1 Department of Woman and Child Health, Karolinska Institute, Astrid Lindgren Children's Hospital, S-171 76 Stockholm, Sweden; 2 Centro de Investigaciones Endocrinologicas, Consejo Nacional de Investigaciones Científicas y Técnicas, 1425 Buenos Aires; and 3 Facultad de Ciencias Biomedicas, Universidad Austral, 1063 Buenos Aires, Argentina
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ABSTRACT |
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Short-term regulation of sodium
metabolism is dependent on the modulation of the activity of sodium
transporters by first and second messengers. In understanding diseases
associated with sodium retention, it is necessary to identify the
coupling between these messengers. We have examined whether dopamine,
an important first messenger in tubular cells, activates and
translocates various protein kinase C (PKC) isoforms. We used a
proximal tubular-like cell line, LLCPK-1 cells, in which dopamine was
found to inhibit Na+-K+-ATPase in a
PKC-dependent manner. Translocation of PKC isoforms was studied with
both subcellular fractionation and confocal microscopy. Both techniques
revealed a dopamine-induced translocation from cytosol to plasma
membrane of PKC- and -
, but not of PKC-
, -
, and -
. The
process of subcellular fractionation resulted in partial translocation
of PKC-
. This artifact was eliminated in confocal studies. Confocal
imaging permitted detection of translocation within 20 s.
Translocation was abolished by a phospholipase C inhibitor and by an
antagonist against the dopamine 1 subtype (D1) but not the
2 subtype of receptor (D2). In conclusion, this study
visualizes in renal epithelial cells a very rapid activation of the
PKC-
and -
isoforms by the D1 receptor subtype.
kidney; Na+-K+-ATPase; phospholipase C; dopamine
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INTRODUCTION |
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RENAL DOPAMINE PLAYS A KEY ROLE for the regulation of sodium metabolism. There are principally two types of dopamine receptors, which from the beginning were characterized with regard to their capacity to activate (D1) or inhibit (D2) the activity of adenylate cyclase (9, 13). More recent evidence suggests that dopamine receptors can also signal via other pathways. Thus there are a number of observations from studies on renal tissue indicating that dopamine receptors can couple to phospholipase C (PLC) (22, 23) and that the physiological effects of dopamine are abolished in the presence of specific protein kinase C (PKC) inhibitors (3, 7, 17). Direct evidence that dopamine activates PKC is, however, still lacking.
Several PKC isoforms have been identified. On the basis of their different activators they have been subdivided into three families, which are designated as classic, novel, and atypical PKC. Activation of PKC is generally associated with a translocation of the enzyme to the plasma membrane or to specific binding sites within the cell (14-16). The advent of confocal microscopy has greatly improved the possibilities to visualize cellular processes that involve transport of proteins. In the present study, performed on a proximal tubule-like cell line, we used this technique to examine the effect of dopamine, and the role of its receptors, on the localization of PKC isoforms from the three families.
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METHODS |
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Cells. LLCPK-1 is a renal epithelial cell line, which has several characteristics of proximal tubular cells. Cells were grown to confluence for 4-5 days in 10-cm dishes at 37°C in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; pH 7.6) supplemented with 10% fetal bovine serum (FBS), penicillin (100 mg/ml), and streptomycin (100 mg/ml).
Immunofluorescence.
Cells were harvested by trypsinization, replated on 12-mm coverslips
coated with fibronectin, and incubated for 1 h at 37°C in a
humidified atmosphere with 5% CO2 in modified
phosphate-buffered saline (mPBS; pH 7.4; in mM: 137 NaCl, 2.7 KCl, 80 Na2HPO4, 21 NaH2PO4,
2.4 CaCl2, and 0.4 MgSO4) supplemented with
0.1% bovine serum albumin (mPBS-BSA). Coverslips with adherent cells
were washed once in mPBS-BSA, incubated with drug or vehicle for the desired time, and fixed for 20 min in ice-cold 2% paraformaldehyde. Cells were washed with mPBS, blocked, and permeabilized in mPBS with
7% normal goat serum (NGS), 5% nonfat dry milk (NFDM), and 0.1%
saponin for 10 min at 37°C. The cells were incubated overnight at
4°C in mPBS containing 1.4% NGS, 1% NFDM, 0.1% saponin, and affinity-purified polyclonal antibodies directed against peptide sequences of PKC that reacted specifically with the -,
-,
-,
-, and
-isoforms of PKC. After being washed with mPBS containing 0.1% saponin, the cells were incubated for 1 h at room
temperature with secondary goat anti-rabbit Texas Red X antibody. The
cells were washed again and mounted in Prolong antifade. Each
experiment was repeated at least three times. Negative controls
included cells incubated with primary antibodies preabsorbed with their respective immunizing peptides and cells incubated with secondary antibodies alone.
