Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1

J. Reinhardt1, M. Kosch2, M. Lerner1, H. Bertram1, D. Lemke1, and H. Oberleithner1

1 Institute of Physiology and 2 Department of Internal Medicine, University of Münster, D-48149 Münster, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+ in exchange for H+ or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.

nongastric hydrogen-potassium-adenosine triphosphatase; Madin-Darby canine kidney cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IN EPITHELIAL CELLS OF THE renal collecting duct, luminal K+ reabsorption and H+ secretion are mediated by two subgroups of H+-K+-ATPases, the gastric and the nongastric H+-K+-ATPases. Both ion transporters belong to the gene family of P-type ATPases and are therefore structurally related to the Na+-K+-ATPases and Ca+-ATPases (3). The pharmacological profiles and ion transport properties of H+-K+-ATPases were extensively studied using various heterologous expression systems (e.g., Xenopus laevis oocytes, SF9 cells, HEK-293 cells) and in isolated kidney tubules from various species (8, 9, 11, 19, 24, 29, 30, 43). On the basis of these studies, it is presumed that the gastric H+-K+-ATPase is constitutively expressed in the apical plasma membrane of kidney epithelial cells and mediates proton secretion in exchange for K+ (for a review, see Refs. 23 and 37). In contrast, several nongastric ATPase isoforms from various species exchange K+ for intracellular protons or Na+, classifying these transporters as (H+-Na+)/K+ exchangers (9, 19, 20). Moreover, it has been shown that the nongastric H+-K+-ATPases are primarily active under certain pathophysiological conditions. Studies focusing on the rat colonic isoform (HKcalpha ) demonstrated that mRNAs of the alpha - and beta -subunits are upregulated in response to systemic Na+ or K+ depletion, respectively (36). In addition, HKcalpha was found to be upregulated during acid-base disorders (for a review, see Refs. 1, 27, and 37). However, an understanding of the mechanisms that regulate H+-K+-ATPase activity in response to dietary manipulation or acid-base irregularities is only beginning to emerge.

Studies focusing on HKcalpha showed that in rat kidney, splice variants of the alpha -subunit are expressed (28). One splice variant lacks the entire intracellularly localized NH2-terminal region where potential protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites are localized, indicating a possible role for PKA or PKC in the regulation of the ion pump. In this context, it is interesting that plasma membrane expression of the highly homologous Na+-K+-ATPase was shown to be regulated by NH2-terminal, PKC-dependent phosphorylation (5-7, 12). Phosphorylation of PKC consensus signals in the NH2-terminal region has been shown to trigger Na+ pump internalization but also ion pump insertion, presumably in a cell- or species-specific manner, which could also depend on the activation of different PKC isoforms (16, 38). In contrast, until now there have not been any studies focusing on the regulation of nongastric H+-K+-ATPases by the activation of PKC. Nevertheless, a previous study indicates that renal H+-K+-ATPase activity could be regulated through cycles of regulated membrane insertion and internalization (41).

To investigate the influence of PKC on plasma membrane expression of nongastric H+-K+-ATPases, we analyzed stably transfected Madin-Darby canine kidney (MDCK) cells. The cells were cotransfected with the human nongastric H+-K+-ATPase alpha -subunit, ATP1AL1, together with the gastric H+-K+ beta -subunit (34). In agreement with earlier expression studies in Xenopus laevis oocytes and HEK-293 cells (19, 30), both transfected subunits interact specifically and their coexpression is necessary for trafficking of the ion pump to the plasma membrane in MDCK cells. Analogous to the postulated luminal expression of nongastric H+-K+-ATPases in vivo, ATP1AL1 is polarized to the apical plasma membrane of transfected MDCK cells and is functionally expressed, as shown by 86Rb+ transport measurements (34). Immunofluorescence analysis showed that the H+-K+-ATPase is not intracellularly localized but is totally plasma membrane associated. Therefore, alterations of PKC-mediated H+-K+-ATPase plasma membrane expression in the apical plasma membrane can be analyzed. In the present study, we combined immunofluorescence with biochemical and functional analyses to evaluate the influence of PKC on plasma membrane expression of the human nongastric H+-K+-ATPase.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue culture. MDCK wild-type cells were harvested in minimal essential medium containing 10% fetal bovine serum (Sigma), 2 mM L-glutamine, and 50 U/ml each of penicillin and streptomycin (Life Technologies). Cotransfected MDCK cells expressing ATP1AL1, the human nongastric H+-K+-ATPase alpha -subunit, and the rat gastric H+-K+ beta -subunit were grown in the presence of 0.9 g/l Geneticin (PAA Laboratories, Linz, Austria). Cells were cultured in a 37°C humidified incubator with 5% CO2. For all experiments, the medium was changed daily.

