(Received for publication, January 28, 1997, and in revised form, March 31, 1997)
From the Department of Woman and Child Health,
Karolinska Institute, S-112 81 Stockholm, Sweden, the ¶ Laboratory
of Molecular and Cellular Neuroscience, The Rockefeller University, New
York, New York 10021, and the
La Jolla Cancer Research Center,
The Burnham Institute, La Jolla, California 92037
The enzyme
Na+,K+-ATPase creates the transmembrane
Na+ gradient that is of vital importance for functioning of
all eukaryotic cells. Na+,K+-ATPase can be
phosphorylated by protein kinase A (PKA) and protein kinase C (PKC),
and these sites of phosphorylation have been identified. In the present
study, we have examined the physiological significance of PKC
phosphorylation of rat Na+,K+-ATPase. In COS
cells transfected with wild type rat
Na+,K+-ATPase 1, intracellular
Na+ was higher and pH was lower than in cells transfected
with rat Na+,K+-ATPase
1 in which the PKC
phosphorylation site, Ser-23, had been mutated into alanine. Phorbol
dibutyrate inhibited
Na+,K+-ATPase-dependent ATP
hydrolysis and Rb+ uptake in cells expressing wild type
Na+,K+-ATPase but not in cells expressing S23A
Na+,K+-ATPase. Cells expressing the S23A mutant
had a more rounded appearance and attached less well to fibronectin
than did untransfected cells or cells transfected with wild type rat
Na+,K+-ATPase
1. These results indicate a
functional role for PKC-mediated phosphorylation of rat
Na+,K+-ATPase
1 and suggest a connection
between this enzyme and cell adhesion.
A precise intracellular cation composition is of vital importance for cell homeostasis and function. The maintenance of high K+ and low Na+ concentrations, characteristic of the eukaryotic cell, is critically dependent on Na+,K+-ATPase (1, 2). The intracellular Na+ concentration is such that Na+,K+-ATPase is not saturated with regard to this substrate. Therefore, Na+,K+-ATPase activity is dependent on the activity of the Na+ influx (leak) pathway (3). For this reason the pump has often been considered to have a passive role in the regulation of cellular Na+ and K+ homeostasis (4). Recently it has been shown that Na+,K+-ATPase activity can be regulated by hormones, second messengers, protein kinase A (PKA)1 and protein kinase C (PKC) (5-8). Together, these various results suggest that the response of Na+,K+-ATPase to altered ion levels is hormonally modulated allowing for greater versatility in the regulation of cellular homeostasis.
Serine 23 has recently been identified as the major PKC phosphorylation
site in rat Na+,K+-ATPase 1 (9, 10). In this
study Ser-23 was mutated to alanine and the functional consequences
examined in intact cells.
A pCMV/ouabain-resistant vector (Pharmingen, San
Diego, CA) with the rat Na+,K+-ATPase 1
subunit was used, and the S23A mutation was introduced using a
polymerase chain reaction mutation strategy (Pfu DNA
polymerase from Stratagene, La Jolla, CA). To produce the two fragments
needed to create the mutation, polymerase chain reaction was carried out with the following primer pairs: pair 1, antisense primer 5
-CTCTTTCTAGTCTCCAGCCACAGG-3
and sense primer
5
-CCCATGTTCTGATACAGCTGC-3
; pair 2, antisense primer
5
-GACAAGAAGGCCAAGAAGGCGAA-3
and sense primer
5
-CAGATCACCAACGACGACATC-3
. The following polymerase chain reaction
reaction cycles were used: 96 °C for 3 min followed by 30 cycles of
57 °C for 30 s, 72 °C for 1 min, and 95 °C for 20 s
and finally 72 °C for 5 min. The two fragments were subjected to a
polynucleotide kinase reaction (Boehringer Mannheim) for 30 min at
37 °C and then ligated with the ligase chain reaction (Pfu DNA ligase, Stratagene) with wild type rat
Na+,K+-ATPase
1 cDNA as a template. The
following ligase chain reaction reaction cycles were used: 96 °C for
2 min and 60 °C for 2 min followed by 30 cycles of 96 °C for
20 s and 60 °C for 20 s.
