Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present studies were
undertaken to examine the possible regulation of apical membrane
Cl/OH
exchanger in Caco-2 cells by protein
kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate
13-acetate (PMA), an in vitro PKC agonist, on OH
gradient-driven 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed.
The results demonstrated that PMA decreased apical
Cl
/OH
exchanger activity via
phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of
PKC
. The data consistent with these conclusions are as follows:
1) short-term treatment of cells for 1-2 h with PMA
(100 nM) significantly decreased Cl
/OH
exchange activity compared with control (4
-PMA); 2)
pretreatment of cells with specific PKC inhibitors chelerythrine
chloride, calphostin C, and GF-109203X completely blocked the
inhibition of Cl
/OH
exchange activity by
PMA; 3) specific inhibitors for PKC
(Ro-318220) but not
PKC
(Go-6976) significantly blocked the PMA-mediated inhibition;
4) specific PI3-kinase inhibitors wortmannin and LY-294002 significantly attenuated the inhibitory effect of PMA; and
5) PI3-kinase activators IRS-1 peptide and
phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] mimicked the effects of PMA.
These findings provide the first evidence for PKC
-mediated
inhibition of Cl
/OH
exchange activity in
Caco-2 cells and indicate the involvement of the PI3-kinase-mediated
pathways in the regulation of Cl
absorption in intestinal
epithelial cells.
phosphatidylinositol 3-kinase; protein kinase C epsilon; human intestine; chloride absorption; phorbol 12-myristate 13-acetate
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN THE MAMMALIAN ILEUM
and colon, the major mechanism of Na+ and Cl
uptake has been suggested to be via an electroneutral process involving
the operation of the dual ion exchangers Na+/H+
and Cl
/HCO
)
(36). In this regard, recent studies from our laboratory
(3) characterized the luminal membrane
Cl
/OH
(HCO
/OH
(HCO
/OH
(HCO
Previous functional studies demonstrated that recombinant AE2-mediated
Cl/HCO
/HCO
/HCO
secretion (13). Studies from our laboratory
(1) and by others (14) demonstrated the
regulation of Na+/H+ exchanger (NHE) isoforms
by various protein kinases. To date, however, almost nothing is known
about the regulation of apical anion exchangers by PKC or any other
signal cascades in the human intestine.
In this regard, phorbol esters, analogs of diacylglycerol (DAG, a lipid produced during membrane phosphatidylinositol turnover), activate PKC by translocating it from the cytosol to the membrane (34). A number of cellular studies showed phorbol esters to induce a variety of responses including effects on ionic channels, second messenger production, cell-cell communication, membrane transport, protein phosphorylation, cellular growth, morphology, differentiation, and transformation (34, 40, 48). PMA has also been shown to induce phosphatidylinositol 3-kinase (PI3-kinase) activity and to increase the levels of lipid products of PI3-kinase, phosphoinositides, in mouse epidermal JB6 cells (20). PI3-kinase signaling pathway has been suggested to play an important regulatory role in the translocation of insulin-regulatable glucose transporters (11, 48) and NHE (28, 45).
The current studies were undertaken to determine 1) the
possible regulation of Cl/OH
exchange
activity in apical membranes of Caco-2 cells, the human colonic
adenocarcinoma cell line, by PMA and 2) the role of specific PKC isoform(s) and also the signal transduction pathways involved in
this process. Our data demonstrate that PMA decreases the apical Cl
/OH
exchange activity in Caco-2 cells and
provide evidence for the involvement of PI3-kinase and PKC
-mediated
pathways in the regulation of Cl
transport.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was obtained
from Sigma (St. Louis, MO). Radionuclide 35S-sulfuric acid
and 36Cl were obtained from NEN Life Science Products
(Boston, MA). Caco-2 cells were obtained from American Type Culture
Collection (ATCC, Manassas, VA). PMA, 4-phorbol 12-myristate
13-acetate (4
-PMA, inactive form), chelerythrine chloride,
calphostin C, GF-109203X, wortmannin, LY-294002, and IRS-1 (Y608)
peptide were obtained from Biomol (Plymouth Meeting, PA). Ro-318220,
Go-6796, and phosphatidylinositol 3,4,5-trisphosphate
[PI(3,4,5)P3] were obtained from Calbiochem
(San Diego, CA). Affinity-purified rabbit polyclonal antibody against
PKC
and goat-anti-rabbit antibody conjugated to horseradish
peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). All other chemicals were of at least reagent grade and were
obtained from Sigma or Fisher Scientific (Pittsburgh, PA).
