Receptor-mediated inhibition of renal
Na+-K+-ATPase
is associated with endocytosis of its
- and
-subunits
Alexander V.
Chibalin,
Adrian I.
Katz,
Per-Olof
Berggren, and
Alejandro M.
Bertorello
Department of Molecular Medicine, Karolinska Institutet, Rolf Luft
Center for Diabetes Research, Karolinska Hospital, 171 76 Stockholm,
Sweden; and Department of Medicine, The University of Chicago,
Chicago, Illinois 60637
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ABSTRACT |
The mechanisms involved in receptor-mediated inhibition of
Na+-K+-ATPase
remain poorly understood. In this study, we evaluate whether inhibition
of proximal tubule
Na+-K+-ATPase
activity by dopamine is linked to its removal from the plasma membrane
and internalization into defined intracellular compartments.
Clathrin-coated vesicles were isolated by sucrose gradient
centrifugation and negative lectin selection, and early and late
endosomes were separated on a flotation gradient. Inhibition of
Na+-K+-ATPase
activity by dopamine, in contrast to its inhibition by ouabain, was
accompanied by a sequential increase in the abundance of the
-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation
between these organelles. A similar pattern was found for the
-subunit. The increased incorporation of both subunits in all
compartments was blocked by calphostin C. The results demonstrate that
the dopamine-induced decrease in
Na+-K+-ATPase
activity in proximal tubules is associated with internalization of its
- and
-subunits into early and late endosomes via a
clathrin-dependent pathway and that this process is protein kinase C
dependent. The presence of
Na+-K+-ATPase
subunits in endosomes suggests that these compartments may constitute
normal traffic reservoirs during pump degradation and/or
synthesis.
dopamine; proximal tubules; clathrin vesicles; endosomes; protein
kinase C; actin-microtubule cytoskeleton
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INTRODUCTION |
IN RENAL EPITHELIAL CELLS (7, 28, 31) and medium spiny
neurons (6), dopamine inhibits
Na+-K+-ATPase
activity by mechanisms that include binding to dopaminergic receptors
(4, 6) and the sequential activation and integration of several
intracellular signaling networks, which ultimately activate protein
kinases (reviewed in Ref. 7). Studies using both cell-free preparations
(5, 10) and intact cells (8, 12, 23) suggest that phosphorylation of
Na+-K+-ATPase
-subunit by protein kinase A (PKA) or protein kinase C (PKC) may be
an important step in this regulation, although how phosphorylation
leads to decreased enzyme activity in intact cells is unclear.
Inhibition could either occur through a kinase-mediated conformational
change of
Na+-K+
pump molecules that may result in a change of affinity for one or more
of its substrates or, alternatively, such conformational change could
serve as a triggering signal for the pump's internalization and
eventual degradation in organelles within the cell. For example, inhibition of
Na+-K+-ATPase
activity by phorbol esters in Xenopus
oocytes is associated with a decreased number of pump units in the
plasma membrane (32). Whether phosphorylation and endocytosis in
response to dopamine take place in epithelial cells and, if they do,
whether they occur independently or as part of a common mechanism are
unknown.
The purpose of this study was to further examine the cellular
mechanisms of
Na+-K+
pump inhibition in renal proximal tubules and specifically whether such
inhibition by dopamine involves internalization of its subunits into
the cell.
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EXPERIMENTAL PROCEDURES |
Preparation of proximal convoluted tubule cells.
Proximal convoluted tubule (PCT) cells were prepared as described
previously (2, 31). Briefly, male Sprague-Dawley rats (BK Universal,
Sollentuna, Sweden) weighing between 150 and 200 g were used. After the
kidneys were removed and the cortex was isolated, the tissue was minced
on ice to a paste-like consistency. The cortical minceate was incubated
with 0.075 g/100 ml collagenase (type I, Sigma) in 50 ml Hanks' medium
(Life Technologies, Gaithersburg, MD). The incubation was carried out
at 37°C for 60 min. The solution was continously exposed to 95%
O2-5%
CO2. The incubation was terminated by placing the tissue on ice and pouring it through graded sieves (180, 75, 53, and 38 µm in pore size) to obtain a cell suspension. The PCT
cells were washed three to four times by centrifugation at 100 g for 4 min to separate the remaining
blood cells and traces of collagenase. The cells were kept on ice until
studied and were used immediately after preparation. Cells were
resuspended in Hanks' medium to yield a protein concentration of
~1-3 mg/ml.
