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INTRODUCTION |
The molecular mechanism by which hormone receptors coupled
to stimulation of protein kinase C
(PKC)1 regulate sodium
reabsorption in renal proximal convoluted tubules is not well
understood. The Na+,K+-ATPase, located within
the basolateral membrane of tubule epithelial cells, maintains a
transmembrane concentration gradient for sodium, ensuring the net
reabsorption of this cation. Hormonal short term regulation of
Na+,K+-ATPase activity may contribute to the
ability of the kidney to adjust sodium reabsorption. In recent years,
an increasing number of publications (1-4) have reported the short
term regulation of kidney Na+,K+-ATPase by
hormones and intracellular second messengers that modulate proximal
tubule sodium reabsorption. Renal Na+,K+-ATPase
activity is regulated by phosphorylation/dephosphorylation processes,
and both cAMP-dependent protein kinase and protein kinase C
(PKC) phosphorylate the Na+,K+-ATPase (1-10).
We have demonstrated that Ser-18 of the 
-subunit is essential for
the inhibition of the Na+-pump activity by dopamine and
that both Ser-18 and Ser-11 are essential for the stimulation of this
activity by PMA (10-15). These amino acids are phosphorylated by
different PKC isoforms during these processes (10-15). We also
described that although dopamine inhibition of
Na+,K+-ATPase is mediated by endocytosis of
plasma membrane Na+,K+-ATPase molecules, PMA
stimulation is due to recruitment of
Na+,K+-ATPase molecules from intracellular
compartments to the plasma membrane (10, 15).
Decreased Na+,K+-ATPase activity induced by
dopamine is partly responsible for reduced sodium reabsorption during a
high salt diet, and impaired regulation of the
Na+,K+-ATPase activity in renal tubules has
been linked to the development of high blood pressure (1-4). In
vivo, increases in dietary sodium intake or acute sodium loading
lead to natriuresis accompanied by elevated urinary dopamine excretion,
which suggested that dopamine produced endogenously by the epithelial
proximal tubule cells might contribute to the natriuretic response
(2-4). In this model, endogenously produced dopamine would be
transported outside the proximal tubule cells where it binds to
specific cell membrane receptors. The question arising is how an
external effect, the acute sodium load, is translated into activation
of the intracellular dopaminergic system. Our hypothesis is that an
increased filtered load of sodium may lead to a transient elevation in
intracellular sodium concentration that triggers the dopaminergic
response. The present study was performed to test the hypothesis that
the level of intracellular sodium modulates the tight control of
Na+,K+-ATPase activity by different agonists.
We present evidence that the intracellular sodium concentration of
kidney cells determines the level of inhibition of the
Na+,K+-ATPase activity by dopamine. We also
demonstrate that direct stimulation of cellular protein kinase C by the
phorbol ester PMA leads to either activation or inhibition of
Na+,K+-ATPase activity depending on the
intracellular sodium concentration.
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture supplies were purchased from
Invitrogen and HyClone Laboratories (Logan, UT). Molecular biology
reagents were from New England Biolabs (Beverly, MA), Promega (Madison,
WI), Stratagene (La Jolla, CA), and Sigma. Ouabain was purchased from Calbiochem. PMA, ethoxyresorufin, and dopamine were obtained from Sigma. [86Rb+]RbCl was obtained from
PerkinElmer Life Sciences. Other reagents were of the highest quality available.
Cell Culture and Transfection--
Opossum kidney (OK) cells
were maintained at 37 °C (10% CO2) in Dulbecco's
modified Eagle's medium with 10% calf serum and antibiotics
(DMEM-10). The expression vector pCMV containing the rodent
Na+-pump 1-subunit cDNA was obtained from
PharMingen. Mutants of 1 were prepared, as previously described
(12-15), from a plasmid containing the wild type -subunit sequence
and complementary oligonucleotides containing the desired change.
Briefly, annealed plasmid and oligonucleotides were subjected to PCR
amplification with Pfu polymerase, followed by restriction
of the original wild type template with DpnI. After
transformation of bacteria, the recovered mutant plasmids were
evaluated by restriction analysis and direct sequencing of the altered
region. Plasmids containing the wild type and mutated -subunit
cDNAs were transfected into OK cells using liposomes, as described
previously (12-15). Selection for cells expressing the highest level
of rodent -subunit was achieved by exposing them to a medium
containing 3 µM ouabain. Because the endogenous
Na+-pump of OK cells is completely inhibited by this
concentration of ouabain, only successful recipients of transfected
rodent -subunit would be able to survive. Resistant colonies were
expanded and maintained in DMEM-10 containing 3 µM
ouabain. Experiments were performed with a mix of at least 20 independent clones for each cell line. The
Na+,K+-ATPase of mock-transfected cells (vector
alone, vector plus liposomes, or liposomes alone) had the same activity
and sensitivity to ouabain as non-transfected host cells.
Determination of Protein Concentration--
Cells were
solubilized with SDS, and aliquots were used for protein determination.
Protein concentration was determined by the bicinchoninic acid method
(Pierce) using bovine serum albumin as a standard.