The mutated fragment was purified by agarose gel electrophoresis, digested with ApaI (Boehringer Mannheim) and BstBI (New England Biolabs Inc.) restriction enzymes and substituted for the wild type fragment. Each complete clone was sequenced with an Applied Biosystems Taq Dye Deoxy terminator cycle sequencing kit in a Perkin-Elmer DNA thermal cycler 9600 to confirm the mutation and exclude other mutations. The S23A mutation produced a BglI restriction site in the mutated clone.
TransfectionWild type and mutant
Na+,K+-ATPase 1 were stably transfected into
COS7 cells using the calcium phosphate method (11). Cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum and 5% penicillin/streptomycin in 37 °C humidified
air with 5.3% CO2. COS cells, which are derived from early
embryonic monkey kidneys, express a highly ouabain-sensitive Na+,K+-ATPase. Selection of transfected clones
was achieved as described (12-14). Briefly, cells were grown in 10 µM ouabain for 3-4 weeks, and the medium was changed
every 3rd day. Untransfected COS cells died within 2 days in ouabain
medium, while cells expressing the relatively ouabain-insensitive rat
Na+,K+-ATPase
1 subunit survived. Following
this ouabain selection procedure, 200-300 single clones with
approximately the same growth rate were pooled and replated in DMEM
containing 10 µM ouabain. With the exception of the study
shown in Fig. 3B, all experiments were performed on this
mixture of clones.
We selected clones of cells, expressing wild type and S23A
Na+,K+-ATPase, that had similar levels of
transfected Na+,K+-ATPase activity. The
abundance of Na+,K+-ATPase, as determined by
immunoblotting, was also within the same range in both cell types. In
pilot studies we could clearly distinguish between ouabain-sensitive
(endogenous) and ouabain-insensitive (transfected)
Na+,K+-ATPase populations that were completely
inhibited by ouabain concentrations of 105 M
and 5 × 10
3 M, respectively.
Approximately 60% of Na+,K+-ATPase was
resistant to 10
5 M ouabain and was considered
to be rat Na+,K+-ATPase. Furthermore, the
expression of wild type and S23A Na+,K+-ATPase
mRNA was in each protocol verified by reverse
transcriptase-polymerase chain reaction with RNase-free DNase
(Pharmacia Biotech Inc., Uppsala, Sweden)-treated total RNA. By
measuring Rb+ flux (see below), we could verify the
presence of functioning Na+,K+-ATPase in the
plasma membrane. Rb+ flux sensitive to 5 × 10
3 M, but not to 10
5
M, ouabain was not significantly different in cells
expressing wild type and S23A
Na+,K+-ATPase.
Cells expressing wild type and S23A
Na+,K+-ATPase were seeded (3.0 × 105 cells/well) in 30-mm diameter wells and grown to
confluence. The cells were incubated for 20 min at 37 °C in DMEM
with or without 1 µM PDBu. Cells were lysed by treatment
with 1 mM Tris-HCl, pH 7.5, on ice for 15 min, and a crude
membrane fraction was prepared as described (15). The membrane fraction
was frozen in aliquots and stored overnight at 70 °C.
Na+,K+-ATPase-dependent ATP
hydrolysis was measured in triplicate as described (12). Transfected
Na+,K+-ATPase activity was determined as the
difference between ATPase activity at 10
5 M
and 5 × 10
3 M ouabain.
All experiments were performed
in phosphate-buffered saline (PBS), which contained (in mM)
130 NaCl, 4 KCl, 1.5 CaCl2, 1 MgCl2, 4 Trizma
phosphate, 10 D-glucose. The osmolality of this solution was adjusted to 315 mosM/liter by choline chloride
addition, and the pH was adjusted to 7.4 at 37 °C. Experiments were
performed at 37 °C in a water bath under a humidified atmosphere
with 5% CO2, 95% air. After removal of the culture
medium, cells were washed twice and preincubated for 40 min in the
presence of ouabain (105 M) to inhibit the
activity of endogenous Na+,K+-ATPase or 5 × 10
3 M to inhibit both endogenous and
transfected Na+,K+-ATPase. PDBu, when used, was
added after 20 min of preincubation. 86Rb+ flux
measurements were initiated by adding to each well 0.1 ml of PBS
containing 0.25 µCi/ml 86Rb+.
86Rb+ uptake was linear for 5 min (data not
shown), and recordings were made after 2 min. Reactions were stopped by
rinsing cells three times with ice-cold PBS, containing 5 mM BaCl2. Cells were then lysed by the addition
of 0.6 ml of 1 mM NaOH. Radioactivity in cell lysates were
measured using a scintillation counter (LKB, Wallac, Turku, Finland).
