From the Department of Molecular Medicine, Karolinska Institute,
Karolinska Hospital, S-171 76 Stockholm, Sweden; the
Department of Pharmacological and Pharmaceutical
Sciences, College of Pharmacy, University of Houston, Houston, Texas
77204; the § Department of Medicine, University of Chicago,
Chicago, Illinois 60637, and the ¶ Division de Néphrologie,
Hôpital Cantonal Universitaire, CH-1211 Geneva 14, Switzerland
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ABSTRACT |
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Inhibition of
Na+,K+-ATPase activity by dopamine is an
important mechanism by which renal tubules modulate urine sodium
excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of
Na+,K+-ATPase activity in response to dopamine
is associated with endocytosis of its - and
-subunits, an effect
that is protein kinase C-dependent. In this study we used
isolated proximal tubule cells and a cell line derived from opossum
kidney and demonstrate that dopamine-induced endocytosis of
Na+,K+-ATPase and inhibition of its activity
were accompanied by phosphorylation of the
-subunit. Inhibition of
both the enzyme activity and its phosphorylation were blocked by the
protein kinase C inhibitor bisindolylmaleimide. The early time
dependence of these processes suggests a causal link between
phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the
-subunit was no longer
phosphorylated, whereas enzyme activity remained inhibited due to its
removal from the plasma membrane. Dephosphorylation occurred in the
late endosomal compartment. To further examine whether phosphorylation
was a prerequisite for subunit endocytosis, we used the opossum kidney
cell line transfected with the rodent
-subunit cDNA. Treatment
of this cell line with dopamine resulted in phosphorylation and
endocytosis of the
-subunit with a concomitant decrease in
Na+,K+-ATPase activity. In contrast, none of
these effects were observed in cells transfected with the rodent
-subunit that lacks the putative protein kinase C-phosphorylation
sites (Ser11 and Ser18). Our results support
the hypothesis that protein kinase C-dependent phosphorylation of the
-subunit is essential for
Na+,K+-ATPase endocytosis and that both events
are responsible for the decreased enzyme activity in response to
dopamine.
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INTRODUCTION |
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The natriuretic effect of dopamine (DA)1 depends on its ability to increase the glomerular filtration rate and/or to modulate directly tubular sodium reabsorption (1-3). Changes in vectorial transport of sodium induced by DA in renal tubules are largely mediated by inhibition of Na+,K+-ATPase (4, 5) and Na+/H+-exchanger activity (6). At the cellular level, DA triggers a specific signaling cascade that ultimately activates protein kinase C (PKC) (7), a process postulated to be responsible for the decreased Na+,K+-ATPase activity.
Activators of PKC, such as phorbol esters and diacylglycerol analogs,
decrease Na+,K+-ATPase activity in isolated rat
renal PCT segments (7, 8) as well as the vectorial transport of sodium
by isolated perfused PCTs (9). In isolated renal PCT cells, another
activator of PKC,
L-1-oleoyl-2-acetoyl-sn,n-acetoyl-glycerol,
decreased Na+,K+-ATPase activity determined as
the rate of ouabain-sensitive oxygen consumption (10). In a renal cell
line derived from opossum kidney (OK cells), but not from pig kidney
(LLC-PK1 cells), incubation with phorbol esters resulted in
phosphorylation of the Na+,K+-ATPase
-subunit and inhibition of its activity (11). However, stimulation
of Na+,K+-ATPase by phorbol esters has also
been reported (12, 13). Although phosphorylation of the
-subunit by
PKC in a cell-free preparation was associated with a decrease in
enzymatic activity (14-16), it is not clear whether this effect occurs
in intact cells in response to phorbol esters (11, 17).
DA is produced locally in renal PCT cells (18-20) where its synthesis
is regulated physiologically during ingestion of a high salt diet (21).
Contrary to the diverse effects of PKC stimulation by phorbol esters
and diacylglycerols on Na+,K+-ATPase activity,
there is a consensus on the inhibitory action of DA on the enzyme.
Moreover, we have recently demonstrated that inhibition of PCT
Na+,K+-ATPase activity by DA is associated with
endocytosis of its - and
-subunits into early- (EE) and late (LE)
endosomes via a clathrin-coated vesicle (CCV)-dependent
mechanism (22). Nevertheless, despite the information gained during the
last few years on the regulation of
Na+,K+-ATPase activity, it is not known whether
inhibition of enzyme activity in intact cells depends on the
phosphorylation of the catalytic subunit, or whether such
phosphorylation is necessary for subunit endocytosis in response to a
physiologic agonist such as DA.