Determination of Rb+ Transport--
Measurements of
Na+,K+-ATPase-mediated transport by
Rb+ uptake were performed with attached cells as described
previously (12, 13, 15). Transfected cells grown in DMEM-10 were
exposed for 2 h to 2 mM EGTA to facilitate access of
ligands to Na+,K+-ATPase. To measure
Rb+ transport, cells were transferred to serum-free DMEM
containing 50 mM HEPES, pH 7.4 (DMEM/HEPES), and either 3 µM or 5 mM ouabain (incubation medium). Cells
were incubated with these amounts of ouabain first for 20 min at
37 °C in an air atmosphere and then for 10 min at 25 °C. All
treatments and determinations were performed at 25 °C. After
treatment, a trace amount of [86Rb+]RbCl was
added to the cell medium. After 20 min, cells were washed four times
with ice-cold saline and dissolved with SDS, and accumulated radioactivity was determined.
Na+,K+-ATPase-mediated Rb+
transport was calculated from the difference in tracer uptake between
samples incubated in 3 µM and 5 mM ouabain.
The ouabain-insensitive Rb+ transport was 25-30% of the
total Rb+ transport measured. As PMA was dissolved in
Me2SO, the same volume of solvent was added to control
samples. For the experiments with ethoxyresorufin, the drug was
dissolved in ethanol. The volume of solvent used did not alter the
Rb+ transport of control samples. Each experiment was made
in triplicate, and results are the mean ± S.D. of at least three
independent experiments. Data are expressed as nanomoles of
Rb+ transported per mg of protein per min, or as the
percentage of Rb+ transported with respect to a control
sample. When monensin was used, control sample was the
ouabain-sensitive Rb+ transport of cells treated with monensin.
Experiments in Low Calcium Medium--
Several experiments were
performed with cells in a low calcium medium. DMEM has a concentration
of calcium of 1.8 mM. To reduce calcium concentration, 4 mM EGTA was added to the medium. Assuming that the total
calcium in DMEM is free calcium, 4 mM EGTA would reduce the
free calcium concentration to 0.12 µM. Several hours of
incubation in this low calcium medium does not affect the attachment of
the cells or their morphology as observed by microscopy. To chelate
intracellular calcium,
1,2-bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid (BAPTA) was used. The membrane-permeant acetoxymethyl form of
BAPTA (BAPTA-AM) was added to the cell incubation solution at the
concentration of 100 µM and incubated for 1 h at
25 °C, as indicated by the manufacturer (Molecular Probes, Eugene, OR).
Monitoring Ionic Changes in OK Cells--
Optical determinations
of intracellular sodium with a sodium-sensitive dye were performed as
described previously (12, 13). Fluorescence measurements of
[Na+]i were performed using the membrane-permeant
tetra(acetoxymethyl) ester of the sodium-binding
benzofuran-isophthalate (SBFI-AM, Molecular Probes, Eugene, OR)
following standard protocols (16, 17). Cells were loaded for 3 h
with the dye at room temperature in DMEM/HEPES medium containing 2-5
µM SBFI-AM and 0.1% w/v Pluronic F-127 (Molecular
Probes, Eugene, OR). After loading, the cells were washed several times
with DMEM/HEPES medium and incubated for 30 min in the same medium to
allow de-esterification of SBFI-AM. The complete hydrolysis of SBFI-AM
to SBFI was judged by changes in the excitation and emission spectra
(16). Optical measurements were performed in DMEM/HEPES medium at room
temperature. Fluorescence measurements of [Ca2+]i
were performed using
1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fura-2). OK cells were loaded with the membrane-permeant tetra(acetoxymethyl) ester of this dye (Fura-2-AM, Molecular Probes, Eugene, OR) following similar protocols as with the SBFI-AM dye. Cells
were loaded for 1-2 h with the dye at room temperature in DMEM/HEPES
medium containing 2 µM Fura-2-AM and 0.1% w/v Pluronic F-127. Cells were then washed several times with DMEM/HEPES and incubated for 30 min in the same medium to allow de-esterification of
the dye. Optical signals were acquired in DMEM/HEPES medium at room temperature.
Optical Setup--
The general plan of the optical setup used to
monitor Na+ and Ca2+ cytosolic levels in OK
cells was based on standard methods using a dual-excitation
fluorescence imaging system. Essentially the optical system consists of
an inverted fluorescence microscope (Olympus, Melville, NY) with a
video camera (Pentamax, Princeton Instruments, Trenton, NJ) attached to
its video port. Light from a 75-watt xenon lamp (model 1600, Optic
Quip, Highland Mills, NY) is collimated and rendered
quasi-monochromatic by one of several interference filters, focused by
means of a quartz UV-grade condenser and reflected to the preparation
by a dichroic mirror. Excitation wavelengths were selected through a
computer-controlled filter changer using excitation filters having
wavelengths of 340 and 380 nm (5 nm bandwidth, Omega Optical,
Brattleboro, VT). These excitation filters were selected because they
are adequate for ratio measurements using either SBFI or Fura-2
indicators. By using these indicators, the fluorescence emission was
detected above 420 nm after passing through a dichroic mirror (400 nM, Omega Optical, Brattleboro, VT) and a 420 nm highpass
filter (Omega Optical, Brattleboro, VT). To ensure optical stability in
the recordings and avoid possible photobleaching effects, the
excitation light levels were reduced by neutral density filters until
the emitted fluorescence intensity remained constant for 200 s of illumination. No significant levels of auto-fluorescence were observed,
and drugs at the concentrations used did not affect or quench
fluorescence levels. No detectable change in the SBFI or Fura-2 ratios
was observed in the pH range (7.4-7.1) tested in the present study. To
improve efficiency, fluorescent light from the cells was collected by
high numerical aperture (×20 or 40, Fluo; Nikon), which formed a real
image on the CCD sensor of the video camera located in the image plane
of the microscope. The camera sensitivity was optimized by controlling
exposure times according to background fluorescence levels of the cells
and the size of the fluorescence changes to be detected. By using the protocol previously described, fluorescence measurements of
Na+ levels in cells loaded with SBFI usually required image
exposures of 250-300 ms. For free Ca2+ determinations with
Fura-2, exposures in the order of 100-120 ms were needed. The chambers
containing loaded cells were alternately excited at 340 and 380 nm by
rapidly switching optical filters, and ratiometric determinations
usually corresponded to image pairs taken within 800 ms. Sequential
image pairs were usually collected every 6 s, although in some
experiments faster time resolution was used.
