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J Biol Chem, Vol. 273, Issue 42, 27111-27117, October 16, 1998
From the Department of Medicine, University of California, San
Diego, School of Medicine, San Diego, California 92103
We have examined the role of tyrosine
phosphorylation in regulation of calcium-dependent chloride
secretion across T84 colonic epithelial cells. The
calcium-mediated agonist carbachol (CCh, 100 µM)
stimulated a time-dependent increase in tyrosine
phosphorylation of a range of proteins (with molecular masses ranging
up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor,
genistein (5 µM), significantly potentiated chloride
secretory responses to CCh, indicating a role for CCh-stimulated
tyrosine phosphorylation in negative regulation of CCh-stimulated
secretory responses. Further studies revealed that CCh stimulated an
increase in both phosphorylation and activity of the extracellular
signal-regulated kinase (ERK) isoforms of mitogen-activated protein
kinase. Chloride secretory responses to CCh were also potentiated by
the mitogen-activated protein kinase inhibitor, PD98059 (20 µM). Phosphorylation of ERK in response to CCh was
mimicked by the protein kinase C (PKC) activator, phorbol myristate
acetate (100 nM), but was not altered by the PKC inhibitor
GF 109203X (1 µM). ERK phosphorylation was also induced
by epidermal growth factor (EGF) (100 ng/ml).
Immunoprecipitation/Western blot studies revealed that CCh stimulated
tyrosine phosphorylation of the EGF receptor (EGFr) and increased
co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the
EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 µM), reversed CCh-stimulated phosphorylation of both EGFr
and ERK. Tyrphostin AG1478 also potentiated chloride secretory
responses to CCh. We conclude that CCh activates ERK in T84
cells via a mechanism involving transactivation of the EGFr, and that
this pathway constitutes an inhibitory signaling pathway by which
chloride secretory responses to CCh may be negatively regulated.
Chloride secretion across intestinal epithelial cells is under
close regulation by hormonal, neural, paracrine, and endocrine factors.
Such factors stimulate chloride secretion by binding to specific
receptors on the surface of epithelial cells and consequently stimulating elevations in the levels of intracellular second
messengers, such as cyclic nucleotides and calcium. In turn, increases
in the levels of intracellular messengers activate the transport proteins that comprise the chloride secretory mechanism (1). However,
even though both calcium and cyclic nucleotides are both capable of
stimulating epithelial chloride secretion, it has been known for many
years that these two classes of intracellular messengers stimulate
temporally distinctive chloride secretory responses. While responses to
cyclic nucleotide-mediated agonists, such as vasoactive intestinal
peptide, are sustained, secretory responses to calcium-mediated
agonists, such as the muscarinic agonist, carbachol
(CCh),1 are transient, even
though levels of intracellular calcium remain elevated after the
secretory response has resolved (2-4). This implies that negative
signals exist within intestinal epithelial cells that limit, or
"switch off" epithelial secretory responses to calcium-mediated
agonists. Since epithelial cells are continuously exposed, in
vivo, to endogenously generated hormones and neurotransmitters that stimulate epithelial secretion via calcium-dependent
mechanisms (5), such inhibitory signals may represent an intrinsic
epithelial "braking" mechanism by which excessive chloride, and
consequently water, secretion may be prevented.
Previous work from our laboratory has identified several intracellular
messengers which appear to negatively influence calcium-mediated chloride secretion. Influx of extracellular calcium, generation of
inositol (3,4,5,6)-tetrakisphosphate, and activation of protein kinase
C (PKC) have all been shown to inhibit secretory responses to
calcium-dependent agonists (1). More recently we have shown that receptor tyrosine kinase-dependent signaling pathways,
such as those activated by epidermal growth factor (EGF) also activate intracellular pathways that are inhibitory to
calcium-dependent secretion (6, 7). Furthermore, since
influx of extracellular calcium (8) and stimulation of inositol
(3,4,5,6)-tetrakisphosphate production (9) in response to
calcium-dependent secretagogues have both been demonstrated
to be dependent upon stimulation of tyrosine kinase activity, tyrosine
kinases appear to play important role(s) in negative regulation of
epithelial secretory processes. Indeed, recent studies demonstrate that
tyrosine kinases may function in the regulation of apical cystic
fibrosis transmembrane conductance regulator Cl Although it has been demonstrated previously that
calcium-dependent secretagogues stimulate intestinal
epithelial tyrosine kinase activity (8), there is little
known of the signaling pathways involved in such processes.
