Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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Cytoplasmic pH (pHi) was evaluated during Na+-glucose cotransport in Caco-2 intestinal epithelial cell monolayers. The pHi increased by 0.069 ± 0.002 within 150 s after initiation of Na+-glucose cotransport. This increase occurred in parallel with glucose uptake and required expression of the intestinal Na+-glucose cotransporter SGLT1. S-3226, a preferential inhibitor of Na+/H+ exchanger (NHE) isoform 3 (NHE3), prevented cytoplasmic alkalinization after initiation of Na+-glucose cotransport with an ED50 of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. In contrast, HOE-694, a poor NHE3 inhibitor, failed to significantly inhibit pHi increases at <500 µM. Na+-glucose cotransport was also associated with activation of p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pHi increases by 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely, activation of p38 MAP kinase with anisomycin induced NHE3-dependent cytoplasmic alkalinization in the absence of Na+-glucose cotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediated Na+-glucose cotransport and that the mechanism of this NHE3 activation requires p38 MAP kinase activity. This coordinated regulation of glucose (SGLT1) and Na+ (NHE3) absorptive processes may represent a functional activation of absorptive enterocytes by luminal nutrients.
SGLT1; p38 mitogen-activated protein kinase; nutrient absorption; Na+/H+ exchanger isoform 3
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INTRODUCTION |
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IN THE MAMMALIAN SMALL INTESTINE, Na+ absorption provides the driving force for transcellular absorption of glucose and many amino acids. Deficiency of the apical Na+-glucose cotransporter SGLT1 in humans leads to glucose/galactose malabsorption, with severe diarrhea and dehydration (21). Similarly, a genetically engineered mouse lacking functional Na+/H+ exchanger isoform 3 (NHE3) suffers from diarrhea, intestinal malabsorption, dehydration, mild acidosis, and increased volume and alkalinity of the intestinal contents (16). Thus loss of NHE3, the primary Na+ absorptive pathway in the intestine and renal tubules, leads to fluid and pH imbalances (16, 27). Conversely, increased renal tubular NHE3 activity has been suggested as a disease mechanism in spontaneously hypertensive rats (3), perhaps because of increased Na+ and volume retention.
Recently, NHE3 regulation has been the subject of intense investigation. For example, it is clear that NHE3 activity can be acutely increased by recruitment of NHE3 from intracellular stores to the plasma membrane (1, 6). NHE3 can also be regulated by the actin cytoskeleton (8, 18), an effect at least partly mediated by a family of Na+/H+ exchanger regulatory factors (26, 28) that interact with NHE3 and cytoskeletal proteins. In renal thick ascending limb, NHE3 can also be activated by hyposmolality (25).
Although NHE3 is highly enriched in the brush border of small intestinal absorptive (villus) enterocytes (4), only one study has examined coordination of NHE3 activity with the activity of absorptive pathways. This study demonstrated preferential activation of apical Na+/H+ exchange after cytoplasmic acidification induced by apical H+-solute cotransport (19). This led to the conclusion that activation of apical Na+/H+ exchange by H+-solute cotransport resulted in optimal absorption of Na+ and nutrients in conjunction with maintenance of intracellular pH (pHi) (19). Such regulation is likely critical in absorptive epithelia, inasmuch as it is a necessary component of cell volume and ion channel regulatory pathways (11, 12, 15).
We recently developed an in vitro model of intestinal Na+-glucose cotransport using Caco-2 cells stably transfected with intestinal SGLT1 (24). We showed that monolayers of these cells are capable of vectorial Na+-glucose cotransport with kinetic properties similar to those observed in native small intestine (23, 24). Moreover, like native small intestinal mucosae, monolayers of these cells regulate paracellular transport after activation of SGLT1-mediated Na+-glucose cotransport (24). Further characterization of the signaling mechanisms necessary for this process demonstrated that, in the presence of ongoing Na+-glucose cotransport, inhibition of NHE3 prevented SGLT1-dependent regulation of paracellular permeability (22). Thus we hypothesized that SGLT1- and NHE3-mediated transcellular transport might be coordinately regulated.
