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Originally published In Press as doi:10.1074/jbc.M303741200 on May 20, 2003
J. Biol. Chem., Vol. 278, Issue 31, 28719-28726, August 1, 2003
Intracellular Na+ Regulates Dopamine and Angiotensin II Receptors Availability at the Plasma Membrane and Their Cellular Responses in Renal Epithelia*
Riad Efendiev ,
Claudia E. Budu ,
Angel R. Cinelli ¶,
Alejandro M. Bertorello || and
Carlos H. Pedemonte **
From the
College of Pharmacy, University of
Houston, Houston, Texas 77204, ¶Department of
Anatomy and Cell Biology, State University of New York, Brooklyn, New York
11203, and ||Department of Medicine,
Atherosclerosis Research Unit, Karolinska Institutet, Karolinska Hospital,
Stockholm S-17176, Sweden
Received for publication, April 10, 2003
, and in revised form, May 16, 2003.
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ABSTRACT
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The balance and cross-talk between natruretic and antinatruretic hormone
receptors plays a critical role in the regulation of renal Na+
homeostasis, which is a major determinant of blood pressure. Dopamine and
angiotensin II have antagonistic effects on renal Na+ and water
excretion, which involves regulation of the
Na+,K+-ATPase activity. Herein we demonstrate that
angiotensin II (Ang II) stimulation of AT1 receptors in proximal tubule cells
induces the recruitment of Na+,K+-ATPase molecules to
the plasmalemma, in a process mediated by protein kinase C and
interaction of the Na+,K+-ATPase with adaptor protein 1.
Ang II stimulation led to phosphorylation of the subunit Ser-11 and
Ser-18 residues, and substitution of these amino acids with alanine residues
completely abolished the Ang II-induced stimulation of
Na+,K+-ATPase-mediated Rb+ transport. Thus,
for Ang II-dependent stimulation of Na+,K+-ATPase
activity, phosphorylation of these serine residues is essential and may
constitute a triggering signal for recruitment of
Na+,K+-ATPase molecules to the plasma membrane. When
cells were treated simultaneously with saturating concentrations of dopamine
and Ang II, either activation or inhibition of the
Na+,K+-ATPase activity was produced dependent on the
intracellular Na+ concentration, which was varied in a very narrow
physiological range (919 mM). A small increase in
intracellular Na+ concentrations induces the recruitment of D1
receptors to the plasma membrane and a reduction in plasma membrane AT1
receptors. Thus, one or more proteins may act as an intracellular
Na+ concentration sensor and play a major regulatory role on the
effect of hormones that regulate proximal tubule Na+
reabsorption.
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INTRODUCTION
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Sodium excretion by the kidney is a tightly regulated process, and several
cardiovascular diseases are usually associated with its abnormal regulation.
Variations in salt intake may affect total body Na+ balance and
induce a rise in blood pressure unless homeostasis is maintained
(1). The balance between
natruretic and antinatruretic hormones plays a critical role in the regulation
of renal Na+ transport and excretion
(1,
2). In proximal tubules, where
more than 70% of filtered Na+ reabsorption occurs, the interplay
between the antagonistic actions of the natruretic dopamine
(DA)1 and the
anti-natruretic angiotensin II (Ang II) represents an important mechanism to
regulate renal Na+ and water excretion
(3). During salt deprivation,
enhanced Ang II production and AT1 receptor expression in proximal tubules
increase Na+ and water reabsorption
(47).
Conversely, Na+ load causes increased production of DA and DA
receptor expression in proximal tubule epithelial cells promoting renal
Na+ and water excretion
(811).
Ang II antagonizes the natriuretic response elicited by DA stimulation of D1
receptors (3,
12), and DA opposes the Ang
II-induced stimulation of Na+ uptake in proximal tubules
(13,
14). Therefore, the balanced
action of DA and Ang II has a primary impact on proximal tubule Na+
reabsorption. The process that determines the balance of hormonal action, and
how DA can reduce proximal tubule Na+ reabsorption in the presence
of saturating concentrations of the antagonistic Ang II, are some of the
mechanistic aspects that remain to be elucidated. Although some insights
regarding the regulation by DA have been reported by us and other researchers,
the molecular mechanism by which Ang II regulates the activity of the proteins
involved in proximal tubule Na+ reabsorption is still unknown
(10,
11,
1518).
In this study, we have investigated the cellular mechanisms beyond the
antagonistic actions of Ang II and DA and how these mechanisms are dependent
on changes in intracellular Na+ concentration.
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EXPERIMENTAL PROCEDURES
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MaterialsCells culture supplies were purchased from
Invitrogen and Hyclone Laboratories (Logan, UT). Molecular biology reagents
were obtained from New England Biolabs (Beverly, MA), Promega (Madison, WI),
Stratagene (La Jolla, CA), and Sigma. Angiotensin II, dopamine, PMA, and the
anti-phosphoserine antibody were purchased from Sigma. Sulfo-NHS-biotin was
obtained from Pierce (Rockford, IL). Antibodies against AP1 and AP2 were
purchased from Upstate Biotechnology (Lake Placid, NY). Antibody against D1
was obtained from Alpha Diagnostics (San Antonio, TX). Antibody against AT1
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
[86Rb+]RbCl was obtained from PerkinElmer Life Sciences.
LY333531 was a kind gift from Eli Lilly (Indianapolis, IN). Protein kinase C
(PKC) isozyme inhibitor peptides were a generous gift from Dr. Mochly-Rosen
(Stanford University, Stanford, CA). All other reagents were of the highest
grade available.
Cell Culture and TransfectionStudies were performed with
the established opossum kidney (OK) epithelial cell line, which is often used
as a physiological model system of renal proximal tubule function
(1921).
OK cells were maintained at 37 °C (10% CO2) in Dulbecco's
modified Eagle's medium with 10% calf serum and antibiotics (DMEM-10). Mutants
of rodent Na+,K+-ATPase (NKA) 1 subunit cDNA were
prepared as described previously
(2225)
from a plasmid containing the wild type 1 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 1
subunit cDNAs were transfected into OK cells using LipofectAMINE 2000
liposomes (Invitrogen) as described previously
(2225).
