Role of caveolae in signal-transducing function of cardiac
Na+/K+-ATPase
Lijun
Liu*,
Kamiar
Mohammadi*,
Behrouz
Aynafshar,
Haojie
Wang,
Daxiang
Li,
Jiang
Liu,
Alexander V.
Ivanov,
Zijian
Xie, and
Amir
Askari
Departments of Pharmacology and Medicine, Medical College
of Ohio, Toledo, Ohio 43614
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ABSTRACT |
Ouabain binding to
Na+/K+-ATPase activates Src/epidermal growth
factor receptor (EGFR) to initiate multiple signal pathways that
regulate growth. In cardiac myocytes and the intact heart, the early
ouabain-induced pathways that cause rapid activations of ERK1/2 also
regulate intracellular Ca2+ concentration
([Ca2+]i) and contractility. The goal of this
study was to explore the role of caveolae in these early signaling
events. Subunits of Na+/K+-ATPase were detected
by immunoblot analysis in caveolae isolated from cardiac myocytes,
cardiac ventricles, kidney cell lines, and kidney outer medulla by
established detergent-free procedures. Isolated rat cardiac caveolae
contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of
1- and
2-isoforms of
Na+/K+-ATPase, along with nearly all of
cellular caveolin-3. Immunofluorescence microscopy of adult cardiac
myocytes showed the presence of caveolin-3 and
-isoforms in
peripheral sarcolemma and T tubules and suggested their partial
colocalization. Exposure of contracting isolated rat hearts to a
positive inotropic dose of ouabain and analysis of isolated cardiac
caveolae showed that ouabain caused 1) no change in total
caveolar ERK1/2, but a two- to threefold increase in caveolar
phosphorylated/activated ERK1/2; 2) no change in caveolar
1-isoform and caveolin-3; and 3) 50-60%
increases in caveolar Src and
2-isoform. These findings,
in conjunction with previous observations, show that components of the
pathways that link Na+/K+-ATPase to ERK1/2 and
[Ca2+]i are organized within cardiac caveolae
microdomains. They also suggest that ouabain-induced recruitments of
Src and
2-isoform to caveolae are involved in the
manifestation of the positive inotropic effect of ouabain.
digitalis; heart failure; rafts; sodium pump; Na+/Ca2+ exchanger
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INTRODUCTION |
THE
ENERGY-TRANSDUCING ion pump Na+/K+-ATPase
maintains the normal gradients of Na+ and K+
across the plasma membrane of most eucaryotic cells (34).
It has recently been shown that in cardiac myocytes and several other cell types, Na+/K+-ATPase also acts as a signal
transducer; i.e., in response to ouabain and related cardiac
glycosides, the enzyme interacts with neighboring membrane proteins to
relay messages to intracellular signaling complexes, the mitochondria,
and the nucleus (45). The most proximal ouabain-induced
interaction seems to be between Na+/K+- ATPase and Src, leading to Src
activation and epidermal growth factor receptor (EGFR) transactivation
(7, 8), activations of protein kinase C (PKC) and the
Ras/Raf/MEK/ ERK1/2 cascade (13, 25, 26), and a host of
subsequent downstream effects emanating from these rapid proximal
signaling events. To date, the downstream consequences of
ouabain-induced signaling through Na+/K+-ATPase
have been studied in some detail only in cardiac myocytes, where
ouabain and related cardiac glycosides exert their well-known effects
on cardiac contractility. In these cells, such consequences include
increased mitochondrial generation of reactive oxygen species,
activation of transcription factors activator protein-1 and nuclear
factor-
B, transcriptional regulation of early- and late-response
growth-related genes, increased rate of protein synthesis, and myocyte
hypertrophy (45). Recent evidence (41) indicates that the rapid ouabain-induced signaling that leads to the
activation of ERK1/2 is also essential to the manifestation of the
classical effects of ouabain on intracellular Ca2+
concentration ([Ca2+]i) and cardiac
contractility; i.e., the positive inotropic effect of the drug.
It is generally accepted that the plasma membrane contains microdomains
(rafts) that are rich in cholesterol and sphingolipids relative to the
remainder of the plasma membrane and that caveolae are specific forms
of rafts that contain the marker proteins caveolins (1, 30,
35). Because such microdomains have been implicated as sites of
assembly and regulation of signaling complexes associated with a
variety of plasma membrane receptors (1, 30, 35), we were
prompted to explore their possible involvement in the newly appreciated
signal-transducing functions of Na+/K+-ATPase.
Specifically, the primary goals of this study were twofold. First, in
view of the ambiguities of previous reports on the caveolar localization of Na+/K+-ATPase, we wanted to
determine whether or not this enzyme is a normal resident of caveolae.
Our findings establish that it is. Second, because of the central role
of ERK1/2 in the manifestation of ouabain effects on cardiac myocytes
and the intact heart (26, 45), we assessed whether or not
the caveolar pool of cardiac Na+/K+-ATPase
isoforms are involved in the ouabain-induced activation of signal
pathways that link Na+/K+- ATPase to ERK1/2
concomitant with the development of the positive inotropic effect of
ouabain on the heart.
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MATERIALS AND METHODS |
Materials.
Chemicals of highest purity and culture media were purchased from Sigma
(St. Louis, MO). Anti-caveolin-3 monoclonal antibody (clone 26), and
anti-caveolin-1 monoclonal antibody (clone C060) were obtained from BD
Transduction Laboratories (Lexington, KY). Anti-caveolin-3 polyclonal
antibody was purchased from Affinity BioReagents (Golden, CO).
