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INTRODUCTION |
Signal transduction by cell-surface receptors is critical for the
regulation of cell growth and differentiation, but may serve a variety
of other functions, including induction of programmed cell death or
apoptosis. The cell-surface receptor Fas (CD95), which belongs to the
tumor necrosis factor/nerve growth factor receptor family, is the most
extensively studied of the so-called death receptors (1, 2).
Cross-linking Fas molecules on the cell surface either by Fas ligand or
by anti-Fas monoclonal antibody (mAb)1 results in the
apoptotic death of the cell. There have been a number of studies of the
phenotypic changes that occur early in apoptosis. Among those events,
the disruption of the mitochondrial transmembrane potential
(
m) (3, 4) and the aberrant exposure of
phosphatidylserine (PS) have been studied extensively (5, 6). It has
also been reported that a sustained increase in intracellular
Ca2+ concentration ([Ca2+]i) can
trigger lethal processes (7) and that increased intracellular
Mg2+ concentration ([Mg2+]i) promotes
apoptosis in rat hepatocytes (8). Most recently, Eskes et
al. (9) demonstrated that the release of cytochrome c
from mitochondria (induced by overexpression of the apoptosis-promoting
molecule Bax) is independent of 
m, but dependent on
Mg2+. Although Mg2+ has often been considered a
messenger in signal transduction because of its ability to regulate a
variety of cellular processes, changes in [Mg2+]i
following induction of apoptosis via receptor ligation in lymphocytes
have not been demonstrated.
The susceptibility of primary B cells to Fas-mediated apoptosis is
dependent on whether the cell has received signals in the absence of
antigen through CD40, another member of the tumor necrosis factor
receptor superfamily. Resting B cells express very low levels of Fas
and are not prone to apoptosis induced by this receptor (10, 11).
Previous investigators (12-15) have shown that engagement of CD40 can
induce high levels of Fas expression and Fas sensitivity in primary B
cells. In this study, we have used Fas-positive, CD40-primed primary B
cells or the M12.C3 B cell lymphoma, which also expresses high levels
of Fas, to explore the role of [Mg2+]i in
Fas-induced apoptosis. Our results demonstrate that B cells undergoing
apoptosis have elevated [Mg2+]i and that this
increase in free cytosolic Mg2+ appears to serve as a
"second messenger" for downstream events in apoptosis.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemicals were purchased from Sigma unless stated
otherwise. Mag-indo-1 AM and indo-1 AM were purchased from Molecular
Probes, Inc. (Eugene, OR). The ApoAlert annexin V apoptosis kit was
obtained from CLONTECH (Palo Alto, CA). Baby rabbit
complement and Lympholyte-M were purchased from Accurate Chemical & Scientific Corp. (Westbury, NY). Fetal bovine serum was purchased from
Life Technologies, Inc. Ionomycin was obtained from Calbiochem.
Anti-Fas mAb (Jo2) was purchased from Pharmingen (San Diego, CA). The
hybridoma secreting the anti-CD40 mAb 1C10 was a gift from Maureen
Howard (16). Hybridomas secreting anti-CD4 (GK 1.5) and anti-CD8 (HO = 2.2) antibodies were obtained from American Type Culture Collection. The hybridoma secreting anti-Thy 1.2 antibody (T24) was obtained from
Dr. Uwe Staerz (National Jewish Medical and Research Center). All mAbs
were purified from culture supernatants by passage over protein
A-Sepharose (Sigma). RPMI 1640 complete medium (Cellgro, Herndon,
VA) was supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 40 µg/ml gentamicin, 100 units/ml
penicillin G, 100 µg/ml streptomycin, and 50 µM
2-mercaptoethanol. Hanks' balanced salt solution (HBSS) and modified
HBSS lacking Mg2+ and Ca2+ were obtained from Sigma.
Cells--
AKR/J mice were purchased from the Jackson Laboratory
(Bar Harbor, ME) and were used at 6-10 weeks of age. Splenic B cells were prepared from murine splenocytes after the depletion of T cells by
a mixture of anti-CD4 (GK 1.5), anti-CD8 (HO = 2.2), and anti-Thy 1.2 (T24) mAbs with baby rabbit complement as described previously (17).
