Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

Vera A. Golovina1, Hong Song1, Paul F. James2, Jerry B. Lingrel3, and Mordecai P. Blaustein1

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
INTRODUCTION
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The role of the Na+ pump alpha 2-subunit in Ca2+ signaling was examined in primary cultured astrocytes from wild-type (alpha 2+/+ = WT) mouse fetuses and those with a null mutation in one [alpha 2+/- = heterozygote (Het)] or both [alpha 2-/- = knockout (KO)] alpha 2 genes. Na+ pump catalytic (alpha ) 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 alpha 1- (approx 80% of total alpha ) and alpha 2- (approx 20% of total alpha ) subunits. Het astrocytes express approx 50% of normal alpha 2; those from KO express none. Expression of alpha 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 alpha 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 alpha 2 Na+ pumps and Na+/Ca2+ exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 alpha - and beta -subunits in a 1:1 ratio (5, 24). The beta -subunit is a highly glycosylated 40- to 60-kDa protein that may be involved in chaperoning and membrane trafficking of the larger (approx 112 kDa) alpha -subunit (12). The alpha - (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 alpha -subunit isoforms have been identified: alpha 1, alpha 2, alpha 3 (41, 44, 46), and alpha 4 (47). The latter is found only in the testis and will not be discussed here. Most cells express alpha 1 and one of the other isoforms, all of which have different kinetic properties. The alpha 1 has a higher affinity for Na+ [KNa(alpha 1) = 12mM] than alpha 2 and alpha 3 [KNa(alpha 2) and KNa(alpha 3) = 22 and 33mM, respectively] (Ref. 48; see also Refs. 40 and 45). In rodents, the alpha 1-isoform has an especially low affinity [KI(alpha 1) > 10µM] for ouabain; in contrast, the alpha 2- and alpha 3-isoforms have high affinity [K I(alpha 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), alpha 2 and alpha 3 are confined to PM microdomains that overlie sarcoplasmic reticulum or ER (S/ER). In contrast, alpha 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 alpha 2 or alpha 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 alpha 2- or alpha 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 alpha 1 and alpha 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(alpha 2) alleles (23).


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Knockout Mice

Mice with null mutations in one or both alpha 2 genes [i.e., heterozygotes (Het) = alpha 2+/-, and homozygote knockouts (KO) = alpha 2-/-, respectively] were generated as described (23). WT (alpha 2+/+) mice from the same litters were also studied. The mice were genotyped by Southern blot analysis of genomic DNA prepared from embryo tails (23). The Het mice appeared normal and developed normally into adults. The KO mice die very shortly after birth (23), but the fetuses appeared to be normal. All astrocytes were cultured from near-term fetuses.

Astrocyte 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 (approx 50,000 cells/coverslip). The medium was changed on days 4 and 7. The cells were characterized as protoplasmic (type 1) astroglial cells (4). Experiments were performed on subconfluent cultures on days 7-9 in vitro.

Immunoblot Analysis of Expressed Na+ Pump alpha -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 alpha -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 alpha 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 alpha 1- or alpha 2-subunit isoforms, or with an alpha -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; pi 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).

The nitrocellulose membranes were washed with Tris-buffered saline with Tween 20 and then incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 1 h. The immune complexes on the membranes were detected by enhanced chemiluminescence-plus (Amersham) and exposure to X-ray film (Eastman Kodak, Rochester, NY) for 30-60 s.

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 alpha 1 and alpha 2 were measured with isoform-specific polyclonal antibodies and were correlated with changes in alpha 1 + alpha 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 alpha -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 alpha 1- or alpha 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).

In some experiments, the cells were identified as astrocytes by labeling with polyclonal antibodies raised against glial fibrillary acidic protein (Boehringer Mannheim). In these experiments, nuclei also were identified by labeling for 5 min with a 50 µM solution of the nucleic acid stain, 4',6'-diamidino-2-phenylindole (DAPI).