The goal of these studies was to determine whether a functional interaction exists between SGLT1 and NHE3. Our studies show that initiation of SGLT1-dependent Na+-glucose cotransport leads to cytoplasmic alkalinization and that this alkalinization is dependent on NHE3. Moreover, the data suggest that p38 mitogen-activated protein (MAP) kinase is a critical intermediate in this SGLT1-dependent NHE3 activation.
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METHODS |
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Materials. Tissue culture media and serum were obtained from GIBCO (Life Technologies, Gaithersburg, MD). HOE-694 and S-3226 were the kind gifts of Dr. Hans-Jochen Lang (Hoechst-Marion Roussel) (2, 17). Other Na+/H+ exchange inhibitors were obtained from Sigma (St. Louis, MO). The rho kinase inhibitor Y-27632 was generously provided by Yoshitomi Pharmaceutical Industries (Saitama, Japan).
Cell culture.
Clonal populations of Caco-2 cells with active physiological
Na+-glucose cotransport were generated by stable
transfection with native intestinal SGLT1 and maintained in
high-glucose (25 mM) DMEM with 10% fetal calf serum, 15 mM HEPES, pH
7.4, and 0.25 mg/ml geneticin, as described previously
(23). For fluorometry, cells from a confluent flask were
lightly trypsinized, replated onto collagen-coated coverslips, and used
at 100% confluence after 4-5 days. For p38 MAP kinase assays,
cells were grown to confluence in 35-mm-diameter tissue culture dishes
(Corning-Costar). Whether grown on coverslips or tissue culture dishes,
cells were cultured in low-glucose (5.5 mM) DMEM supplemented with 19.5 mM mannitol (to maintain overall osmolarity), 10% fetal calf serum,
and 15 mM HEPES, pH 7.4 (without geneticin), for 18 h before use
in experiments. All experiments were performed in nominally
HCO
Sugar uptake assays.
Sugar uptake assays were done in triplicate using cells grown on
collagen-coated 1.9-cm2 surface area tissue culture dishes
(Corning-Costar). Briefly, wells were washed three times with
glucose-free medium, incubated for 15 min at 37°C with glucose-free
medium containing the drugs indicated, and then incubated for the
indicated time at 37°C with 0.4 ml of glucose-free medium containing
14C-labeled -D-methyl glucoside
(23) and the indicated drugs. For the assays evaluating
drug inhibitions (see Fig. 8), uptake was determined after 15 min of
incubation in glucose-free medium containing 14C-labeled
-D-methyl glucoside. Wells were then washed at 4°C in
medium with 25 mM glucose, and cells were solubilized with 0.1 ml of
0.1 N NaOH. Specificity was confirmed by >97% reduction in
14C-labeled
-D-methyl glucoside uptake when
0.1 mM phloridzin was added and by >98% reduction when 10 mM glucose
was added.
Measurement of pHi. Confluent monolayers were washed with medium (with 25 mM mannose) and incubated for 15 min at room temperature with 3.5 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR). After they were washed, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded cells were warmed to 37°C and analyzed using a fluorometer equipped with dual emission/excitation monochromators (model RC-M, Photon Technology International, Monmouth Junction, NJ). Fluorescence was measured at excitations of 439 and 502 nm and emission of 535 nm. Fluorometric ratios corresponding to pH 7.00, 7.25, 7.50, and 7.75 were determined by clamping pHi using medium (at the designated pH) containing 110 mM KCl (in place of NaCl) and 10 µg/ml nigericin. Standard values were obtained for each sample set, and standard curves were generated and experimental data were analyzed using Felix software (version 1.21, Photon Technology International). Initial pHi values averaged 7.48 ± 0.005. Unless otherwise indicated, drugs were added in medium containing 25 mM mannose 15 min before exchange for medium containing 25 mM glucose and the drug. LY-294002, wortmannin, and ML-7 were added 30 min before the pH response to glucose was tested, cytochalasin D was added 3 h before the pH response to glucose was tested, and cells were preloaded with phalloidin for 18 h before the pH response to glucose was tested.
Immunoblot and in vitro p38 MAP kinase activity
assay.