However, some insight into how G-protein-coupled receptors (GPCRs),
such as the muscarinic receptor for CCh on colonic epithelia, might be
linked to alterations in intracellular tyrosine kinase activity comes
from studies in other models. In recent years, there has been a growing
body of evidence to suggest that many GPCR agonists can activate the
MAPK signaling cascade in a variety of cell types including epithelial cells (14-19). The MAPK signaling cascade is classically involved in
mediating the mitogenic actions of growth factors, such as EGF, and the
mechanisms by which the EGF receptor (EGFr) is coupled to MAPK
activation have been well elucidated (20). Upon ligand binding the EGFr
becomes autophosphorylated and recruits the adapter molecules, Shc and
Grb2, which bind to tyrosine-phosphorylated residues on the receptor by
virtue of their SH2 domains. Formation of the EGFr-Shc-Grb2 complex
leads to activation of a guanine nucleotide exchange factor, mSOS,
which in turn activates the low molecular weight G-protein,
p21ras. This, in turn brings about activation of the upstream
components of the MAPK cascade, Raf and MEK, ultimately leading to the
stimulation of the ERK isoforms of MAPK. The mechanisms by which GPCRs
stimulate ERK activation are less well understood but current evidence
suggests the involvement of two predominant pathways which may be
differentially activated depending on the nature of receptor coupling
to heterotrimeric G-proteins (21-23). Gi protein-coupled
receptors (GiPCR) appear to stimulate ERK activation
predominantly by a mechanism involving increases in intracellular
tyrosine kinase activity, and which may be mediated by transactivation
of the EGF receptor. In contrast, Gq protein-coupled
receptors (GqPCR) are believed to stimulate ERK
predominantly via a tyrosine kinase-independent pathway mediated by
PKC. However, recent studies indicate that these two pathways for ERK
activation may not be mutually exclusive and that in some systems they
may converge into a common p21ras-mediated pathway (24).
In the present study we sought to elucidate further the involvement of
tyrosine kinase-dependent signaling pathways in negative regulation of calcium-dependent chloride secretion across
T84 colonic epithelial cells. We employed CCh as a
prototypic calcium-mediated secretagogue, which acts via
Gq-protein coupled M3 muscarinic receptors on
colonic epithelial cells to stimulate chloride secretion (25, 26). In
particular, we set out to investigate a possible role for the ERK
isoforms of MAPK in regulation of CCh-stimulated secretion, and to
determine possible mechanisms underlying CCh stimulation of ERK
activity in T84 cells.
Materials--
Carbachol, vasoactive intestinal polypeptide, and
genistein were obtained from Sigma. Tyrphostin AG1478, GF 902103X, and
PD 98059 were obtained from Calbiochem, San Diego, CA. Epidermal growth
factor was obtained from Genzyme, Cambridge, MA. Mouse anti-human EGF
receptor and mouse anti-phosphotyrosine antibodies were obtained from
Upstate Biotechnology Inc., Lake Placid, NY. Rabbit anti-phospho-ERK
antibodies were obtained from New England Biolabs. Tris glycine
electrophoresis gels were obtained from Bio-Rad. All other reagents
were of analytical grade and were obtained commercially.
Cell Culture--
Methods for maintenance of T84
cells in culture were as described previously (27). Briefly,
T84 cells were grown in Dulbecco's modified Eagle's/F-12
media (JRH, Lenexa, KS) supplemented with 5% newborn calf serum. Cells
were passaged by trypsinization. For Ussing chamber/voltage clamp
experiments, approximately 106 cells were seeded onto
collagen-coated polycarbonate filters (Nuclepore, Pleasanton, CA) glued
onto Lexan rings, as described previously (27). For Western blotting
experiments and MAPK assays, 106 cells were seeded onto
30-mm Millicell transwell polycarbonate filters. When grown on
polycarbonate filters T84 cells are known to retain the
polarized phenotype of native colonic epithelia. Cells seeded onto
filters were cultured for 7-10 days prior to use.
