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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
endocytosis; recycling; ion transporters
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-, 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-
in model human intestinal epithelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies.
Anti-NKCC1 monoclonal antibody T4 (27) and anti-Na+-K+-ATPase monoclonal antibody -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
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 03 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.
|
|
|
|
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- but not PKC-
or PKC-
(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).
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-, PKC-
, and PKC-
, while carbachol, acting via muscarinic receptors to stimulate DAG production, activates only the
-isoform. The lack of NKCC1 recycling with PMA may be due to costimulation of PKC-
or PKC-
along with PKC-
, overstimulation of PKC-
, 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- appears to be the isoform responsible for functional regulation of NKCC1 (23, 24). We have no evidence for an effect of PKC-
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-
and PKC-
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- 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Barrett KE. Positive and negative regulation of chloride secretion in T84 cells. Am J Physiol Cell Physiol 265: C859C868, 1993.
3. Barthelson RA, Jacoby DB, and Widdicombe JH. Regulation of chloride secretion in dog tracheal epithelium by protein kinase C. Am J Physiol Cell Physiol 253: C802C808, 1987.
4. Bertrand CA and Frizzell RA. The role of regulated CFTR trafficking in epithelial secretion. Am J Physiol Cell Physiol 285: C1C18, 2003.
5. Brown D. The ins and outs of aquaporin-2 trafficking. Am J Physiol Renal Physiol 284: F893F901, 2003.
6. Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, and Alper S. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261267, 1996.[ISI][Medline]
7. Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R, Dorn GW II, and Mochly-Rosen D. Opposing cardioprotective actions and parallel hypertrophic effects of PKC and
PKC. Proc Natl Acad Sci USA 98: 1111411119, 2001.
8. D'Andrea L, Lytle C, Matthews JB, Hofman P, Forbush B III, and Madara JL. Na:K:2Cl cotransporter (NKCC) of intestinal epithelial cells: surface expression in response to cAMP. J Biol Chem 271: 2896928976, 1996.
9. Darman RB, Flemmer A, and Forbush B. Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter. J Biol Chem 276: 3435934362, 2001.
10. Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, and Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 279: L429L438, 2000.
11. Dharmsathaphorn K and Pandol SJ. Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest 77: 348354, 1986.[ISI][Medline]
12. 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: G509G517, 1986.
13. Dowd BFS and Forbush B. PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1). J Biol Chem 278: 2734727353, 2003.
14. Farokhzad OC, Mun EC, Sicklick JK, Smith JA, and Matthews JB. Effects of bryostatin 1, a novel anticancer agent, on intestinal transport and barrier function: role of protein kinase C. Surgery 124: 380387, 1998.[CrossRef][ISI][Medline]
15. Farokhzad OC, Vivek Sagar GD, Mun EC, Sicklick JK, Lotz M, Smith JA, Song JC, O'Brien TC, Sharma CP, Kinane TB, Hodin RA, and Matthews JB. Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl cotransporter. J Cell Physiol 181: 489498, 1999.[CrossRef][ISI][Medline]
16. Fournier KM, González MI, and Robinson MB. Rapid trafficking of the neuronal glutamate transporter, EAAC1: evidence for distinct trafficking pathways differentially regulated by protein kinase C and platelet-derived growth factor. J Biol Chem 279: 3450534513, 2004.
17. Galan A and Cervero F. Painful stimuli induce in vivo phosphorylation and membrane mobilization of mouse spinal cord NKCC1 co-transporter. Neuroscience 133: 245252, 2005.[CrossRef][ISI][Medline]
18. Giménez I and Forbush B. Short-term stimulation of the renal Na-K-Cl cotransporter (NKCC2) by vasopressin involves phosphorylation and membrane translocation of the protein. J Biol Chem 278: 2694626951, 2003.
19. Gottardi CJ, Dunbar LA, and Caplan MJ. Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol Renal Fluid Electrolyte Physiol 268: F285F295, 1995.
20. Graeve L, Drickamer K, and Rodriguez-Boulan E. Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor. J Cell Biol 109: 28092816, 1989.[Abstract]
21. Haas M and Forbush B III. The Na-K-Cl cotransporter of secretory epithelia. Annu Rev Physiol 62: 515534, 2000.[CrossRef][ISI][Medline]
22. Hoekstra D, Tyteca D, and van IJzendoorn SCD. The subapical compartment: a traffic center in membrane polarity development. J Cell Sci 117: 21832192, 2004.
23. Liedtke CM and Cole TS. Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC- and ERK. Biochim Biophys Acta 1589: 7788, 2002.[CrossRef][ISI][Medline]
24. Liedtke CM, Papay R, and Cole TS. Modulation of Na-K-2Cl cotransport by intracellular Cl and protein kinase C- in Calu-3 cells. Am J Physiol Lung Cell Mol Physiol 282: L1151L1159, 2002.[Medline]
25. Lock JG and Stow JL. Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin. Mol Biol Cell 16: 17441755, 2005.
26. Loder MK and Melikian HE. The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines. J Biol Chem 278: 2216822174, 2003.