The isolated primary B cells (5 × 106/ml) were
incubated with anti-CD40 antibody (1C10) at 8 µg/ml of RPMI 1640 complete medium in six-well tissue culture plates (5.5 ml/well) at
37 °C and 5% CO2 for 3 days (13). The CD40-stimulated B
cells were washed with phosphate-buffered saline, and the viable cells
were obtained by sedimentation of dead cells with Lympholyte-M. The
isolated activated B cells had high Fas expression as determined by
staining with fluorescein isothiocyanate-labeled anti-Fas mAb Jo2. The
cell line used in this study is the B cell lymphoma M12.C3, which is
maintained in RPMI 1640 complete medium and also has high Fas
expression. Both types of cells were treated with varying concentrations of anti-Fas mAb in RPMI 1640 complete medium at 37 °C
and 5% CO2.
Determination of Mg2+ and Ca2+
Mobilization--
Untreated and anti-Fas mAb-treated cells were washed
with HBSS, loaded with 20 µM mag-indo-1 AM or indo-1 AM
in HBSS (pH 7.0) containing 10 mM HEPES for 30 min at
37 °C, diluted 2-fold with prewarmed HBSS (pH 7.4) containing 5%
fetal bovine serum and 10 mM HEPES; and incubated for an
additional 30 min as described previously (18). The fluorochrome-loaded
cells were then washed and resuspended in HBSS (pH 7.2) containing 5%
fetal bovine serum and 10 mM HEPES for flow cytometric
analysis. In the experiments in which the role of extracellular
Ca2+ and Mg2+ was explored, the cells were
washed, resuspended, and analyzed in modified HBSS containing only the
appropriate divalent cation(s). An Ortho 50H cytometer with a 5-watt
argon laser set for 364 nm excitation at 50 milliwatts was used.
Fluorescence emission data were collected through a 390/490-nm
band-pass filter using Phoenix Flow Acquisition software. The ratio of
emissions for 390/490 nm was determined, and a cytogram of
[Mg2+]i or [Ca2+]i was
constructed by reference to standard curves of 390/490 nm emission
ratio versus cation concentration (19). Readily mobilized
pools of Mg2+ and Ca2+ in the cells
(106/ml/sample) were measured by treating the mag-indo-1-
and indo-1-loaded cells with 1 µM ionomycin. Flow
cytometric analysis was begun before addition of ionomycin to obtain
base-line values. The cell flow was halted for addition of ionomycin,
resulting in a gap in the profiles. The data were analyzed using
Phoenix Flow System MTIMENEW software, and mean
[Mg2+]i and [Ca2+]i were
calculated as functions of time. In addition, the percentage of cells
mobilizing Mg2+ after ionomycin treatment was determined
for each time point, and maximum values were calculated.
Phosphatidylserine Expression--
PS expression was determined
using an ApoAlert annexin V apoptosis kit. The untreated and anti-Fas
mAb-treated cells were washed with phosphate-buffered saline, and the
washed cells (5 × 105) were resuspended in 200 µl
of 1× binding buffer. After adding 5 µl each of fluorescein-labeled
annexin V (0.5 µg/ml final concentration) and propidium iodide (PI; 1 µg/ml final concentration), the treated cells were incubated at room
temperature for 15 min in the dark; the cells were then analyzed on a
FACSCalibur flow cytometer (Becton Dickinson Advanced Cellular Biology,
San Jose, CA). Two-color analysis allowed the resolution of viable
(PS
PI
), early apoptotic
(PS+PI
), and late apoptotic and necrotic
(PS+PI+) populations.
DNA Fragmentation--
Cells (5 × 105) were
incubated in 200 µl of a solution containing PI (5 µg/ml), saponin
(0.3%), RNase (50 µg/ml), and EDTA (5 mM) for 30 min at
room temperature (20). Apoptotic cells were identified on a FACSCalibur
flow cytometer as a distinct hypodiploid population that showed a
diminished staining relative to the G0/G1
population of the normal viable cells.
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RESULTS |
Fas-mediated Mg2+ and Ca2+ Mobilization in
B Cell Lymphoma Cells--
To study the effect of Fas ligation on
[Mg2+]i and [Ca2+]i, we
conducted a flow cytometric analysis of M12.C3 B cell lymphomas that
had been incubated with anti-Fas mAb and then loaded with the
Mg2+ and Ca2+ indicators mag-indo-1 and indo-1,
respectively. Untreated cells appeared as a homogeneous population by
forward side scatter (data not shown) with uniform base-line
distributions of [Mg2+]i and
[Ca2+]i (Fig. 1,
A and B, respectively; 0~2 min). The mean value
of [Mg2+]i in the untreated cells was 500 µM (Fig. 1E, dashed line at 0~2
min), in good agreement with the range of values reported for
hepatocytes (21), vascular smooth muscle cells (22), and cardiac muscle
cells (23). The mean value for [Ca2+]i was 100 nM (Fig. 1F, dashed line at 0~2
min), in agreement with the published value of 90 nM for
[Ca2+]i in murine splenic B cells (19).
