©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Apoptosis in an Interleukin-2-dependent Cytotoxic T Lymphocyte Cell Line Is Associated with Intracellular Acidification
ROLE OF THE Na/H-ANTIPORT (*)

(Received for publication, December 19, 1994)

Jinfang Li Alan Eastman (§)

From the Department of Pharmacology and Toxicology and the Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire 03755

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apoptosis is a form of cell death associated with DNA fragmentation and chromatin condensation. We recently established that intracellular acidification occurred during apoptosis following cytotoxic insult. The current studies were designed to determine whether intracellular acidification was more generally associated with apoptosis, specifically in a model of growth factor withdrawal. Upon withdrawal of interleukin-2, CTLL-2 cells accumulated in the G(1) phase of the cell cycle and started to fragment their DNA around 12 h concurrent with both decreased pH and increased Ca. Chelation of Ca did not inhibit DNA digestion, whereas incubation with a calcium ionophore prevented both acidification and DNA digestion. Hence, acidification rather than increased Ca was associated with apoptosis. The acidified cells represented a discrete population up to 0.7 pH units below normal. The extent of acidification depended upon the extracellular pH; above pH 6.3, intracellular pH was significantly below extracellular pH, whereas below pH 6.3, the cells still regulated their pH. Inhibition of the Na/H-antiport prevented the apoptotic cells from regulating their intracellular pH under these acidic conditions. These results demonstrate that apoptotic cells retain a functional antiport but that its set-point has changed. Many survival factors are known to phosphorylate and activate the antiport, hence apoptosis is likely to be associated with dephosphorylation. Although acidification always occurred during apoptosis, maintaining intracellular pH above 7.2 did not prevent apoptosis, suggesting that an acid pH is not essential for apoptosis. We hypothesize that other critical regulators of apoptosis must be subject to dephosphorylation.


INTRODUCTION

Apoptosis is a form of cell death defined by morphological criteria(1, 2) . The earliest event observed in a cell undergoing apoptosis is chromatin condensation at the nuclear membrane, whereas other organelles remain intact. This is generally followed by nuclear fragmentation, cell surface blebbing and cell shrinkage. Loss of membrane integrity is a late event. These changes occur as a consequence of many initiating stimuli such as glucocorticoid-mediated killing of thymocytes(3) , growth factor withdrawal(4) , and cytotoxic T-cell killing(5) . In addition, a wide variety of cytotoxic agents induce apoptosis(6, 7) .

Much interest in apoptosis has been generated by the observation that genes such as bcl-2 can prevent cells from dying(8) . There are now many genes and drugs that can either cause or prevent apoptosis, but there is little understanding of the pathways that induce this form of cell death. Accordingly, there is no framework for understanding how any gene or drug modulates the eventual cell death. The current experiments were designed to address this important gap in our understanding.

The origin of the chromatin condensation and therefore an initiating event in apoptosis is thought to be DNA fragmentation. Usually, this fragmentation arises from internucleosomal digestion of genomic DNA (1) . Several reports that apoptosis occurs without such DNA digestion can be explained by the finding that DNA breaks at >50-kilobase pair frequency are sufficient to induce the morphological changes of apoptosis(9, 10) . To understand the signal transduction pathways leading to apoptosis, we have attempted to identify an endonuclease that produces the DNA digestion, so that we can subsequently understand the upstream events that regulate its activation.

Mammalian cells contain a variety of endonucleases any of which could be involved in apoptosis(11) . A Ca/Mg-dependent endonuclease has frequently been implicated in apoptosis, but many cells appear to lack this endonuclease or lack the increase in Ca required to activate it. Even former proponents of a Ca/Mg-dependent endonuclease have recently suggested that Ca may be involved in a signal transduction pathway rather than as a direct activator of the endonuclease(12) . Our laboratory identified an alternate endonuclease, deoxyribonuclease II, that was activated by decreased intracellular pH with no requirement for Ca(13) . In human HL-60 cells, apoptosis can be induced rapidly by many drugs. Using the topoisomerase II inhibitor etoposide, we established that apoptotic cells underwent selective inhibition of pH regulation leading to a 0.8 pH unit acidification with no change in intracellular Ca(14) . Cells were sorted on a flow cytometer, and only the acidic cells had the morphology and DNA digestion of apoptosis. These results were consistent with apoptosis resulting from intracellular acidification.