Selection for cells expressing the highest level of rodent subunit was
achieved by exposing the cells to a medium containing 3 µM
ouabain. Because the endogenous Na+ pump of OK cells is completely
inhibited by this concentration of ouabain
(2225),
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 NKA 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 Rb+
TransportMeasurements of NKA-mediated transport by
Rb+ uptake were performed with attached cells as described
previously
(2224).
Briefly, 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). All treatments and determinations were performed
at 23 °C. Then, a trace amount of [86Rb+]RbCl was
added to the cell medium. After 20 min, cells were washed three times with
ice-cold saline and dissolved with SDS, and accumulated radioactivity was
determined. NKA-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
(measured in the presence of 5 mM ouabain) was 2530% of the
total Rb+ transport measured. In some experiments, cells were
treated with hormones, activators, and inhibitors before the Rb+
transport determination. The concentrations used and the time of treatment are
described in the respective figures.
Protein Biotin Labeling to Separate the Plasma Membrane Pool of
NKAThe experiments were performed with OK cells expressing the
rodent wild type 1 subunit and grown to 8090% confluence in
six-well plates. After treatment of the cells with Ang II, the medium was
changed to ice-cold 10 mM Tris-HCl, pH 7.5, 2 mM
CaCl2, 150 mM NaCl, 1.5 mg/ml sulfo-NHS-biotin. After
incubation for 1 h at 4 °C, the cells were scraped in immunoprecipitation
buffer (20 mM Tris, 2 mM EDTA, 2 mM EGTA, 30
mM sodium pyrophosphate, pH 7.3) containing a protease inhibitor
mixture, frozen in liquid nitrogen, thawed rapidly, probe-sonicated twice on
ice-water bath, and frozen-thawed again. The cell suspension was centrifuged
at 14,000 x g at 4 °C for 5 min. The supernatant was
separated, and protein concentration was determined. Aliquots containing equal
amounts of protein were transferred to clean tubes, and 1% Triton X-100 and
0.2% SDS were added. Anti- 1 antibody was added, and the suspension was
incubated for 1 h at 4 °C with end-over-end shaking and overnight with
protein A/G-agarose, which had been pre-washed three times with
phosphate-buffered saline and once with immunoprecipitation buffer containing
1% Triton X-100. After separation, the agarose beads were washed four times
with immunoprecipitation buffer containing 1% Triton X-100 and 0.1% SDS and
once with 50 mM Tris-HCl, pH 7.4, and finally resuspended in
Laemmli sample buffer. Electrophoresis, Western blot analysis with
extravidin-peroxide conjugate, and densitometric analysis were performed as
described previously (24).
Determinations of DA and Ang II Receptors at the Plasma
MembraneThe experiments were the same as described for the
determination of plasma membrane NKA 1, except that antibodies against
D1 and AT1 were used. Other details are described in the respective
figures.
Monitoring Ionic Changes in OK CellsOptical determinations
of the intracellular Na+ concentration
([Na+]i) with the Na+-binding
benzofuran-isophthalate were performed as described previously
(22,
23,
26). Based on the changes in
intracellular Na+ produced by different concentrations of monensin,
the following equation was deduced: [Na+]i =
(2.2 ± 0.1)·103 [monensin] + (8.9 ± 0.7)
mM (26). This
equation was used to calculate the [Na+]i that
corresponds to the concentration of monensin in the cell medium.
Determination of AP1 and AP2 Co-precipitation with NKA
1After treatment with Ang II, OK cells were dissolved,
and the NKA 1 was immunoprecipitated with anti- 1 antibody. The
precipitated material was separated by SDS-PAGE, and the proteins were
transferred to a piece of polyvinylidene difluoride membrane. This was assayed
by Western blot analysis with anti-AP1 antibody. After development and
scanning, the membrane was stripped and tested successively with anti-AP2 and
anti- 1 antibodies. The protein bands were developed and scanned. Each
experiment was repeated three times.
Other Determinations and Data AnalysisUnless indicated
otherwise, all treatments were performed at 23 °C. Reagents were dissolved
in water, except for DA, which was dissolved in 0.5% sodium metabisulfite, PMA
in 100% dimethyl sulfoxide, and monensin in 95% ethanol. For each assay, equal
amounts of solvents were added to control and test samples. However, these
amounts were minimal, and they did not appreciably change the
ouabain-sensitive NKA-mediated Rb+ transport. Determinations of
protein concentration and immunoprecipitation of NKA were performed as
described previously (24).
Comparisons between groups were performed by either Student's t test
for unpaired data or analysis of variance, as indicated in the figure
legends.
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RESULTS
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Ang II Has a Biphasic Action on Proximal Tubule NKA
Picomolar concentrations of the hormone stimulate, whereas micromolar
concentrations inhibit proximal tubule Na+ reabsorption
(7,
18,
27). A similar effect was
described for the Ang II-induced activation of proximal tubule NKA
(3,
28). Importantly, under
physiological conditions, only stimulating picomolar concentrations of Ang II
are produced (18). We observed
that treatment of OK cells with Ang II induced a concentration-dependent
increase in Rb+ transport, and a maximal activation was achieved
with 1 pM Ang II (Fig.
1A). The basal Rb+ transport was 7.6 ±
0.9 nmol/mg/min, and it was stimulated to a maximum of 10.6 ± 0.5
nmol/mg/min by 1 pM Ang II. Higher picomolar concentrations of Ang
II resulted in lower stimulation of Rb+ transport
(Fig. 1A). As reported
by other researchers (7,
18,
27), we also observed that
micromolar concentrations of Ang II inhibited Rb+ transport (data
not shown). At all tested picomolar concentrations of Ang II, activation of
NKA was prevented by addition to the cell medium of 10 nM of the
PKC inhibitor LY333531 (Fig.
1A). This inhibitor had no effect on the basal
NKA-mediated Rb+ transport by itself. At the concentration used (10
nM), LY333531 is a very specific inhibitor of the -isoforms
of PKC
(2931).