Electrophysiological Studies--
Monolayers of T84
cells were mounted in Ussing chambers (window area = 2cm2) and bathed in oxygenated (95% O2, 5%
CO2) Ringers' solution at 37 °C. The composition of the
Ringers' solution was (in mM): 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 120 Cl Immunoprecipitations and Western Blotting--
Polarized
T84 cell monolayers grown on Millicell filters were washed
(twice) with Ringers' solution, allowed to equilibrate for 30 min at
37 °C, and were then stimulated with agonists (± antagonists) for
the times indicated. The reaction was stopped by washing in ice-cold
phosphate-buffered saline and the cells were then lysed in ice-cold
lysis buffer (1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 µg/ml antipain, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM Na+-vanadate, 1 mM
sodium fluoride, and 1 mM EDTA in phosphate-buffered saline) for 45 min. Cells were then scraped into microcentrifuge tubes,
spun at 12,000 rpm for 10 min and the pellet was discarded. Samples
were assayed for protein content (Bio-Rad protein assay kit) and
adjusted so that each sample contained an equal amount of protein. For
immunoprecipitation studies lysates were incubated with mouse
anti-human EGFr for 1 h at 4 °C followed by another 1-h
incubation at 4 °C with protein A-Sepharose. Lysates were then
centrifuged for 3 min at 15,000 rpm and the supernatant was discarded.
The pellets were then washed twice in lysis buffer and twice in
phosphate-buffered saline and were then resuspended in 2 × gel
loading buffer (50 mM Tris, pH 6.8, 2% SDS, 200 mM dithiothreitol, 20% glycerol, 0.2% bromphenol blue).
Samples were boiled for 3 min and were then loaded onto a
polyacrylamide gel and proteins were separated by electrophoresis.
Resolved proteins were transferred overnight at 4 °C onto a
polyvinylidene difluoride membrane (NEN Life Science Products Inc.,
Boston, MA). After transfer the membrane was preblocked with a 1%
solution of blocking buffer (Upstate Biotechnology Inc., Lake Placid,
NY) for 30 min followed by a 1-h incubation with the appropriate
concentration of primary antibody in 1% blocking buffer. After washing
(3 × 10 min) in Tris-buffered saline with 1% Tween (TBST),
membranes were then incubated for 30 min in horseradish
peroxidase-conjugated secondary antibody (anti-mouse or anti-rabbit
IgG; Transduction Laboratories, Lexington, KY) in 1% blocking buffer.
This was followed by three 10-min washes in TBST. Proteins were then
detected using an enhanced chemiluminescence detection kit (Boehringer
Mannheim, Indianapolis, IN). Densitometric analysis of Western blots
was carried out using NIH Image software.
MAP Kinase Assays--
Cells were stimulated, lysed, and
immunoprecipitated with anti-phospho-ERK as described above in the
immunoprecipitation and Western blotting protocol. ERK activity was
determined using a commercially available assay kit (New England
Biolabs). Briefly, immunoprecipates were washed twice in lysis buffer
and twice in MAPK assay buffer. Washed immunoprecipates were then
incubated for 30 min at 30 °C with 100 µM ATP and 1 µg of Elk1 substrate in 50 µl of assay buffer. Reactions were
stopped by the addition of 50 µl of 2 × gel loading buffer and
samples were boiled for 3 min prior to SDS-PAGE. Phosphorylation of
Elk1 by MAPK was determined by Western blotting (as described above)
with antibodies specific for the phosphorylated form of Elk1.
Statistical Analyses--
All data are expressed as mean ± S.E. for a series of n experiments. Student's t
tests or analysis of variance (ANOVA) with the Student-Newman-Keuls
post-test were used to compare mean values as appropriate. p
values < 0.05 were considered to represent significant differences.
