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Endocrinology Vol. 142, No. 12 5059-5068
Copyright © 2001 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Glucocorticoid-Induced Plasma Membrane Depolarization during Thymocyte Apoptosis: Association with Cell Shrinkage and Degradation of the Na+/K+-Adenosine Triphosphatase

Cynthia L. Mann, Carl D. Bortner, Christine M. Jewell and John A. Cidlowski

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health (C.L.M., C.D.B., J.A.C.), Research Triangle Park, North Carolina 27709; and Curriculum in Toxicology, University of North Carolina (C.L.M.), Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Dr. John A. Cidlowski, P.O. Box 12233, MD F3-07, 111 Alexander Drive, Research Triangle Park, North Carolina 27709. E-mail: cidlowski{at}niehs.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Multiple signaling pathways are known to induce apoptosis in thymocytes through mechanisms that include the loss of mitochondrial membrane potential, cell shrinkage, caspase activation, and DNA degradation but little is known about the consequences of apoptosis on the properties of the plasma membrane. We have previously shown that apoptotic signals, including survival factor withdrawal and glucocorticoids, induce plasma membrane depolarization during rat thymocyte apoptosis, but the mechanisms involved in this process are unknown. We report here that inhibition of the Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) with ouabain similarly depolarized control thymocytes and enhanced glucocorticoid-induced membrane depolarization, suggesting a link between Na+/K+-ATPase and plasma membrane depolarization of thymocytes. To determine whether repression of Na+/K+-ATPase levels within cells can account for the loss of plasma membrane potential, we assessed protein levels of the Na+/K+-ATPase in apoptotic thymocytes. Spontaneously dying thymocytes had decreased levels of both catalytic and regulatory subunits of Na+/K+-ATPase, and glucocorticoid treatment enhanced the loss of Na+/K+-ATPase protein. The pan caspase inhibitor (z-VAD) blocked both cellular depolarization and repression of Na+/K+-ATPase in both spontaneously dying and glucocorticoid-treated thymocytes; however, specific inhibitors of caspase 8, 9, and caspase 3 did not. Interestingly, glucocorticoid treatment simultaneously induced cell shrinkage and depolarization. Furthermore, depolarization and the loss of Na+/K+-ATPase protein were limited to the shrunken population of cells. The data indicate an important role for Na+/K+-ATPase in both spontaneous and glucocorticoid-induced apoptosis of rat thymocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EARLY STUDIES OF the effects of glucocorticoids on the immune system showed that glucocorticoids caused a profound reduction in thymic mass and volume due primarily to thymocyte apoptosis (1, 2). Glucocorticoids induce a cell death program in thymocytes characterized by the loss of mitochondrial membrane potential, cell shrinkage, caspase activation, and DNA degradation (3, 4, 5). The structure and function of the plasma membrane are also altered by glucocorticoids during apoptosis, as is evidenced by alterations in the transport of glucose and amino acids (6) and the distribution of ions across the membrane (7, 8, 9, 10). Phosphatidylserine residues also reorient to the exterior of the cell during apoptosis (11). Finally, late in apoptosis, the plasma membrane blebs and pinches off to form apoptotic bodies (12, 13).

The loss of water and ions from the cell during apoptosis, particularly potassium (7, 10), results in cell shrinkage, a characteristic feature of apoptosis (1, 13). In thymocytes, glucocorticoids induce cell shrinkage through a receptor-dependent and gene expression-dependent pathway (5, 14). In addition, studies with the pan-caspase inhibitor z-VAD showed that glucocorticoid-induced cell shrinkage is dependent upon activation of the caspase cascade (5, 15). Interestingly, the loss of potassium has been shown to occur only in the shrunken population of cells (10, 16), and the hypotonic intracellular environment caused by the loss of potassium is required for subsequent activation of caspase 3-like enzymes and DNA degradation (15, 16, 17).

Recent studies from our laboratory have shown that anti-Fas antibody treatment of human Jurkat T cells led to sustained depolarization of the plasma membrane and other characteristics of apoptosis (18). We have also observed that glucocorticoids induce receptor-dependent depolarization of the plasma membrane of primary isolated rat thymocytes both in vivo and in vitro (19). The ability of glucocorticoids to depolarize the membrane correlated with their ability to induce apoptosis in the target cell. Interestingly, apoptotic stimuli, such as survival factor withdrawal, also lead to depolarization of thymocytes and subsequent detection of apoptotic characteristics, but the mechanisms defining this cellular apoptosis are largely unknown.

