1 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201; 2 Department of Zoology, Miami University, Oxford, Ohio 45056; and 3 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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The role of the Na+ pump
2-subunit in Ca2+ signaling was examined in
primary cultured astrocytes from wild-type
(
2+/+ = WT) mouse fetuses and those
with a null mutation in one [
2+/
= heterozygote (Het)] or both [
2
/
= knockout (KO)]
2 genes. Na+ pump catalytic
(
) subunit expression was measured by immunoblot; cytosol
[Na+] ([Na+]cyt) and
[Ca2+] ([Ca2+]cyt) were
measured with sodium-binding benzofuran isophthalate and fura 2 by
using digital imaging. Astrocytes express Na+ pumps
with both
1- (
80% of total
) and
2- (
20% of total
) subunits. Het astrocytes
express
50% of normal
2; those from KO express none.
Expression of
1 is normal in both Het and KO cells.
Resting [Na+]cyt = 6.5 mM in WT, 6.8 mM
in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells
(P < 0.001); 500 nM ouabain (inhibits only
2) equalized [Na+]cyt at 8 mM
in all three cell types. Resting
[Ca2+]cyt = 132 nM in WT, 162 nM in Het,
and 196 nM in KO cells (both P < 0.001 vs. WT).
Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)
Ca2+ pumps and unloads the ER, induces transient (in
Ca2+-free media) or sustained (in Ca2+-replete
media) elevation of [Ca2+]cyt. These
Ca2+ responses to 10 µM CPA were augmented in Het as well
as KO cells. When CPA was applied in Ca2+-free media, the
reintroduction of Ca2+ induced significantly larger
transient rises in [Ca2+]cyt (due to
Ca2+ entry through store-operated channels) in Het and KO
cells than in WT cells. These results correlate with published evidence
that
2 Na+ pumps and
Na+/Ca2+ exchangers are confined to plasma
membrane microdomains that overlie the ER. The data suggest that
selective reduction of
2 Na+ pump activity
can elevate local [Na+] and, via
Na+/Ca2+ exchange, [Ca2+] in the
tiny volume of cytosol between the plasma membrane and ER. This, in
turn, augments adjacent ER Ca2+ stores and thereby
amplifies Ca2+ signaling without elevating bulk
[Na+]cyt.
astrocytes; catalytic subunit; fura 2; sodium-binding benzofuran isophthalate; sodium-potassium-adenosine 5'-triphosphatase isoforms; transgenic mice
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INTRODUCTION |
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NUMEROUS PHYSIOLOGICAL PROCESSES are regulated by cytosolic Ca2+ signals in all cells. It is, therefore, important to understand how these signals are controlled. To this end, we studied the influence of Na+ pump (Na+-K+-ATPase) expression on the regulation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) and the control of Ca2+ signaling in primary cultured mouse cortical astrocytes.
The rationale for these studies is the evidence that both a plasma membrane (PM) ATP-driven Ca2+ pump (PMCA) (19) and a PM Na+/Ca2+ exchanger (NCX) (10) help to control resting [Ca2+]cyt in astrocytes (7) as in many other types of cells. The NCX is regulated by the Na+ pump via its influence on the Na+ electrochemical gradient across the PM. Most of the intracellular Ca2+ in quiescent cells is stored in the endoplasmic reticulum (ER). By controlling [Ca2+]cyt, the PMCA and NCX indirectly influence the ER Ca2+ store content. During cell activity, much of the "signal Ca2+" comes from the ER stores, although some also enters the cells through PM Ca2+-permeable channels.
The Na+ pump consists of - and
-subunits in a 1:1
ratio (5, 24). The
-subunit is a highly glycosylated
40- to 60-kDa protein that may be involved in chaperoning and membrane
trafficking of the larger (
112 kDa)
-subunit (12).
