1 Institute of Physiology and 2 Department of Internal Medicine, University of Münster, D-48149 Münster, Germany
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ABSTRACT |
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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
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INTRODUCTION |
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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 (HKc) demonstrated that mRNAs of the
- and
-subunits are upregulated in response to systemic Na+ or
K+ depletion, respectively (36). In addition,
HKc
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 HKc showed that in rat kidney, splice variants
of the
-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 -subunit, ATP1AL1, together
with the gastric H+-K+
-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.
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EXPERIMENTAL PROCEDURES |
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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
-subunit, and the rat gastric H+-K+
-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--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+ -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+
-subunit, 1:500; clathrin heavy
chain (1:50, Transduction Laboratories); H+-K+
-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+
-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+
-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+
-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
-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
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RESULTS |
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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
-subunit- and gastric H+-K+-ATPase
-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 -subunit and the
gastric H+-K+-ATPase
-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-
-PMA (100 nM) or HEPES did not change apical
H+-K+-ATPase distribution (Fig. 1G,
20-min 4-
-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-
-PMA) and K (HEPES)] and can also not be assigned to
comparable intracellular structures in the middle of the cells [Fig.
1, I (4-
-PMA) and L (HEPES)].
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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 -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-
-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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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+-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+
-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-
-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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DISCUSSION |
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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+ -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+ - and gastric
H+-K+
-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+
-subunit. The
-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
-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--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 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 (HK2a and
HK
2b) are expressed. The shorter variant
(HK
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
HK
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 - and
-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.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. Michael Caplan for the generous gift of several antibodies.
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FOOTNOTES |
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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.
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