Therefore, Ang II induces the stimulation of NKA activity via activation of
PKC .

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FIG. 1. A, effect of the PKC inhibitor LY333531 on Ang II-induced
activation of NKA-mediated Rb+ transport. Cells were treated with
10 nM LY333531 for 30 min before treatment with various
concentrations of Ang II for 10 min. The percentage of change for each
experimental condition was calculated with respect to a control measured in
the absence of Ang II and LY333531. Data were analyzed using analysis of
variance (p < 0.01) and t tests (*, p < 0.01
with respect to control values; #, p < 0.01 with
respect to basal value, not treated with Ang II). B, the AT1 receptor
antagonist candesartan blocks the Ang II-induced activation of NKA. Cells were
treated with different concentrations of candesartan for 30 min before Ang II
treatment. The percentage of change for each experimental condition was
calculated with respect to a control in the absence of Ang II and candesartan.
*, p < 0.05 with respect to control.
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On the basis of pharmacological studies, the effects of Ang II on
Na+ and fluid transport were attributed to AT1 receptors
(32,
33). Consistent with this
observation, Fig. 1B
shows that the AT1 receptor antagonist candesartan
(34) antagonized, in a
concentration-dependent manner, the NKA stimulatory effect of a maximally
effective dose (1 pM) of Ang II. In the absence of Ang II,
candesartan had no effect on the basal Rb+ transport. Therefore,
Ang II acting through AT1 receptors induced the stimulation of OK cell
NKA-mediated Rb+ transport.
Treatment of the cells with either 1 pM Ang II or 1
µM PMA produced about the same level of stimulation of
Rb+ transport (Fig.
2A). This level of NKA activation was not further
increased when cells were treated simultaneously with PMA and Ang II. The fact
that the stimulatory effects of PMA and Ang II on Rb+ transport
were not additive (PMA, 34.6 ± 7.5%; Ang II, 32.2 ± 4.5%; PMA
plus Ang II, 36.8 ± 7.5%) suggests that PMA and Ang II share a common
signaling pathway to activate the NKA. Consistent with this conclusion, we
have demonstrated previously that PMA-induced activation of NKA is also
mediated by the -isoform of PKC
(35).
Fig. 2A also shows
that 0.1 µM staurosporine prevented the stimulation of
Rb+ transport by either PMA or Ang II. At the concentration of 0.1
µM, staurosporine inhibits all of the classic and novel PKC
isoforms but not the atypical PKC
(29,
31).
Ang II Induces Phosphorylation of the NKA
SubunitAng II induces a significant increase in the
phosphorylation level of NKA subunit
(Fig. 2B). As 1
Ser-11 and Ser-18 are targets for phosphorylation by PKC
(15,
36,
37), the level of 1
phosphorylation induced by Ang II was determined in cells expressing 1
mutants in which either Ser-11 (S11A cells) or Ser-18 (S18A cells) residues
were substituted by alanine residues. The basic level (without Ang II) of
1 phosphorylation was not significantly different between the mutants
and wild type subunits (Fig.
2B). Ang II induced an increased phosphorylation of S11A
and S18A 1 mutants, but the final level of phosphorylation was lower in
either mutant than in wild type 1
(Fig. 2B). Taking into
consideration the variations inherent to these measurements, the sum of the
levels of phosphorylation in Ser-11 and Ser-18 approximately corresponds to
the level of phosphorylation in wild type 1. Therefore, Ang II
treatment induced the phosphorylation of both Ser-11 and Ser-18 in NKA
1.
1 Ser-11 and Ser-18 Are Essential for Ang II-induced Stimulation
of NKA-mediated Rb+ TransportActivation of NKA
by Ang II was not observed in cells expressing a truncated 1 in which
the first 26 NH2-terminal amino acids of 1 were eliminated
( 126; see Fig.
3A). To further characterize 1 amino acids
involved in Ang II activation of NKA, experiments were performed in cells
expressing the 1 S11A and S18A mutants. The basal ouabain-sensitive
Rb+ transport was the same in cells transfected with wild type and
mutant rodent 1 cDNAs (Fig.
3A). In cells expressing the rodent wild type 1,
treatment with Ang II resulted in increased levels of ouabain-sensitive
Rb+ transport. However, substitution of either Ser-11 or Ser-18
with alanine residues (S11A and S18A) greatly impaired the stimulation of
Rb+ transport (Fig.
3A). The same results were observed with PMA
(Fig. 3A). Because
none of the mutations altered the basal Rb+ transport measured in
the absence of either Ang II or PMA, the results illustrated in
Fig. 3A suggest that
either S11A or S18A mutations have affected specifically the mechanism of NKA
activation and not the intrinsic mechanism of NKA activity. We have
demonstrated previously that the effect of PMA was specific and mediated by
PKC and that 4 -phorbol 12,13-didecanoate, a phorbol ester that does not
stimulate PKC, had no effect on the level of ouabain-sensitive Rb+
transport (22,
23).

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FIG. 3. A, both Ser-11 and Ser-18 residues of 1 are essential for
the stimulation of NKA-mediated Rb+ transport induced by either Ang
II or PMA. Rb+ transport mediated by NKA was determined in OK cells
expressing the wild type and S11A, S18A, and 126 mutants of
1. S11A and S18A indicate substitutions by alanine
residues of 1 Ser-11 and Ser-18. 126 represents
a mutant in which amino acids 126 of the mature 1 subunit were
deleted. The percentage of change in Rb+ transport for each
condition was calculated with respect to a control in the absence of either
Ang II or PMA. *, p < 0.05 with respect to control. B,
Ang II induces the recruitment of NKA molecules to the plasma membrane. Cells
were treated with 1 pM Ang II for 10 min and then the abundance of
NKA molecules at the plasma membrane was determined by biotinylation as
described under "Experimental Procedures." A representative
Western blot is shown in the upper panel. Quantitation data of
biotinylated NKA 1 are presented in the lower panel as the
percentage change of Ang II-induced biotinylation with respect to a
non-treated control. C, AP1, but not AP2, is involved in the Ang
II-induced recruitment of NKA molecules to the plasma membrane. Cells were
treated with 1 pM Ang II for 10 min and then NKA 1 was
immunoprecipitated, and Western blot analysis was performed with AP1, AP2, and
NKA 1 antibodies. As described under "Experimental
Procedures," each blotted membrane was tested with the three antibodies.