Incubating M12.C3 cells for 3 h with anti-Fas mAb (0.5 µg/ml)
produced two distinguishable subpopulations with regard to
[Mg2+]i. In the anti-Fas mAb-treated cells, one
population had a base-line [Mg2+]i of 500 µM (Fig. 1C, arrowheads), and a
second population had a base-line [Mg2+]i of 900 µM (Fig. 1C, 0~2 min). The existence of
these two populations is reflected in the mean base-line
[Mg2+]i of 700 µM for the treated
cells (Fig. 1E, solid line, 0~2 min).
Intracellular Ca2+ was monitored in parallel by flow
cytometric analysis of indo-1-loaded cells. Fas cross-linking also gave
rise to two separate populations, a well defined major one with a
[Ca2+]i of 100 nM and a broad
"tail" with Ca2+i levels ranging up to 1000 nM or higher (Fig. 1D, 0~2 min). These
populations produced an increase in the mean base-line [Ca2+]i from 100 nM in the untreated
cells to 170 nM in the anti-Fas mAb-treated cells (Fig.
1F, solid line, 0~2 min).

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Fig. 1.
Fas-mediated Mg2+ and
Ca2+ mobilization in B cell lymphoma cells. M12.C3
cells (106/ml) were incubated with 0.5 µg/ml anti-Fas mAb
for 3 h at 37 °C, and then untreated control and Fas-treated
cells were loaded with mag-indo-1/AM or indo-1/AM. Shown is an
isometric display of [Mg2+]i and
[Ca2+]i of the control (A and
B) and anti-Fas (0.5 µg/ml)-treated (C and
D) M12.C3 cells. Once base-line levels of
[Mg2+]i and [Ca2+]i were
established, 1 µM ionomycin was added at the time
indicated by the arrows, and [Mg2+]i
and [Ca2+]i were monitored for an additional 8 min. Mean cation concentrations were calculated from the isometric data
(E and F). The dip indicates the addition of
ionomycin to the flow cells. Values before the dip are base-line
values, and differences between the treated and control samples
indicate cation mobilization induced by anti-Fas mAb treatment alone.
Values after the dip indicate the extent of further cation mobilization
induced by ionomycin. The solid lines show the response of
cells treated with anti-Fas mAb, and the dashed
lines show that of the control cells. The percentage of
cells mobilizing Mg2+ in response to treatment with
ionomycin was calculated from the isometric data (G).
Ionomycin was added at the times indicated in E. The
solid line shows the response of cells after treatment with
anti-Fas mAb, and the dashed line shows that of
the untreated control cells. Cells piling up on the left-hand
axis in B-D are not shown for clarity. They are,
however, included in the calculations for E-G.
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After the base-line values for [Mg2+]i and
[Ca2+]i were established, the cells were treated
in the flow cytometer with 1 µM ionomycin and were
monitored for several minutes longer (~2-10 min) to determine if
there are pool(s) of divalent cations that can be mobilized by
ionophore treatment. In the untreated cells, ionomycin produced the
classical Ca2+ mobilization pattern with an initial sharp
rise followed by a gradual decrease to a plateau value somewhat above
base-line levels (Fig. 1, B and F, dashed
line). Untreated cells did not have a pool of Mg2+
that could be mobilized by this same dose of ionomycin and showed an
insignificant increase in the mean [Mg2+]i (Fig.
1E, dashed line) as well as a
transient response by no more than 22% of the cells in the population
(Fig. 1G, dashed line). Ionomycin
treatment of cells that had been incubated with anti-Fas mAb revealed
significant differences from the control cells. The vast majority of
the high [Mg2+]i cells clearly had a pool of
Mg2+ that could be further mobilized by ionomycin treatment
(Fig. 1, C and G, solid line), which
is reflected in the gradual rise in the mean
[Mg2+]i, reaching maximum values by 8-10 min
after ionomycin treatment (Fig. 1E, solid line).