The current experiments were designed to investigate whether intracellular acidification was more generally associated with the induction of apoptosis. We have therefore extended our studies to a common physiological model of apoptosis; specifically, a system in which apoptosis is induced by removal of a growth factor. We report on an IL-2(^1)-dependent cytotoxic T lymphocyte cell line CTLL-2 and establish that intracellular acidification, but not increased Ca, also correlates with apoptosis in this model. We further demonstrate that the acidification can be explained by alteration in the set-point of the Na/H-antiport. However, we also show that an acidic pH is not required for apoptosis. This has led to a hypothesis that may explain the regulation of apoptosis.


EXPERIMENTAL PROCEDURES

Materials

Hoechst 33342 and the acetoxymethyl esters of carboxyl-SNARF-1, indo-1, and EGTA were purchased from Molecular Probes (Eugene, OR). Other chemicals were obtained from Sigma. Cell culture supplies were purchased from Life Technologies, Inc.

Cell Culture

IL-2-dependent murine cytotoxic T-lymphocyte CTLL-2 cells (15) were maintained at a density of 0.1-2.0 times 10^5 cells/ml. The cells were maintained in 5% CO(2) at 37 °C in Dulbecco's modified Eagle's medium with 10% heat-inactivated (56 °C, 30 min) fetal bovine serum, penicillin, streptomycin, 90 pM recombinant IL-2, 100 µM 2-mercaptoethanol, 25 mM HEPES, 30 mM NaHCO(3), 0.12 µM Na(2)SeO(4), and 0.1 mM cystine HCl at pH 7.4. In several experiments, cells were incubated in medium at various pH. This was achieved by making complete medium at either pH 8.0 with 50 mM HEPES or 5.5 with 50 mM Mes. These media were mixed to obtain the required final pH. Sodium bicarbonate was added to 20 mM as required. The pH values were always measured after mixing, as well as at the end of the experiment to confirm that no drift had occurred.

DNA Digestion

DNA digestion in cells was measured by electrophoresis as described previously(13, 16) . Briefly, 10^6 cells are lysed directly in the wells of the gel and high molecular weight genomic DNA remains trapped in or near the well. Smaller fragments down to 180 base pairs are resolved in the gel. DNA is visualized with ethidium bromide.

Flow Cytometry

Cell cycle analysis was performed on cells fixed in ethanol and stained with propidium iodide as described previously(17) . Intracellular pH (pH(i)) and Ca were measured as described previously(14, 16) . For pH(i) measurements, cells were loaded for 1 h with 2 µM carboxyl-SNARF-1 AM and analyzed on a flow cytometer with excitation at 488 nm and emission measured at 585 and 640 nm. For Ca measurements, cells were loaded with 1 µM indo-1 AM and analyzed on a flow cytometer with excitation at 355 nm and emission measured at 405 and 485 nm. For both measurements, cells were maintained at 37 °C in complete medium, including bicarbonate during analysis unless otherwise noted. Quantitation of the number of cells in various populations was obtained by drawing regions on the profiles and excluding cells with fluorescence <50 to avoid cells that either do not load with dye or that have lost membrane integrity. The pH(i) and Ca measurements were obtained by ratioing the fluorescence emissions at the two appropriate wavelengths and generating a pH calibration curve or using a Ca dissociation equation(18) . Cells were also sorted based on these ratios and collected into separate tubes on ice and analyzed for DNA digestion.

As an additional assay for apoptosis, cells were incubated with 1 µg/ml Hoechst 33342 for 5 min and analyzed on a flow cytometer with excitation at 355 nm and emission measured at 440 nm. This assay detects an increased rate of accumulation of Hoechst 33342 into apoptotic cells which is thought to be due to very early changes in membrane permeability(19) . pH(i) and Hoechst 33342 fluorescence were measured simultaneously by first incubating cells with carboxyl-SNARF-1 AM for 1 h in complete medium, then washing once and resuspending the cells in medium at defined pH(e) containing Hoechst 33342. As needed, the medium also contained 20 µM EIPA to inhibit the Na/H-antiport. Analysis was performed after 5 min, a time shown in initial experiments to equilibrate the pH(i), and results were displayed as a two-dimensional contour plot of 585/640 ratio for pH versus Hoechst 33342 fluorescence.


RESULTS

DNA Digestion and Apoptosis

Cytotoxic T lymphocyte CTLL-2 cells require IL-2 for survival and proliferation. Upon removal of IL-2, these cells degrade their DNA in a manner characteristic of apoptosis. To determine the kinetics of appearance of DNA digestion, cells were harvested at various times after removal of IL-2, and the DNA was analyzed by electrophoresis for the appearance of 180-base pair DNA fragments (Fig. 1A). In the presence of IL-2, no digested DNA was observed. Following removal of IL-2, DNA digestion was significantly above background at 12 h, reaching a maximum around 16 h.


Figure 1: DNA digestion induced in CTLL-2 cells following withdrawal of IL-2. 10^6 cells were electrophoresed in each lane except where stated. A, time course of appearance of DNA fragments following withdrawal of IL-2. B, following 16 h without IL-2, cells were analyzed by flow cytometry for intracellular pH and Ca and sorted into the 40% most alkaline and 40% most acidic cells (see Fig. 3) or the 40% lowest Ca and 40% highest Ca (see Fig. 4). In the case of the sorted cells, 2 times 10^5 cells were analyzed in each lane. C, cells were incubated with either 5 mM EGTA or 1 µM ionomycin for 16 h, with or without IL-2.




Figure 3: Flow cytometric analysis of intracellular pH changes in individual CTLL-2 cells following withdrawal of IL-2. At the indicated times following withdrawal of IL-2, cells were loaded with carboxyl-SNARF-1, then analyzed by flow cytometry, and displayed as a two-dimensional scatter plot according to the fluorescence intensities at emission wavelengths of 585 and 640 nm. Quantitation was performed by drawing regions around each population excluding the cells with fluorescence <50 and presenting the results as histograms: A, cells with normal pH; B, acidic cells.




Figure 4: Flow cytometric analysis of intracellular Ca changes in individual CTLL-2 cells following withdrawal of IL-2. At the indicated times following withdrawal of IL-2, cells were loaded with indo-1, then analyzed by flow cytometry, and displayed as a two-dimensional scatter plot according to the fluorescence intensities at emission wavelengths of 405 and 485 nm. Quantitation was performed by drawing regions around each population, excluding the few cells with fluorescence <50, and presenting the results as histograms: A, cells with normal Ca; B, cells with increased Ca.



The cell cycle phase dependence of apoptosis following removal of IL-2 was assessed. Cells were analyzed for DNA content by flow cytometry (Fig. 2). By 4 h following removal of IL-2, cells began to accumulate in the G(1) phase, and by 8 h, most of the cells had exited the S phase. At 16 h, the time at which DNA digestion appeared maximum, most of the cells remained arrested in the G(1) phase. Not until 20 h did a significant number of cells begin to appear in the sub-G(1) population as is frequently observed in apoptotic cells. However, these sub-G(1) cells rapidly disintegrate into many fragments, so quantitation of this population is not possible. Up to the time that the cells disintegrate, they also exclude trypan blue. These results show that DNA digestion occurs several hours before loss of membrane integrity and cell disintegration.


Figure 2: Cell cycle analysis of CTLL-2 cells following withdrawal of IL-2. Relative DNA content was assessed by fixing cells, staining with propidium iodide, and analyzing by flow cytometry.



Intracellular pH

We have previously observed intracellular acidification in human HL-60 and ML-1 cells following incubation with etoposide(14, 20) . For those studies, we adapted the flow cytometer to measure pH(i) which facilitated detection of large pH shifts in individual cells as opposed to only small shifts observed when the average of a total population was measured in a spectrofluorimeter. CTLL-2 cells were loaded with the pH-sensitive fluorescent dye, carboxyl-SNARF-1, and then analyzed by flow cytometry. The data are displayed in a two-dimensional scatter plot according to the fluorescent intensities at two emission wavelengths (Fig. 3). The cells exhibit a large variation in loading of the fluorescent dye, but in a control population, all the cells lie close to a single line extending outward from the origin. By comparison with a standard curve, the mean pH(i) in these cells was 7.4 when the pH(e) was 7.40. Cells to the right of this line would be more acidic, whereas cells to the left would be more alkaline.