Cell surface biotinylation and Western blotting. Transfected MDCK cells were plated (1 × 105 cells) on 24-mm polycarbonate filter inserts (0.4-µm pore, Costar) and grown to confluency for at least 5 days. Expression of the transfected cDNAs was enhanced with sodium butyrate stimulation. After the butyrate treatment (10 mM, 12 h), the cells were cooled down on ice and washed twice with cold HEPES buffer (in mM: 122.5 NaCl, 5.4 KCl, 0.8 MgCl2, 1.2 CaCl2, 1 NaH2PO4, 5.5 glucose, and 10 HEPES). For activation of PKC (5-30 min), the filter inserts were incubated in a heating chamber with prewarmed HEPES buffer (37°C, pH 7.4) containing freshly added phorbol 12-myristate 13-acetate (PMA; 100 nM) or 1,2-dicapryloyl-sn-glycerol (DOG; 50 µM). For each time point (5-30 min), two filters were analyzed and each experiment was repeated five times. The control cells were either incubated with the inactive PMA analog 4-alpha -PMA (100 nM) or preincubated (30 min) with the PKC blocker bisindoylmaleimide (BIM; 500 nM), which was also used in combination with PMA. Incubations were stopped by washing the cells three times in ice-cold biotinylation buffer. Thereafter, the cells were apically biotinylated (30 min, pH 9.0, 4°C) using N-hydroxysuccinimide bound to biotin (NHS-SS-biotin; Pierce, Rockford, IL) as previously described (18). The protein concentration of whole cell lysates was determined. Similar amounts of cell lysate protein were used for precipitation of biotinylated proteins using streptavidin-agarose beads (Sigma). Biotinylated proteins were separated from streptavidin-agarose beads in SDS sample buffer (80 mM dithiothreitol, 5.6% SDS, 0.008% bromophenol blue, 0.24 M Tris, pH 8.9, 16% glycerol) by heating (10 min, 95°C). The concentration of biotinylated proteins was measured as described (10). Briefly, aliquots of biotinylated proteins in SDS sample buffer were spotted on nitrocellulose membranes, air-dried, and stained with amido black solution [0.5% amido black (wt/vol)-45% (vol/vol) methanol-45% (vol/vol) H2O-10% (vol/vol) acidic acid]. Afterward, the nitrocellulose membrane was incubated in destaining solution [47% (vol/vol) methanol-47.5% (vol/vol)-H2O-5% (vol/vol) acetic acid], which removes unspecific amido black binding. The membrane was dissolved in solubilization buffer [80% (vol/vol) formic acid, 10% (vol/vol) acedic acid, 10% (wt/vol) trichloroacetic acid], and the protein concentration was measured with a photometer. Similar amounts of biotinylated proteins were analyzed by SDS-PAGE and Western blotting using a specific rabbit polyclonal, affinity-purified anti-ATP1AL1 antibody (1:1,000) (19). Detection was performed using goat anti-rabbit antibodies (1:1,000) conjugated to horseradish peroxidase (Sigma) and developed by the enhanced chemiluminescence technique (Amersham Pharmacia Biotech).

