Regulation of intracellular calcium in N1E-115
neuroblastoma cells: the role of Na+/Ca2+
exchange
Kara L.
Kopper and
Joseph
S.
Adorante
Allergan, Inc., Department of Biological Sciences, Irvine,
California 92612
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ABSTRACT |
In fura 2-loaded N1E-115 cells, regulation
of intracellular Ca2+ concentration
([Ca2+]i) following a Ca2+ load
induced by 1 µM thapsigargin and 10 µM carbonylcyanide
p-trifluoromethyoxyphenylhydrazone (FCCP) was
Na+ dependent and inhibited by 5 mM Ni2+. In
cells with normal intracellular Na+ concentration
([Na+]i), removal of bath Na+,
which should result in reversal of Na+/Ca2+
exchange, did not increase [Ca2+]i unless
cell Ca2+ buffer capacity was reduced. When N1E-115 cells
were Na+ loaded using 100 µM veratridine and 4 µg/ml
scorpion venom, the rate of the reverse mode of the
Na+/Ca2+ exchanger was apparently enhanced,
since an ~4- to 6-fold increase in [Ca2+]i
occurred despite normal cell Ca2+ buffering. In SBFI-loaded
cells, we were able to demonstrate forward operation of the
Na+/Ca2+ exchanger (net efflux of
Ca2+) by observing increases (~ 6 mM) in
[Na+]i. These Ni2+ (5 mM)-inhibited increases in [Na+]i could only
be observed when a continuous ionomycin-induced influx of
Ca2+ occurred. The voltage-sensitive dye
bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to
measure changes in membrane potential. Ionomycin (1 µM) depolarized
N1E-115 cells (~25 mV). This depolarization was Na+
dependent and blocked by 5 mM Ni2+ and 250-500 µM
benzamil. These data provide evidence for the presence of an
electrogenic Na+/Ca2+ exchanger that is capable
of regulating [Ca2+]i after release of
Ca2+ from cell stores.
calcium flux; membrane transport; neuronal calcium regulation; sodium/calcium antiport
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INTRODUCTION |
THE REGULATION of
intracellular Ca2+ concentration
([Ca2+]i) in mammalian cells is of
fundamental importance in physiological and pathophysiological
conditions. Ca2+ is an important regulator of a variety of
physiological functions including processes such as
excitation-contraction coupling, fluid secretion in epithelia, and
release of neurotransmitters (15). [Ca2+]i must be tightly regulated to achieve
transient or sustained levels required to trigger specific
physiological processes.
Cells have several mechanisms in place to return elevated free
[Ca2+]i to resting levels. These transport
mechanisms may be located at the level of the plasma membrane or reside
in the cytosol within organelles such as mitochondria and endoplasmic
reticulum (ER). Ca2+-ATPases (Ca2+ pumps)
located at the plasma membrane (8) and ER
(32) are primary active transport mechanisms capable of
lowering [Ca2+]i following a stimulus-induced
increase and can also help maintain steady-state
[Ca2+]i levels (8, 20). In
addition, many animal cells contain a Na+/Ca2+
exchanger, which, depending on the sign of the electrochemical potential (driving force), can operate in the forward or reverse mode.
In the forward mode, Ca
is extruded from the cell. In the reverse mode, the cell is loaded with
Ca2+ (3, 6).
Although regulation of [Ca2+]i is important
for normal cell functioning, its disregulation has been linked to
cellular pathologies and even cell death (31). For
example, in some central nervous system (CNS) neurons, anoxia results
in uncontrolled increases in [Ca2+]i that, if
unchecked, can eventually lead to cell death (29). Because
of the importance of [Ca2+]i in neuronal
health and disease, a relatively simple cell model system, one where
[Ca2+]i regulation can be studied fairly
easily, is desirable.
N1E-115 neuroblastoma cells have been used for many years as a neuronal
model system. They grow very easily and can be used in a variety of
assays and cellular investigations. Like other CNS neurons, N1E-115
cells contain voltage-gated Na+, K+, and
Ca2+ channels (21, 24, 25). In addition, these
cells have also been used to investigate Ca2+ signaling
pathways (7, 23, 33). However, very little is known about
how these cells regulate [Ca2+]i. In this
study, we examined how N1E-115 neuroblastoma cells regulate
[Ca2+]i following the release of
Ca2+ from intracellular stores. We show that these cells
possess 1) an electrogenic Na+/Ca2+
exchange mechanism that is responsible for restoring elevated [Ca2+]i to control level following depletion
of Ca2+ stores and 2) a
Na+-independent Ca2+ extrusion mechanism that
is most evident following [Ca2+]i elevation
in Na+-free media.
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METHODS |
Cell preparation.
N1E-115 mouse neuroblastoma cells were purchased from the University of
California, San Francisco Cell Culture Facility. They were
grown in Dulbecco's modified Eagle's medium (DMEM) high glucose without phenol red supplemented with 2 mM L-glutamine, 50 units penicillin/50 µg streptomycin, 0.2 mM sodium hypoxanthine, 400 nM aminopterin plus 16 µM thymidine (1× HAT; GIBCO BRL, Rockville, MD), and 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT)
at 37°C in a humidified atmosphere of 5% CO2. The cells
were split 1:3 twice a week and were plated on glass coverslips up to 2 days before use. Data are expressed as means ± SE where
applicable. Statistical significance was tested, where appropriate,
using a paired or unpaired t-test. To determine significance
between control and experimental groups, a one-way analysis of variance (one-way ANOVA) followed by Dunnnett's test was performed. Where stated, n is the number of experiments performed.
