC
(22) was a gift from R. Treisman (London, UK). SRE-Luc (39) was
obtained from J. Pessin (Iowa City, IA). pCMV-
Gal was a
gift from M. Uhler (Ann Arbor, MI).
Primary parietal cell preparation and culture.
For preparation of primary parietal cells, we utilized a modification
of the method of Soll (33; see also Refs. 4, 5, 13). The mucosal layer
of freshly obtained canine gastric fundus was bluntly separated from
the submucosa and rinsed in Hanks' balanced salt solution containing
0.1% bovine serum albumin (BSA). The cells were then dispersed by
sequential exposure to collagenase (0.35 µg/ml) and 1 mM EDTA, and
parietal cells were enriched by centrifugal elutriation using a Beckman
JE-6B elutriation rotor. Our best preparations contained 70% parietal
cells as determined by hematoxylin and eosin and periodic acid-Schiff
reagent staining. The parietal cells were further purified by
centrifugation through density gradients generated by 50% Percoll
(Pharmacia Biotech, Piscataway, NJ) at 30,000 g for 20 min. The cell fraction at
density = 1.05 consisted of 95-100% parietal cells.
In some experiments, the isolated parietal cells (0.8 × 106 cells/well) were cultured for
16 h according to the method of Chew et al. (6) with some modifications
(25). Briefly, the cells were cultured in Ham's F-12-Dulbecco's
modified Eagle's medium (1:1) containing 50 µg/ml gentamycin, 50 U/ml penicillin G, and 2% dimethyl sulfoxide (DMSO; Sigma, St. Louis,
MO) on 12-well culture dishes (Coster, MA) coated with 150 µl of
H2O-diluted (1:5) growth
factor-reduced Matrigel (Becton Dickinson, Bedford, MA). For our
studies, the parietal cells were incubated with carbachol (1-100
µM; Sigma), histamine (100 µM; Sigma), gastrin (10 nM; G17; Bachem,
Torrance, CA), EGF (0.1-100 nM; Becton Dickinson), and ionomycin
(1 µM; Calbiochem, La Jolla, CA) for various time periods. In some
experiments, either PD-98059 (10-100 µM; a gift from Dr. Alan
Saltiel, Parke Davis Pharmaceutical Research, Ann Arbor, MI) or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 10 µM; Calbiochem) was added before the addition of the secretagogues. BAPTA-AM, ionomycin, and
PD-98059 were dissolved in DMSO. All other test substances were
dissolved in culture medium. Control experiments with untreated cells
were performed by incubating the cells in either vehicle (0.1% DMSO)
or incubation buffer without the test substances. ERK2 induction and
aminopyrine uptake in parietal cells obtained from the enriched
elutriated fractions were identical to those detected in cells further
purified by Percoll density gradients.
Transfection of primary cultured parietal cells.
Before transfection, the cells were washed once with 1 ml Opti-MEM I
serum-reduced media (GIBCO, Gaithersburg, MD) and fed with 400 µl
Opti-MEM I medium supplemented with 2% DMSO. The cells were
transfected with 5 µg of the luciferase reporter plasmids and, where
indicated, with 0.5 µg of the expression vectors. Transfections were
carried out using Lipofectin (GIBCO, Grand Island, NY) as previously
described (25). The day after transfection, the medium was removed and
the cells were fed with serum-free media for 24 h, then incubated for 5 h with the test substances. At the end of the incubation period, the
cells were washed twice with cold calcium-free phosphate-buffered
saline. Then, 100 µl cell lysis buffer [25 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.8, 2 mM EDTA, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100] were added and
incubated at room temperature for 15 min. The cells were then scraped
and transferred to Eppendorf tubes. After quick centrifugation to pellet large debris, the supernatant was transferred to a new tube. An
aliquot of cell lysate (20 µl) was mixed with 100 µl luciferase
assay reagent [20 mM tricine, 1.07 mM
(MgCO3)4Mg(OH)2 · 5H2O,
2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 µM coenzyme A, 470 µM luciferin, and 530 µM ATP, final pH 7.8], and luminescent intensity was measured
for 10 s utilizing a Lumat LB9501 luminometer (Berthold, Germany).
Luciferase activity was expressed as relative light units and
normalized for
-galactosidase activity.
-Galactosidase activity
was measured by the luminescent light derived from 10 µl of each
sample incubated in 100 µl of Lumi-Gal 530 (Lumigen, Southfield, MI)
and used to correct the luciferase assay data for transfection
efficiency.
