Dynamic regulation of Na+-K+-2Cl cotransporter surface expression by PKC-{epsilon} in Cl-secretory epithelia

Isabel Calvo Del Castillo,1 Mary Fedor-Chaiken,1 J. Cecilia Song,1 Veronika Starlinger,1,2 James Yoo,1 Karl S. Matlin,1 and Jeffrey B. Matthews1

1Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 2University of Vienna Medical School, Vienna, Austria

Submitted 29 November 2004 ; accepted in final form 29 June 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na+-K+-2Cl cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the {epsilon}-isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na+-K+-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC-{epsilon}-dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC-{epsilon}-dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl secretion.

endocytosis; recycling; ion transporters


ACTIVE TRANSCELLULAR TRANSPORT of Cl represents the primary mechanism of fluid secretion by epithelial cells lining the intestine, airway, and many exocrine organs. Dysregulation of this process underlies diseases ranging from cystic fibrosis to secretory diarrhea. Cl secretion requires the coordinated activities of specific ion transporters, pumps, and channels restricted to the apical or basolateral membrane of polarized epithelial cells. Basolateral Cl entry is driven by a Na+-K+-2Cl cotransporter (NKCC1), which uses the Na+ gradient established by the Na+-K+-ATPase, while K+ channels mediate basolateral K+ recycling or, in some instances, apical membrane K+ exit. Cl exits across the apical membrane through Cl channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), which classically have been considered the primary site of regulation of epithelial secretion. However, it is increasingly appreciated that basolateral transport pathways can also serve as independent regulatory sites that control net secretory output and hence the capacity for diarrhea (28).

The bumetanide-sensitive Na+-K+-2Cl cotransporter NKCC1 is a member of a family of cation-Cl cotransporters (21, 36). In addition to its housekeeping role in the regulation of cell volume and ion composition, NKCC1 is the basolateral isoform that is largely responsible for Cl entry in secretory epithelia (21). NKCC1 spans the membrane 12 times and displays multiple potential phosphorylation sites on its cytoplasmic COOH and NH2 tails. The COOH terminus is highly conserved between NKCC1 and NKCC2 (a renal system-specific, absorptive isoform that localizes to the apical membrane); variability in the NH2 terminus may confer cell-specific regulatory behavior (21). Although the driving force for NKCC1 function is set by transmembrane gradients of the three cotransported ions, its optimal activation also requires an increase in NKCC1 phosphorylation (21). Additional factors that modulate NKCC1 function include associated regulatory proteins, including protein phosphatase 1 (9), the kinases Ste20-related proline-alanine-rich kinase and oxidative stress response 1 (13, 34), and heat shock protein 90 (38), as well as cell volume, intracellular Cl concentration, and the cytoskeleton (21, 36). We previously suggested that short-term modulation of NKCC1 function in intestinal epithelial cells may also occur through regulated changes in its cell surface expression via transfer to or from vesicular pools (30), although direct evidence for this proposal is lacking. Membrane recycling as a means of transporter regulation has been described for several apical membrane channels and transporters, including CFTR (4), NKCC2 (18, 32), and aquaporin-2 (5). However, comparatively little is known about basolateral pathways.

PKC is a family of serine-threonine kinases sharing a common catalytic domain that is subdivided into conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC) isoforms by certain structural features and activation requirements (10). The effects of activation of PKC on epithelial Cl secretion are complex and depend on the organ/epithelial system, the nature of the stimulus, and the time course of observation. Activation of PKC by phorbol ester induces a transient increase in electrogenic Cl secretion, a response that is much smaller in magnitude than that elicited by stimulation of the cAMP-PKA pathway in various epithelial tissues, including intestinal (12, 46), tracheal (3), and conjunctival epithelium (45), as well as in cultured cell lines (30, 43). This early transient activation is generally followed by a prolonged and profound inhibition of transepithelial Cl secretion. For example, in the intestinal epithelial cell lines T84 and HCT29cl.19A, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (PMA) inhibits cAMP-dependent Cl secretion in a time- and dose-dependent manner (29, 30, 37, 43). We have shown that both the transient stimulatory effect and the long-term inhibitory effect of PMA are exerted primarily at the level of basolateral transporters (29, 30) with specific effects on NKCC1 (14, 15). PMA treatment inhibits NKCC1 activity (14, 30) and causes a decrease in bumetanide binding, indicating a decrease in NKCC1 at the cell surface (30). Inhibition of NKCC1 function was shown to parallel a selective increase in basolateral fluid phase endocytosis mediated by PKC-{epsilon}, suggesting that activation of this nPKC may induce endocytic retrieval of NKCC1 (40, 41). In the present study, we have used cell surface biotinylation, immunolocalization, and selective pharmacological manipulation to demonstrate regulated trafficking of NKCC1 mediated by PKC-{epsilon} in model human intestinal epithelial cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. T84 human intestinal epithelial cells were obtained from Dr. Kim E. Barrett (University of California, San Diego, CA) and cultured as described previously (40). Cells used were from passage 30 (the earliest passage available to us) to passage 40. At later passages, cell growth rate decreased and cells showed a diminished response to PKC agonists. Monolayers grown to confluence on collagen-coated permeable filters mounted on supports for 12- or 24-well culture dishes (Transwell inserts; Costar, Cambridge, MA) were used for experiments. Cells were seeded at 4 x 105 cells/cm2 and grown for ~14 days, with fresh medium added every 3 days, until the transepithelial electrical resistance of the monolayer exceeded 1,000 {Omega}/cm2.

Antibodies. Anti-NKCC1 monoclonal antibody T4 (27) and anti-Na+-K+-ATPase monoclonal antibody {alpha}-6F (42) were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Anti-NKCC1 polyclonal antibody TEFS-2 was a gift from Dr. C. Lytle (University of California, Riverside, CA). Anti-human E-cadherin monoclonal antibody was obtained from Zymed.