In the present study, using intact renal PCT cells metabolically
labeled with [32P]orthophosphate, we have examined
whether dopamine phosphorylates the
Na+,K+-ATPase -subunit and whether this
effect is responsible for the decreased enzymatic activity and subunit
endocytosis.
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EXPERIMENTAL PROCEDURES |
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Materials--
The cAMP analog Rp-cAMPS was obtained from
BioLog, Bremen, Germany. Bisindolylmaleimide was purchased from
Calbiochem, San Diego, CA. All other chemicals were from Sigma. A
monoclonal antibody, kindly provided by Dr. M. Caplan (Yale
University), was used against the Na+,K+-ATPase
-subunit (antibody A, which recognizes only the N-terminal first
five residues of the
-subunit). Immunoprecipitation of the
Na+,K+-ATPase in the phosphorylation
experiments and Western blots were performed using a polyclonal
antibody (B) raised against the rat Na+,K+-ATPase
-subunit (23). The identity of
EE was determined with a polyclonal antibody raised against a
rab5 synthetic peptide (Santa Cruz Biotechnology Inc., Santa
Cruz, CA). The late endosome fraction was identified with a
mannose-6-phosphate receptor antibody (courtesy of Dr. B. Hoflack, EMBL, Heidelberg, Germany).
Preparation of PCT Cells--
PCT cells were prepared as
described before (10, 24). Briefly, male Sprague-Dawley rats (BK
Universal, Sollentuna, Sweden) weighing between 150-200 g were used.
After the kidneys were removed and the cortex isolated, the tissue was
minced on ice to a paste-like consistency. The cortical minceate was
incubated with 0.7 mg/ml collagenase (Type I, Sigma) in 50 ml of
Hanks' medium (Life Technologies,Inc., Gaithersburg, MD). The
incubation was carried out at 37 °C for 60 min, where the solution
was continuously exposed to 95% O2, 5% CO2
and was terminated by placing the tissue on ice and pouring through
graded sieves (180-75-53-38 µM pore size) to obtain a cell suspension. It has been reported that phorbol esters regulate Na,K-ATPase differently depending on whether the tissue has been continously oxygenated during its preparation and incubation with the
PKC activator. Although this effect was not reported to be involved in
the DA response, we had taken the precaution to incubate cells in
oxygenated solutions in all steps until the tissue was disrupted to
immunoprecipitate the -subunit or for preparation of BLM, EE, and
LE.
Cell Culture and Transfection--
The expression vector pCMV
containing the rodent Na+,K+-ATPase
1-subunit cDNA was obtained from PharMingen (San
Diego, CA). Preparation of the expression vector (myc/1.32) that
encodes a shortened mutant of the
1-subunit was as
described by Shanbaky and Pressley (25). This vector expresses a rodent
-subunit in which the first 31 amino acids of the nascent
polypeptide are replaced by an initiation methionine and a sequence of
10 amino acids (EQKLISEEDL) from the human c-myc oncogene product.
Transfection of OK cells and selection of ouabain-resistant colonies
were performed as described previously (26).
Determination of Na+,K+-ATPase
Activity--
Cells were incubated in modified Hanks' medium in the
presence or absence of 1 µM DA at room temperature for
different periods of time. The incubation was terminated by placing the
samples on ice. Cell aliquots (approximately 10-20 µg of protein)
were transferred to the Na+,K+-ATPase assay
medium (final volume of 100 µl) containing in mM NaCl,
50; KCl, 5; MgCl2, 10; EGTA, 1; Tris-HCl, 50;
Na2ATP, 7 (Calbiochem, La Jolla, CA); and
[-32P]ATP (NEN Life Science Products, specific
activity 3000 Ci/mmol) in tracer amounts (3.3 nCi/µl).
Na+,K+-ATPase activity was determined in
permeabilized cells as described before (27, 28).