Solutions.
HEPES Ringer contained (in mM) 120 NaCl, 10 glucose, 10 HEPES, 5 KCl,
1.2 CaCl2, 0.6 MgCl2, and 12 Na-cyclamate. pH
was adjusted to 7.40 at 37°C with NaOH, and the osmolarity was
adjusted to 291-294 mosM. Na+-free solutions were
prepared by isosmotic replacement of NaCl with K+ or
N-methyl-D-glucamine (NMDG) salts of Cl. All
chemicals were purchased from Sigma Chemical (St. Louis, MO) unless
indicated otherwise.
Intracellular Ca2+ measurements.
Fura 2-AM (5 mM) was mixed at a 1:1 ratio with 20% Pluronic in DMSO
(Molecular Probes, Eugene, OR). This mixture was added to HEPES Ringer
to a final concentration of 5 µM fura 2-AM (loading solution). The
coverslips with adhering N1E-115 cells were placed in the loading
solution for 45-60 min at 37°C. After loading, the cells were
washed with fura-free HEPES Ringer.
[Ca2+]i measurements were made by using
either a microscope-based imaging system with an intensified
charge-coupled device (CCD) camera or a microscope-based photon
counting system with a photomultiplier tube (Photon Technology
International, Monmouth Junction, NJ). Coverslips were mounted in a
chamber (Warner Instrument, Hamden, CT) and perfused at 37°C with the
appropriate Ringer. As a result of the dead space in our perfusion
system, there was a 60- to 90-s delay from the time a solution was
changed to the time it reached the chamber in all experiments. The
samples were excited at wavelengths of 340 and 380 nm, and the
resulting emissions were band-pass filtered at 490-530 nm.
The methods used to estimate [Ca2+]i
employing fura 2 ratio fluorescence were described previously
(13). Briefly, fura 2 was calibrated by using the
Ca2+ ionophore ionomycin (10 µM; Calbiochem, San Diego,
CA); a Ca2+-free buffer containing 2.0 mM EGTA was used to
deplete the cells of Ca
and thus
obtain the minimum signal (Rmin). The same cells were then
perfused with a 10 mM Ca2+ buffer to saturate the fura 2 dye and obtain a maximum signal (Rmax). To obtain
background fluorescence, 5 mM manganese was used to quench the dye.
Values of Kd, Rmax, and
Rmin were 224 nM, 10.8, and 0.42, respectively.
Intracellular Na+ measurements.
Sodium-binding benzofuran isophthalate-acetoxymethyl ester (5 mM,
SBFI-AM; Molecular Probes) was mixed at a 1:1 ratio with 20% Pluronic
in DMSO. This mixture was added to HEPES Ringer for a final
concentration of 7 µM SBFI-AM (loading solution). The coverslips with
adhering N1E-115 cells were placed in the loading solution for
60-80 min at 37°C. After loading, the cells were washed with
SBFI-free HEPES Ringer.
The [Na+]i measurements were made by using a
microscope-based imaging system with an intensified CCD camera from
Photon Technology International. The coverslips were mounted in a
chamber to allow perfusion at 37°C (Warner Instrument). The samples
were excited at wavelengths of 345 and 380 nm. A band-pass filter of
490-530 nm determined the emission.
The methods used to estimate [Na+]i by
employing SBFI ratio fluorescence were described previously
(35). Briefly, SBFI was calibrated by using the cation
ionophores monensin (1 µM), nigericin (5 µM), and gramicidin (1 µM) in HEPES buffers containing either 0, 25, or 50 mM
Na+ (balancing cation K+). Plotting
fluorescence ratio vs. [Na+] generated a calibration
curve. Digitonin (0.33 mg/ml) was used to determine background
fluorescence and dye compartmentalization.
Membrane potential measurements.
Changes in the membrane potential (Em) were made
by using the voltage-sensitive fluorescent anionic dye
bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (bis-oxonol;
Molecular Probes). Bis-oxonol partitions into the cell membrane as a
Nernstian function of the membrane potential. After depolarization,
bis-oxonol enters the membrane and the fluorescence intensity
increases. After membrane hyperpolarization, dye leaves the cell and
fluorescence intensity decreases (1, 26).
A cell preparation for Em experiments was
established as follows: cells were removed from their culture flasks
and counted by using trypan blue (Sigma) exclusion to assess viability,
which was typically 15-25% dead cells. The cells were divided
into aliquots of ~0.42 × 106 living cells per
sample, and these were stored on ice for a minimum of 30 min. The
pellets were resuspended in 3 ml of HEPES Ringer (37°C) for 10 min in
a cuvette before an experiment. Bis-oxonol (30 nM) was added 2 min
before the beginning of the run to allow for equilibration.
Measurements were made by using an LS-50B spectrofluorometer (Perkin
Elmer, Norwalk, CT) at an excitation of 540 nm (±10-nm slit widths)
and emission at 580 nm (±5.0-nm slit widths).
Calibration of bis-oxonol was performed as described by Grinstein et
al. (12). Calibration solutions were made by substituting Ringer Na+ with NMDG. Cells were resuspended in a cuvette
containing Ringer with Na+ ranging from 0 to 140 mM and
were subsequently exposed to 0.5 µM gramicidin. After gramicidin
addition, intensity was measured for 2-3 min. Because gramicidin
is a nonselective monovalent cation ionophore, permeable to both
Na+ and K+ but not to NMDG, the monovalent
cation ratio determines the membrane potential.