A representative Western blot for each antibody is illustrated in left
panel. Quantitation data of precipitated AP1, AP2, and NKA 1 are
presented in the bar graph as a percentage change of Ang II-induced
co-precipitation with respect to a non-treated control.
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Ang II-induced NKA Stimulation Is the Result of Recruitment of NKA
Molecules to the Plasma MembraneThe Ang II-induced increase in
NKA-mediated Rb+ transport may result from a more rapid ATPase rate
of enzyme molecules already present at the plasma membrane or from the
translocation of NKA molecules from intracellular compartments to the plasma
membrane. Thus, we studied the effect of Ang II on the size of the
plasmalemmal pool of NKA molecules. After treatment with Ang II, the
temperature of the cell medium was reduced to 4 °C to label plasma
membrane proteins with sulfo-NHS-biotin. The low temperature impeded the
trafficking of NKA molecules between the plasma membrane and intracellular
compartments locking the recruited molecules at the cell plasma membrane
during treatment with sulfo-NHS-biotin. This reagent reacts with primary amino
groups and does not permeate across biological membranes; thus, protein side
chains containing primary amines that are exposed to the extracellular medium
were biotinylated. Then, cells were lysed, and NKA 1 was
immunoprecipitated with an anti- 1 monoclonal antibody and protein
A/G-agarose. Precipitated proteins were separated by SDS-PAGE and blotted onto
a piece of polyvinylidene difluoride membrane, and the biotinylated NKA
1 was identified by Western blotting with extravidin-peroxidase. The
anti- 1 antibody was a kind gift from Dr. Robert Mercer (Washington
University, St. Louis, MO). Results illustrated in
Fig. 3B indicate a
significant increase of biotinylated 1 produced by treatment of the
cells with Ang II. The increase of the plasma membrane pool of NKA molecules
elicited by Ang II treatment (Fig.
3B) is consistent with the hormone-induced activation of
Rb+ transport illustrated in
Fig. 3A. Therefore,
Ang II-induced stimulation of NKA activity is produced by recruitment of NKA
molecules to the plasma membrane.
Ang II Induces the Interaction between Adaptor Protein 1 and NKA
MoleculesRecruitment of plasma membrane proteins occurs by
selective recognition of the target protein, located in intracellular
compartments, by interaction with AP1 followed by the protein translocation
into the plasma membrane via clathrin-coated vesicles
(38). To determine whether
this mechanism is involved in the recruitment of NKA molecules elicited by Ang
II, the level of co-precipitation of AP1 with NKA molecules was determined. As
shown in Fig. 3C,
there was no significant difference in the amount of NKA immunoprecipitated
from samples treated or not with Ang II. However, the co-precipitation of AP1
and NKA molecules is increased by Ang II treatment. On the contrary, Ang II
has no effect on the level of co-precipitation of NKA molecules and AP2
(Fig. 3C), which is
responsible for clathrin vesicle formation during plasma membrane endocytosis.
Indeed, we have demonstrated previously
(3942)
that DA induces the interaction of AP2 with NKA molecules that are retrieved
from the plasma membrane by clathrin-vesicle-mediated endocytosis.
Small Changes of Intracellular Na+ Concentration
Modulate the Ang II-induced Stimulation of NKA ActivityThe
Na+ ionophore monensin was used to produce stable incremental
concentrations of [Na+]i. Monensin has been
used extensively as an Na+ ionophore, and stable incremental
increases in [Na+]i by graded concentrations of
monensin have been described in several tissues and cell lines
(4345).
Monensin works as an Na+ transporter when it binds to the cell
membrane. Then, maintaining extracellular Na+ at a physiological
concentration (155 mM), Na+ enters the cell in a
saturable fashion that depends on the concentration of monensin added to the
cell medium (26). In contrast,
other Na+ ionophores (e.g. gramicidin D) equilibrate
Na+ across the membrane and dissipate the Na+ gradient.
Because we are studying a process that depends on the maintenance (and
modulation) of the Na+ gradient across the cell membrane, it was
important to perform the experiments under conditions in which the
Na+ gradient was maintained. Changes in
[Na+]i were monitored by digital fluorescence
microscopy of cells loaded with the Na+ indicator
Na+-binding benzofuran-isophthalate
(26). Then, keeping
extracellular Na+ at 155 mM, cells were treated with 1,
2, 3, and 5 µM monensin to increase the
[Na+]i from 9 mM (basal) to 11, 13,
15, and 19 mM, respectively. Therefore, the maximal change of
[Na+]i was 10 mM. As the NKA
activity is limited by the availability of intracellular Na+, the
elevated [Na+]i elicited by monensin produced
stimulation of basal NKA-mediated Rb+ transport
(26). Because of this, data
presented in Fig. 4 represent
the change in Rb+ transport produced by DA and/or Ang II in the
presence of different monensin concentrations, expressed as a percent of the
Rb+ transport measured in the presence of the corresponding
concentration of monensin alone (no DA or Ang II). Although NKA inhibition
induced by DA was higher at increasing [Na+]i,
NKA activation induced by Ang II was reduced as the
[Na+]i was raised
(Fig. 4). When the cells were
treated simultaneously with DA and Ang II, NKA stimulation was observed at
basal [Na+]i (9 mM). However, this
stimulation was stepwise reduced to become an inhibition at increasing
[Na+]i. It can be argued that in the presence
of both DA and Ang II, the NKA inhibition observed at 19 mM
[Na+]i is because of a shift of the action of
Ang II to an inhibitory effect. This is clearly not the case, because at 19
mM [Na+]i, Ang II alone has no
significant effect on NKA-mediated Rb+ transport
(Fig. 4). Furthermore, in the
presence of both Ang II and DA, the level of NKA inhibition at 19
mM [Na+]i is the same as that
produced by DA alone. Between 11 and 13 mM
[Na+]i, treatment of the cells with both DA and
Ang II would not translate into a significant modification of Rb+
transport. It is likely that at these [Na+]i
the stimulatory effect of Ang II is compensated by the inhibitory effect of
DA. Although we determined that the increased
[Na+]i was accompanied by a transient elevation
in intracellular free calcium (Ca2+) concentration, we
have also demonstrated that the intracellular Ca2+
concentration was at or below the basal level when determinations of
Rb+ transport were performed, and the same results were obtained
when a Ca2+ chelator was introduced into the cells
(26). Therefore, modulation of
the actions of DA and Ang II as illustrated in
Fig. 4 is not produced by
changes in intracellular Ca2+ but the result of
different [Na+]i.