Immunoprecipitations and in-gel ERK2 assay.
Immunoprecipitations and in-gel ERK2 assays were performed according to
previously described techniques (30, 35) with minor modifications. The
parietal cells were lysed in 500 µl of lysis buffer [50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1.5 mM
MgCl2, 1 mM
Na3VO4,
10 mM NaF, 10 mM
Na4P2O7 · 10H20,
1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF; ICN
Biomedicals, Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml
aprotinin], transferred into Microfuge tubes, and spun at 16,000 g for 20 min at 4°C. Equal amounts
of protein from each treatment group (1,000 µg) were incubated with
an anti-ERK2 specific antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) and mixed on a rotating platform for 3 h at 4°C. Protein
concentrations were measured by the Bradford method (3). Control
experiments were conducted by mixing aliquots of the samples with
identical volumes of nonimmune sera. Aliquots of protein A-Sepharose
(50 µl) (Pharmacia Biotech) were then added, and the solutions were
mixed for one additional hour. After centrifugation, the pellets were
washed four times with lysis buffer. The samples were resuspended in 20 µl of electrophoresis buffer [for 10 ml: 1 ml glycerol, 0.5 ml
2-mercaptoethanol, 3 ml of 10% sodium dodecyl sulfate (SDS), 1.25 ml
of 1 M Tris buffer, 2 ml of 0.1% bromphenol blue, and 0.6 g
urea], boiled for 5 min, and applied to a 10% SDS-polyacrylamide
gel containing 0.5 mg/ml myelin basic protein (Sigma). After
electrophoresis, the gel was washed with two changes of 20% 2-propanol
in 50 mM Tris (pH 8.0) for 1 h and then with two changes of 50 mM Tris
(pH 8.0) containing 5 mM 2-mercaptoethanol for 1 h. The
enzyme was denatured by incubating the gel with two changes of 6 M
guanidine HCl for 1 h and then renaturated with five changes of 50 mM
Tris (pH 8.0) containing 0.04% Tween 40 and 5 mM 2-mercaptoethanol for
1 h. The kinase reaction was performed in conditions inhibitory to
cyclic nucleotide-dependent protein kinase and
Ca2+-dependent protein kinases, by
incubating the gel at 25°C for 1 h with 40 mM HEPES (pH 8.0)
containing 0.5 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 10 mM MgCl2, 2 µM
cAMP-dependent protein kinase inhibitor peptide (Sigma), 40 µM ATP,
and 2.5 µCi/ml [
-32P]ATP (6,000 Ci/mmol) (Amersham Life Science, Arlington Heights, IL). After
incubation, the gel was washed with a 5% (wt/vol) trichloroacetic acid
solution containing 1% (wt/vol) sodium pyrophosphate, dried, and
subjected to autoradiography.
Western blots.
Equal amounts of immunoprecipitated ERK2 from each treatment group were
loaded on a 10% SDS-polyacrylamide gel and run at 20 A for 8 h. The
gel was transferred on an Immobilon-P transfer membrane (Millipore,
Bedford, MA) using a TE 42 Transphor Electro-Transfer Unit (Hoefer
Scientific Instruments, San Francisco, CA) in 25 mM Tris, 150 mM
glycine, and 20% methanol. After transfer, the membrane was blocked in
100 ml TBST (20 mM Tris, 0.15 M NaCl, and 0.3% Tween) and 5% BSA for
2 h and incubated for 20 min at 37°C in TBST and 1% BSA containing
a specific antiphosphotyrosine antibody directly conjugated to
horseradish peroxidase (RC20, Transduction Laboratories, Lexington, KY)
(1:2,500). At the end of the incubation period, the membrane was washed
in TBST for 15 min at room temperature and then exposed to the Amersham
enhanced chemiluminescence detection system (Amersham Life Science)
according to the manufacturer's instructions.
Aminopyrine uptake.
The accumulation of
[14C]aminopyrine
(Amersham Life Science) was used as an indicator of acid production by
parietal cells. A 1-ml aliquot of acutely isolated parietal cells (2 × 106) suspended in
Earle's balanced salt solution (EBSS) was incubated with 0.1 µCi
[14C]aminopyrine and
the various substances to be tested. In some experiments, the cells
were resuspended in Ca2+-free EBSS
supplemented with 1 mM EGTA. After a 20-min incubation at 37°C, the
cells were centrifuged and the radioactivity of the pellet was
quantified in a liquid scintillation counter as previously reported
(18). In some experiments, the parietal cells were cultured in Ham's
F-12-Dulbecco's modified Eagle's medium (1:1) (6, 18, 25) with or
without EGF (10 nM) for 18 h in the presence or absence of PD-98059 (50 µM). At the end of the incubation period, the cells were washed once
with EBSS, preincubated with 0.1 µCi
[14C]aminopyrine for
30 min, and then stimulated with carbachol (100 µM) for 30 min.