Confocal microscopy. Confocal microscopy recordings were done with the use of a Zeiss LSM410 inverted confocal scanning laser microscope with excitation at 543 nm and long-pass detection at 570 nm. Optical sections through the center of the cells were used for localization of the immunofluorescent signal. Between 50 and 100 cells from each coverslip were analyzed. From those, five representative cells were recorded.
Immunoblotting studies.
Cell monolayers were washed twice with prewarmed (37°C) mPBS (pH 7.4)
and incubated for 10 min at room temperature with mPBS in the absence
or presence of dopamine (final concentration 105 M) or
phorbol 12,13-dibutyrate (PDBu; final concentration 10
6
M). Incubation was stopped by aspiration of the medium. The cell monolayer was covered with 2 ml of ice-cold homogenization buffer (20 mM Tris · HCl, containing a protease inhibitor cocktail tablet, final concentration in µg/ml: 60 antipain dihydrochloride, 10 bestatin, 20 chymostatin, 60 E-64, 10 leupeptin, 10 pepstatin, 60 phosphoramidon, 400 pefabloc SC, 200 EDTA disodium salt, 10 aprotinin,
pH 7.4). Cells were disrupted by three cycles of freezing (dry ice, 5 min) and thawing (37°C water bath, 5 min). To obtain cell cytosol and
membrane fractions, we centrifuged the lysate at 28,000 rpm (4°C, 60 min). After protein measurements were obtained, the supernatant
containing the cytosolic fraction was diluted in loading buffer (1%
SDS, 0.25 mM Tris · HCl, 10% glycerol, 5%
-mercaptoethanol), boiled for 3 min, and stored at
70°C. The pellet was resuspended in homogenization buffer plus 1% (vol/vol) Triton X-100, incubated at room temperature for 30 min, and centrifuged at 28,000 rpm (4°C, 60 min). The supernatant containing the membrane fraction was treated in the same way as that containing the cytosol extracts. Immediately after centrifugation, an aliquot of the cytosolic
or membrane fraction dissolved in homogenization buffer or
homogenization buffer plus 1% (vol/vol) Triton X-100, respectively, was used for the measurement of protein concentration. In both fractions protein concentration was determined according to Bradford (4). Cytosolic and membrane samples (stored in loading
buffer), containing equal amounts of protein (10 µg), were separated
by electrophoresis on SDS-polyacrylamide gels (8% acrylamide in the running gel), transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with PKC isoenzyme-specific antibodies. After being washed, membranes were incubated with peroxidase-conjugated anti-rabbit polyclonal antibodies and examined with an enhanced chemiluminescence method (ECL Plus Western blotting analysis system). The control, dopamine, and PDBu samples were always prepared together in a single experiment and run on the same gels. After immunodetection, the PVDF membranes were stained with amido black for 5 min at room
temperature and with constant shaking [0.1% (wt/vol) amido black-10B,
10% (vol/vol) methanol, 2% (vol/vol) acetic acid]. The membranes
were washed three to four times for 5 min in destaining solution [45%
(vol/vol) methanol, 7% (vol/vol) acetic acid] as a control for
protein loading.
Ouabain-sensitive rubidium uptake.
Ouabain-sensitive 86Rb+ uptake was used as an
index of Na+-K+-ATPase activity. Since the
intracellular sodium concentration is substantially lower than that
required for half-maximal activation of
Na+-K+-ATPase, any changes in intracellular
sodium will affect Na+-K+-ATPase activity. To
ensure that the recorded effect of dopamine on
86Rb+ uptake was due to a direct effect on
Na+-K+-ATPase, measurements were performed in
the presence of the sodium ionophore monensin (2 × 105 M, 20 min). Ouabain-sensitive
86Rb+ uptake was determined in the following
groups: 1) control (incubation with PBS only;
n = 19), 2) monensin (n = 12), 3) monensin plus the PKC inhibitor Ro-318220
(10
7 M, 10 min; n = 7), 4)
monensin plus dopamine (10
5 M, 3 min; n = 5), and 5) monensin plus Ro-318220 plus dopamine (n = 7). LLCPK-1 cells were washed twice with PBS and
incubated at 37°C with the different drugs for the indicated periods.