CCh Stimulated Tyrosine-phosphorylation Negatively Regulates
CCh-stimulated Chloride Secretion in T84 Cells--
We
first set out to examine the effects of CCh on protein tyrosine
phosphorylation in T84 cells. Western blot analysis of T84 cell lysates revealed that, when stimulated with CCh
(100 µM) on the basolateral domain, a rapid-onset
tyrosine phosphorylation of several T84 cell proteins, with
molecular masses ranging up to approximately 180 kDa, occurred (Fig.
1).
Carbachol Stimulates Transactivation of Epidermal Growth Factor
Receptor and Mitogen-activated Protein Kinase in T84
Cells
IMPLICATIONS FOR CARBACHOL-STIMULATED CHLORIDE SECRETION*
, and
ABSTRACT
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Introduction
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Discussion
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INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
channels
(10-13) and basolateral K+ channels (13), both of which
are transport proteins essential for epithelial chloride secretion.
EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References
, 25 HCO3,
2.4 H2PO4, 0.4 HPO42, and 10 glucose. Monolayers were
voltage-clamped to zero potential difference by the continuous
application of short-circuit current (Isc).
Under these conditions, changes in Isc
(
Isc) in response to agonists are wholly
reflective of electrogenic chloride secretion (28).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (52K):
[in a new window]
Fig. 1.
CCh stimulates tyrosine phosphorylation in
T84 cells. Cells were stimulated with basolateral CCh
(100 µM) for the times indicated and lysates were
separated by SDS-PAGE. After transfer to polyvinylidene difluoride
membrane, Western blots were probed with anti-phosphotyrosine and
antibody binding was detected using enhanced chemiluminescence.
Densitometric analysis of data pooled from several different
experiments is presented in the inset (mean ± S.E.,
n = 4) with values presented as fold stimulation over
basal (unstimulated cells).
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CCh Stimulates ERK Activity in T84 Cells-- Two of the proteins showing increased tyrosine phosphorylation in response to CCh had calculated molecular masses of 44 and 42 kDa, corresponding to the molecular masses of the ERK1 and ERK2 isoforms of MAPK, respectively. We therefore decided to investigate a possible role for ERK in negative regulation of CCh-stimulated secretory responses. Western blot experiments were first conducted which verified that ERK is phosphorylated in T84 epithelial cells in response to CCh stimulation (Fig. 3A). Furthermore, kinase assays, in which we measured the ability of anti-phospho-ERK immunoprecipitates to phosphorylate the ERK substrate, Elk, demonstrated that CCh-stimulated increases in ERK phosphorylation were closely accompanied by increases in ERK activity (Fig. 3B).
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CCh-stimulated ERK Activation Is Mediated via Muscarinic M3 Receptors-- In order to determine if CCh stimulation of ERK phosphorylation is a specific effect mediated by activation of cholinergic receptors, we examined the effect of the muscarinic M3 receptor antagonist, 4-DAMP, on CCh-stimulated responses. 4-DAMP (100 nM) abolished Isc responses to CCh across voltage-clamped T84 cells. Control responses to CCh were 21.5 ± 7.5 µA/cm2 compared with 0.4 ± 0.2 µA/cm2 in 4-DAMP-pretreated cells (n = 4). 4-DAMP (100 nM) also abolished ERK activation in response to CCh. ERK phosphorylation in CCh-stimuated cells was 27.5 ± 8.2-fold that in unstimulated cells compared with a 1.4 ± 1.0-fold stimulation in 4-DAMP-pretreated cells (n = 3).
CCh-stimulated ERK Activity Negatively Regulates CCh-stimulated Chloride Secretion in T84 Cells-- In order to test the hypothesis that CCh-stimulated ERK activation may be involved in regulation of CCh-stimulated chloride secretion, we examined the effects of a specific inhibitor of ERK activation, PD 98059, on both ERK phosphorylation and Isc responses to CCh. Pretreatment of T84 cell monolayers with PD 98059 caused a concentration-dependent reduction in phosphorylation of ERK in response to CCh (Fig. 4A). Furthermore, PD 98059 (20 µM), which reduced CCh-stimulated ERK phosphorylation by 53.7 ± 19.9% (n = 3; p < 0.05), potentiated subsequent Isc responses to CCh across voltage-clamped monolayers of T84 cells (Fig. 4B). Maximal responses to CCh were 31.2 ± 5.3 µA/cm2 in control cells compared with 50.7 ± 7.4 µA/cm2 in PD90859-treated cells (n = 7; p < 0.05). PD 98059 also prolonged CCh-stimulated Isc responses so that Isc values in PD 98059-treated monolayers remained signficantly higher (p < 0.01 or better) than those in control monolayers up to 15 min after addition of the agonist.
