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Departments of Woman and Child Health, 1 Pediatric Unit, and 2 Neuroscience, Karolinska Institute, S-171 76 Stockholm, Sweden; and 3 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Several indirect lines of evidence suggest that protein kinases
and phosphatases modulate the activity of renal
Na+-K+-ATPase.
The aim of this study was to examine whether such regulation may occur
via modulation of the state of phosphorylation of
Na+-K+-ATPase.
Slices from rat renal cortex were prelabeled with
[32P]orthophosphate
and incubated with the inhibitors of protein phosphatase (PP)-1 and
PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the
protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or
the PP-2B inhibitor, FK-506. Phosphorylation of
Na+-K+-ATPase
-subunit was evaluated by measuring the amount of
[32P]phosphate
incorporation into the immunoprecipitated protein. Incubation with
either OA, CL-A, or PDBu caused four- to fivefold increases in the
amount of
[32P]phosphate
incorporation into immunoprecipitated
Na+-K+-ATPase
-subunit. OA and PDBu had a synergistic effect on the state of
phosphorylation of
Na+-K+-ATPase
-subunit. FK-506 did not affect
Na+-K+-ATPase
phosphorylation, neither alone nor in the presence of PDBu. Each of the
drugs, OA, CL-A, and PDBu, inhibited the activity of
Na+-K+-ATPase
in microdissected proximal tubules. PDBu potentiated OA-induced inhibition of
Na+-K+-ATPase
activity. Inhibition of
Na+-K+-ATPase
required a lower dose of CL-A than of OA. On the basis of the
inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is
concluded that the tubular effect is mainly due to inhibition of PP-1.
The PP-1 activity in rat renal cortex was ~1.5 nmol
Pi · mg
protein
1 · min1.
Using a monoclonal anti- antibody that fails to recognize the subunit when Ser23 is
phosphorylated by PKC, we demonstrated that the dose response of PDBu
inhibition of
Na+-K+-ATPase
correlated with the dose response of phosphorylation of the enzyme. The
results suggest that the state of phosphorylation and activity of
proximal tubular
Na+-K+-ATPase
are determined by the balance between the activities of protein kinases
and phosphatases.
renal cortical tissue; proximal convoluted tubule; phorbol 12,13-dibutyrate
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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RENAL Na+-K+-ATPase plays a pivotal role for tubular Na+ reabsorption by generating the electrochemical gradient necessary for transcellular Na+ transport. It is well established that the activity of renal tubular Na+-K+-ATPase is bidirectionally regulated by natriuretic and antinatriuretic hormones, which, via G protein-coupled receptors, act on a common intracellular signaling pathway (2). Several indirect lines of evidence suggest that activation/deactivation of Na+-K+-ATPase by reversible phosphorylation is the final step in this pathway (4, 6, 25).
To test this hypothesis, we have examined whether, in intact renal tissue, the phosphorylation state of Na+-K+-ATPase can be affected by various inhibitors of protein phosphatases such as okadaic acid (OA) and calyculin A (CL-A), which inhibit protein phosphatase (PP)-1 and PP-2A (14, 23), and FK-506, which inhibits protein phosphatase-2B (PP-2B) (26), or by an activator of protein kinase C (PKC), phorbol 12,13-dibutyrate (PDBu). The studies were performed on slices from rat outer renal cortex, a tissue that mainly consists of proximal tubular cells. The effect of OA on PP-1 activity in renal cortex was also evaluated. In separate protocols, we determined the effects of OA, CL-A, or PDBu on Na+-K+-ATPase activity in microdissected renal proximal tubules. The results support the concept that in intact renal tissue, the activity of Na+-K+-ATPase is dynamically modulated by phosphorylation/dephosphorylation reactions.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Chemicals. OA was purchased from
Scientific Marketing (Barnet Herts, UK); CL-A and BSA were from
Boehringer (Mannheim, Germany); FK-506 was from Fujisawa Pharmaceutical
(Osaka, Japan); DMSO was from LabKemi (Stockholm, Sweden); PDBu, EDTA,
EGTA, NaF, phenylmethylsulfonyl fluoride (PMSF), benzamidine,
leupeptin, antipain, pepstatin A, chymostatin, 
-mercaptoethanol,
SDS, collagenase, and disodium ATP grade II were from Sigma (St. Louis,
MO); ouabain was from Merck (Darmstadt, Germany);
[32P]orthophosphoric
acid (specific activity 8,500-9,120 Ci/mmol) and
[
-32P]ATP (specific
activity 10 Ci/mmol) were from New England Nuclear (Boston, MA);
Protein A Sepharose CL-4B was from Pharmacia (Uppsala, Sweden); rabbit
affinity-purified antibody to mouse IgG was from Cappel (Durham, NC);
and Malachite Green phosphatase assay kit was from Upstate
Biotechnology (Lake Placid, NY).