Cell fractionation using sucrose density gradients.
Renal proximal tubules (~0.7-0.9 mg protein) were suspended in
400 µl homogenization buffer containing (in mM) 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 MgCl2, 2 EDTA, 250 sucrose, and 1 phenylmethylsulfonyl fluoride (PMSF) as well as
0.5 µg/ml each of leupeptin, pepstatin, aprotinin, and
antipain. The tissue was homogenized (15-20 strokes) in a
Dounce homogenizer. The homogenate was centrifuged (1,000 g for 5 min), and the pellet was
resuspended in 200 µl homogenization buffer and centrifuged again, as
above. Supernatants from the two centrifugations were pooled, and this
postnuclear supernatant (PNS) was layered on a 4-ml linear sucrose
gradient (1.074-1.242 g/ml in 20 mM HEPES, 2 mM EDTA, and 1 mM
PMSF) and was centrifuged in a SW50.1 Beckman rotor at 110,000 g and 4°C for 18 h. Fractions (290 µl each) were collected from the top of the gradient, and their
density was determined by refractometry.
Preparation of endosomes.
Endosomes were fractionated on a flotation gradient, using essentially
the technique described by Gorvel et al. (14). Cells in suspension (1.5 mg protein/ml) were incubated with dopamine (1 µM) or vehicle.
Incubation was terminated by transferring the samples to ice and by
addition of cold homogenization buffer containing 250 mM sucrose and 3 mM imidazole, pH 7.4. The 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).
The PNS was adjusted to 40.6% sucrose and loaded (1.5 ml) at the
bottom of a 5.0-ml centrifuge tube, to which were added sequentially
16% sucrose (1.5 ml) in 3 mM imidazole and 0.5 mM EDTA in
2H2O (Cambridge
Isotope Laboratories, Andover, MA), 10% sucrose in the same buffer (1 ml), and finally homogenization buffer (1 ml). The samples were
centrifuged (1 h; 110,000 g) in a
Beckmann SW 50.1 rotor. Early endosomes were collected at the
homogenization buffer and 10% sucrose interface, whereas the late
endosomes were collected at the 10 and 16% sucrose
interface.
Preparation of clathrin-coated vesicles.
Isolation of clathrin-coated vesicles (CCV) was performed as described
by Hammond and Verroust (17). Briefly, after incubation with dopamine,
PCT cells were homogenized using a Potter homogenizer (3 strokes, 30 s)
in 1 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.5 mM MgCl2, 0.1 M
2-(N-morpholino)ethanesulfonic acid, and 0.2 mg/ml
NaN3, titrated to pH 6.5 with
NaOH. The homogenate was centrifuged at 15,000 g for 15 min, and the supernatant was further centrifuged at 85,000 g for 1 h. The pellet was resuspended in the same buffer and applied to a
discontinuous sucrose gradient (wt/vol): 60, 50, 40, 10, and 5%.
Samples were centrifuged at 80,000 g
for 75 min, collected from the 10-40% interface, and then washed
in homogenization buffer and pelleted at 85,000 g for 1 h. Wheat germ agglutinin was
added to a concentration of 1 mg/10 mg protein and incubated overnight
at 4°C. The agglutinated material was sedimented at 20,000 g for 15 min. The resultant CCV
preparation was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and subjected to either silver staining or
Western blotting. It is important to note that although this method
will isolate all CCV, it may also leave inside-out vesicles in the
supernatant.
Determination of
Na+-K+-ATPase
activity.
Na+-K+-ATPase
activity was determined as described before (5). Briefly, aliquots
(~3 µg protein) were transferred to the Na+-K+-ATPase
assay medium (final volume 100 µl) containing (in mM) 50 NaCl, 5 KCl, 10 MgCl2, 1 EGTA,
50 tris- (hydroxymethyl)aminomethane · HCl,
7 Na2ATP (Calbiochem, La Jolla,
CA), and [
-32P]ATP
(Amersham; sp act 3,000 Ci/mmol) in tracer amounts (3.3 nCi/µl).