86Rb+ uptake, which can be used as an index of
K+ uptake (6), was initiated by addition of
0.1 ml/well PBS containing 25 µCi/ml 86Rb+.
After a 2-min incubation period, the reactions were stopped by
aspirating the medium and rinsing the cells three times with 1 ml/well
ice-cold PBS containing 5 × 10
3 M
BaCl2. Previous experiments have shown that
86Rb+ uptake is linear for at least 5 min.
Cells were then lysed twice with 0.55 ml of 1 × 10
3
M NaOH. Radioactivity in cell lysates was measured by liquid scintillation counting (LKB Wallac, Sweden). Protein content was determined by the Bradford method (4).
Chemicals. The following drugs were used: dopamine (Giludop, Hannover, Germany), Sch-23390 (Shering-Plough, Bloomfield, NJ), and S-sulpiride (Ravizza, Milan, Italy). Ro-318220 was kindly donated by Roche (London, UK). DMEM, FBS, trypsin, penicillin, and streptomycin were purchased from Life Technologies (Rockville, MD). Polyclonal rabbit antibodies against PKC isoforms and the blocking peptides were from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti rabbit-Texas Red X antibody and Prolong antifade were purchased from Molecular Probes (Eugene, OR). Protease inhibitor cocktail tablets were from Roche Diagnostics Scandinavia (Bromma, Sweden). 86Rb+, peroxidase-conjugated anti-rabbit polyclonal antibodies, the ECL Plus Western blotting analysis system, and Hybond-P-PVDF membranes were obtained from Amersham-Pharmacia Biotech (Uppsala, Sweden). All other chemicals of the highest purity available were purchased from Sigma Chemical (St. Louis, MO).
The following drugs were prepared as stock solutions: S-sulpiride was dissolved in methanol, monensin and PDBu were dissolved in ethanol, and U-73122, U-73343, and Ro-318220 were dissolved in DMSO. Further dilutions were performed in the corresponding incubation buffers.Results
Results from studies of PKC translocation with confocal imaging are shown in Figs. 1-3. In control LLCPK-1 cells, PKC-
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Translocation of PKC- was abolished by the D1 receptor
subtype antagonist Sch-23390 (10
5 M, 10 min) but not by
the D2 receptor subtype antagonist S-sulpiride (10
5 M, 10 min). Translocation was also abolished in
cells preincubated with the PLC inhibitor U-73122 (5 × 10
6 M, 10 min) but not in cells incubated with the same
concentration of the inactive analog U-73343 (Fig.
3).
PKC translocation was also studied with subcellular fractionation and
immunoblotting of the cytosolic and membrane fractions. It was found
that, under resting conditions, PKC- appeared mainly in the cytosol
fraction, whereas PKC-
was found mainly in the membrane fraction.
Densitometric analysis of PKC-
in the cytosol and membrane fractions
showed a significant increase in the ratio of membrane to cytosol
signal after 10-min incubation with dopamine (10
5 M). An
even more pronounced increase was observed in fractions from cells
incubated with phorbol ester (10
6 M). Similar increases
in the ratio of membrane to cytosol signal were found for PKC-
(Fig.
4).
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The functional importance of the dopamine-PKC pathway in LLCPK cells is
shown in Fig. 5. Dopamine inhibition of
the activity of Na+-K+-ATPase was measured as
ouabain-dependent 86Rb+ uptake and was
completely abolished by the PKC inhibitor Ro-318220 in sodium-loaded
cells (see METHODS for details). Previous results from our
and other laboratories have demonstrated that, in rat renal epithelial
cells, dopamine inhibits Na+-K+-ATPase through
a PKC-mediated pathway (3, 7). The serine residue 23 in
the NH2-terminal of the catalytic -subunit of rat Na+-K+-ATPase is a substrate for PKC
phosphorylation (2, 8, 12). So far there is no evidence
for a PKC phosphorylation motif in the
-subunit of pig
Na+-K+-ATPase. Hence, it is possible that, in
LLCPK-1 cells, PKC phosphorylates an intermediate protein that
regulates Na+-K+-ATPase activity.
