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J. Biol. Chem., Vol. 278, Issue 31, 28719-28726, August 1, 2003
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Intracellular Na+ Regulates Dopamine and Angiotensin II Receptors Availability at the Plasma Membrane and Their Cellular Responses in Renal Epithelia*
**
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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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (9–19 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (4–7). Conversely, Na+ load causes increased production of DA and DA receptor expression in proximal tubule epithelial cells promoting renal Na+ and water excretion (8–11). 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, 15–18). 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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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—Cells 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 Transfection—Studies 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
(19–21).
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
(22–25)
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
(22–25).
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
(22–25),
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+ Transport—Measurements of NKA-mediated transport by Rb+ uptake were performed with attached cells as described previously (22–24). 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 25–30% 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
NKA—The experiments were performed with OK cells expressing the
rodent wild type 1 subunit and grown to 80–90% 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
Membrane—The 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 Cells—Optical 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
1—After 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 Analysis—Unless 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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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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
(29–31).
Therefore, Ang II induces the stimulation of NKA activity via activation of
PKC.
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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).
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Ang II Induces Phosphorylation of the NKA
Subunit—Ang 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+ Transport—Activation 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
(
1–26; 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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Ang II-induced NKA Stimulation Is the Result of Recruitment of NKA
Molecules to the Plasma Membrane—The 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 Molecules—Recruitment 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 (39–42) 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 Activity—The 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 (43–45). 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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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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The Level of [Na+]i Modulates the Plasma Membrane Pool of Ang II and DA Receptors—It 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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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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (1–26) of the
NKA 1NH2 terminus. LY333531 prevented the Ang II-dependent
activation of NKA. It has been demonstrated previously
(29–31)
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 0.1–1 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.
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FOOTNOTES |
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* 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.
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ACKNOWLEDGMENTS |
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We thank Drs. Peter Doris, Mustafa F. Lokhandwala, and Douglas Eikenburg for critical reading of the manuscript.
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