Protein was determined using a Bio-Rad assay with bovine serum albumin
as standard.
Rat wild type and S23A
Na+,K+-ATPase-dependent
86Rb+ uptake was determined as the difference
between the accumulated 86Rb+ uptake rate in
the presence of 105 M and 5 × 10
3 M ouabain.
Cells grown on
25-mm coverslips and placed in Petri dishes were incubated in DMEM
culture medium containing a membrane-permeable acetoxymethyl (AM) ester
of the Na+-sensitive fluorescent indicator,
Na+-binding benzofuran isophthalate (SBFI/AM)
105 M (16, 17) for 3 h at 37 °C and
equilibrated with 5% CO2, in air. Probenecid (0.6 mM) was added to block the transport of SBFI out of the
cells. The coverslips were mounted on a microscope stage. The DMEM was
replaced, and the cells were superperfused with physiological salt
solution containing (in mM): 110 NaCl, 5 KCl, 1 CaCl2, 1.2 MgCl2, 1 Na2HPO4, 25 NaHCO3, 20 HEPES, 5 glucose, 1 sodium butyrate, pH 7.4, at 37 °C. In K+-free
solutions, the KCl in physiological salt solution was omitted.
Fluorescence was measured using a SPEX dual-beam excitation spectrofluorometer (18). The excitation monochromators were set at 345 and 380 nm, the emission at 510 nm, and fluorescence was recorded as the 345/380 nm ratio. After loading the cells, Petri dishes were placed on the stage of a Zeiss Neoflurar X40, 1.75 objective, at 37 °C and UV measurements selected using a computerized spectrophotometer system (DM3000 cm, SPEX Inc., New York). Emitted light was measured by photon counting (Hamamatsu R928). All experimental solutions were kept at 37 °C and equilibrated with 5% CO2 in air.
At the end of each experiment, the following calibration procedure was
carried out. The SBFI-loaded cells were superperfused with a solution
containing (in mM): 5 KCl, 1 CaCl2, 1.2 MgCl2, 1 KH2PO4, 20 HEPES, 5 glucose, pH 7.4, at 37 °C. The Na+ concentration ranged
from 0 to 137 mM. Osmolality was kept constant by the
addition of choline chloride. The solution also contained ouabain
(5 × 103 M) and the Na+
ionophores, gramicidin (10
5 M),
monensin (10
5 M), and nigericin
(10
5 M) (16, 19). The intracellular
Na+ (Na+i) calibration curve was found
to be almost linear from about 1 to 20 mM, the
physiological intracellular range, and a change of less than 0.5 mM Na+i could be detected (data not
shown).
Cells were cultured in DMEM
and incubated with the membrane permeable AM ester of fluorescent probe
2,7
-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM),
(10
5 M) for 30-60 min at 37 °C. The
coverslips were mounted on a microscope stage. The DMEM was replaced,
and the cells were superfused with standard physiological salt
solution.
Fluorescence was measured in a SPEX dual-beam excitation spectrofluorometer as described (18). The excitation monochromators were set at 485 and 445 nm, the emission at 520 nm, and cell fluorescence was recorded as the 485/445 nm ratio. Calibration was performed at the end of each experiment as described (18). Cells were superfused with a solution containing (in mM): 120 KCl, 20 NaCl, 20 HEPES, and 0.025 nigericin. The pH of the extracellular medium was varied between 6.0 and 8.0.
Cell MorphologyAfter transfection, dilution series were
made on the cell population. The cell suspension was aliquoted in
microtiter wells (Costar) and grown in medium containing
105 M ouabain. After 2-4 weeks single cell
clones were visible. Their morphology was characterized using light
microscopy. They could be divided into two categories, cells with long
cellular extensions and cells with short or no cellular extensions.
This difference in shape was most obvious when the cells were
subconfluent. In two independent experiments 150 clones of subconfluent
cells expressing wild type Na+,K+-ATPase and
120 clones of cells expressing S23A
Na+,K+-ATPase were characterized in each
experiment.