Antibody. Mouse monoclonal antibody
(MAb) 6H raised against
1-subunit of
Na+-K+-ATPase
was used for immunoprecipitation. It was produced using microsomal preparations of outer renal medulla of dog and rat enriched
for
Na+-K+-ATPase
(30). As shown in Fig.
1A,
phosphorylation of
Na+-K+-ATPase
does not affect the ability of the antibody to immunoprecipitate the
protein. Accordingly, immunoblots obtained from samples treated with or
without PDBu yielded equivalent
1-subunit immunoreactive signal.
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32P-labeling and drug incubation
of renal outer cortical slices.
Male Sprague-Dawley rats (39-45 days, 150-200 g body wt) were
anesthetized with an intraperitoneal injection of pentobarbital sodium
(60 mg/kg). The animals were bled by cutting the abdominal aorta, and
the kidneys were removed and decapsulated. A 200-µm-thick slice was
taken from the superficial cortex using a Stadie-Riggs microtome. Each
slice was preincubated at 30°C for 15 min in 2 ml of low-phosphate
Krebs bicarbonate buffer (in mM: 124 NaCl, 4 KCl, 26 NaHCO3, 1.5 CaCl2, 1.5 MgSO4, 0.25 KH2PO4,
and 10 D-glucose), bubbled with
95% O2-5%
CO2. The slice was then incubated
with 2.5 mCi of
[32P]orthophosphoric
acid in 2 ml of the same buffer at 30°C for 60 min to radiolabel
the intracellular ATP pool. At the end of the labeling, the buffer was
removed, and the 32P-labeled renal
cortical slice was rinsed twice with 2 ml of fresh buffer. Slices from
each sample were then incubated for the time indicated in the legends
to Figs. 2 and 3 with drugs or DMSO (final concentration
was less than 0.5%). The reaction was terminated by removing the
buffer and rapidly freezing the tissues in dry ice and ethanol. The
samples were stored at 
70°C.
Immunoprecipitation of
Na+-K+-ATPase.
Each slice was sonicated in 1 ml of cold lysis buffer (20 mM
Tris hydrochloride, 150 mM NaCl, 5 mM EDTA, 1% Triton
X-100, and 0.2% BSA; pH 8.0) containing 50 mM NaF, 1 mM EGTA, 25 mM
benzamidine, 0.1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml antipain, 5 µg/ml pepstatin A, and 5 µg/ml chymostatin. Aliquots from each
sample containing equal amounts of protein were used for
immunoprecipitation. Lysates were subsequently precleared at 4°C
for 30 min with 10 mg of preswollen protein A-Sepharose CL-4B (final
concentration of protein A-Sepharose was 1%) to get rid of nonspecific
IgG and labeled proteins that bind nonspecifically to the beads. The
beads were spun down for 15 s at 10,000 g. The supernatants were incubated at
4°C for 1.5 h on a rotatory shaker with 15 µl of MAb 6H antibody
(final dilution ~1:100), followed by incubation with 25 µl of
affinity-purified rabbit anti-mouse antibody (final dilution ~1:80)
at 4°C for 1 h. The resultant immunocomplexes were incubated with
protein A-Sepharose beads at 4°C for 1 h. The beads were collected
by centrifugation and washed at 4°C first with 1 ml of lysis
buffer, then three times with 1 ml of a buffer containing 20 mM
Tris · HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Triton
X-100, 0.1% SDS, and 0.2% BSA (pH 8.0); three times with 1 ml of a
buffer containing 20 mM Tris · HCl, 500 mM NaCl,
0.5% Triton X-100, and 0.2% BSA (pH 8.0); and finally with 1 ml of a
buffer containing 50 mM Tris · HCl (pH 8.0). After
the final wash, the beads were resuspended in 50 µl of 2×
Laemmli sample buffer, vortexed, and centrifuged. The supernatants were
resolved by electrophoresis on 7.5% SDS-polyacrylamide gel (24). Gels
were dried and subjected to autoradiography. 32P-labeled immunoprecipitated
Na+-K+-ATPase
1-subunit was identified by its
ability to migrate to the same position on SDS-PAGE as radiolabeled
purified renal
Na+-K+-ATPase
1-subunit (Fig.