Parietal cells were lysed with 500 µl of 1% Triton X-100, and the
radioactivity of lysate was quantified in a liquid scintillation
counter.
Data analysis.
Data are expressed as means ± SE;
n is equal to the number of separate
dog preparations from which the parietal cells were obtained.
Statistical analysis was performed using Student's
t-test. P values <0.05 were considered to be
significant.
 |
RESULTS |
The ERKs are known to be rapidly induced in response to a wide variety
of stimuli. Accordingly, we examined the effect of gastric acid
secretagogues on ERK activation using isolated gastric parietal cells.
For these experiments, we immunoprecipitated the ERKs using an antibody
that preferentially recognized ERK2 from parietal cell lysates, and we
quantitated its activity using in-gel kinase assays. As shown in Fig.
1, of the primary gastric acid secretagogues, carbachol (100 µM) was the most potent inducer of ERK2
activity (26.5 ± 7.3-fold induction over control,
n = 15). Gastrin (10 nM) and EGF (10 nM) had weaker stimulatory effects (3.4 ± 1.1-fold induction over
control, n = 8, and 13.0 ± 2.9-fold induction over control, n = 11, in the presence of gastrin and EGF, respectively), whereas no
induction was noted in response to histamine (100 µM) (1.0 ± 0.2-fold induction over control, n = 7). The effect of carbachol was dose (1-100 µM) and time
dependent, with a maximal stimulatory effect detected after 5 min of
incubation (data not shown). Similarly, EGF (0.1-100 nM) time- and
dose-dependently induced ERK2 activity with a maximal stimulatory
effect detected after 5 min of incubation. No difference in ERK2
induction was detected between 10 and 100 nM EGF (data not shown).

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Fig. 1.
Effect of gastric acid secretagogues on extracellular signal-regulated
protein kinase 2 (ERK2) activity. ERK2 in lysates from parietal cells
stimulated with histamine (100 µM), carbachol (100 µM), gastrin (10 nM), and epidermal growth factor (EGF; 10 nM) was immunoprecipitated,
and its activity was measured by in-gel kinase assays.
A: representative assays obtained with
a single parietal cell preparation. B:
results obtained from densitometric analysis of blots from at least 7 parietal cell preparations. Data are expressed as fold induction over
control and are means ± SE. OD, optical density.
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Activation of the ERKs requires phosphorylation on both tyrosine and
threonine residues by the dual specificity protein kinase MEK (8, 11).
Accordingly, we first tested the effect of the recently described,
highly specific MEK inhibitor PD-98059 on EGF-stimulated ERK2 activity.
For these experiments, the parietal cells were pretreated for 15 min
with either different doses of PD-98059 or vehicle (0.1% DMSO) before
the addition of 10 nM EGF. PD-98059 did not affect parietal cell
viability measured by trypan blue exclusion at any of the doses tested
(data not shown). As shown in Fig. 2,
PD-98059 (10-50 µM) dose-dependently inhibited EGF-stimulated
ERK2 activity with a complete inhibitory effect observed at the dose of
50 µM. We then tested the effect of PD-98059 on carbachol-stimulated
ERK2 activity. For these experiments, the parietal cells were
pretreated for 15 min with either vehicle (0.1% DMSO) or different
doses of PD-98059 before the addition of 100 µM carbachol. As shown
in Fig. 3, PD-98059 (10-100 µM) dose-dependently inhibited, although not completely,
carbachol-stimulated ERK2 activity, with a maximal effect observed
between 50 and 100 µM (44.9 ± 8.5 and 34.7 ± 8.2%
of carbachol-stimulated ERK2 activity in the presence of 50 and 100 µM PD-98059, respectively, n = 3). Neither PD-98059 nor vehicle (0.1% DMSO) had any effect on ERK2 activity (Fig. 4). In a similar fashion, 50 µM PD-98059 inhibited carbachol-stimulated ERK2 phosphorylation as
measured by Western blot analysis using an antiphosphotyrosine
antibody, confirming the involvement of MEK in ERK2 activation and
phosphorylation (Fig. 5).

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Fig. 2.