Cell attachment on fibronectin-coated substrates was assayed in microtiter wells (Costar). All wells were preincubated overnight at 4 °C with varying concentrations of fibronectin. Before adding cells, each well was preincubated with 1% bovine serum albumin for 30 min in 37 °C and later washed with serum-free DMEM. Two alternative methods were used to assess the earliest stages of cell attachment. 1) Cells expressing wild type or S23A Na+,K+-ATPase were grown in a 3 µCi/ml [3H]thymidine containing medium supplemented with 10% fetal calf serum and harvested after approximately 72 h at 60-80% confluence. 2.5 × 104 cells/well were seeded in serum-free DMEM. Cells were allowed to attach to fibronectin (5 µg/ml)-coated substrates during 40 min of incubation in 37 °C and later washed with serum-free DMEM. Radioactivity was measured in a Wallac scintillation counter. Results are presented as percent remaining radioactivity of total. 2) Cells expressing wild type or S23A Na+,K+-ATPase were harvested at 60-80% confluence, and 2.5 × 104 cells/well were seeded in serum-free DMEM. After different incubation times the wells were washed with serum-free DMEM and fixed in PBS containing 4% paraformaldehyde and stained with a 20% methanol cresyl-violet solution. Cells were counted in three randomly selected areas (each area containing >60 cells) within the center of the well. Two types of cells were recognized: cells that had started to spread and had a flattened appearance and cells that were rounded and appeared loosely attached. The ratio between cells that had a flattened appearance and the total amount of cells was plotted against the concentration of the surface coating or time of incubation.
ChemicalsOuabain was obtained from Merck (Darmstadt,
Germany), 86Rb+ from Amersham Life
Science, Inc., bovine serum albumin from Boehringer Mannheim,
[-32P]ATP from NEN Life Science Products, and
Dulbecco's modified Eagle's medium from Life Technologies, Inc. Human
fibronectin was from the Finnish Red Cross Blood Service. SBFI and
BCECF were obtained from Molecular Probes (Eugene, OR). Nigericin,
gramicidin, monensin, probenecid, PDBu, and vitronectin were from
Sigma. All other reagents were of analytic grade or of highest
available purity.
Values are given as means ± S.E. Data were analyzed by Student's t test and analysis of variance test. p values < 0.05 are considered significant.
Na+,K+-ATPase activity was
determined in microsomal preparations from transfected cells as
described (12). Determinations were made both at saturating (70 mM) and nonsaturating Na+ (10 mM)
concentrations (Table I). As with the other protocols, ouabain (105 M) was present in all media to
inhibit endogenous Na+,K+-ATPase. In cells
expressing wild type Na+,K+-ATPase, the PKC
activator, PDBu, inhibited enzyme activity by 24 ± 7% at 70 mM Na+ and 27 ± 5% at 10 mM
Na+. In cells expressing S23A
Na+,K+-ATPase, PDBu had no effect at either 10 mM or 70 mM Na+.
|
It is generally accepted
that 86Rb+ can be used as a tracer for
K+.
Na+,K+-ATPase-dependent
K+ uptake was therefore assessed by determination of the
difference in cellular uptake of 86Rb+ in the
presence and absence of 5 × 103 M
ouabain. PDBu (5 × 10
6 M) significantly
reduced Na+,K+-ATPase-dependent
86Rb+ uptake to 77 ± 6%
(n = 8) of the control value in cells expressing wild
type Na+,K+-ATPase (p < 0.01 by paired t test). PDBu stimulated
Na+,K+-ATPase-dependent
86Rb+ uptake in cells expressing S23A
Na+,K+-ATPase to 140 ± 10% of the
control value (n = 6), p < 0.02 (see "Discussion").
Under basal conditions,
Na+i was significantly lower in cells expressing
S23A Na+,K+-ATPase than in cells expressing
wild type Na+,K+-ATPase (Fig.
1A). Values of Na+i are
generally lower, when determined with fluorescent probes, which measure
free Na+i, than with indirect methods that use, for
example, isotopes or an electron probe to measure total
Na+i (16, 17, 19). Intracellular pH was slightly
but significantly higher in cells expressing S23A
Na+,K+-ATPase compared with those expressing
wild type Na+,K+-ATPase.
Exposure of cells to ouabain or K+-free medium caused
similar increases in Na+i in cells expressing wild
type or S23A Na+,K+-ATPase (data not shown). In
both cell types, Na+i increased linearly with
incubation time for at least 15 min. Addition of PDBu caused an
increase in Na+i in cells expressing wild type
Na+,K+-ATPase after a lag period of a few
minutes (Fig. 2A). After 15 min of PDBu
incubation, the increase in Na+i averaged 7.6 ± 0.4 mM (p < 0.01). PDBu had no effect on Na+i in cells expressing S23A
Na+,K+-ATPase.