The following equation was used to calculate Em
at each extracellular Na+ concentration
([Na+]o): Em =
60 log([cation]i/[cation]o), where i and
o denote the intra- and extracellular compartments, respectively. It
was assumed that [cation]i
145 mM. Plotting the
intensity for each sample against its calculated
Em then generated a calibration curve, which was
linear to approximately
75 mV. The equation of this curve allowed the
conversion of intensity units to millivolts (see Fig. 2B).
 |
RESULTS |
Regulation following Ca2+ loading.
Regulation of [Ca2+]i in fura 2-loaded
N1E-115 cells was investigated following release of Ca2+
from intracellular stores and mitochondria by using 1 µM thapsigargin and 10 µM carbonylcyanide
p-trifluoromethoxyphenylhydrazone (FCCP), respectively (Fig.
1A). These two compounds
allowed sequestered Ca
to flood
into the cytoplasm and also prevented the Ca2+ from
reloading into the intracellular stores (10). After
perfusion of 1 µM thapsigargin and 10 µM FCCP,
[Ca2+]i rose from 52.9 ± 2.5 nM (215 cells, n = 15) to a peak of 105.3 ± 2.5 nM (153 cells, n = 15) and relaxed toward control levels in
~5 min. However, in the absence of extracellular Na+
(Na
), [Ca2+]i
rose to 129.5 ± 15.4 nM (34 cells, n = 8) and
remained elevated (Fig. 1A). In the absence of
Na
, the increase in
[Ca2+]i following 1 µM thapsigargin and 10 µM FCCP was statistically greater than that observed with
Na
present (P < 0.05, unpaired t-test). When the extracellular solution was
replaced with one containing Na+,
[Ca2+]i levels returned to baseline.

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Fig. 1.
Recovery from an intracellular Ca2+
concentration ([Ca2+]i) increase was
dependent on external Na+ and inhibited by
Ni2+. N1E-115 cells were loaded with fura 2 and perfused
with control HEPES Ringer before being switched to an experimental
solution. A: the perfusate was changed to one containing
thapsigargin and carbonylcyanide p-trimethoxyphenylhydrazone
(FCCP) in the presence and in the absence of extracellular
Na+ (filled arrows). At the line arrow, the solution was
switched back to control Na+-containing medium. The
Na+-free curve was obtained by averaging the results of
responses obtained from 20 cells and is representative of 8 similar
experiments. The curve obtained from experiments performed in
Na+-containing media represents the average response of 12 cells and is representative of 15 similar experiments. B:
recovery from the [Ca2+]i increase was
inhibited by 5 mM Ni2+. At the horizontal bar, the
perfusate was changed to a Na+-free Ringer containing 1 µM thapsigargin and 10 µM FCCP. At the filled arrow, the perfusate
was changed to one containing Na+. For another cell the
perfusate was changed to one containing extracellular Na+
and 5 mM Ni2+ (line arrow). Data were obtained from single
cells by using photon counting techniques. Each curve was generated
from 1 cell and is representative of at least 5 similar experiments.
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The Na+ dependence of recovery suggested that
Na+/Ca2+ exchange may play a role in the
recovery process. To determine whether Na+/Ca2+
exchange was responsible for the return of
[Ca2+]i toward control level, 5 mM
Ni2+, an inhibitor of the Na+/Ca2+
exchange, was utilized. As in Fig. 1A, cells exposed to 1 µM thapsigargin and 10 µM FCCP in the absence of
Na
(NMDG replacement) failed to regulate
the increase in [Ca2+]i caused by these
agents until Na
was added back to the
medium. However, in the presence of 5 mM Ni2+ the rate of
regulation was slowed following addition of Na+ to the bath
(Fig. 1B). The rate of regulation of Ca2+ in the
presence of 5 mM Ni2+ (0.4 × 10
9 M/s,
n = 6) was significantly slower than the rate in its
absence (1.4 × 10
9 M/s, n = 5;
P < 0.05, unpaired t-test). Substitution of
Li+ for Na+ was also effective at inhibiting
the rate of [Ca2+]i recovery following
exposure to 1 µM thapsigargin and 10 µM FCCP (not shown).
Membrane potential measurements.
It has been previously reported that Na+/Ca2+
exchange is electrogenic because 3 Na+ exchange for every
Ca2+, resulting in a net movement of positive charge
(3). Under appropriate conditions, this mechanism should
be able to alter the membrane potential of the cells during its
operation. In the forward mode with net influx of Na+ and
efflux of Ca2+, operation of the exchanger should
measurably depolarize the cell provided its relative conductance is
significant. To see whether we could detect its contribution to
the membrane potential, we employed the voltage-sensitive dye
bis-oxonol.
Intracellular Ca2+ is both a substrate and modulator of
Na+/Ca2+ exchange with a
K1/2 for activation of Na+ dependent
Ca2+ efflux of 1-3 µM (28). To maximize
Na+/Ca2+ exchange rate and thus current
density, ionomycin, a calcium ionophore, was used to increase
[Ca2+]i in a sustained manner. This maneuver
should optimize the chances for detecting the electrogenicity of the exchanger.
Addition of 1 µM ionomycin to the cell suspension caused an increase
in fluorescence corresponding to a depolarization of ~25 mV. The
ionomycin-induced depolarization was reduced by ~80% by the absence
of Na+ in the media and was also blocked by 5 mM
Ni2+ (Fig. 2A). In
addition, 1 µM tetrodotoxin (TTX), an inhibitor of voltage-gated
Na+ channels, had no effect on the depolarization
(Table 1), nor did removal of chloride
from the media (data not shown). Furthermore, the removal of
Ca2+ from the extracellular media completely inhibited the
depolarization, indicating that it was dependent on Ca2+
entering the cell. Benzamil (250-500 µM), a
Na+/Ca2+ exchange inhibitor (16),
also blocked the ionomycin-induced depolarization (Table 1).