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Transactivation of the EGFr Mediates CCh-stimulated ERK Activation in T84 Cells-- To date two mechanisms have been proposed by which GPCRs may bring about ERK activation. One is mediated by a tyrosine kinase-dependent pathway, which may involve transactivation of the EGFr, while the other is a PKC-mediated tyrosine kinase-independent pathway (21-23). Both pathways for ERK activation appear to be present and functional in T84 cells since both EGF (100 ng/ml) and the phorbol ester, PMA (100 nM), stimulate T84 cell ERK phosphorylation, albeit with different kinetics and to different extents than does CCh (Fig. 5, compare with Fig. 3). In the next series of experiments we set out to investigate the extent to which either, if any, of these pathways is involved in mediating the effects of CCh on ERK activity. Since one of the proteins displaying increased phosphorylation in response to CCh was a 180-kDa protein corresponding to the molecular mass of the EGFr (Fig. 1), we first examined a possible role for the EGFr in mediating the effects of CCh on ERK activation. Immunoprecipitation studies were carried out which verified that CCh (100 µM) stimulated a time-dependent increase in tyrosine phosphorylation of EGFr (Fig. 6A). The time course of the response was similar to that observed for CCh stimulation of ERK phosphorylation (cf. Fig. 3). CCh also stimulated an increase in co-immunoprecipitation of the adapter proteins, Shc and Grb2 with EGFr (Fig. 6, B and C). As expected, EGF itself (100 ng/ml) also stimulated phosphorylation of the EGFr and increased co-immunoprecipitation of both Shc and Grb2 with the EGFr (Fig. 6, A-C). Taken together, these data imply that CCh stimulates transactivation of EGFr in T84 cells, and, in a fashion similar to that of EGF, stimulates the formation of Shc-Grb2-EGFr complexes. Formation of such complexes is normally a prerequisite for ERK activation in many cell types.
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PKC Is Not Involved in CCh-stimulated ERK Activation in T84 Cells-- In order to determine a possible role for PKC in the effects of CCh on ERK activation, we examined the effects of a cell-permeable inhibitor of PKC activity, GF109203X, on CCh-stimulated ERK phosphorylation. Pretreatment of T84 cells with GF109203X (1 µM) significantly inhibited ERK phosphorylation responses to PMA. Responses in cells treated with PMA in the presence of GF109203X were 58.8 ± 5.0% of those in cells stimulated with PMA alone (n = 3; p < 0.05). In contrast, GF109203X did not alter CCh stimulation of ERK phosphorylation when used alone, nor did it have any additional effect above that induced by the EGFr inhibitor, tyrphostin AG1478 when both inhibitors were used in combination (Fig. 8). These data imply that neither GF109203X-sensitive isoforms of PKC nor other potential nonspecific targets of GF109203X, are involved in mediating the effects of CCh on ERK activity in T84 epithelial cells.
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In the present study we have investigated a possible role for tyrosine kinase-dependent signaling pathways in negative regulation of calcium-mediated epithelial chloride secretion. In agreement with previous studies in another colonic epithelial cell line, HT-29 (8), we found that the prototypic calcium-mediated secretagogue, CCh, stimulated tyrosine phosphorylation of several proteins in T84 cells. Increased tyrosine phosphorylation was rapid in onset, occurring within 1 min, with maximal tyrosine phosphorylation occurring after approximately 5 min, a time corresponding to that at which CCh-stimulated secretory responses return to basal levels. This increase in tyrosine kinase activity appears to be involved in regulation of CCh-stimulated secretion since the broad-spectrum tyrosine kinase inhibitor, genistein, significantly potentiated both the magnitude and duration of CCh-stimulated Isc responses. These data suggest that, concommitant to stimulation of chloride secretion, CCh stimulates epithelial tyrosine kinase activity which appears to function in limiting the extent of CCh-stimulated secretory responses.