The
- (catalytic) subunit contains the Na+,
K+, and ATP binding (and hydrolytic) sites, as well as a
binding site for cardiotonic steroids such as ouabain, which inhibits the pump (5, 31). Four Na+ pump
-subunit
isoforms have been identified:
1,
2,
3 (41, 44, 46), and
4
(47). The latter is found only in the testis and will not
be discussed here. Most cells express
1 and one of the
other isoforms, all of which have different kinetic properties. The
1 has a higher affinity for Na+
[KNa(
1) = 12mM]
than
2 and
3
[KNa(
2) and
KNa(
3) = 22 and 33mM, respectively] (Ref. 48; see also Refs. 40
and 45). In rodents, the
1-isoform has an
especially low affinity
[KI(
1) > 10µM] for ouabain; in contrast, the
2- and
3-isoforms have high affinity [K I(
2) < 0.1 µ M] for ouabain (5, 35). Moreover, these isoforms are
differently distributed in the PM (25, 26). In at least
several types of cells (astrocytes, neurons, and arterial myocytes),
2 and
3 are confined to PM microdomains
that overlie sarcoplasmic reticulum or ER (S/ER). In contrast,
1 is more uniformly distributed in the PM of these cells
(25, 26). It is noteworthy that the NCX also is confined
to PM microdomains that overlie the S/ER, whereas the PMCA is
more uniformly distributed (25, 28).
These observations led to the suggestion (6, 8) that
low-dose ouabain might regulate cell Ca2+ signaling by
inhibiting only 2 or
3 and controlling
the [Na+] primarily in the tiny ("junctional") space
between the aforementioned PM microdomains and the subjacent junctional
S/ER. Thus Na+ pumps with
2- or
3-subunits would be expected to regulate, via NCX, not
only the local [Ca2+] in this junctional space (JS), but
also the [Ca2+] in the junctional S/ER that plays a key
role in Ca2+ signaling. To test this hypothesis, we
measured the bulk cytosolic concentrations of Na+
([Na+]cyt) and
[Ca2+]cyt in resting astrocytes and the rise
in [Ca2+]cyt induced by blocking the S/ER
Ca2+ pump (SERCA). Astrocytes express only
1 and
2 Na+ pump isoforms
(26, 46). Therefore, these parameters were studied in
astrocytes from normal [wild-type (WT)] mice and from mice missing
one or both of the high
KI(
2) alleles (23).
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METHODS |
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Knockout Mice
Mice with null mutations in one or bothAstrocyte Cell Cultures
Astrocyte primary cultures were initiated from embryonic day 18-19 mouse cerebral cortex by using a modification of the method of Booher and Sensenbrenner (11) as described (17). The cortex was separated from the meninges and the hippocampus and was placed in culture medium [DMEM-F12 (1:1) with 10% FBS, penicillin G (50 U/ml), and streptomycin (50 µg/ml)]. The cells from each mouse cortex were mechanically dissociated by sequential passage of the cortex through 80- and 10-µm nylon mesh. The resulting cell suspension was plated onto poly-L-lysine-coated 25-mm glass coverslips (Immunoblot Analysis of Expressed
Na+ Pump -Subunit Isoforms
Membrane preparation.
Mouse astrocytes were cultured in 10-cm dishes for 2 wk. Cells were
then harvested with buffer containing 140 mM NaCl and 25 mM
imidazole-HCl (pH 7.4) and were pelleted (3,000 g, 4°C, 20 min). The cell pellet was resuspended in lysis buffer containing (in
mM) 140 NaCl, 2 EDTA, 10 sodium azide, 20 Tris base, and 250 sucrose;
the buffer also included four tablets per 100 ml of a complete protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The
resuspended cells were homogenized with a Polytron (Brinkmann,
Westbury, NY), and the homogenate was centrifuged at 480 g
(4°C, 30 min). The supernate was centrifuged at 17,200 g
(4°C, 30 min) to pellet membrane fragments and vesicles. The membrane
pellet was then resuspended in lysis buffer with 1% deoxycholate and
1% Triton X-100 and incubated on ice for 30 min. After centrifugation (17,200 g, 4°C, 20 min), the supernatant fluid containing
membrane proteins was collected and stored at 80°C. The protein
concentration was determined with the bicinchoninic acid assay (Bio-Rad
Laboratories, Richmond, CA) by using bovine serum albumin as a standard.
Skeletal muscle membrane preparation.