Immunofluorescence. Transfected MDCK cells were grown on 24-mm polycarbonate Transwell filter inserts for at least 5 days. The medium was changed daily and 12 h before fixation was supplemented with 10 mM sodium butyrate. Cells were washed with PBS+ (0.1 mM CaCl2, 1 mM MgCl2, 4°C) and, after PMA or DOG stimulation (diluted in HEPES buffer, 37°C), the cells were fixed with ice-cold methanol for 7 min. The clathrin/H+-K+ alpha -subunit colocalization was performed with paraformaldehyde-fixed cells (4%, 2 h, 4°C). Blocking (30 min) and antibody dilution were performed with 16% goat serum (Sigma), 0.3% Triton X-100, 0.1% bovine serum albumin (Sigma), 0.45 M NaCl, and 20 mM NaPi, pH 7.4 (2). Diluted primary antibodies [H+-K+ alpha -subunit, 1:500; clathrin heavy chain (1:50, Transduction Laboratories); H+-K+ beta -subunit (1:1,000, Sigma)] were incubated for 2 h at room temperature. After several washing steps with PBS+, secondary anti-mouse fluorescein-labeled and anti-rabbit rhodamine red-labeled antibodies (1:1,000, Sigma) were incubated for 1 h at room temperature. Internalization of the apically colocalized H+-K+ beta -subunit (see Fig. 2, A-C) was analyzed using an antibody against an extracellularly localized epitope. After sodium butyrate stimulation, filter-grown cells were cooled to 4°C and the H+-K+ beta -subunit antibody was exclusively applied from the apical side (1:50 in HEPES, pH 7.2, 30 min, 4°C). Unspecific binding sites were blocked by preincubation with HEPES/BSA (2% BSA, 30 min, 4°C). After a washing with HEPES buffer, secondary antibodies were also applied from the apical side (30 min) and afterward unbound antibodies were removed by several washing steps. Internalization of the H+-K+ beta -subunit and the specifically bound antibodies was stimulated by PMA incubation (in HEPES buffer, pH 7.2, 30 min, 37°C) and stopped by methanol fixation. Filters were mounted with Mowiol (Calbiochem). Confocal images were generated with a laser scanning fluorescence microscope (Fluoview, Olympus). Images are the product of threefold averaging. The total cell height of fixed cells was estimated by analyzing stacks of confocal sections in the xyz direction. Shown are xy sections from an uppermost view, corresponding to the apical plasma membrane; confocal sections (xy), representing the subapical region (800 nm below the apical plasma membrane); and sections focusing on the middle of the cells (4 µm below the apical membrane). Confocal sections of cells with internalized beta -subunit were taken from the apical plasma membrane and from the middle of the cells.

Intracellular pH measurement. Cells were grown to confluence on gelatinase-coated coverslips for 2-4 days. Sixteen hours before the experiment, 10 mM sodium butyrate was added to the culture medium. Intracellular pH (pHi) was measured as previously described, using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (19, 42). Cells were loaded with the membrane-permeable acetoxymethyl ester of BCECF (BCECF-AM; Molecular Probes, Eugene, OR) by incubation for 10 min at room temperature in a standard HEPES-buffered solution and 8.5 µmol BCECF-AM. After incubation, cells were washed three times by a BCECF-free solution delivered at 37°C. The coverslips were mounted in a thermostated microscopic tissue chamber that allows continuous perfusion at a rate of 3 ml/min. The solution delivery lines, as well as the cuvette, was water jacketed to maintain the temperature of the cuvette at 37°C. Fields of ~10-15 cells were visualized with a Nikon (Diaphot 300) inverted microscope and excited alternately at 490 and 439.5 nm. The emission-light intensities were measured at 535 nm using a photon-counting detector (PTI-Photon, Photon Technology) with a sampling interval of 1 s. The ratio of the emitted intensities at the excitation wavelengths, the fluorescence-to-excitation ratio, was corrected for background intensity. Cells were continuously perfused (3 ml/min) by HEPES-buffered and sodium-free solution, pH 7.4, at 37°C. Sodium was replaced by an equimolar concentration of N-methyl-D-glucammonium. Cells were acid loaded using the NH4Cl prepulse technique (19, 25). Briefly, cells were incubated for 5 min in a NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-containing solution in which 20 mM NaCl was replaced with 20 mM NH4Cl. Then, NH4Cl was removed from the perfusion solution in 3-5 s by a bolus injection of sodium-free solution, and the pHi recovery was analyzed. Curves of pHi were stored on the hard disk of a personal computer and analyzed offline. A line was fitted to the initial slope of the pHi curve (1-3 min after acidification) to quantify pHi recovery rates. Calibration of pHi measured by BCECF fluorescence was carried out by the high-potassium-nigericin calibration technique using solutions containing 10 µmol of the ionophore nigericin, pH 6.2, 6.5, 6.8, 7.1, and 7.4 (39). Data are reported as means ± SE. We used an unpaired Student's t-test with Bonferroni's correction for comparison of groups. P < 0.05 is considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We analyzed the influence of PKC on plasma membrane expression of the human nongastric H+-K+-ATPase. We used our previously established nongastric H+-K+-ATPase alpha -subunit- and gastric H+-K+-ATPase beta -subunit-cotransfected MDCK cells. Both proteins were coexpressed to guarantee trafficking of the ion pump to the apical plasma membrane and functional expression of the H+-K+-ATPase (34).