Dynabeads coated with goat anti-mouse IgG were obtained from Dynal
(catalog no. M-450; Lake Success, NY).
Anti-Na+/K+-ATPase
1 monoclonal
antibody, anti-Na+/K+-ATPase
2
polyclonal antibody, anti-Na+/K+-ATPase
1 monoclonal antibody, anti-Src (clone GD11) monoclonal antibody, polyclonal anti-EGFR antibody, and rabbit anti-sheep secondary antibody were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies against ERK1/2, phosphorylated ERK1/2, PKA
catalytic subunit, goat anti-rabbit secondary antibody, and goat
anti-mouse secondary antibody were obtained by Santa Cruz Biotechnology
(Santa Cruz, CA). Monoclonal antibody against
Na+/K+-ATPase
1 (
6F) was
obtained from Developmental Studies Hybridoma Bank, The University of
Iowa (Iowa City, IA). Anti-Na+/K+-ATPase
3 polyclonal antibody against a synthetic peptide
corresponding to the NH2 terminus of the rat subunit was a
gift from Dr. R. W. Mercer (Washington University, St. Louis, MO).
Peptide N-glycosidase F was purchased from New England
Biolabs (Beverly, MA). Kidney medullas were obtained by dissection from
frozen kidneys. Pig kidneys and beef hearts were obtained from
slaughterhouses. Rats used as sources of organs were housed and
euthanized according to institutional policies.
Cell preparation and culture.
Neonatal rat cardiac myocytes were cultured from the ventricles of 1- to 2-day-old rats, as described previously (13, 25), and
used after 24 h of serum starvation. Calcium-tolerant adult rat
cardiac myocytes were also prepared as described before
(41). Freshly made suspensions were either used for
caveolae isolation or seeded on laminin-coated coverslips and used for
immunocytochemistry. Pig kidney LLC-PK1 cells and human
transformed primary embryonic kidney cells (HEK-293) (American Type
Culture Collection) were cultured in Dulbecco's modified Eagle's
medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. When 80-90% confluence was reached, cells were
serum starved for 24 h and used for caveolae preparation from cell lysates.
Ouabain-induced positive inotropy in isolated hearts.
Conventional isolated Langendorff preparations of rat heart were set up
for the measurement of cardiac contractility and perfused with normal
Krebs-Henseleit solution as described before (26). Each
heart was perfused for 30 min with the control buffer before contractility measurements were recorded. To induce the effects of
ouabain, the perfusion solution was switched to the same buffer containing 50 µM ouabain. As shown before (26), this
dose of ouabain causes a significant and sustained doubling of the rate of left ventricular pressure increase without producing toxic arrhythmias. After 10 min of perfusion, when the peak positive inotropic effect of ouabain was obtained (26), hearts were
quick-frozen in liquid nitrogen and lysates of powdered ventricular
samples were used for caveolae preparation.
Fractionation of cell/tissue lysates for caveolae preparation by
carbonate-based procedure.
This procedure was done by slight modification of the procedure of Song
et al. (37). A sample of intact cells (myocytes, LLC-PK1 cells, and HEK-293 cells) or tissue (cardiac
ventricle, kidney outer medulla) with a total protein content of
8-16 mg was placed at 4°C in 2 ml of 0.5 M
Na2CO3 solution (pH 11) containing 1 mM EDTA, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 1 mM NaF, 10 nM
okadaic acid, 10 µg/ml aprotonin, and 10 µg/ml leupeptin. In
specified experiments, this solution also contained 1% Triton X-100.
All subsequent steps were done on ice or at 4°C. The sample was
homogenized at setting 5 of a Polytron homogenizer (three
6-s bursts) and sonicated at setting 3 of a Branson Sonifier
model 250 (three 40-s bursts). We added 2 ml of 90% sucrose to the
homogenate, prepared in 25 mM MES (pH 6.5) plus 150 mM NaCl (MBS). This
suspension was placed in the bottom of the centrifuge tube and was
overlaid with 4 ml of 35% sucrose and then 4 ml of 5% sucrose, each
prepared in MBS containing 250 mM Na2CO3. The
sample was centrifuged with a rotor (model SW41, Beckman) at 39,000 revolutions/min for 17-18 h, and twelve 1-ml fractions (numbered
from top to bottom) were collected.
Preparation of caveolae from cardiac sarcolemma by OptiPrep
procedure.
Highly purified cardiac sarcolemma were prepared from bovine or rat
heart ventricles, following the method of Jones (11). A
sample of these plasma membranes (0.7-0.8 mg total protein) was
suspended in 2 ml of 0.25 M sucrose, 1 mM EDTA, and 20 mM Tricine (pH
7.6) and sonicated on ice with three series of two consecutive bursts,
6-8 s each, at setting 4 of a Branson Sonifier 250, with 2-min intervals between the series. The sample was then fractionated on OptiPrep gradients by the procedures of Smart et al.
(36), as subsequently modified (42). The
opaque band at the 5% OptiPrep overlay after the second fractionation
step was designated as caveolae.
Immunoblot analysis.
Samples were subjected to 10 or 12% SDS-PAGE, transferred to
nitrocellulose membrane, and probed with appropriate antibodies by
standard procedures. The immunoreactive bands were developed and
detected using enhanced chemiluminescence. For quantitative comparisons, images were scanned with a densitometer. Different dilutions of samples were subjected to SDS-PAGE, and multiple exposures
of the films were used to ensure that quantitations were made within
the linear range of the assays. Quantitative comparisons of blots that
are not subjected to such procedures may be misleading.