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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Expression of Na+ Pump alpha -Subunit Isoforms in Cultured Astrocytes

The expression of Na+ pump alpha 1- and alpha 2-isoforms in astrocytes cultured from the brains of WT, Het, and KO mouse embryos is compared in Fig. 1. Figure 1A shows the alpha 2 genotype data from Southern blot analysis of the genomic DNA from the 12 embryos in a single litter. Figure 1B shows the Western blot analysis of the expressed alpha 2 protein from these same embryos. Astrocytes from Het mice express about one-half as much alpha 2 as do cells from WT mice, and cells from KO mice do not express any alpha 2. Figure 1C, which shows alpha 1 expression data from the same litter, indicates that astrocytes with all three genotypes express approximately equal amounts of the Na+ pump alpha 1-isoform. These Western blot data from primary cultured astrocytes are comparable to the results obtained in fresh cardiac tissue from alpha 2 WT, Het, and KO mice (23).


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Fig. 1.   A: Southern blot of genomic DNA from 12 mouse fetuses (numbered 1 through 12) from a pregnant mouse. The parents of these fetuses were both heterozygous for a null mutation of the Na+ pump alpha 2-subunit isoform (i.e., alpha 2+/-). B and C: Western blots of membranes from cortical astrocytes cultured from the 12 mouse fetuses analyzed in A. Membrane proteins (10 µg/lane) were separated by SDS-PAGE, blotted, and probed with anti-alpha 2 (B) or anti-alpha 1 (C) isoform polyclonal (PC) antibodies. The alpha 2- and alpha 1-isoform proteins (112 kDa) are indicated. The 2 right-hand lanes are controls from wild-type (WT) mouse brain (expresses alpha 1 and alpha 2) and kidney (expresses alpha 1, only). B: the alpha 2 knockout (KO; alpha 2-/-) astrocytes express no alpha 2 protein (lanes 5 and 8); alpha 2 heterozygote (Het; alpha 2+/-) cells express about one-half as much alpha 2 protein as do WT (alpha 2+/+) astrocytes. C: all 3 types of astrocytes express similar amounts of alpha 1. (Note: there was insufficient sample to test for alpha 1 expression in mouse 1.)

The Na+ pump alpha 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 alpha 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 alpha 2 Het and KO mice. Surprisingly, NCX expression was not significantly upregulated in Het and KO astrocytes (Fig. 2).


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Fig. 2.   Na+/Ca2+ exchanger (NCX) expression in astrocytes cultured from WT, Het, and KO mice. A: Western blot of data from cells from 8 fetuses; genotypes are given at the top. The membrane proteins were cross-reacted with a monoclonal (MC) antibody (R3F1) raised against NCX1 (approx 120 kDa, top row) and a PC antibody raised against actin (approx 48 kDa, bottom row). Right-hand lane is a "control" from mouse brain. B: averaged data from the experiment in A, corrected for protein loading and 2 other, similar experiments. Values are means of data from 5 WT, 11 Het, and 8 KO fetuses, normalized to the data from the WT fetuses (= 100%) in each experiment; error bars are SE. There is no significant difference in the expression of NCX in astrocytes from the three genotypes.

Quantitation of Na+ Pump alpha 2-Isoform Expression

To interpret the functional effects of reduced alpha 2-isoform expression, it is important to know what fraction of the total Na+ pump alpha -subunit (alpha 1 + alpha 2) is alpha 2. This information cannot be obtained directly from Western blots cross-reacted with the isoform-specific antibodies. It should be possible, however, to obtain some quantitative information by also using an antibody that is not isoform specific and that recognizes both alpha 1 and alpha 2 (LEAVE) (37). In control experiments on mouse skeletal muscle (which also expresses only alpha 1 and alpha 2), we found that, in alpha 2 Hets, total alpha -subunit expression was decreased by ~30% (Fig. 3). Expression of alpha 2 was decreased by ~50% in the EDL of alpha 2 Het mice, compared with WT (Fig. 3), whereas an ~30% increase in alpha 1 expression was observed. These results are comparable to those reported by He and colleagues (20), despite the use of different antibodies. The data suggest that ~80% of total expressed alpha  in EDL is alpha 2 (Fig. 3B); this is close to the published value of 87% (20).