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Fig. 2.
The ionomycin-induced depolarization is Na+
dependent and inhibited by Ni2+. Bis-oxonol was used to
measure changes in the membrane potential in N1E-115 cells.
A: at the filled arrows, 1 µM ionomycin was added to a
cuvette containing cells in control HEPES Ringer. At the open arrow, 5 mM Ni2+ was added to 1 set of cells. Note: 5 mM
Ni2+ alone caused an 8-mV depolarization (this
depolarization was not Na+ dependent). Each curve is from a
population of cells and is representative of at least 5 similar
experiments. B: calibration curve for bis-oxonol
fluorescence (arbitrary units) vs. membrane potential
(Em). Em was set by using
0.5 µM gramicidin and various ratios of extracellular Na+
and N-methyl-D-glucamine (NMDG) as described in
METHODS. The line represents the best-fit linear regression
(I = 0.73Em + 142.38;
R = 0.99). I, intensity.
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In addition, removal of Na
before 1 µM
ionomycin addition resulted in an ~25-mV hyperpolarization (Fig.
2A) consistent with reversal of an electrogenic
Na+/Ca2+ exchanger. However, this
hyperpolarization was transient and a continuous depolarization
followed. On addition of 1 µM ionomycin, this depolarization
was halted and a slight hyperpolarization followed. Thus the extent
of hyperpolarization following ionomycin addition was masked,
presumably, by another conductive mechanism activated by removal of
media Na+ (see DISCUSSION).
Measurement of intracellular Na+.
In the forward mode of the Na+/Ca2+ exchanger,
a decrease in [Ca2+]i is accompanied by an
increase in [Na+]i. However, since under
physiological conditions nanomolar to micromolar amounts of
Ca2+ are removed following a rise in
[Ca2+]i, the increase in
[Na+]i is not detectable using conventional
Na+ dyes such as SBFI, which has a
Kd of 18 mM Na+ (19).
However, if a continuous source of Ca2+ from an infinite
reservoir is supplied intracellularly, it should be possible to measure
such an increase in [Na+]i (see
DISCUSSION and Fig. 3
legend). In the experiments shown in Fig.
3, a continuous source of Ca2+ was supplied to the cells
via ionomycin. Because in our experiments the bath can be considered
infinite, in the presence of ionomycin a continuous influx of
Ca2+ occurred.

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Fig. 3.
Continuous influx of Ca2+ resulted in a
measurable increase in [Na+]i. Sodium-binding
benzofuran isophthalate (SBFI)-loaded N1E-115 cells were perfused with
control HEPES buffer, and at the arrows the solution was changed to one
containing 2 µM ionomycin with or without 5 mM Ni2+.
Ionomycin was used to elevate [Ca2+]i and to
allow for continuous influx of Ca2+ into cells. Under these
experimental conditions, a sustained level of ~475 nM
[Ca2+]i was maintained (see text for added
details). The ionomycin and the ionomycin + Ni2+
curves were obtained by averaging the response from 26 and 24 cells,
respectively, and are representative of at least 3 similar
experiments.
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Addition of ionomycin (2 µM) to the bath increased
[Ca2+]i to ~475 nM (data not shown) and
depolarized Em (see Fig. 2A and Table 1). After the addition of ionomycin, there was an increase in [Na+]i. In paired experiments, 5 mM
Ni2+ blocked the elevation of
[Na+]i following addition of ionomycin. In
the absence of Ni2+, following ionomycin addition,
[Na+]i increased by 6.3 ± 0.4 mM,
whereas in the presence of 5 mM Ni2+ there was no increase
(
0.2 ± 2.6 mM, n = 3; P < 0.05, see Fig. 3). These observations are consistent with the
continuous operation of the Na+/Ca2+ exchanger
in the forward mode.
Effect of Na
removal.
The direction of the Na+/Ca2+ exchanger is
determined by the sign of the driving force and therefore is a function
of the membrane potential and the ion gradients for Na+ and
Ca2+. One way of reversing the driving force for
Na+/Ca2+ exchange to one favoring net efflux of
Na+ and influx of Ca2+ is to remove
Na
. Figure
4A shows the effect of
removing Na
on
[Ca2+]i. The removal of Na+ from
the media resulted in little (2.6 ± 1.9 nM, n = 6), if any, increase in [Ca2+]i (Fig.
4A). One explanation for the insignificant
[Ca2+]i increase was that the cell buffered
the Ca2+ as it was brought in by the exchanger. To test
this hypothesis, the cells were exposed to 1 µM thapsigargin and 10 µM FCCP to reduce their ability to buffer
[Ca2+]i. Thapsigargin plus FCCP caused
[Ca2+]i to rise transiently and then recover.
After recovery, Na
was removed from the
media in the presence of 1 µM thapsigargin and 10 µM FCCP. This
maneuver resulted in a significant increase in
[Ca2+]i to a final concentration of 79.8 ± 6.8 nM (69 cells, n = 5). This increase in
[Ca2+]i was significantly greater
(P < 0.05, paired t-test) than that seen in
the absence of 1 µM thapsigargin and 10 µM FCCP. Reperfusion with
Na
returned
[Ca2+]i to its previous level. These data
suggest that the buffer capacity of the cell was able to keep pace with
the influx of Ca2+ following Na
removal (Fig. 4A).