Effect of PKC stimulation analyzed by confocal immunofluorescence microscopy. PKC activation of confluent filter-grown cells was performed with PMA (100 nM) or with the PKC activator DOG (50 µM) for 0-20 min. We compared stacks of sections scanned in the xy direction (400 nm each), which were taken from the uppermost apical plasma membrane to the basal membrane of the cells. The antibodies applied against the H+-K+-ATPase alpha -subunit and the gastric H+-K+-ATPase beta -subunit do not recognize endogenously expressed proteins in untransfected MDCK cells (Figs. 1M and 2D, respectively) as previously documented (34). Confocal immunofluorescence analysis focusing on the apical plasma membrane (Fig. 1A) showed that as early as 10 min poststimulation, the H+-K+-ATPase is clustered in a few c-shaped or ringlike structures (arrow) of variable sizes, which are further increased by longer PMA stimulation (Fig. 1D, 20-min PMA). These clusters of H+-K+-ATPase appear to reside predominantly in intracellular compartments. Confocal sections of the subapical region (Fig. 1, B and E; 800 nm below the apical plasma membrane) and the middle of the same cells (Fig. 1, C and F) showed strong intracellular accumulation of the H+-K+-ATPase (arrows). Similar results were obtained with the synthetic diacylglycerol analogon DOG (50 µM, 20 min, 37°C; data not shown). In contrast, treatment with the inactive PMA analog 4-alpha -PMA (100 nM) or HEPES did not change apical H+-K+-ATPase distribution (Fig. 1G, 20-min 4-alpha -PMA; Fig. 1J, 20-min HEPES). In both control experiments, the H+-K+ ATPase is almost undetectable 800 nm below the apical membrane [Fig. 1, H (4-alpha -PMA) and K (HEPES)] and can also not be assigned to comparable intracellular structures in the middle of the cells [Fig. 1, I (4-alpha -PMA) and L (HEPES)].


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Fig. 1.   Confocal immunofluorescence analysis of H+-K+-alpha /H+-K+-beta -cotransfected Madin-Darby canine kidney (MDCK) cells after phorbol 12-myristate 13-acetate (PMA) or 4-alpha -PMA stimulation. Shown are sections scanned in the xy direction focusing on the apical plasma membrane (A, D, G, J, M), 800 nm below the apical membrane (B, E, H, K), and the middle of the cells (C, F, I, L). PMA incubation (100 nM, 37°C) for 10 min leads to strong clustering of the H+-K+-ATPase in c-shaped or ringlike structures (A, arrow) within and underneath the apical plasma membrane. Sections taken from the subapical region and 3 µm below the apical plane (B and C, respectively) show intracellular localization of the H+-K+-ATPase. Clustering and internalization further increase during longer PMA incubation (20 min; D, E, F). Control cells without PMA treatment (HEPES, 20 min; G, H, I) and 4-alpha -PMA-treated cells (J, K, L) show unclustered apical staining and no internalized H+-K+-ATPase. As a control, untransfected cells show no cross-reaction with the antibody (M). Bar: 15 µm.



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Fig. 2.   Intracellular localization of the H+-K+ATPase is shown using an H+-K+ATPase beta -subunit antibody that recognizes an extracellular epitope. Filter-grown living cells are labeled with primary and secondary antibodies from the apical side. Filter-grown living cells are labeled with primary and secondary antibodies from the apical side and either while fixed (A, B) or before fixation are treated with PMA (100 nM, 30 min, 37°C; C). In untreated cells, the beta -subunit is apically localized (focused on the apical plasma membrane; A) and shows no intracellular H+-K+ beta -subunit staining (xy section taken 800 nm below the apical membrane; B). In contrast, after PMA treatment the beta -subunit is intracellularly accumulated in vesicular structures (C, arrow; same plane as in B). Untransfected cells show no signal (D). Bar: 15 µm.

Internalization of the H+-K+-ATPase could also be shown for the associated H+-K+-ATPase beta -subunit. Therefore, a beta -subunit-specific antibody, which recognizes an extracellulary localized epitope, was applied to the apical plasma membrane of filter-grown MDCK cells labeled with a secondary antibody and afterward treated with PMA (100 nM in HEPES, 30 min, 37°C). After fixation, the internalized H+-K+ beta -subunit-antibody complex was further processed for immunofluorescence. Confocal sections (xy direction) taken from the middle of the cells clearly showed internalized H+-K+ beta -subunit-positive vesicular structures (Fig. 2C, arrow). In contrast, untreated cells (Fig. 2A, apical view) showed no intracellularly localized H+-K+-ATPase beta -subunit in analyzed sections from the middle of the cells (Fig. 2B). These results indicate that PMA-mediated PKC activation leads to H+-K+-ATPase plasma membrane clustering and internalization of the apically expressed ion pump.

