The role of the Na+ pump α2-subunit in Ca2+ signaling was examined in primary cultured astrocytes from wild-type (α2 +/+ = WT) mouse fetuses and those with a null mutation in one [α2 +/− = heterozygote (Het)] or both [α2 −/− = knockout (KO)] α2 genes. Na+ pump catalytic (α) 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 α1- (≈80% of total α) and α2- (≈20% of total α) subunits. Het astrocytes express ≈50% of normal α2; those from KO express none. Expression of α1 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 α2) 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 α2 Na+ pumps and Na+/Ca2+ exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of α2 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.
- catalytic subunit
- fura 2
- sodium-binding benzofuran isophthalate
- sodium-potassium-adenosine 5′-triphosphatase isoforms
- transgenic mice
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 α- and β-subunits in a 1:1 ratio (5, 24). The β-subunit is a highly glycosylated 40- to 60-kDa protein that may be involved in chaperoning and membrane trafficking of the larger (≈112 kDa) α-subunit (12). The α- (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 α-subunit isoforms have been identified: α1, α2, α3 (41, 44, 46), and α4(47). The latter is found only in the testis and will not be discussed here. Most cells express α1 and one of the other isoforms, all of which have different kinetic properties. The α1 has a higher affinity for Na+ than α2 and α3 and respectively] (Ref. 48; see also Refs. 40and 45). In rodents, the α1-isoform has an especially low affinity for ouabain; in contrast, the α2- and α3-isoforms have high affinity 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), α2 and α3 are confined to PM microdomains that overlie sarcoplasmic reticulum or ER (S/ER). In contrast, α1 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 α2 or α3 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 α2- or α3-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 α1 and α2 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 alleles (23).
Mice with null mutations in one or both α2 genes [i.e., heterozygotes (Het) = α2 +/−, and homozygote knockouts (KO) = α2 −/−, respectively] were generated as described (23). WT (α2 +/+) 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 (≈50,000 cells/coverslip). The medium was changed ondays 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 α-Subunit Isoforms
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 α-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 α2 +/−) 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 3,000g, 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.
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 α1- or α2-subunit isoforms, or with an α-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; π11–13 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 α1 and α2 were measured with isoform-specific polyclonal antibodies and were correlated with changes in α1 + α2 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 α-subunit isoforms after they are unfolded in SDS buffer.
Primary cultured mouse cortical astrocytes were fixed and cross-reacted with polyclonal or monoclonal antibodies raised against Na+pump α1- or α2-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).
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.
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.
The numerical data presented in results are the means fromn 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'st-tests for paired or unpaired data were used to calculate the significance of the differences between means.
Expression of Na+ Pump α-Subunit Isoforms in Cultured Astrocytes
The expression of Na+ pump α1- and α2-isoforms in astrocytes cultured from the brains of WT, Het, and KO mouse embryos is compared in Fig.1. Figure 1 A shows the α2 genotype data from Southern blot analysis of the genomic DNA from the 12 embryos in a single litter. Figure1 B shows the Western blot analysis of the expressed α2 protein from these same embryos. Astrocytes from Het mice express about one-half as much α2 as do cells from WT mice, and cells from KO mice do not express any α2. Figure 1 C, which shows α1 expression data from the same litter, indicates that astrocytes with all three genotypes express approximately equal amounts of the Na+ pump α1-isoform. These Western blot data from primary cultured astrocytes are comparable to the results obtained in fresh cardiac tissue from α2 WT, Het, and KO mice (23).
The Na+ pump α2-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 α2-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 α2 Het and KO mice. Surprisingly, NCX expression was not significantly upregulated in Het and KO astrocytes (Fig. 2).