Na+i is critically dependent on the relationship
between Na+ entry into the cell, the leak, and
Na+,K+-ATPase, the pump. Activation of PKC by
phorbols may stimulate one of the most important Na+ entry
pathways, the amiloride-sensitive Na+/H+
exchanger (NHE). To test whether PDBu had dual and opposite effects in
this system on the NHE and Na+,K+-ATPase,
recordings were also made of intracellular pH, and the studies were
repeated in the presence of amiloride, 105 M
(20). PDBu increased intracellular pH in both types of cells, but the
increase was of longer duration in cells expressing S23A Na+,K+-ATPase (Fig. 2B). Three min
after PDBu addition, the pH started to fall in cells expressing wild
type Na+,K+-ATPase but not in cells expressing
S23A Na+,K+-ATPase. Amiloride abolished the pH
response in both types of cell (data not shown). Preincubation with
amiloride (10
5 M) abolished the PDBu-induced
rise in Na+i in cells expressing wild type
Na+,K+-ATPase (data not shown). Amiloride alone
had no significant effect on basal Na+i in either
type of cells.
COS cells normally start to
spread out and appeared flattened within 30 min after they have been
seeded to fibronectin coated plates. After 60 min all cells have
started to spread. During the next 24-48 h they continued to spread
and form long extensions. Most COS cells expressing wild type
Na+,K+-ATPase also develop extensions within
48 h, while most cells expressing S23A
Na+,K+-ATPase fail to do so. Fig.
3A illustrates the difference in shape between subconfluent cells expressing wild type and S23A
Na+,K+-ATPase. Analysis of 100-150 individual
clones indicated that more than 80% of the wild type cells had long
extensions, while more than 85% of the S23A cells had a round
appearance (Fig. 3B). To semiquantitatively compare the
capacity of cells expressing wild type and S23A
Na+,K+-ATPase to attach,
[3H]thymidine-loaded cells were plated on nonadhesive
plastic dishes coated with fibronectin. Forty min after plating,
48 ± 4.9% cells expressing wild type
Na+,K+-ATPase, but only 18 ± 2.7% of
cells expressing S23A Na+,K+-ATPase, were
attached to fibronectin (Fig. 4A)
(p < 0.01). The rate of cell spreading and flattening
was visually estimated in cells that had been fixed and stained at
various time points after plating. Sixty min after plating,
approximately 80-90% of cells expressing wild type
Na+,K+-ATPase, but only 40-50% of the cells
expressing S23A Na+,K+-ATPase, appeared spread
and flattened. After approximately 120 min, both cell types appeared
spread and flattened to a similar extent (Fig. 4B). The
difference in spreading between cells expressing wild type and S23A
Na+,K+-ATPase at 60 min was similar when the
fibronectin concentration varied between 2.5 and 25 µg/ml (Fig.
4C).
All mammalian cells are characterized by low Na+i and a high Na+ gradient across the plasma membrane that is generated by vectorial transport of Na+ mediated by Na+,K+-ATPase. Since the coupling ratio for outward Na+ transport and inward K+ transport is 3:2, Na+,K+-ATPase activity also generates a difference in membrane potential. The Na+,K+-ATPase-dependent electrochemical gradient is necessary for many vital cell functions, and total inhibition of Na+,K+-ATPase results in cell death (2, 21).
Na+i is generally too low to saturate
Na+,K+-ATPase (Refs. 21 and 22 and this study),
and the activity of the Na+,K+-ATPase is
dependent on Na+i. Since the affinity for
Na+ of the various subunits appears to vary,
Na+i concentration is also expected to be dependent
on which isoform(s) a given cell type expresses (21-23). The results
of the present study indicate that other mechanisms are also involved in the regulation of Na+i. Thus we have shown using
a variety of methods that cells transfected with S23A
Na+,K+-ATPase 1 subunit were no longer able
to respond to PKC activation with the inhibition of ATP hydrolysis,
decrease in Rb+ uptake, and increase in
Na+i exhibited by cells expressing wild type rat
Na+,K+-ATPase. These results highlight the
significance of phosphorylation of Ser-23 of rat
Na+,K+-ATPase
1 subunit in response to
activation of PKC in intact cells. Furthermore, our results suggest
that this regulatory phosphorylation site may play a direct role in
modulating basal Na+i, as well as an indirect role
in modulating intracellular pH and other cellular phenotypes.