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Fig. 4.
Reversal of the Na+/Ca2+
exchanger: Ca2+ uptake was masked by cell Ca2+
buffers and was dependent on Ca . Fura 2-loaded
N1E-115 cells were perfused in control HEPES Ringer and, in
A, were subsequently perfused with a Na+-free
Ringer. Upon return to control HEPES Ringer, the cells were then
perfused with buffer containing 1 µM thapsigargin and 10 µM FCCP.
Na was again removed and replaced with NMDG
in the presence 1 µM thapsigargin and 10 µM FCCP. After 3.8 min,
the Na+ was added back to the external media. The curve was
obtained by averaging the results from 10 cells. Data are
representative of at least 5 similar experiments. B:
elevation of [Ca2+] following
Na removal required Ca .
The cells were perfused with buffer containing 1 µM thapsigargin plus
10 µM FCCP. Na (replaced with NMDG) or
Na and Ca were removed
in the presence of 1 µM thapsigargin and 10 µM FCCP.
Ca2+-free solutions contained 100 µM EGTA. After
5.5 min the Na+, or after 4.5 min the Na+ plus
Ca2+, were added back to the external media. The curve
representing cells in Ca2+-containing media was obtained by
averaging the results from 12 cells and is representative of 5 similar
experiments. The Ca2+-free curve was obtained by averaging
the results from 13 cells and is representative of 5 similar
experiments.
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The increase in [Ca2+]i following removal of
Na
is consistent with reversal of a
Na+/Ca2+ exchanger such that net
Na+ efflux results in net Ca2+ influx, thus the
countercoupling of Na+ and Ca2+. If
Na+ and Ca2+ fluxes are countercoupled as
predicted, then the increase in [Ca2+]i
following Na
removal should be dependent on
Ca
. Figure 4B shows that, after recovery
of the [Ca2+]i increase induced by 1 µM
thapsigargin and 10 µM FCCP, simultaneous removal of
Na
and Ca
did not cause
[Ca2+]i to increase; in fact, it decreased,
an effect most likely due to the presence of EGTA in the bath solution.
In experiments conducted in Ca2+-containing Ringer, removal
of media Na+ following a 1 µM thapsigargin/10 µM FCCP
pulse was always followed by an increase in
[Ca2+]i (126.8 ± 19.3 nM,
n = 6). On the other hand, this same protocol in the
absence of media Ca2+ caused
[Ca2+]i to decrease by
37.6 ± 12.9 nM
(n = 5). This decrease was significantly different
(P < 0.01, paired t-test) from the
[Ca2+]i increase seen following
Na+ removal in a Ca2+-containing Ringer. This
result strongly suggests that the increase in
[Ca2+]i following
Na
removal is due to a Na+/Ca2+ exchange mechanism operating in a
reverse mode favoring net Na+ efflux and Ca2+ influx.
Effect of Na
loading.
The intracellular Ca2+ buffers were able to prevent a
detectable increase in [Ca2+]i
following Na+ removal (Fig. 4A).
Such intracellular buffers were therefore able to keep pace with
the influx of Ca2+ via the exchanger following
Na
removal. However, it is possible that
the putative Na+/Ca2+ exchanger was not running
at maximum velocity because of insufficient concentration of a kinetic
modulator. For example, [Na+]i is known to
modulate the reverse mode of the Na+/Ca2+
exchanger with a K1/2 of ~25-60 mM
(28).
To test this hypothesis, neuroblastoma cells were loaded with
Na+ by using the voltage-gated Na+ channel
activator veratridine (100 µM) and scorpion venom (4 µg/ml,
Leiurus quinquestriatus from North Africa), a
toxin that prevents Na+ channels from inactivating
(5). To prevent net efflux of
Na
via active transport, ouabain
(100 µM), which poisons the Na+-K+-ATPase,
was also added. This treatment increased
[Na+]i from 11.5 ± 1.4 mM (188 cells,
n = 11) to 25.8 ± 2.1 mM (18 cells,
n = 3) as measured using SBFI (not shown) and was
statistically significant (P < 0.01, unpaired
t-test).
The increase in [Ca2+]i following the removal
of Na
in Na+-loaded cells was
transient (Fig. 5A). This
[Ca2+]i increase (~4- to 6-fold over
resting level) peaked at 410.5 ± 95.4 nM (43 cells,
n = 4) and was inhibited by 5 mM Ni2+ (Fig.
5B) but not by 1 µM TTX (Fig. 5C). In the
presence of 5 mM Ni2+, the rate of rise of
[Ca2+]i following
Na
removal (0.5 ± 0.3 × 10
9 M/s, n = 4) was significantly less
than that in its absence (5.0 ± 1.1 × 10
9
M/s; P < 0.01, paired t-test). In the
presence of 1 µM TTX, the increase in
[Ca2+]i
(
[Ca2+]i = 178.2 ± 18.1 nM;
n = 5) was not significantly different from that seen
in its absence (
[Ca2+]i = 198.5 ± 28.8 nM, n = 5; unpaired
t-test). Compared with the [Ca2+]i
increase following Na
removal after Ca2+ store depletion (Fig. 4A), the increase in
[Ca2+]i shown in Fig. 5A was
significantly greater (P < 0.05, unpaired t-test).

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Fig. 5.