Quantitation of Na+ Pump α2-Isoform Expression
To interpret the functional effects of reduced α2-isoform expression, it is important to know what fraction of the total Na+ pump α-subunit (α1 + α2) is α2. 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 α1 and α2 (LEAVE) (37). In control experiments on mouse skeletal muscle (which also expresses only α1 and α2), we found that, in α2 Hets, total α-subunit expression was decreased by ∼30% (Fig. 3). Expression of α2 was decreased by ∼50% in the EDL of α2 Het mice, compared with WT (Fig. 3), whereas an ∼30% increase in α1 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 α in EDL is α2(Fig. 3 B); this is close to the published value of 87% (20).
When this isoform-nonselective antibody was tested on astrocytes, however, there was no detectable decline in the Western blot total α-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 α1-isoform in α2 Het and KO astrocytes (Figs. 1 C and 3), the implication is that the α2-isoform accounts for no more than ∼20% of the total α-subunit in these cells.
Localization of α1 and α2
It is important to ask whether astrocytes, which normally express both the α1- and α2-isoforms, but not α3 (26, 34), are able to grow normally when α2 is knocked out. Information on this point is provided by the immunocytochemical data from WT and α2 KO cells in Fig. 4 and the fura 2 images in Fig.5 A. As illustrated, WT and KO cells have a similar appearance. Cells of both genotypes express Na+ pumps with α1-subunits distributed uniformly over the cell surface, as detected with either polyclonal or monoclonal antibodies (Fig. 4) (25, 26). In contrast, the α2-isoform is expressed in WT but not KO cells (Fig. 4). Despite the absence of α2, the cells could be identified as astrocytes by cross-reaction with anti-glial fibrillary acidic protein antibodies (Fig. 4 B,middle). Also, cell nuclei could be detected by staining with DAPI (Fig. 4 B, right). In WT cells, α2 is distributed in a lacy, reticular pattern.
The black-and-white images in Fig. 5 A (top) show fura 2-stained astrocytes from WT, Het, and KO mice (left toright). The morphology of the cells from the three genotypes are all very similar.
Immunocytochemical data (26) reveal that the α2 epitope “colocalizes” with SERCA in astrocytes. As shown below (Fig. 6), functional α2 is located in the PM in WT astrocytes because the α2 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.
[Na+]cytin 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. 6 B,left).
To test further the roles of the α1- and α2-isoforms in maintaining [Na+]cyt, the effects of 500 nM and 1 mM ouabain were compared. The lower dose should block only the α2-isoform (IC50 = 20–100 nM), whereas 1 mM ouabain should block both isoforms in rodents (α1 IC50 = 50–100 μM) (5,35). As revealed by the representative data in Fig.6 A (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 α2, 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. 6 B,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.6 A. Data for a 10-min exposure to 1 mM ouabain are summarized in Fig. 6 B, right. Thus the α1-isoform is the primary determinant of bulk [Na+]cyt, and α2 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. 5 A. 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+]cytvalues in WT and KO cells is presented in Fig. 5 B. There is considerable overlap between the [Ca2+]cytvalues 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. 5 A 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.5 B), the data from Het cells are intermediate between those of WT and KO cells.
Resting [Ca2+]cyt data are summarized in Fig.7 B, 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+]cytis significantly elevated in the Het cells (P < 0.001; Fig. 7 B), despite a normal [Na+]cyt (Fig. 6).
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. 7 A 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.8 A) and on continued block of SERCA by CPA (Fig. 7 A). The summary data in Fig.7 B (right) reveal that the mean peak [Ca2+]cyt transient elevation is significantly greater in Het cells and KO cells than in WT cells.
Comparable data for cells incubated in Ca2+-free medium are illustrated by the initial responses (leftCa2+ transients) in Fig. 8 A. There is no plateau in this case, because the plateau (Fig. 7 A) 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.8 B, 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. 8 A,second Ca2+ transients; Fig. 8 B,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).