Many pumps and other ion transporters are involved in the
regulation of Na+i and pH. The results of the
present study illustrate that it is important not only to consider the
regulation of Na+,K+-ATPase, but also its
impact on other Na+-coupled transporters such as the NHE
(Fig. 5). Activation of PKC stimulates NHE, resulting in
an increase of intracellular pH (24, 25) and presumably an increase of
Na+ influx (Fig. 5, A-D). Our results suggest
that in cells expressing wild type rat
Na+,K+-ATPase 1, activation of PKC also
inhibits the activity of Na+,K+-ATPase (Fig. 5,
A and B). At early time points (<5 min, Fig. 5A), it appears that any increase in Na+ influx
mediated by stimulation of NHE is compensated for by increased Na+i-dependent
Na+,K+-ATPase activity. However, at later time
points (>5 min, Fig. 5B) the combined effects of PKC on
stimulation of Na+ entry and inhibition of Na+
exit pathways results in a measurable increase in
Na+i (Fig. 5B). This increase in
Na+i appears ultimately to create such an
unfavorable Na+ gradient that down-regulates NHE activity,
resulting in the transient nature of the pH increase observed. In cells
expressing S23A Na+,K+-ATPase, PKC activation
will lead only to activation of NHE (Fig. 5, C and
D). In these cells, as observed at early time points in
cells expressing wild type Na+,K+-ATPase, any
increase in Na+i mediated by stimulation of NHE is
compensated for by increased Na+,K+-ATPase
activity (Fig. 5C). The observation that PDBu stimulated 86Rb+ uptake in cells expressing S23A
Na+,K+-ATPase, which was measured in intact
cells with low, nonsaturating Na+ concentration, is
consistent with this conclusion. Moreover, in cells expressing S23A
Na+,K+-ATPase, the increased pH induced by PDBu
does not return to the basal level, because Na+i
does not increase to a level sufficient to inhibit NHE (Fig.
5D). The regulation of Na+,K+-ATPase
activity by phosphorylation of Ser-23 also appears to influence the
basal ionic status of cells. Presumably because of a basal PKC
activity, basal Na+i is higher in cells expressing
wild type rat Na+,K+-ATPase
1. Furthermore,
most likely because of the relationship between the NHE leak and the
Na+,K+-ATPase pump activity, the basal
intracellular pH is lower in cells expressing wild type enzyme.
Since mutation of S23A of rat Na+,K+-ATPase
1 altered such important parameters as the concentration of
Na+i and pH, it might also influence the phenotype
of the cell in other ways. Such an effect was indeed observed, in that the shape of the cells expressing S23A
Na+,K+-ATPase indicated that the attachment to
substrate was perturbed. Furthermore, the ability of these cells to
attach and spread on fibronectin was impaired. As cell attachment to
this protein is mediated by integrins (27, 28), this raises the
possibility of a change in the expression and/or function of integrins.
The relationship between this phenomenon and the resetting of
Na+i and pH will be a topic for future studies.
Notably, an increase in intracellular pH following overexpression of a H+-ATPase has previously been shown to lead to a change in
cell shape (29).
There is compelling evidence indicating that the activity of
Na+,K+-ATPase can be modulated by hormones and
that regulation of the enzyme is likely to be mediated by
phosphorylation/dephosphorylation (6, 8, 12, 26). Recently, it was
shown in rat choroid plexus that serotonin stimulated the
phosphorylation of the 1 subunit (at a site subsequently identified
as Ser-23 (10)) and that this was accompanied by inhibition of
Na+,K+-ATPase activity (30). In the rat renal
tubule, Na+,K+-ATPase is inhibited by several
natriuretic hormones, the effect of which may be mediated via PKC (31).
By inhibiting the activity of Na+,K+-ATPase
they should increase Na+i and thereby decrease the
transcellular driving force for Na+. This may explain why
these hormones inhibit Na+ reabsorption in the renal
tubules. In conclusion, the results of the present study demonstrate
the importance of PKC in the regulation of rat
1
Na+,K+-ATPase and suggest that regulation of
Na+,K+-ATPase by
phosphorylation/dephosphorylation plays an important role in cell and
body Na+ homeostasis.