The plasma membrane potential of lymphocytes is maintained predominantly by the electrogenic action of Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) (20, 21). The Na+/K+-ATPase is an ATP-dependent membrane enzyme that exchanges 3Na+ for 2K+ against an electrochemical gradient (22) and thus maintains high potassium and low sodium levels within the cell (23, 24). Inhibition of Na+/K+-ATPase leads to depolarization of the plasma membrane in a variety of cells, including Jurkat cells (25). Furthermore, inhibition of Na+/K+-ATPase enhances the sensitivity of Jurkat cells to the death-inducing actions of anti-Fas antibody. However, it is unknown whether Na+/K+-ATPase is regulated during spontaneous or glucocorticoid-induced apoptosis of primary rat thymocytes. Additionally, it is unclear whther caspases, which are known to be activated in both spontaneous and glucocorticoid-induced apoptosis (5), are involved in modulation of cellular Na+/K+-ATPase.

The present study examines the role of Na+/K+-ATPase in glucocorticoid-mediated apoptosis of primary thymocytes. We report that thymocytes depolarize during spontaneous and glucocorticoid-induced cell death and that inhibition of Na+/K+-ATPase enhances both cellular depolarization and apoptosis. Interestingly, although nonselective inhibition of the caspase cascade blocks cellular depolarization in response to both apoptotic stimuli, caspase specific inhibitors do not. We show that Na+/K+-ATPase protein levels selectively decrease in both spontaneous and glucocorticoid-induced death. Finally, examination of the relationship between cell shrinkage, depolarization, and Na+/K+-ATPase repression reveals that depolarization and repression of Na+/K+-ATPase protein levels occur in the shrunken population of cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
FCS was purchased from Summit Biotechnology (Fort Collins, CO), and dexamethasone was purchased from Steraloids (Wilton, NH). Z-Ile-Glu-Thr-Asp-fluoromethylketone (IETD-fmk), Z-Ala-Glu (Ome)-Val-Asp (Ome)-FMK (AEVD-fmk), Z-Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk), and Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) were purchased from Kamiya Biomedical Co. (Seattle, WA). DiBAC4(3) was purchased from Molecular Probes, Inc. (Eugene, OR). Ouabain and propidium iodide (PI) were purchased from Sigma (St. Louis, MO).

Animals
Male Sprague Dawley rats (2–3 months of age) were used in all experiments. The animals were bilaterally adrenalectomized by the provider at least 5 d before use, maintained under controlled conditions of temperature (25 C) and lighting, and allowed free access to food and 0.85% saline. All experimental protocols were approved by the animal review committee at the institute and were performed in accordance with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals published by the USPHS. Animals were killed by decapitation, and the thymus was surgically removed.

Thymocyte cultures
Thymocytes were prepared from freshly isolated thymus as previously described (16, 26, 27). Briefly, thymocytes were dispersed by gentle homogenization in a glass/glass homogenizer (no. 22, Kontes Co., Vineland, NJ), filtered, washed in cold PBS, and counted on a hemocytometer. Cells were cultured (5 x 106 cells/ml) at 37 C in 5% CO2, in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 4 mM glutamine, 100 U/ml penicillin, and 75 U/ml streptomycin sulfate (prepared in house).

FACS analysis
The plasma membrane of isolated thymocytes was measured with the anionic oxonal dye DiBAC4(3) (Molecular Probes, Inc.) as described previously (18). DiBAC4(3) was prepared in dimethylsulfoxide according to the manufacturer’s instructions. Briefly, cells were incubated with 150 nM DiBAC4(3) for 30 min at 37 C in 5% CO2. PI was added to a final concentration of 10 µg/ml immediately before flow cytometric analysis to exclude nonviable cells (28). Cells were examined as changes in their plasma membrane potential and cell size by flow cytometry using a FACSort (Becton Dickinson and Co., Mountain View, CA) equipped with an argon (488-nm) laser. Fifteen thousand cells were examined under each condition, and all flow cytometric analysis was accomplished using CellQuest software. Cell size was monitored by alterations in the forward light-scattering properties of the cells as described previously (5). Fluorescent emission of DiBAC4(3) and PI were detected at 530 and 650 nm, respectively. An increase in DiBAC4(3) fluorescence at 530 nm indicates cellular depolarization. The percentage of cells with increased DiBAC4(3) fluorescence was determined by gating on the fresh, viable population of cells. Cells with DiBAC4(3) fluorescence greater that that for the fresh population of cells were quantified for each treatment. The average ± SEM for each treatment represent at least three independent experiments. Statistical analyses were performed using t test with {alpha} = 0.05.