Cell culture.
Caco-2 cells obtained from ATCC were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 4.5 g/l glucose, 2 mM
glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 mM HEPES,
1% essential and nonessential amino acids, and 20% fetal bovine
serum, pH 7.4, in 5% CO2-95% O2 at 37°C.
For the uptake experiments, cells from between passages 20 and 25 were plated in 24-well plates at a density of 2 × 104 cells/ml. Confluent monolayers were then used for
transport experiments on the 10th to 12th days after plating (i.e.,
6-8 days after confluence). To study the effect of phorbol esters
4-PMA and PMA on Cl
/OH
exchange
activity, cells were rendered quiescent by serum removal overnight
before study. Cells were then acutely exposed to 100 nM PMA for 15, 30, 60, and 120 min. In a separate set of experiments, cells were exposed
to 1 µM PMA for 24 h. Control cells were treated with an
equivalent amount of 4
-PMA (inactive form).
35SO uptake.
SO
uptake experiments were
performed essentially as described by Olsnes et al. (35) with some modifications (2). Caco-2 cells were incubated
with DMEM base medium containing 20 mM HEPES-KOH, pH 8.5, for 30 min at
room temperature. The medium was removed, and the cells were rapidly
washed with 1 ml of tracer-free uptake mannitol buffer containing 260 mM mannitol and 20 mM Tris-MES, pH 7.0. The cells were then incubated
with the uptake buffer for a 2-min time period. For
35SO
uptake studies, the uptake buffer was the mannitol buffer containing 1.3 µCi of 36Cl
(2.7 mM) of hydrochloric
acid per milliliter. The uptake was terminated by removing the buffer
and washing the cells rapidly two times with 1 ml of ice-cold
phosphate-buffered saline (PBS), pH 7.2. Finally, the cells were
solubilized by incubation with 0.5 N NaOH for 4 h. The
protein concentration was measured by the method of Bradford
(9), and the radioactivity was measured by Packard Liquid
Scintillation Analyzer TRI-CARB 1600-TR (Packard Instrument, Downers
Grove, IL). The uptake values are expressed as picomoles per milligram
per 2 minutes for SO
.
Subcellular fractionation. Caco-2 cells grown to confluence in 25-cm2 flasks (Corning Costar) were washed with ice-cold PBS three times and scraped into 400 µl of the cold homogenization buffer (HB) containing (in mM) 20 Tris · HCl, pH 7.5, 250 sucrose, 4 EDTA, and 2 EGTA with complete protease inhibitor cocktail tablets. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer. The resulting homogenate was ultracentrifuged at 59,000 rpm for 50 min at 4°C (Optima TLX ultracentrifuge; Beckman). The supernatant was designated the cytosolic fraction. The pellet was resuspended in 150 µl of the HB containing 0.5% (vol/vol) Triton X-100 by brief sonication and incubated on ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 rpm for 20 min at 4°C. The resulting supernatant was designated the membrane fraction.
Gel electrophoresis and Western blotting.
Equal amounts (~75 µg/sample) of protein, as determined by the
Bradford assay, were combined with Laemmli's sample buffer containing
5% (vol/vol) -mercaptoethanol and boiled for 5 min. Proteins were
separated by electrophoresis on 8% SDS-PAGE gels and transblotted to
nitrocellulose membranes. The protein-bound nitrocellulose membranes
were first incubated for 1 h at room temperature in blocking
buffer containing 1× PBS, 0.1% Tween 20, and 5% nonfat dry milk.
Nitrocellulose membranes were then incubated with the polyclonal
antibody specific to PKC
(1:800 dilution) in the blocking buffer
containing 1× PBS, 0.1% Tween 20, and 1% nonfat dry milk for 1 h at room temperature and rinsed for 30 min with a wash buffer
containing 1× PBS and 0.1% Tween 20. Finally, the membranes were
incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:2,000
dilution) for 1 h at room temperature and washed for 45 min with
agitation, during which the wash buffer was changed every 5 min. PKC
bands were visualized with enhanced chemiluminescence (ECL) detection reagents.
Statistical analysis. Results are expressed as means ± SE. Each independent set represents means ± SE of data from at least nine wells used on three separate occasions. Student's t-test was used for statistical analysis. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acute and chronic effects of PMA.
To examine the effect of PMA on Cl/OH
or
SO
exchange activity, Caco-2 cells
were serum deprived overnight and then incubated with 100 nM PMA or its
inactive form (4
-PMA) at different time points and DIDS-sensitive
Cl
/OH
and
SO
exchange activities
were assessed as described in MATERIALS AND METHODS. As shown in Fig.
1, no change in
Cl
/OH
exchange activity was observed over
15- and 30-min time periods, whereas incubation of Caco-2 cells with
PMA for 1 h decreased the activity by ~50% compared with
control (4
-PMA treated) and the inhibition remained about the same
even at 2 h. However, similar treatment of Caco-2 cells with PMA
(100 nM, 1 h) showed no significant change in
SO
exchange activity (1,626 ± 144 vs. 1,351 ± 87 pmol · mg
1 · 2 min
1 in PMA
treated and 4
-PMA treated, respectively). Exposure to PMA (1 µM)
for 24 h to downregulate PKC also did not alter
Cl
/OH
exchange activity (data not shown).
|
Effect of PKC inhibitors on ability of PMA to inhibit
DIDS-sensitive Cl uptake in Caco-2
cells.
To confirm that the PMA-mediated effect on
Cl
/OH
exchange activity was mediated via
PKC activation, the effects of specific PKC inhibitors on PMA-mediated
changes in Cl
/OH
exchange activity were
examined. Overnight serum-deprived cells were pretreated with specific
PKC inhibitors for 1 h before addition of PMA or 4
-PMA (100 nM), followed by coincubation for another 1 h. As shown in Fig.
2, pretreatment of
cells with the specific PKC inhibitors, for example, chelerythrine
chloride (2 µM; Fig. 2A), calphostin C (200 nM; Fig.
2B), or GF-109203X (50 nM; Fig. 2C) completely
abolished the PMA-mediated inhibition of
Cl
/OH
exchange activity in Caco-2 cells.
These results suggested that the observed decrease in
Cl
/OH
exchange activity involved PKC
activation.
|
Effect of isoform-specific inhibitors of PKC on PMA-induced
inhibition of
Cl/OH
exchange activity in Caco-2 cells.
To test which particular PKC isoform is involved in this effect of PMA,
we used two PKC inhibitors, Go-6976 and Ro-318820, which have been
shown to differentially inhibit classic and novel PKCs, namely,
-
and
-isoforms, respectively. The reported IC50 of
Go-6976 for PKC
is 5 nM (30) and that of Ro-318220 for
PKC
is 24-48 nM (26) depending on the cell type
studied. As shown in Fig. 3A,
Go-6976 (5 nM; 1 h before PMA addition, followed by coincubation
for another 1 h) failed to block the inhibitory effect of PMA on
Cl
/OH
exchange activity. Similar results
were obtained with 10 and 50 nM concentrations of Go-6976 (data not
shown). However, Ro-318220 (100 nM; 1 h before PMA addition,
followed by coincubation for another 1 h) significantly attenuated
the inhibitory effect of PMA on Cl
/OH
exchange activity in Caco-2 cells (Fig. 3B). The data
suggest that PKC
or other isoforms sensitive to Go-6976 do not
appear to mediate the inhibitory effect of PMA on Cl
uptake. Conversely, the PKC
isoform, sensitive to Ro-318220, was
observed as the potential candidate to mediate the inhibitory effect of
PMA on Cl
uptake.
|
Effect of PI3-kinase inhibitors wortmannin and LY-294002 on
PMA-mediated inhibition of
Cl/OH
exchange activity in Caco-2 cells.
PKC
, the isoform sensitive to RO-318220, was previously shown to lie
downstream of PI3-kinase in signal transduction cascades in other
systems (10). It was therefore of interest to determine whether PI3-kinase pathway is involved in the PMA-mediated inhibition of Cl
/OH
exchange process in Caco-2 cells.
Wortmannin (100 nM; 1 h before PMA addition, followed by
coincubation for another 1 h) suppressed the PMA-mediated
inhibition of Cl
/OH
exchange activity (Fig.
4A). LY-294002 (5 µM), a
more specific PI3-kinase inhibitor that acts through a mechanism
distinct from that of wortmannin (47), also significantly
attenuated the inhibitory effect of PMA on the
Cl
/OH
exchange process in Caco-2 cells
(Fig. 4B). These findings implicate the activation of
PI3-kinase and subsequent activation of its downstream effector PKC
in the effects of PMA on apical Cl
/OH
exchange process in Caco-2 cells.
|
Effect of PI3-kinase activator IRS-1 peptide on inhibition of
Cl/OH
exchange activity in Caco-2 cells.