Phosphorylation and Immunoprecipitation of
Na+,K+-ATPase in Intact Cells--
Renal PCT
cells (4-6 mg of protein/3 ml) were labeled during 2 h at
32 °C in a buffer containing (in mM) NaCl, 120; KCl, 5; NaHCO3, 4; CaCl2, 1; MgSO4, 1;
NaH2PO4, 0.2; Na2HPO4,
0.15; glucose, 5; lactate, 10; pyruvate, 1; HEPES, 20; and 1% bovine
serum albumin, pH 7.45, with the addition of 250 µCi/ml
[32P]orthophosphate (NEN Life Science Products). OK cells
(2.0-2.5 mg of protein/dish) were labeled in the same buffer (2.5 ml/dish) containing 100 µCi/ml [32P]orthophosphate for
2.5 h at 37 °C. All incubations with different agonists were
performed at room temperature. The incubation was terminated by
removing the medium and adding cold immunoprecipitation buffer.
Immunoprecipitation of the Na+,K+-ATPase
-subunit was performed as described by Carranza et al. (12). Briefly, aliquots (200 µg of protein) were incubated overnight at 4 °C with 50 µl of rabbit polyclonal antibody and with the simultaneous addition of excess protein A-Sepharose beads (Pharmacia Biotech Inc., Uppsala, Sweden). Samples were analyzed by SDS-PAGE using
the Laemmli buffer system (29). Proteins were transferred to
polyvinylidene difluoride membranes (Immobilon-P, Millipore) and
subjected to autoradiography. Phosphoproteins were also analyzed using
a phosphoimager (Fuji, Japan), and quantitation was performed as
described (22).
Preparation of Endosomes-- Cells were labeled with 32P as detailed above. Cells in suspension (1.5 mg of protein/ml) were incubated under different protocols at room temperature. Incubation was terminated by transferring the samples to ice and adding cold homogenization buffer containing 250 mM sucrose and 3 mM imidazole, 2 mM EGTA, 10 mM NaF, 30 mM Na4O7P2, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 4 µg/ml aprotinin, pH 7.4. Cells were gently homogenized (15-20 strokes) to minimize damage of the endosomes, using a Dounce homogenizer, and the samples were subjected to a brief (5 min) centrifugation (4 °C, 3,000 × g). Endosomes were fractionated on a flotation gradient as described (22), using essentially the technique of Gorvel et al. (30).
Preparation of Basolateral Plasma Membranes-- After separation of EE and LE, another fraction (500 µl) was collected at the 16 and 42% sucrose interface corresponding to cell ghosts, mitochondria, and plasma membranes. Basolateral membranes (BLM) were further purified according to Hammond et al. (31), using a Percoll gradient. Briefly, the collected material was diluted by adding 500 µl of imidazole (3 mM, pH 7.4) buffer containing protease inhibitors (final sucrose concentration 25-26% w/w), and spun at 20,000 × g for 20 min. The yellow layer was resuspended again in the supernatant (carefully removed from the brown pellet containing mitochondria and cell ghosts) and centrifuged at 48,000 × g for 30 min. The supernatant was discarded, and the pellet was resuspended in 1 ml of buffer (300 mM mannitol and 12 mM HEPES, pH 7.6, adjusted with Tris) by gentle pipetting. To form a Percoll gradient, 0.19 g of undiluted Percoll (Pharmacia Biotech Inc.) was added to a 1-ml suspension (0.2-1 mg of protein). The suspension was gently mixed and centrifuged at 48,000 × g for 30 min, and the ring of BLM was collected.
Miscellaneous--
Protein content was determined
according to Bradford (32). Western blots were developed with
an ECL (Amersham, UK) detection kit. Scans were performed using a
ScanJet IIc scanner (Hewlett Packard, Palo Alto, CA). Quantitation of
the phosphorylated Na+,K+-ATPase -subunit
was performed using a Fuji Bas 1000 Bio-imaging analyzer (Fuji, Japan),
and the data (arbitrary units) were analyzed using Tina 2.07 ray test
software (Isotopenmessyeräte GmbH, Staulenhardt, Germany).
Statistics-- Comparison between two experimental groups were made by the unpaired Student's t test. For multiple comparisons, one-way ANOVA with Sheffe's correction was used. p < 0.05 was considered significant.