Further evidence for PMA-induced H+-K+-ATPase internalization can be shown by colocalization experiments with an antibody against clathrin (clathrin heavy chain). Although it has been demonstrated that clathrin-dependent endocytosis is much more pronounced at the basolateral plasma membrane, apical clathrin-dependent internalization has been shown to be highly regulated in polarized MDCK cells (35). Confocal immunofluorescence analysis focusing on the apical plasma membrane of PMA-treated cells (100 nM, 5 min, 37°C) showed that H+-K+-ATPase (Fig. 3A, rhodamine red labeled, arrow) is partially colocalized (Fig. 3C, merge) with clathrin-positive vesicles or invaginations (Fig. 3B, fluorescein labeled). Colocalization could also be seen in untreated cells, but short-term PMA treatment (5 min) increased the total number of colocalized structures. However, the H+-K+-ATPase is also found in clathrin-negative structures and vice versa (Fig. 3C, arrowheads). These findings suggest that the H+-K+-ATPase is internalized by a clathrin-dependent mechanism.


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Fig. 3.   Colocalization of the H+-K+-ATPase alpha -subunit and clathrin by confocal immunofluorescence analysis in PMA-treated cells (100 nm, 5 min, 37°C). The confocal sections (xy plane) were scanned from the apical cell region. The specific H+-K+-ATPase alpha -subunit staining (rhodamine red) appears in dotlike structures (A, arrow). Clathrin staining (FITC) can be seen in B (arrow). In the merged picture (C), H+-K+-alpha and clathrin are partially colocalized (arrow). The H+-K+-ATPase alpha -subunit and clathrin can also be detected separately (C, arrowheads). Bar: 15 µm.

PKC-mediated internalization of the H+-K+-ATPase shown by surface biotinylation experiments. Whether PKC activation induces H+-K+-ATPase endocytosis can also be tested by surface biotinylation experiments. The filter-grown MDCK cells were stimulated for various time points with PMA or DOG (100 nM, 37°C, and 50 µM, respectively) and afterward were selectively biotinylated from the apical side (NHS-SS-biotin, 30 min, 4°C). Before precipitation, the protein concentration of all total cell lysates was measured and normalized to a similar level. For Western blot analysis, the protein concentration of the precipitated and concentrated biotinylated protein fractions was additionally measured as described in EXPERIMENTAL PROCEDURES and in previous studies (18, 34). Therefore, we could always separate and compare similar amounts of apically biotinylated proteins from each PMA incubation time point. The Western blot in Fig. 4A shows the results from one of five similar experiments using the H+-K+-ATPase alpha -subunit-specific antibody after 5-30 min of PMA stimulation. Already after 5 min of PMA treatment (100 nM, 37°C), the amount of apically expressed H+-K+-ATPase is decreased and further declines with time. After 20 min, the nongastric H+-K+-ATPase is undetectable in the apically biotinylated protein fraction. In contrast, incubation of the cells with inactive 4-alpha -PMA (100 nM, 37°C) does not result in a time-dependent decrease (Fig. 4B). To provide additional information that phorbol ester-induced H+-K+-ATPase endocytosis was due to PKC activation, we preincubated and coincubated the cells with the commonly used PKC blocker BIM. The Western blot in Fig. 4C shows that the PMA-induced decrease in apically expressed H+-K+-ATPase can be totally blocked by BIM (500 nM). These findings indicate that PMA-induced H+-K+-ATPase endocytosis was specifically due to activation of the PKC pathway.


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Fig. 4.   Western blot analysis of PMA-treated and apically biotinylated MDCK cells. Western blots were probed with the H+-K+-alpha subunit antibody (n = 5). The antibody recognizes a prominent 110-kDa band and a weak smear just above. PMA stimulation leads to a drastic decrease in apically biotinylated H+-K+-ATPase alpha -subunit (A), beginning between 5 and 15 min after stimulation. After 20 min, the alpha -subunit could no longer be detected by surface biotinylation. In contrast, incubation with the inactive PMA analogon 4-alpha -PMA had no effect (B). The internalization of the H+-K+-ATPase by PMA treatment could be totally blocked by preincubation (500 nM, 30 min) and coincubation of the cells with the protein kinase (PKC) blocker bisindolylmaleimide (BIM; C).