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FIG. 4. The intracellular Na+ concentration modulates the activation
or inhibition of NKA-mediated Rb+ transport induced by Ang II or
DA, respectively. Cells were treated with different concentrations of
monensin for 30 min to increase [Na+]i to
levels indicated in the figure. Then, the cells were treated with 1
µM DA (5 min) and/or 1 pM Ang II (10 min) or 1
µM PMA (10 min) followed by the Rb+ transport assay.
The percentage of change in Rb+ transport for each experimental
condition was calculated with respect to the corresponding control at the same
[Na+]i and in the absence of DA/Ang II/PMA. *,
p < 0.05 with respect to control.
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The intracellular signaling pathways associated with D1 or AT1 receptors
can be stimulated directly by treatment of the cells with PMA (see
Fig. 2A and
Fig. 3A and Ref.
24). As illustrated in
Fig. 4, whereas 1
µM PMA activates NKA at basal
[Na+]i, the phorbol ester inhibits
the NKA at 19 mM [Na+]i.
Therefore, [Na+]i modulates the stimulatory and
inhibitory hormonal actions on NKA even when the hormonal receptors are
by-passed by direct stimulation of the signaling pathways. We have
demonstrated previously (35)
that PKC mediates the NKA inhibition induced by DA. However, PMA cannot
activate PKC (31). To
determine the PKC isoform involved in this inhibition, peptides that inhibit
the interaction of the PKC isoforms and their anchoring proteins (RACKS) were
used (46). As illustrated in
Table I, although PKC is
the PKC isoform involved in DA-induced inhibition of NKA, both PKC and
PKC participate in the PMA-elicited inhibition of NKA at 19
mM [Na+]i. So far, we do not know
the molecular mechanism involved in PMA stimulation of the DA pathway, but the
above results suggest that PMA activates PKC , which may then activate
PKC molecules that are components of the DA intracellular pathway.
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TABLE I Effects of PKC isozyme peptide inhibitors on PMA and DA modulation of
NKA-mediated Rb+ -transport at normal and elevated
[Na+]
Experiments were performed as described under "Experimental
Procedures." The percentage of change for each experimental condition
was calculated with respect to a control in the absence of either PMA or
DA.
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The Level of [Na+]i
Modulates the Plasma Membrane Pool of Ang II and DA
ReceptorsIt has been described
(47) that DA induced the
recruitment of D1 receptors to the plasma membrane. Thus, we studied the
effect of increased [Na+]i on the level of
plasma membrane D1 receptors. After the cells were treated with 5
µM monensin to increase [Na+]i
from9to19mM, the abundance of plasma membrane D1 receptors was
determined at different times (Fig.
5A). This produced a steady increase of D1 receptors, and
after 30 min of monensin treatment (the maximum time measured), a four to five
times increase in plasma membrane D1 receptors was determined. Addition of 1
µM DA for 5 min produced a constant increase in the number of
plasma membrane D1 receptors, and the level of increase (on top of those
increased by monensin) was the same at the different times
(Fig. 5A). Therefore,
treatment for 5 min with 1 µM DA increased a fixed amount of D1
receptors independently of how many receptors were already at the plasma
membrane.

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FIG. 5. A, a small increase in [Na+]i
induces the recruitment of D1 receptors to the plasma membrane. Cells were
treated with 5 µM monensin for the indicated periods of time
(solid line) and then with 1 µM DA for 5 min
(dashed line). The abundance of D1 receptors at the plasma membrane
was determined by cell surface protein biotinylation and Western blot analysis
with an anti-D1 antibody as described under "Experimental
Procedures." A representative Western blot is shown in the upper
panel. Quantitation data are presented in the lower panel as
linear regressions of plasma membrane D1 receptors in the presence or absence
of DA with respect to controls that were not treated with monensin.
B, DA also induces the recruitment of D1 receptors to the plasma
membrane. Cells were treated with 5 µM monensin for 30 min total
and 1 µM DA for the times indicated in the figure. Then, the
abundance of D1 receptors at the plasma membrane was determined by cell
surface biotinylation and Western blot analysis with an anti-D1 antibody as
described under "Experimental Procedures." A representative
Western blot is shown in the upper panel. Quantitation data are
presented in the lower panel as linear regressions of plasma membrane
D1 receptors in the presence or absence of monensin with respect to controls
that were not treated with DA. The dashed line represents the plasma
membrane abundance of D1 receptors in cells treated with DA in the absence of
monensin. C, increased [Na+]i induces
endocytosis of AT1 receptors. Cells were treated with 5 µM
monensin for the indicated times. Then, the abundance of AT1 receptors at the
plasma membrane was determined by cell surface biotinylation and Western blot
analysis with an anti-AT1 antibody as described under "Experimental
Procedures." A representative Western blot is shown in the upper
panel. Quantitation data are presented in the lower panel as
linear regression of plasma membrane AT1 receptors with respect to a control
non-treated with monensin.
|
|
In the absence of monensin treatment, DA produced a very small change in
the plasma membrane abundance of D1 receptors
(Fig. 5B). When added
after an [Na+]i increase from 9 to 19
mM, DA steadily increases the plasma membrane abundance of D1
receptors at the plasma membrane that reaches maximum at 20 min of DA
treatment (Fig. 5B).