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RESULTS |
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In this study we sought to determine whether inhibition of
Na+,K+-ATPase activity and endocytosis was
associated with phosphorylation of the -subunit. In isolated renal
PCT cells, incubation with DA decreased
Na+,K+-ATPase activity (nmol Pi/mg
prot/min, vehicle: 112 ± 8 versus DA, 1 µM: 60 ± 2, n = 4, p < 0.05), and this effect was blocked by PKC
inhibitors (7, 8). Intact renal PCT cells were metabolically labeled
with 32P and thereafter incubated for 2.5 min at room
temperature with or without DA (Fig.
1A). The
-subunit was
immunoprecipitated, separated by SDS-PAGE, and transferred to
polyvinylidene difluoride membranes. In every experiment the amount of
radioactivity (autoradiography or phosphoimager) incorporated into the
-subunit was corrected for the amount of protein present (Western
blot), and the quantitative data are shown as percent of control at the
bottom of each panel. DA increased the state of
phosphorylation (to ~165% of control) of the
-subunit, as
illustrated in Fig. 1A. This increased phosphorylation was
inhibited by bisindolylmaleimide, a specific PKC inhibitor, but not by
a cAMP-dependent protein kinase (PKA) inhibitor, suggesting that phosphorylation of the
-subunit induced by DA is mediated by
PKC. Neither inhibitor affected the state of
-subunit
phosphorylation in non-stimulated PCT cells.
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Phosphorylation of the Na+,K+-ATPase
-subunit by DA was time-dependent (Fig. 1B).
It increased significantly after 1 min and was maximal at 2.5 min
(178% of control), whereas it was no longer evident at 10 min.
However, while the initial (1.0 and 2.5 min) increase in
-subunit
phosphorylation corresponded to the decrease in enzyme activity, this
correlation was no longer present at 10 min, i.e. enzyme
activity remained inhibited (percent of control, 60 ± 3, p < 0.05), whereas phosphorylation was similar to that of control cells.
To determine whether the Na+,K+-ATPase has been
dephosphorylated, we examined the effect of DA in the presence of a
phosphatase inhibitor, 1 µM okadaic acid (OKD) (Fig.
1C). Basal phosphorylation (resting condition = control, C) was moderately higher (~1.5-fold) in the OKD-treated cells. As hypothesized, in OKD-treated cells, DA (10 min) did increase the state of -subunit phosphorylation, suggesting
that at this time period it had been dephosphorylated by the action of
protein phosphatases.
Because after 10 min the -subunit has been dephosphorylated yet the
decreased enzymatic activity persisted, it is possible that the
dephosphorylated
-subunits no longer reside in the plasma membrane.
To test this hypothesis, we evaluated the state of phosphorylation of
the
-subunit in BLM and in LE. In BLM prepared from cells that have
been preincubated with DA for 10 min, the state of phosphorylation of
the immunoprecipitated
-subunit remained unchanged regardless of
whether the PCT cells were previously treated with 1 µM
OKD or not, whereas it increased significantly in LE. This was evident, however, only if PCT cells had been preincubated with OKD
(phosphorylation (percent of control): 104 ± 6 without OKD
versus 126 ± 5 OKD-treated cells, n = 3), supporting the notion that the
-subunit is dephosphorylated in
LE.
In BLM prepared from cells that have been preincubated with DA for 2.5 min (Fig. 2A, left
panel) the state of phosphorylation of the immunoprecipitated
-subunit was significantly increased, regardless of whether the PCT
cells were previously treated with 1 µM OKD or not
(phosphorylation (percent of control): 130 ± 2.0 without OKD,
131 ± 1.2 with OKD). These results suggest that the phosphorylated subunits (2.5 min) in the BLM are not affected by
protein phosphatases
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Although the results described above support the concept that in
response to DA the Na+,K+-ATPase -subunits
are phosphorylated in the plasma membrane and then internalized and
dephosphorylated in LE, the link between these two processes
(i.e. whether phosphorylation is a requisite for
endocytosis) is not clear. Therefore, we next used an epithelial cell
line from OK transfected with the rat
Na+,K+-ATPase
-subunit cDNA carrying a
deletion in the nascent 28 amino acids in which Ser11 and
Ser18, the putative phosphorylation sites for PKC (33, 34),
are absent. OK cells (non-transfected) behaved similarly to native PCT
cells in their response to DA: DA decreased the
Na+,K+-ATPase activity, and this inhibition was
associated with endocytosis of the
-subunit. Thus, they constitute a
useful model to study the mechanisms of action of DA.