pHi measurements. Correlated with the confocal immunofluorescence and biotinylation results, PKC-mediated endocytosis, and, as a result, inactivation of the transfected nongastric H+-K+-ATPase, was additionally analyzed by using pHi measurements. Although it has been previously shown that several isoforms of nongastric H+-K+ ion pumps are working as (Na+/H+)-K+-ATPases, functional expression of the ion pump can be measured by its proton extrusion capacity (19). We used the NH4Cl prepulse technique and the pH-sensitive dye BCECF to acidify the cells and monitor the pHi recovery rates (42). In all experiments, the proton extrusion capacity of the endogenously expressed Na+/H+ exchangers was blocked or reversed by using Na+-free solutions. Baseline pHi was not different between MDCK wild-type cells and ATP1AL1/gastric H+-K+beta -subunit-cotransfected MDCK cells (7.28 ± 0.02 vs. 7.29 ± 0.02 pH units). Moreover, exposure to PMA did not change the baseline pHi of either group. The minimal pHi after acidification by the ammonium pulse was comparable in all groups (6.54 ± 0.1 for wild-type cells, 6.60 ± 0.1 for ATP1AL1-transfected cells, 6.56 ± 0.2 for PMA-treated ATP1AL1-transfected cells; not significant, respectively). As shown in Fig. 5, ATP1AL1/gastric H+-K+beta -subunit-cotransfected MDCK cells show a strongly increased pHi recovery rate compared with untransfected MDCK wild-type cells. The accelerated realkalinization of cotransfected cells compared with wild-type cells documents functional expression of the ion pump. In contrast, a 20-min incubation with PMA (100 nM) abolishes H+-K+-ATPase-mediated pHi recovery and adjusts the proton extrusion capacity to the level of that of untransfected MDCK wild-type cells, which are not influenced by PMA incubation. Incubation of ATP1AL1-transfected cells with the inactive phorbol ester 4-alpha -PMA (100 nM) did not affect baseline pHi or pHi after acidification (7.28 ± 0.02 and 6.52 ± 0.2, respectively) and did not change pHi recovery (realkalinization rate 0.091 ± 0.018 pHi units/min; not significantly different from cells not incubated with PMA). Analogous to the biotinylation experiments, we tested whether ion pump inactivation is based on specific PKC activation. With (pre)incubation of the cells with the PKC-blocker BIM (500 nM, 30 min), PMA induced PKC activation and, as a result, H+-K+-ATPase inactivation was almost completely prevented (Fig. 5).


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Fig. 5.   Shown are intracellular pH (pHi) recovery rates after acid load by 5-min ammonium pulse (20 mM NH4Cl) in MDCK wild-type (WT) and human nongastric H+-K+-ATPase (ATP1AL1)-transfected cells (HK). In cells expressing ATP1AL1, pHi recovery was significantly higher compared with wild-type cells (P < 0.05). The pHi recovery in wild-type cells was not changed after 20-min incubation with 100 nM PMA; however, in ATP1AL1-transfected cells pHi recovery significantly decreased after PMA incubation (P < 0.05). The effect of PMA exposure was blunted by 30-min preincubation of the cells with 500 nM bisindolylmaleimide (BIM) and coincubation with PMA (100 nM). Stimulation of PKA by a combination of forskolin (10 µM) and IBMX (0.2 mM) did not significantly reduce H+ secretion capacity of transfected MDCK cells. Values are means ± SD of 5 experiments each. All experiments were performed in Na+-free solution in which Na+ was replaced by equimolar amounts of N-methyl-D-glucammonium. PMA, forskolin, and IBMX, respectively, were present before (15 min) and during the ammonium pulse (5 min). Inset: original tracing showing the starting pH (pH 7.3), the initial alkalinization followed by acidification to pH 6.6, and the realkalinization of H+-K+-ATPase-transfected MDCK cells.

In contrast to the results after PMA incubation, stimulation of PKA by incubation of ATP1AL1/gastric H+-K+beta -subunit-cotransfected MDCK cells with forskolin (10 µM) and blockade of the phosphodiesterase with IBMX (0.2 mM) for 20 min did not change pHi recovery after acidification (Fig. 5). This was also true for PKA incubation of MDCK wild-type cells (Fig. 5). Baseline pHi as well as pHi after acidification by the ammonium pulse in BIM-incubated and PKA-stimulated cells was comparable and not significantly different from pHi values in nonpreincubated cells (7.27 ± 0.02 and 6.55 ± 0.2; 7.29 ± 0.02 and 6.60 ± 0.2, respectively).