Effect of PD-98059 on EGF-stimulated ERK2 activity. ERK2 in lysates
from parietal cells stimulated with EGF (10 nM) in presence of either
vehicle (0.1% DMSO) or PD-98059 (10-50 µM) was
immunoprecipitated, and its activity was measured by in-gel kinase
assays (A). A linear transformation
of densitometric analysis of autoradiograms is depicted
(B). Data are expressed as
percentage of 10 nM EGF-stimulated ERK2 activity in absence of
PD-98059. Identical results were obtained in 1 other separate
experiment.
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Fig. 3.
Effect of PD-98059 on carbachol-stimulated ERK2 activity. ERK2 in
lysates from parietal cells stimulated with carbachol (100 µM) in
presence of either vehicle (0.1% DMSO) or PD-98059 (10-100 µM)
was immunoprecipitated, and its activity was measured by in-gel kinase
assays. A: representative assays
obtained with a single parietal cell preparation.
B: results obtained from densitometric
analysis of blots from 3 parietal cell preparations. Data are expressed
as percentage of 100 µM carbachol-stimulated ERK2 activity in absence
of PD-98059 and are means ± SE.
* P < 0.05.
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Fig. 4.
Effect of PD-98059 and DMSO on ERK2 activity. ERK2 in lysates from
parietal cells treated with carbachol (100 µM) and either DMSO
(0.1%) (A) or PD-98059 (50 µM)
(B) was immunoprecipitated, and its
activity was measured by in-gel kinase assays. These data represent
results from a single parietal cell preparation.
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Fig. 5.
Effect of PD-98059 on carbachol-stimulated ERK2 phosphorylation. ERK2
in lysates from parietal cells stimulated with carbachol (100 µM) in
presence of either vehicle (0.1% DMSO) or PD-98059 (50 µM) was
immunoprecipitated, and its phosphorylation was studied by Western
blots using an antiphosphotyrosine antibody. These data represent
results from a single parietal cell preparation. Identical results were
obtained in experiments with 3 other separate parietal cell
preparations. IgG, immunoglobulin G.
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Because Ca2+ is an important
mediator of carbachol actions on gastric parietal cells (12, 13), we
undertook studies to examine the role of both intra- and extracellular
Ca2+ on carbachol induction of
ERK2 activity. As shown in Fig. 6, both
chelation of intracellular Ca2+ by
preincubation of the cells for 10 min with either vehicle (0.1% DMSO)
or with the cell-permeable Ca2+
chelator BAPTA-AM (10 µM) and deprivation of extracellular
Ca2+ by incubation of the cells in
Ca2+-free EBSS containing 1 mM
EGTA failed to affect the stimulatory action of carbachol on ERK2
activity. Similarly, no induction was observed in the presence of 1 µM ionomycin, an agent known to induce
Ca2+ influx into cells. Thus
carbachol appears to activate ERK2 in parietal cells via
Ca2+-independent pathways. In
contrast, incubation of the parietal cells in either
Ca2+-free medium or in the
presence of BAPTA-AM completely inhibited 100 µM carbachol-stimulated
aminopyrine uptake [8.1 ± 1.0- vs. 1.5 ± 0.4-fold induction over control in the presence of either vehicle
(0.1% DMSO) or BAPTA-AM and 7.4 ± 1.7- vs. 1.3 ± 0.1-fold induction over control in the absence and presence of
Ca2+-free medium, respectively,
n = 3]. These results confirm
the notion that Ca2+ signaling is
of crucial importance in the stimulatory action of carbachol on gastric
acid secretion but not on ERK2 activation (Fig. 7).

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Fig. 6.
Effect of intra- and extracellular
Ca2+ on carbachol-stimulated ERK2
activity. ERK2 in lysates from parietal cells stimulated with either
ionomycin (1 µM) or carbachol (100 µM) in presence of vehicle
(0.1% DMSO), cell-permeable Ca2+
chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester (BAPTA-AM; 10 µM), or
Ca2+-free medium was
immunoprecipitated, and its activity was measured by in-gel kinase
assays. These data represent results from a single parietal cell
preparation. Identical results were obtained in experiments with 3 other separate parietal cell preparations.
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Fig. 7.
Effect of intra- and extracellular
Ca2+ on carbachol (C)-stimulated
[14C]aminopyrine
uptake. Canine gastric parietal cells were incubated with 100 µM
carbachol, in combination with either vehicle (0.1% DMSO) or BAPTA-AM
(10 µM) (A). Identical results
were obtained when cells were treated with 100 µM carbachol in
presence of Ca2+-free medium, as
shown in B. Data are expressed as fold
induction over control and are means ± SE
(n = 3).