Astrocytes cultured from the brains of rodents normally express both the α1- and α2-isoforms of the Na+ pump catalytic subunit (26, 46). The fact that α1 and α2 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 α2 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 α2 Expression Has Little Effect on “Bulk” [Na+]cyt
The SBFI data demonstrate that reduction of α2expression by ≈50% in the α2 Het astrocytes has a negligible effect on measured (“bulk”) [Na+]cyt. In fact, complete KO of α2 expression increased [Na+]cyt by only ∼1.5 mM (from 6.5 to 8 mM). These small effects on bulk [Na+]cytmight be attributed simply to a small reduction in total α-subunit expression, because α2 accounts for only ∼20% of the total α-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 α2 expression are isoform specific. Moreover, the changes in [Na+]cyt are clearly attributable to reduced Na+ extrusion by the Na+ pumps with α2-subunits, because the effects are mimicked by 500 nM ouabain, which does not block α1 in rodents. Indeed, it is noteworthy that the Na+ content in EDL muscles from both α1 Hets (mice with one α1 null mutation) and α2 Hets is indistinguishable from that of WT muscles (20).
Reduced α2 Expression Elevates Resting [Ca2+]cyt and Augments Ca2+ Transients and CCE in Astrocytes
Reduction of α2 expression by ≈50% in α2 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 α2-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 α3-isoform.
The hearts and skeletal muscle of α2 Het mice exhibit increased contractility (20, 23). In contrast, in mice with a null mutation in one α1 gene and one-half the normal α1 expression, cardiac and skeletal muscle contractility are reduced. Inhibition of α2 activity with low-dose ouabain in the α1 Het then enhances the contractility in both types of muscles, despite the further reduction of total α-subunit activity (20, 23).
In summary, the aforementioned observations all demonstrate that selective reduction of the activity of Na+ pumps with α2- or α3-subunits augments Ca2+ signaling in a variety of cell types. The implication is that a major role of these high-ouabain-affinity α-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 α2- (or α3-) subunits in astrocytes and other cell types (25) is consistent with other evidence that the NCX and α2/α3 Na+ pumps are tightly coupled (reviewed in Refs. 2,10).
Reduction of α2 Activity Augments Ca2+ Signaling by Regulating [Na+] in a Sub-PM Cytosolic Compartment
How does reduced activity of the high-ouabain affinity Na+ pump α-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, α1 = 12 mM] operates at 14% of its maximal rate, whereas α2 = 22 mM] operates at only 2.5% of its maximal rate (48). Moreover, the turnover number (i.e., molecular cycling rate) for α2 is only 0.6 times that of α1(40). Thus the expected relative α2-to-α1 flux ratio is only ∼0.02:1, because only ≤20% of the expressed α in astrocytes is α2 (Fig. 3). That is, only ∼2% [or 5%, using the Na+ affinity (K Na) values of Segall et al. (40)] of the total Na+ pump flux in resting astrocytes would be mediated by α2! This seems unlikely.
To explain how reduction of Na+ pump α2/α3-subunit activity can modify Ca2+ homeostasis via NCX without detectable elevation of bulk [Na+]cyt (in α2 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 lowK Na(α2 ) andK Na(α3 ) can function while the “housekeeping” highK Na(α1 ) 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).
Immunocytochemical data (16, 25, 26), as well as preliminary coimmunoprecipitation data (29), indicate that these PM microdomains contain Na+ pumps with α2- or α3- (but not α1-) subunits, NCX, and transient receptor potential channel proteins, which may be components of SOCs (22) (Fig. 9 A). 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 α1-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 9 B depicts the elevation of [Na+] and [Ca2+] in the JS and the consequent rise in ER [Ca2+] as a result of α2 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. 7 A 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 α2Na+ 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 α2 has a very small effect on bulk [Na+]cyt (a rise of <2 mM). Thus Na+ pumps with α2-subunits play only a minor role in maintaining the low resting [Na+]cytin astrocytes. In contrast, even a 50% reduction in α2expression, 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 α2 (and α3; Ref. 2) subunits is the modulation of Ca2+ signals.
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.
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:).
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
- Copyright © 2003 the American Physiological Society