Ionic Determination--
Fluorescence measurements of
[Na+]i and [Ca2+]i were
performed using traditional ratiometric determination protocols (12,
13, 18, 19). Terms of the equation were assessed by in situ
calibration at the end of each experiment with solutions of known ionic
concentrations. Cytosolic Na+ levels were calculated
according to the original equation described previously (18) with a
Kd value for SBFI-Na+ of 18 mM. Calibrations of the excitation ratio were performed with cells permeabilized with gramicidin D (10 µM) and
superfused with different standard Na+ concentrations.
Similarly, intracellular free Ca2+ concentration was
measured by determining the ratio of Fura-2 fluorescence at 340 nm
(F340) and 380 nm (F380) excitations. There was
no detectable photobleaching during measurements as determined by the
isosbestic wavelength for both dyes. The fluorescence ratio F340/F380 of Fura-2 was calibrated in
situ according to standard protocols using the same equation (18).
In this case, calibrations were performed in OK cells permeabilized
with the Ca2+ ionophore ionomycin. These calibrations were
confirmed with cells permeabilized with either digitonin or saponin.
Fmax and Fmin were
determined in Ringer's solution (1 mM Ca2+) to
saturate the Ca2+ indicator and then bathing the cell in
low Ca2+ Ringer's solution supplemented with 5 mM EGTA.
Image Processing and Statistical Analysis of Optical
Data--
Standard computer-based image analysis software was used for
the analysis of Na+ and Ca2+ images. Video
images were acquired at 8 bits resolution and stored in real time in a
Pentium IBM-compatible computer system. Final ionic determinations were
obtained applying standard ratiometric processing algorithms. In the
figures, ionic changes in single cells are illustrated by pseudocolors
reflecting ionic concentration ranges as determined according to
ratiometric determinations (see above). Temporal plots of
Na+ and Ca2+ transients were obtained from
averaged values over 6 × 6 pixel kernels. To improve signal-noise
ratio of SBFI measurements, [Na+]i determinations
at each time results from the averaging of multiple samples acquired at
faster time resolutions. Usually, individual points represent the
average of 6 ratio determinations taken every 10 s (6 paired-frame/min), although in some experiments faster time resolution
was used.
Statistical Analysis--
Comparisons between groups were
performed by Student's t test for unpaired data.
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RESULTS |
Monensin Induces a Dose-dependent Increase of
Intracellular Sodium in OK Cells--
The sodium ionophore monensin
was used to produce stable incremental elevations of intracellular
sodium in OK cells. To determine the intracellular concentration of
free sodium, OK cells were loaded with a sodium-sensitive dye, and the
level of emitted fluorescence was monitored using a video imaging
system, as described previously (12, 13). Fluorescent images of OK
cells loaded with SBFI and excited at 380 nm are shown at the
left of Fig. 1,
A
C. Upon excitation at 340 and 380 nm, the level of
intracellular sodium was calculated from the ratio of emitted
fluorescence that was calibrated by loading the cells with standard
sodium concentrations. Ratiometric images of SBFI-loaded cells,
displayed in pseudocolor, were obtained at different times of treatment
and concentrations of monensin. Images in the center of Fig.
1, A-C (X1), illustrate the basal sodium
concentration (no monensin treatment). Basal levels of intracellular
sodium ranged from 5.3 to 13.1 mM with an average of
7.8 ± 3.3 mM (n = 24). Images on the
right of Fig. 1, A-C (X2) illustrate
the intracellular sodium concentration 5 min after the addition of 6, 9, or 12 µM monensin, respectively, to the cell medium.
Fig. 1, D and E, illustrate intracellular sodium
levels at various times after addition of different monensin concentrations to the cell medium. In situ calibration of
the excitation ratio of SBFI at various intracellular sodium
concentrations indicated that monensin produced a steady increase in
the intracellular sodium concentration of OK cells up to about 30 min,
and then the concentration of sodium was stable for at least another 40 min (Fig. 1E). As expected, higher intracellular
concentrations of sodium were determined at higher levels of monensin
in the cell medium. Once the steady state of intracellular sodium
concentration was reached, a linear relation between the concentration
of monensin in the cell medium and the concentration of intracellular
sodium was calculated (Fig. 1F). The linear equation from
this plot ([Na+]i = (2.25 ± 0.11)[monensin] 103 + (8.92 ± 0.71) mM)
was used to calculate the intracellular sodium concentrations that
correspond to the concentrations of monensin in the cell medium.
Because the new steady state of intracellular sodium produced by
monensin is reached at about 30 min, determinations of Rb+
transport were always started at this time.
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Fig. 1.