Protein extraction and immunoblot analysis
Total cellular protein was recovered by lysis in high detergent buffer [20 mM Tris (pH 7.5), 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate] with protease inhibitors (Roche, Indianapolis, IN). Protein was quantified by the method of Bradford (Bio-Rad Laboratories, Inc., Hercules, CA) and denatured in 5 x Laemmli buffer [250 mM Tris (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, and 100 mM ß-mercaptoethanol] at 100 C for 5 min and then stored at -70 C. Proteins were resolved by SDS-PAGE through 4–20% gradient gels (Novex, San Diego, CA) and transferred to nitrocellulose in Tris-glycine buffer [12 mM Tris-HCl (pH 8.3), 75 mM glycine, and 20% methanol]. Membranes were stained with Ponceau S (0.5% in 1% acetic acid) to confirm equal loading and to evaluate transfer efficiency. Next, membranes were preincubated overnight at 4 C in Tris-buffered saline [TBS; 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20] with 10% nonfat dry milk. The blots were then washed with TBS and incubated overnight at 4 C with a 1:250 dilution of either antirabbit Na+/K+-ATPase-{alpha} monoclonal antibody or antirabbit Na+/K+-ATPase-ß monoclonal antibody (Upstate Biotechnology, Inc., Saranac Lake, NY). To assess GR levels, blots were incubated with a 1:1,000 dilution of an antipeptide GR antibody (29). Actin levels were determined by incubation with a 1:10,000 dilution of a mouse antiactin antibody (Chemicon, Temecula, CA). Membranes were then washed according to the manufacturer’s instructions and incubated with a horseradish peroxidase-labeled goat antimouse or goat antirabbit secondary antibody (1:10,000 in TBS; Amersham Pharmacia Biotech, Arlington Heights, IL), washed again, and reacted with chemiluminescent reagents for autoradiography (Amersham Pharmacia Biotech).

Cell sorting
To analyze Na+/K+-ATPase protein levels in normal vs. shrunken cells, thymocytes were treated with dexamethasone for 3 h to induce cell shrinkage. After this time the cells were stained with PI to eliminate from further analysis cells that had lost their membrane integrity. Simultaneous sorting of both normal and shrunken populations of cells was accomplished using a Becton Dickinson and Co. FACSVantage SE equipped with CellQuest software. A gate was set on a forward scatter vs. PI fluorescence plot based on an untreated control sample to isolate the normal population of cells. A second gate has then set to the left of this normal cell population denoting cells that had a decreased ability to scatter light the forward direction, indicating cells that were of smaller or shrunken cell size. Sorted cells were maintained at 4 C to maintain their integrity after the sort. Eight million cells were immediately processed for protein analysis by immunoblot analysis as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma membrane depolarizes during spontaneous and glucocorticoid-induced thymocyte apoptosis
We have recently shown that in rat thymocytes, glucocorticoids depolarize the plasma membrane in a dose-dependent manner that is dependent upon the interaction of glucocorticoids with the GR and subsequent gene expression (19). These studies were conducted with the plasma membrane potential-sensitive dye DiBAC4(3), an oxonal anionic dye that responds to changes in plasma membrane potential (18, 30). Figure 1Go provides an example of DiBAC4(3) fluorescence in freshly isolated thymocytes, spontaneously dying thymocytes, and thymocytes treated with dexamethasone for 6 h. Freshly isolated thymocytes have low DiBAC4(3) fluorescence, indicating that plasma membrane potential is intact. Furthermore, addition of high extracellular potassium to the media in these cells as well as human lymphocytes results in cellular depolarization (18), indicating that this dye is reliable for measurement of plasma membrane potential. After 6 h in culture, there is a population of cells with increased DiBAC4(3) fluorescence (19.5 ± 1.8% vs. 9.1 ± 1.7% in the fresh population; P < 0.05 vs. freshly isolated cells). These data are consistent with our previous observations that primary thymocytes undergo spontaneous death in the absence of an apoptotic stimulus (19). Glucocorticoid treatment exacerbates this effect by dramatically increasing the percentage of depolarized cells after 6 h (41.2 ± 6.2%; P < 0.05 vs. time-matched control cells). It is important to note that glucocorticoids do not increase the magnitude of the depolarization, only the percentage of cells that have a depolarized plasma membrane, suggesting that apoptosis is stochastic in this model system. Similarly, examples of cellular depolarization have been observed in Jurkat cells treated with other apoptotic stimuli (18). These data in combination with our previous observations demonstrate that rat thymocytes depolarize their plasma membrane during both spontaneous and glucocorticoid-induced apoptosis, but the mechanisms underlying this effect and the role of caspases in this process are currently unknown.



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  		Figure 1. Dexamethasone depolarizes rat thymocytes. Fresh thymocytes or thymocytes cultured for 6 h in the presence or absence of 100 nM dexamethasone were incubated with DiBAC4(3 ) to evaluate plasma membrane potential. Before flow cytometric analysis, PI was added to exclude nonviable cells. Ten thousand viable cells for each treatment were evaluated for DiBAC4(3 ) fluorescence as described in Materials and Methods. The figure shows representative histograms for DiBAC4(3 ) fluorescence in freshly isolated thymocytes (FRESH) or thymocytes cultured for 6 h alone (CON) and with dexamethasone (DEX).

 
Inhibition of Na+/K+-ATPase depolarizes thymocytes and potentiates glucocorticoid-induced loss of plasma membrane potential
In thymocytes and lymphocytes, in general, Na+/K+-ATPase is the primary pump that maintains the electrochemical gradient that is responsible for establishment of plasma membrane potential (20, 21, 22). Disruption of Na+/K+-ATPase by ouabain is well known to lead to depolarization the plasma membrane (18). To determine whether Na+/K+-ATPase may be involved in the loss of plasma membrane potential during glucocorticoid-induced apoptosis of thymocytes, we blocked the activity of Na+/K+-ATPase with ouabain, a specific inhibitor of Na+/K+-ATPase in rats as well as other species (31). As shown in Fig. 2Go, ouabain treatment alone depolarizes primary thymocytes, as evidenced by the increase in the number of cells with high DiBAC4(3) fluorescence (47.9 ± 5.3%; P < 0.05 vs. time-matched control cells) vs. the control cells (19.5 ± 1.8%; fresh, 9.1 ± 1.7%; P < 0.05 vs. freshly isolated cells). Additionally, ouabain potentiated the effects of glucocorticoids alone (38.5 ± 4.7%; P < 0.05 vs. time-matched control cells) by significantly increasing the number of cells with high DiBAC4(3) fluorescence (60.0 ± 1.6%; P < 0.05 vs. time-matched control cells). These results demonstrate that inhibition of Na+/K+-ATPase potentiates glucocorticoid-induced depolarization and suggests a potential role for Na+/K+-ATPase in the depolarization of thymocytes during apoptosis.



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  		Figure 2. Inhibition of Na+/K+-ATPase potentiates glucocorticoid- induced loss of plasma membrane potential. Primary isolated thymocytes were cultured with ouabain (10 mM) in the presence or absence of dexamethasone (100 nM) for 6 h. After treatment, cells were incubated with DiBAC4(3 ). PI was added before flow cytometric analysis to exclude nonviable cells. Ten thousand viable cells for each treatment were evaluated for DiBAC4(3 ) fluorescence as described in Materials and Methods. Representative DiBAC4(3 ) fluorescence histograms are shown for each treatment.

 
Glucocorticoids repress Na+/K+-ATPase protein levels during apoptosis in thymocytes
The fact that inhibition of Na+/K+-ATPase resulted in the loss of plasma membrane potential suggested that Na+/K+-ATPase might be a target for inhibition or perhaps degradation during glucocorticoid-induced apoptosis. In models of Fas-induced apoptosis of Jurkat cells it has been shown that decreased levels of both catalytic ({alpha}) and regulatory (ß) subunits of Na+/K+-ATPase occur (18). To determine whether glucocorticoid treatment alters the protein levels of Na+/K+-ATPase in primary thymocytes, we examined the protein levels of both catalytic and regulatory subunits of Na+/K+-ATPase (Fig. 3Go). Interestingly, we similarly found that the protein levels of both the catalytic and regulatory subunits decreased in untreated cells after 6 h compared with freshly isolated cells, findings consistent with the fact that all of these isolated cells will eventually depolarize and undergo spontaneous apoptosis. Glucocorticoid treatment augmented the loss of Na+/K+-ATPase protein to negligibly detectable levels. In contrast, under the same experimental conditions, GR protein decreased only minimally under these in vitro incubation conditions, and cellular actin levels did not change. These results demonstrate that Na+/K+-ATPase protein levels are selectively decreased during cell death and are consistent with the observed depolarization of the plasma membrane in both spontaneous and glucocorticoid-induced thymocyte apoptosis. The extent of decrease in Na+/K+-ATPase protein levels largely reflects the number of cells in spontaneous and glucocorticoid-induced death that depolarize their plasma membrane, but the assays we have used do not directly measure activity.



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  		Figure 3. Glucocorticoids repress Na+/K+-ATPase protein levels in primary thymocytes. Total cellular protein was extracted from fresh thymocytes (FRESH) or thymocytes cultured for 6 h in the presence (DEX) or absence (CON) of 100 nM dexamethasone. Twenty-five micrograms of total cellular protein was resolved by SDS-PAGE. Protein levels of the {alpha}1- and ß1-subunits of Na+/K+-ATPase as well as levels of GR and actin were evaluated by immunoblot analysis as described in Materials and Methods.