Twelve hours following removal of IL-2, a second population of cells began to appear to the right of the control population. As previously seen with HL-60 and ML-1 cells(14, 20) , this population was not a continuum of cells with decreasing pH(i), but rather a distinct population with a more acidic pH(i). The mean pH(i) of this acidic population was 6.7. The fluorescence intensity of the acidic cells was lower than the normal cells, suggesting that the acidic cells have a reduced uptake or increased efflux of carboxyl SNARF-1. However, the measurement of pH(i) is independent of the intracellular dye concentration as it is the ratio of fluorescence emissions that is used to calculate pH. Quantitation of the number of cells in each population was obtained by drawing regions around these cells; these values are presented as histograms in Fig. 3.

The number of acidic cells increased throughout the experiment with concomitant decrease in the control population. At 12 h, approximately 7% of the cells were in the acidic population. The number of acidic cells increased to 20, 30, and 48% at 16, 20, and 24 h, respectively. The time of acidification correlated closely with the time of the DNA digestion and occurred prior to fragmentation of the cells or loss of membrane integrity as assayed by trypan blue.

Intracellular Ca

The flow cytometer was also used to investigate whether increased intracellular Ca occurred following withdrawal of IL-2 (Fig. 4). The intracellular Ca concentration of the control cells was about 90 nM when the cells were incubated in normal medium with an extracellular Ca concentration of 1.8 mM. At 16 h following removal of IL-2, about 12% of the cells had shifted to the right which represents an increased intracellular Ca. This number increased to 44% by 24 h. Unlike the pH shift in which a second population was observed, these cells showed a heterogeneous increase. Of the cells with increased Ca, the average Ca concentration was 600 nM, whereas the maximum was around 1 µM.

Cell Sorting

To determine whether the cells with low pH(i) and high Ca were the same cells, and to determine which cells had undergone DNA digestion, the flow cytometer was used to sort the cells based on either parameter. Cells were analyzed 16 h after removal of IL-2. The cells were separated into the 40% most acidic and 40% most alkaline or those with the 40% highest and 40% lowest intracellular Ca. Cells were collected directly on to ice and then analyzed for DNA digestion (Fig. 1B). Almost all of the DNA digestion was observed in the acidic cells as well as in the cells with high Ca. This demonstrated that the acidified cells were also those with increased Ca, so it did not resolve the question as to which of these signals might be associated with DNA digestion.

Modulation of Intracellular pH and Ca

CTLL-2 cells were incubated with the Ca chelator EGTA to determine whether this would block the intracellular Ca increase and suppress DNA digestion following removal of IL-2. Incubation of CTLL-2 cells in the presence of IL-2 and 5 mM EGTA for 16 h caused a slight shift to the left in the two-dimensional dot plot consistent with a decrease in intracellular Ca to near zero (Fig. 5). Following removal of IL-2 for 16 h, 26% of the cells shifted to the high Ca region, but this increase was completely inhibited by the presence of EGTA. Incubation of cells with the acetoxymethyl ester form of EGTA to chelate intracellular Ca was unable to prevent the increase in Ca, suggesting that the source of Ca was the extracellular medium. Parallel samples were also analyzed for changes in intracellular pH (Fig. 6). Following removal of IL-2, about 30% of the cells underwent intracellular acidification, but the addition of EGTA did not reduce this number. When the cellular DNA was analyzed following removal of IL-2, DNA digestion still occurred in the presence of EGTA (Fig. 1C), demonstrating that intracellular acidification rather than increases in Ca correlated with DNA digestion.


Figure 5: Modulation of intracellular Ca by EGTA and ionomycin in CTLL-2 cells following withdrawal of IL-2. Cells were incubated with 5 mM EGTA or 1 µM ionomycin for 16 h in the presence or absence of IL-2 and analyzed by flow cytometry for intracellular Ca. Histograms are as described in legend to Fig. 4.




Figure 6: Modulation of intracellular pH by EGTA and ionomycin in CTLL-2 cells following withdrawal of IL-2. Cells were incubated with 5 mM EGTA or 1 µM ionomycin for 16 h in the presence or absence of IL-2 and analyzed by flow cytometry for intracellular pH. Histograms are as described in legend to Fig. 3.