We next examined whether the PI3-kinase activator IRS-1 peptide could
mimic the effects of PMA on Cl
/OH
exchange activity. IRS-1 peptide, a
tyrosine-phosphorylated peptide, activates the PI3-kinase enzyme by
binding to its SH2 domain. IRS-1 peptide (0.1-10 µM, 1 h)
significantly decreased Cl
/OH
exchange
activity in a dose-dependent manner (Fig.
5). These results suggest that IRS-1
peptide could mimic the effects of PMA on the
Cl
/OH
exchange process in Caco-2 cells.
|
Effect of PI3-kinase activator PI(3,4,5)P3
on inhibition of
Cl/OH
exchange activity in Caco-2 cells.
The products of PI3-kinase activity include the 3-phosphorylated lipids
phosphatidylinositol 3,4-bisphosphate
[PI(3,4)P2] and
PI(3,4,5)P3. It was shown previously that PMA
can induce PI3-kinase activity and increase the levels of
PI(3,4)P2 and
PI(3,4,5)P3 (second messengers) in mouse
epidermal JB6 cells (21). Hence, to further establish the
role of PI3-kinase in PMA-mediated inhibition of
Cl
/OH
exchange activity, Caco-2 cells were
incubated with 10 µM of PI(3,4,5)P3 for
1 h. PI(3,4,5)P3 significantly decreased
the Cl
/OH
exchange activity by ~50%
(Fig. 6). These findings further confirm that the PMA-mediated inhibition of Cl
/OH
exchange activity involves the activation of PI3-kinase, which in turn
can activate PKC
.
|
Time course of translocation of PKC in Caco-2 cells after PMA
addition.
In light of the data of PKC isoform inhibitors presented above
indicating that PKC
may be the major isoform responsible for the
regulation of the Cl
/OH
exchanger in Caco-2
cells, we further characterized the time course of PKC
membrane
translocation on PMA addition. With Western blot analysis, it is clear
that an increase in PKC
density in the membrane fraction was
apparent as early as 15 min after PMA (100 nM) addition and continued
to increase up to at least 1 h (Fig.
7). The translocation/activation of
PKC
within 1 h of PMA treatment is consistent with our
functional data showing inhibition of Cl
/OH
exchange activity in Caco-2 cells.
|
Effect of LY-294002 and wortmannin on PMA-induced PKC activation
in Caco-2 cells.
Previous studies showed that PI3-kinase can activate novel PKCs
(30, 31). PI3-kinase activity was also found to be
involved in the inhibitory action of EGF on carbachol (CCh)-induced
Cl
secretion (46). To determine whether
PKC
is a downstream effector of PI3-kinase, specific inhibitors of
PI3-kinase, LY-294002 (5 µM) and wortmannin (100 nM), were used. We
found that both inhibitors inhibited the PMA-induced translocation of
PKC
by ~40% (Fig. 7). These data further confirm our findings
that PKC
is a downstream effector of PI3-kinase in the PMA-mediated
inhibition of Cl
/OH
exchange activity in
Caco-2 cells.
Effect of IRS-1 peptide on PMA-induced PKC activation in Caco-2
cells.
To further substantiate our findings that PI3-kinase is upstream to
PKC
, we used the PI3-kinase activator IRS-1 peptide. IRS-1 peptide
(1 µM, 1 h) significantly induced the translocation/activation of PKC
from the cytosol to the membrane fractions (Fig.
8) compared with the control. These
findings suggest that IRS-1 peptide could mimic the effects of PMA on
PKC
translocation and further confirm that PI3-kinase is the
upstream effector of PKC
in the PMA-mediated inhibition of
Cl
/OH
exchange activity in Caco-2 cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data demonstrate a novel pathway for the regulation of
Cl/OH
exchange by PMA occurring via
activation of a PI3-kinase and the involvement of a downstream
calcium-independent PKC isoform, namely, PKC
.
PKC was previously shown to directly regulate the activity of the
Cl/HCO
/HCO
secretion (13). Activation of PKC by
pretreatment with 100 nM PMA has also been shown to inhibit CCh-induced
Cl
secretion in T84 cells (39). Studies on
the regulation of NHE3 by PMA also showed that the PMA-induced acute
inhibition of endogenous NHE3 in Caco-2 cells is mediated by PKC
(25). Our data, complementing the above studies of
inhibition of NHE3 by PMA, showed that short-term treatment of Caco-2
cells with PMA at 100 nM for 1 h resulted in a significant
decrease in Cl
/OH
exchange activity.