Cells were transiently exposed to a thermic shock (10 min at
20°C) to render membranes permeable to ATP. The samples were
then incubated at 37°C for 15 min, and the reaction was terminated by rapid cooling to 4°C and addition of 700 µl trichloroacetic acid-charcoal (5%/10% wt/vol) suspension. After separation of the
charcoal phase (12,000 g for 5 min)
containing the unhydrolyzed nucleotide, the liberated
32P was counted in an aliquot (200 µl) from the supernatant.
Na+-K+-ATPase
activity was calculated as the difference between test samples (total
ATPase activity) and samples assayed in the same medium but devoid of
Na+ and
K+ and in the presence of 1 mM
ouabain (ouabain-insensitive ATPase activity).
Miscellaneous.
Identification of clathrin heavy chain was performed using a monoclonal
antibody (Harlan Sera-Lab, Sussex, UK). The presence of glucose
transporter GLUT-2 was evaluated with a polyclonal antibody
(Biogenesis, Stinsford Road Poole, UK). The identity of early endosomes
was determined with a polyclonal antibody raised against a
rab5 synthetic peptide corresponding
to amino acids 193-211 within the carboxy terminal of human
rab5 (Santa Cruz Biotechnology, Santa
Cruz, CA). The late endosome fraction was identified with a mannose
6-phosphate receptor (MPR) antibody (courtesy of Dr. B. Hoflack, EMBL,
Heidelberg, Germany). Identification of the
Na+-K+-ATPase
- and
-subunits was performed using monoclonal antibodies raised
against the
1- and
1-subunits (25) (courtesy of
Dr. M. Caplan, Yale University, New Haven, CT). Proteins were analyzed by SDS-PAGE (7.5-15%) using the Laemmli buffer system (21). Protein content was determined according to Bradford (9). Western blots
were developed with an ECL detection kit (Amersham, UK), as recommended
by the manufacturer. Measurements were performed using multiple
exposures of autoradiograms to ensure that signals were within the
linear range of the film. Scans were performed using a Scan Jet IIc
scanner (Hewlett Packard, Palo Alto, CA). Each band was scanned two
times in different regions, the scans were averaged, and the area of
the peak minus the background (in arbitrary units) was quantitated.
Statistics.
Comparisons between two experimental groups were made by the unpaired
Student's t-test. For multiple
comparisons, one-way analysis of variance with Sheffé's
correction was used. P < 0.05 was
considered significant.
 |
RESULTS |
In this study, we used rat renal PCT cells to assess the mechanisms
involved in the rapid inhibition of
Na+-K+-ATPase
activity by dopamine. Cell fractionation applying linear sucrose
gradients has been used successfully to determine the subcellular
localization of
Na+-K+-ATPase
-subunit during Xenopus oocyte
maturation (27, 30). To identify the distribution pattern of
Na+-K+-ATPase,
homogenates were therefore fractionated on a continuous sucrose
gradient (1.074-1.242 g/ml). Fractions were grouped into three
samples
(A-C,
see Fig. 1).
Na+-K+-ATPase
-subunit immunoreactivity was higher in fraction
B (enriched in plasma membrane fragments) than in
fraction C. Incubation of PCT cells
with 1 µM dopamine for 15 min caused a shift in the
-subunit
immunoreactivity away from fraction B,
while increasing markedly its abundance in fraction
C. MPR immunoreactivity, denoting the presence of late
endosomes and to a lesser extent plasma membrane fragments (15), was
present in fractions B and
C (Fig. 1, 2nd panel),
whereas rab5 immunoreactivity, which
indicates the presence of early endosomes (14, 16), was found
predominantly in fraction C (Fig. 1,
3rd panel). These results suggested that the
-subunit may
have been incorporated into endosomal compartments. Indeed, Western
blot analysis revealed increased incorporation of the
-subunit in
early and late endosomes prepared from PCT cells exposed to dopamine (1 µM; 15 min) as compared with PCT cells incubated with vehicle
(Fig.2A). The
increased incorporation of Na+-K+-ATPase
-subunits into early endosomes was time dependent and became clearly
apparent at 5 min (Fig. 2B,
top panel), whereas increased
incorporation into late endosomes occurred after a lag of 15 min (Fig.