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Fig. 3.   Quantitation of alpha -subunit isoform expression in skeletal muscle and astrocytes. A: Western blots of Na+ pump alpha 1-, alpha 2-, and total alpha -subunit expression in the extensor digitorum longus (EDL) of 12-wk-old male WT and alpha 2 Het mice and in cultured astrocytes. The nitrocellulose membranes were cross-reacted with PC antisera raised against alpha 1- (NASE), alpha 2- (HERED), and isoform nonspecific alpha -subunit (LEAVE; detects both alpha 1 and alpha 2) (37). All wells contained 5 µg of protein. B: relative band densities for alpha 1-, alpha 2-, and total alpha -subunit data from WT and alpha 2 Het EDL averaged from 4-5 bands similar to those in A, and from WT, alpha 2 Het, and alpha 2 KO astrocytes (see A and Fig. 1, B and C). The changes in total alpha  band density are the measured relative values. As illustrated by the total bar heights, the data for the isoform-specific antibodies are consistent with the data for the nonspecific antibody if, for WT EDL, alpha 2 is assumed to be 80% of total alpha  and, for WT astrocytes, alpha 2 is 20% of total alpha . Band density standard errors averaged ±12%.

When this isoform-nonselective antibody was tested on astrocytes, however, there was no detectable decline in the Western blot total alpha -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 alpha 1-isoform in alpha 2 Het and KO astrocytes (Figs. 1C and 3), the implication is that the alpha 2-isoform accounts for no more than ~20% of the total alpha -subunit in these cells.

Localization of alpha 1 and alpha 2

It is important to ask whether astrocytes, which normally express both the alpha 1- and alpha 2-isoforms, but not alpha 3 (26, 34), are able to grow normally when alpha 2 is knocked out. Information on this point is provided by the immunocytochemical data from WT and alpha 2 KO cells in Fig. 4 and the fura 2 images in Fig. 5A. As illustrated, WT and KO cells have a similar appearance. Cells of both genotypes express Na+ pumps with alpha 1-subunits distributed uniformly over the cell surface, as detected with either polyclonal or monoclonal antibodies (Fig. 4) (25, 26). In contrast, the alpha 2-isoform is expressed in WT but not KO cells (Fig. 4). Despite the absence of alpha 2, the cells could be identified as astrocytes by cross-reaction with anti-glial fibrillary acidic protein antibodies (Fig. 4B, middle). Also, cell nuclei could be detected by staining with DAPI (Fig. 4B, right). In WT cells, alpha 2 is distributed in a lacy, reticular pattern.


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Fig. 4.   A: immunocytochemical determination of the distribution of plasma membrane (PM) Na+ pump alpha 1- (left-hand pairs of images) and alpha 2- (right-hand pairs of images) subunits in primary cultured mouse brain WT (top row) and KO (bottom row) astrocytes. The cells were labeled with PC (NASE, HERED) or MC (McK1, McB2) antibodies raised against either the alpha 1 (NASE, McK1) or alpha 2 (HERED, McB2) Na+ pump alpha -subunit isoform (13, 37, 45). Both anti-alpha 1 antibodies distribute uniformly, resulting in a "ground-glass" appearance of the labeling in both WT and KO cells. In contrast, both anti-alpha 2 antibodies distribute in a reticular, lacy pattern in the WT cells; the KO cells are not labeled by the anti-alpha 2 antibodies. B: comparison of WT (top) and KO cells (bottom) probed with PC anti-alpha 2 antibodies (HERED, left), MC anti-glial fibrillary acidic protein (GFAP) antibodies (center), and stained with 4',6'-diamidino-2-phenylindole (DAPI) to visualize nuclei (right). All scale bars in A and B = 10 µm.