Increase of intracellular
Na+ levels induced by monensin and measured by optical
techniques. A-C illustrate fluorescence determinations
of [Na+]i in three representative OK cells
incubated with different concentrations of monensin (6, 9, and 12 µM, respectively). The left image in each
panel shows fluorescence video images of SBFI-loaded OK cells excited
at 380 nm. The fluorescence emission in these pictures illustrates the
adequate level of dye loading and the distribution of the dye within
the cell cytosol. The center and right images in
each panel correspond to the ratiometric determination of
[Na+]i in SBFI-loaded cells performed immediately
before and 5 min after the addition of monensin to the cell medium.
Pseudocolor represents ratio fluorescent intensity values proportional
to cytosolic Na+ concentrations as determined by in
situ calibration after the end of the experiments. The
calibration bar corresponds to 0-10 µm. Traces in
D show the time courses of cytosolic sodium changes taken
from the individual OK cells shown in A-C. Each plot
displays [Na+]i changes determined from a 6 × 6 pixel area outside the cell nuclear region and corresponds to
ratio determinations from image pairs taken every 6 s (10 pair-frames/min). The arrows labeled X1 and
X2 indicate time points corresponding to the ratio images
illustrated in A-C. E illustrates dose-response
curves of cytosolic Na+ changes obtained at (from
bottom to top) 0.75, 1.5, 3, 6, 9, and 12 µM monensin. Each trace corresponds to the mean ± S.D. responses calculated from 4 to 6 cells. Individual points in each
plot represent the average of 6 ratio determinations taken every
10 s (6 pair-frames/min). F illustrates the linear
dependence between the concentration of monensin in the cell medium and
the final steady state reached by intracellular sodium. The linear
trace and the corresponding equation were calculated by linear
regression. Assays were performed as indicated under "Experimental
Procedures" and in the text.
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Elevated Intracellular Sodium Concentrations Promote the Inhibition
of the Na+,K+-ATPase by Dopamine and
PMA--
Because the activity of the
Na+,K+-ATPase is limited by the level of
intracellular sodium (3), the elevated intracellular sodium
concentration elicited by monensin produced stimulation of the
Na+,K+-ATPase activity (Fig.
2A). The basal
Na+,K+-ATPase activity was 9.8 ± 0.7 nmol/mg/min, and it was stimulated to 18.5 ± 1.0 by 5 µM monensin (Fig. 2A).
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Fig. 2.
A, monensin increases
Na+,K+-ATPase activity. Cells were incubated
with the indicated concentrations of monensin for 30 min before
Rb+ transport assay. *, p < 0.02 with
respect to non-treated cells. B, intracellular sodium level
modulates the effects of dopamine and PMA on the
Na+,K+-ATPase activity. Cells were incubated
with the indicated concentrations of monensin for 30 min before
treatment with 1 µM dopamine for 5 min or 1 µM PMA for 10 min. The percentage of change for each
concentration of monensin was calculated with respect to a control in
the absence of either dopamine or PMA. *, p < 0.02 with respect to control. Assays were performed as indicated under
"Experimental Procedures" and in the text.
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To determine the effect of intracellular sodium concentration on the
inhibition of Na+,K+-ATPase activity by
dopamine, cells were treated with monensin for 30 min before the
addition of 1 µM dopamine. Five minutes later,
radioactive Rb+ was added to the cell medium to determine
the ouabain-sensitive ion transport for 20 min. At basal concentrations
of intracellular sodium, dopamine has no effect on the
Na+,K+-ATPase activity, but a steady increase
in the inhibition of the Na+,K+-ATPase activity
by dopamine was observed at increasing concentrations of intracellular
sodium (Fig. 2B, left panel). The inhibitory effect illustrated in Fig. 2B is due to dopamine and not to
monensin because in the range of concentrations this reagent was used
it only produced "activation" of the
Na+,K+-ATPase activity (Fig. 2A).
Data presented in Fig. 2B, (left panel) represent
the change in Na+,K+-ATPase activity produced
by dopamine in the presence of different monensin concentrations,
expressed as a percent of the Na+,K+-ATPase
activity measured in the presence of that concentration of monensin alone.
We have demonstrated previously (11-13, 15) that treatment of OK cells
with phorbol esters promoted a significant stimulation of
Na+,K+-ATPase activity. This stimulatory
effect, which occurs with cells containing basal concentrations of
sodium, is illustrated in Fig. 2B. However, when increasing
the intracellular sodium concentration, the activation of
Na+,K+-ATPase induced by treatment with 1 µM PMA was gradually reduced to become a significant
inhibition at concentrations of monensin higher than 3 µM
(16 mM intracellular sodium) (Fig. 2B,
right panel). Interestingly, as shown in Fig. 2B
(right panel), there is a range of monensin concentration
(1-2 µM) corresponding to 11-13 mM
intracellular sodium concentration in which the treatment of the cells
with PMA did not translate into any significant modification of the
Na+,K+-ATPase activity. It is likely that at
these concentrations of intracellular sodium the stimulatory and
inhibitory effects of PMA are compensated. Data presented in Fig.
2B (left panel) represent the change in
Na+,K+-ATPase activity produced by PMA in the
presence of different monensin concentrations, expressed as a percent
of the Na+,K+-ATPase activity measured in the
presence of that concentration of monensin alone.