Immunofluorescence microscopy.
Myocytes attached to coverslips were washed two times in
phosphate-buffered saline (PBS) containing 0.1 mM CaCl2 and
1 mM MgCl2 (PBS-Ca-Mg) and fixed in
20°C methanol for
10 min. Cells were rinsed one time in PBS-Ca-Mg, incubated for 15 min
in cell permeabilization buffer (PBS-Ca-Mg, 0.3% Triton X-100, and
0.1% bovine serum albumin), and then incubated in goat serum dilution buffer (GSDB; 16% filtered goat serum, 0.3% Triton X-100, 20 mM sodium phosphate, pH 7.4, and 150 mM NaCl) for 30 min at room temperature to block nonspecific IgG binding sites. Myocytes were incubated overnight at 4°C with
anti-Na+/K+-ATPase
1 monoclonal
antibody (1:100) and anti-caveolin-3 polyclonal antibody (1:100) in
GSDB or anti-Na+/K+-ATPase
2
polyclonal antibody (1:100) and anti-caveolin-3 monoclonal antibody
(1:100) in GSDB, respectively. The same procedure was also performed by
using normal goat serum or PBS instead of primary antibody as a
negative control, to ensure the specificity of the double-staining
procedure and the specificity of fluorescent secondary antibodies. On
the following day, myocytes were washed three times for 5 min each in
permeabilization buffer and then incubated for 2 h at room
temperature with Alexa Fluor 488-conjugated anti-mouse IgG(H+L) and
Alexa Fluor 546-conjugated anti-rabbit IgG(H+L) antibodies (Molecular
Probes, Eugene, OR) in GSDB or with Alexa Fluor 488-conjugated anti-rabbit IgG(H+L) and Alexa Fluor 546-conjugated anti-mouse IgG
(H+L) antibodies in GSDB, respectively. Myocytes were washed three
times for 5 min each in permeabilization buffer and once in 10 mM
sodium phosphate (pH 7.5). The coverslips were mounted with
Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
Images were acquired by confocal laser scanning microscope (Bio-Rad
Radiance 2000) with the use of the 488- and 543-nm lines of Ar-Kr and
He-Ne lasers. An Olympus UplanApo water-immersion objective
×60/1.2 NA was used, and the software LaserSharp 2000 (Bio-Rad) was
used for image acquisition, storage, and visualization.
Immunoaffinity isolation of caveolae.
This was done by modification of previously described procedures
(38, 43). Caveolae samples isolated by the detergent-free carbonate-based procedure were precleared with Dynal M450 beads coated
with goat anti-mouse IgG. Additional Dynabeads were incubated with
anti-caveolin-3 monoclonal antibody overnight at 4°C, and the cleared
sample was then added and incubated for another 1 h at 4°C.
Immune complexes were collected by magnetic separation and washed four
times in 20 mM Tris · HCl (pH 7.4). Two
fractions, material bound to beads (bound) and material not bound to
beads (unbound), were subjected to SDS-PAGE and Western analysis.
Other assays.
Na+/K+-ATPase activity was assayed
(46) at 37°C by measuring the initial rate of release of
32Pi from [
-32P]ATP in a medium containing
100 mM NaCl, 25 mM KCl, 3 mM MgCl2, 1 mM EGTA, 2 mM ATP,
and 20 mM Tris · HCl (pH 7.4). Each assay was
done in the presence and absence of 1 mM ouabain to assess the
ouabain-sensitive component of the activity. Protein was determined by
the Bio-Rad DC protein colorimetric assay.
Analysis of data.
Data are means ± SE of the results of a minimum of three
experiments. Student's t-test was used and significance was
accepted at P < 0.05.
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RESULTS |
Colocalization of
Na+/K+-ATPase
subunits and related signaling proteins in cardiac caveolae.
Several previous studies (5, 9, 18, 33, 48) had concluded
that caveolae/rafts prepared from several cell types, including cardiac
myocytes, do not contain Na+/K+-ATPase. Because
our preliminary experiments suggested otherwise, we set out to resolve
this issue. First, we focused on cardiac caveolae because most of our
studies on the signal-transducing role of
Na+/K+-ATPase had been done in cardiac myocytes
and the intact heart (26, 45). The widely used
detergent-free and carbonate-based procedure of Song et al.
(37) had been used successfully in numerous studies on
cardiac caveolae prepared from cultured cardiac myocytes and ventricles
(4, 5, 27, 28, 31, 32). Hence, we applied this density
gradient fractionation procedure to homogenized/sonicated samples of
rat heart ventricles, purified adult rat heart myocytes, and cultured
neonatal rat cardiac myocytes. The fractions were then assayed for
protein content and subjected to Western blot analyses for the
muscle-specific caveolin-3 and the other indicated proteins. Typical
distribution patterns of total protein and caveolin-3 for a
fractionated sample of ventricle are shown in Fig.
1. Nearly identical patterns were
obtained after fractionations of the adult or neonatal myocyte
preparations. These findings are in agreement with previous
observations on cardiac preparations fractionated by the same procedure
(4, 5, 28, 32), showing that most of the caveolin-3
content of the homogenate is located in light fractions
(fractions 4 and 5) that contain <3% of the
total protein. Such cardiac light fractions that contain caveolin-3,
and are prepared by a procedure similar to that used here, have been
shown (32) by electron microscopic examination to contain
vesicles and membrane fragments that resemble the "cave-like"
caveolae vesicles that are noted on the plasma membrane of the intact
cardiac cells (6, 16).