Male mice, 12 wk old, were used for quantitation of Na+
pump -isoform expression. Extensor digitorum longus (EDL) muscles from both legs were dissected and frozen (
80°C) for later use. EDL
muscles (4-6 from each genotype: WT and
2+/
) were homogenized with a Polytron in
1-ml ice-cold homogenization buffer [in mM: 250 sucrose, 30 imidazole
(pH 7.5), and 1 EDTA]. The homogenates were centrifuged at 3,000 g, 4°C, for 20 min to remove cellular debris. The
supernatants were centrifuged at 17,200 g, 4°C, for 30 min. The membrane pellet was resuspended in lysis buffer containing
protease inhibitors, 1% deoxycholate, and 1% Triton X-100 and treated
as in the preceding section.
Immunoblot analysis.
Membrane (PM) proteins were solubilized in sodium dodecyl sulfate (SDS)
buffer containing 5% 2-mercaptoethanol and were separated by 7.5%
polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then
transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ);
transfer was checked by Ponceau S staining. The membranes were blocked
with 5% nonfat dry milk in Tris-buffered saline with 137 mM NaCl, 20 mM Tris, pH 7.6, and 0.1% Tween 20 for at least 2 h at room
temperature. Nitrocellulose membranes were incubated overnight at room
temperature with polyclonal antibodies raised against Na+
pump 1- or
2-subunit isoforms, or with an
-subunit isoform nonspecific antibody (gifts of Dr. Thomas
Pressley). Some membranes were probed with monoclonal or polyclonal
antibodies raised against the cardiac/neuronal NCX, NCX1 (R3F1, a gift
from Dr. Kenneth Philipson;
11-13 from Swant, Bellinzona,
Switzerland). In some cases, gel loading was controlled with polyclonal
or monoclonal anti-actin antibodies (Sigma Chemical, St. Louis, MO).
Quantitation of Na-K-ATPase isoform levels.
The band intensities of the immune complexes on the film were scanned
(Epson Expressions, Epson America, Long Beach, CA) and quantified by
densitometry with the use of Kodak ID image analysis software (Kodak
Digital Science, Eastman Kodak). Samples containing various amounts of
membrane protein were analyzed in a single blot, and each blot was
exposed for two to three different times to ensure linearity of signal
intensity. Changes in band densities (relative to WT band densities)
for 1 and
2 were measured with isoform-specific polyclonal antibodies and were correlated with changes
in
1 +
2 measured with a
nonselective polyclonal antibody ("LEAVE"; see Ref.
37). We assume, as did He et al. (20), who
used different antibodies, that the nonselective antibody cross-reacts
equally well with the two
-subunit isoforms after they are unfolded
in SDS buffer.
Immunocytochemistry.
Primary cultured mouse cortical astrocytes were fixed and cross-reacted
with polyclonal or monoclonal antibodies raised against Na+
pump 1- or
2-subunit isoforms (gifts of
Drs. Thomas Pressley and Kathleen Sweadner; Refs. 13,
37, 46). The primary antibodies were then
cross-reacted with fluorescent-labeled secondary antibodies: Alexa-Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for monoclonal antibodies and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).
This enabled us to visualize the distribution of the primary label with
a fluorescence microscope (Nikon Diaphot TMD; Nikon, Melville, NY).
Details are published (26).
Fluorescent dye loading. Astrocytes on coverslips were loaded with the Ca2+-sensitive fluorochrome fura 2 by incubation for 30 min in medium containing 3.3 µM fura 2-AM (22-24°C, 5% CO2-95% air). Alternatively, cells were loaded with the Na+-sensitive fluorescent dye sodium-binding benzofuran isophthalate (SBFI) by incubation for 1 h at 22-24°C in medium containing 10 µM SBFI-AM. After loading with either dye, the coverslips were transferred to a tissue chamber mounted on a microscope stage, where they were superfused for 15-20 min (35-36°C) with standard physiological salt solution to wash away extracellular dye.