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Fig. 5.   Images showing the resting cytosolic free [Ca2+] ([Ca2+]cyt) measured with fura 2 in astrocytes cultured from fetuses with different Na+ pump alpha 2-isoform alleles (WT, Het, and KO). A: representative fura 2 (360-nm excitation) images and Ca2+ images of resting cells. Scale bars = 10 µm. B: histogram of resting [Ca2+]cyt distribution in cells from WT (blue bars) and KO (red bars) fetuses. Note that the distribution is skewed to the right in the cells from the KO fetuses. Data were obtained from a total of 14 fetuses from 3 litters.

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 alpha 2 epitope "colocalizes" with SERCA in astrocytes. As shown below (Fig. 6), functional alpha 2 is located in the PM in WT astrocytes because the alpha 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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Fig. 6.   Sodium metabolism in astrocytes cultured from fetuses with different Na+ pump alpha 2-isoform alleles (alpha 2+/+, alpha 2+/-, and alpha 2-/- = WT, Het, and KO, respectively) measured with sodium-binding benzofuran isophthalate. A: time course of changes in cytosolic [Na+] ([Na+]cyt) in representative WT cells (blue lines) and KO cells (red lines). The cells were treated with either 500 nM ouabain (solid lines and inset with expanded ordinate scale) or 1 mM ouabain (dashed lines). B: summarized data showing the resting [Na+]cyt (control) and the effects of 10 min of treatment with either 500 nM or 1 mM ouabain on [Na+]cyt in cells from WT (69 cells), Het (40 cells), or KO (43 cells) fetuses. Values are means ± SE. * P < 0.001 vs. WT; # P < 0.001 vs. controls. Each bar shows data from 4-5 fetuses from 2 litters (2-3 fetuses/litter).

[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 alpha 1- and alpha 2-isoforms in maintaining [Na+]cyt, the effects of 500 nM and 1 mM ouabain were compared. The lower dose should block only the alpha 2-isoform (IC50 = 20-100 nM), whereas 1 mM ouabain should block both isoforms in rodents (alpha 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 alpha 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 alpha 1-isoform is the primary determinant of bulk [Na+]cyt, and alpha 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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Fig. 7.   Effect of cyclopiazonic acid (CPA) on [Ca2+]cyt in astrocytes cultured from fetuses with different Na+ pump alpha 2-isoform alleles (WT, Het, and KO). The media in these experiments contained 1.8 mM Ca2+. A: representative data from single cells show the time course of changes in [Ca2+]cyt in astrocytes from a WT fetus (blue line), and from Het (green line) and KO (red line) fetuses. The bar below the graph indicates the period of exposure to 10 µM CPA. B: summarized data showing resting [Ca2+]cyt (control) and the peak amplitude of the CPA-induced Ca2+ transient. Resting [Ca2+]cyt data are from 670 WT cells, 244 Het cells, and 465 KO cells. Peak CPA-induced Ca2+ transients were studied in 223 WT cells (blue bars), 138 Het cells (green bars), and 167 KO cells (red bars); each bar corresponds to data from a total of 4-5 fetuses from 2 litters. Values are means ± SE. * P < 0.001 vs. WT.

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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Fig. 8.   Demonstration of capacitative Ca2+ entry in astrocytes cultured from fetuses with different Na+ pump alpha 2-isoform alleles (WT, Het, and KO). Ca2+ was removed from the bathing medium 1 min before application of 10 µM CPA and was restored 6 min later during the continued exposure to CPA as indicated by the bars below the graph in A. A: representative data from single cells show the time course of changes in [Ca2+]cyt in astrocytes from a WT fetus (blue line) and from Het (green line) and KO (red line) fetuses. B: summarized data showing resting [Ca2+]cyt (control) and the peak amplitudes of the CPA-induced Ca2+ transient (middle bars) and the response to restoration of external Ca2+ in cells from WT (139 cells; blue bars), Het (124 cells; green bars) and KO (155 cells; red bars) fetuses. Values are means ± SE. + P < 0.05, # P < 0.01, and * P < 0.001 vs. WT. Each bar represents data from 6-8 fetuses from 2 litters.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Astrocytes cultured from the brains of rodents normally express both the alpha 1- and alpha 2-isoforms of the Na+ pump catalytic subunit (26, 46). The fact that alpha 1 and alpha 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 alpha 2 Expression Does Not Affect Cell Morphology