Kinetic stimulation of the reverse mode of
Na+/Ca2+ exchange by elevating
[Na+]i. Fura 2-loaded N1E-115 cells were
perfused with control HEPES buffer and then were exposed to a solution
containing 100 µM veratridine (Verat), 4 µg/ml scorpion venom (SV),
and 100 µM ouabain (Ouab). This solution was used to load the cells
with Na+ in A-C. A:
after 5 min of Na+ loading, the perfusate was changed to a
Na+-free (NMDG replacement) buffer containing 100 µM
ouabain. After an additional 5 min, Na+ was returned to the
media. The curve was generated by data averaged from 6 cells and is
representative of 4 experiments. B: Ni2+ blocked
the [Ca2+]i increase. After 5 min of
Na+-loading, the perfusate was changed to a
Na+-free (NMDG replacement) buffer with 100 µM ouabain.
For the first 5 min, the buffer also contained 5 mM NiCl2.
After the 5 min, the Ni2+ was washed out. At the end of the
experiment, the medium was changed to one containing Na+
(average response from 7 cells). Data are representative of 4 similar
experiments. C: Ca2+ does not move into the cell
through the Na+ channels. After 5 min of Na+
loading, Na+ was isosmotically replaced with NMDG and 1 µM TTX was added to the perfusate. At the end of the experiment, the
medium was changed to one containing Na+ (average response
from 12 cells). Data are representative of 5 similar experiments.
|
|
These results suggest that the [Ca2+]i
increase was caused by the reversal of the
Na+/Ca2+ exchanger and not by the influx of
Ca2+ through Na+ channels following perfusion
with Na+-free media. Moreover, these results are consistent
with the notion that increasing [Na+]i
kinetically stimulates the putative Na+/Ca2+
exchanger in N1E-115 cells.
However, Fig. 5A also revealed that
[Ca2+]i returned to near baseline levels of
99.0 ± 14.9 nM (43 cells, n = 4) after a few minutes despite the absence of Na+. The recovery of
[Ca2+]i in the absence of
Na
was apparently the result of a
Na+-independent extrusion mechanism that was activated or
kinetically stimulated by the relatively large increase in
[Ca2+]i following
Na
removal (compare Figs. 1A and
4A with Fig. 5A; see DISCUSSION).
On the basis of results shown in Figs. 4A and 5A,
we predicted that reducing N1E-115 cell Ca2+ buffering and
kinetically stimulating the putative Na+/Ca2+
exchanger by elevating [Na+]i should give the
most robust increase in [Ca2+]i seen so far.
To reveal the full magnitude of the [Ca2+]i
increase due to the reversal of the Na+/Ca2+
exchange, we utilized 1 µM thapsigargin and 10 µM FCCP to
compromise the cell's buffer capacity and a 100 µM veratridine/4
µg/ml scorpion venom cocktail to elevate
[Na+]i (Fig.
6). After Na
removal, the increase in [Ca2+]i was dramatic
with [Ca2+]i peaking at 607.3 ± 207.4 nM (47 cells, n = 3). However, compared with the peak
increase in [Ca2+]i shown in Fig.
5A where cells were simply loaded with Na+
(~400 nM), the increase was not significant, as indicated by the
relatively large SE. Again, a significant portion of the
[Ca2+]i increase recovered in the absence of
Na
, indicating the presence of a
Na+-independent Ca2+ regulatory mechanism.

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Fig. 6.
A: effect of
Na loading and decreased
Ca2+ buffer capacity on the reverse mode of
Na+/Ca2+ exchange. Fura 2-loaded N1E-115 cells
were perfused with control HEPES buffer and then were exposed to 1 µM
thapsigargin and 10 µM FCCP to compromise the Ca2+ buffer
capacity. After 4 min the cells were loaded with Na+ by
using 100 µM veratridine, 4 µg/ml scorpion venom, and 100 µM
ouabain. After 6 min of Na+ loading, Na+ was
removed from the medium (isosmotically replaced with NMDG). At the end
of the experiment, the medium was changed to one containing
Na+. B: part of the recovery was Na+
dependent. The scale has been magnified and starts 4 min into the
Na+ load after the Ca2+ buffer capacity has
been reduced (average response from 12 cells and representative of 3 similar experiments).
|
|
Although significant, the recovery in Na+-free media was
not complete because [Ca2+]i remained
elevated. This sustained phase of the [Ca2+]i
increase (144 ± 11 nM; 47 cells, n = 3) was
significantly greater than resting level (P < 0.05, paired t-test) and Na
dependent because introduction of Na
into the bath
reduced [Ca2+]i to control levels (See Fig.
6B).
 |
DISCUSSION |
Under normal physiological conditions, the
Na+/Ca2+ exchanger runs in the forward
direction, extruding one Ca2+ for three Na+
taken in (3). By elevating
[Ca2+]i, one can observe the cells' ability
to return [Ca2+]i to control levels. After
addition of 10 µM FCCP and 1 µM thapsigargin, N1E-115 cells were
able to recover from a [Ca2+]i load induced
by releasing Ca2+ from intracellular stores. Because
Ca2+ sequestering mechanisms were inhibited (22,
32) in the presence of 10 µM FCCP and 1 µM thapsigargin, the
[Ca2+]i recovery was presumably achieved
through extrusion mechanisms. The restoration of
[Ca2+]i to control level following its
elevation with FCCP and thapsigargin was exclusively
Na
dependent (Fig. 1A).
Consistent with a Na+/Ca2+ exchanger, the rate
of recovery was slowed by 5 mM Ni2+ and blocked by
replacing Na
with NMDG (Fig.
1B).