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Fig. 1.
Localization of the 1-subunit of
Na+/K+-ATPase in caveolin-3 (Cav-3)-rich
domains of rat heart ventricles and adult myocytes. Samples of lysates
(8-16 mg of protein) were fractionated by the detergent-free
carbonate-based procedure, and 12 fractions of equal volume (1 ml each)
were collected (see MATERIALS AND METHODS). Each fraction
was assayed for protein, and an equal amount of protein from each
fraction was subjected to SDS-PAGE and Western blot analysis with
specific antibodies. The protein distribution pattern and the Cav-3
blots were similar for the samples from the two sources.
Fractions 1-3 did not contain protein.
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The
1-isoform of the catalytic subunit of
Na+/K+-ATPase is the predominant isoform
(~80% of the total
-content) of the adult rat heart and the rat
cardiac myocytes (20, 22, 23, 39). Typical blots of the
1-subunit for the fractionated samples of ventricles and
adult myocytes are shown in Fig. 1. These blots were obtained by using
an equal amount of protein from each fraction and equal film exposure
time for image development from each fraction. Considering this, and
the pattern of distribution of total protein in the 12 fractions (Fig.
1, top), we may reach the following semiquantitative
conclusions: 1) most of the
1 content of the fractionated lysate is recovered in heavy fractions
8-12; 2) the caveolin-3-rich light
fractions 4 and 5 also contain significant quantities of the
1-subunit; and 3) per-unit
protein fractions 4 and 5 are enriched in
1 relative to heavy fractions
8-12.
In Fig. 2, several representative blots
of a number of other proteins of interest in the fractionated samples
of the cardiac preparations are presented. The results show that like
the
1-isoform, the less abundant cardiac isoforms of the
rat (adult
2 and neonatal
3), Src, and
ERK1/2 are also enriched per unit protein in caveolar fractions. The
blot on the catalytic subunit of protein kinase A (Fig. 2) exemplifies
the fact that not all fractionated proteins are enriched in the light
fractions obtained by this procedure.

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Fig. 2.
Localization of the 2- and 3-isoforms
of Na+/K+-ATPase, Src, and ERK1/2 in Cav-3-rich
domains of cardiac ventricles and neonatal myocytes. Fractionations and
immunoblots were performed as indicated in Fig. 1 and MATERIALS
AND METHODS. PKA, protein kinase A.
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We used immunostaining and confocal microscopy to explore the
subcellular localizations of caveolin-3 and the
1 and
2 isoforms of Na+/K+-ATPase in
adult cardiac myocytes. In agreement with previous observations
(23), the
-subunits were clearly localized in peripheral sarcolemma, T tubules, and intercalated disks (Fig. 3). Caveolar vesicles have been noted
before by electron microscopic examination of the myocardium on both
peripheral plasma membrane and T tubules (6, 16).
Immunostaining also showed the presence of caveolin-3 in peripheral
sarcolemma, T tubules, and intercalated disks (Fig. 3). Significantly,
our findings indicated considerable overlap in the immunostaining of
the
-subunits and caveolin-3 (Fig. 3). The combined data of Figs.
1-3, in conjunction with previous findings, strongly support the
colocalization of caveolin-3 and a significant portion of
Na+/K+-ATPase in cardiac caveolae.

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Fig. 3.
Colocalization of Na+/K+-ATPase
-isoforms and Cav-3 in adult cardiac myocytes. Cultured myocytes
were fixed for immunostaining and confocal microscopy (see
MATERIALS AND METHODS). A: staining of
1 (green) and Cav-3 (red); B: staining of
2 (green) and Cav-3 (red) in greater
detail.
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Although the experiments of Figs. 1 and 2 showed the presence of
significant amounts of Na+/K+-ATPase
-isoforms and several related signaling proteins in cardiac caveolae
isolated from lysates, it was necessary to make a better quantitative
assessment of the relative distributions of the proteins between
caveolae and the remaining cellular compartments. Such determinations,
based on single composite blots (e.g., those of Figs. 1 and 2), may be
misleading because the relative intensities of bands in such blots are
often over- or underestimated. It is therefore necessary to subject
different dilutions of the various fractions to immunoblot analysis,
and to quantitate multiple exposures of the luminescent images, to
ensure the appropriate comparison of the contents of the various
fractions. This procedure was done on each protein (shown in Fig.
4) by using ventricular samples from four
hearts. After fractionation of each lysate sample by the
carbonate-based procedure, and the assay of total protein content of
each fraction, the content of the immunoreactive protein in each
fraction was determined by procedures that optimize such comparative
quantitations (as indicated above and in MATERIALS AND
METHODS) and expressed in arbitrary relative units.
Caveolar content of each protein, as a percentage of the total, was
then calculated. The results of these experiments as summarized in Fig.