1B).
[32P]phosphate
incorporation into the catalytic -subunit of
Na+-K+-ATPase
was quantified with a LKB UltroScan XL Laser Densitometer interfaced to
an IBM PC. Uneven background from lane to lane was corrected by
subtraction. Results were expressed as a ratio of integrated absorbance
units (IAU) of treated group vs. IAU of control group.
1-subunit was assessed with
Mck1, an antibody that detected specifically the PKC dephosphorylated
form, but not phosphorylated form, of rat
Na+-K+-ATPase
(18). The antibody was a kind gift from Dr. Kathleen J. Sweadner
(Laboratory of Membrane Biology, Massachusetts General Hospital).
Immunoblotting and determination of
Na+-K+-ATPase
1-subunit phosphorylation were
performed as described (12). Results were expressed as a ratio of IAU
of treated group vs. IAU of control group.
Determination of calcium/magnesium-independent,
serine/threonine protein phosphatase (PP)
activity. Renal outer cortical
slices (200 µm thick) were homogenized in cold PP assay buffer
composed of 50% glycerol, 20 mM MOPS (pH 7.5 at room temperature), 60 mM -mercaptoethanol, 0.1 M NaCl, and 1 mg/ml BSA. The homogenates were centrifuged at 12,000 g for 10 min at 4°C. The supernatants, termed "crude extracts," were
collected. PP activity in the crude extracts was measured with
Malachite Green phosphatase assay kit, in which a serine/threonine
phosphatase substrate, phosphopeptide (KRpTIRR), was used as the
substrate. In preliminary experiments, crude extracts were checked for
linearity of PP activity with respect to protein concentration and
reaction time. Under these conditions, the amount of dephosphorylated
substrate never exceeded 30% of the phosphorylated substrate present
at the beginning of the reaction. About 1.5 mg/ml of protein from crude
extracts was used in the assay, and the reaction time was 10 min. After
incubation with vehicle or with 10 nM of OA at 4°C for 5 min,
aliquots of 15 µl were transferred from each sample to a multi-well
microtiter plate. The reaction was started by addition of
phosphopeptide substrate (KRpTIRR) and carried out in a final volume of
25 µl at room temperature for 10 min in the presence or absence of
phosphorylated Thiol-Cys34-D-32
peptide (40 nM), a potent and selective inhibitor of PP-1 (3, 22). This peptide contains the PP-1 binding site as well as the
cAMP-dependent protein kinase (PKA) phosphorylation site of DARPP-32, a
dopamine- and cAMP-dependent phosphoprotein of 32 kDa. DARPP-32 is,
following phosphorylation by PKA, converted into a selective PP-1
inhibitor (21). The peptide was synthesized by solid-phase method using
a model 430A Applied Biosystems peptide synthesizer and was
phosphorylated by incubation with PKA. The characteristics of the
phosphorylated DARPP-32 peptide has been described (22). The
concentration of phosphorylated DARPP-32 peptide used is sufficient to
block PP-1 (22). The reaction was then terminated by adding 100 µl of
Malachite Green solution, and the samples were let to stand for 15 min
at room temperature for color development. The absorbance of each well
was measured in a microtiter plate reader at 650 nm. Absorbance from
PP-1 was calculated as the difference between the absorbances from the total PP and the phosphorylated DARPP-32 peptide-insensitive PP. The
amount of phosphate released was determined by comparing the absorbance
to a standard curve prepared by incubating a set of phosphate standards
of known concentration with Malachite Green solution. PP-1 activity was
expressed as nanomoles of Pi
released per milligram protein per minute. Results are given as percent of control.