The low [Mg2+]i population (Fig. 1C,
arrowheads) did not further mobilize Mg2+ in
response to ionomycin. Ionomycin treatment also revealed that the cells
that had been incubated with anti-Fas mAb fell into two populations
with regard to Ca2+ mobilization. Although both
subpopulations responded to ionomycin treatment, one continued to show
the "classic" mobilization profile of the untreated cells, whereas
the other reached and maintained extremely high
Ca2+i levels (Fig. 1D). The behavior of
these populations is reflected in the increased mean
[Ca2+]i response (Fig. 1F, solid
line). Because both subpopulations mobilized Ca2+, it
was not possible to use the flow cytometer software to calculate a
percent response for Ca2+ in order to discriminate between them.
The Percentage of Cells in the High [Mg2+]i
State, Which Further Mobilize Mg2+ upon Ionomycin
Treatment, Correlates with the Percentage of Cells That Die by
Apoptosis--
Treatment of M12.C3 cells with increasing
concentrations of anti-Fas mAb for 16 h caused a
dose-dependent increase in the percentage of cells in the
high [Mg2+]i state (Fig.
2A). This is also reflected in
a dose-dependent increase in the mean
[Mg2+]i, which reached a plateau value of 700 µM with 0.03 µg/ml anti-Fas mAb (Fig. 2C,
closed squares). Similarly, the percentage of cells in the
high [Ca2+]i state increased in a
dose-dependent manner (Fig. 2B), and the
base-line [Ca2+]i increased from 100 to 170 nM (Fig. 2D, closed circles). These
latter observations are consistent with the generally accepted model
that elevation of [Ca2+]i is required for
apoptotic death. Ionomycin was used to determine the additional
Ca2+ and Mg2+ mobilization potential at each
dose of anti-Fas mAb (Fig. 2, C and D, open
symbols). With ionomycin treatment, [Ca2+]i
increased ~100 nM regardless of the dose of anti-Fas mAb
used. In contrast, ionomycin caused [Mg2+]i to
increase markedly only at anti-Fas mAb concentrations above 0.03 µg/ml. We used the Mg2+ response to ionomycin as a
readout for cells in the high [Mg2+]i state
because these data (Figs. 1C and 2, A and
C) suggest that the cells that are in the high
[Mg2+]i state are the same cells that further
mobilize Mg2+ in response to ionomycin treatment and
because the available cytometric analysis software provides a direct
calculation of the percentage of cells mobilizing Mg2+ in
response to ionomycin.

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Fig. 2.
The percentage of cells in the high
[Mg2+]i state, which further mobilize
Mg2+ upon ionomycin treatment, correlates with the
percentage of cells that die by apoptosis. M12.C3 cells were
treated with various concentrations of anti-Fas mAb for 16 h at
37 °C. Shown is the percentage of cells in the high
[Mg2+]i state (A) or the high
[Ca2+]i state (B) calculated from
base-line values (before addition of ionomycin) by looking at the
distribution of [Mg2+]i or
[Ca2+]i at a single time point and setting gates
to determine the percentage of high [Mg2+]i or
high [Ca2+]i cells. The base-line levels of the
mean [Mg2+]i ( ) and
[Ca2+]i ( ) in cells incubated with increasing
doses of anti-Fas mAb are indicated (C and D).
After the base-line levels of [Mg2+]i and
[Ca2+]i were established, the cells were treated
in the cytometer with ionomycin (1 µM), and the resultant
[Mg2+]i ( ) and [Ca2+]i
( ) were determined. Duplicate cultures from these experiments were
evaluated for apoptotic death by measuring the percentage of cells with
hypodiploid levels of DNA using saponin/PI and flow cytometry
(E). The maximum percentage of cells mobilizing
Mg2+ at each dose of anti-Fas mAb ( ) is compared with
the percentage of apoptotic cells at that dose ( ).
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Duplicate wells from the experiments shown in Fig. 2 (A-D)
were examined for apoptotic cells using PI staining and flow cytometry. In saponin-permeabilized cells, PI uptake is proportional to DNA content, and apoptotic cells show diminished ("hypodiploid")
staining below the G0/G1 population of viable
cells (20, 24, 25). The results of this analysis indicate that the
percentage of cells mobilizing Mg2+ correlates with the
apoptotic (hypodiploid) cells (Fig. 2E).