4 show the caveolar contents of caveolin-3,
1,
2, Src, ERK1, and ERK2. These data indicate that
~20-30% of total cellular contents of
1- and
2-isoforms are located in caveolae. The remainder must
be in the bulk plasma membrane and the internal membranes. In skeletal
muscle myocytes, it has been estimated that about half of the cellular
content of Na+/K+-ATPase is in the plasma
membrane and the other in the internal membranes (44). If
we assume about the same distribution for rat cardiac myocytes, the
data of Fig. 4 would suggest that 40-60% of the total plasma
membrane pool is in caveolae and the remainder in the rest of the bulk
plasma membrane. Because quantitative analysis of electron micrographs
has suggested that caveolae microdomains constitute ~20-30% of
the plasma membrane area of myocytes of the adult heart (6,
16), we may conclude tentatively that the
-subunits of
cardiac Na+/K+-ATPase are concentrated in the
caveolae microdomains relative to the remaining areas of the cardiac
plasma membrane.

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Fig. 4.
Relative distributions of Cav-3, 1- and
2-isoforms of Na+/K+-ATPase,
Src, and ERK1/2 in caveolar and noncaveolar fractions of cardiac
ventricle lysates. Fractionations were performed as in Fig. 1 on
samples from 4 different hearts. Immunoblots of the indicated proteins
were obtained from each of the 12 fractions under conditions that are
optimal for the quantitation of such blots, as described in
MATERIALS AND METHODS. On the basis of these determinations
and the assay of total protein content of each fraction, the content of
each indicated protein in fractions 4 and 5,
relative to total sample content, was calculated. Values are means ± SE (n = 4).
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Caveolae preparations may be mixed with low buoyant density noncaveolar
rafts (43). Using the approach of Stan et al.
(38), which involved the isolation of caveolae on
anticaveolin-coated magnetic beads, we sedimented/purified the cardiac
caveolae on anticaveolin-3-coated beads. The results shown in Fig.
5 indicate that the subunits of
Na+/K+-ATPase (
1 and
1), Src, and EGFR are indeed colocalized in the same
microdomains that contain caveolin-3. We verified that the band
reacting with the monoclonal anti-
1 antibody is, indeed, the
1-subunit by performing experiments showing that
incubation of caveolae with N-glycosidase F converted the
band to one with mobility well in accord with that of the
unglycosylated
1 protein (Fig. 5).

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Fig. 5.
Localization of Na+/K+-ATPase subunits
( 1 and 1), Src, and epidermal growth
factor receptor (EGFR) in caveolae that are immunoseparated from
possible contaminating rafts. A and B: caveolae
(fractions 4 and 5) prepared from cardiac
ventricles (see Fig. 1) were purified on anti-Cav-3-coated magnetic
beads (see MATERIALS AND METHODS) and immunoblotted for the
indicated proteins. In B, the results with control beads,
which were similar to those in A, are not shown.
C: caveolae were treated with peptide
N-glycosidase F (PNGase F) (40) before being
subjected to immunoblot analysis with monoclonal anti- 1
antibody.
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Caveolin-containing membranes of low buoyant density may also be
obtained by fractionation of homogenates in the presence of Triton
X-100, because of the insolubility of caveolins in this detergent at
4°C (1, 36). It is now widely recognized, however, that
detergents may remove some caveolar proteins (1, 36, 37).
When cardiac caveolae were prepared by the carbonate-based procedure in
the presence and absence of 1% Triton X-100, comparable levels of
caveolin-3 were detected in both preparations as expected, but
Na+/K+-ATPase subunits were absent from the
detergent-treated caveolae (Fig. 6). This
explains why some studies (9, 18, 33), in which Triton
X-100 was used, failed to detect Na+/K+-ATPase
subunits in caveolae (see DISCUSSION).

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Fig. 6.
Effects of Triton X-100 on the caveolar contents of Cav-3
and Na+/K+-ATPase subunits. Cardiac ventricular
samples were fractionated by the carbonate-based procedure in the
presence and absence of Triton X-100 (see MATERIALS AND
METHODS). The combined fractions 4 and 5 were immunoblotted for the indicated proteins.
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Another widely used detergent-free procedure for the preparation of
caveolae involves the fractionation of sonicated plasma membrane
preparations on OptiPrep gradients (36, 42). We deemed it
necessary, therefore, to test for the presence of
Na+/K+-ATPase in cardiac caveolae prepared by
this procedure. Immunoblots of such caveolae made from the plasma
membranes of rat and bovine cardiac ventricles, prepared as described
in MATERIALS AND METHODS, showed the presence of
Na+/K+-ATPase subunits along with caveolin-3
(not presented).
Showing the presence of Na+/K+-ATPase subunits
in caveolae does not establish that this pool is catalytically
competent, especially because the lipid composition of the caveolae is
known to be significantly different from that of the bulk plasma
membrane (1, 35), and because
Na+/K+-ATPase activity is known to be dependent
on the nature of phospholipids and the level of membrane cholesterol
(3, 34, 49). It was important, therefore, to know whether
the Na+/K+-ATPase subunits detected in caveolar
fractions exhibit enzyme activity. Caveolae prepared by the
carbonate-based procedure are not suitable for the assay of
Na+/K+-ATPase activity due to the high
alkalinity of the preparative medium and its content of vanadate and
fluoride (see MATERIALS AND METHODS), all of which have
inhibitory effects on this activity. However, having established that
the OptiPrep-containing media do not inhibit
Na+/K+-ATPase (not shown), we were able to
detect ouabain-sensitive ATPase activity in cardiac caveolae prepared
by the OptiPrep procedure (Fig. 7). We
also compared this activity and the
-subunit content of caveolae
with those of the sarcolemma used for the preparation of caveolae. The
-subunit was enriched in caveolae relative to sarcolemma, but the
specific activity of caveolar ouabain-sensitive activity was lower than
that of sarcolemma (Fig. 7). The cause of this remains to be determined
(see DISCUSSION).