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One of the best-characterized pathways leading to induction of the
early response gene c-fos involves
ERK2 activation and phosphorylation of Elk-1, a transcription factor
known to bind to the c-fos SRE (36).
We therefore sought to investigate the effects of carbachol on
c-fos gene transcription regulated by the SRE. For these experiments, we transfected Percoll-purified cultured parietal cells with luciferase reporter plasmids containing the c-fos SRE upstream of the
thymidine kinase (TK) gene minimal promoter and the firefly luciferase
reporter gene together with the pCMV-
Gal expression vector. As
depicted in Fig. 8, 10 µM carbachol stimulated a
twofold increase in luciferase activity, and this induction was
completely inhibited by 50 µM PD-98059 (2.19 ± 0.11- vs. 0.97 ± 0.12-fold induction over control in the absence and presence of
50 µM PD-98059, respectively, n = 4). In contrast, parietal cells transfected with a plasmid containing the TK gene minimal promoter but devoid of the SRE exhibited no induction in response to carbachol (data not shown). To examine if
carbachol induction of the c-fos SRE
was mediated by stimulation of Elk-1 transcriptional activity, we used
a yeast hybrid system involving cotransfection of the parietal cells
with the Gal4-ElkC and the pCMV-
Gal expression vectors and the
5×Gal luciferase reporter plasmids. In this system, Gal4-ElkC
transactivates and stimulates luciferase activity only if the carboxy
terminus of Elk-1 is phosphorylated by the ERKs (22). As shown on Fig.
9, carbachol (10 µM) stimulated a threefold increase
in luciferase activity, and this effect was blocked by 50 µM PD-98059
(3.53 ± 0.54- vs. 1.42 ± 0.24-fold induction over control in
the absence and presence of 50 µM PD-98059, respectively,
n = 6). Cotransfection of the cells
with the expression vector lacking Gal4-ElkC coding sequences and the
5×Gal luciferase reporter plasmid gave only background luciferase
activity (data not shown). Taken together, these data indicate that
carbachol activates a cascade of phosphorylation reactions that targets
MEK and the ERKs, leading to induction of early gene transcription via
phosphorylation and transcriptional activation of Elk-1.

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Fig. 8.
Effect of PD-98059 (PD) on carbachol (C)-stimulated serum response
element (SRE) transcriptional activity. Canine gastric parietal cells
were transfected with plasmids SRE-Luc and pCMV- Gal and treated with
10 µM carbachol alone or in combination with 50 µM PD-98059. Data
are expressed as fold induction over control and are means ± SE
(n = 4). RLU, relative light units.
* P < 0.05.
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Fig. 9.
Effect of carbachol (C) and PD-98059 (PD) on Elk-1 transcriptional
activity. Canine gastric parietal cells were transfected with Gal4-ElkC
expression vector, 5×Gal luciferase reporter plasmid, and pCMV- Gal
vector and treated with 10 µM carbachol alone or in combination with
50 µM PD-98059. Data are expressed as fold induction over control and
are means ± SE (n = 6).
* P < 0.01.
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We then examined the functional significance of the ERK pathway in the
acid secretory function of the parietal cells. For these experiments,
we tested the effects of PD-98059 on carbachol-stimulated uptake of
[14C]aminopyrine. As
depicted in Fig. 10, addition of 50 µM
PD-98059 led to a small but reproducible and significant increase in
aminopyrine uptake induced by 100 µM carbachol [9.4 ± 1.3- vs. 10.7 ± 1.6-fold induction over control in the presence of
either vehicle (0.1% DMSO) or 50 µM PD-98059, respectively,
n = 9]. Thus acute activation of
the ERKs may not be important for stimulation of gastric acid secretion
by carbachol but may, in fact, have a modest inhibitory effect. Because
EGF has been shown to have acute inhibitory and chronic stimulatory
effects on gastric acid secretion (21-23) and the ERKs are known
to mediate some of the cellular effects of EGF, we conducted studies to
examine whether PD-98059 influenced EGF inhibition of parietal cell
activity stimulated by either carbachol or histamine. For these
studies, the parietal cells were pretreated for 15 min with either
vehicle (0.1% DMSO) or PD-98059 (50 µM) before the addition of EGF
(10 nM). After 30 min of incubation, the cells were stimulated with
carbachol (100 µM) or histamine (100 µM) for an additional 20 min.