27. Lytle C, Xu JC, Biemesderfer D, and Forbush B III. Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269: C1496C1505, 1995.
28. Matthews JB. Molecular regulation of Na+-K+-2Cl cotransporter (NKCC1) and epithelial chloride secretion. World J Surg 26: 826830, 2002.[CrossRef][ISI][Medline]
29. Matthews JB, Awtrey CS, Hecht G, Tally KJ, Thompson RS, and Madara JL. Phorbol ester sequentially downregulates cAMP-regulated basolateral and apical Cl transport pathways in T84 cells. Am J Physiol Cell Physiol 265: C1109C1117, 1993.
30. Matthews JB, Smith JA, and Nguyen H. Modulation of intestinal chloride secretion at basolateral transport sites: opposing effects of cyclic adenosine monophosphate and phorbol ester. Surgery 118: 147153, 1995.[ISI][Medline]
31. McDaniel N and Lytle C. Parietal cells express high levels of Na-K-2Cl cotransporter on migrating into the gastric gland neck. Am J Physiol Gastrointest Liver Physiol 276: G1273G1278, 1999.
32. Meade P, Hoover RS, Plata C, Vázquez N, Bobadilla NA, Gamba G, and Hebert SC. cAMP-dependent activation of the renal-specific Na+-K+-2Cl cotransporter is mediated by regulation of cotransporter trafficking. Am J Physiol Renal Physiol 284: F1145F1154, 2003.
33. Myat MM, Anderson S, Allen LA, and Aderem A. MARCKS regulates membrane ruffling and cell spreading. Curr Biol 7: 611614, 1997.[CrossRef][ISI][Medline]
34. Piechotta K, Garbarini N, England R, and Delpire E. Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl cotransporter in the nervous system: evidence for a scaffolding role of the kinase. J Biol Chem 278: 5284852856, 2003.
35. Reinhardt J, Kosch M, Lerner M, Bertram H, Lemke D, and Oberleithner H. Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1. Am J Physiol Renal Physiol 283: F335F343, 2002.
36. Russell JM. Sodium-potassium-chloride cotransport. Physiol Rev 80: 211276, 2000.
37. Shen BQ, Barthelson RA, Skach W, Gruenert DC, Sigal E, Mrsny RJ, and Widdicombe JH. Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters. J Biol Chem 268: 1907019075, 1993.
38. Simard CF, Daigle ND, Bergeron MJ, Brunet GM, Caron L, Noël M, Montminy V, and Isenring P. Characterization of a novel interaction between the secretory Na+-K+-Cl cotransporter and the chaperone hsp90. J Biol Chem 279: 4844948456, 2004.
39. Slater SJ, Milano SK, Stagliano BA, Gergich KJ, Curry JP, Taddeo FJ, and Stubbs CD. Interaction of protein kinase C with filamentous actin: isozyme specificity resulting from divergent phorbol ester and calcium dependencies. Biochemistry 39: 271280, 2000.[CrossRef][ISI][Medline]
40. Song JC, Hanson CM, Tsai V, Farokhzad OC, Lotz M, and Matthews JB. Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms. Am J Physiol Cell Physiol 281: C649C661, 2001.
41. Song JC, Hrnjez BJ, Farokhzad OC, and Matthews JB. PKC- regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS. Am J Physiol Cell Physiol 277: C1239C1249, 1999.
42. Takeyasu K, Tamkun MM, Renaud KJ, and Fambrough DM. Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the -subunit of an avian sodium pump. J Biol Chem 263: 43474354, 1988.
43. Vaandrager AB, van den Berghe N, Bot AG, and de Jonge HR. Phorbol esters stimulate and inhibit Cl secretion by different mechanisms in a colonic cell line. Am J Physiol Gastrointest Liver Physiol 262: G249G256, 1992.
44. Verstovsek G, Byrd A, Frey MR, Petrelli NJ, and Black JD. Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes. Gastroenterology 115: 7585, 1998.[ISI][Medline]
45. Von Brauchitsch DK and Crook RB. Protein kinase C regulation of a Na+, K+, Cl cotransporter in fetal human pigmented ciliary epithelial cells. Exp Eye Res 57: 699708, 1993.[CrossRef][ISI][Medline]
46. Weikel CS, Sando JJ, and Guerrant RL. Stimulation of porcine jejunal ion secretion in vivo by protein kinase-C activators. J Clin Invest 76: 24302435, 1985.[ISI][Medline]
47. Yarbrough TL, Lu T, Lee HC, and Shibata EF. Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude. Circ Res 90: 443449, 2002.[Medline]
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |