INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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⁺ [K_Na(1) = 12mM] than ₂ and ₃ [K_Na(2) and K_Na(3) = 22 and 33mM, respectively] (Ref. 48; see also Refs. 40 and 45). In rodents, the ₁-isoform has an especially low affinity [K_I(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 K_I(2) alleles (23).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Knockout Mice

+/

/

Astrocyte Cell Cultures

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.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Expression of Na+ Pump -Subunit Isoforms in Cultured Astrocytes

View larger version (48K): [in this window] [in a new window]   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 2-subunit isoform (i.e., 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-2 (B) or anti-1 (C) isoform polyclonal (PC) antibodies. The 2- and 1-isoform proteins (112 kDa) are indicated. The 2 right-hand lanes are controls from wild-type (WT) mouse brain (expresses 1 and 2) and kidney (expresses 1, only). B: the 2 knockout (KO; 2/) astrocytes express no 2 protein (lanes 5 and 8); 2 heterozygote (Het; 2+/) cells express about one-half as much 2 protein as do WT (2+/+) astrocytes. C: all 3 types of astrocytes express similar amounts of 1. (Note: there was insufficient sample to test for 1 expression in mouse 1.)

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).

View larger version (35K): [in this window] [in a new window]   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 (120 kDa, top row) and a PC antibody raised against actin (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 ₂-Isoform Expression

View larger version (52K): [in this window] [in a new window]   Fig. 3.   Quantitation of -subunit isoform expression in skeletal muscle and astrocytes. A: Western blots of Na+ pump 1-, 2-, and total -subunit expression in the extensor digitorum longus (EDL) of 12-wk-old male WT and 2 Het mice and in cultured astrocytes. The nitrocellulose membranes were cross-reacted with PC antisera raised against 1- (NASE), 2- (HERED), and isoform nonspecific -subunit (LEAVE; detects both 1 and 2) (37). All wells contained 5 µg of protein. B: relative band densities for 1-, 2-, and total -subunit data from WT and 2 Het EDL averaged from 4-5 bands similar to those in A, and from WT, 2 Het, and 2 KO astrocytes (see A and Fig. 1, B and C). The changes in total  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, 2 is assumed to be 80% of total  and, for WT astrocytes, 2 is 20% of total . 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 -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

View larger version (92K): [in this window] [in a new window]   Fig. 4.   A: immunocytochemical determination of the distribution of plasma membrane (PM) Na+ pump 1- (left-hand pairs of images) and 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 1 (NASE, McK1) or 2 (HERED, McB2) Na+ pump -subunit isoform (13, 37, 45). Both anti-1 antibodies distribute uniformly, resulting in a "ground-glass" appearance of the labeling in both WT and KO cells. In contrast, both anti-2 antibodies distribute in a reticular, lacy pattern in the WT cells; the KO cells are not labeled by the anti-2 antibodies. B: comparison of WT (top) and KO cells (bottom) probed with PC anti-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.

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Images showing the resting cytosolic free [Ca2+] ([Ca2+]cyt) measured with fura 2 in astrocytes cultured from fetuses with different Na+ pump 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 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.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Sodium metabolism in astrocytes cultured from fetuses with different Na+ pump 2-isoform alleles (2+/+, 2+/, and 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

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

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).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effect of cyclopiazonic acid (CPA) on [Ca2+]cyt in astrocytes cultured from fetuses with different Na+ pump 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

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.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Demonstration of capacitative Ca2+ entry in astrocytes cultured from fetuses with different Na+ pump 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 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

Na(₁)

Na(₂)

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 K_Na(2) and K_Na(3) can function while the "housekeeping" high K_Na(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).

View larger version (51K): [in this window] [in a new window]   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 1 Na+ pumps and PM Ca2+ pumps (PMCA). The PM microdomain overlying junctional ER contains 2/3 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 × 1019 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 2-subunit (40). A: normal conditions. B: the situation in astrocytes from 2 KO mice (or after complete inhibition of 2 with 500 nM ouabain). Shading indicates relative [Na+] and/or [Ca2+] extracellular fluid (ECF); 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 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