At this point, D1 receptors at the plasma membrane have increased two and a
half times. The DA-induced plasma membrane increase of D1 receptors is in
addition to those receptors already recruited by monensin treatment.
Contrary to the effect on D1 receptors, an increase of
[Na+]i induced a reduction in AT1 receptors,
and after 30 min about 50% of the AT1 receptors have been internalized
(Fig. 5C). The
opposite effects of [Na+]i on D1 and AT1
receptors (Fig. 5) is
consistent with the effect of [Na+]i on the
action of DA and Ang II on NKA activity
(Fig. 4).
 |
DISCUSSION
|
|---|
The present study demonstrates that changes in
[Na+]i modulate the number of DA and Ang II
receptors present at the plasma membrane. These changes lead to significant
differences in cellular responses. Increases in
[Na+]i lead to a higher number of D1 receptors
in the plasma membrane that is paralleled by a reduced abundance of AT1
receptors. Whereas at basal [Na+]i, Ang II
stimulates NKA, this effect is blunted by rising the
[Na+]i. Conversely, at higher
[Na+]i there is an increased abundance of D1
receptors at the plasma membrane associated with a reduction in NKA-mediated
Rb+ transport.
We demonstrate that stimulation of OK cell AT1 receptors by Ang II leads to
recruitment of NKA molecules to the plasma membrane in a process mediated by
PKC and an increased interaction between NKA and AP1 molecules. The
recruitment of NKA molecules to the plasma membrane is responsible for the
increased NKA activity. This conclusion is supported by the observation that
Ang II stimulation of AT1 receptors produced comparable increases in both the
plasma membrane pool of NKA molecules and the ouabain-sensitive Rb+
transport. That the stimulation of NKA activity by Ang II is because of a
direct effect on NKA and not a consequence of increased Na+
permeability is shown by the fact that the stimulation is prevented by amino
acid substitutions (S11A or S18A) or the deletion ( 126) of the
NKA 1NH2 terminus. LY333531 prevented the Ang II-dependent
activation of NKA. It has been demonstrated previously
(2931)
that 10 nM LY333531 inhibits (in vivo and in
vitro) the activity of the PKC without any effect on other PKC
isoforms, protein kinase A, Ca2+-calmodulin kinase,
casein kinase, and Src tyrosine kinase. Therefore, the Ang II-induced
activation of NKA is mediated by PKC . We have demonstrated previously
that the - and -isoforms of PKC are present in OK cells and that
PKC is involved in the DA-dependent inhibition of NKA activity
(35).
Determinations of the levels of Rb+ transport and
phosphorylation with S11A and S18A mutants suggest that Ang II-dependent
stimulation of Rb+ transport is exclusively dependent on
PKC-mediated phosphorylation of Ser-11 and Ser-18. The fact that the presence
of both serine residues is essential and that they are phosphorylated by
stimulation of AT1 receptors suggests that phosphorylation is indeed involved
in the mechanism of Ang II activation of NKA. Because activation of NKA
produced by stimulation of AT1 receptors results from recruitment of NKA
molecules to the plasma membrane, phosphorylation of Ser-11 and Ser-18 may be
the signal that triggers this process, and thereby, only phosphorylated NKA
molecules may be translocated from intracellular compartments to the plasma
membrane. The fact that Ang II increases the co-precipitation of AP1 and NKA
molecules suggests that translocation of NKA from intracellular compartments
to the plasma membrane is a clathrin vesicle-mediated process.
DA treatment of proximal tubule cells results in inhibition of NKA activity
(22,
23,
25,
48). DA acts through PKC
and endocytosis of NKA molecules, and only phosphorylation of 1 Ser-18
is essential (24). Although DA
inhibition of NKA is increased, stimulation of this activity by Ang II is
reduced at higher [Na+]i. That inhibition or
activation of NKA may be observed when both DA and Ang II are added to the
cell medium at maximal activating concentrations is dependent on the level of
[Na+]i. Therefore, the effect of hormones that
regulate the rate of Na+ translocation across the proximal tubule
epithelial cells, and thereby Na+ excretion, may be modulated by
the level of [Na+]i. The regulation of hormonal
action by [Na+]i may explain the observation
that DA is able to reduce proximal tubule Na+ reabsorption in the
presence of concentrations of Ang II that should totally override the effect
of DA (18). Although luminal
proximal tubule Ang II concentrations are in the 0.11 nM
range, plasma concentrations are in the picomolar range
(18,
49,
50). How can low
concentrations of DA inhibit Na+ reabsorption in the presence of
saturating concentrations of the antagonistic Ang II? Our results suggest
that, even in the presence of maximal activating concentrations of the
hormones, it is the level of [Na+]i that may
determine whether the DA or Ang II signaling pathway is activated. One
possible mechanism by which [Na+]i modulates
the effects of these antagonistic hormones is the abundance of their receptors
present at the plasma membrane. Consistent with this, we have shown that
increasing [Na+]i from 9 to 19 mM
leads to an increased abundance of plasma membrane D1 receptors with a
parallel decrease in AT1 receptors.
We have observed that the DA-induced recruitment of D1 receptors is very
small in the absence of an increased
[Na+]i. Thus, an increased
[Na+]i is not only more effective than DA, but
also a pre-existent increased [Na+]i is
required for DA to induce the recruitment of D1 receptors to plasma membrane.
These results represent the first report that
[Na+]i modulates the plasma membrane level of
hormones receptors that have antagonistic effects on Na+ excretion.
Importantly, in addition to regulating the level of D1 and AT1 receptor
expression at the plasma membrane, [Na+]i
modulates the action of Ang II and DA by affecting other steps in the
signaling pathways of these hormones. The receptors can be bypassed by direct
activation of PKC molecules involved in these processes. We have demonstrated
that treatment of the cells with PMA may lead to either activation or
inhibition of NKA and that which effect is observed is dependent on the level
of [Na+]i. Although at basal
[Na+]i, PMA activates NKA, the phorbol ester
inhibits the NKA at 19 mM [Na+]i.