Confluent monolayers were preincubated in medium with 25 mM mannose and
0.5 mM phloridzin for 20 min at 37°C. The buffer was then exchanged
isosmotically for medium with 25 mM glucose at 37°C. After incubation
at 37°C for the times indicated, cells were rapidly rinsed with PBS
at 4°C and lysed in 0.4 ml of lysis buffer (20 mM Tris, 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM -glycerophosphate, 1 mM
Na3VO4, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride, pH 7.5). After incubation on ice for 5 min, lysates were centrifuged at 14,000 g for 10 min, and
supernatants were used for subsequent assays. Protein content was
determined using the bicinchoninic acid protein assay (Pierce Chemical,
Rockford, IL).
Statistical analysis. All experiments were performed multiple times with two or more samples in each individual experiment. Results are expressed as means ± SE. Conditions were compared using Student's t-test (Excel, Microsoft, Redmond, WA). In pHi experiments, the change in pHi at 120 s after buffer exchange was compared using Student's t-test, and pHi response curves over the interval from 0 to 300 s were compared by analysis of variance (SigmaStat, SPSS).
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RESULTS |
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Cytoplasmic alkalinization occurs rapidly after Na+-glucose cotransport. The Caco-2 cell line used for these studies was stably transfected with the native intestinal Na+-glucose cotransporter SGLT1. We previously showed that these cells take up sugar with kinetics typical for native SGLT1 and that the transfected native SGLT1 is appropriately localized to the apical membrane domain in polarized monolayers (23). Such monolayers demonstrate vectorial Na+ and glucose transport, which coincides with the development of an Na+-dependent short-circuit current (24). We have also shown that these cells express all three intestinal Na+/H+ exchanger isoforms: basolateral NHE1 and apical NHE2 and NHE3 (22).
To evaluate the potential for a functional interaction between Na+/H+ exchange and Na+-glucose cotransport, we first evaluated pHi during initiation and termination of Na+-glucose cotransport (Fig. 1). Initiation of SGLT1-mediated Na+-glucose cotransport (by isosmotic substitution of mannose with glucose) resulted in a rapid pHi increase of 0.069 ± 0.002 that was complete within 150 s (P < 0.001). The pHi was then stable at this new elevated level for
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Na+-glucose
cotransport-dependent alkalinization requires NHE3 but not NHE1 or
NHE2.
As summarized above, our previous data led to the hypothesis that
Na+/H+ exchange might be activated after
Na+-glucose cotransport (22). The observation
that pHi increases after initiation of
Na+-glucose cotransport is consistent with activation of
Na+/H+ exchange. Because we previously showed
that the Caco-2 cells used in this study express all three intestinal
Na+/H+ exchanger isoforms (22), we
sought to determine which, if any, of the
Na+/H+ exchangers were responsible for the
observed increases in pHi. We compared the effects of two
Na+/H+ exchange inhibitors, S-3226 and HOE-694,
on pHi increases after initiation of
Na+-glucose cotransport. S-3226 has been reported to
inhibit NHE3 with an IC50 of 0.02 µM (human NHE3
expressed in fibroblasts) or 0.2 µM (porcine kidney brush-border
preparations) (17). In contrast, S-3226 inhibits NHE1 and
NHE2 relatively poorly, with IC50 of 3.5 µM for NHE1 and
80 µM for NHE2 (17). At 0.1 µM, S-3226 prevented
alkalinization after Na+-glucose cotransport by 41 ± 8% (P < 0.03), while 1 µM S-3226 prevented
alkalinization by 66 ± 1% (P < 0.001; Fig.
4). Further inhibition was not apparent
at higher S-3226 concentrations. If a linear relationship between the
inhibitory effects of S-3226 at 0.1 and 1 µM is assumed, these data
correspond to an ED50 of 0.35 µM, a value comparable to
the IC50 of 0.2 µM reported for NHE3 from porcine kidney
brush-border preparations (17). Thus pharmacological
inhibition of NHE3 can prevent the majority of cytoplasmic
alkalinization after Na+-glucose cotransport.
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Cytoplasmic alkalinization can be prevented by p38
MAP kinase inhibitors.