Calcium ionophores often induce apoptosis(21, 22) , but in some systems they have also been shown to prevent apoptosis(23, 24) . Similarly, calcium ionophores can cause either intracellular acidification or alkalinization(22, 25) . Accordingly, we investigated the effect of ionomycin on intracellular pH and Ca, as well as on DNA digestion. Ionomycin (1 µM) in the presence of IL-2 increased intracellular Ca in the majority of cells to 400 nM, and after removal of IL-2, a subpopulation was observed with even higher Ca (Fig. 5). In contrast, ionomycin inhibited intracellular acidification following removal of IL-2 (Fig. 6). Ionomycin appeared to decrease the carboxyl-SNARF-1 fluorescence in many cells, but there was clearly no cells in the acidic population. Ionomycin also markedly inhibited DNA digestion (Fig. 1C). These results confirm that acidification rather than increase in intracellular Ca concentration correlates with apoptotic DNA digestion.

Activation of protein kinase C by phorbol esters TPA can also protect cells from apoptosis(26, 27) . The addition of 10 nM TPA to CTLL-2 cells at the time of removal of IL-2 inhibited both the acidification and DNA digestion up to at least 24 h (data not shown). These cells still accumulated in the G(1) phase but produced no sub-G(1) population of apoptotic cells. Intracellular pH could also be modulated by the readdition of IL-2 at any time point, which immediately prevented more cells from undergoing intracellular acidification as well as preventing further DNA digestion, although readdition of IL-2 could not reverse acidification (data not shown).

pH Regulation

The origin of the intracellular acidification was investigated further. We previously hypothesized that the intracellular acidification was due to selective loss of pH regulation while maintaining an electrochemical gradient across the cytoplasmic membrane (14) (see ``Discussion''). Accordingly, we determined which proton regulators were responsible for normal regulation of pH in these cells. CTLL-2 cells were acid loaded by transient exposure, then removal of ammonium chloride. The rate of pH recovery was then measured continuously on the flow cytometer (Fig. 7). The intracellular pH returned to 7.3 in about 10 min in bicarbonate-free medium, and this recovery could be inhibited by concurrent incubation with the Na/H-antiport inhibitor EIPA (20 µM). Parallel studies with the H-ATPase inhibitor bafilomycin (1 µM) had little effect on the rate of pH recovery. Hence, these cells appear to use primarily the Na/H-antiport to protect against intracellular acidification.


Figure 7: Identification of proton transport mechanisms operating in CTLL-2 cells. Cells were loaded with carboxyl-SNARF-1 for 1 h, then analyzed continuously by flow cytometry in the absence of bicarbonate; the lines represent the mean pH of the cell population. Ammonium chloride (10 mM) was added to cells at the indicated time, then removed rapidly by centrifugation after 10 min followed by one wash. The first part of the curve is representative of the pH changes that occur prior to the acid-loading step. Following acid loading, cells were allowed to recover either in control medium (Cont) or medium containing 1 µM bafilomycin (Baf) or 20 µM EIPA.



A more detailed analysis of the pH changes that occur in CTLL-2 cells was performed. Following the induction of apoptosis, cells were equilibrated for 5 min at different pH(e) and the pH(i) was measured (Fig. 8). During the final 5 min, the cells were also incubated with Hoechst 33342; this helps to discriminate the normal from apoptotic cells as the latter show enhanced Hoechst fluorescence. When pH(e) was between 7 and 7.9, the undamaged cells were able to maintain their pH(i) close to 7.4; at lower pH(e), these cells still maintained their pH(i) above pH(e). In contrast, the pH(i) in apoptotic cells was lower than pH(e) except under the most acidic conditions. If these apoptotic cells were completely unable to regulate pH(i), then the line for pH(i) would be parallel to, but more acidic than the pH(e) by an amount dependent upon the electrochemical gradient across the cytoplasmic membrane. However, the two lines were not parallel, rather they converged around pH 6.3. To determine whether the convergence of these two lines was due to the Na/H-antiport, cells were equilibrated in the presence of EIPA; under these conditions, the pH(i) dropped dramatically at acidic pH(e). In contrast, incubation of control cells with EIPA for 5 min had no effect on the pH(i) because EIPA only prevents recovery from an acid load as occurs in the apoptotic cells, but does not itself cause acidification. Parallel experiments in the presence of bicarbonate had negligible effect on these profiles (data not shown). These results demonstrate that the apoptotic cells still possess a functional Na/H-antiport, but that its set-point has changed.