All of the following experiments to determine Rb+ transport
were performed with cells treated with 5 µM monensin,
which increased the intracellular sodium concentration from ~9
to ~ 20 mM. This concentration of monensin (5 µM) was chosen because the increase of intracellular
sodium (~11 mM) produced is believed to be in the
physiological range. The inhibition of
Na+,K+-ATPase elicited by PMA resembles that
produced by dopamine (Fig. 2B). Therefore, we performed
several experiments to determine whether both reagents stimulate the
same signaling pathway. We determined whether the effects of both
agonists are additive. Also we determined whether phosphorylation of
Na+,K+-ATPase -subunit Ser-18 and production
of 20-HETE are essential for the inhibition of
Na+,K+-ATPase activity by both PMA and
dopamine. Determination of Rb+ transport in cells treated
simultaneously with both PMA and dopamine in the presence of 5 µM monensin (~20 mM intracellular sodium) showed that the inhibitory effects of PMA and dopamine on the Na+,K+-ATPase activity were not additive (PMA,
45 ± 7%; dopamine, 52 ± 9%; PMA and dopamine,
56 ± 7%) (Fig. 3). Note that
data in Fig. 3 represent the change of activity produced by PMA and/or dopamine, expressed as a percentage of the activity measured in cells
treated with monensin alone.
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Fig. 3.
The inhibitory effects of PMA and dopamine on
the Na+,K+-ATPase activity of cells treated
with monensin are not additive. Cells were incubated with 5 µM monensin for 30 min before treatment with 1 µM PMA for 10 min and/or 1 µM dopamine for
5 min. In the experiment with both PMA and dopamine, dopamine was added
5 min after the beginning of PMA treatment. The percentage of change
for each experimental condition was calculated with respect to a
control in the absence of dopamine or/and PMA. *, p < 0.05 with respect to control. Assays were performed as indicated under
"Experimental Procedures" and in the text.
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We have demonstrated previously (10, 14) that whereas phosphorylation
of the -subunit Ser-18 is essential for dopamine inhibition of
the Na+,K+-ATPase, activation of the
Na+,K+-ATPase in response to PMA requires the
phosphorylation of both Ser-11 and Ser-18 (15). Fig.
4 illustrates that the inhibition of
Na+,K+-ATPase by PMA plus monensin requires the
integrity of Ser-18 and that substitution of Ser-11 by an alanine
residue prevented the stimulation of
Na+,K+-ATPase by PMA, but it did not affect the
inhibition of this activity elicited by PMA plus monensin. Elimination
of the first 26 amino acids of the -subunit totally blunted the
inhibitory effects of both PMA and dopamine. None of these mutations
affected the basal Na+,K+-ATPase activity.
Thus, the inhibition of Na+,K+-ATPase by either
PMA or dopamine requires the integrity of Ser-18.
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Fig. 4.
Ser-18 is essential for both PMA- and
dopamine-elicited inhibition of Na+,K+-ATPase
in cells treated with monensin. Rb+ transport mediated
by the Na+,K+-ATPase of cells expressing the
wild type rodent 1-subunit and three 1 mutants was determined.
 (1-26) is the mutant in which amino acids
1-26 of the mature 1-subunit were deleted. The percentage of change
for each condition was calculated with respect to a control in the
absence of either dopamine or PMA. *, p < 0.05 with
respect to control. Assays were performed as indicated under
"Experimental Procedures" and in the text.
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The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid
(20-HETE) is an important component of the signaling pathway stimulated
by dopamine for inhibition of the Na+,K+-ATPase
activity (20, 21). Preincubation of the cells with ethoxyresorufin, an
inhibitor of the cytochrome P450-dependent monooxygenase
which produces 20-HETE (20), totally prevented the inhibition of
Na+,K+-ATPase activity induced by either
dopamine plus monensin (control, 49 ± 7%;
+ethoxyresorufin, 4 ± 9%) or PMA plus monensin (control, 40 ± 5%; +ethoxyresorufin, 6 ± 9%) (Fig.
5). The effect of ethoxyresorufin appears
to be specific for the inhibitory pathway because it has no effect
on the basal Na+,K+-ATPase or on the
stimulation of this activity produced by PMA in the absence of
monensin. Therefore, results illustrated in Figs. 2-5 support the
hypothesis that PMA and dopamine stimulate the same intracellular
messenger pathway to inhibit the
Na+,K+-ATPase.
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Fig. 5.
Effect of ethoxyresorufin on both PMA- and
dopamine-elicited inhibition of Na+,K+-ATPase
activity in cells treated with monensin. Cells were incubated with
0.1 µM ethoxyresorufin for 30 min and with 5 µM monensin for another 30 min before treatment with 1 µM PMA for 10 min or 1 µM dopamine for 5 min. The percentage of change for each experimental condition was
calculated with respect to a control in the absence of either dopamine
or PMA. *, p < 0.02 with respect to control. Assays
were performed as indicated under "Experimental Procedures" and in
the text.
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Calcium Does Not Affect the Inhibition of
Na+,K+-ATPase by Dopamine and PMA--
So far
our results indicate that an increased intracellular sodium
concentration is essential for the inhibition of the
Na+,K+-ATPase. Because changes in intracellular
sodium produced by the monensin treatment may be associated with
changes in intracellular calcium, we determined whether the effects
described above were due to alterations in the intracellular calcium
concentration. To reduce the entry of calcium into the cells, in some
experiments, 4 mM EGTA was added to the cell medium.
Because the incubation medium has a calcium concentration of 1.8 mM, a concentration of 0.12 µM free calcium
was calculated assuming that the initial calcium concentration
corresponded to free calcium. However, if some of the calcium in the
cell medium was forming complexes with other molecules, the free
calcium concentration in the presence of 4 mM EGTA should
then be lower than 0.12 µM.