 
Glucocorticoid-induced loss of plasma membrane potential is modulated by caspases
Our observation that plasma membrane depolarization and the loss of Na+/K+-ATPase protein are observed in both spontaneously dying thymocytes and glucocorticoid-treated thymocytes suggests that a common pathway is activated during both signaling cascades necessary for the induction of apoptosis. We have previously shown that activation of the caspase cascade is a common feature of both spontaneous and glucocorticoid-induced apoptosis (5). Furthermore, we have shown that z-VAD, a pan-caspase inhibitor, modulates both spontaneous cell shrinkage and blocks glucocorticoid-induced cell shrinkage in thymocytes (5). In addition, cell shrinkage and depolarization appeared to be associated in glucocorticoid-induced apoptosis (19). To determine whether caspases are involved in glucocorticoid-induced membrane depolarization, we next evaluated the effects of caspase inhibition on glucocorticoid-induced membrane depolarization and cell shrinkage. After 6 h in culture, 16.2 ± 1.4% (P < 0.05 vs. freshly isolated cells) of thymocytes undergoing spontaneous cell death were depolarized, and 44.8 ± 4.3% (P < 0.05 vs. time-matched control cells) of glucocorticoid-treated thymocytes were depolarized compared with freshly isolated thymocytes (8.6 ± 2.0%; Fig. 4Go). Cellular depolarization was accompanied by concomitant cell shrinkage, indicating that thymocyte depolarization and shrinkage are tightly coupled. The pan-caspase inhibitor z-VAD-fmk inhibited cell shrinkage and blocked plasma membrane depolarization (11.4 ± 2.3%), such that a statistically significant difference could not be observed between the percentage of depolarized cells in freshly isolated thymocytes or thymocytes treated with z-VAD. Glucocorticoid-induced depolarization of thymocytes was also blocked by z-VAD (14.9 ± 3.2%) as was glucocorticoid-induced cell shrinkage. Interestingly, IETD-fmk, a specific inhibitor of caspase 8-like enzymes, did not block cell shrinkage or the spontaneous depolarization of thymocytes (15.2 ± 0.9%; P < 0.05 vs. freshly isolated cells). Inhibition of caspase 8-like activity also did not block the glucocorticoid-induced cell shrinkage or the loss of plasma membrane potential (37.0 ± 4.5%; P < 0.05 vs. time-matched control cells). Inhibition of caspase 9-like activity by the specific inhibitor AEVD also was ineffective in blocking spontaneous (16.5 ± 0.5%; P < 0.05 vs. freshly isolated cells) or glucocorticoid-induced apoptosis (33.4 ± 1.0%; P < 0.05 vs. time-matched control cells). Furthermore, DEVD, a more specific inhibitor of caspase 3-like enzymes, was even less effective at blocking cell shrinkage and the loss of plasma membrane potential in spontaneous (25.0 ± 3.2%; P < 0.05 vs. time-matched control cells) or glucocorticoid-induced (39.5 ± 7.2%; P < 0.05 vs. time-matched control cells) cell death. These results suggest that the individual activation of caspase 8-like enzymes, caspase 9-like enzymes, and caspase 3-like enzymes is not sufficient for cell shrinkage or membrane depolarization in spontaneous or glucocorticoid-induced death. These specific caspase inhibitor studies suggest that either an unusual caspase is mediating membrane depolarization or perhaps z-VAD-fmk may inhibit proteases other than caspases, which are involved in the generation of a cellular depolarized state.



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  		Figure 4. Caspases modulate glucocorticoid-induced plasma membrane depolarization. Primary thymocytes were cultured with 100 µM z-VAD-fmk, 100 µM IETD-fmk, or 100 µM DEVD-fmk in the presence or absence of 100 nM dexamethasone for 6 h. After treatment, cells were incubated with DiBAC4(3 ). PI was added before flow cytometric analysis to exclude nonviable cells. DiBAC4(3 ) fluorescence was evaluated in 10,000 viable cells as described in Materials and Methods. To compare DiBAC4(3 ) fluorescence vs. cell size, cells were analyzed on a representative DiBAC4(3 ) fluorescence vs. forward scatter contour plot.