Since two of the proteins displaying increased tyrosine phosphorylation in response to CCh had molecular weights corresponding to those of the ERK 1 and 2 isoforms of MAPK, we extended our studies to investigate a possible role for these enzymes in regulation of epithelial secretion. Although MAPK activation is classically associated with regulation of cell growth and differentiation, our studies also reveal an apparent acute role for ERK in negative regulation of calcium-mediated chloride secretion across intestinal epithelial cells. This conclusion stems from the observations that not only does CCh rapidly stimulate increases in ERK phosphorylation and activity in T84 cells, but pretreatment of T84 cells with PD 98059 leads to increases in both the magnitude and duration of CCh-stimulated secretory responses. PD 98059 is reported to be a highly specific inhibitor of the upstream component of the MAPK cascade, MEK, and has been shown reliably to inhibit ERK activation in a variety of systems (15, 29, 30). The idea that CCh simultaneously activates both prosecretory and antisecretory signaling pathways is supported by the finding that the muscarinic M3 receptor antagonist, 4-DAMP, not only inhibited CCh-stimulated Isc responses, but also abolished the effects of CCh on ERK phosphorylation. Thus, it appears that net CCh-stimulated secretory responses are determined not only by the extent of stimulation of prosecretory pathways, but also by the extent to which negative signaling intermediates, such as ERK, are activated.
The mechanisms by which GPCRs stimulate ERK activation are complex and appear to be dependent not only on the nature of receptor coupling to heterotrimeric G-proteins, but also upon cell type (21-24). However, in most cell types studied to date, ERK activation in response to GqPCR stimulation (14, 31, 32), including M3 receptor activation in neuroblastoma cells (33), is a PKC-mediated process. Even though our data with PMA indicate that stimulation of PKC can be linked to ERK activation in T84 cells, this pathway does not appear to represent a mechanism by which CCh stimulates ERK activity in these cells. Thus, GF 902103X, which inhibited PMA-stimulated T84 cell ERK activation, and which has been shown to inhibit GqPCR-stimulated ERK activation in other cells (14), had no effect on ERK phosphorylation in response to CCh. Rather, our data strongly support a role for transactivation of the EGFr, a pathway normally favored by Gi-PCR (21-23), in mediating the effects of CCh on ERK activation. Several lines of evidence support this hypothesis. First, CCh stimulates tyrosine phosphorylation of the EGFr with a time course similar to that for ERK activation. Second, CCh stimulates recruitment of the adapter proteins, Shc and Grb2, to the EGFr. Association of these proteins with receptor tyrosine kinases is well documented as being an early step in growth factor-mediated activation of the MAPK cascade (20). Finally, at concentrations which have been demonstrated to block autophosphorylation of the EGFr, but not that of other EGFr family members (34, 35), we found that tyrphostin AG1478 not only inhibtited CCh-stimulated EGFr phosphorylation in T84 cells, but also significantly reduced CCh-stimulated ERK phosphorylation. Furthermore, similar to the ERK inhibitor, PD 98059, tyrphostin AG1478 potentiated Isc responses to CCh across voltage-clamped T84 cell monolayers. Thus, our data support a role for a CCh-stimulated signaling pathway, involving transactivation of the EGFr and subsequent ERK activation, which negatively influences simultaneous CCh-stimulated chloride secretory responses. Of note, our studies were conducted on polarized cells where the EGFr and M3 muscarinic receptor are known to be localized to the basolateral membrane. This may imply that particular spatial interrelationships are required to mediate the receptor transactivation events discussed here. It is also of note that even though tyrphostin AG1478 abolished CCh-stimulated transactivation of the EGFr, significant ERK phosphorylation remained in the presence of the antagonist, implying more than one pathway may exist by which CCh stimulates ERK activation. This residual, EGFr-independent ERK phosphorylation does not appear to be mediated by PKC since GF 902103X was not only ineffective in reducing CCh-stimulated ERK phosphorylation when used alone, but was also without effect when used in combination with tyrphostin AG1478. Further studies are required in order to identify this apparent PKC- and EGFr-independent mode of T84 cell ERK activation in response to CCh.