Digital imaging methods. Fura 2 fluorescence (510 nm emission; 380 and 360 nm excitation) and SBFI fluorescence (510 nm emission; 340 and 380 nm excitation) were imaged with a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY). The dye-loaded cells were illuminated with a diffraction grating-based system (Polychrome II, Applied Scientific Instruments, Eugene, OR) (18). Fluorescent images were recorded by using a Gen III ultrablue intensified charge-coupled device camera (Stanford Photonics, Palo Alto, CA). Image acquisition and analysis were performed with a MetaFluor/MetaMorph Imaging System (Universal Imaging, Chester, PA). Images were captured at rates of one per minute (under resting conditions) to one per second (when Ca2+ was changing rapidly); eight frames were averaged to improve the signal-to-noise ratio in each image. [Ca2+]cyt was calculated by determining the ratio of fura 2 fluorescence excited at 380 and 360 nm as described (17). [Na+]cyt was calculated by determining the ratio of SBFI fluorescence excited at 340 and 380 nm. SBFI calibration was carried out in the cells, in situ, at the end of each experiment, as described (17).
Solutions. The standard physiological salt solution contained (in mM) 140 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES (titrated to pH 7.4 with NaOH). In Ca2+-free solutions, CaCl2 was omitted, and 50 µM EGTA was added. Stock solutions of fura 2-AM (1 mM) and SBFI-AM (10 mM) were prepared in DMSO.
Materials. FBS was obtained from Paragon Bioservices (Baltimore, MD); all other tissue culture reagents were obtained from GIBCO-BRL (Grand Island, NY). Fura 2-AM and SBFI were obtained from TefLabs (Austin, TX). Ouabain, cyclopiazonic acid (CPA), DMSO, poly-L-lysine, DAPI, penicillin G, and streptomycin were purchased from Sigma. All other reagents were analytic grade or the highest purity available.
Data analysis. The numerical data presented in RESULTS are the means from n single cells (one value per cell). The numbers of different animals and different litters are also presented, where appropriate. Data from two to three litters were obtained for most protocols and were consistent from litter to litter. Student's t-tests for paired or unpaired data were used to calculate the significance of the differences between means.
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RESULTS |
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Expression of Na+ Pump -Subunit
Isoforms in Cultured Astrocytes
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The Na+ pump 2-isoform in astrocytes is
confined to PM microdomains that overlie ER, where it colocalizes with
the NCX (24), with which it is functionally coupled (see
below). Several groups have reported that the Na+ pump
2-isoform and the NCX are reciprocally regulated under some conditions (e.g., Ref. 32; reviewed in Ref.
10). Therefore, we also compared NCX expression in the
astrocytes from WT and
2 Het and KO mice. Surprisingly,
NCX expression was not significantly upregulated in Het and KO
astrocytes (Fig. 2).
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Quantitation of Na+ Pump
2-Isoform Expression
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When this isoform-nonselective antibody was tested on astrocytes,
however, there was no detectable decline in the Western blot total
-subunit band density in Het astrocytes and a 20% decline in KO
astrocytes, compared with that in controls (Fig. 3). Because there is
little or no upregulation of the
1-isoform in
2 Het and KO astrocytes (Figs. 1C and 3), the
implication is that the
2-isoform accounts for no more
than ~20% of the total
-subunit in these cells.
Localization of 1 and
2
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The black-and-white images in Fig. 5A (top) show fura 2-stained astrocytes from WT, Het, and KO mice (left to right). The morphology of the cells from the three genotypes are all very similar.
Immunocytochemical data (26) reveal that the
2 epitope "colocalizes" with SERCA in astrocytes. As
shown below (Fig. 6), functional
2 is located in the PM in WT astrocytes because the
2 can be blocked with low-dose (500 nM) ouabain, a
hydrophilic, membrane-impermeant cardiotonic steroid. Thus these
Na+ pumps must be located in PM microdomains that
overlie sub-PM ER. Even with image deconvolution and reconstruction,
the z-axis resolution is only ~0.7 µm, vs. 0.25 µm in
the x- and y-axes (25). Therefore,
epitopes in the PM and in the underlying ER, <0.1 µm away, will
appear to colocalize (26), even though they are in different membranes.