The relative expression of Na+,K+-ATPase
-subunits in transfected OK cells was determined using antibody A
raised against the first five amino acids of the
-subunit (which
should not recognize the truncated form, OK
rat-t) and
compared with antibody B, raised against the holoenzyme. While antibody
B recognized the Na+,K+-ATPase
-subunit from
both the full-length (OK
rat) and OK
rat-t
cells, antibody A detected only a slight presence of
Na+,K+-ATPase
-subunits in
OK
rat-t (Fig.
3A), indicating that in
OK
rat-t most of the
-subunits correspond to the
truncated form.
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We next evaluated the Na+,K+-ATPase activity
and its response to DA in OKrat and
OK
rat-t (Fig. 3B). While basal
Na+,K+-ATPase activity was similar in both
groups of cells and comparable with that in earlier reports (13, 26),
incubation with DA resulted in a significant decrease in
Na+,K+-ATPase activity from
OK
rat (p < 0.01), but not from
OK
rat-t (p = 0.567). The inhibitory
effect of DA in OK
rat was abolished by coincubation with
a PKC inhibitor, bisindolylmaleimide (percent of control: 99.3 ± 7, n = 3). We further examined whether this inhibition
was associated with phosphorylation of the
-subunit (Fig.
3C). 1 µM DA (3 min; room temperature)
increased the state of phosphorylation of the
-subunit in
OK
rat but not in OK
rat-t cells.
Last, to determine whether phosphorylation of the -subunit was
necessary for endocytosis, early and late endosomes were prepared from
OK
rat and OK
rat-t cells incubated with DA
(Fig. 4). DA stimulated the incorporation of
-subunits into EE and LE from OK
rat, and this
effect was blocked by bisindolylmaleimide (percent of control, EE:
105 ± 12, n = 3; and LE: 96 ± 17, n = 3) or calphostin C (percent of control, EE: 98 ± 13, n = 3; and LE: 86 ± 11, n = 3). However, DA did not increase the incorporation of
-subunits from OK
rat-t.
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DISCUSSION |
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In this report we have demonstrated that DA treatment of both
isolated proximal tubule cells and OK cells transfected with the rodent
-subunit leads to inhibition of
Na+,K+-ATPase activity and phosphorylation and
endocytosis of the
-subunit. In contrast, when the DA effect was
examined in OK cells expressing the
Na+,K+-ATPase
-subunit isoform in which the
putative PKC-phosphorylation sites were removed, DA-treatment neither
inhibited the enzyme activity nor induced any significant
phosphorylation or endocytosis of the
-subunit. These observations
strongly suggest a causal link between PKC-dependent
phosphorylation of amino acids at the
-subunit N terminus and
Na+,K+-ATPase inhibition and endocytosis in
response to a physiological agonist.
Inhibition of Na+,K+-ATPase activity by DA in
renal PCT involves the sequential activation of arachidonic acid,
20-HETE, and PKC (35). Although cAMP stimulation has been suggested to
contribute to the action of DA (36, 37), it is unlikely that it would be directly involved in Na+,K+-ATPase
regulation (phosphorylation of the -subunit in renal PCT cells)
because increased cAMP in this segment does not inhibit (7) but is
rather associated with stimulation of
Na+,K+-ATPase activity (38). Accordingly, in
this study phosphorylation of Na+,K+-ATPase
-subunits was blocked by PKC-, but not cAMP-K, inhibition. Our
observation differs from that reported by Beguin et al.
(39), perhaps reflecting differences in the preparations used. We
examined isolated PCT cells, where DA is synthetized and
physiologically regulates Na+,K+-ATPase
activity, whereas Beguin et al. (39) used a reconstituted system in which the receptor (human dopaminergic DA1A) and
the target (Bufo marinus
Na+,K+-ATPase
-subunit) were expressed in a
cell line (COS-7) that normally does not express this regulatory
system.
The present results suggest that inhibition of total cell
Na+,K+-ATPase activity is initially
accomplished by phosphorylation of the -subunit and that the
activity remains decreased because the inhibited units no longer reside
in the plasma membrane. Once the
-subunits become phosphorylated,
they are internalized by sequential translocation into CCV, EE, and
finally LE, where they may be dephosphorylated. Because in CCV and EE
the increased Na+,K+-ATPase
-subunit
abundance is not associated with increased enzymatic activity (22), it
is unlikely that it could have been dephosphorylated in these
compartments.