The intracellular free calcium concentration was monitored in cells
loaded with the specific free calcium indicator Fura-2-AM, the
membrane-permeant acetoxymethyl form of Fura-2. Fluorescent images of
OK cells loaded with Fura-2-AM are shown on the left of Fig.
6, A and B.
Determinations were performed with cells in a medium containing either
1.8 mM (Fig. 6A) or 0.12 µM (Fig.
6B) calcium. The intracellular level of free calcium (Fig.
6) and the effect of external calcium on the intracellular sodium
concentration (Fig. 7) were determined in
cells treated with 12 µM monensin to have the "worst
case" scenario. To determine the intracellular free calcium
concentration, the level of emitted fluorescence upon excitation at 340 and 380 nm was monitored. Images of Fura-2-AM loaded cells (displayed
in pseudocolor) obtained at different times and concentrations of
monensin illustrate the change in intracellular free calcium levels.
Images labeled X1 (Fig. 6, A and
B) illustrate the basal free calcium concentration (no
monensin treatment). Basal level of cytosolic free calcium ranged from 27.5 to 50.2 nM with an average of 34.0 ± 5.1 nM (n = 12). In Fig. 6,
A and B, images labeled X2 and
X3 illustrate the intracellular free calcium concentration
at 1.5 and 6 min, respectively, after the addition of 12 µM monensin to the cell medium. Fig. 6C
illustrates the intracellular free calcium levels in either 1.8 mM (curve A) or 0.12 µM
(curve B) extracellular free calcium, at various times after
addition of monensin. With cells in a medium containing 1.8 mM calcium, addition of monensin produced a rapid increase in intracellular free calcium that reached a maximum of ~200
nM (Fig. 6C, curve A). Peak latency was in the
range 1-3 min with an average of 2.4 ± 1.5 min
(n = 8). Afterward, the intracellular free calcium
concentration progressively decays to basal levels. The elevated
intracellular free calcium concentration had a total duration from 7 to
13 min with an average of 9.3 ± 4.3 min (n = 9).
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Fig. 6.
Monensin produces a transient increase in
cytosolic Ca2+. Images in A and
B illustrate cytosolic Ca2+ levels in individual
OK cells in a medium containing either 1.8 mM or 0.12 µM free calcium, respectively. The left image
in each panel shows fluorescence video images of Fura-2-AM-loaded OK
cells excited at 380 nm. Subsequent pseudocolored images correspond to
ratiometric [Ca2+]i determinations performed
immediately before and 1.5 and 6 min after the addition of 12 µM monensin to the cell medium. Pseudocolored images in
A and B represent ratio fluorescent intensity
values proportional to cytosolic Ca2+ concentrations as
determined by in situ calibration at the end of the
experiments. Calibration bar corresponds to 15 µm. Plots
on C illustrate the time course of
[Ca2+]i changes elicited by 12 µM
monensin in OK cells in a medium containing either 1.8 mM
(curve A) or 0.12 µM (curve B) free
calcium. Arrows (X1, X2 and X3)
indicate the time points when the individual images shown in
A and B were taken. Measurements correspond to
the mean ± S.D. of 6 ratio determinations taken at 1-min
intervals and performed in a 6 × 6 pixel region of each cell.
Assays were performed as indicated under "Experimental Procedures"
and in the text.
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Fig. 7.
The rate of cytosolic Na+
increase induced by monensin depends on the level of extracellular
Ca2+. Images in A and B
illustrate cytosolic Na+ levels in individual OK cells in a
medium containing either 1.8 mM or 0.12 µM
free calcium, respectively. The left image in each panel
shows fluorescence video images of these cells following loading of the
Na+ indicator SBFI. Subsequent pseudocolored images
correspond to ratiometric [Na+]i determinations
performed immediately before and 4 and 8 min after the addition of 12 µM monensin to the cell medium. Pseudocolors in
A and B represent ratio fluorescent intensity
values proportional to cytosolic Na+ concentrations as
determined by in situ calibration at the end of the
experiments. Calibration bar corresponds to 15 µm. Plots
on C illustrate the time course of
[Na+]i changes elicited by 12 µM
monensin in OK cells in a medium containing either 1.8 mM
(curve A) or 0.12 µM (curve B) free
calcium. Arrows (X1, X2, and X3)
indicate the time points when the individual images shown in
A and B were taken. Measurements correspond to
the mean ± S.D. of 6 ratio determinations taken at 10-s
intervals. Assays were performed as indicated under the "Experimental
Procedures" and in the text.
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The rise in intracellular free calcium elicited by monensin
was totally dependent on the presence of extracellular calcium. When
the determination of intracellular free calcium was repeated with cells
in a medium with very low calcium concentration (0.12 µM), the raise in intracellular free calcium elicited by
monensin was blunted (Fig. 6C, curve B). Although a small
"bump" is apparent in curve B, the change in
intracellular free calcium was not significant with respect to the
basal concentration measured in the absence of monensin. After the
bump, there was a steady reduction in intracellular free calcium
concentration, and 13 min after the addition of monensin, the
intracellular free calcium concentration was ~1/4 of that measured in
the absence of monensin (Fig. 6C, curve B). Therefore, independent of the concentration of extracellular calcium (0.12 µM or 1.8 mM), intracellular calcium was at
or below the basal level when the determinations of Rb+
transport were performed.