Gel electrophoresis and Western blotting. SDS-PAGE and Western blot analysis were performed as described previously (40). Equal amounts of protein (~20 µg/sample) were run on 6% gels and transferred to nitrocellulose membranes. Membranes were blocked and then incubated with appropriate antibodies. After being washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and washed again, and then antibody binding was visualized using ECL detection reagent.

Cell surface biotinylation. T84 monolayers grown to confluence on 4.7-cm2 Transwell inserts were treated with 100 nM PMA or 100 µM carbachol for various times at 37°C in HEPES-phosphate-buffered Ringer (HPBR) solution containing (in mM) 135 NaCl, 5.0 KCl, 3.33 NaH2PO4, 1.0 CaCl2, 1.0 MgCl2, 10 glucose, and 5.0 HEPES, pH 7.4. Cells were washed three times with ice-cold PBS supplemented with (in mM) 0.1 CaCl2 and 1.0 MgCl2 (PBS+). Fresh biotinylation buffer [BB; 1.0 mg/ml N-hydroxysulfosuccinimidobiotin (Sulfo-NHS-Biotin; Pierce) in PBS+] was prepared for every experiment. Cells were incubated with BB basolaterally for 15 min on ice, and then a fresh aliquot of BB was added for another 15 min. Cells were then washed five times with ice-cold PBS+ supplemented with 100 mM glycine as a quenching agent (19). Filters were excised and incubated in 1 ml of extraction buffer containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5, and Complete protease inhibitor cocktail (Roche) for 30 min on ice. Cells were scraped from the filter and centrifuged at 14,000 g at 4°C for 10 min. Supernatants were filtered through disposable centrifugal microfilters at 1,500 g for 10 min at 4°C. Samples containing equal amounts of protein were incubated with 100-µl streptavidin-agarose beads (Pierce) overnight at 4°C. Beads were collected using brief centrifugation and washed three times with ice-cold extraction buffer, twice with a high-salt wash buffer (0.1% Triton X-100, 500 mM NaCl, and 50 mM Tris, pH 7.5), and once with a low-salt wash buffer (10 mM Tris, pH 7.5). Aspirated pellets were mixed with 40 µl of sample buffer (20 mM Tris, 0.2 M DTT, 0.01% bromophenol blue, and 1.0% SDS), boiled for 5 min, and subjected to SDS-PAGE and Western blot analysis.

Internalization protocol. Dynamic internalization of NKCC1 was assessed by initial "pulse" biotin labeling at 4°C, followed by "chase" labeling at 37°C and then surface glutathione stripping of residual biotin (20). Cells were incubated with BB containing 1 mg/ml cleavable biotinylation reagent sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin; Pierce) basolaterally for 15 min on ice, and then a fresh aliquot of BB was added for another 15 min. Cells were washed five times with ice-cold PBS+ supplemented with 100 mM glycine as a quenching agent (19). Cells were washed twice with PBS+, and then inserts were treated with 100 nM PMA or 100 µM carbachol in HPBR solution for various times as indicated at 37°C. After two washes with PBS+, biotin was removed from the basolateral cell surface by performing two incubations with biotin stripping medium containing 155 mg of reduced glutathione (Sigma), 9 ml of 83 mM NaCl, 1 ml of fetal bovine serum, and 60 µl of 50% NaOH for 20 min at 4°C, followed by two washes with PBS+. Cell lysates were prepared, and biotinylated proteins were isolated and subjected to immunoblotting as described above. To assess NKCC1 recycling after internalization, a two-stage stripping procedure was used. After the initial stripping reaction, cells were returned to HPBR solution with or without PMA or carbachol at 37°C for 30 min. A second glutathione stripping procedure was then performed at 4°C, and samples were processed as described above.

Immunofluorescence microscopy. Monolayers grown on 0.33-cm2 permeable filters were prepared for confocal microscopy as previously described (40), with the addition of an epitope retrieval step to facilitate the binding of NKCC1 antibodies (31). After fixation and permeabilization, cells were washed with K+-free PBS and then incubated for 5 min in K+-free PBS containing 1% SDS. This step compromised cellular morphology and reduced filamentous actin (F-actin) staining; but without it, the T4 and TEFS-2 antibodies did not label NKCC1. After being washed with PBS, filters were excised and incubated with blocking buffer (1% normal goat serum and 3% BSA in PBS) for 30 min, followed by incubation with primary antibody in a humidified chamber at 4°C. Filters were then washed with PBS and incubated with Alexa Fluor 555-conjugated secondary antibody along with 1 U (~165 nM) of Alexa Fluor 488-phalloidin for F-actin staining. Filters were washed and mounted using Vectashield Hardset mounting medium (Vector Laboratories, Burlingame, CA). Confocal images were collected with a Zeiss LSM510 confocal scanning microscope using a x63 magnification/1.4 numerical aperture Plan Apochromat objective.

Materials. Tissue culture reagents were obtained from Invitrogen. Sulfo-NHS-Biotin, Sulfo-NHS-SS-Biotin, and ImmunoPure-immobilized streptavidin beads were purchased from Pierce. Gel electrophoresis and Western blotting reagents were obtained from Bio-Rad (Hercules, CA). ECL detection reagent was purchased from Amersham. Complete protease inhibitor cocktail tablets were obtained from Roche. Horseradish peroxidase-conjugated secondary antibodies used for Western blot analysis were obtained from Bio-Rad. Alexa Fluor 555-conjugated secondary antibodies used for immunostaining and Alexa Fluor 488-phalloidin were from Molecular Probes (Eugene, OR). Proteasome inhibitor I (PSI) and the PKC inhibitors Gö6976, Gö6850, and rottlerin were purchased from Calbiochem. All other chemicals, including carbachol, were obtained from Sigma.