Parallel studies were also carried out with recently demonstrated
SO
anion exchange activity in our
laboratory (2) in Caco-2 cells to examine whether PMA
effects were specific for Cl
/OH
exchange.
The results demonstrated that, in contrast to the inhibition of
Cl
/OH
exchange,
SO
exchange activity remained
unaltered by PMA treatment, suggesting that the effects of PMA were
specific to the Cl
/OH
exchanger.
The PMA-mediated inhibition of Cl/OH
exchange activity was completely blocked by specific inhibitors of PKC,
chelerythrine chloride (2 µM), calphostin C (200 nM), and GF-109203X
(50 nM), thereby confirming the involvement of PKC in PMA-mediated
inhibition of Cl
/OH
exchange activity in
Caco-2 cells. Because PMA-induced acute inhibition of
Cl
/OH
exchange activity is mediated by PKC,
it is thus possible that one or several isoforms of PKC could mediate
the divergent inhibitory effect of PMA. Thus PKCs are grouped into
three major classes, conventional PKC isoforms (
,
1,
2,
), novel PKCs (
,
,
,
), and atypical
PKCs (
,
) (22). Conventional PKCs are activated by
DAG in a Ca2+-dependent manner. In contrast, activation of
novel PKCs is Ca2+ independent (40). In
addition to the natural activation, conventional and novel PKCs are
also activated with high specificity by PMA (8). For this
reason, PMA is often used in the study of mechanisms of conventional
and novel PKC activation and their function (12).
Previous studies demonstrated that both PKC and PI3-kinase play
important roles in many different cell regulatory pathways as well as
in a broad range of biological effects (19, 42). Several
studies suggested that PMA can induce PI3-kinase activity (27,
33, 43) and increase the level of
PI(3,4,5)P3 and
PI(3,4)P2, lipid products of PI3-kinase
(second messengers), as well as having a markedly synergistic effect
with insulin on PI3-kinase activation in mouse epidermal JB6 cells
(21). Earlier studies of Toker et al. (44)
showed that the calcium-independent PKC isoforms were activated by the
lipid products of PI3-kinase. Therefore, in the current study, we also
attempted to examine the PKC isotype(s) involved in the regulation of
Cl/OH
exchange activity in Caco-2 cells by
PMA. Our results showed that Ro-318220, but not Go-6976, completely
blocked the inhibitory effect of PMA on
Cl
/OH
exchange activity, suggesting that
PKC
, but not PKC
, was likely to play a pivotal role in the
inhibitory effect on Cl
uptake by PMA. The results are
well correlated with the Western blot analysis, showing that PMA
treatment (100 nM, 1 h) resulted in the translocation of PKC
from the cytosol to the membrane fractions in Caco-2 cells. In this
regard, PI3-kinase has also been shown to activate novel PKCs in other
systems (31). For example, PI3-kinase was found to be
associated with PKC
in a human hematopoietic cell line and platelets
(15) and incubation of HepG2 cells with platelet-derived
growth factor led to the translocation of PKC
via the activation of
PI3-kinase (30). However, in contrast, Huang et al.
(20) showed that the effect of PMA-induced PI3-kinase
activity was mediated by the novel PKC
in mouse epidermal JB6 cells,
suggesting that the PKC
effect was upstream to PI3 kinase. In our
system, the current data also indicate a link between PKC
and
PI3-kinase in Caco-2 cells and suggest PI3-kinase to be the upstream
effector of PKC
, as both wortmannin (100 nM) and LY-294002 (5 µM)
blocked the inhibitory effect of PMA on
Cl
/OH
exchange activity. In this regard,
wortmannin is a somewhat nonspecific inhibitor of PI3-kinase, as it was
also shown to inhibit phosphatidylinositol 4-kinase (PI 4-kinase),
myosin light chain kinase, phospholipase A2, and
phospholipase D (32). In contrast, LY-294002 is a more specific PI3-kinase inhibitor and therefore was also used to exclude the possibilities of nonspecific effects of wortmannin. Our data showed
similar results with both inhibitors. Thus, in our study, the
inhibitory effect of wortmannin on PMA-induced activation of PKC
favors an essential role of the second messengers of PI3-kinase pathway
and suggests a downstream position of PKC
related to PI3-kinase. Our
data are also in accordance with the previous findings of Chow et al.