Under these conditions, EGF inhibited both carbachol- and
histamine-stimulated aminopyrine uptake according to previously
published observations (38), and PD-98059 completely reversed this
effect [10.02 ± 1.89- vs. 8.08 ± 1.52- and 11.28 ± 1.97-fold induction over control in the presence of carbachol,
carbachol after preincubation with EGF, and carbachol after
preincubation with EGF in combination with PD-98059, respectively,
n = 6; and 2.57 ± 0.4- vs. 1.91 ± 0.23- and 6.23 ± 0.93-fold induction over control in the
presence of histamine, histamine after preincubation with EGF, or
histamine after preincubation with EGF in combination with PD-98059,
respectively, n = 6]. PD-98059
(50 µM) had no effect on basal aminopyrine uptake (1.03 ± 0.13-fold induction over control,
n = 8; Fig.
11). Similarly, addition of 0.1% DMSO, EGF, or EGF in
combination with PD-98059 had no effect on basal aminopyrine uptake
(data not shown). These data confirm the notion that rapid activation
of the ERKs results in inhibition of gastric acid secretion stimulated
by different classes of gastric acid secretagogues. We then examined
whether PD-98059 influenced the chronic acid-stimulatory effect of EGF. For these experiments, the parietal cells were cultured in 10 nM EGF in
the presence of either vehicle (0.1% DMSO) or PD-98059 (50 µM) for
16 h and then stimulated with carbachol (100 µM) for 30 min. As shown
in Fig. 12, EGF enhancement of carbachol-stimulated aminopyrine uptake was completely blocked by pretreatment of the cells
with 50 µM PD-98059 (4.16 ± 1- vs. 5.06 ± 1- and 3.25 ± 0.79-fold induction over control in the presence of carbachol, carbachol after preincubation with EGF, or carbachol after
preincubation with EGF in combination with PD-98059, respectively,
n = 5). Exposure of the parietal cells
to PD-98059 (50 µM) alone for 16 h had no effect on basal aminopyrine
uptake (0.81 ± 0.23-fold induction over control,
n = 5). These data suggest that
prolonged activation of the ERKs could be responsible for stimulatory
effects on gastric acid
production.

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Fig. 10.
Effect of PD-98059 (PD) on carbachol (C)-stimulated
[14C]aminopyrine
uptake. Canine gastric parietal cells were incubated with 100 µM
carbachol after preincubation of cells with either vehicle (0.1% DMSO)
or 50 µM PD-98059. Data are expressed as fold induction over control
and are means ± SE (n = 9).
* P < 0.01.
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Fig. 11.
Effect of PD-98059 (PD) and EGF on carbachol (C)- and histamine
(H)-stimulated
[14C]aminopyrine
uptake. Canine gastric parietal cells were incubated for 20 min with 50 µM PD-98059 (n = 8) or with either
100 µM carbachol or 100 µM histamine, alone or after
preincubation for 30 min with 10 nM EGF in presence of either vehicle
(0.1% DMSO) or 50 µM PD-98059 (n = 6). Data are expressed as fold induction over control and are means ± SE. * P < 0.05.
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Fig. 12.
Effect of PD-98059 (PD) and EGF on carbachol (C)-stimulated
[14C]aminopyrine
uptake. Canine gastric parietal cells were incubated for 16 h with 50 µM PD-98059 or for 20 min with 100 µM carbachol, alone or after
preincubation for 16 h with 10 nM EGF in presence of either vehicle
(0.1% DMSO) or 50 µM PD-98059. Data are expressed as fold induction
over control and are means ± SE (n = 5). * P < 0.05.
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DISCUSSION |
The ERKs or MAPKs are key elements in a cascade of phosphorylation
reactions that is triggered by the interaction of hormones, growth
factors, and neurotransmitters with their specific cellular receptors
(8, 11). Numerous studies have shown that these enzymes are responsible
for the regulation of a wide variety of physiological functions.
Activation of the ERKs is in fact necessary for both the induction of
cellular proliferation and for the expression of highly differentiated
cellular phenotypes (8, 11, 23). Recent studies have shown that seven
transmembrane receptors, known to interact with heterotrimeric
GTP-binding proteins to transduce extracellular signals to downstream
effector molecules (14, 27), are able to activate Ras, Raf, MEK, ERKs,
and early response genes in a fashion similar to that employed by
receptor tyrosine kinases (2, 9, 10, 15, 28, 29, 30, 31, 35). These
findings suggest that common signaling pathways are shared by a diverse
array of extracellular messengers that are likely to act in concert at
the level of the cell surface to control the expression of diverse
physiological functions.