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Fig. 7.
Na+/K+-ATPase activities (A) and
the 1-subunit contents (B) of cardiac
sarcolemma and caveolae prepared from the sarcolemma by the OptiPrep
procedure. Partially purified sarcolemma were prepared from bovine
heart ventricles and fractionated by the OptiPrep procedure to obtain a
light caveolae fraction (see MATERIALS AND METHODS).
Multiple caveolae preparations from the same batch of sarcolemma were
combined to allow the assay of Na+/K+-ATPase
activity. This activity and quantitation of the immunoblots were also
performed (see MATERIALS AND METHODS). *P < 0.05 compared with sarcolemma.
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Caveolar localization of
Na+/K+-ATPase
in cells other than cardiac myocytes.
To determine whether the caveolar localization of
Na+/K+-ATPase is a peculiarity of cardiac
myocytes, we used the carbonate-based procedure to fractionate samples
of LLC-PK1 cells, HEK-293 cells, and the outer medullas of
rat kidney and pig kidney. Fraction immunoblots (see Fig.
8) for HEK-293 cells and the pig kidney outer medulla clearly indicate the presence of
Na+/K+-ATPase subunits and Src in the light
fractions containing caveolin-1. As expected, no muscle-specific
caveolin-3 was detected in these preparations (not shown). In these
experiments, like those with cardiac preparations,
Na+/K+-ATPase subunits were not as restricted
to the light fractions as caveolin (Fig. 8). The results with
LLC-PK1 cells and the rat kidney medulla (not shown) were
similar to those of Fig. 8, A and B,
respectively, demonstrating the presence of significant amounts of
Na+/K+-ATPase subunits in caveolin-containing
light fractions 4 and 5.

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Fig. 8.
Localization of Na+/K+-ATPase subunits in
Cav-1-rich domains of human embryonic kidney (HEK)-293 cells
(A) and pig kidney outer medulla (B). Sample
fractionations were done as indicated in Fig. 1. Total protein
distribution patterns (not shown) were similar to those shown in Fig.
1. For the immunoblots of the HEK-293 cell lysate, an equal volume from
each fraction was used; for the pig kidney lysate, an equal amount of
protein from each fraction was blotted.
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|
Kidney epithelial cells are known to be rich sources of
Na+/K+-ATPase. It was of interest, therefore,
to estimate the fraction of the total cellular content of
Na+/K+-ATPase that is localized in the caveolar
fractions of such cells. In experiments similar to those of Fig. 4,
fractionations of four different samples of HEK-293 cells were done,
and distribution of the
1-subunit in various fractions
was quantitated from blots and protein contents of the fractions also
as described for experiments of Fig. 4. The light fractions
4 and 5 contained 55.9 ± 3.3% (means ± SE;
n = 4) of the total cellular content of the subunit.
Comparison of this with the significantly lower value found in cardiac
preparations (Fig. 4) indicates cell-specific differences in caveolar
contents of Na+/K+-ATPase and suggests that the
abundance of a membrane protein may be a significant factor in its
relative distribution between the caveolae/raft microdomains, the bulk
plasma membrane, and the internal membranes (27).
Ouabain effects on cardiac caveolar pools of ERK1/2, Src, and
Na+/K+-ATPase.
To begin the assessment of the role of the caveolar pool of
Na+/K+-ATPase in the signal-transducing
function of the enzyme, we focused our initial studies on the cardiac
enzyme. We had shown recently (26) that ouabain-induced
increase in the contractility of the intact heart is accompanied by the
activation of the proximal signaling events leading to ERK1/2
activation. Therefore, we exposed the isolated rat hearts to an ouabain
dose that produces positive inotropy but no toxicity, froze the control
and the exposed hearts at the peak of the ouabain effect on
contractility, prepared the caveolae from ventricular samples, and
determined the caveolar contents of ERK1/2. The results (Fig.
9) showed that ouabain did not change the
caveolar contents of ERK1/2 proteins but that the caveolar contents of
the phosphorylated ERK1/2 were significantly increased by ouabain. It
has been previously demonstrated (13, 15) that such an
increase in the ratio of phosphorylated ERK1/2 to total ERK1/2 is
indicative of ERK1/2 activation. We also compared the caveolar contents
of caveolin-3, Src, and
1- and
2-subunits of Na+/K+-ATPase in the ouabain-treated and the
control hearts. Ouabain did not change the caveolar contents of
caveolin-3 and the
1-subunit, but it caused significant
increases in the caveolar contents of the
2-subunit and
Src (Fig. 10).

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Fig. 9.
Effects of ouabain-induced positive inotropy on the
cardiac caveolar contents of phosphorylated ERK1/2 and total ERK1/2.
Isolated rat hearts were exposed to 50 µM ouabain for 10 min to
induce maximal increase in contractility (see MATERIALS AND
METHODS). Ventricular samples of control and treated hearts (8 pairs) were fractionated as in Fig. 1, and caveolae (fractions
4 and 5) were quantitated for total immunoreactive
ERK1/2 protein and immunoreactive phosphorylated ERK1/2 (active ERK1/2)
as indicated in MATERIALS AND METHODS. *P < 0.05 compared with control.
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Fig. 10.
Effects of ouabain-induced positive inotropy on the
cardiac caveolar contents of Cav-3, Src, and the 1- and
2-isoforms of Na+/K+-ATPase.