Examination of Fas-mediated Apoptosis in Anti-CD40 Antibody-treated
B Cells--
Incubation of murine splenic B cells with CD40 ligand
leads to enhanced Fas expression and increased sensitivity to
Fas-mediated apoptosis, as seen in other systems (13, 26). In a similar manner, we treated splenic B cells (which had been pretreated with
anti-CD40 mAb for 72 h) with anti-Fas mAb for 2 h and then determined Mg2+ mobilization, PS externalization, and DNA
fragmentation. The dose dependence of Mg2+ mobilization in
anti-Fas mAb-treated B cells (Fig.
3A) was similar to that of the
B cell lymphoma M12.C3 (cf. Fig. 2C). Thus, the base-line [Mg2+]i of the primary B cells also
reached a plateau value of 700 µM with 0.1 µg/ml
anti-Fas mAb (Fig. 3A, closed squares), and
ionomycin (1 µM) treatment during the flow cytometric
analysis increased [Mg2+]i 2-fold at anti-Fas mAb
concentrations above 0.03 µg/ml (open squares). As with
M12.C3 B cells, the percentage of cells mobilizing Mg2+ in
response to ionomycin correlates with the high [Mg2+]
population (data not shown).

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Fig. 3.
Examination of Fas-mediated apoptosis in
CD40-stimulated B cells. Splenic B cells were obtained from AKR/J
mice and incubated with anti-CD40 mAb (1C10) for 3 days. The
CD40-stimulated B cells were treated with various concentrations of
anti-Fas mAb as triplicate cultures for 2 h at 37 °C. The
anti-Fas mAb-treated cells were loaded with mag-indo-1 and other
reagents separately. A, base-line levels of the mean
[Mg2+]i ( ) in cells treated with increasing
doses of anti-Fas mAb. After the base-line levels of
[Mg2+]i were determined, the cells were treated
in the cytometer with 1 µM ionomycin, and
[Mg2+]i levels were re-determined ( ).
B, triplicate cultures from the same experiment evaluated
for apoptotic death. The percentage of cells mobilizing
Mg2+ at each dose of anti-Fas mAb ( ) was compared with
the percentage of apoptotic cells as determined by DNA fragmentation
( ) and PS externalization ( ). Curves were normalized by setting
the maximum value for each parameter to 100%.
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We also examined DNA fragmentation and PS externalization in the same
experiment. PS is largely excluded from the external leaflet of the
plasma membrane in viable cells, but becomes expressed on the external
leaflet during apoptosis (5). Therefore, we used staining with annexin
V, a naturally occurring PS-binding protein, to detect cells in early
stages of apoptosis. Cells from parallel cultures used for
Mg2+ mobilization were doubly stained with fluorescein
isothiocyanate-labeled annexin V and PI, without permeabilizing the
cells (5). This technique allows the definition of three
subpopulations: viable cells are PS
PI
,
early apoptotic cells are PS+PI
, and late
apoptotic cells (and necrotic cells) are
PS+PI+. In our experiments, we considered only
the early apoptotic (PS+PI
) cells. The
results (Fig. 3B) show that each of the responses reaches a
plateau above 0.03 µg/ml anti-Fas mAb and also indicate, for each
dose of mAb, that the relative percentage of cells that mobilize
Mg2+ is same as the percentage of cells with increased PS
externalization and DNA fragmentation. These results further
demonstrate that Mg2+ mobilization is a property of primary
B cells undergoing apoptosis and not just an artifact associated with
the use of the B cell lymphoma M12.C3.
Kinetics of Anti-Fas mAb-induced Mg2+ Mobilization, DNA
Fragmentation, and PS Externalization in B Cells--
We used kinetic
analysis to determine whether Mg2+ mobilization is an early
or late event in apoptosis. In M12.C3 cells treated with 0.5 µg/ml
anti-Fas mAb, the mean base-line [Mg2+]i
increased with time, reaching a value of 1000 µM after 1 h of incubation (Fig.
4A). At each time point (other
than t = 0), ionomycin caused a significant
Mg2+ mobilization in the high [Mg2+]i
population of cells that were committed to die by apoptosis as
previously demonstrated. The kinetics of Mg2+ mobilization
versus DNA fragmentation were also measured in cells treated
with 0.5 µg/ml anti-Fas mAb (Fig. 4B). The percentage of
cells showing Fas-potentiated, ionomycin-induced Mg2+
mobilization was significantly higher than the percentage of those
fragmenting DNA at 1-3 h after mAb treatment, but by 16 h, DNA
fragmentation exceeded Mg2+ mobilization. These results
suggest that Mg2+ mobilization precedes DNA fragmentation
in B cells.