Endocytosis of the -subunit requires phosphorylation by PKC because
mutants lacking the PKC phosphorylation sites do not internalize in
response to dopamine. The mechanisms by which membrane proteins are
internalized have been studied extensively, and the consensus sequences
of interaction with adaptins have been described. The amino acids that
were deleted by the truncation do not bear any homology with known
endocytic sequences. Thus, it appears that it is the lack of
phosphorylation rather than impairment of a putative
Na+,K+-ATPase
-subunit-adaptin
interaction that precludes the dopamine-dependent endocytosis in
cells transfected with the truncated isoform. However, Beron et
al. (40), using phorbol esters to stimulate PKC, have recently
postulated that PKC activation is not necessary for
Na+,K+-ATPase
-subunit endocytosis in A6
cells. These observations, however, are not comparable with the effect
of DA in PCT cells. Phorbol esters increased fluid phase endocytosis,
and the signal could thus have occurred at any other target in the
plasma membrane. In PCT cells, by contrast, DA selectively internalized
the Na+,K+-ATPase
/
-subunits while the
distribution of other basolateral membrane markers such as the glucose
transporter GLUT-2 and the mannose 6-phosphate receptor remained
unchanged (22). Incubation of PCT cells with phorbol esters, on the
other hand, resulted in internalization of the GLUT-2 transporter as
well as the Na+,K+-ATPase
-subunit and also
induced a significant change in the actin cytoskeleton
organization.2 Thus,
endocytosis and phosphorylation of the
Na+,K+-ATPase
-subunit, as well as
inhibition of its activity in response to DA (Ref. 22 and present
study), require activation of PKC.
It has also been reported that phorbol esters stimulate
Na+,K+-ATPase activity (12, 13, 26) and that
this effect is accompanied by phosphorylation of the -subunit (12).
Thus, although both effects (that of DA and of phorbol esters) share a
common target, PKC, they are clearly different. For example,
stimulation by phorbol esters of Na+,K+-ATPase
activity and phosphorylation of the
-subunit were significant after
15 min of incubation (12). In contrast, the effect of DA occurs already
at 1 min, and after 10 min, the
-subunits are no longer
phosphorylated and, in addition, they no longer reside in the plasma
membrane. Finally, another reason why the effects of phorbol esters and
DA are different in nature may be that the effect of DA on
Na+,K+-ATPase activity is mediated via a PKC
isoform that can be activated by arachidonic acid metabolism and
generation (in the PCT) of the cytochrome P-450 metabolite, 20-HETE, an
eicosanoid that activates PKC (41). The action of DA might therefore
involve an atypical PKC isoform that is not responsive to phorbol
esters (42) but whose activation is rather dependent on membrane
lipids.
In conclusion, while in intact cells the use of phorbol esters has not
been proved to be an efficient probe to demostrate the relationship
between phosphorylation of the Na+,K+-ATPase
-subunit and inhibition of its activity (17), by using an agonist
such as dopamine in cells where it is produced and exerts its
physiologic action it was possible to demonstrate that phosphorylation
of the
-subunit is associated with inhibition of
Na+,K+-ATPase activity and that this step is
required for subunit endocytosis.
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FOOTNOTES |
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* This study was supported in part by Grants 10860 (to A. M. B) and 09890 (to P.-O. B) from the Swedish Medical Research Council, DK 52273 from the National Institutes of Health (to C. H. P), and 9650139N from the American Heart Association (C. H. P) and by the Swedish Natural Science Research Council (to A. I. K.).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.
To whom correspondence should be addressed: Rolf Luft Centrum
L6B:01, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel.: 46 8 517-75727, Fax: 46-8-517-73658, E-mail:
alejan{at}enk.ks.se.
1 The abbreviations used are: DA, dopamine; PKC, protein kinase C; PCT, proximal convoluted tubules; EE, early endosomes; LE, late endosomes; BLM, basolateral plasma membrane; CCV, clathrin-coated vesicles; OKD, okadaic acid; 20-HETE, 20-hydroxyeicosatetraenoic acid; OK cells, opossum kidney cells.
2 A. V. Chibalin, A. I. Katz, and A. M. Bertorello, unpublished observations.
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REFERENCES |
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