These results strongly support our immunofluorescence and biotinylation data presented here and indicate that apical plasma membrane expression of human H+-K+-ATPase is downregulated by the PKC pathway. Activation of PKC leads to fast clathrin-dependent internalization of the apically expressed human nongastric H+-K+-ATPase.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In kidney epithelial cells, the activity of several transport systems appears to be governed by cycles of regulated membrane insertion and internalization (17, 21, 26, 38). Retrieval of plasma membrane proteins is often coupled to PKA- or PKC-mediated phosphorylation of the target or of associated partners. Among the family of P-type ATPases, the Na+-K+-ATPase was shown to be regulated by PKC activation. Phosphorylation of PKC sites in the intracellular NH2-terminal domain of the Na+ pump is a prerequisite for receptor-mediated ion pump internalization in epithelial cells of the renal proximal tubule (5-7, 32). Although it was shown that gastric and nongastric H+-K+-ATPases are regulated differently, it is not known whether their activity in kidney epithelial cells is controlled by endocytosis. Indirect evidence for a recycling mechanism comes from transgenic mouse studies (41). The animals express a mutant gastric H+-K+ beta -subunit that lacks an intracellularly localized internalization signal. Functional studies in mice show that renal K+ reabsorption is enhanced, possible due to constitutive H+-K+-ATPase activity.

The purpose of our study was to analyze the influence of PKC on plasma membrane surface expression of the human nongastric H+-K+-ATPase, ATP1AL1. Stably H+-K+ alpha - and gastric H+-K+ beta -subunit-cotransfected MDCK cells served as an expression system for the analysis of the influence of PKC. We previously showed that coexpression of both subunits is necessary for targeting and apical polarization of the H+-K+-ATPase in transfected MDCK cells. The expressed H+-K+-ATPase is functionally active, as previously shown by 86Rb+ uptake measurements, and strictly polarized to the apical plasma membrane (19, 20, 34). It has been demonstrated that MDCK wild-type cells endogenously express an omeprazole-sensitive H+-K+-ATPase in the apical plasma membrane, which is upregulated by aldosterone (31). However, our MDCK cells do not express ATP1AL1, as shown by Western blot analysis (34), immunofluorescence (Figs. 1 and 2), and by ATP1AL1-specific RT-PCR experiments (not shown). The molecular identity of the endogenously expressed H+-K+-ATPase is not known, and we cannot exclude expression of a related nongastric H+-K+-ATPase. More recently, functional analysis showed that a subclone of MDCK cells (MDCK-C11), the characteristics of which resemble those of intercalated cells, expresses a homologue to gastric H+-K+-ATPase (15). However, independent of the nature of the potential endogenous protein, the ion pump should be expressed in our transfected and in untransfected control MDCK cells. Therefore, our biotinylation, immunofluorescence analysis, and the pHi measurements should not have been influenced. Stimulation of the PKC pathway was performed with PMA or the DAG analogon DOG. Both PKC activators stimulate apical endocytosis in MDCK cells (22), and short-term incubation (10-30 min) has no influence on cell polarity of filter-grown MDCK cells. The confocal immunofluorescence data show that the apical H+-K+-ATPase is clustered in a few ringlike structures, starting 5-10 min after PMA treatment. This signal accumulation is strongly enhanced by further incubation with PMA (20-30 min) and could be localized to intracellular vesicular structures. So far, we have no clear evidence whether these ringlike structures correspond to early endosomal compartments, to an apical recycling compartment, or to lysosomes. To track the route of internalized ATPases, colocalization experiments with endosomal marker proteins are necessary. Here, we show that the H+-K+-ATPase can be colocalized with clathrin. A potential target for the clathrin-dependent endocytosis machinery is the cotransfected H+-K+ beta -subunit. The beta -subunit features a tyrosine-containing motif in its cytoplasmic domain, which is necessary for internalization of the related gastric ATPase isoform. This cytoplasmic motif in the beta -subunit interacts with clathrin and adapter protein-2 in gastric parietal cells (33, 41).

The specificity of PKC-driven internalization was demonstrated in our study by Western blot analysis of surface biotinylated MDCK cells and pHi measurements. Whereas the amount of the apically accessible H+-K+-ATPase declines with increasing PMA incubation, the ion pump is not internalized by incubation with the inactive 4-alpha -PMA. Moreover, internalization of the ion pump could be completely prevented by the specific PKC blocker BIM (40). The immunofluorescence and biotinylation data are confirmed by pHi measurements. The outward proton extrusion capacity of intracellularly acidified and H+-K+-ATPase-transfected MDCK cells is strongly influenced by PMA treatment. The reduced realkalinization rate after 20 min of PKC stimulation and the blockade of this effect after pre- and coincubation with the PKC blocker BIM strongly support the hypothesis that the H+-K+-ATPase is rapidly endocytosed after activation of the PKC pathway.