(10) showing that the ability of EGF to inhibit
CCh-induced Cl
secretion in T84 cells was completely
reversed by the PI3-kinase inhibitor wortmannin and that PKC
acts as
a downstream effector to PI3-kinase in T84 cells stimulated with EGF.
Moreover, we have also shown the increased translocation of PKC
from
the cytosol to the membrane fractions in Caco-2 cells in the presence
of the PI3-kinase activator IRS-1 peptide. Thus our results with the PI3-kinase activators IRS-1 peptide and
PI(3,4,5)P3 further confirm that the observed
effects of PMA on the Cl
/OH
exchange
activity occur via PI3-kinase, which in turn activates PKC
, rather
than a direct activation of PKC
by PMA.
Several earlier studies showed that agents like phorbol esters and CCh
that increase PKC activity decrease Vmax of NHE3
in the Chinese hamster lung fibroblast cell line PS120
(45), the rabbit gall bladder epithelium
(41), and the opossum kidney cell line (5).
Because the inhibition occurs via decrease in Vmax and is observed within hours, this type of
regulation might theoretically be achieved by a decrease in the number
of active molecules at the membrane due to endocytic retrieval from
and/or exocytic insertion into the plasma membrane and/or rapid
degradation by changes in the turnover number of individual exchanger
molecules. Multiple plasma membrane transport proteins (7, 16,
29, 37, 48) were also shown to be regulated, at least in part, by cellular redistribution (endocytic retrieval from and/or exocytic insertion into the plasma membrane). PKC was reported to generally stimulate apical but not basolateral endocytosis in Caco-2 cells (18). Hence, in our study, certainly effects of PKC via
PI3-kinase pathway on endocytosis could account for the ability of PMA
to inhibit Cl
/OH
exchange activity in
Caco-2 cells by retrieving apical membrane transporter molecules to the
cytoplasm. Future studies will address this important question
regarding the mechanism of Cl
/OH
exchange attenuation.
In conclusion, the present studies demonstrate for the first time that
PMA inhibits the Cl/OH
exchange process in
Caco-2 cells via the activation of PI3-kinase, which in turn stimulates
PKC
via its lipid products. Future studies to elucidate the detailed
signal transduction pathways and molecular mechanism of this
Cl
transport regulation would be important to better
understand the role of PKC in intestinal electrolyte transport.
![]() |
ACKNOWLEDGEMENTS |
---|
These studies were supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016 (P. K. Dudeja), DK-33349 (K. Ramaswamy), and DK-09930 (W. A. Alrefai).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), Chicago VA West Side Division, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.edu).
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.
July 24, 2002;10.1152/ajpcell.00473.2001
Received 4 October 2001; accepted in final form 17 July 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alrefai, WA,
Scaglione-Sewell B,
Tyagi S,
Wartman L,
Brasitus TA,
Ramaswamy K,
and
Dudeja PK.
Differential regulation of the expression of Na+/H+ exchanger isoform NHE3 by PKC- in Caco-2 cells.
Am J Physiol Cell Physiol
281:
C1551-C1558,
2001
2.
Alrefai, WA,
Tyagi S,
Mansour F,
Saksena S,
Syed I,
Ramaswamy K,
and
Dudeja PK.
Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRA gene.
Am J Physiol Gastrointest Liver Physiol
280:
G603-G613,
2001
3.
Alrefai, WA,
Tyagi S,
Nazir TM,
Barakat J,
Anwar SS,
Hadjiagapiou C,
Bavishi D,
Sahi J,
Malik P,
Goldstein J,
Layden TJ,
Ramaswamy K,
and
Dudeja PK.
Human intestinal anion exchanger isoforms: expression, distribution, and membrane localization.
Biochim Biophys Acta
1511:
17-27,
2001[ISI][Medline].
4.
Alvaro, D,
Della Guardia P,
Bini A,
Gigliozzi A,
Furfaro S,
La Rosa T,
Piat C,
and
Capocaccia L.
Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.
J Clin Invest
96:
665-675,
1995[ISI][Medline].
5.
Azarani, A,
Orlowski J,
and
Goltzman D.
Parathyroid hormone and parathyroid hormone-related peptide activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic cells (UMR-106) via a cAMP-dependent pathway.
J Biol Chem
270:
23166-23172,
1995
6.
Benedetti, A,
Strazzabosco M,
Ng OC,
and
Boyer JL.
Regulation of activity and apical targeting of the Cl/HCO
7.
Beron, J,
Forster I,
Beguin P,
Geering K,
and
Verrey F.
Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.
Mol Biol Cell
8:
387-398,
1997[Abstract].
8.
Blumberg, PM.
Complexities of the protein kinase C pathway.
Mol Carcinog
4:
339-344,
1991[ISI][Medline].
9.
Bradford, M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
10.
Chow, JY,
Uribe JM,
and
Barrett KE.
A role for protein kinase C epsilon in the inhibitory effect of epidermal growth factor on calcium-stimulated chloride secretion in human colonic epithelial cells.
J Biol Chem
275:
21169-21176,
2000
11.
Czech, MP,
and
Corvera S.
Signaling mechanisms that regulate glucose transport.
J Biol Chem
274:
1865-1868,
1999
12.
Danielsen, AG,
Liu F,
Hosomi Y,
Shii K,
and
Roth RA.
Activation of protein kinase C alpha inhibits signaling by members of the insulin receptor family.
J Biol Chem
270:
21600-21605,
1995
13.
Donowitz, M,
Cheng HY,
and
Sharp GW.
Effects of phorbol esters on sodium and chloride transport in rat colon.
Am J Physiol Gastrointest Liver Physiol
251:
G509-G517,
1986
14.
Donowitz, M,
Janecki A,
Akhter S,
Cavet ME,
Sanchez F,
Lamprecht G,
Zizak M,
Kwon WL,
Khurana S,
Yun CH,
and
Tse CM.
Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.
Ann NY Acad Sci
915:
30-42,
2000
15.
Ettinger, SL,
Lauener RW,
and
Duronio V.
Protein kinase C delta specifically associates with phosphatidylinositol 3-kinase following cytokine stimulation.
J Biol Chem
271:
14514-14518,
1996
16.
Forte, TM,
Machen TE,
and
Forte JG.
Ultrastructural changes in oxyntic cells associated with secretory function: a membrane-recycling hypothesis.
Gastroenterology
73:
941-955,
1977[ISI][Medline].
17.
Hoglund, P,
Haila S,
Socha J,
Tomaszewski L,
Saarialho-kere U,
Lindsberg MLK,
Chapelle A,
and
Kere J.
Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea.
Nat Genet
14:
316-319,
1996[ISI][Medline].
18.
Holm, PK,
Eker P,
Sandvig K,
and
van Deurs B.
Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells.
Exp Cell Res
217:
157-168,
1995[ISI][Medline].
19.
Hu, Q,
Klippel A,
Muslin AJ,
Fantl WJ,
and
Williams LT.
Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase.
Science
268:
100-102,
1995[ISI][Medline].
20.
Huang, C,
Ma WY,
and
Dong Z.
Potentiation of insulin-induced phosphatidylinositol-3 kinase activity by phorbol ester is mediated by protein kinase C epsilon.
Cell Signal
10:
185-190,
1998[ISI][Medline].
21.
Huang, C,
Schmid PC,
Ma WY,
Schmid HH,
and
Dong Z.
Phosphatidylinositol-3 kinase is necessary for 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and activated protein 1 activation.
J Biol Chem
272:
4187-4194,
1997
22.
Hug, H,
and
Sarre TF.
Protein kinase C isoenzymes: divergence in signal transduction?
Biochem J
291:
329-343,
1993[ISI][Medline].
23.
Humphreys, BD,
Jiang L,
Chernova MN,
and
Alper SL.
Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes.
Am J Physiol Cell Physiol
267:
C1295-C1307,
1994
24.
Humphreys, BD,
Jiang L,
Chernova MN,
and
Alper SL.
Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalization.
Am J Physiol Cell Physiol
268:
C201-C209,
1995
25.
Janecki, AJ,
Montrose MH,
Zimniak P,
Zweibaum A,
Tse CM,
Khurana S,
and
Donowitz M.
Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger.
J Biol Chem
273:
8790-8798,
1998
26.
Johnson, MS,
MacEwan DJ,
Simpson J,
and
Mitchell R.
Characterisation of protein kinase C isoforms and enzymic activity from the alpha T3-1 gonadotroph-derived cell line.
FEBS Lett
333:
67-72,
1993[ISI][Medline].
27.
King, WG,
Kucera GL,
Sorisky A,
Zhang J,
and
Rittenhouse SE.
Protein kinase C regulates the stimulated accumulation of 3-phosphorylated phosphoinositides in platelets.
Biochem J
278:
475-480,
1991[ISI][Medline].
28.
Liedtke, CM.
The role of protein kinase C in -adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
268:
L414-L423,
1995
29.