As shown above, hypotonic swelling did not cause pHi
increases comparable to those following Na+-glucose
cotransport. However, it is clear that cell volume increases after
Na+-glucose cotransport (10, 13). Thus we
considered the hypothesis that activation of the osmotically responsive
p38 MAP kinase could be involved in the NHE3-dependent cytoplasmic
alkalinization after initiation of Na+-glucose cotransport.
Two separate structurally related p38 MAP kinase inhibitors, PD-169316
(5 µM) and SB-202190 (10 µM), inhibited cytoplasmic alkalinization
after initiation of Na+-glucose cotransport by 100 ± 0.1 and 86 ± 0.1%, respectively (Fig.
7; P < 0.01). In
contrast, the structurally similar inactive compound SB-202474 (10 µM) did not affect cytoplasmic alkalinization after initiation of
Na+-glucose cotransport. Thus these data suggest that p38
MAP kinase may be an intermediate in the signal transduction pathway
between Na+-glucose cotransport and NHE3-dependent
cytoplasmic alkalinization. Notably, the effects of p38 MAP kinase
inhibitors and Na+/H+ exchange inhibitors on
cytoplasmic alkalinization are not due to inhibition of
Na+-glucose cotransport, since none of the compounds used
in these studies inhibited the Na+-glucose cotransporter
SGLT1 (Fig. 8).
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p38 MAP kinase is rapidly activated
after initiation of
Na+-glucose cotransport.
We sought to confirm that activation of p38 MAP kinase did indeed
follow the initiation of Na+-glucose cotransport with a
time course consistent with that of NHE3-dependent alkalinization.
Intracellular activation of p38 MAP kinase is accomplished by the p38
MAP kinase kinase that phosphorylates p38 MAP kinase at threonine-180
and tyrosine-182. Thus immunoblot using antisera specific for the
diphosphorylated form of p38 MAP kinase can be used to infer kinase
activation. Such immunoblots showed a 3.7 ± 0.3-fold increase in
diphosphorylated p38 MAP kinase (Fig. 9;
P < 0.01). This was comparable to the 4.1 ± 0.2-fold increase in p38 MAP kinase activity we measured using
immunoprecipitated p38 MAP kinase and an in vitro kinase assay (Fig. 9;
P < 0.05). Thus p38 MAP kinase activity is rapidly
increased after initiation of Na+-glucose cotransport. We
also examined p38 MAP kinase activation, by diphosphorylation, after
cell swelling in 10% hypotonic medium, even though this stimulus did
not cause cytoplasmic alkalinization (Fig. 3). p38 MAP kinase was
activated after osmotic swelling of Caco-2 cells with 10% hypotonic
medium in the absence of Na+-glucose cotransport. However,
this p38 MAP kinase activation occurred more slowly than that following
initiation of Na+-glucose cotransport, with a lag of
60-90 s in hypotonic medium-induced p38 MAP kinase activation
relative to Na+-glucose cotransport-induced p38 MAP kinase
activation. Moreover, the maximal degree of p38 MAP kinase activation
by 10% hypotonic medium (at 240 s) was only 20 ± 2% of
that induced by Na+-glucose cotransport.
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Pharmacological activation of p38 MAP
kinase induces cytoplasmic alkalinization.
The data above show that p38 MAP kinase inhibitors can prevent
NHE3-dependent cytoplasmic alkalinization after initiation of
Na+-glucose cotransport and that p38 MAP kinase activity is
increased after initiation of Na+-glucose cotransport. To
test whether p38 MAP kinase activation alone could cause NHE3-dependent
cytoplasmic alkalinization without Na+-glucose cotransport,
we used the chemical stressor anisomycin to activate p38 MAP kinase.
Anisomycin (0.3 µM) caused a 4.3 ± 0.4-fold increase in p38 MAP
kinase diphosphorylation and a 3.7 ± 0.4-fold increase in p38 MAP
kinase activity. In the absence of Na+-glucose cotransport,
addition of anisomycin caused cytoplasmic alkalinization of 0.047 ± 0.006 pH unit within 120 s (Fig.