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Fig. 4.
Kinetics of anti-Fas mAb-induced
Mg2+ mobilization, DNA fragmentation, and PS
externalization in B cells. M12.C3 cells were treated with 0.5 µg/ml anti-Fas mAb at 37 °C for varying times. A, the
mean [Mg2+]i was determined before ( ) and
after ( ) treatment with ionomycin. B, comparison of the
percentage of cells that mobilized Mg2+ after ionomycin
treatment ( ) with the percentage of apoptotic cells as determined by
DNA fragmentation ( ). C, comparison of the percentage of
cells that mobilized Mg2+ after ionomycin treatment ( )
with the percentage of apoptotic cells as determined by PS
externalization ( ).
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We decided to examine loss of plasma membrane asymmetry on these cells
because, whereas DNA fragmentation is considered to be a late stage
apoptotic event in many systems, PS externalization occurs early in the
process. Kinetic analysis of PS externalization versus
Fas-potentiated, ionomycin-mediated Mg2+ mobilization
suggested that Mg2+ mobilization preceded PS
externalization (Fig. 4C), although the lag in PS response
was not as striking as with DNA fragmentation (Fig. 4B).
Because the percentage of cells in the PS+PI
population never reached the same level of those mobilizing
Mg2+, we checked to see if we were underestimating
apoptosis by excluding the PS+PI+ population.
The PS+PI+ population did not increase
significantly over the 0-h time point except at the 16-h time point
(data not shown), suggesting that the apparent decrease in
PS+PI
cells at that time point may be due to
their conversion to PS+PI+, late apoptotic (or
necrotic) cells. However, at the earlier time points (1-3 h), loss of
PS+PI
cells to the
PS+PI+ population is not an explanation for
lower PS+PI
numbers, and thus, PS
externalization clearly lags behind Mg2+ mobilization.
Intracellular Stores Are the Primary Source of
[Mg2+]i in B Cells Undergoing
Apoptosis--
Calcium mobilization in B cells involves both release
from intracellular stores (primarily endoplasmic reticulum) and the influx of extracellular Ca2+ (see Fig. 1 and Ref. 27).
Significantly less is known about the relative roles of intracellular
versus extracellular sources of Mg2+ for
Mg2+ mobilization during apoptosis. We therefore examined
the source of [Mg2+]i in cells undergoing
apoptosis. The experiments described above were repeated with M12.C3
cells in defined media lacking Mg2+, Ca2+, or
both ions. The increase in the base-line [Mg2+]i
caused by anti-Fas mAb was not dependent on Mg2+ in the
extracellular milieu (Fig.
5A). Likewise, the percentage of cells possessing a readily mobilizable store of Mg2+ was
independent of extracellular Mg2+ (Fig. 5B). In
contrast, the production of a high [Mg2+]i state
and the generation of a pool of readily mobilized Mg2+ were
completely dependent on extracellular Ca2+ (Fig. 5). These
data support the concept that Ca2+ mobilization is required
for the induction of the apoptosis signaling pathway and place the
Ca2+-dependent event(s) upstream of the release
of Mg2+ from intracellular stores.

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Fig. 5.
Mg2+ mobilization in B cells
undergoing apoptosis does not require extracellular Mg2+,
but does require extracellular Ca2+. M12.C3 cells were
washed and resuspended in HBSS, either complete or modified to lack
Ca2+, Mg2+, or both ions. Triplicate samples in
each medium were cultured alone ( ) or were treated for 3 h with
anti-Fas mAb ( ). A, base-line levels of the mean
[Mg2+]i of cells. B, percentage of
cells with a pool of Mg2+ that can be mobilized by
treatment with 1 µM ionomycin in the flow cytometer. The
means ± S.E. of triplicate cultures are shown.