Preparation of tubules. Kidney
perfusion and tubular microdissection were performed as described (15).
Briefly, after a midline incision, the left kidney was exposed and
perfused with 10 ml of cold Ringer solution and then with 40 ml of cold
collagenase solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4,
1 CaCl2, 1 MgCl2, and 10 Tris · HCl, as well as 0.05% collagenase and 0.1%
BSA, pH 7.4, at 4°C. Kidney blood flow was not interrupted before
the perfusion. The kidney was removed and cut along the
corticopapillary axis into small pyramids, which were incubated at
35°C for 20 min in 10 ml of the same collagenase solution bubbled
with 95% O2-5%
CO2. After incubation, the
pyramids were rinsed three times with cold microdissection solution,
which was identical to the collagenase solution except that collagenase and BSA were omitted and CaCl2
concentration was lowered to 0.25 mM.
The proximal convoluted tubule (PCT) segments were manually dissected
from the superficial cortex at 4°C with the help of a
stereomicroscope. They were individually transferred to the concavity
of a bacteriological slide and photographed for length determination in
an inverted microscope at ×100 magnification. The slides were
stored on ice until assay.
Preincubation of tubules with drugs.
The tubule segments were preincubated at room temperature for 20 min in
1 µl of microdissection solution with the addition of CL-A, OA,
and/or PDBu, or vehicle and were transferred to ice. The
segments were permeabilized with hypotonic shock, rapid freezing, and
thawing to ensure that Na+ and ATP
entered the cell. The Na+
concentration in the medium
([Na+]m)
was 70 mM. Permeabilization equalizes the intracellular
Na+ concentration
([Na+]i)
with the
[Na+]m,
thereby eliminating the transmembrane
Na+ gradient and the possibility
that changes in
Na+-K+-ATPase
activity are secondary to changes in
[Na+]i.
Determination of
Na+-K+-ATPase
activity.
Na+-K+-ATPase
activity was measured as described (15). All
Na+-K+-ATPase
assays were performed in the presence of saturating concentrations of
all major substrates (70 mM Na+, 5 mM K+, and 10 mM ATP). After
preincubation and permeabilization, the tubule segments were incubated
at 37°C for 15 min in the following solution (in mM): 50 NaCl, 5 KCl, 10 MgCl2, 1 EGTA, 100 Tris · HCl, 10 disodium ATP, and 2-5 Ci/mmol
[-32P]ATP in tracer
amount (5 nCi/ml), pH 7.4, at 37°C, with or without 2 mM ouabain.
When ouabain was present, NaCl and KCl were omitted, and
Tris · HCl was 150 mM. The phosphate liberated by
hydrolysis of
[-32P]ATP was
separated by filtration through a Millipore filter after absorption of
the unhydrolyzed ATP on activated charcoal. The radioactivity was
measured in a liquid scintillation spectrophotometer. Total ATPase and
ouabain-insensitive ATPase activity were measured in separate samples,
each consisting of five to eight segments. Na+-K+-ATPase
activity was calculated as the difference between total ATPase and
ouabain-insensitive ATPase activity and was expressed as picomoles of
[32P]phosphate
hydrolyzed per millimeter tubule per hour. Results are given as percent
of control.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Incubation of renal slices with an inhibitor of PP-1 and PP-2A, OA (5 µM) or CL-A (5 µM), for 30 min, increased
Na+-K+-ATPase
1-subunit phosphorylation by
5.4- and 5.5-fold, respectively (Fig. 2,
A and
B). The time course of the effect of
OA is shown in Fig. 2C. The increase
in the amount of
[32P]phosphate
incorporation into
Na+-K+-ATPase
1-subunit was maximal after 30 min of incubation and still present after 60 min.