 
Inhibition of caspase activity blocks the decrease in Na+/K+-ATPase protein levels during thymocyte apoptosis
To determine the effect of caspases on Na+/K+-ATPase protein levels during apoptosis, we examined the effect of z-VAD-fmk on the loss of Na+/K+-ATPase protein levels during both spontaneous and glucocorticoid-induced apoptosis. As shown earlier (Fig. 3Go), the catalytic and regulatory subunits of Na+/K+-ATPase were both decreased in spontaneously dying and glucocorticoid-treated thymocytes compared with freshly isolated thymocytes (Fig. 5Go). Inhibition of the caspase cascade by the pan-caspase inhibitor z-VAD blocked the loss of in Na+/K+-ATPase levels in both spontaneous and glucocorticoid-induced apoptosis (Fig. 5Go) without affecting the level of cellular actin. These results are consistent with the observation that z-VAD blocked plasma membrane depolarization and suggest that a common pathway, perhaps mediated by novel caspases, is activated in both spontaneously dying and glucocorticoid-treated thymocytes.



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  		Figure 5. z-VAD blocks glucocorticoid-induced repression of Na+/K+-ATPase protein levels. Total cellular protein was extracted from fresh thymocytes or thymocytes cultured with 100 µM z-VAD-fmk in the presence or absence of 100 nM dexamethasone for 6 h. Twenty-five micrograms of total cellular protein were resolved by SDS-PAGE. Protein levels of the {alpha}1- and ß1-subunits of Na+/K+-ATPase and actin were evaluated by immunoblot analysis as described in Materials and Methods.

 
Cells with a loss of plasma membrane potential comprise the shrunken population of cells
The experiments described thus far were performed in a population of cells that included both shrunken, apoptotic cells and normal cells. We have previously shown that shrinkage is a defining feature of apoptosis that discriminates committed and noncommitted cells, and in Fas-treated Jurkat cells, depolarization precedes cell shrinkage (18). To determine whether depolarization precedes cell shrinkage in glucocorticoid-treated thymocytes, we analyzed thymocytes by flow cytometry to simultaneously determine cell size and relative plasma membrane potential (Fig. 6Go). Freshly isolated cells form a population with a uniform size distribution that has low DiBAC4(3) fluorescence, indicating that the plasma membrane potential is intact. Spontaneous cell death results in the appearance of a small population of shrunken cells, which is consistent with our previous observation that spontaneously dying thymocytes shrink (5). In spontaneous apoptosis, the population of cells that are still of normal size has low DiBAC4(3) fluorescence, indicating that these cells have not lost their plasma membrane potential. However, the shrunken population of cells has high DiBAC4(3) fluorescence, indicating that all of these cells have lost plasma membrane potential. Glucocorticoids dramatically increase the shrunken population of cells, as we have reported previously (5). However, there remains a subpopulation with a normal cell volume. This population, similar to normal cells in the fresh and spontaneously dying samples, still has low DiBAC4(3) fluorescence. All of the shrunken cells, however, have high DiBAC4(3) fluorescence, indicating they have lost their plasma membrane potential. These results demonstrate that in spontaneously dying and glucocorticoid-treated thymocytes, plasma membrane depolarization is limited to the shrunken population of cells.



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  		Figure 6. Simultaneous depolarization and shrinkage in rat thymocytes. Fresh thymocytes (FRESH) or thymocytes cultured in the presence (DEX) or absence (CON) of dexamethasone for 6 h were analyzed for their light-scattering properties and DiBAC4(3 ) fluorescence. The center column shows forward scatter vs. side scatter dot plots for each treatment. Gates were set based on the distribution of the normal population in the freshly isolated cells (right gate). Shrunken cells have a decrease in forward scatter and a concomitant increase in side scatter and are shown in the left gate. The left column shows the DiBAC4(3 ) fluorescence for the shrunken population. The right column shows the DiBAC4(3 ) fluorescence for the normal population.

 
The decrease in Na+/K+-ATPase protein levels is limited to the shrunken, depolarized population of cells
The results presented thus far suggest that cell shrinkage and depolarization correlate with a decrease in Na+/K+-ATPase protein levels. However, the analyses of Na+/K+-ATPase protein levels were only conducted on the entire population of cells, which contains a mixture of polarized normal cells and depolarized shrunken cells. Therefore, we were interested in determining whether the repression of Na+/K+-ATPase protein levels was restricted to either the normal or shrunken population of apoptotic cells. To accomplish this goal, glucocorticoid-treated cells were physically sorted into shrunken and normal populations and evaluated for changes in Na+/K+-ATPase levels. ATPase levels of the catalytic and regulatory subunits of Na+/K+-ATPase were also evaluated in freshly isolated thymocytes and in the entire population of glucocorticoid-treated thymocytes (Fig. 7Go). Cells sorted into normal and shrunken populations showed a dramatic difference in protein levels of the Na+/K+-ATPase. In the normal population, Na+/K+-ATPase protein levels were the same as those for freshly isolated thymocytes. In contrast, the shrunken population showed dramatically diminished levels of both catalytic and regulatory subunits of Na+/K+-ATPase, whereas no significant difference in the cellular levels of actin was seen between the normal and shrunken populations of cells. These results demonstrate that the repression of Na+/K+-ATPase protein levels, like depolarization, is limited to the shrunken population of cells.