Numerous reports indicate that Na+ pump inhibition by both low-dose and high-dose ouabain modulates early response genes and transcription factors and promotes mitogenesis (e.g., Refs. 1, 15, 30, 36). Although issues of mitogenesis were not directly addressed in the present study, some relevant data were obtained. First, the 18- to 19-day-old Het and KO fetuses all appeared normal [not shown, but see James et al. (23), who also reported that Het mice develop normally into adults]. Second, the morphology of primary cultured cortical astrocytes from Het and KO fetuses was indistinguishable from that of WT astrocytes. Third, the rates of proliferation of Het and KO cells were not significantly different from those of WT cells (not shown). This is not surprising because the KO fetuses appeared to develop normally.

Reduction of alpha 2 Expression Has Little Effect on "Bulk" [Na+]cyt

The SBFI data demonstrate that reduction of alpha 2 expression by approx 50% in the alpha 2 Het astrocytes has a negligible effect on measured ("bulk") [Na+]cyt. In fact, complete KO of alpha 2 expression increased [Na+]cyt by only ~1.5 mM (from 6.5 to 8 mM). These small effects on bulk [Na+]cyt might be attributed simply to a small reduction in total alpha -subunit expression, because alpha 2 accounts for only ~20% of the total alpha -subunit and an even smaller fraction of the total Na+ pump flux in astrocytes (see below). Other evidence discussed below, however, indicates that the consequences of reduced alpha 2 expression are isoform specific. Moreover, the changes in [Na+]cyt are clearly attributable to reduced Na+ extrusion by the Na+ pumps with alpha 2-subunits, because the effects are mimicked by 500 nM ouabain, which does not block alpha 1 in rodents. Indeed, it is noteworthy that the Na+ content in EDL muscles from both alpha 1 Hets (mice with one alpha 1 null mutation) and alpha 2 Hets is indistinguishable from that of WT muscles (20).

Reduced alpha 2 Expression Elevates Resting [Ca2+]cyt and Augments Ca2+ Transients and CCE in Astrocytes

Reduction of alpha 2 expression by approx 50% in alpha 2 Hets significantly increased resting [Ca2+]cyt and augmented Ca2+ transients and CCE. While similar, but more pronounced, effects were observed in KO astrocytes, the data in Het cells are particularly noteworthy because bulk [Na+]cyt was not elevated significantly in the Het cells. These effects on [Ca2+]cyt are clearly attributable to inhibition of Na+ pumps with alpha 2-subunits because low-dose (100-500 nM) ouabain has a comparable effect in astrocytes (9). Low-dose ouabain also augments Ca2+ transients without elevating bulk [Na+]cyt in arterial myocytes (2), which express the high ouabain affinity alpha 3-isoform.

The hearts and skeletal muscle of alpha 2 Het mice exhibit increased contractility (20, 23). In contrast, in mice with a null mutation in one alpha 1 gene and one-half the normal alpha 1 expression, cardiac and skeletal muscle contractility are reduced. Inhibition of alpha 2 activity with low-dose ouabain in the alpha 1 Het then enhances the contractility in both types of muscles, despite the further reduction of total alpha -subunit activity (20, 23).

In summary, the aforementioned observations all demonstrate that selective reduction of the activity of Na+ pumps with alpha 2- or alpha 3-subunits augments Ca2+ signaling in a variety of cell types. The implication is that a major role of these high-ouabain-affinity alpha -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 alpha 2- (or alpha 3-) subunits in astrocytes and other cell types (25) is consistent with other evidence that the NCX and alpha 2/alpha 3 Na+ pumps are tightly coupled (reviewed in Refs. 2, 10).