The change in intracellular sodium produced by monensin was dependent
on the level of extracellular calcium (Fig. 7). The new steady state
level of intracellular sodium elicited by 12 µM monensin
was reached faster when the concentration of free-calcium in the cell
medium was reduced from 1.8 mM to 0.12 µM
(Fig. 7C). Four minutes after the addition of monensin,
cells in 0.12 µM free calcium medium had about twice the
concentration of intracellular sodium as compared with cells in medium
containing 1.8 mM calcium. Although the level of
extracellular calcium affects the rate of intracellular sodium
increase, the final steady state levels of intracellular sodium
elicited by monensin appears not to be significantly different at the
two extracellular calcium concentrations.
To determine whether the transient increase in intracellular free
Ca2+ level produced by monensin is involved in the
inhibition of Na+,K+-ATPase by dopamine, the
assay of ouabain-sensitive Rb+ transport was performed with
cells in the presence of either 1.8 mM or 0.12 µM external calcium. As illustrated in Fig.
8, the inhibition of
Na+,K+-ATPase by dopamine was not significantly
affected by the changes in intracellular Ca2+
concentration. Under the same condition, the inhibition of
Na+,K+-ATPase by PMA plus monensin was the same
at the two extracellular Ca2+ concentrations (48 ± 7 and 43 ± 12%, respectively. These data are not presented in
the figure). Determinations of Rb+ transport were also
performed with cells in a 0.12 µM extracellular free
calcium medium and loaded with the calcium chelator BAPTA. The
inhibition of Na+,K+-ATPase activity promoted
by dopamine plus monensin was not affected by the simultaneous
depletion of extracellular calcium and the intracellular loading of the
calcium chelator (Fig. 8). Thus, the basal
Na+,K+-ATPase activity and its inhibition by
either PMA or dopamine were not affected by changes of extracellular or
intracellular calcium.
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Fig. 8.
Inhibition of
Na+,K+-ATPase activity by dopamine is not
affected by the level of extra- and intracellular calcium. For the
determinations, cells were incubated in a medium containing either 1.8 mM or 0.12 µM extracellular calcium
(+EGTA). The right bar illustrates results
obtained with cells incubated in a 0.12 µM extracellular
calcium medium and loaded with 100 µM BAPTA-AM. Cells
were incubated with 5 µM monensin for 30 min before
treatment with 1 µM dopamine for 5 min. The percentage of
change for each experimental condition was calculated with respect to a
control in the absence of dopamine. *, p < 0.02 with
respect to control. Assays were performed as indicated under
"Experimental Procedures" and in the text.
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DISCUSSION |
This is the first time that changes in intracellular sodium were
measured to correlate accurately changes in this ion concentration with
the regulation of the Na+,K+-ATPase activity by
dopamine and PMA. The present results demonstrate that regulation of
the Na+,K+-ATPase activity by dopamine and PMA
is modulated by changes of the cell intracellular sodium concentration.
Several lines of evidence indicate the modulation of the regulation of
the Na+,K+-ATPase activity by the level of
intracellular sodium. This conclusion is supported by several lines of
evidence. 1) Dopamine does not inhibit the
Na+,K+-ATPase activity unless intracellular
sodium is increased (Fig. 2). 2) Basal intracellular sodium stimulation
of PKC with PMA activates the Na+,K+-ATPase,
and the phorbol ester inhibits the
Na+,K+-ATPase activity at elevated
intracellular sodium concentration (Fig. 2). 3) The level of the
inhibition of Na+,K+-ATPase activity by either
PMA or dopamine is increased at increasing intracellular sodium
concentrations (Fig. 2). Moreover, the inhibitory action by either PMA
or dopamine occurs by triggering the same intracellular signaling
pathway (Figs. 3-5). Thus, we observed a direct correlation between
the level of intracellular sodium and the regulation of
Na+,K+-ATPase activity by PMA and dopamine.
The change in intracellular sodium concentration is likely to be
exerting a permissive role on a particular signaling pathway involved
in cellular homeostasis regulation. Effects of the level of
intracellular sodium concentration on cell homeostasis have been
suggested previously. A stimulation of the synthesis de novo of Na+,K+-ATPase molecules by elevated
intracellular sodium has been described in several tissues and cells
(27-29), and a specific mechanism that involves a transcriptional
regulation stimulated by elevated sodium has been reported (24). These
effects, however, are long term; the ones we describe in this report
are short term regulations that do not involve the synthesis of new
Na+,K+-ATPase molecules (9-15). More related
to short term regulation is the report that elevated intracellular
sodium stimulates the production of dopamine in proximal tubule cells
(30-32). The experiments described in this report relied on
exogenously added dopamine and PMA. Hence, it is likely that inhibition
of Na+,K+-ATPase by dopamine or PMA in the
presence of monensin is the result of a permissive effect of elevated
intracellular sodium on signaling molecule(s) that are downstream of
the dopamine receptor. Finally, reports from other researchers (30-32)
and this work suggest that intracellular sodium acts in several
different signaling pathways and at different levels to regulate
cellular homeostasis.
The free intracellular sodium concentration of OK cells was determined
in situ by digital imaging fluorescence microscopy from the
changes in fluorescence produced by the sodium indicator SBFI (22). On
the basis of these determinations, the effect of the various drugs on
the Na+,K+-ATPase activity was tested with
cells treated with 5 µM monensin which produced an
increase in intracellular sodium from ~9 to ~20 mM.