The gastric parietal cell is a highly differentiated cell that secretes
gastric acid in response to a broad range of physiological stimuli.
Carbachol, gastrin, and histamine are among the major gastric acid
secretagogues (12). These agents are known to interact with specific
seven-transmembrane receptors present on the surface of the parietal
cell to stimulate gastric acid secretion. Recent studies have indicated
that mammalian parietal cells have receptors for EGF and that this
agent regulates gastric acid secretion in a complex manner. Under acute
conditions, EGF has a long-recognized inhibitory effect on acid
secretion, whereas prolonged administration of EGF increases both basal
and maximal acid secretion in vivo and in vitro (7, 18, 37). The
mechanisms responsible for this phenomenon are currently only partially
understood. Some studies have suggested that these effects of EGF could
be mediated by the activation of protein tyrosine kinases since they
are fully reversed by the addition of protein tyrosine kinase
inhibitors (37). In our study we have examined the physiological
regulation of the ERKs in response to carbachol, gastrin, histamine,
and EGF and found that of these agents, carbachol was the most potent inducer. Accordingly, we opted to focus our efforts primarily on the
understanding of the intracellular pathways that target the ERKs in
response to carbachol and to analyze the functional relevance of this
phenomenon. For these experiments, we took advantage of the recent
discovery of PD-98059, a highly selective MEK inhibitor. This compound
is known to prevent the activation of MEK1 and to a lesser degree of
MEK2 in response to a wide variety of agonists (1). The extent of
inhibition, however, seems to depend on how potently MEK or c-Raf is
activated by any particular agonist. In fact, in Swiss 3T3 cells, 50 µM PD-98059 almost completely prevents the activation of ERK2 in
response to insulin (the weakest activator of MEK in these cells),
whereas it inhibits the strong stimulatory effect of platelet-derived
growth factor, serum,
12-O-tetradecanoylphorbol 13-acetate, and bombesin by only 60-80% (1, 31). In
our study, we demonstrated that PD-98059 dose dependently inhibited
both carbachol- and EGF-stimulated ERK2 induction, with a maximal
effect detected between 50 and 100 µM. In agreement with previous
observations, we found that PD-98059 led to a partial inhibition of
carbachol-stimulated ERK2 activity even in the presence of lower doses
of carbachol (10 µM) (data not shown), whereas it completely
inhibited ERK2 activity stimulated by EGF, a weaker inducer of the ERKs
in the parietal cells.
Numerous studies have examined the role of
Ca2+ signaling in activation of
the ERKs. Although in some systems, such as cardiac myocytes or PC12
cells, induction of the ERKs requires
Ca2+ influx or
Ca2+ release from intracellular
stores (21), in pancreatic acinar cells this process appears to occur
via Ca2+-independent pathways
(15). In agreement with these findings, we observed that activation of
the ERKs in the canine parietal cells is not affected by changes in
either intra- or extracellular Ca2+, although this pathway is
known to play a crucial role in the process of gastric acid secretion
in these cells. Thus carbachol activates the ERKs in gastric canine
parietal cells through MEK-dependent, Ca2+-independent
mechanisms.
In previous studies we have shown that carbachol stimulates the
expression of the early response gene
c-fos in isolated gastric canine
parietal cells (34). Accordingly, we examined whether carbachol
induction of the ERKs would result in
c-fos induction. Transcription of the
c-fos gene is under the control of
numerous DNA-regulatory elements. The SRE, in particular, is known to
be the target of growth factor-activated signal transduction pathways (36). Transcriptional activation of the SRE is under the control of
numerous nuclear proteins that are known to assemble on the gene
forming specific DNA-protein complexes. Some of these transcription factors are thought to be bound constitutively to the DNA and to be
phosphorylated rapidly and activated in response to cellular stimulation by growth factors. Elk-1 and SAP-1 are members of a
well-characterized family of transcription factors that are known to
bind to the SRE and to interact with the serum response factor to form
a ternary complex that plays an important role in the activation of
c-fos transcription (36). In response
to a wide variety of extracellular signals, these nuclear proteins are
rapidly phosphorylated and transcriptionally activated by the ERKs,
resulting in stimulation of c-fos gene
expression (24, 36). In contrast to these findings, ternary complex
formation by a third member of this family, SAP-2, seems to be weak and unaffected by serum stimulation (36). In our study, we have shown that
in response to carbachol, the ERKs are rapidly activated and that this
effect translates into induction of
c-fos expression via phosphorylation
and transcriptional activation of Elk-1. Thus carbachol initiates a
program of cellular activation that targets the ERKs and
c-fos and that is likely, in the end,
to induce the transcription of numerous other yet unidentified genes
that might play a crucial role in a broad array of physiological
functions of the gastric parietal cell. Further studies will be
required to elucidate the role of SAP-1 and other nuclear proteins in
the regulation of SRE transcriptional activation in these cells.