Figure 8: The dependence of pH on pH in normal and apoptotic CTLL-2 cells. Fifteen hours after withdrawal of IL-2, cells were loaded with carboxyl-SNARF-1 for 1 h, then equilibrated in medium of various pH containing 1 µg/ml Hoechst 33342 and as required, 20 µM EIPA. After 5 min, pH was measured by flow cytometry. A-F represent examples of the experimental results. Cells at pH = 7.40 (A, C, E) or 6.45 (B, D, F) were equilibrated either in the absence (A, B) or presence (C, D) of EIPA. pH is recorded on the abscissa and Hoechst fluorescence on the ordinate. The pH profiles are overlapped in E and F, to emphasize that EIPA only influences pH in apoptotic cells at low pH. The summary of the complete experiment is shown in G; the diagonal line represents pH = pH.



Role of Acidification in DNA Digestion

The experiment described above and in Fig. 8provides an experimental strategy for testing whether the observed intracellular acidification is required for DNA digestion. If pH(e) is raised to 7.9, the pH(i) in apoptotic cells is approximately 7.2. Under these conditions, DNA digestion would not be expected to occur if acidification was required either to activate deoxyribonuclease II or any other endonuclease. CTLL-2 cells were incubated in either the presence or absence of IL-2 at pH(e) values from 5.8 to 8.0. In the presence of IL-2, DNA digestion was only observed at low pH, probably due to deoxyribonuclease II activity (Fig. 9). In the absence of IL-2, DNA digestion was observed at every pH(e) tested, including pH(e) 8, suggesting that a low intracellular pH was not required for apoptosis.


Figure 9: The influence of pH on DNA digestion in CTLL-2 cells following withdrawal of IL-2. Cells were incubated at the indicated pH in the presence or absence of IL-2 for 16 h. DNA digestion was then analyzed by gel electrophoresis.




DISCUSSION

Growth factors are usually required for passage of cells through the G(1) phase of the cell cycle. Upon removal of IL-2, CTLL-2 cells initially accumulated in the G(1) phase. Between 8 and 12 h after IL-2 withdrawal, these cells began to digest their DNA in a manner characteristic of apoptosis. No other indicator of toxicity was evident until around 20 h at which time a population of cells was observed with sub-G(1) DNA content. Until this time, the cells maintained membrane integrity.

The flow cytometer was used to measure intracellular H and Ca as these two ions have been variously implicated in apoptosis. Although the apoptotic cells exhibited increases in both of these ions, only increased H consistently correlated with the appearance of DNA digestion. In particular, when EGTA was used to chelate extracellular Ca, no increase in intracellular Ca was observed even though the cells still underwent intracellular acidification and apoptotic DNA digestion. Furthermore, prevention of intracellular acidification with ionomycin prevented DNA digestion.

Perhaps the most significant observation is that the intracellular acidification produced a discrete population of cells with a pH about 0.7 below that for control cells. This same observation was made when HL-60 and ML-1 cells underwent apoptosis following incubation with the anti-cancer drug etoposide(14, 20) . These acidic cells remain viable as judged by their retention of the fluorescent dyes, exclusion of trypan blue, and by their ability to maintain low intracellular Ca against a Ca gradient which requires metabolic activity. It is assumed that an electrochemical gradient is maintained across the cytoplasmic membrane as this provides the only explanation for the observations. Under conditions of a normal electrochemical gradient (-60 mV), the Nernst equation calculates that complete inhibition of pH regulation will cause the pH(i) to drop 1 pH unit(28) . This is consistent with the intracellular acidification observed in these studies. Any other origin of intracellular acidification such as through metabolic production of protons, would lead to cells exhibiting a continuum of decreasing pH. Furthermore, complete loss of ion regulation would lead to equalization of the intracellular and extracellular pH.