10; P < 0.01). S-3226
reduced anisomycin-induced cytoplasmic alkalinization to 0.001 ± 0.004 pH unit (Fig. 10; P < 0.01), verifying the role of NHE3 in anisomycin-induced cytoplasmic alkalinization. In contrast, 50 µM HOE-604 did not inhibit anisomycin-induced cytoplasmic
alkalinization. Finally, anisomycin-induced cytoplasmic alkalinization
was completely prevented by PD-169316, confirming the role of p38 MAP
kinase in this alkalinization (Fig. 10; P < 0.01).
Thus p38 MAP kinase activation is sufficient to induce NHE3-dependent
cytoplasmic alkalinization, although to a slightly lesser degree than
the alkalinization following initiation of Na+-glucose
cotransport.
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NHE3-dependent alkalinization following Na+-glucose cotransport is not mediated by phosphatidylinositol 3-kinase or actomyosin function. Small intestinal NHE3-dependent Na+ absorption is acutely upregulated by epidermal growth factor (7). Inhibition of phosphatidylinositol 3-kinase with wortmannin prevents this epidermal growth factor-induced NHE3 activation (7). Thus it was concluded that phosphatidylinositol 3-kinase is responsible for this activation of NHE3 (7). To evaluate the potential role of phosphatidylinositol 3-kinase in NHE3-dependent cytoplasmic alkalinization after the initiation of Na+-glucose cotransport, we tested the effect of two different phosphatidylinositol 3-kinase inhibitors, LY-294002 (50 µM) and wortmannin (100 and 500 nM), each in at least four independent trials. Neither LY-294002 nor wortmannin prevented cytoplasmic alkalinization after initiation of Na+-glucose cotransport. Furthermore, in the absence of Na+-glucose cotransport, epidermal growth factor (200 nM) induced only a small degree of cytoplasmic alkalinization of 0.016 ± 0.001 pH unit at 120 s (P = 0.05). Subsequent initiation of Na+-glucose cotransport, in the continued presence of epidermal growth factor, resulted in typical cytoplasmic alkalinization that was not different from that induced by initiation of Na+-glucose cotransport in the absence of epidermal growth factor. Thus, although phosphatidylinositol 3-kinase can activate NHE3 after epidermal growth factor stimulation of serum-starved NHE3-transfected Caco-2 cells (7), phosphatidylinositol 3-kinase activity does not appear to be required for Na+-glucose cotransport-induced NHE3-dependent cytoplasmic alkalinization.
NHE3 can also be regulated by agents that disrupt actin polymerization (8) or modify myosin regulatory light chain phosphorylation (18). Therefore, we considered the possibility that actomyosin contraction could be an intermediate in NHE3 activation due to Na+-glucose cotransport. The effects of disrupting actin structure (20 µM cytochalasin D), stabilizing actin filaments (10 µM phalloidin), inhibiting myosin light chain kinase (20 µM ML-7), inhibiting rho kinase (30 µM Y-27632), or inhibiting actomyosin contraction (10 mM 2,3-butanedione monoxime) were evaluated. In at least three independent trials, each of these treatments failed to inhibit NHE3-dependent cytoplasmic alkalinization after initiation of Na+-glucose cotransport (data not shown). Thus these data suggest that the Na+-glucose cotransport-dependent activation of NHE3 is independent of actomyosin function. ![]() |
DISCUSSION |
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In the mammalian small intestine, absorption of Na+ and glucose is essential for maintenance of fluid and electrolyte balance. However, the coordinated regulation of Na+ absorption and Na+-glucose cotransport has not been studied previously. Previous studies of cell volume responses after swelling induced by Na+-glucose cotransport or hyposmotic stimuli suggest that Na+/H+ exchange is necessary for regulatory volume decreases (10-12). Moreover, separate studies of pHi regulation after H+-solute cotransport also suggest a role for Na+/H+ exchange in the regulation of intestinal absorption and enterocyte pHi (19). We have developed a model of Na+-glucose cotransport in monolayers of the human intestinal epithelial cell line Caco-2 (23), which we now use to ask whether Na+/H+ exchange is activated by Na+-glucose cotransport.