Because proximal tubule epithelial cells should normally support
changes in this range of intracellular sodium concentration, we assumed
that 5 µM monensin produced an elevation of intracellular sodium concentration within the physiological range. This is very important because it is likely that many changes in protein function are produced when the intracellular ionic concentration is changed to
limits in which the integrity and survival of the cell are in question.
The increased intracellular sodium level produced by monensin was
accompanied by a transient elevation in intracellular free calcium
concentration, and the rate of increase of intracellular sodium
concentration promoted by monensin was greater in cells assayed in the
absence of extracellular calcium. Although interesting, the study of
the mechanism responsible for increased calcium entry into the cell was
not the object of this project and was not pursued further.
Nevertheless, independent of the mechanism involved, at the time the
Rb+ transport assay was performed (30 min after monensin
addition), the intracellular calcium concentration was at or lower than
the basal level, and intracellular sodium had reached its new steady state concentration. Therefore, no effect due to calcium should be
expected under these conditions. That calcium is not involved in the
modulation of the hormonal regulation of
Na+,K+-ATPase was also supported by the
observations that the inhibition of
Na+,K+-ATPase by either PMA or dopamine was not
affected by removal of external calcium, and by loading the cells with
the calcium chelator BAPTA. Therefore, modulation of the PMA and
dopamine effects on Na+,K+-ATPase appears to be
dependent exclusively on the level of intracellular sodium.
We and other investigators have described previously (4, 11-13, 15)
that treatment of OK cells and rat renal proximal tubules with PMA
results in stimulation of the Na+,K+-ATPase
activity. The present results demonstrate that the level of activation
of Na+,K+-ATPase by PMA is reduced and even
reversed to an inhibition by an increase of the OK cell intracellular
sodium concentration. On the basis of these results, we hypothesized
that PMA can activate two different pathways for either inhibition or
stimulation of Na+,K+-ATPase activity. Which
one is activated would depend on the intracellular sodium
concentration. While at basal intracellular sodium concentration PMA
stimulated the signaling pathway that leads to
Na+,K+-ATPase activation; at higher
intracellular sodium concentrations PMA stimulated a different pathway
that leads to inhibition of Na+,K+-ATPase.
Under the latter conditions, PMA appears to stimulate the signaling
pathway that is normally activated by dopamine to inhibit the
Na+,K+-ATPase activity. Several lines of
evidence support this conclusion. 1) The higher the intracellular
sodium concentration, the greater the inhibition of
Na+,K+-ATPase activity by both PMA and
dopamine. 2) The inhibition produced by PMA and dopamine is not
additive when both reagents were added simultaneously to the cell
medium. 3) The integrity of the -subunit Ser-18, but not of Ser-11,
is essential for the inhibition of the
Na+,K+-ATPase activity by both PMA and
dopamine. 4) Inhibition of the production of the arachidonic acid
metabolite 20-HETE blunted the inhibitory effect of both PMA and
dopamine on the Na+,K+-ATPase activity (26).
The involvement of 20-HETE in the signaling pathway stimulated by
dopamine to inhibit the Na+,K+-ATPase has been
characterized previously (20, 21).
Monensin has been extensively used as a sodium ionophore, and stable
incremental elevations of intracellular sodium by graded concentrations
of monensin have been described in several tissues and cell lines
(23-25). Unlike other sodium ionophores (e.g. gramicidin D)
that work as a sodium channel, monensin works as a sodium transporter when it binds to the membrane. Then, maintaining extracellular sodium
at the normal high concentration, the amount of sodium that enters the
cell depends on the concentration of monensin added to the cell medium,
as shown in Fig. 1. On the contrary, other sodium ionophores
equilibrate sodium across the membrane and dissipate the sodium
gradient. Because we are studying a process that depends on the
maintenance (and modulation) of the sodium gradient across the
membrane, it was important to perform the experiments under conditions
where the sodium gradient was present. It has been described that high
monensin concentrations lead to impairment of the intracellular traffic
of proteins, which may be associated with elevation of intracellular
calcium (24, 25). However, this effect has not been observed at the low
concentrations of monensin we have used, which produced only a
transient increase of intracellular calcium concentration (Fig. 6).
The results presented in this report describe a novel component of
physiological significance to the regulation of sodium reabsorption by
dopamine in the kidney. Whereas the local actions of dopamine within
proximal tubule cells have been described with some detail in the last
few years, the factors that trigger the peripheral dopaminergic
response still remained to be identified. A large increase in the
content of sodium within the diet is directly related to the
natriuretic response, and in vivo experiments have shown
that dopamine does not inhibit proximal tubule sodium reabsorption unless there is a sodium load (33-36). Because the dopaminergic response is initiated by synthesis of dopamine by the proximal tubule
cells and the translocation of dopamine receptors from cytosolic
storage compartments to the plasma membrane (37), there must be an
intracellular mechanism that senses the presence of the sodium load.
Because sodium enters the kidney proximal tubule cells through the
apical domain by a gradient-dependent mechanism, an
elevation of luminal sodium should translate in increased intracellular
sodium. This, alone or in combination with other cellular factors, may
determine and modulate the hormonal regulation of proteins involved in
sodium reabsorption. Understanding the mechanisms of such intracellular
networks would provide alternative strategies to treat disorders
associated with altered sodium homeostasis such as salt-sensitive hypertension.
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ABSTRACT
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