2B, bottom
panel). Even earlier times (2.5 and 5 min,
respectively) were revealed by scanning densitometry (Fig. 2E).

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Fig. 1.
Cellular distribution of
Na+-K+-ATPase
catalytic -subunit in renal proximal convoluted tubule (PCT)
cells and effect of incubation with dopamine (1 µM) during 15 min at
room temperature (top panel). After incubation with dopamine or vehicle, homogenates of PCT suspensions were layered on a 4-ml linear sucrose gradient and processed as described in EXPERIMENTAL
PROCEDURES. After fractionation, samples were pooled as
follows: A, fractions 1.083 to 1.129 g/ml; B, fractions 1.139 to 1.183 g/ml; and C, fractions 1.194 to 1.240 g/ml. Samples [3 µg
Na+-K+-ATPase,
7.5 µg mannose 6-phosphate receptor (MPR) and
rab5, and 50 µg GLUT-2] of
protein were separated by SDS-PAGE (7.5-15%) and transferred to
nitrocellulose membranes. Qualitative determination of
Na+-K+-ATPase
-subunit immunoreactivity, GLUT-2,
rab5, and MPR, as indicators of early
and late endosomes, respectively, was performed by Western blot
analysis. Data are representative of 3 experiments performed
independently.
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Fig. 2.
Na+-K+-ATPase
-subunit abundance in endosomes prepared from PCT cells incubated
with dopamine.
Na+-K+-ATPase
-subunit (A),
rab5 and MPR immunoreactivity
(C and
D) in early endosomes (early e.) and
late endosomes (late e.) prepared from PCT cells that were previously
incubated with and without [control (C)] 1 µM dopamine (DA) for 5 min. Increased -subunit abundance in early and late endosomes
induced by DA (1 µM) is time dependent
(B). Total protein loaded for
SDS-PAGE separation was 1 µg for
Na+-K+-ATPase
and 8 µg for MPR and rab5; Western
blots are representative of 6 separate experiments. Densitometric scans
of time course experiments (E) were
expressed as -subunit enrichment (percentage of control at
time 0), and mean ± SE
values are shown. Asterisks represent significant differences of
P < 0.05.
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The early and late endosomes were separated on a flotation gradient
(14), and their identities were verified by their immunoreactivity to
rab5 and MPR antibody, respectively
(Fig. 2, C and
D). Late endosomes were rich in MPR,
whereas early endosomes were rich in
rab5 but showed no MPR
immunoreactivity, indicating that they were not contaminated with late
endosomes or plasma membrane fragments. Unlike the translocation of the
- and
-subunits (see also Fig. 5), the cell distribution of MPR
did not change in response to dopamine (Figs. 1,
middle panels, and
2D). In separate experiments, a
specific marker of basolateral membranes, GLUT-2, also did not change
its distribution in early and late endosomes in response to 1 µM
dopamine (Fig. 3), in contrast to the
-subunit
abundance.

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Fig. 3.
GLUT-2 immunoreactivity in early endosomes
(A) and late endosomes
(B) from PCT cells incubated with 1 µM dopamine for 5 min. Equal amount of protein (5 µg) was loaded in
each lane. Representative autoradiograms
(A and B) and a
densitometric scan (C) (means ± SE) of 4 experiments
performed independently are shown. EE, early endosome; LE, late
endosome.
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Receptor-mediated endocytosis generally occurs by formation of
clathrin-coated pits and CCV, followed by vesicle transport (22). In
this study, CCV were isolated from PCT cells, utilizing sucrose density
gradients and negative lectin selection (17). The latter takes
advantage of the fact that CCV are the only intracellular organelles
lacking mannose sugars (17). Silver staining of CCV revealed both the
heavy chains (160 kDa) and light chains (~30 kDa) of clathrin, as
well as an ~100-kDa protein corresponding in size to the
-subunit
(Fig. 4A). The
identity of CCV was further documented by Western blot analysis, using
a specific antibody against the clathrin heavy chain in the CCV
preparation, which was compared with brain and kidney homogenates (Fig.