Experiments were performed as indicated in Fig. 9 and MATERIALS
AND METHODS. Representative blots are shown at bottom,
and quantitation of such blots from experiments on multiple hearts is
shown at top. The number of pairs of control (Con) and
ouabain (Oua)-treated caveolar preparations immunoassayed for the
indicated proteins were as follows: Cav-3, n = 4;
1-subunit, n = 9;
2-subunit, n = 13; and Src,
n = 9. *P < 0.05 compared with
control.
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|
 |
DISCUSSION |
A major new finding reported here is the demonstration of the
presence of a significant pool of Na+/K+-ATPase
in the caveolae microdomains of the plasma membrane of several
different cell types. It is appropriate, therefore, that at the outset
we address the apparent discrepancy between this finding and those of
previous reports. In several studies where caveolae were reported not
to contain Na+/K+-ATPase (9, 18,
33), the caveolar fractions were prepared in the presence of
Triton X-100. Our experiments (Fig. 6) show that
Na+/K+-ATPase, like numerous other caveolar
proteins (1, 36, 37), is indeed solubilized by Triton
X-100 and removed from caveolin-containing fractions. In two other
studies (5, 48), caveolae that were prepared from cardiac
myocytes by the same detergent-free and carbonate-based procedure that
we have used here were reported not to contain
Na+/K+-ATPase. Detection of the enzyme in one
of these studies (5) was attempted through the assay of
[3H]ouabain binding. Because rat cardiac myocytes were
used, and the predominant Na+/K+-ATPase isoform
of these cells (
1) is known to be relatively insensitive
to ouabain (its Kd being ~10 µM; Refs.
20, 46), it is not surprising that the
caveolar presence of the enzyme was missed. In the other study
(48), also on rat cardiac myocytes, we suspect that
Na+/K+-ATPase was not detected in caveolae
because of the inadequate sensitivity of the particular Western blot
analysis used.
Cardiac caveolar
Na+/K+-ATPase
and signal transduction.
To explore the role of caveolar
Na+/K+- ATPase in signal transduction, we
limited our initial studies to cardiac
Na+/K+-ATPase because most of our previous work
on ouabain-induced signaling had been done on cardiac myocytes and the
intact heart (26, 45). Because it is now evident that the
rapid proximal signal pathways emanating from cardiac
Na+/K+- ATPase regulate the effect of ouabain
on cardiac contractility (26, 41), we sought evidence for
the occurrence of these proximal pathways in the caveolae of the
isolated Langendorff heart where the unambiguous positive inotropic
effect of ouabain may be demonstrated.
Our previous studies on cultured cardiac myocytes and isolated heart
preparations have identified the proximal pathways that link
Na+/K+-ATPase to ERK1/2, as depicted in Fig.
11, and have established that
activation of these pathways accompanies ouabain-induced positive
inotropy (26). Significantly, our studies have
also shown the existence of a positive feedback mechanism within this pathway: the requirement of the ouabain-induced rise in
[Ca2+]i for ERK1/2 activation (13,
25), and the necessity of ouabain-induced ERK1/2 activation for
the rise in [Ca2+]i (41).
Because a ouabain-induced rise in [Ca2+]i is
known to be due to the cooperation of the pumping function of
Na+/K+-ATPase and the transport functions of
the neighboring Na+/Ca2+ exchanger (22,
23, 41) and the voltage-regulated Ca2+ channels
(21, 41), we must conclude that the signal-transducing function of cardiac Na+/K+-ATPase, its ion
pumping function, and the effects of these on the regulation of
[Ca2+]i are tightly coupled within the cycle
shown in Fig. 11. It is intuitively obvious that colocalization of the
multiple protein components of this cycle within a restricted
microdomain would be more conducive to rapid and specific
stimulus-induced activation of the cycle than the random dispersion of
the component proteins within the plasma membrane. Of the indicated
proteins of Fig. 11, localization of the following in the caveolae of
cardiac myocytes has been indicated by previous studies: PKC, Raf, MEK,
and ERK1/2 (31, 32); Ras, growth factor rececptor-bound
protein (4); Na+/Ca2+ exchanger
(2); and Ca2+ channels (19). Our
data (Figs. 1, 2, and 5) add the subunits of cardiac
Na+/K+-ATPase, Src, and EGFR to this list and
confirm the previous findings on the caveolar presence of ERK1/2. More
importantly, our data show that the constant caveolar pool of ERK1/2 is
indeed activated in response to ouabain (Fig. 9), clearly indicating
that concomitant with the development of the positive inotropy in the
intact heart, the activation of the entire cycle of Fig. 11 may occur
within the caveolae.

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Fig. 11.
The postulated integration of the signal-transducing and
ion-pumping functions of Na+/K+-ATPase and the
control of intracellular Ca2+ concentration
([Ca2+]i) by these functions within cardiac
caveolae microdomains (see DISCUSSION). Grb2, growth factor
receptor-bound protein-2; SOS, mammalian homologue of
son-of-sevenless.