Reduction of alpha 2 Activity Augments Ca2+ Signaling by Regulating [Na+] in a Sub-PM Cytosolic Compartment

How does reduced activity of the high-ouabain affinity Na+ pump alpha -subunit isoforms have such large effects on Ca2+ homeostasis and Ca2+ signaling, despite little or no effect on bulk [Na+]cyt? Consider that, at [Na+]cyt = 6.5 mM, alpha 1 [KNa(alpha 1) = 12 mM] operates at 14% of its maximal rate, whereas alpha 2 [KNa(alpha 2) = 22 mM] operates at only 2.5% of its maximal rate (48). Moreover, the turnover number (i.e., molecular cycling rate) for alpha 2 is only 0.6 times that of alpha 1 (40). Thus the expected relative alpha 2-to-alpha 1 flux ratio is only ~0.02:1, because only <= 20% of the expressed alpha  in astrocytes is alpha 2 (Fig. 3). That is, only ~2% [or 5%, using the Na+ affinity (KNa) values of Segall et al. (40)] of the total Na+ pump flux in resting astrocytes would be mediated by alpha 2! This seems unlikely.

To explain how reduction of Na+ pump alpha 2/alpha 3-subunit activity can modify Ca2+ homeostasis via NCX without detectable elevation of bulk [Na+]cyt (in alpha 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(alpha 2) and KNa(alpha 3) can function while the "housekeeping" high KNa(alpha 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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Fig. 9.   Model of the PM-junctional endoplasmic reticulum (ER) region (PLasmERosome) showing key transport proteins involved in local control of junctional ER Ca2+ stores and modulation of Ca2+ signaling. Model shows a PM containing alpha 1 Na+ pumps and PM Ca2+ pumps (PMCA). The PM microdomain overlying junctional ER contains alpha 2/alpha 3 Na+ pumps, NCX, and store-operated channels (SOCs). The adjacent junctional ER contains sarcoplasmic/ER (S/ER) Ca2+-ATPase (SERCA), inositol trisphosphate (IP3) receptors (IP3R), and ryanodine receptors (RYR). The tiny, "diffusion-restricted" junctional cytosolic space (JS) lies between the PM microdomains and the junctional ER; the distance between the PM and ER is ~10-15 nm. Thus, if the local [Na+] is 6.5 mM, a JS volume of 100 × 100 × 15 nm (=1.5 × 10-19 liters) will contain only ~600 Na ions (see Ref. 2). This is fewer than the number of Na ions transported in 3 s by a single Na+ pump molecule with an alpha 2-subunit (40). A: normal conditions. B: the situation in astrocytes from alpha 2 KO mice (or after complete inhibition of alpha 2 with 500 nM ouabain). Shading indicates relative [Na+] and/or [Ca2+] extracellular fluid (ECF); alpha 1 Na+ pumps are widely distributed in the PM but may be excluded from these microdomains.

Immunocytochemical data (16, 25, 26), as well as preliminary coimmunoprecipitation data (29), indicate that these PM microdomains contain Na+ pumps with alpha 2- or alpha 3- (but not alpha 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 alpha 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 alpha 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 alpha 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 alpha 2 has a very small effect on bulk [Na+]cyt (a rise of <2 mM). Thus Na+ pumps with alpha 2-subunits play only a minor role in maintaining the low resting [Na+]cyt in astrocytes. In contrast, even a 50% reduction in alpha 2 expression, which does not affect bulk [Na+]cyt, is associated with significant elevation of resting [Ca2+]cyt and augmentation of Ca2+ signaling. The implication is that a major role of Na+ pumps with alpha 2 (and alpha 3; Ref. 2) subunits is the modulation of Ca2+ signals.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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