4B). Contamination of CCV with early
endosomal vesicles in this preparation was excluded by the lack of
immunoreactivity with rab5 (data not
shown). The
-subunit abundance increased in CCV treated with
dopamine (1 µM) in a time-dependent manner: it became evident at 1 min, peaked at 2.5 min, and lasted up to 30 min of incubation (Fig. 4,
C and D). This time course coincides with
the early presence of the
-subunit in CCV and its subsequent
appearance (at 5 min) in early endosomes (see Fig.
2B). Despite the increase in
Na+-K+-ATPase
-subunit abundance in CCV and early endosomes from dopamine-treated cells, the enzymatic activity remained unchanged (Table
1), indicating that the endocytosed
subunits became inactive.

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Fig. 4.
Na+-K+-ATPase
-subunit immunoreactivity in clathrin-coated vesicles (CCV) from PCT
cells incubated with dopamine. A:
silver stain of CCV protein content.
B: identification of clathrin heavy chain (CHC) in CCV. K, kidney homogenate control; B, brain homogenate control. C: time-dependent
incorporation of -subunits in CCV induced by dopamine (2 µg
protein loaded in each lane). Western blots are representative of 5 separate experiments. D: densitometric scans of time course experiment were expressed as -subunit abundance (percentage of control at time 0),
and mean ± SE values are shown. Asterisks represent significant
differences of P < 0.05.
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Table 1.
Na+-K+-ATPase activity in basolateral
membranes, clathrin-coated vesicles, and early endosomes prepared from
vehicle- and dopamine-treated PCT cells
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In PCT cells, dopamine inhibits
Na+-K+-ATPase
activity by activation of PKC (3, 29), which may also be involved in
endocytosis. Consistent with this hypothesis, the ability of dopamine
to increase incorporation of
-subunits in CCV was abolished by
calphostin C, a specific PKC inhibitor found by us to block dopamine
inhibition of the pump in intact tubules (24, 26) (Fig.
5, A and
D, left panel). Similar to CCV, calphostin C also prevented
the increased
-subunit incorporation induced by dopamine in early
and late endosomes (Fig. 5, A and
D, left
panel), whereas calphostin C alone had no effect on
-subunit distribution (data not shown). In addition to the
-subunit, dopamine also increased the incorporation of
Na+-K+-ATPase
-subunit into early and late endosomes, and this effect as well was
blocked by calphostin C (Fig. 5, B and
D, right
panel). Similar inhibition of
-subunit endocytosis
(data not shown) was obtained with another specific PKC inhibitor,
bisindolylmaleimide.

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Fig. 5.
Internalization of
Na+-K+-ATPase
- and -subunits in PCT cells incubated with dopamine is protein
kinase C dependent. A and B: PCT cells were incubated with
dopamine at room temperature for 2.5 min (CCV) and 15 min (EE and LE).
Dopamine (1 µM)-induced incorporation of -
(A) and -subunits
(B) in CCV, EE, and LE is blocked by
coincubation with calphostin C (1 µM).
C: in contrast, inhibition of
Na+-K+-ATPase
activity with ouabain (Ouab; 1 mM) is not associated with increased
endocytosis of its -subunit in endosomes. Amount of protein loaded
in each lane was 1 µg
(Na+-K+-ATPase
-subunit) and 5 µg
(Na+-K+-ATPase
-subunit). Experiments were independently performed 4 times.
Densitometric scans (D) were
expressed as -subunit enrichment (percentage of control), and mean ± SE values are shown. Asterisks represent values statistically
(P < 0.05) different from
dopamine-treated cells without calphostin C (open bars).
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Is the activation of PKC a requisite for endocytosis of pump subunits?