Statistics. Data are expressed as means ± SE. Differences among groups were determined using one-way ANOVA and the Holm-Sidak method of multiple comparisons, and differences between two groups were determined using the Mann-Whitney rank-sum test. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NKCC1 expression is polarized in T84 cells. To confirm that NKCC1 is expressed exclusively on the basolateral epithelial surface in T84 cells, we cultured cells to confluence on collagen-coated permeable supports and biotinylated the apical and basolateral surfaces selectively. In agreement with previous results (8), biotinylated NKCC1 was detectable in samples biotinylated from either the basolateral side or both sides, but not from the apical side, indicating exclusive basolateral expression (Fig. 1A). When the overall distribution of NKCC1 in T84 cells was examined using indirect immunofluorescence with the NKCC1 antibody T4 (27, 31), significant staining was observed on the basolateral surface (Fig. 1B). Fluorescence also was observed near the apical surface in a submembranous compartment just below apical actin (Fig. 1B, x-z section), suggesting that a fraction of NKCC1 in T84 cells could reside in intracellular vesicles at the steady state (Fig. 1B). To verify this unexpected observation, we repeated the staining with a polyclonal NKCC1 antibody (TEFS-2) and obtained the same result (data not shown).



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Fig. 1. Na+-K+-2Cl cotransporter (NKCC1) is present on the basolateral plasma membrane and in intracellular compartments in T84 cells. A: confluent T84 cells grown on collagen-coated Transwell permeable supports were biotinylated from the apical side (Ap; lane 2), the basolateral side (BL; lane 3), or both sides (Ap/BL; lane 1) and extracted. Biotinylated proteins were recovered using precipitation with streptavidin-agarose, and NKCC1 on the cell surface was detected using Western blot analysis with the T4 antibody. B: confluent cultures of T84 cells on permeable supports were fixed with formaldehyde, permeabilized with Triton X-100, treated with SDS, and stained with the NKCC1 antibody T4, followed by an Alexa Fluor 555-conjugated secondary antibody (red). To visualize filamentous actin (F-actin), the cultures were also stained with Alexa Fluor 488-conjugated phalloidin (green). Optical sections were collected throughout the entire cell at 1.0-µm intervals with the use of a confocal fluorescence microscope. In an orthogonal (x-z) section, NKCC1 staining colocalized with actin along the lateral cell borders (arrows) but not at the apical surface (arrowheads). A large amount of staining was also present in the subapical cytoplasm below the level of actin in the apical cortex. Some NKCC1 staining was also present on the basal surface and protruded into pores of the support. In x-y sections, most NKCC1 staining was evident in the apical part of the cell and on the lateral borders (arrows in apical and middle sections). In a basal x-y section, actin stress fibers were apparent (arrows with asterisks). Scale bar, 10 µm.

 
PMA reduces surface expression of NKCC1 in a time-dependent fashion. The phorbol ester PMA is a nonspecific, irreversible activator of conventional and novel PKC isoforms. We have previously shown that PMA inhibits NKCC1 function (bumetanide-sensitive 86Rb uptake and [3H]bumetanide binding) as well as transepithelial Cl secretion in a time- and dose-dependent fashion (29, 30). We also have shown that PMA increases basolateral endocytosis in T84 cells in a time- and dose-dependent fashion (41). On the basis of these previously published reports, a 100 nM concentration of PMA, which has a nearly maximal effect, and a 30-min to 4-h time window were used in the present study. To determine whether activation of PKC with PMA would affect cell surface expression of NKCC1, cultures on permeable supports were treated with vehicle or with 100 nM PMA, and the amount of NKCC1 on the basolateral surface was then determined using selective basolateral biotinylation. As shown in Fig. 2A, treatment with PMA for 1 h decreased the amount of cell surface biotinylated NKCC1, while treatment for 2 h had an even more dramatic effect. Total NKCC1 in the cell did not change significantly within this time frame. As shown in Fig. 2B, cells treated with PMA for 1 h had <50% the amount of NKCC1 at the surface compared with controls (42.9 ± 6.2%; n = 3), while cells treated for 2 h had only ~10% that of control cells (10.5 ± 3.1%; n = 3).



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Fig. 2. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) stimulates loss of NKCC1 from the basolateral surface. A: confluent T84 cells grown on collagen-coated permeable supports were incubated without (C) or with 100 nM PMA in HEPES-phosphate-buffered Ringer (HPBR) solution at 37°C for 1 or 2 h and then were biotinylated from the basolateral surface. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 on the cell surface was detected using Western blot analysis with the T4 antibody. An aliquot of the total cell extract from each sample was also run on a parallel SDS gel and studied using Western blot analysis with anti-NKCC1 to provide a measure of total NKCC1 expression. B: band intensities from blots of biotinylated NKCC1 from untreated control cells, cells treated with PMA for 1 h, and cells treated with PMA for 2 h were measured using densitometric scanning. Data represent the average percentage of the control (100%) from 3 separate experiments. *P < 0.001, significantly different from control.

 
PMA induces internalization of NKCC1. To determine whether NKCC1 was internalized after PMA stimulation, the basolateral surface of T84 cells grown on permeable supports was incubated with Sulfo-NHS-SS-Biotin and then incubated with or without 100 nM PMA for 0–90 min (Fig. 3A). The cell surface was then stripped of biotin by treatment with glutathione, proteins were extracted, biotinylated proteins were captured on streptavidin beads, and biotinylated NKCC1 was detected using Western blot analysis. Under these conditions, only those proteins that were protected from glutathione stripping by internalization during the incubation period were detected. As shown in Fig. 3A, glutathione stripping eliminated much of the biotinylated NKCC1 at time 0 and at later times of incubation in the absence of PMA (Fig. 3A, lanes 2, 3, 5, and 7). In contrast, 30- to 90-min incubation with PMA resulted in a two- to threefold increase in biotinylated (and glutathione resistant) NKCC1, indicating that internalization had occurred (Fig. 3A, lanes 4, 6, and 8). During the incubation period, the total amount of NKCC1 did not change significantly. Treating cells for 60 min with 100 nM 4-{alpha}-phorbol, which does not activate PKC, did not cause an increase in glutathione-resistant, biotinylated NKCC1 (data not shown).



