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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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 approx  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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP>), [Ca2+]i rose to 129.5 ± 15.4 nM (34 cells, n = 8) and remained elevated (Fig. 1A). In the absence of Na<UP><SUB>o</SUB><SUP>+</SUP></UP>, the increase in [Ca2+]i following 1 µM thapsigargin and 10 µM FCCP was statistically greater than that observed with Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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.

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<UP><SUB>o</SUB><SUP>+</SUP></UP> (NMDG replacement) failed to regulate the increase in [Ca2+]i caused by these agents until Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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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Table 1.   Effect of inhibitors on the ionomycin-induced depolarization

In addition, removal of Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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.

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<UP><SUB>o</SUB><SUP><UP>+</UP></SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP>. Figure 4A shows the effect of removing Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>2+</SUP></UP>. 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> removal required Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP>. The cells were perfused with buffer containing 1 µM thapsigargin plus 10 µM FCCP. Na<UP><SUB>o</SUB><SUP>+</SUP></UP> (replaced with NMDG) or Na<UP><SUB>o</SUB><SUP>+</SUP></UP> and Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP> 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.

The increase in [Ca2+]i following removal of Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> removal should be dependent on Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP>. Figure 4B shows that, after recovery of the [Ca2+]i increase induced by 1 µM thapsigargin and 10 µM FCCP, simultaneous removal of Na<UP><SUB>o</SUB><SUP>+</SUP></UP> and Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> removal is due to a Na+/Ca2+ exchange mechanism operating in a reverse mode favoring net Na+ efflux and Ca2+ influx.

Effect of Na<UP><SUB>i</SUB><SUP><UP>+</UP></SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>i</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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 (Delta [Ca2+]i = 178.2 ± 18.1 nM; n = 5) was not significantly different from that seen in its absence (Delta [Ca2+]i = 198.5 ± 28.8 nM, n = 5; unpaired t-test). Compared with the [Ca2+]i increase following Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP>, indicating the presence of a Na+-independent Ca2+ regulatory mechanism.


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Fig. 6.   A: effect of Na<UP><SUB>i</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> dependent because introduction of Na<UP><SUB>o</SUB><SUP>+</SUP></UP> into the bath reduced [Ca2+]i to control levels (See Fig. 6B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>o</SUB><SUP>+</SUP></UP> dependent (Fig. 1A). Consistent with a Na+/Ca2+ exchanger, the rate of recovery was slowed by 5 mM Ni2+ and blocked by replacing Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>2+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP>, Em did not depolarize over time. Thus the cells hyperpolarize by as much as 25 mV following Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> exchange for one Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (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
&Dgr;<A><AC>&mgr;</AC><AC>˜</AC></A><SUB>Na-Ca</SUB><IT>=RT/F</IT>[log ([Na<SUP>+</SUP>]<SUB>i</SUB>/[Na<SUP>+</SUP>]<SUB>o</SUB>)<SUP>3</SUP> 

− log ([Ca<SUP>2+</SUP>]<SUB>i</SUB>/[Ca<SUP>2+</SUP>]<SUB>o</SUB>)]<IT>+E</IT><SUB>m</SUB>
where Delta <A><AC>&mgr;</AC><AC>˜</AC></A>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: Delta <A><AC>&mgr;</AC><AC>˜</AC></A>Na-Ca = RT/F[log ([Na+]i/[Na+]o)3 - log ([Ca2+]i/[Ca2+]o)] + Em, where Delta <A><AC>&mgr;</AC><AC>˜</AC></A>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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP> removal caused [Ca2+]i to increase. This increase was completely dependent on the presence of Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP> (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<UP><SUB>i</SUB><SUP>2+</SUP></UP> buffer capacity.

Interestingly, [Ca2+]i returned to near baseline levels even in the absence of Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>i</SUB><SUP>+</SUP></UP> loading) caused [Ca2+]i to increase fivefold during Na<UP><SUB>o</SUB><SUP>+</SUP></UP> 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<UP><SUB>i</SUB><SUP>2+</SUP></UP> 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<UP><SUB>o</SUB><SUP>+</SUP></UP>. 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<UP><SUB>o</SUB><SUP>+</SUP></UP> 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(5):C1000-C1008
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