To address this question, we next examined their distribution after
exposure to ouabain, a specific
Na+-K+
pump inhibitor that lacks the PKC effect. Although ouabain (1 mM, 10 min) inhibited pump activity, it had no effect on the internalization of the
-subunit into endosomes (Fig.
5C) (
-subunit was not examined). This observation further supports the concept that receptor-mediated inhibition/endocytosis of the pump in epithelial cells requires activation of a specific intracellular signaling system.
The trafficking of proteins between the plasma membrane and several
subcellular compartments is dependent on the integrity of the cortical
actin cytoskeleton and the microtubule system. Therefore, we also
examined the role of the actin/microtubule system in this process by
using microtubule depolymerizing agents, nocodazole (Noc) and
phallacidin (Phall), which stabilize the cortical actin cytoskeleton
but do not affect the
-subunit distribution [Noc:
-subunit
abundance (% of control): CCV, 107 ± 3, n = 4; early endosomes, 98.5 ± 5.2, n = 4; late endosomes, 105.8 ± 2.3, n = 4; Phall:
-subunit
abundance (% of control): CCV, 95.2 ± 7.3, n = 4; early endosomes, 98.8 ± 9.3, n = 5; late endosomes, 106.6 ± 6.3, n = 5]. In the
presence of Noc, 1 µM dopamine failed to increase
Na+-K+-ATPase
-subunit endocytosis into early and late endosomes (similar results
were obtained with colchicine, another microtubule depolymerizing agent), but not into CCV, whereas Phall treatment prevented their incorporation into CCV as well (Fig. 6).
The ability of dopamine to modulate
Na+-K+-ATPase
activity was also affected by Phall and Noc treatment. Although Phall
prevented the inhibitory action of dopamine (97 ± 5% of control,
n = 4), Noc only attenuated the
inhibition (72.6 ± 5.3% of control,
n = 11;
P < 0.01). In parallel experiments without Noc, dopamine inhibited
Na+-K+-ATPase
activity maximally by 65 ± 5% of control
(n = 4).

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Fig. 6.
Endocytosis of
Na+-K+-ATPase
-subunits from PCT cells that have been previously incubated with
nocodazole (5 µM) for 30 min at room temperature or phallacidin (1 µM). To ensure incorporation of phallacidin, cells were subjected to
a thermic shock (10 min at 20°C), and, after a recovery
period of 10 min at room temperature, dopamine was added for an
additional 15 min. Thermic shock did not affect endocytosis of
Na+-K+-ATPase
subunits induced by dopamine as seen in PCT cells that have not
received phallacidin treatment but were subjected for 10 min to
20°C. Neither phallacidin (% of control of -subunit abundance: CCV, 95.2 ± 7.3, n = 4;
EE, 98.8 ± 9.3, n = 5; LE, 106.6 ± 6.3, n = 5) nor nocodazole (% of control of -subunit abundance: CCV, 107.0 ± 3.0, n = 4; EE, 98.5 ± 5.2, n = 4; LE, 105.8 ± 2.3, n = 4) affects sedimentation
properties of CCV, EE, and LE. Total amount of protein subjected to
SDS-PAGE separation was 1 µg. Western blots
(A) are representative of 4 experiments, and statistical comparison of densitometry scans is shown
in B.
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DISCUSSION |
In contrast to the wealth of information about its structure and
kinetic properties, the regulation of
Na+-K+
pump activity and abundance is less well understood. Recent progress in
this area led to definition of two types of regulation: short and long
term, or rapid and sustained, which are mediated by different mechanisms (reviewed in Refs. 7 and 11). It is generally accepted that
rapid modulation of
Na+-K+-ATPase
activity involves stimulation of serine/threonine protein kinases,
primarily PKA and PKC (1, 3, 5, 8, 12, 23, 28, 29), leading to
activation of other intracellular mediators (e.g., eicosanoids)
and/or the reversible phosphorylation of its catalytic (
)
subunit. However, the molecular mechanisms that translate the
phosphorylation effect into modulation of pump activity remain obscure.