We saw no evidence of a sustained Ca2+ entry following the
initial rise in [Ca2+]i with 1 µM
thapsigargin/10 µM FCCP addition (see Figs. 1A and 4A). Thus a capacitive Ca2+ entry (CCE)
following Ca2+ release from stores was apparently absent or
negligible. However, Mathes and Thompson (18) did find
evidence for CCE in DMSO-differentiated N1E-115 cells. In addition, in
those authors' studies, addition of thapsigargin (1 µM) was found to
increase [Ca2+]i by ~150 nM
(18), whereas in our hands 1 µM thapsigargin/10 µM
FCCP led only to a ~60 nM increase in
[Ca2+]i. Unlike the studies of Mathes and
Thompson (18), our N1E-115 cells were not differentiated,
and this difference may account for the apparent absence of CCE and the
smaller [Ca2+]i elevation in response to
thapsigargin/FCCP. In support of this notion, it has been shown that
the Ca2+-gated K+ channel in N1E-115 cells is
only expressed following differentiation using DMSO (27).
Effect of ionomycin on membrane potential.
Because the Na+/Ca2+ exchanger has been
reported to be electrogenic (3), operation of the
exchanger is expected to result in current flow across the cell
membrane. In addition, depending on the relative conductance of the
exchanger, a measurable change in membrane potential may also occur.
When [Ca2+]i was raised by using 1 µM
ionomycin, the membrane potential depolarized by ~27 mV. In the
absence of Ca
there was no depolarization,
which indicates the necessity for Ca2+ to enter the cell
for the depolarization to occur. The ionomycin-induced depolarization
was Na
dependent and inhibited by 5 mM
Ni2+ and 250-500 µM benzamil (Table 1). The
depolarization was not blocked by 1 µM TTX, ruling out a
Na+ channel contribution. These results suggest that
depolarization was most likely mediated by the
Na+/Ca2+ exchanger.
After Na
removal, Em
went from approximately
60 mV to
85 mV (at t = 0 min,
see Fig. 2A) and then slowly drifted upward. In normal
Na
, Em
did not depolarize over time. Thus the cells hyperpolarize by as much
as 25 mV following Na
removal. The
upward drift in Em in Na+-free
Ringer is not understood. One possibility is that removal of
Na+ acidifies the cells via reverse
Na+/H+ exchange and that this decrease in pH
blocks a K+ conductance (34). On addition of
ionomycin in Na+-free Ringer, the linear increase in
Em was halted and, in fact, there was a slight
hyperpolarization. Two factors may be responsible for this small effect
on Em following ionomycin addition in
Na+-free media. First, the secondary depolarization in
Na+-free Ringer following the initial hyperpolarization
(see Fig. 2A) would tend to offset the ionomycin-induced
hyperpolarization. Second, after Na
removal, intracellular Na+ (10-15 mM) drops
precipitously within 1-2 min to a few millimoles per liter (data
not shown). Thus, when ionomycin was added some minutes after
the cells were in Na+-free Ringer,
[Na+]i most likely was greatly reduced, and
this would tend to reduce the large expected ionomycin-induced hyperpolarization.
In any event, N1E-115 cells do, in fact, hyperpolarize following
Na+ removal as expected for reversal of a
Na+/Ca2+ exchange mechanism. However, without
knowing the relative conductances of all
current-generating/electrogenic mechanisms in these cells, there is no
way a priori to predict what the magnitude of the hyperpolarization
should be.
Detection of the forward mode of the
Na+/Ca2+
exchanger during continuous influx of
Ca2+ using SBFI.
During forward operation of the Na+/Ca2+
transporter, three Na
exchange for one
Ca
(3). Because
Ca2+ is a tightly regulated second messenger, it is highly
unlikely that, under physiological conditions, millimolar amounts of
Ca2+ are moved by the Na+/Ca2+
exchange. Therefore, detecting less-than-micromolar increases in
[Na+]i during the forward operation of the
Na+/Ca2+ exchanger is not technically possible.
However, under special conditions (see Fig. 3), it is possible to
measure millimolar increases in [Na+]i
provided 1) the exchanger is fed with a continuous supply of Ca2+ and 2) the parameters determining the
driving force favor net influx of Na+ (forward mode of the exchanger).
To determine what values of [Na+]i,
[Ca2+]i, and Em were
needed to allow forward operation of the exchanger, the equation below was used to calculate the driving force
where 
Na-Ca is the electrochemical
potential (driving force, in mV)and R, T, and
F are the gas constant, the absolute temperature, and
Faraday's constant, respectively. Em and the subscripts o and i denote the membrane potential (mV) and the extra-
and intracellular ion concentrations, respectively. A plot of the
driving force as a function of Em at
[Na+]i ranging from 10 to 25 mM shows what
values are required, at [Ca2+]i of 475 nM,
for net Na+ influx to occur (Fig.
7).

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Fig. 7.
Driving force for a Na+/Ca2+
exchanger as a function of membrane potential at fixed
[Ca2+]i (475 nM). The electrochemical
potential (driving force) was calculated for four different
[Na+]i by the following equation:
 Na-Ca = RT/F[log ([Na+]i/[Na+]o)3 log ([Ca2+]i/[Ca2+]o)] + Em, where  Na-Ca is the
electrochemical potential (driving force for 3 Na+/Ca2+ exchange), and the subscripts o and i
denote extracellular and intracellular compartments, respectively.
R, T, and F have their usual meaning,
and Em denotes the membrane potential.