On the basis of sensitivity to the Na+/H+
exchange inhibitor S-3226 and resistance to HOE-694, we concluded that
the isoform responsible for cytoplasmic alkalinization after the
initiation of apical Na+-glucose cotransport was the apical
brush-border Na+/H+ exchanger NHE3. However, a
minority of alkalinization was not inhibitable by even 10 µM S-3226.
This S-3226-resistant fraction of alkalinization is not due to NHE1 or
NHE2, since S-3226 in combination with HOE-694 also failed to
completely prevent cytoplasmic alkalinization after initiation of
Na+-glucose cotransport. Because our studies were performed
in nominally HCO
To characterize the mechanism by which Na+-glucose cotransport leads to NHE3-dependent cytoplasmic alkalinization, we considered a variety of stimuli known to regulate NHE3. These included epidermal growth factor, phosphatidylinositol 3-kinase (7), myosin II regulatory light chain phosphorylation (18), and actin assembly (8). However, none of these signaling pathways appear to be involved in NHE3 activation after Na+-glucose cotransport. In the case of myosin II regulatory light chain, this result is consistent with our previous data showing that NHE3 inhibition leads to reduced phosphorylation of that protein (22).
Although the buffers used in these studies were isosmotic, modest cell swelling is an established consequence of Na+-glucose cotransport in absorptive enterocytes (10). Thus we evaluated the effects of cell swelling and found that hypotonic media-induced cell volume increases alone were insufficient to trigger the NHE3-dependent pHi increases. Despite the failure of hypotonic cell swelling alone to trigger cytoplasmic alkalinization, we also considered the possibility that p38 MAP kinase might be an intermediate in NHE3 activation after Na+-glucose cotransport. The osmotically sensitive p38 MAP kinase has been shown to be activated by cell swelling in renal and intestinal epithelial cell lines (14, 20). We found that inhibitors of p38 MAP kinase prevented NHE3-dependent cytoplasmic alkalinization after initiation of Na+-glucose cotransport and that initiation of Na+-glucose cotransport led to activation of p38 MAP kinase. Nonetheless, we considered the possibility that the activation of p38 MAP kinase could be a peripheral event unrelated to the observed NHE3-dependent cytoplasmic alkalinization. However, activation of p38 MAP kinase by anisomycin in the absence of Na+-glucose cotransport also caused NHE3-dependent cytoplasmic alkalinization, although to a quantitatively lesser degree than Na+-glucose cotransport. Finally, we confirmed that, although hypotonic cell swelling caused p38 MAP kinase activation, this p38 MAP kinase was blunted in both response rate and magnitude relative to p38 MAP kinase activation after initiation of Na+-glucose cotransport. We conclude that SGLT1-mediated Na+-glucose cotransport leads to activation of p38 MAP kinase and that subsequent increases in NHE3-mediated Na+/H+ exchange are mediated through this p38 MAP kinase activation. The failure of hypotonic cell swelling to activate NHE3-mediated cytoplasmic alkalinization, despite activation of p38 MAP kinase, likely relates to differences in the rapidity and extent of the response. The mechanism by which SGLT1-mediated Na+-glucose cotransport leads to activation of p38 MAP kinase is unknown. However, because intracellular Ca2+ signaling occurs after Na+-glucose cotransport-induced and hypotonic media-induced cell swelling and is necessary for volume regulation in both cases (13), one possibility may be that such increases in intracellular Ca2+ are responsible for p38 MAP kinase activation (5, 9). Although we have not measured intracellular Na+ directly, one might anticipate that increases in intracellular Na+ concentration caused by Na+-glucose cotransport and Na+/H+ exchange alter the driving force for Na+/H+ exchange. These intracellular Na+ concentration increases could represent the compensatory mechanism that limits the magnitude of pHi increases and establishes the new steady-state pHi.
It is possible that the NHE3 activation we have identified after Na+-glucose cotransport allows the coordinated activation of Na+-absorptive pathways in vivo. Because activation of Na+-nutrient, e.g., Na+-glucose, cotransport signals the presence of luminal nutrients, a system where this in turn activates other absorptive pathways could be reasonably envisioned. Thus Na+-glucose cotransport may lead to increased Na+ absorption via NHE3, the major mechanism of Na+ absorption in mammalian small intestine. In this manner, the initiation of Na+-glucose cotransport could trigger a shift of the cell from a quiescent to an active state with regard to nutrient and ion absorption.