Lostao, MP,
Hirayama BA,
Panayotova-Heiermann M,
Sampogna SL,
Bok D,
and
Wright EM.
Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.
FEBS Lett
377:
181-184,
1995[ISI][Medline].
30.
Moriya, S,
Kazlauskas A,
Akimoto K,
Hirai S,
Mizuno K,
Takenawa T,
Fukui Y,
Watanabe Y,
Ozaki S,
and
Ohno S.
Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.
Proc Natl Acad Sci USA
93:
151-155,
1996
31.
Nakanishi, H,
Brewer KA,
and
Exton JH.
Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate.
J Biol Chem
268:
13-16,
1993
32.
Nakanishi, S,
Catt KJ,
and
Balla T.
A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids.
Proc Natl Acad Sci USA
92:
5317-5321,
1995[Abstract].
33.
Nave, BT,
Shepherd PR,
and
Siddle K.
Effect of phorbol esters on phosphatidylinositol 3-kinase activity in adipocytes.
Biochem Soc Trans
23:
183S,
1995[Medline].
34.
Nishizuka, Y.
The molecular heterogeneity of protein kinase C and its implications for cellular regulation.
Nature
334:
661-665,
1988[ISI][Medline].
35.
Olsnes, S,
Tonnessen TI,
Ludt J,
and
Sandvig K.
Effect of intracellular pH on the rate of chloride uptake and efflux in different mammalian cell lines.
Biochemistry
26:
2778-2785,
1987[ISI][Medline].
36.
Powell, DW.
Intestinal water and electrolyte transport.
In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by Johnson LR.. New York: Raven, 1987, p. 1267-1305.
37.
Prince, LS,
Workman RB, Jr,
and
Marchase RB.
Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.
Proc Natl Acad Sci USA
91:
5192-5196,
1994[Abstract].
38.
Rajendran, VM,
Black J,
Ardito TA,
Sangan P,
Alper SL,
Schweinfest C,
Kashgarian M,
and
Binder HJ.
Regulation of DRA and AE1 in rat colon by dietary Na depletion.
Am J Physiol Gastrointest Liver Physiol
279:
G931-G942,
2000
39.
Reenstra, WW.
Inhibition of cAMP- and Ca-dependent Cl secretion by phorbol esters: inhibition of basolateral K+ channels.
Am J Physiol Cell Physiol
264:
C161-C168,
1993
40.
Selbie, LA,
Schmitz-Peiffer C,
Sheng Y,
and
Biden TJ.
Molecular cloning and characterization of PKC iota, an atypical isoform of protein kinase C derived from insulin-secreting cells.
J Biol Chem
268:
24296-24302,
1993
41.
Silviani, V,
Colombani V,
Heyries L,
Gerolami A,
Cartouzou G,
and
Marteau C.
Role of the NHE3 isoform of the Na+/H+ exchanger in sodium absorption by the rabbit gallbladder.
Pflügers Arch
432:
791-796,
1996[ISI][Medline].
42.
Slaga, TJ,
Fischer SM,
Weeks CE,
Klein-Szanto AJ,
and
Reiners J.
Studies on the mechanisms involved in multistage carcinogenesis in mouse skin.
J Cell Biochem
18:
99-119,
1982[ISI][Medline].
43.
Standaert, ML,
Avignon A,
Yamada K,
Bandyopadhyay G,
and
Farese RV.
The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.
Biochem J
313:
1039-1046,
1996[ISI][Medline].
44.
Toker, A,
Meyer M,
Reddy KK,
Falck JR,
Aneja R,
Aneja S,
Parra A,
Burns DJ,
Ballas LM,
and
Cantley LC.
Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3.
J Biol Chem
269:
32358-32367,
1994
45.
Tse, CM,
Levine SA,
Yun CH,
Brant SR,
Pouyssegur J,
Montrose MH,
and
Donowitz M.
Functional characteristics of a cloned epithelial Na+/H+ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C.
Proc Natl Acad Sci USA
90:
9110-9114,
1993[Abstract].
46.
Uribe, JM,
Keely SJ,
Traynor-Kaplan AE,
and
Barrett KE.
Phosphatidylinositol 3-kinase mediates the inhibitory effect of epidermal growth factor on calcium-dependent chloride secretion.
J Biol Chem
271:
26588-26595,
1996
47.
Vlahos, CJ,
Matter WF,
Hui KY,
and
Brown RF.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem
269:
5241-5248,
1994
48.
Yang, J,
and
Holman GD.
Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells.
J Biol Chem
268:
4600-4603,
1993