This indicates that depending on the level of
[Na+]i either the activating or inhibiting
pathway is facilitated. There is also evidence that increased
[Na+]i induces the production of DA, which is
then transported out of the cell to stimulate D1 receptors
(47,
51). Therefore, there are at
least three levels at which [Na+]i modulates
the antagonistic effects of DA and Ang II on proximal tubule NKA: the cell
membrane abundance of hormone receptors, the intracellular signaling pathway,
and the synthesis of DA. Thus, one or more proteins that are part of these
intracellular signaling pathways may act as
[Na+]i sensors and contribute to modulate the
effect of hormones that regulate proximal tubule Na+ excretion.
Most cases of hypertension are associated with an inability of the kidney
to regulate Na+ reabsorption
(1,
2,
52). Even small changes in
Na+ intake in the absence of compensatory changes in tubular
Na+ reabsorption rate would rapidly lead to life-threatening salt
retention (3). The results of
the present report suggest that, despite of the availability of hormones, the
level of [Na+]i may represent a major
determinant that balances the action of such hormones having antagonistic
effects on the regulation of proximal tubule Na+ reabsorption. The
regulation of hormonal action by the level of proximal tubule epithelial cell
[Na+]i may provide the organism with a first
step mechanism to control tubular Na+ reabsorption.
 |
FOOTNOTES
|
|---|
* This work was supported in part by National Institutes of Health Grant
DK53460, American Heart Association Grant 0050801Y, and Swedish Research
Council Grant 10860. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
Contributed equally to this publication. 
**
To whom correspondence should be addressed: University of Houston, College of
Pharmacy, 4800 Calhoun Rd., Bldg. SR2, Rm. 555, Houston, TX 77204. Tel.:
713-743-1211; Fax: 713-743-1229; E-mail:
cpedemonte{at}uh.edu.
1 The abbreviations used are: DA, dopamine; Ang II, angiotensin II; NKA,
Na+,K+-ATPase; DMEM, Dulbecco's modified Eagle's medium;
[Na+]i, intracellular Na+
concentration; OK, opossum kidney; PKC, protein kinase C; PMA, phorbol
12-myristate 13-acetate; AP, adaptor protein; sulfo-NHS,
N-hydroxysulfosuccinimide. 
 |
ACKNOWLEDGMENTS
|
|---|
We thank Drs. Peter Doris, Mustafa F. Lokhandwala, and Douglas Eikenburg
for critical reading of the manuscript.
 |
REFERENCES
|
|---|
- Guyton, A. C. (1991) Science
252,
18131816[Abstract/Free Full Text]
- Dahl, L. K. (1972) Am. J. Clin.
Nutr. 25,
231244[Medline]
[Order article via Infotrieve]
- Aperia, A., Hotback, U., Syren, M. L., Svensson, L. B., Fryckstedt,
J., and Greengard, P. (1994) FASEB J.
8,
436439[Abstract]
- Cheng, H. F., Becker, B. N., Burns, K. D., and Harris, R. C.
(1995) J. Clin. Invest.
95,
20122019[Medline]
[Order article via Infotrieve]
- Ingelfinger, J. R., Zhou, W. M., Fon, E. A., Ellison, K. E., and
Dzau, V. J. (1990) J. Clin. Invest.
85,
417423[Medline]
[Order article via Infotrieve]
- Garvin, J. L. (1991) J. Am. Soc.
Nephrol. 1,
11461152[Abstract]
- Harris, P. J., Hiranyachattada, S., Antoine, A. M., Walker, L.,
Reilly, A. M., and Eitle, E. (1996) Clin. Exp.
Pharmacol. Physiol. 23, Suppl. 3,
S112S118
- Hegde, S. S., Jadhav, A. L., and Lokhandwala, M. F.
(1989) Hypertension
13,
828834[Abstract/Free Full Text]
- Bertorello, A. M., and Katz, A. I. (1993)
Am. J. Physiol. 265,
F743F755[Medline]
[Order article via Infotrieve]
- Hussain, T., and Lokhandwala, M. F. (1998)
Hypertension 32,
187197[Abstract/Free Full Text]
- Aperia, A. C. (2000) Annu. Rev.
Physiol. 62,
621647[CrossRef][Medline]
[Order article via Infotrieve]
- Chen, C. J., Apparsundarum, S., and Lokhandwala, M. F.
(1991) J. Pharmacol. Exp. Ther.
256,
486491[Abstract/Free Full Text]
- Sheikch-Hamad, D., Wang, Y. P., Jo, O. D., and Yanagawa, N.
(1993) Am. J. Physiol.
264,
F737F743[Medline]
[Order article via Infotrieve]
- Cheng, H. F., Becker, B. N., and Harris, R. C. (1996)
J. Clin. Invest. 97,
27452752[Medline]
[Order article via Infotrieve]
- Pedemonte, C. H., and Bertorello, A. M. (2001)
J. Bioenerg. Biomembr.
33,
439447[CrossRef][Medline]
[Order article via Infotrieve]
- Carey, R. M. (2001)
Hypertension 38,
297302[Abstract/Free Full Text]
- Ewart, H. S., and Klip, A. (1995) Am. J.
Physiol. 269,
C205C311
- Féraille, E., and Doucet, A. (2001)
Physiol. Rev. 81,
345418[Abstract/Free Full Text]
- Malstrom, K., Stange, G., and Murer, H. (1987)
Biochim. Biophys. Acta.
902,
269277[Medline]
[Order article via Infotrieve]
- Nash, S. R., Godinot, N., and Caron, M. G. (1993)
Mol. Pharmacol. 44,
918925[Abstract]
- Guimaraes, J. T., Vieira-Coelho, M. A., Serrao, M. P., and
Soares-da-Silva, P. (1997) Int. J.