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DISCUSSION |
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Intrarenal dopamine is a key modulator of sodium metabolism and
blood pressure (9). It is therefore of the utmost
importance to understand the intracellular pathways by which dopamine
can produce its physiological read out. Previous studies have provided circumstantial evidence that renal dopamine receptors may, in addition
to their well-known effects on cAMP and protein kinase A, also activate
PKC (10, 20, 21). We now provide direct evidence that
dopamine selectively activates the PKC - and
-isoforms in renal
epithelial cells.
The effects of dopamine on PKC translocation were mediated via activation of the D1 receptor subtype in LLCPK-1. We also found that dopamine-mediated PKC translocation was abolished in the presence of the PLC inhibitor U-73122. Together, these findings corroborate the observation by José and collaborators (9, 13, 22, 23) that renal D1 receptor subtype can activate PLC.
The results from immunocytochemical and subcellular fractionation
studies both demonstrated that dopamine can translocate PKC in renal
cells. They did, however, differ in some aspects. The immunoblot
studies indicated that, even in the absence of hormone stimulation,
PKC- was predominantly found in the membrane fraction. This is in
line with previous studies using subcellular fractionation (1,
11). On the contrary, the confocal recordings indicated that
PKC-
was randomly located intracellularly under basal conditions.
This discrepancy may be due to the fact that alterations in
membrane-bound PKC-
can occur during cell disruption and isolation
of subcellular fractions (19), a step that is avoided with
the use of the confocal imaging technique.
By using confocal imaging, we could detect a dopamine-induced
translocation of PKC- and PKC-
to the region of the plasma membrane within 20 s. It is generally assumed that activation of
intracellular messengers occurs within seconds or fraction of seconds.
In previous studies, performed with the use of subcellular fractionation, the effect of first messengers on PKC translocation was
recorded after at least 10 min of incubation (5, 18). Such
a long delay may include a series of alternative biochemical events.
The effects of phorbol esters were more pronounced than the effects of dopamine in the subcellular fractionation studies. If dopamine-induced translocation is mediated by endogenous diacylglycerol, it will be more readily reversed than translocation mediated by exogenous phorbol esters. Thus, because of the rapid metabolism of phospholipids and diacylglycerol, hormone-induced increases in PKC-membrane binding are more transient and less pronounced than the prolonged activation observed with phorbol ester treatment.
The PKC isoforms are subdivided into three families that are
differently regulated and designated as classic, atypical, and novel
PKCs. The classic PKCs are dependent on calcium for their activation,
while the novel PKCs are independent of calcium. Both PKC subfamilies
are activated by diacylglycerol (14-16). In the present study we chose PKC- and -
as representatives of the classic PKCs, PKC-
and -
as representatives of the novel PKCs, and PKC-
as a representative of the atypical PKCs. Dopamine was found to translocate both the classic
- and the novel
-isoform. Interestingly, dopamine differentially regulated two members of the
novel PKC family, namely, PKC-
and -
. The differential regulation of different PKC isoforms is an important topic, since there is a need
for drugs that can selectively inhibit a certain isoform.
In conclusion, dopamine-induced translocation of PKC toward the plasma membrane will allow PKC to act on integral membrane protein. This is an attractive hypothetical model to explain dopamine-PKC-mediated regulation of ion pumps, channels, and other transporters in the plasma membrane of renal cells.
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ACKNOWLEDGEMENTS |
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We thank Ann-Christine Eklöf, Mona Agrén, Eivor Zettergren, and Louise Gustafsson for expert experimental assistance.
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FOOTNOTES |
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This work was funded by grants from the Swedish Foundation for International Cooperation in Research and Higher Education (A. Aperia and S. Nowicki), the Swedish Medical Research Council (Project 03644, 013114) (A. Aperia and H. Brismar), and the Märta and Gunnar V. Philipson Foundation (H. Brismar).
Address for reprint requests and other correspondence: H. Brismar, Astrid Lindgren Children's Hospital, Q2:09, Karolinska Hospital, S-171 76 Stockholm, Sweden (E-mail: hjalmar.brismar{at}ks.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 April 2000; accepted in final form 14 June 2000.
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