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Inhibition of Mg2+ Mobilization and Apoptosis in B
Cells by CCCP--
Mitochondria are one of the major storage sites of
intracellular Mg2+ (28) and play a central role in
intracellular signaling by releasing Mg2+ in response to a
variety of agents (22, 29). Therefore, we investigated Mg2+
release from mitochondria during Fas-induced, ionomycin-potentiated Mg2+ mobilization using CCCP. This agent uncouples
oxidative phosphorylation in mitochondria (30), and short-term
treatment of cells with CCCP has been reported to disrupt

m (4). We treated M12.C3 B lymphoma cells overnight with
20 µM CCCP. Although the cells remained viable, they did
show disrupted 
m (as measured by decreased uptake of the
cationic fluorochrome DiOC6(3); data not shown). Cells
treated with CCCP for 16 h and with anti-Fas mAb for the last
3 h were examined for their ability to mobilize Mg2+
and Ca2+ in response to 1 µM ionomycin.
Pretreatment of the cells with CCCP did not alter their ability to
mobilize Ca2+ relative to controls not treated with CCCP
(Fig. 6A, shaded
bars). The slight (6%) decrease in ability to mobilize
Ca2+ between cells treated with anti-Fas mAb plus CCCP and
those treated with anti-Fas mAb alone was not statistically
significant. These results suggest that pretreatment of the cells with
CCCP did not significantly alter their ability to flux
Ca2+. Conversely, whereas pretreatment of the cells with
CCCP did not alter the ability of the control cells to mobilize
Mg2+, it did cause a marked (40%) and highly significant
decrease in the ability of the anti-Fas mAb-treated cells to mobilize
Mg2+.

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Fig. 6.
Inhibition of Mg2+ mobilization
and apoptosis in B cells by long-term treatment with CCCP. M12.C3
cells that had been preincubated overnight with 20 µM
CCCP were treated for 3 h with 1 µg/ml anti-Fas mAb. Parallel
cultures were loaded separately with mag-indo-1 or indo-1 to measure
Mg2+ or Ca2+ mobilization, respectively;
treated with saponin/PI to determine DNA fragmentation; or labeled with
fluorescein isothiocyanate-labeled annexin V/PI to determine PS
externalization. A, mean [Mg2+]i ( )
and [Ca2+]i ( ) produced in response to
treatment with 1 µM ionomycin. Preincubation of cells
with CCCP prior to anti-Fas mAb treatment did not significantly reduce
Ca2+ mobilization (p < 0.46), but markedly
reduced Mg2+ mobilization (p < 0.0001).
B, percentage of cells mobilizing Mg2+ ( ) in
response to treatment with 1 µM ionomycin, externalizing
PS ( ), or fragmenting DNA ( ). Preincubation of cells with CCCP
prior to anti-Fas mAb treatment caused significant reductions in
Mg2+ mobilization (p < 0.0405), PS
externalization (p < 0.0024), and DNA fragmentation
(p < 0.0103). The means ± S.E. of triplicate
cultures are shown.
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In a subsequent experiment, parallel cultures were assayed for
Mg2+ mobilization, PS externalization, and DNA
fragmentation (Fig. 6B). In the anti-Fas mAb-treated cells,
pretreatment with CCCP caused a 37% inhibition of Mg2+
mobilization relative to the untreated cells, a 41% inhibition of PS
externalization, and a 45% inhibition of DNA fragmentation. In each
case, the decrease in response was statistically significant, and the
decreases in cells showing PS externalization and DNA fragmentation
were proportional to the decrease in the number of cells mobilizing
Mg2+. These data identify the mitochondria as a major
source of intracellular Mg2+ and, furthermore, are
consistent with a role for Mg2+ as a second messenger for
apoptosis in B cells. The data also clearly separate the role of
Mg2+ from that of Ca2+ since long-term
treatment with CCCP does not significantly inhibit Ca2+
mobilization in response to ionomycin treatment even while
Mg2+ mobilization and apoptosis are decreased.
 |
DISCUSSION |
In this study, we have used the fluorescent Mg2+
chelator mag-indo-1 to determine [Mg2+]i.
Although this chelator is the indicator of choice for determining
[Mg2+]i by flow cytometry, it is capable of
binding Ca2+ as well. However, other studies have indicated
that [Ca2+]i in the ~1 µM range
causes >7% overestimation of [Mg2+]i in the 500 µM range (31). Because the mean
[Ca2+]i rarely, if ever, achieved these levels in
our experiments, it is unlikely that interference from
[Ca2+]i plays a significant role in our
measurements of [Mg2+]i.