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|
Stimulus-induced movements of several receptors into or out of caveolae
have been demonstrated both in cardiac myocytes and other cell types
(5, 14, 24), and in some cases such traffic has been shown
to be relevant to signaling by the receptor. Our data show no effect of
ouabain on the caveolar traffic of the predominant housekeeping isoform
of Na+/K+-ATPase (
1) but clearly
indicate the ouabain-induced recruitments of Src and the
2-isoform of Na+/K+-ATPase to
caveolae (Fig. 10). This preferential ouabain-induced movement of the
less abundant
2-isoform is of particular interest in
view of the previous suggestions on the possibility of a special role
of the minor isoforms in the manifestation of the classic effects of
ouabain on cardiac contractility. Because the positive inotropic effect
of ouabain involves the cooperative functions of
Na+/K+-ATPase and
Na+/Ca2+ exchanger, and because several studies
have indicated the preferential concentration of the exchanger in the T
tubules of the adult myocyte (23, 47), the possibility was
considered (23) that a similar localization of the
2-isoform, but not that of the
1-isoform, in T tubules would make the minor isoform the preferred partner for the
Na+/Ca2+ exchanger in the regulation of
[Na+]i and [Ca2+]i.
The findings of immunocytochemical studies, however, did not support
this attractive hypothesis, showing the uniform distribution of the
2-isoform in T tubules and the peripheral plasma
membrane of the adult cardiac myocytes (23). On the other
hand, similar studies on cultured cells other than cardiac myocytes
(12) have indicated the preferential localization of the
minor isoforms (
2 and
3) of
Na+/K+-ATPase in areas of plasma membrane close
to endoplasmic reticuli/sarcoplasmic reticuli (ER/SR), leading to the
suggestion that ouabain effects on [Ca2+]i in
these cells may be through the inhibition of the minor isoforms that
communicate with ER/SR within a restricted space. Evidence for
a specific role of the
2-isoform in the regulation of
cardiac contractility has also been obtained from studies on cardiac
function of transgenic mice with altered levels of the cardiac
Na+/K+-ATPase isoforms (10), and
the localization of the
2-isoform in a restricted space
of cardiac myocyte plasma membrane has been postulated despite of the
lack of evidence for the preferential concentration of this isoform in
T tubules. Perhaps in adult cardiac myocytes the fraction of the
caveolae that is located on the T tubules is indeed the restricted
space from where the caveolar Na+/K+-ATPase
communicates with the SR. If so, our findings (Fig. 10) suggest that
the preferential localization of the
2-isoform in these
caveolae may be induced by ouabain or other stimuli rather than being
representative of the unstimulated state. The testing of this
hypothesis requires the extension of the present work using
experimental approaches different from those used here. Clearly, our
findings open new avenues to further studies on the differential roles
of the Na+/K+-ATPase isoforms in the control of
cardiac function.
The ouabain-induced recruitment of excess Src to caveolae (Fig. 10),
along with that of the
2-isoform, is not surprising
because Src seems to be the closest partner of the ouabain-stimulated Na+/K+-ATPase (7, 8). The evidence
that Src and other Src family kinases are normal residents of caveolae
in cells other than cardiac myocytes was established long ago (1,
30, 35). There is evidence to suggest that caveolar Src is
inhibited by its interaction with caveolins (17), but it
is also known that activated caveolar Src participates in signaling by
some caveolar receptors (15, 24). To our knowledge,
however, there is no prior evidence for stimulus-induced recruitment of
Src to caveolae similar to that noted here. Though ouabain-induced
activation of Src is essential to signaling by
Na+/K+-ATPase (7, 8), it is not
evident why the resting level of caveolar Src is not sufficient for
interaction with caveolar Na+/K+-ATPase and why
an excess of Src is recruited. Clarification of the mechanism of
interaction of Src with caveolar Na+/K+-ATPase
isoforms also requires further study.
The caveolar pool of Na+/K+-ATPase that is
postulated to participate in the ouabain-induced events of Fig. 11
should have Na+/K+-ATPase activity and the
associated pumping function in the absence of ouabain. This is
supported by the limited but important data of Fig. 7 showing that the
cardiac caveolar pool indeed exhibits ouabain-sensitive
Na+/K+-ATPase activity. This pool, however,
also has a lower specific activity and a higher
-subunit content
than the total plasma membrane pool (Fig. 7). Although the cause of
this may prove to be trivial, an intriguing possibility is that the
different pools within the bulk plasma membrane and the caveolae
microdomains may have different ATPase and transport properties. This
raises the question of whether there is independent evidence for the functional heterogeneity of Na+/K+-ATPase. In
fact, even in the highly purified preparations of the membrane-bound
Na+/K+-ATPase that have been used extensively
for studies on the reaction mechanism, there is ample evidence for such
heterogeneity and some support for lipid-phase heterogeneity being the
cause of this (29). Because cholesterol has been shown to
have a biphasic effect (activating followed by inhibitory) on
Na+/K+-ATPase activity (49), and
because caveolae are known to have significantly higher cholesterol
content than the bulk plasma membrane (1, 35), it is
reasonable to suspect a role of cholesterol, and/or caveolin-3, in the
regulation of the pumping function of cardiac caveolar
Na+/K+-ATPase. Studies on the possibility of
detecting the transport function of this pool and on the potential
differences between the hydrolytic and the transport functions of the
caveolar and the noncaveolar pools of cardiac
Na+/K+-ATPase are in progress.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Heart, Lung, and Blood
Institute Grants HL-36573, HL-67963, and HL-63238.
 |
FOOTNOTES |
*
L. Liu and K. Mohammadi contributed equally to this work.
Address for reprint requests and other correspondence: A. Askari, Dept. of Pharmacology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804 (E-mail:
mheck{at}mco.edu).
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.
First published February 26, 2003;10.1152/ajpcell.00555.2002
Received 26 November 2002; accepted in final form 13 February 2003.
 |
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Am J Physiol Cell Physiol 284(6):C1550-C1560
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