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Fig. 3. PMA induces internalization of NKCC1. Confluent T84 cells grown on collagen-coated permeable supports were biotinylated at 4°C with the cleavable biotinylation reagent sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin; Pierce). A: to evaluate the internalization of NKCC1 after biotinylation, control samples were either processed immediately without stripping (lane 1) or stripped of biotin with glutathione at 4°C and then processed (lane 2). Other biotinylated samples were reincubated at 37°C for 30 min (lanes 3 and 4), 60 min (lanes 5 and 6), or 90 min (lanes 7 and 8), without PMA (lanes 3, 5, and 7) or with PMA (lanes 4, 6, and 8), and then stripped with glutathione at 4°C. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 was detected using Western blot analysis with the T4 antibody. Using this protocol, we found that only biotinylated NKCC1 that had been internalized was resistant to glutathione stripping. Densitometric analysis of biotinylated NKCC1 remaining after stripping in 4 experiments is shown as the ratio of biotinylated NKCC1 to total, compared with the untreated control ratio at each time point, and normalized to 1. *P = 0.029, significantly different from control. B: to evaluate internalization and recycling of NKCC1, a two-stage stripping protocol was used. After biotinylation, control samples were either processed immediately without stripping (lane 1) or stripped at 4°C and then processed (lane 2). Other samples were reincubated at 37°C for 60 min without or with PMA (lanes 3 and 4) and then stripped at 4°C. After this first round of stripping (1°), two samples were reincubated for an additional 30 min with PMA at 37°C (lanes 5 and 6), and one of these was stripped a second time (2°; lane 6). Biotinylated proteins were then recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 was detected using Western blot analysis with the T4 antibody. Densitometric analysis of biotinylated NKCC1 remaining after stripping in 4 experiments is shown as the ratio of biotinylated NKCC1 to total, compared with untreated control ratio at 60 min, and normalized to 1. *P = 0.029, significantly different from control.

 
In some experiments in which we used the glutathione stripping procedure to measure internalization, it appeared that the amount of biotinylated NKCC1 protected from stripping began to decline after longer treatment with PMA. One possible explanation for this observation is that internalized and biotinylated NKCC1 recycled back to the basolateral surface, where it would again be accessible to the stripping reagents. To rule out this possibility, a two-stage stripping experiment was performed. As in the previous experiment, surface proteins were biotinylated with Sulfo-NHS-SS Biotin and then incubated for 60 min with or without PMA. After the cultures were cooled on ice, the surface was stripped with glutathione (first-round stripping). The cells were then reincubated for an additional 30 min, and a second round of stripping was performed. Under these conditions, biotinylated and internalized NKCC1 protected inside the cell from the first round of stripping would be stripped in the second round only if it had recycled to the basolateral surface. Recycling would be apparent by comparing samples that had undergone the second round of stripping with those that had not. As shown in Fig. 3B, the amount of internalized NKCC1 in PMA-treated cells subjected to two rounds of stripping was not significantly different from that of cells stripped only once (Fig. 3B, compare lanes 5 and 6) and was still significantly higher than that observed in untreated cells, indicating that NKCC1 that internalized after PMA stimulation did not recycle to the basolateral surface. Instead, the decrease in biotinylated NKCC1 protected from stripping after longer PMA treatment might be due to degradation.

In the experiments shown in Figs. 2 and 3, only the basolateral surface was biotinylated and stripped. To exclude the possibility that a significant fraction of the internalized NKCC1 underwent transcytosis or lateral diffusion to the apical membrane domain, subsets of monolayers were treated with PMA for 90 min and then selectively biotinylated from either the apical or the basolateral aspect. Under these conditions, NKCC1 was not detected at the apical surface, demonstrating that transcytosis did not occur (data not shown).

Morphological confirmation of these biochemical data was obtained by performing indirect immunofluorescence. T84 monolayers were treated with or without 100 nM PMA for 0–3 h, NKCC1 was localized using antibody staining (Fig. 4, red), and F-actin was visualized using fluorescein isothiocyanate-phalloidin (Fig. 4, green). In control cells, NKCC1 staining was visible on the lateral cell surfaces and in small, poorly resolved vesicles extending beneath the apical surface (Fig. 4, top; see also Fig. 1B). As shown in Fig. 4, left, after 1 h of PMA treatment, NKCC staining appeared in both small and medium-sized vesicles; the lateral membrane also continued to stain for NKCC1. After 2 h of PMA treatment, the number of medium-sized vesicles increased, while the lateral membrane appeared to be almost devoid of staining. At the same time, some large vacuoles staining for NKCC1 also became evident. By 3 h of PMA treatment, many more large NKCC1-positive vacuoles were visible, along with some slight reactivity of lateral membranes. These results were verified using a polyclonal NKCC1 antibody (TEFS-2; data not shown). Overall, immunolocalization of NKCC1 after PMA treatment was consistent with the biochemical results and suggested that internalized NKCC1 progressively moved through a number of vesicular compartments.



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Fig. 4. NKCC1 appears in intracellular vesicles after PMA and carbachol treatment. Confluent cultures of T84 cells on permeable supports were treated with PMA or carbachol (CCh) for 1–3 h and then fixed with formaldehyde, permeabilized with Triton X-100, treated with SDS, and stained with the NKCC1 antibody T4, followed by an Alexa Fluor 555-conjugated secondary antibody (red). To visualize F-actin, the cultures were also stained with Alexa Fluor 488-conjugated phalloidin (green). Optical sections were collected throughout the entire cell at 1.0-µm intervals using a confocal fluorescence microscope. Only single sections from the subapical region containing the bulk of NKCC1 staining are shown. Staining at the lateral membrane (arrows with asterisks), small vesicles (arrowheads with asterisks), medium-sized vesicles (arrows), and large vacuoles (arrowheads) is highlighted. Scale bar, 10 µm.