[Na+]o = 137 mM and
[Ca2+]o = 1.2 mM.
|
|
Under our experimental conditions (see Fig. 7 legend), driving force
calculations show that the exchanger will reverse when [Na+]i reaches ~20 mM and/or when
Em depolarizes to
15 mV (Fig. 7). Because both
[Na+]i increases and
Em depolarizes following ionomycin
addition, the increase in [Na+]i
was, as expected, short lived (Fig. 3). After
[Ca2+]i was increased and held at ~475 nM,
[Na+]i levels increased ~6 mM, whereas
cells treated with 5 mM Ni2+ had no
[Na+]i gain (Fig. 3).
Reversal of
Na+/Ca2+
exchange following removal of media Na+.
The Na+ and Ca2+ transmembrane gradients and
Em determine whether net Ca2+ efflux
(forward mode) or influx (reverse mode) is occurring via the
Na+/Ca2+ exchanger. Altering any of these
components can change the direction of the exchanger. With the removal
of external Na+, the driving force favoring the reverse
mode of the exchanger is infinite. However, simply removing
Na
did not increase
[Ca2+]i (Fig. 4A). In contrast,
when the buffer capacity was compromised before Na+
removal, with 10 µM FCCP and 1 µM thapsigargin, the contribution of
the Na+/Ca2+ exchange was readily seen.
Reducing the Ca2+ buffer capacity of the cell followed by
Na
removal caused
[Ca2+]i to increase. This increase was
completely dependent on the presence of Ca
(Fig. 4B).
To investigate a possible kinetic effect of
[Na+]i as demonstrated by others
(28), veratridine, scorpion venom, and ouabain were used
to increase [Na+]i. This cocktail increased
[Na+]i by ~14 mM. When bath Na+
was removed following [Na+]i loading,
[Ca2+]i increased, transiently peaking to
~410 nM (Fig. 5A). Comparing Fig. 4A with Fig.
5A demonstrates that [Na+]i can
kinetically modulate the Na+/Ca2+ exchanger
because an increase in [Ca2+]i was seen only
after [Na+]i was elevated. The kinetic
modulation increased Ca2+ uptake via the putative
Na+/Ca2+ exchanger such that it was able to
swamp the cells' Ca
buffer capacity.
Interestingly, [Ca2+]i returned to near
baseline levels even in the absence of Na
in the experiments shown in Fig. 5 but not in those of Fig. 4. One
difference in these two experiments was the level of
[Ca2+]i increase. On average, kinetically
stimulating the Na+/Ca2+ exchanger
(Na
loading) caused
[Ca2+]i to increase fivefold during
Na
removal compared with simply reducing
the cells' buffer capacity. It appears that increasing
[Ca2+]i above ~200 nM stimulates or
kinetically modulates another Ca2+ extrusion mechanism that
is Na+ independent and functioning at a much lower rate at
lower [Ca2+]i.
It is possible that the Na+-independent recovery may be due
to an ATP-driven Ca2+ extrusion mechanism. Others have
reported that Ca2+-ATPases (pumps) may play an important
role in [Ca2+]i regulation (20).
That is, instead of the traditional role of maintaining basal levels of
[Ca2+]i, new evidence suggests that some
isoforms of the plasma membrane Ca2+-ATPase may play an
active role in [Ca2+]i regulation after
cellular stimulation and may be involved in shaping
Ca
signals (8). The
Km for the Ca2+-ATPase (<0.5-1
µM; Ref. 9) is within the range of the Ca2+
increases shown in Fig. 5, A and B, and this may
explain why regulation is so rapid in the above experiments despite the
absence of media Na+.
Because the stimulatory effect of elevating
[Na+]i allows the
Na+/Ca2+ exchanger to exceed the cells'
Ca2+ buffer capacity, one would predict that reducing the
buffer capacity and kinetically stimulating the exchanger would also
yield a large [Ca2+]i increase. Figure 6
shows when bath Na+ was removed following FCCP/thapsigargin
and veratridine/scorpion toxin treatments,
[Ca2+]i increased to a peak value that was
significantly greater than that following FCCP/thapsigargin treatment
alone (P < 0.05). Here again, there was a significant
recovery in the absence of Na
. However,
there was also a significant sustained increase of ~90 nM above
baseline [Ca2+]i (see Fig. 5C).
The sustained increase in [Ca2+]i was
Na+ dependent because addition of
Na
caused [Ca2+]i
to return to control levels. This Na+-dependent decrease in
[Ca2+]i is presumably the result of
activation of the Na+/Ca2+ exchanger in the
forward mode.
In summary, our data strongly suggest that there are at least two
extrusion mechanisms involved in [Ca2+]i
regulation in N1E-115 neuroblastoma cells: a
Na+/Ca2+ exchanger, which has been our primary
focus; and a second, Na+-independent mechanism (possibly a
Ca2+-ATPase) that is further stimulated/kinetically
modulated following relatively large increases in
[Ca2+]i. Under pathophysiological conditions,
increasing [Na+]i may set into motion a
destructive pathway of events (29). [Na+]i overload via voltage-gated
Na+ channels can lead to [Ca2+]i
overload via reversal of the Na+/Ca2+ exchanger
(30). The existence of both a
Na+/Ca2+ exchanger and Na+ channels
may make the N1E-115 neuroblastoma a potentially valuable model for
studying neurotoxicity from [Na+]i and
[Ca2+]i overload.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. Adorante, Mail stop RD-2C, 2525 Dupont Dr., Irvine, CA 92612 (E-mail: adorante_joseph{at}allergan.com).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00182.2001
Received 19 April 2001; accepted in final form 21 October 2001.
 |
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