In this paper, we propose the concept (summarized in schematic fashion
in Fig. 7) that receptor-mediated inhibition of
Na+-K+-ATPase
activity by dopamine in PCT cells is associated with a stepwise
endocytosis of its
- and
-subunits. The proposed mechanism appears to be analogous to that of other systems such as
vasopressin-dependent regulation of aquaporin-2 water channels (20) in
collecting duct cells, or insulin-dependent regulation of the glucose
transporter GLUT-4 in skeletal muscle (18) and, in the opposite
direction, of
Na+-K+-ATPase
in lacrimal glands (33) and skeletal muscle (19). Further evidence for
the proposed sequence of events is provided by the lack of endocytosis
in the presence of a microtubule depolymerizing agent. Failure of Noc
to inhibit the incorporation of
-subunits in CCV is consistent with
the fact that this process is microtubule independent (16). In
contrast, stabilizing the cortical actin cytoskeleton with Phall
inhibited the incorporation of
-subunits into CCV. Although
inhibition of
Na+-K+-ATPase
activity is strongly associated with formation of CCV (Phall treatment
blocked the effect of dopamine), disrupting microtubules affect
transport to endosomes but only partially the inhibition of enzyme
activity.

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Fig. 7.
Proposed schema of intracellular traffic sequence of
Na+-K+-ATPase
subunits after inhibitory action of dopamine. For explanation, see
text.
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The endocytosis of
Na+-K+-ATPase
subunits observed in response to dopamine is not likely to be due to
increased fluid-phase endocytosis, because other membrane proteins such
as GLUT-2 and MPR did not change their cellular distribution in the
presence of dopamine.
The results described above constitute in our view persuasive
biochemical evidence for the increased abundance of enzyme subunits in
defined intracellular organelles during
Na+-K+-ATPase
inhibition. Localization of
Na+-K+-ATPase
subunits in early endosomes has previously been suggested in Chinese
hamster ovary cells, where they could contribute to the interior
membrane potential and thereby affect
H+ transport and
intravesicular pH (13). Because enzyme subunits capable of ATP
hydrolysis are present in endosomes under basal conditions, the present
study also raises the possibility that these compartments might
constitute traffic reservoirs during pump synthesis as well as during
endocytosis, serving, for example, as intracellular storage sites from
which the enzyme's subunits may shuttle rapidly to the plasma
membrane. As demonstrated in this paper, receptor-mediated endocytosis
likely increases the number of
Na+-K+-ATPase
subunits in this preexisting pool of organelles, a process dependent on
PKC activation. It is unclear at present whether the endocytosed units
are eventually recycled to the plasma membrane or follow a degradative
pathway. That
Na+-K+-ATPase
activity in early and late endosomes could be modulated by okadaic acid
(unpublished observations) suggests, however, that this compartment is
endowed with protein phosphatase activity, and that dephosphorylation
may be a necessary step if the subunits are to be returned to the
plasma membrane. It should be emphasized that although our results
underscore the importance of PKC, they do not allow an assessment of
whether activation of PKC is needed to phosphorylate the
-subunit
itself or at other stages during endocytosis, such as regulation of CCV
formation, or for actin or microtubule function.
Finally, preliminary evidence from our laboratory that endocytosis of
membrane pumps also occurs in Madin-Darby canine kidney and opossum
kidney cells, where enzyme activity can apparently be inhibited by
either PKA or PKC, and in pancreatic
-cells, where the signal is not
receptor mediated but rather dependent on glucose metabolism, suggests
that the mechanism described here in PCT cells may constitute a general
mode of
Na+-K+-ATPase
regulation.
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ACKNOWLEDGEMENTS |
We thank P. C. Pinto-do-O for assistance in preparing the PCT cells
in some of these experiments.
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FOOTNOTES |
This study was supported by grants from the Swedish Medical Research
Council and Emil and Vera Cornells Stiftelse (to A. M. Bertorello) and
by the Swedish Natural Science Council (to A. I. Katz). A. Chibalin is
a recipient of a postdoctoral scholarship from the Karolinska
Institute.
Address for reprint requests: A. M. Bertorello, The Rolf Luft Center
for Diabetes Research L6B:02, Dept. of Molecular Medicine, Karolinska
Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Received 10 March 1997; accepted in final form 30 June 1997.
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