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Incubation with an activator of PKC, PDBu (5 µM), for 20 min,
resulted in a 5.5-fold increase in the amount of
[32P]phosphate
incorporation into
Na+-K+-ATPase
1-subunit (Fig.
3, A and
C). In slices incubated for 20 min
with PDBu (5 µM) and OA (5 µM), a synergistic, 29-fold increase in
the state of phosphorylation of
Na+-K+-ATPase
1-subunit was observed (Fig. 3,
A and
C). The specific inhibitor of the
calcium/calmodulin-dependent PP-2B, FK-506 (26), did not increase the
state of phosphorylation of
Na+-K+-ATPase
1-subunit and did not enhance
the phosphorylation induced by PDBu (Fig. 3,
B and
C).
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To evaluate the effect of OA on PP-1 activity, crude extracts from renal outer cortical slices were incubated with vehicle alone or with 10 nM OA. This method does not distinguish very well between PP-1 and PP-2A activity. To get an approximate measurement of PP-1 activity, we added to the assay the specific endogenous PP-1 inhibitor, DARPP-32, which in its phosphorylated state inhibits PP-1 (21). In this assay a synthetically produced phosphorylated DARPP-32 peptide was used. Using this approach, we found that OA inhibited PP-1 activity by 25.4 ± 3.9% (P < 0.05 vs. control by Student's t-test). The contribution of PP-1 to total serine/threonine PP activity in renal cortex was ~30%.
To determine the functional consequences of phosphorylation, Na+-K+-ATPase activity was measured in microdissected PCT in the presence or absence of OA, CL-A, or PDBu. All three substances inhibited PCT Na+-K+-ATPase activity in a concentration-dependent manner. OA and CL-A caused maximal inhibition at the concentration of 1 µM and 10 nM, respectively. The apparent half-maximal inhibitory concentration was ~3.6 nM for OA and ~0.14 nM for CL-A. The threshold concentration for inhibition was 1 nM for OA and 0.1 nM for CL-A (Fig. 4). PDBu inhibited PCT Na+-K+-ATPase activity maximally at the concentration of 5 µM. The threshold concentration was 0.1 µM (Fig. 5A). A subthreshold concentration (10 nM) of PDBu significantly enhanced the inhibitory effect of OA (Fig. 6).
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To compare the dose responses between inhibition of
Na+-K+-ATPase
activity and phosphorylation of the -subunit, we studied PKC-induced
phosphorylation using a PKC site-selective dephosphorylation-specific MAb developed by Feschenko and Sweadner (18). One advantage of this
method is that it is not necessary to deprive the renal tissue of
phosphate. The method also minimizes the risks for protein degradation
and dephosphorylation. In addition, it identifies the specific
phosphorylation site. As shown in Fig.
5B, PDBu triggered dose-dependent
phosphorylation of the
Na+-K+-ATPase
1-subunit. The dose response of
PDBu inhibition of
Na+-K+-ATPase
activity correlated with the dose response of phosphorylation of the
-subunit (Fig. 5, A and
B).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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It is well documented that
Na+-K+-ATPase
purified from rat renal cortex can be phosphorylated in vitro by PKC
(7, 17, 27). Phosphorylation occurs on
Ser23 of the
1-subunit and is accompanied by
a shift in the equilibrium between the E1 and the E2 forms of the
enzyme (27). It is shown here that activation of PKC leads to
phosphorylation of
Na+-K+-ATPase
1-subunit in intact rat renal
tissue as well as inhibition of the enzyme activity.