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[Na+]cyt in Astrocytes From Het and KO Mice
The normal bulk [Na+]cyt was 6.5 ± 0.1 mM in quiescent WT astrocytes. The level was not significantly higher (6.8 ± 0.4 mM; P > 0.05) in Het cells. [Na+]cyt was, however, modestly, but significantly, elevated (8.0 ± 0.3 mM; P < 0.001) in quiescent cells from KO mice (Fig. 6B, left).To test further the roles of the 1- and
2-isoforms in maintaining
[Na+]cyt, the effects of 500 nM and 1 mM
ouabain were compared. The lower dose should block only the
2-isoform (IC50 = 20-100 nM), whereas 1 mM ouabain should block both isoforms in rodents
(
1 IC50 = 50-100 µM) (5,
35). As revealed by the representative data in Fig.
6A (solid lines and inset), 500 nM ouabain
increased [Na+]cyt only to 8.0 mM in WT
astrocytes after a 10-min incubation (blue line). It had no effect on
[Na+]cyt in the KO cells (red line) because
there was no
2, and the [Na+]cyt was already at this level. Data from
a number of such cells, and from Het cells, in which the findings were
similar to those in WT cells, are summarized in Fig. 6B,
left (controls) and middle (+500 nM ouabain).
In contrast to the low-dose ouabain, 1 mM ouabain increased
[Na+]cyt at comparable initial rates in all
three cell types. Representative data for a WT cell and a KO cell are
indicated by the dashed blue and red lines, respectively, in Fig.
6A. Data for a 10-min exposure to 1 mM ouabain are
summarized in Fig. 6B, right. Thus the
1-isoform is the primary determinant of bulk
[Na+]cyt, and
2 apparently has
only a minor influence on bulk [Na+]cyt.
Resting [Ca2+]cyt in Cells From Het and KO Mice
The distribution of [Ca2+] in quiescent cells from WT, Het, and KO cells is illustrated by the representative Ca2+ images in Fig. 5A. These images indicate that, on the average, resting [Ca2+]cyt is slightly elevated in cells from Het mice and even more so in cells from KO mice. A frequency histogram of [Ca2+]cyt values in WT and KO cells is presented in Fig. 5B. There is considerable overlap between the [Ca2+]cyt values in the two types of cells; nevertheless, it is clear that [Ca2+]cyt is skewed toward higher levels in the KO cells. The cell images in Fig. 5A reveal that the lowest [Ca2+]cyt values are observed in some WT cells, but not in any of the KO cells. Conversely, some KO cells have higher resting [Ca2+]cyt levels than do any WT cells. Although not illustrated in the histogram (Fig. 5B), the data from Het cells are intermediate between those of WT and KO cells.Resting [Ca2+]cyt data are summarized in Fig.
7B, left. The
average resting [Ca2+]cyt is 132 ± 2 nM
in the WT astrocytes (n = 670) and is elevated by
~23% in Het cells and by ~49% in KO cells. Particularly
noteworthy is the fact that resting [Ca2+]cyt
is significantly elevated in the Het cells (P < 0.001;
Fig. 7B), despite a normal
[Na+]cyt (Fig. 6).
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Releasable Ca2+ and Ca2+ Signaling in Cells From Het and KO Mice
Storage of Ca2+ in the ER is governed by the ambient [Ca2+]cyt and by SERCA. The [Ca2+] gradient across the ER membrane is maintained by SERCA. Thus, if resting [Ca2+]cyt is elevated in cells from Het and KO mice, we would expect to observe increased storage of (releasable) Ca2+ in the ER. Inhibition of SERCA by agents such as thapsigargin and CPA promotes the unloading of ER Ca2+ in astrocytes and thereby induces large, transient elevation of [Ca2+]cyt (17, 18; and see Ref. 38).Figure 7 illustrates the effects of a maximal effective
concentration of CPA (10 µM) on the elevation of
[Ca2+]cyt in cells from WT, Het, and KO mice
incubated in the presence of normal (1.8 mM) extracellular
Ca2+. The representative time course data from individual
cells in Fig. 7A show that the peak of the
[Ca2+]cyt transient is augmented, as is the
amplitude of the plateau (until CPA is washed out) in cells from Het as
well as KO mice. The initial peak Ca2+ transient
corresponds to Ca2+ release from the ER. The plateau
represents the balance between Ca2+ entry through
store-operated Ca2+ channels (SOCs) and removal of free
Ca2+ from the cytosol by PMCA and NCX and by mitochondria
and perhaps a CPA-insensitive SERCA (but see Ref. 18). The
SOCs are opened by ER Ca2+ store depletion (18,
38). The plateau depends on extracellular Ca2+
(Ref. 17, and see Fig.