It is not yet known how CCh-stimulated transactivation of the EGFr and subsequent ERK activation might interact with epithelial transport pathways to bring about inhibition of chloride secretion. Some studies implicate cystic fibrosis transmembrane conductance regulator chloride channels, which are believed to be an important exit pathway for chloride in response to calcium-dependent secretagogues, as a possible target for inhibitory tyrosine kinase-dependent signals (36, 37). Moreover, recent studies demonstrate roles for M1 muscarinic receptor-mediated transactivation of the EGFr in regulation of K+ channels in kidney cells (38), and for ERK in regulation of K+ channel function in neuronal cells (39). Further studies are required to determine if similar processes are involved in ERK-mediated inhibition of CCh-stimulated secretion in T84 cells.
Likewise, additional studies are required to define possible interactions between CCh-stimulated ERK activity and other signals believed to be involved in negative regulation of calcium-dependent chloride secretion, such as agonist-stimulated influx of extracellular calcium (4). Of particular note in this regard are the recent findings of Rosen and Greenberg (40) who have demonstrated that influx of calcium through voltage-sensitive calcium channels stimulates EGFr phosphorylation, association of Grb2 and Shc with the EGFr, and ERK activation in PC12 neuronal cells. Similarly, it has been demonstrated that calcium influx and a novel calcium-dependent tyrosine kinase, PYK-2, are important in mediating the effects of GPCR receptor activation on ERK activity and subsequent K+ channel inhibition in HEK-293 kidney cells (24, 39, 41). Although a possible role for CCh-stimulated calcium influx in mediating the effects of the agonist on ERK activation in T84 cells has yet to be investigated, preliminary studies from our laboratory indicate that T84 cells not only express PYK-2, but that the enzyme becomes associated with the EGFr upon stimulation with CCh.2 Thus, CCh-stimulated influx of extracellular calcium may represent an early step in a signaling cascade which ultimately results in stimulation of ERK activity via transactivation of the EGFr.
In summary, we have shown that CCh stimulates activation of the ERK isoforms of MAPK in colonic epithelial cells, and that this appears to be involved in limiting the extent of CCh-stimulated chloride secretory responses. Stimulation of ERK activity by CCh occurs via a pathway independent of PKC, but which involves transactivation of the EGFr. We speculate that since chloride is the predominant ion driving fluid secretion in the intestine (5), agonist-stimulated transactivation of the EGFr and subsequent activation of ERK may represent a physiological braking mechanism to prevent excessive electrolyte and fluid loss when the levels of calcium-dependent secretagogues are elevated within the intestinal mucosa.
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ACKNOWLEDGEMENT |
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We thank Glenda Wheeler for assistance with manuscript submission.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK28305 (to K. E. B.). These studies were presented in part at the 1997 annual meeting of the American Gastroenterological Association, and have been published in abstract form (Keely, S. J., Uribe, J. M., and Barrett, K. E. Gastroenterology (1997) 112, A375).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.
Recipient of a Predoctoral Fellowship DK07202 from an
Institutional Training Grant in Digestive Diseases.
§ Faculty member, Biomedical Science Ph.D. Program, University of California, San Diego, School of Medicine. To whom correspondence and reprint requests should be addressed: UCSD Medical Center, 8414, 200 West Arbor Dr., San Diego, CA 92103. Tel.: 619-543-3726; Fax: 619-543-6969; E-mail: kbarrett{at}ucsd.edu.
The abbreviations used are: CCh, carbachol; GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; EGFr, EGF receptor: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; Isc, short circuit currentPMA, phorbol myristate acetatePKC, protein kinase CPAGE, polyacrylamide gel electrophorsis4-DAMP, 4-diphenylacetoxy-N-methylpiperidine.
2 S. J. Keely, L. S. Bertelsen, and K. E. Barrett, unpublished observations.
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