Biochem. 29,
681688
- Pedemonte, C. H., Pressley, T. A., Cinelli, A. R., and Lokhandwala,
M. F. (1997) Mol. Pharmacol.
52,
8897[Abstract/Free Full Text]
- Pedemonte, C. H., Pressley, T. A., Lokhandwala, M. F., and Cinelli,
A. R. (1997) J. Membr. Biol.
155,
219227[CrossRef][Medline]
[Order article via Infotrieve]
- Efendiev, R., Bertorello, A. M., Pressley, T. A., Rousselot, M.,
Féraille, E., and Pedemonte, C. H. (2000)
Biochemistry 39,
98849892[CrossRef][Medline]
[Order article via Infotrieve]
- Chibalin, A. V., Ogimoto, G., Pedemonte, C. H., Pressley, T. A.,
Katz, A. I., Féraille, E., Berggren, P.-O., and Bertorello, A. M.
(1999) J. Biol. Chem.
274,
19201927[Abstract/Free Full Text]
- Efendiev, R., Bertorello, A. M., Zandomeni, R., Cinelli, A. R., and
Pedemonte, C. H. (2002) J. Biol. Chem.
277,
1148911496[Abstract/Free Full Text]
- Harris, P. J., and Young, J. A. (1977)
Pflugers Arch. 367,
295297[CrossRef][Medline]
[Order article via Infotrieve]
- Bharatula, M., Hussain, T., and Lokandwala, M. F.
(1998) Clin. Exp. Hypertens. [A]
20,
465480
- Ishii, H., Jirousek, M. R., Koya, K., Takagi, C., Xia, P.,
Clermont, A., Bursell, S. E., Kern, T. W., Ballas, L. M., Heath, W. F.,
Stranum, L. E., Feener, E. P., and King, G. L. (1996)
Science 272,
728731[Abstract]
- Kowluru, R. A., Jirousek, M. R., Stranum, L., Farid, N., Engerman,
R. L., and Kern, R. S. (1998) Diabetes
47,
464469[Abstract]
- Hofmann, J. (1997) FASEB J.
11,
649669[Abstract]
- Quan, A., and Baum, M. (1998) Am. J.
Physiol. Renal Physiol. 275,
F74F78[Abstract/Free Full Text]
- Wong, P. S., and Johns, E. J. (1998) Br. J.
Pharmacol. 124,
4146[CrossRef][Medline]
[Order article via Infotrieve]
- Zhuo, J. L., Imig, J. D., Hammond, T. G., Orengo, S., Benes, E.,
and Navar, L. G. (2002) Hypertension
39,
116121[Abstract/Free Full Text]
- Efendiev, R., Bertorello, A. M., and Pedemonte, C. H.
(1999) FEBS Lett.
456,
4548[CrossRef][Medline]
[Order article via Infotrieve]
- Feschenko, M. S., and Sweadner, K. J. (1997)
J. Biol. Chem. 272,
1772617733[Abstract/Free Full Text]
- Chibalin, A. V., Vasilets, L. A., Hennekes, H., Pralong, D., and
Geering, K. (1992) J. Biol. Chem.
267,
2237822384[Abstract/Free Full Text]
- Ohno, H., Stewart, J., Fournier, M.-C., Bosshart, H., Rhee, I.,
Miyatake, S., Saito, T., Gallusser, A., Kirchhausen, T., and Bonifacino, J. S.
(1995) Science
269,
18721875[Abstract/Free Full Text]
- Ogimoto, G., Yudowski, G. A., Barker, C. J., Köhler, M., Katz,
A. I., Féraille, E., Pedemonte, C. H., Berggren, P.-O., and Bertorello,
A. M. (2000) Proc. Natl. Acad. Sci. U. S.
A. 97,
32423247[Abstract/Free Full Text]
- Yudowski, G. A., Efendiev, R., Pedemonte, C. H., Katz, A. I.,
Berggren, P.-O., and Bertorello, A. M. (2000) Proc.
Natl. Acad. Sci. U. S. A. 97,
65566561[Abstract/Free Full Text]
- Efendiev, R., Yudowski, G. A., Zwiller, J., Leibiger, B., Katz, A.
I., Berggren, P.-O., Pedemonte, C. H., Leibiger, I. B., and Bertorello A. M.
(2002) J. Biol. Chem.
277,
4410844114[Abstract/Free Full Text]
- Done, S. C., Leibiger, I. B., Efendiev, R., Katz, A. I., Leibiger,
B., Berggren, P.-O., Pedemonte, C. H., and Bertorello A. M.
(2002) J. Biol. Chem.
277,
1710817111[Abstract/Free Full Text]
- Haber, R. S., Pressley, T. A., Loeb, J. N., and Ismail-Beigi, F.
(1987) Am. J. Physiol.
253,
F26F33[Medline]
[Order article via Infotrieve]
- Ruiz-Opazo, N., Cloix, J. F., Melis, M. G., Xiang, X. H., and
Herrera, V. L. (1997) Hypertension
30,
191198[Abstract/Free Full Text]
- Pressman, B. C., and Fahim, M. (1983) Adv.
Exp. Med. Biol. 161,
543561[Medline]
[Order article via Infotrieve]
- Mochly-Rosen, D., and Gordon, A. S. (1998)
FASEB J. 12,
3542[Abstract/Free Full Text]
- Brismar, H., Asghar, M., Carey, R. M., Greengard, P., and Aperia,
A. (1998) Proc. Natl. Acad. Sci. U. S. A.
12,
55735578
- Chibalin, A. V., Pedemonte, C. H., Katz, A. I., Féraille,
E., Berggren, P.-O., and Bertorello, A. M. (1998) J.
Biol. Chem. 273,
88148819[Abstract/Free Full Text]
- Navar, L. G., Lewis, L., Hymel, A., Braam, B., and Mitchell, K. D.
(1994) J. Am. Soc. Nephrol.
5,
11531158[Abstract]
- Navar, L. G., Harrison, B. L. M., Imig, J. D., Wang, C. T.,
Crevenka, L., and Mitchell, K. D. J. (1999) Am. Soc.
Nephrol. 10,
S266S272[Medline]
[Order article via Infotrieve]
- Soares-da-Silva, P. (1993) Biochem.
Pharmacol. 45,
17911800[CrossRef][Medline]
[Order article via Infotrieve]
- Kuchel, O. G., and Kuchel, G. A. (1991)
Hypertension 18,
709721[Abstract/Free Full Text]

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