8A) and on continued block of
SERCA by CPA (Fig. 7A). The summary data in Fig.
7B (right) reveal that the mean peak
[Ca2+]cyt transient elevation is
significantly greater in Het cells and KO cells than in WT cells.
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Comparable data for cells incubated in Ca2+-free medium are illustrated by the initial responses (left Ca2+ transients) in Fig. 8A. There is no plateau in this case, because the plateau (Fig. 7A) is maintained by Ca2+ entry through SOCs [i.e., capacitative Ca2+ entry (CCE)] (38). Neverthleless, the peak CPA-induced [Ca2+]cyt transient is augmented in both Het and KO cells, although the mean increase in the peak did not reach statistical significance in the Het cells (Fig. 8B, middle). When external Ca2+ is replaced, however, the Ca2+ transients that result from Ca2+ entry through SOCs are significantly greater in Het cells and KO cells than in WT cells (Fig. 8A, second Ca2+ transients; Fig. 8B, right). This augmentation can be explained if Ca2+ efflux via NCX is impaired and/or Ca2+ entry via NCX is enhanced because of local, sub-PM Na+ accumulation, as discussed below. In addition, there may be augmented Ca2+ entry through SOCs due to increased saturation of the ER Ca2+ stores in the Het and KO astrocytes (21).
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DISCUSSION |
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Astrocytes cultured from the brains of rodents normally express
both the 1- and
2-isoforms of the
Na+ pump catalytic subunit (26, 46). The fact
that
1 and
2 have very different kinetic
properties, especially their different affinities for Na+
and for ouabain (see Introduction), suggests that they have different functions. Here, using data from gene-targeted mice, we provide direct
evidence for this view.
Reduced 2 Expression Does Not Affect Cell
Morphology
Reduction of 2 Expression Has Little Effect on
"Bulk" [Na+]cyt
Reduced 2 Expression Elevates Resting
[Ca2+]cyt and Augments
Ca2+ Transients and CCE in Astrocytes
The hearts and skeletal muscle of 2 Het mice exhibit
increased contractility (20, 23). In contrast, in mice
with a null mutation in one
1 gene and one-half the
normal
1 expression, cardiac and skeletal muscle
contractility are reduced. Inhibition of
2 activity with
low-dose ouabain in the
1 Het then enhances the
contractility in both types of muscles, despite the further reduction
of total
-subunit activity (20, 23).
In summary, the aforementioned observations all demonstrate that
selective reduction of the activity of Na+ pumps with
2- or
3-subunits augments
Ca2+ signaling in a variety of cell types. The implication
is that a major role of these high-ouabain-affinity
-subunit
isoforms is the modulation of Ca2+ homeostasis and
Ca2+ signaling.
It is widely accepted that inhibition of Na+ pumps and
reduction of the [Na+] gradient across the PM
([Na+]o > [Na+]cyt, where
[Na+]o is extracellular [Na+]),
e.g., by ouabain, promotes Ca2+ entry via NCX in most types
of cells (10). Ouabain does not, however, promote
Ca2+ entry or augment Ca2+ signaling when NCX
expression is blocked by antisense oligonucleotides (42)
or by a null mutation (39). Furthermore, the
colocalization of NCX and Na+ pumps with 2-
(or
3-) subunits in astrocytes and other cell types
(25) is consistent with other evidence that the NCX and
2/
3 Na+ pumps are
tightly coupled (reviewed in Refs. 2,
10).