We then examined the functional role of the ERKs on gastric acid
secretion. In the presence of 50 µM PD-98059, we observed a small,
albeit very reproducible, increase in aminopyrine uptake, suggesting
that acute activation of the ERKs does not play any role in stimulation
of gastric acid production but that, on the contrary, it might have a
modest inhibitory function, possibly through the phosphorylation of yet
unidentified cellular substrates. Because the ERKs are known to mediate
some of the cellular effects of EGF, we also examined whether PD-98059
influenced EGF inhibition of parietal cell activity stimulated by
either carbachol or histamine, agents known to stimulate gastric acid
secretion via different signal transduction pathways. In these
experiments, we observed that this compound completely reversed the
acute inhibitory effect of EGF on aminopyrine uptake stimulated by
carbachol, whereas it potentiated the induction observed in response to
histamine. Similar results were reported by Tsunoda et al. (37), who
observed a potentiation of histamine-stimulated aminopyrine uptake in
isolated rabbit parietal cells in the presence of EGF and the protein
tyrosine kinase inhibitor genistein. These data indicate that
activation of both protein tyrosine kinases and ERKs leads to the
induction of an inhibitory pathway that regulates gastric acid
secretion in response to different classes of gastric acid
secretagogues. In addition, the potentiation observed in the presence
of histamine, but not carbachol, in combination with either PD-98059 or
genistein indicates that induction of the MAPK pathway exerts a
specific inhibitory effect on still undefined components of histamine
signaling in the gastric parietal cells. It is possible that inhibition of the MAPK pathway could alter the coupling of the histamine receptor
to Gs, thus regulating receptor
function and coupling to downstream effectors (37). Alternatively, MAPK
could activate one or multiple phosphatases that could in turn
negatively regulate histamine stimulation of gastric acid secretion.
However, the exact mechanisms responsible for this phenomenon are still
unknown, and additional studies will be necessary to further dissect
this interesting observation.
Recent reports have shown that the acute inhibitory effect of EGF on
gastric acid secretion could be mediated by a protein kinase C
(PKC)-dependent pathway (38). In this report, we did not examine the
specific role of PKC on gastric acid production and on ERK activation.
However, because PKC has been shown to activate c-Raf directly (19), it
seems reasonable to speculate that the acute inhibitory effect of EGF
on aminopyrine uptake could be mediated by a signaling cascade
involving activation of PKC, c-Raf, and MEK and leading in the end to
induction of the ERKs. Further studies will be needed to explore this
hypothesis.
In contrast to what was observed in the acute studies, PD-98059
significantly inhibited the chronic stimulatory effect of EGF on
carbachol-stimulated aminopyrine uptake, confirming the notion that
prolonged activation of the ERKs results in induction of gastric acid
secretion. This effect could be attributed to the ability of the ERKs
to induce Elk-1 transcriptional activation and
c-fos gene expression. In fact,
because we previously reported that both EGF and carbachol are able to
stimulate the expression of the
H+-K+-
adenosinetriphosphatase gene in isolated canine gastric parietal cells
(5, 18), it is possible that this gene contains specific DNA regulatory
elements that could receive input from signaling pathways involving the
ERKs and c-fos.
Our data led us to conclude that carbachol induces a cascade of events
in parietal cells that results in ERK activation. Although the acute
effect of the ERKs on gastric acid secretion appears to be inhibitory,
the activation of transcription factors and of early gene expression
could be responsible for its chronic stimulatory effects.
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK) Grants RO1-DK-33500 and R01-DK-34306 and
funds from the University of Michigan Gastrointestinal Peptide Research
Center (NIDDK Grant P30-DK-34933). A. Todisco is a recipient of an
American Gastroenterological Association Industry Research Scholar
Award and a Clinical Investigator Award from the National Institutes of
Health (NIDDK Grant K08-DK-02336).
Address for reprint requests: A. Todisco, 6520 MSRB I, Ann Arbor, MI
48109-0682.