Cells regulate their pH through a variety of ion transport mechanisms, including Na/H-antiports, ATP-driven H-pumps, and several bicarbonate exchangers(28) . The Na/H antiport is a major H-extruding mechanism which is driven by the inward-directed Na gradient. This antiport has a high affinity for H at pH 6, but is inoperative at neutral pH. However, a wide variety of external signals, including growth factors enhance the affinity of the antiport for H at neutral pH leading to alkalinization of the cells. This modulation of intracellular pH is brought about by phosphorylation of the antiport (29, 30, 31) . Activation of protein kinase C by phorbol esters has also been shown to phosphorylate and activate the Na/H-antiport(30, 32) .

CTLL-2 cells appear to regulate their pH(i) primarily with the Na/H-antiport as demonstrated by the ability of EIPA to inhibit recovery from an acid load. In contrast, an inhibitor of the H-ATPase did not prevent recovery of pH(i). A detailed analysis of the pH(i) over a range of pH(e) also suggested that a modification of the Na/H-antiport was responsible for the acidification observed during apoptosis. A similar experiment has been performed in mutant Chinese hamster ovary cells selected for lack of the Na/H-antiport; the pH(i) in these cells was well below the pH(e) even under acidic conditions(33) . These results can only be obtained if the cells retain an electrochemical gradient. This pattern is exactly what was observed here when apoptotic cells were equilibrated with EIPA. However, in the absence of EIPA, the apoptotic cells were still able to regulate their pH(i) under acidic conditions, demonstrating that they still possess a functional antiport but that its set-point has been altered. These observations are consistent with the hypothesis that apoptosis is associated with dephosphorylation of the antiport.

It has previously been suggested that the Na/H-antiport is important in apoptosis. Inhibitors of the antiport have been shown to overcome the protective effect of growth factors or phorbol esters in an IL-3-dependent leukemia cell line as well as inhibiting the alkalinization caused by IL-3 addition(34) . The previous results only observed pH(i) changes of 0.2 units upon addition or removal of growth factor, whereas the current results show a much greater acidification as cells undergo apoptosis. This suggests that previous work observed the fine tuning of the antiport, whereas we suggest that the acidification arises from a complete loss of pH regulation in the cells, possibly as a result of complete dephosphorylation of the antiport. It should be noted that the acidification occurs both in the presence and absence of bicarbonate, so presumably bicarbonate exchangers contribute little to the regulation of intracellular acidification during apoptosis.

Finally, we questioned whether the observed acidification was required for DNA digestion and apoptosis. Cells incubated in the absence of IL-2 at pH(e) 7.9 still underwent intracellular acidification, but only to pH(i) 7.2. Under these conditions, deoxyribonuclease II would not be expected to be active, yet DNA digestion was still observed. These results suggest that an acidic pH(i)per se is not required for activation of any component of an apoptotic pathway, whether it be an endonuclease, a protease, or some other component. Rather, the results suggest that the signal for acidification may be the same as the signal for apoptosis. The signal implicated in these experiments is a phosphatase that might dephosphorylate both the antiport and some other critical regulator of apoptosis.

In summary, these studies have demonstrated that intracellular acidification occurs during apoptosis following withdrawal of an essential survival factor. Together with our previous observations that acidification also occurs during apoptosis following incubation of HL-60 and ML-1 cells with cytotoxins, these results suggest that intracellular acidification is frequently associated with apoptosis. The origin of the acidification appears to be alteration in the set-point of the Na/H-antiport, possibly as a result of its dephosphorylation. This is consistent with the known function of many survival factors that phosphorylate and activate the antiport. However, the acidic pH(i) does not appear essential for apoptosis, so we hypothesize that other critical regulators of apoptosis must be subject to dephosphorylation. In this regard, there are reports that phosphatase inhibitors can protect cells from apoptosis(35, 36) .


FOOTNOTES

*
This study was supported by National Institutes of Health Grant CA50224. Flow cytometry was performed in the Fannie E. Rippel Flow Cytometry Laboratory, supported in part by a Cancer Center Core Grant CA23108. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, 7650 Remsen, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1667; Fax: 603-650-1129.

(^1)
The abbreviations used are: IL-2, interleukin-2; AM, acetoxymethyl ester; EIPA, ethylisopropylamiloride; pH, extracellular pH; pH, intracellular pH; TPA, 12-O-tetradecanoylphorbol-13-acetate; Mes, 2-(N-morpholino)ethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Tom Ciardelli for providing purified recombinant human IL-2.


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