The role of intracellular Ca2+ in apoptosis is generally
accepted (7, 32), and our studies suggest a role for elevated [Ca2+]i in B cell apoptosis as well. Multiple
functions in apoptosis have been ascribed to increased
[Ca2+]i. These include activation of calpain, a
caspase-like protease postulated to be one of the initiators of the
apoptotic cascade (33, 34); inactivation of the PS-specific flippase and activation of the phospholipid scramblase (35); and activation of
Ca2+-dependent endonucleases (36-38).
Mitochondria serve as sinks for Ca2+ released from the
endoplasmic reticulum during intracellular Ca2+
oscillations, and it recently has been postulated that sustained elevated [Ca2+]i may cause the mitochondria to
become overloaded with Ca2+, leading to downstream events
that facilitate apoptosis (39). It should be noted, however, that
although elevations in [Ca2+]i may be necessary
for B cell apoptosis, they are not sufficient, as proliferative
signals such as those produced by ligation of the B cell receptor
(surface immunoglobulin) also result in Ca2+ mobilization.
In contrast to the well studied role of Ca2+ in apoptosis,
the role of Mg2+ has been largely ignored. There is only a
single report that elevating [Mg2+]i in
hepatocytes, using glycodeoxycholates (bile salts) to allow the ingress
of extracellular Mg2+, leads to their death by apoptosis
(8). Intracellular Mg2+ is stored primarily in the
microsomes (50% of total intracellular Mg2+) and
mitochondria (20% of total), with much smaller amounts in the nucleus
and cytosol (28). About 98-99% of total intracellular Mg2+ (23) and 94% of cytosolic Mg2+ (21) are
complexed to nucleic acids, proteins, or membranes. Therefore, complex
equilibria between compartments and between bound and free forms of
Mg2+ contribute to the concentration of free cytosolic
Mg2+, i.e. [Mg2+]i (28).
The relatively slow release of Mg2+ (as compared with
Ca2+) in the response to ionomycin treatment in our studies
may reflect the need for Mg2+ not only to cross the
mitochondrial membrane, but also to dissociate from immobile ligands
within the mitochondria.
Because our data with the mitochondrial oxidative phosphorylation
inhibitor CCCP suggest that mitochondrial stores of Mg2+
are critical for obtaining apoptosis and because our kinetic analysis
suggests that Mg2+ mobilization is an early event in
apoptosis, we are struck by the parallel between disruption of

m and Mg2+ mobilization. On one hand, the
release of Mg2+ from the mitochondria could be merely a
consequence of the opening of mitochondrial pores as 
m is
reduced. On the other hand, there are at least two reports that, taken
together, imply that Mg2+ release from the mitochondria is
independent of the loss of 
m. First, the release of
mitochondrial cytochrome c has been reported to occur prior
to and to be independent of disruption of 
m (40). Second,
when isolated mitochondria are incubated with the pore-forming,
pro-apoptotic molecule Bax, they incorporate Bax and release
cytochrome c in a manner that is independent of 
m, but "highly dependent on Mg2+ ions"
(9). Because of the importance of cytochrome c release in
activating apoptotic protease activating factor-1 (Apaf-1) (41, 42) and
thereby driving post-mitochondrial events in cells undergoing
apoptosis, we suggest that achieving high levels of
[Mg2+]i is central to the apoptotic process.
Although Mg2+ is an important cofactor for a diverse set of
functions in viable and proliferating cells (28), the Mg2+
dependence of a number of endonucleases implicated in apoptosis (36,
43-45) suggests that elevated Mg2+ levels are involved in
apoptosis as well. The role played by Mg2+ in PS
externalization is less clear. One of the best candidates for the
aminophospholipid translocase ("flippase") responsible for
maintaining PS asymmetry in the plasma membrane of erythrocytes is a
Mg2+-ATPase (46-48). One might therefore assume that
elevating [Mg2+]i should decrease PS expression
on the outer leaflet of the plasma membrane. However, although the
Mg2+ dependence of the flippase activity has been well
documented, the effect of supernormal Mg2+ concentrations
is unknown. Thus, it may be possible that elevated [Mg2+]i desensitizes the flippase, thus turning
it off, or that Mg2+ has no direct effect on flippase
activity and that the regulation of PS externalization by
Mg2+ may be indirect, e.g. through the
activation of caspases or other death-signaling molecules. Future
experiments will establish the mechanism of, and the role played by,
mitochondrial Mg2+ in B cells undergoing Fas-initiated apoptosis.