Reduction of 2 Activity Augments
Ca2+ Signaling by Regulating
[Na+] in a Sub-PM Cytosolic
Compartment
To explain how reduction of Na+ pump
2/
3-subunit activity can modify
Ca2+ homeostasis via NCX without detectable elevation of
bulk [Na+]cyt (in
2 Hets), we
must assume that these Na+ pumps regulate the local
[Na+] and, via NCX, the [Ca2+] in a
distinct subcompartment of cytosol. Diffusion of Na+ and
Ca2+ between this compartment and bulk cytosol must be
markedly restricted (2). Several investigators have
provided evidence that cardiotonic steroids exert their cardiotonic
effect by increasing [Na+] in a sub-PM cytosolic
compartment that functionally couples the NCX to the Na+
pump (14, 33, 43a). The presence of such a compartment
also explains how the low
KNa(
2) and
KNa(
3) can function while
the "housekeeping" high
KNa(
1) normally maintains
bulk [Na+]cyt well below 10 mM. This
compartment appears to be located between the PM and adjacent,
junctional ER (jER) (2, 7, 8). The unit, consisting of the
jER, the overlying PM microdomain, and the intervening tiny volume of
cytosol (in the JS; see Fig. 9), has been
named the "PLasmERosome" (8).
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Immunocytochemical data (16, 25, 26), as well as
preliminary coimmunoprecipitation data (29), indicate that
these PM microdomains contain Na+ pumps with
2- or
3- (but not
1-)
subunits, NCX, and transient receptor potential channel proteins, which
may be components of SOCs (22) (Fig. 9A). These
proteins and those in the jER and perhaps other, as yet unidentified
membrane proteins are all involved in Ca2+ signaling. In
contrast, other regions of the PM are rich in PMCA and Na+
pumps with
1-subunits (Fig. 9). Evidence that some SERCA
and inositol trisphosphate receptor isoforms coimmunoprecipitate with NCX or transient receptor potential channel proteins (29)
implies that the jER is structurally coupled to overlying PM microdomains.
This structural and functional coupling is illustrated in Fig. 9.
Figure 9B depicts the elevation of [Na+] and
[Ca2+] in the JS and the consequent rise in ER
[Ca2+] as a result of 2 KO (whether by a
null mutation or by low-dose ouabain).
This model can be used to explain the augmented external
Ca2+-dependent transient and sustained elevations of
[Ca2+]cyt evoked by ER Ca2+ store
depletion in Het and KO astrocytes (Figs. 7A and 8). These external Ca2+-dependent signals are presumably mediated by
Ca2+ entry through SOCs (18, 38). SOCs are
permeable to Na+ as well as Ca2+
(3). Thus, when the SOCs are opened in Het and KO cells,
Na+ will tend to accumulate in the tiny JS between the PM
and jER if Na+ extrusion through nearby 2
Na+ pumps is reduced or abolished. As a consequence,
Ca2+ extrusion should be reduced, and Ca2+
entry increased, through adjacent NCX. The resultant local accumulation of Ca2+ could account for enhanced filling of jER
Ca2+ stores, as well as spillover to bulk cytosol, and thus
the rise in resting [Ca2+]cyt and the
augmented Ca2+ signals.
PLasmERosomes obviously play a key role in regulating Ca2+ signaling. Therefore, it seems appropriate to refer to them as "Ca2+ signaling complexes." Indeed, preliminary Ca2+ imaging studies indicate that SOC-mediated Ca2+ signals are apparently initiated in these Ca2+ signaling complexes (16). Clearly, a major task for future studies is to test this hypothesis directly.
Summary and Conclusion
One of the key results of this study is that knockout of ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Thomas Pressley, Kathleen Sweadner, and Kenneth Philipson for generous supplies of antibodies, and Hugo Gonzales-Serratos for help with the dissection of EDL muscles.
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FOOTNOTES |
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This study was supported by National Institutes of Health grants NS-16106 and HL-45215 (to M. P. Blaustein), HL-41496 (to J. B. Lingrel), a Grant-in-Aid from the American Heart Association Mid-Atlantic Affiliate (to V. A. Golovina), and a Grant to Promote Research from Miami University (to P. F. James).
Address for reprint requests and other correspondence: V. A. Golovina, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201 (E-mail: vgolovin{at}umaryland.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 October 3, 2002;10.1152/ajpcell.00383.2002
Received 26 August 2002; accepted in final form 30 September 2002.
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