In summary, the data demonstrate NHE3 activation and pHi regulation after the initiation of Na+-glucose cotransport in absorptive epithelia and also suggest that p38 MAP kinase is an integral part of this signaling pathway. Thus, in addition to signaling osmotic stress, p38 MAP kinase may be, in part, responsible for the coordinated regulation of Na+-nutrient cotransport and Na+/H+ exchange in absorptive enterocytes.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Mark Donowitz and Marshall Montrose for helpful discussion and Drs. Lucia Schuger and Judith Turner for critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02503 and DK-56121.
Address for reprint requests and other correspondence: J. R. Turner, Dept. of Pathology, The University of Chicago, 5841 S. Maryland Ave., MC 1089, Chicago, IL 60637 (E-mail: jturner{at}bsd.uchicago.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.
Received 5 February 2001; accepted in final form 6 July 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Chow, CW,
Khurana S,
Woodside M,
Grinstein S,
and
Orlowski J.
The epithelial Na+/H+ exchanger, NHE3, is internalized through a clathrin-mediated pathway.
J Biol Chem
274:
37551-37558,
1999
2.
Counillon, L,
Scholz W,
Lang HJ,
and
Pouyssegur J.
Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties.
Mol Pharmacol
44:
1041-1045,
1993[Abstract].
3.
Hayashi, M,
Yoshida T,
Monkawa T,
Yamaji Y,
Sato S,
and
Saruta T.
Na+/H+ exchanger 3 activity and its gene in the spontaneously hypertensive rat kidney.
J Hypertens
15:
43-48,
1997[ISI][Medline].
4.
Hoogerwerf, WA,
Tsao SC,
Devuyst O,
Levine SA,
Yun CH,
Yip JW,
Cohen ME,
Wilson PD,
Lazenby AJ,
Tse CM,
and
Donowitz M.
NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.
Am J Physiol Gastrointest Liver Physiol
270:
G29-G41,
1996
5.
Ikeda, M,
Gunji Y,
Yamasaki S,
and
Takeda Y.
Shiga toxin activates p38 MAP kinase through cellular Ca2+ increase in Vero cells.
FEBS Lett
485:
94-98,
2000[ISI][Medline].
6.
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
7.
Khurana, S,
Nath SK,
Levine SA,
Bowser JM,
Tse CM,
Cohen ME,
and
Donowitz M.
Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na+/H+ exchange.
J Biol Chem
271:
9919-9927,
1996
8.
Kurashima, K,
D'Souza S,
Szaszi K,
Ramjeesingh R,
Orlowski J,
and
Grinstein S.
The apical Na+/H+ exchanger isoform NHE3 is regulated by the actin cytoskeleton.
J Biol Chem
274:
29843-29849,
1999
9.
Lee, SA,
Park JK,
Kang EK,
Bae HR,
Bae KW,
and
Park HT.
Calmodulin-dependent activation of p38 and p42/44 mitogen-activated protein kinases contributes to c-fos expression by calcium in PC12 cells: modulation by nitric oxide.
Brain Res Mol Brain Res
75:
16-24,
2000[ISI][Medline].
10.
MacLeod, RJ,
and
Hamilton JR.
Volume regulation initiated by Na+-nutrient cotransport in isolated mammalian villus enterocytes.
Am J Physiol Gastrointest Liver Physiol
260:
G26-G33,
1991
11.
MacLeod, RJ,
and
Hamilton JR.
Activation of Na+/H+ exchange is required for regulatory volume decrease after modest "physiological" volume increases in jejunal villus epithelial cells.
J Biol Chem
271:
23138-23145,
1996
12.
MacLeod, RJ,
and
Hamilton JR.
Increases in intracellular pH and Ca2+ are essential for K+ channel activation after modest "physiological" swelling in villus epithelial cells.
J Membr Biol
172:
47-58,
1999[ISI][Medline].
13.
MacLeod, RJ,
Lembessis P,
and
Hamilton JR.