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  		Figure 7. Na+/K+-ATPase protein levels decrease in shrunken rat thymocytes. Thymocytes were treated with dexamethasone for 3 h to induce cell shrinkage. After the incubation, thymocytes were physically sorted into normal and shrunken populations, as described in Materials and Methods. Total cellular protein was extracted from freshly isolated thymocytes (FRESH), thymocytes treated with dexamethasone for 3 h just before sorting (PRESORT), normal thymocytes (NORM), and shrunken thymocytes (SHRUNK). Twenty-five micrograms of total cellular protein were resolved by SDS-PAGE. Protein levels of the {alpha}1- and ß1-subunits of Na+/K+-ATPase as well as actin were evaluated by immunoblot analysis as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Research over the last several years has established that cell shrinkage and the movement of ions play important roles in apoptosis (8, 9, 10, 16, 32). The movement of ions and the resulting alterations in the electrical field across the membrane lead to cellular depolarization, which is an important step in many cellular processes ranging from stimulus- secretion coupling (33) to mitogenic T cell activation (34). Work from our laboratory (18, 19) as well as others (35) has suggested that plasma membrane depolarization may be an important component of the apoptotic process. In addition, we recently reported that Na+/K+-ATPase might play a key role in anti-Fas-induced depolarization of Jurkat cells (18). This study has extended these observations to define a role for Na+/K+-ATPase in the depolarization of primary thymocytes during both spontaneous and glucocorticoid- induced apoptosis.

Na+/K+-ATPase is the primary determinant for setting plasma membrane potential in lymphocytes (20, 21, 22). The present study shows that inhibition of Na+/K+-ATPase by ouabain depolarizes thymocytes and potentiates glucocorticoid-induced depolarization of primary thymocytes. These data support our previous observation that inhibition of Na+/K+-ATPase by ouabain potentiates Fas-induced depolarization in Jurkat cells and suggests a role for Na+/K+-ATPase in thymocyte depolarization and apoptosis. An important role for Na+/K+-ATPase in lymphocyte function is also supported by the fact that Na+/K+-ATPase activity increases during mitogenic T cell activation (36, 37). Furthermore, inhibition of Na+/K+-ATPase activity by ouabain has been shown to block mitogenic T cell activation (38). Together, these observations implicate Na+/K+-ATPase as an important moderator of lymphocyte survival.

The plasma membrane depolarization induced during both spontaneous and glucocorticoid-induced apoptosis was associated with a selective decrease in Na+/K+-ATPase protein levels within the cell. The fact that we observed Na+/K+-ATPase degradation in both spontaneous and glucocorticoid-induced death suggests that the repression of Na+/K+-ATPase levels is a shared feature of a common apoptotic pathway induced by disparate signals. Indeed, we also observed that Na+/K+-ATPase levels are decreased during Fas-induced apoptosis of Jurkat cells, and inhibition of this depolarization via activation of PKC blocked apoptotic cell death (18). These results suggested that a pathway common to these diverse apoptotic-signaling pathways results in the repression of Na+/K+-ATPase protein levels.

The activation of the caspase cascade is a common feature to these divergent forms of apoptosis (5, 15, 39, 40, 41, 42). We have previously shown that caspases are activated during spontaneous and glucocorticoid-induced apoptosis, and that caspases mediate glucocorticoid-induced and spontaneous cell shrinkage (5, 15). In this study we found that the pan-caspase inhibitor z-VAD-fmk blocked the loss of plasma membrane potential in both spontaneous and glucocorticoid-induced apoptosis, which suggests that the caspase cascade plays a role in cell membrane depolarization during apoptosis. Furthermore, the inhibition of depolarization by z-VAD occurred simultaneously with the inhibition of cell shrinkage, suggesting a close relation between the two processes, as we have noted previously (18). Inhibition of the caspase cascade by the pan-caspase inhibitor z-VAD-fmk also blocked the repression of Na+/K+-ATPase protein levels in both spontaneous and glucocorticoid-induced apoptosis of thymocytes. These data suggest the involvement of the caspase cascade in the repression of Na+/K+-ATPase levels.