 
PMA-induced internalization of NKCC1 is selective. Our previously published results indicated that treatment of T84 cells with PMA caused significant remodeling of the basolateral membrane and the actin cytoskeleton associated with a selective increase in basolateral fluid phase endocytosis (41). For this reason, it was of interest to determine whether the induction of NKCC1 internalization by PMA was specific or whether a broad group of basolateral proteins was internalized to the same degree in response to PMA. To address this question, T84 cells were treated with PMA for 1 h and then biotinylated from the basolateral surface. After capture of biotinylated proteins on streptavidin beads, the amounts of the adhesion protein E-cadherin, the Na+ pump Na+-K+-ATPase, and NKCC1 at the cell surface were estimated using Western blot analysis and compared with controls that had not been incubated with PMA. As shown in Fig. 5, most E-cadherin and Na+-K+-ATPase remained on the cell surface after 1 h of PMA treatment compared with untreated cells (87.1 ± 4.5% and 93.0 ± 2.2%, respectively), while significantly less of the NKCC1 was at the surface after the same treatment (60.3 ± 4.7%). On this basis, it appeared that the effect of PMA on NKCC1 internalization was more pronounced than the effect on the other integral membrane proteins.



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Fig. 5. NKCC1 is selectively internalized in response to PMA. Confluent T84 cells grown on collagen-coated permeable supports were incubated in HPBR solution with or without 100 nM PMA at 37°C for 60 min and then biotinylated from the basolateral surface. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose and E-cadherin (E-cad), and Na+-K+-ATPase (NaK) and NKCC1 were detected by blotting with specific antibodies. The intensities of the bands from blots of biotinylated E-cadherin, Na+-K+-ATPase, or NKCC1 from untreated control cells (solid bars) and cells treated with PMA for 1 h (shaded bars) were measured using densitometric scanning, and the results are expressed in arbitrary units. Data represent the average of at least 4 separate experiments (n = 5 for E-cad, n = 4 for NaK, n = 8 for NKCC1). *P = 0.008, amount of biotinylated NKCC1 at the cell surface after PMA treatment significantly different from amount without PMA treatment.

 
PMA-induced internalization of NKCC1 is mediated by PKC-{epsilon}. In intestinal epithelia, the major PKC isoforms are cPKC-{alpha} and cPKC-{beta}II, nPKC-{epsilon} and nPKC-{delta}, and aPKC-{zeta} (44). In earlier reports, we showed that in T84 cells, PMA activates cPKC-{alpha}, nPKC-{delta}, and nPKC-{epsilon} (40, 41). To determine which specific PKC isoform mediates NKCC1 internalization, T84 cells were pretreated with the cPKC and nPKC inhibitor Gö6850 (5 µM), the cPKC-specific inhibitor Gö6976 (5 µM), and rottlerin (10 µM), which inhibits PKC-{delta} along with a variety of other non-PKC kinases (40). In our previous report, we used in vitro kinase assays to establish the concentration dependence and isoform selectivity of these three pharmacological PKC inhibitors in T84 cells. In the experiment shown in Fig. 6, T84 cells were pretreated with PKC inhibitors at the determined concentrations, cell surface proteins were biotinylated, and cells were treated with PMA in the constant presence of the PKC inhibitors. After surface biotin was stripped, biotinylated proteins were isolated and internalized NKCC1 was detected using Western blot analysis. PMA treatment increased internalization approximately fivefold, but this effect was blocked by Gö6850 (Fig. 6, lanes 4 and 5). Gö6976 and rottlerin did not significantly block the PMA effect (Fig. 6, lanes 6 and 7). These results suggest that PMA-stimulated internalization of NKCC1 is dependent specifically on PKC-{epsilon}.



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Fig. 6. PMA-stimulated NKCC1 endocytosis is blocked by inhibitors of PKC-{epsilon}. Confluent T84 cells grown on collagen-coated permeable supports were pretreated for 30 min with 5 µM Gö6850, 5 µM Gö6976, or 10 µM rottlerin at 37°C and then biotinylated at 4°C with the cleavable biotinylation reagent Sulfo-NHS-SS-Biotin. Control samples were either processed immediately without stripping (no strip control) or stripped of biotin with glutathione at 4°C and then processed (strip control). Other biotinylated samples were reincubated at 37°C for 1 h in the presence or absence of PMA and PKC inhibitors to allow for the internalization of proteins and then stripped with glutathione at 4°C. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 was detected using Western blot analysis with the T4 antibody. Densitometric analysis of biotinylated NKCC1 remaining after stripping in 3 experiments is shown as the ratio of biotinylated NKCC1 to total, compared with untreated control ratio, and normalized to 1. *P < 0.026, significantly different from control.

 
PMA induces degradation of NKCC1. We have shown previously that long-term treatment of T84 cells with PMA results in a decrease in NKCC1 protein and mRNA (15). In addition, the results presented in Fig. 3 suggest that longer treatment with PMA may lead to degradation of internalized NKCC1. To examine the possibility in the present study that PMA treatment caused a decrease in total NKCC1 protein, cultures were incubated for up to 4 h with and without PMA and then were biotinylated from the basolateral surface. Aliquots of total cell lysate were saved for determination of the total amount of NKCC1 by performing blotting. Biotinylated proteins were captured on streptavidin beads, run on an SDS gel, and blotted with NKCC1-specific antibodies to determine the amount of NKCC1 at the cell surface. As shown in Fig. 7A, after 2-h incubation with PMA, most cell surface NKCC1 was internalized as shown in Fig. 2; at the same time, it was evident that the total amount of NKCC1 had also declined relative to controls not treated with PMA (Fig. 7A, compare lanes 3 and 4). After 4 h of PMA exposure, very little NKCC1 was detectable in the cell lysate on the basis of Western blot analysis, while control levels were only slightly depressed (Fig. 7A, compare lanes 7 and 8).