Differences in Ca2+-mediation of hypotonic and Na+-nutrient regulatory volume decrease in suspensions of jejunal enterocytes.
J Membr Biol
130:
23-31,
1992[ISI][Medline].
14.
Niisato, N,
Post M,
Van Driessche W,
and
Marunaka Y.
Cell swelling activates stress-activated protein kinases, p38 MAP kinase and JNK, in renal epithelial A6 cells.
Biochem Biophys Res Commun
266:
547-550,
1999[ISI][Medline].
15.
Rowe, WA,
Lesho MJ,
and
Montrose MH.
Polarized Na+/H+ exchange function is pliable in response to transepithelial gradients of propionate.
Proc Natl Acad Sci USA
91:
6166-6170,
1994[Abstract].
16.
Schultheis, PJ,
Clarke LL,
Meneton P,
Miller ML,
Soleimani M,
Gawenis LR,
Riddle TM,
Duffy JJ,
Doetschman T,
Wang T,
Giebisch G,
Aronson PS,
Lorenz JN,
and
Shull GE.
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
Nat Genet
19:
282-285,
1998[ISI][Medline].
17.
Schwark, JR,
Jansen HW,
Lang HJ,
Krick W,
Burckhardt G,
and
Hropot M.
S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types.
Pflügers Arch
436:
797-800,
1998[ISI][Medline].
18.
Szaszi, K,
Kurashima K,
Kapus A,
Paulsen A,
Kaibuchi K,
Grinstein S,
and
Orlowski J.
RhoA and rho kinase regulate the epithelial Na+/H+ exchanger NHE3. Role of myosin light chain phosphorylation.
J Biol Chem
275:
28599-28606,
2000
19.
Thwaites, DT,
Ford D,
Glanville M,
and
Simmons NL.
H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells.
J Clin Invest
104:
629-635,
1999
20.
Tilly, BC,
Gaestel M,
Engel K,
Edixhoven MJ,
and
de Jonge HR.
Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade.
FEBS Lett
395:
133-136,
1996[ISI][Medline].
21.
Turk, E,
Zabel B,
Mundlos S,
Dyer J,
and
Wright EM.
Glucose/galactose malabsorption caused by a defect in the Na+/glucose cotransporter.
Nature
350:
354-356,
1991[ISI][Medline].
22.
Turner, JR,
Black ED,
Ward J,
Tse CM,
Uchwat FA,
Alli HA,
Donowitz M,
Madara JL,
and
Angle JM.
Transepithelial resistance can be regulated by the intestinal brush border Na+/H+ exchanger NHE3.
Am J Physiol Cell Physiol
279:
C1918-C1924,
2000
23.
Turner, JR,
Lencer WI,
Carlson S,
and
Madara JL.
Carboxy-terminal vesicular stomatitis virus G protein-tagged intestinal Na+-dependent glucose cotransporter (SGLT1): maintenance of surface expression and global transport function with selective perturbation of transport kinetics and polarized expression.
J Biol Chem
271:
7738-7744,
1996
24.
Turner, JR,
Rill BK,
Carlson SL,
Carnes D,
Kerner R,
Mrsny RJ,
and
Madara JL.
Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.
Am J Physiol Cell Physiol
273:
C1378-C1385,
1997
25.
Watts, BA, III,
and
Good DW.
Hyposmolality stimulates apical membrane Na+/H+ exchange and HCO
26.
Weinman, EJ,
Steplock D,
Wang Y,
and
Shenolikar S.
Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na+-H+ exchanger.
J Clin Invest
95:
2143-2149,
1995[ISI][Medline].
27.
Wormmeester, L,
Sanchez de Medina F,
Kokke F,
Tse CM,
Khurana S,
Bowser J,
Cohen ME,
and
Donowitz M.
Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange.
Am J Physiol Cell Physiol
274:
C1261-C1272,
1998
28.
Yun, CH,
Oh S,
Zizak M,
Steplock D,
Tsao S,
Tse CM,
Weinman EJ,
and
Donowitz M.
cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein.
Proc Natl Acad Sci USA
94:
3010-3015,
1997