Previous studies on the effect of PKC activation on the function of Na+-K+-ATPase in intact cells have given controversial results. In an opossum kidney cell line (29) and in dissected rat renal proximal tubular segments and in rat choroid plexus (19), activation of PKC by phorbol esters was found to cause inhibition of Na+-K+-ATPase. Opposite effects, i.e., stimulation of Na+-K+-ATPase activity, were observed when rat renal proximal tubules in suspension (10) and hepatocytes (28) were incubated with phorbol esters. These controversial results may depend on species and tissue differences as well as on different experimental conditions. One such condition is the level of [Ca2+]i. A stimulatory effect is often observed at experimental conditions where the [Ca2+]i is high (28, 20) or might have been rendered high by manipulations such as using a K+-free solution (31) or hypertonic medium (400 mosmol/kgH2O) (10). K+-free pretreatment will inhibit the activity of Na+-K+-ATPase and then increase [Na+]i. The latter may increase [Ca2+]i via inhibition of the Na+/Ca2+ exchanger (9). Hypertonic medium will result in a decrease in cell volume, which triggers an increase in [Ca2+]i (16, 32). We have found in ongoing studies that in COS cells expressing rat renal Na+-K+-ATPase, activation of PKA or PKC caused inhibition of Na+-K+-ATPase at low [Ca2+]i (125 nM) and stimulation or no change at high [Ca2+]i (450 nM) (11).
In the present study, it was also shown that inhibition of PP-1 and
PP-2A by OA and CL-A increased the state of phosphorylation of the
Na+-K+-ATPase
1-subunit and inhibited the
activity of
Na+-K+-ATPase
in rat kidney. OA and CL-A are known to inhibit PP-2A with the same
potency, but CL-A is a more efficient inhibitor of PP-1 than OA (14,
23). Our concentration-dependence curves indicated that CL-A inhibited
Na+-K+-ATPase
activity more efficiently than OA. This suggests that PP-1 rather than
PP-2A is involved in the regulation of
Na+-K+-ATPase.
The PP-1 activity in rat renal cortex was found to be ~30% of total
calcium/magnesium-independent, serine/threonine protein phosphatase activity.
A large number of first messengers acting on renal tubular cells use
PKC as an intracellular messenger. Thus dopamine regulation of renal
tubular
Na+-K+-ATPase
has been partially attributed to PKC activation (1, 8). We have found
in a preliminary study that the dopamine precursor,
L-dopa, caused a ~2-fold
increase in the phosphorylation of
Na+-K+-ATPase
1-subunit (data not shown).
Since PKC phosphorylation sites are generally good substrates for PP-1
and PP-2A (13), the effects of OA and CL-A on
Na+-K+-ATPase
are likely to be mediated via blockade of dephosphorylation on
Ser23 of the
1-subunit, i.e., the PKC site.
This hypothesis was supported by the observation that PDBu and OA had a
synergistic effect on Na+-K+-ATPase
phosphorylation. A synergistic inhibition of
Na+-K+-ATPase
activity was also observed when renal tubules were incubated with a
subthreshold concentration of PDBu and different concentrations of OA.
The fact that phosphorylation and inhibition by PDBu occurred in the
same dose range supports the concept that there is a direct link
between the state of phosphorylation and the level of activity of the
enzyme. In previous studies from this laboratory, it was shown that
mutation of PKC phosphorylation site abolishes the increase in
[Na+]i
responding to PDBu-induced inhibition of ion transport activity of
Na+-K+-ATPase
(5), and mutation of PKA phosphorylation site blocks the inhibition of
Na+-K+-ATPase
activity caused by cAMP agonists (12); also, a study on purified
Na+-K+-ATPase
showed that phosphorylation of
Na+-K+-ATPase
by PKC occurs on Ser23 of the
1-subunit and is accompanied by
a shift in the equilibrium between the E1 and the E2 forms of the
enzyme, thereby facilitating inhibition of
Na+-K+-ATPase
(27). Taken together, these observations indicate that phosphorylation
of the enzyme may change its function and transporting capacity. It
can, however, not be ruled out that the activity of the enzyme is also
modulated by the state of phosphorylation of an intermediate protein.
In conclusion, the results presented in this study suggest that the state of phosphorylation and activity of renal proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases. PKC and PP-1 appear to play an important role for this balance.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Swedish Medical Research Council (Project no. 03644), the Swedish Heart Lung Foundation and the Foundation of Axel Tielman's Memory (to A. Aperia), and by Swedish Medical Research Council B96-14X-11580-01A (to G. Fisone).
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FOOTNOTES |
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Address for reprint requests: A. Aperia, Dept. of Woman and Child Health, Pediatric Unit, Astrid Lindgren Children's Hospital, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Received 4 June 1996; accepted in final form 17 August 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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