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Fig. 7. Long-term incubation with PMA leads to NKCC1 degradation. A: confluent T84 cells grown on collagen-coated permeable supports were incubated with or without 100 nM PMA in HPBR solution at 37°C for up to 4 h and then biotinylated from the basolateral surface. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 on the cell surface was detected using Western blot analysis with the T4 antibody. An aliquot of the total cell extract from each sample was also run on a parallel SDS gel, and Western blot analysis was performed with anti-NKCC1 to provide a measure of total NKCC1 expression. Densitometric analysis of total NKCC1 after 2 h in HPBR solution (3), 2 h in HPBR solution plus PMA (4), 4 h in HPBR solution (7), and 4 h in HPBR solution plus PMA (8) in 3 experiments is shown, normalized to values for 2 h in HPBR solution. *P < 0.009, significantly different from all other values. B: confluent T84 cells grown on collagen-coated permeable supports were incubated without (0) or with 25 or 50 µM proteasome inhibitor I (PSI; Inhibitor) for 1 h and then without or with PMA, in the continued presence of PSI where appropriate, for 3 h. Total protein was extracted, run on an SDS gel, and subjected to Western blot analysis with anti-NKCC1 antibodies.

 
We next examined whether, in response to PMA, NKCC1 underwent degradation via lysosome or proteasome pathways. Monolayers were preincubated with 2–60 µg/ml leupeptin, an inhibitor of lysosome proteolysis, and then treated with PMA in the continued presence of leupeptin. Under these conditions, leupeptin had no effect on NKCC1 degradation (data not shown). However, incubation with the proteasome inhibitor PSI at 25 or 50 µM blocked degradation of NKCC1 (Fig. 7B). Thus it is likely that PMA-induced degradation is mediated by the proteasome route.

Carbachol induces internalization and recycling of NKCC1. In T84 cells, the acetylcholine analog carbachol acts via muscarinic receptors to transiently stimulate Cl secretion, an effect that involves an inositol 1,4,5-trisphosphate-generated increase in intracellular Ca2+ (2). Carbachol-mediated phospholipid turnover also generates diacylglycerol (DAG) and activates PKC, which itself acts as a negative regulator of secretion (2). After the transient increase in Cl secretion elicited by carbachol, there is a sustained inhibitory phase during which the response to subsequent stimulation by Ca2+ and cAMP-dependent secretagogues is markedly reduced. The late phase of carbachol-mediated inhibition of secretion, like the effect of PMA, correlates with enhanced basolateral endocytosis (41). In T84 cells, carbachol has been shown to induce the activation of PKC-{epsilon} but not PKC-{delta} or PKC-{alpha} (41).

To determine whether carbachol also induced internalization of NKCC1, cultures were incubated with 100 µM carbachol, a concentration shown to stimulate Cl secretion (11) and induce basolateral endocytosis in T84 cells (41), for up to 3 h and then cell surface NKCC1 was detected using biotinylation and Western blot analysis. As with PMA treatment, incubation with carbachol for 1 h caused significant loss of cell surface NKCC1 relative to untreated controls (Fig. 8A, compare lanes 1 and 2). However, unlike PMA, after 2-h incubation, amounts of NKCC1 significantly higher than those observed at 1 h were again detectable on the cell surface (Fig. 8A, lanes 36).



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Fig. 8. Carbachol leads to the disappearance and reappearance of NKCC1 at the basolateral surface in a PKC-{epsilon}-dependent fashion. A: confluent T84 cells grown on collagen-coated permeable supports were incubated with or without 100 µM carbachol in HPBR solution at 37°C for up to 3 h and then biotinylated from the basolateral surface. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 on the cell surface was detected using Western blot analysis with the T4 antibody. An aliquot of the total cell extract from each sample was also run on a parallel SDS gel and subjected to Western blot analysis with anti-NKCC1 to provide a measure of total NKCC1 expression. B: confluent T84 cells grown on collagen-coated permeable supports were pretreated for 30 min with 5 µM Gö6850, 5 µM Gö6976, or 10 µM rottlerin at 37°C and then biotinylated at 4°C with the cleavable biotinylation reagent Sulfo-NHS-SS-Biotin. Biotinylated samples were reincubated at 37°C for 1 h in the presence or absence of 100 µM carbachol and PKC inhibitors to allow for the internalization of proteins and then were stripped with glutathione at 4°C. Biotinylated proteins were recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 was detected using Western blot analysis with the T4 antibody. Densitometric analysis of biotinylated NKCC1 remaining after stripping in 3 experiments is shown as the ratio of biotinylated NKCC1 to total, compared with untreated control ratio, and normalized to 1.

 
To verify that the carbachol effect was a result of specific activation of PKC-{epsilon}, T84 cells were pretreated with the cPKC and nPKC inhibitor Gö6850 (5 µM), the cPKC-specific inhibitor Gö6976 (5 µM), and the PKC-{delta} inhibitor rottlerin (10 µM). If PKC-{epsilon} is specifically activated by carbachol, then only Gö6850 should inhibit the carbachol-induced internalization. Cell surface proteins were biotinylated, and cells were treated with carbachol in the constant presence of the PKC inhibitors. After surface biotin had been stripped, biotinylated proteins were isolated and internalized NKCC1 was detected using Western blot analysis. Carbachol treatment significantly increased internalization, but this effect was blocked by Gö6850 (Fig. 8B, lanes 2 and 3). Gö6976 and rottlerin did not significantly block the PMA effect (Fig. 8B, lanes 4 and 5). These results suggest that carbachol-stimulated internalization of NKCC1, like PMA-stimulated internalization, is dependent specifically on PKC-{epsilon}.

As shown in Fig. 8A, carbachol stimulation initially caused a reduction in the amount of NKCC1 on the basolateral surface; but with continued carbachol incubation, additional NKCC1 seemed to reappear on the cell surface, suggesting that internalized NKCC1 recycled to the plasma membrane. To test this possibility, a two-stage stripping procedure was used. Surface-biotinylated cells were incubated for 1 h with or without carbachol, and the surface was stripped of biotin with glutathione. The cultures were then reincubated for 30 min and stripped a second time or, as a control, were not stripped. Under these conditions, any biotinylated NKCC1 that recycled in the second 30-min incubation would be sensitive to the second stripping procedure. As shown in Fig. 9, incubation of cultures with carbachol for 60 min led to a 2.5-fold increase in internalization of biotinylated NKCC1 relative to untreated controls as demonstrated by the resistance of the biotinylated protein to the first round of stripping (Fig. 9, compare lanes 3 and 4). After an additional 30-min incubation with carbachol and a second round of stripping, the amount of detectable biotinylated NKCC1 was reduced relative to a carbachol-treated sample that was not stripped (Fig. 9, compare lanes 5 and 6). On this basis, we conclude that, like PMA, carbachol induced NKCC1 internalization, but also that, in contrast to PMA, significant amounts of internalized NKCC1 recycled back to the basolateral plasma membrane.



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Fig. 9. Carbachol stimulates internalization and recycling of NKCC1. Confluent T84 cells grown on collagen-coated permeable supports were biotinylated from the basolateral surface at 4°C with a disulfide-cleavable reagent. After biotinylation, control samples were either processed immediately without stripping (lane 1) or stripped of biotin with glutathione at 4°C (lane 2) and then processed. Other biotinylated samples were incubated at 37°C for 60 min, without carbachol (lane 3), or with 100 µM carbachol (lanes 4 and 6) and then stripped with glutathione at 4°C. After this primary round of stripping (1°), two samples were reincubated with carbachol for an additional 30 min at 37°C (lanes 5 and 6), and one of these was stripped again (second round of stripping, 2°; lane 6). Biotinylated proteins were then recovered from cell extracts by performing precipitation with streptavidin-agarose, and NKCC1 was detected using Western blot analysis with the T4 antibody. Densitometric analysis of biotinylated NKCC1 remaining after stripping in 3 experiments is shown as the ratio of biotinylated NKCC1 to total, compared with 60-min untreated control ratio, and normalized to 1. *P = 0.029, significantly different from control.

 
This conclusion was supported by immunolocalization of NKCC1 in carbachol-treated cells. As shown in Fig. 4 (right), 1-h incubation with carbachol led to the appearance of small and some medium-sized vesicles that stained with the NKCC1 antibody, while the lateral surface continued to stain for NKCC1. After 2-h incubation with carbachol, the number of small vesicles increased and some large NKCC1-positive vacuoles were also evident. After 3 h of carbachol treatment, a number of medium-sized vesicles, with some arrayed along the lateral membranes, and a few large vacuoles were also present. In contrast to cells incubated with PMA (Fig. 4, left), at no time in carbachol-treated cells did the lateral surfaces appear depleted of NKCC1 staining. On the basis of these observations, we conclude that in the presence of carbachol, NKCC1 was internalized first in small vesicles and then transported to medium-sized vesicular compartments localized along the lateral plasma membrane. Some NKCC1 also seemed to be routed into large vacuoles, although these were much less evident than after PMA treatment.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Our laboratory previously reported that PMA treatment inhibits NKCC1 activity (14, 30) and markedly reduces the binding of [3H]bumetamide, an NKCC1 inhibitor, to the basolateral membrane of polarized T84 cells (30). Inhibition of NKCC1 function was shown to parallel a selective increase in basolateral fluid phase endocytosis mediated by PKC-{epsilon} (40, 41). These observations led us to suggest that activation of PKC could induce endocytic retrieval or degradation of NKCC1. The present studies clearly indicate that such endocytic retrieval does indeed occur upon PKC stimulation, not only with PMA but also with the physiological PKC agonist carbachol. Using endocytosis and recycling assays based on surface biotinylation and immunoblotting, along with immunocytochemical localization of NKCC1, we have demonstrated that upon PMA stimulation, NKCC1 on the basolateral surface is substantially endocytosed, sequentially enters smaller and then larger vesicular compartments, and eventually is degraded. Although we previously demonstrated that PMA stimulation leads to an increase in fluid phase endocytosis in T84 cells (41), uptake of NKCC1 does not occur as part of a generalized, nonspecific uptake of basolateral membrane, because neither E-cadherin nor the Na+-K+-ATPase, both basolateral proteins in T84 cells, is internalized to the same extent as NKCC1 in response to PMA. When T84 cells are treated with carbachol, which specifically activates PKC-{epsilon} in these cells (41), NKCC1 is also internalized but is recycled to the basolateral surface without apparent degradation. Inhibitor studies have shown that both PMA- and carbachol-induced internalization depend on activation of PKC-{epsilon} and not other PKC isoforms.

PKC has long been known to be a regulator of NKCC1 function, although its effects are highly tissue specific and can be either inhibitory or stimulatory (36). NKCC1 contains several consensus sequences for phosphorylation by PKC, but in no instances have PKC-elicited alterations in NKCC1 function been linked to PKC-dependent phosphorylation of NKCC1 per se. Our finding that PKC activation caused internalization of NKCC1 begins to elucidate the mechanism of PKC-mediated NKCC1 inhibition in T84 cells. However, the precise signal governing PKC-dependent endocytosis is unknown. PKC is known to affect receptor recycling and the surface expression of a number of proteins (16, 26, 35). Although in some instances direct phosphorylation of the protein by PKC is involved, the effects of PKC in many situations appear to be mediated at the level of membrane plasticity and the actin cytoskeleton (33, 39, 41).

We have shown that NKCC1 undergoes recycling in response to carbachol but is degraded in response to PMA. In T84 cells, PMA irreversibly activates PKC-{alpha}, PKC-{delta}, and PKC-{epsilon}, while carbachol, acting via muscarinic receptors to stimulate DAG production, activates only the {epsilon}-isoform. The lack of NKCC1 recycling with PMA may be due to costimulation of PKC-{alpha} or PKC-{delta} along with PKC-{epsilon}, overstimulation of PKC-{epsilon}, or both. The issue of spatially selective stimulation may also be significant. PMA is readily diffusible and may induce activation throughout the cell, whereas carbachol may cause a more limited, regionalized response in the vicinity of muscarinic receptor-mediated DAG generation.

In this article, we report the first data linking a specific PKC isoform to the inhibition of NKCC1 function. In the Calu-3 airway epithelia, cells in which PKC exerts a stimulatory rather than an inhibitory effect on NKCC1, PKC-{delta} appears to be the isoform responsible for functional regulation of NKCC1 (23, 24). We have no evidence for an effect of PKC-{delta} on NKCC1 in T84 cells. However, individual PKC isoforms can exert the same effect, no effect, or even opposing effects on a given biological function (10). Therefore, it is possible that the variable reported effects of PKC on NKCC1 could reflect cell-specific differences in PKC isoform expression or in the pattern of activation elicited by various stimuli. It is also possible that PKC-{delta} and PKC-{epsilon} exert opposing regulatory roles on NKCC1. These two isoforms have previously been reported to play mutually antagonistic roles in the regulation of myocardial protection (7).

It is unclear whether NKCC1 is internalized via clathrin-coated vesicles, a non-clathrin-mediated vesicular mechanism (e.g., caveolin dependent) or macropinocytosis. Several transporter proteins have been shown to be internalized via a clathrin-mediated pathway, including CFTR (4), the nongastric H+-K+-ATPase ATP1AL1 (35), and aquaporin-2 (5). In contrast, certain K+ and Na+ channels localize to caveolin-rich membrane compartments (47). Future studies using both T84 cells and Madin-Darby canine kidney (MDCK) cells stably expressing green fluorescent protein (GFP)-NKCC1 will focus on identifying the endocytic pathway used by NKCC1.

The PMA- and carbachol-induced internalization of NKCC1 that we observed using surface biotinylation was confirmed on the basis of confocal immunofluorescence microscopy. In the course of those experiments, we were surprised to observe a large intracellular pool of NKCC1 in the subapical region of the cell. That this fraction was indeed intracellular and not mislocalized to the apical surface was clear; apical cytoplasmic NKCC1 staining was localized below apical cortical actin in favorable vertical confocal sections, and no apical NKCC1 was detected using surface biotinylation either with or without PMA treatment. While we cannot completely rule it out, we believe that the existence of the subapical pool of NKCC1 in T84 cells is not an artifact of the monoclonal antibody used for immunofluorescence, because a second polyclonal antibody produced a similar distribution. Furthermore, we also have detected such a subapical pool in MDCK cells stably expressing GFP-NKCC1 (Worrell RT, Fedor-Chaiken M, Matlin KS, and Matthews JB, unpublished observations). It is possible, though, that the intensity of staining is due in some manner to the treatment of fixed cells with high concentrations of SDS (so-called antigen retrieval) required to make cellular NKCC1 reactive to both the monoclonal and polyclonal antibodies. While the need for such established antigen retrieval methods is not uncommon with a multispanning membrane protein such as NKCC1 (6), the SDS treatment also led to a reduction of F-actin staining with phalloidin and decreased microscopic resolution. It must also be kept in mind that T84 cells are derived from a colon carcinoma and that cellular changes in concert with carcinogenesis may have abnormally expanded a normally modest intracellular pool of NKCC1.

The effects of SDS antigen retrieval also make it difficult to determine the precise nature of the subapical vesicular compartment containing NKCC1. In many polarized epithelial cells, a subapical recycling endosome participates in both the endocytic recycling pathway and the biosynthetic sorting pathway of apical and basolateral proteins (1, 22, 25). The subapical NKCC1 that we observed may have been trafficking to or from the plasma membrane and may have served as an intracellular reservoir that could be mobilized quickly to increase the number of basolateral transport units. In support of this interpretation, it was recently reported that NKCC1 is rapidly and transiently recruited to the membrane of spinal cord neurons upon painful stimulation (17). In preliminary studies, we have observed movement of vesicular GFP-labeled NKCC1 from the basal side toward the apical side of MDCK cells upon PMA stimulation (Worrell RT, Fedor-Chaiken M, Matlin KS, and Matthews JB, unpublished observations). This system will be used in future studies to further characterize NKCC1 trafficking.

A comprehensive model for NKCC1 regulation has not yet emerged. However, it is increasingly clear that NKCC1 is a key control point for transepithelial Cl transport. NKCC1 function can be envisioned as a rheostat that determines secretory throughput in response to regulated activation of apical Cl conductance. Acute regulation of NKCC1 (from seconds to minutes) appears to be accomplished via phosphorylation-dependent events. Regulation of NKCC1 gene expression may provide a means of long-term control of Cl secretion. The present study provides evidence for an additional mechanism of NKCC1 regulation at the level of cell surface expression, which may be important to the control of Cl secretion over a time course of minutes to hours. PKC-{epsilon} appears to mediate this response and thus appears to be an important negative regulator of NKCC1 and Cl secretion. Isoform-selective pharmacological manipulation of PKC and NKCC1 may thus represent a therapeutic target for diseases of epithelial electrolyte transport.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48010 and DK-51630 (to J. B. Matthews).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Matthews, Dept. of Surgery, Univ. of Cincinnati Medical Center, 231 Albert B. Sabin Way, PO Box 670558, Cincinnati, OH 45267-0558 (e-mail: Jeffrey.Matthews{at}uc.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.


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