Activation of the PKC pathway using the potent, stimulating phorbol esters were shown to trigger fluid-phase endocytosis in polarized kidney epithelium (6) and clathrin-independent endocytosis as well as apical plasma membrane recycling in MDCK cells (4, 22). Therefore, we cannot exclude additional unspecific membrane retrieval after PMA or DOG stimulation. However, the sequence of apical internalization starting within 5 min and the complete disappearance of apically biotinylated H+-K+-ATPase after 20 min of PMA (100 nM) incubation suggest a specific mechanism.

Interestingly, the cloned nongastric H+-K+-ATPase isoforms from humans (ATP1AL1), rats, toads, guinea pigs, and rabbits possess a PKA site in position 955, which is also conserved in the Na+-K+-ATPase alpha 1-subunit. However, it is as yet unknown whether nongastric H+-K+-ATPases are regulated by PKA. Because interactions between PKC and the PKA pathway has been described and because apical endocytosis can also be stimulated by the PKA pathway (13), we analyzed H+-K+-ATPase distribution after PKA stimulation (20 min). The pHi recovery rates after PKA stimulation using forskolin (10 µM) in combination with IBMX (0.2 mM) were unchanged and suggest that ATP1AL1-mediated H+ secretion is not reduced by the PKA pathway. Furthermore, no effects of PKA on apical surface expression of the H+-K+-ATPase as analyzed by confocal immunofluorescence microscopy could be detected (data not shown). However, we cannot completely exclude the influence of PKA on H+-K+-ATPase expression and plasma membrane insertion. We stimulated our transfected cells with sodium butyrate to achieve H+-K+-ATPase expression. Therefore, any additional PKA-mediated insertion of H+-K+-ATPase could have remained undetected. It would be helpful for future experiments to find an epithelial cell system with endogenous ATP1AL1 expression with which to analyze the influence of PKA.

Regulation of the nongastric H+-K+-ATPase ATP1AL1 by PKC can be initiated by phosphorylation of an associated protein or direct phosphorylation of PKC consensus or cryptic sites. It was recently shown that in kidney and colon two alternatively spliced isoforms of the related nongastric rat H+-K+-ATPase (HKalpha 2a and HKalpha 2b) are expressed. The shorter variant (HKalpha 2b) lacks the intracellular NH2-terminal region and therefore the potential PKC phosphorylation site (28). However, it is not known whether the PKC site in HKalpha 2a is phosphorylated or whether the splice variants are differently regulated. Nevertheless, because of the strong functional and structural relationship of our nongastric H+-K+-ATPase to the Na+-K+-ATPase, it is conceivable that the nongastric ion pump is influenced in a similar way by PKC. Regulation of the Na+ pump by PKC is equivocally discussed (for recent reviews, see Refs. 14 and 38). Cell type-specific response and the expression of various PKC isoforms have to be considered. Nevertheless, it was shown that the plasma membrane expression of the Na+ pump is modified by PKC (14, 38). It will be interesting to elucidate which PKC isoforms are involved in the regulation of the human nongastric H+-K+-ATPase. On the basis of the strong homology between the Na+-K+-ATPase and ATP1AL1 and as shown in our PMA and DOG stimulation experiments, we conclude that it is conceivable that members of the classic PKCs or even the novel PKCs participate in H+-K+-ATPase regulation. Furthermore, it has to be tested either whether our human nongastric H+-K+-ATPase is directly phosphorylated using the NH2-terminal region as the target domain or whether phosphorylation of associated proteins is necessary.

The essential finding of our study is that plasma membrane expression of the human nongastric H+-K+-ATPase is regulated by the PKC pathway in transfected MDCK cells. PKC activation triggers the fast internalization of the H+-K+ATPase alpha - and beta -subunits. In future experiments, it will be exciting to study whether this PKC-dependent process is similar for different nongastric H+-K+-ATPase subtypes. It should be interesting to finally detect the physiological signal that initiates PKC activation and subsequently drives H+-K+-ATPase internalization in epithelial cells.


    ACKNOWLEDGEMENTS

The authors are grateful to Dr. Michael Caplan for the generous gift of several antibodies.


    FOOTNOTES

The study was supported by University of Münster Innovative Medizinische Forschung Grant RE110021 (to J. Reinhardt).

Address for reprint requests and other correspondence: J. Reinhardt, Institute of Physiology, Univ. of Münster, Robert Koch Str. 27a, D-48149 Münster, Germany (E-mail: Juergen.Reinhardt{at}pharma.novartis.com).

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.

February 19, 2002;10.1152/ajprenal.00226.2001

Received 20 July 2001; accepted in final form 13 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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