However, selective inhibition of caspase 8-like enzymes, caspase 9-like enzymes, or caspase 3-like enzymes did not block spontaneous or glucocorticoid-induced depolarization in primary thymocytes, suggesting that the activation of these specific enzymes is not required or downstream of plasma membrane depolarization in thymocytes. In anti-Fas-treated Jurkat T cells, other characteristics, such as cell shrinkage, potassium loss, and the loss of mitochondrial membrane potential, were all shown to be independent of caspase 9-like and caspase 3-like enzymes (17). However, the nonspecific caspase inhibitor z-VAD-fmk was able to prevent these characteristics of apoptosis in anti-Fas-treated Jurkat cells. Therefore, either different caspases may be involved in the pathway that leads to plasma membrane depolarization or z-VAD-fmk may be a less specific inhibitor of proteases than was previously thought, perhaps acting outside the caspase pathway.

Activation of the caspase cascade and cell shrinkage are tightly linked. In anti-Fas-treated Jurkat cells, DNA fragmentation, decreased potassium content, and active caspase 3-like enzymes are limited to the shrunken population of cells (10). In fact, the activation of caspase 3-like enzymes is inhibited by physiological concentrations of potassium, and caspase 3-like and nuclease activity are observed only in cells with decreased potassium content (16). The data presented in the present study show that plasma membrane depolarization in thymocytes is limited to the shrunken population of cells in both spontaneously dying cells and cells treated with glucocorticoids. These results indicate that in thymocytes, two stimuli that induce apoptosis through different signaling pathways (5) simultaneously trigger cell shrinkage and depolarization. In contrast, we have observed that Jurkat cells treated with anti-Fas depolarize before the loss of cell volume (18). One possible explanation for this difference is that thymocytes are much smaller than Jurkat cells and respond rapidly to glucocorticoid treatment, which may make it difficult to temporally distinguish sequential activation of depolarization and shrinkage in these cells. Additionally, the difference in cellular responsiveness could be due to differences in the cell types. Whereas thymocytes are a primary cell line and die spontaneously in culture, Jurkat cells are a transformed cell line that has been selected to grow in culture. Alternatively, the difference between Jurkat cells and thymocytes could be due to differences in the pathways that regulate depolarization and shrinkage in these cells.

Although levels of Na+/K+-ATPase decrease during spontaneous and glucocorticoid-induced apoptosis, the protein levels were initially evaluated in a mixed population of cells that included both normal and shrunken cells. When these two populations of cells were subsequently physically separated, we observed that the normal population retains Na+/K+-ATPase expression levels equivalent to freshly isolated cells. The shrunken population, however, had a dramatic decrease in Na+/K+-ATPase protein levels, which demonstrates that the repression of Na+/K+-ATPase protein levels is limited to the shrunken population of cells and supports our previous finding that Na+/K+-ATPase protein levels of both the catalytic and regulatory subunits are repressed in anti-Fas-treated Jurkat cells.

The observation that ion fluxes and depolarization occur early in apoptosis induced by disparate signals in different cell types suggests that these are conserved features of apoptosis that are also important events in other cellular processes. Na+/K+-ATPase plays a central role in maintaining this ionic balance and is conserved across species (43). Given that the potential difference across the plasma membrane results from the asymmetric distribution of ions and is maintained by the electrogenic action of Na+/K+-ATPase (22), it stands to reason that Na+/K+-ATPase may play an important role in apoptosis as it does in other cellular processes. The importance of Na+/K+-ATPase in apoptosis was first demonstrated in Jurkat cells treated with anti-Fas. We have now extended these studies to primary thymocytes and have observed that two disparate apoptotic signals, survival factor withdrawal and glucocorticoids, repress Na+/K+-ATPase protein levels during thymocyte apoptosis. Additionally, we have shown that both depolarization and the repression of Na+/K+-ATPase protein levels are mediated by the caspase cascade and are limited to the shrunken population of cells. Future work in this area will likely show that Na+/K+-ATPase, which is essential to the maintenance of cellular homeostasis, is an important target in other forms of apoptosis as well.


   Footnotes
 
Abbreviations: AEVD-fmk, Z-Ala-Glu (Ome)-Val-Asp (Ome)-FMK; DEVD-fmk, Z-Asp-Glu-Val-Asp-fluoromethylketone; IETD-fmk, Z-Ile-Glu-Thr-Asp-fluoromethylketone; Na+/K+-ATPase, Na+/K+-adenosine triphosphatase; PI, propidium iodide; TBS, Tris-buffered saline; z-VAD-fmk, Z-Val-Ala-Asp-fluoromethylketone.

Received March 12, 2001.

Accepted for publication July 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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