Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes

Gergely Gy Kovacs,1 Akos Zsembery,4 Susan J. Anderson,1 Peter Komlosi,2 G. Yancey Gillespie,3 P. Darwin Bell,1,2 Dale J. Benos,1 and Catherine M. Fuller1

1Department of Physiology and Biophysics, 2Nephrology Research and Training Center, Department of Medicine and 3Division of Neurosurgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama; and 4Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary

Submitted 11 June 2004 ; accepted in final form 22 March 2005


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of [Ca2+]i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca2+]i and pHi in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca2+]i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca2+]i increase than in normal astrocytes. This increase was prevented in nominally Ca2+-free buffer and by 2-APB, an inhibitor of store-operated Ca2+ channels. In addition, TG-activated Ca2+ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pHi was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na+/H+ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca2+ homeostasis and pH regulation in tumor cells compared with normal human astrocytes.

fura-2; BCECF; store-operated calcium channels


THE INCIDENCE RATE of all primary benign and malignant brain tumors is ~14.0 cases per 100,000 person-years (4) in the United States. Gliomas comprise 44.4% of all primary brain tumors, which are mostly astrocytomas and glioblastomas. Despite aggressive treatment strategies, the overall 5-yr survival rate for glioblastoma multiforme (GBM) is only 2.9% (4). Abnormal proliferation and migration, as well as dedifferentiation and escape from apoptosis, are principal features of cancer cells. The poor outcome is largely due to the lack of information regarding the molecular mechanisms that underlie these processes in brain tumor cells and is compounded by the invasiveness of the tumor and the sensitive location.

It is well established in many cell types that intracellular Ca2+ is a crucial participant in processes fundamental for tumor progression, such as cell cycle regulation, apoptosis, migration, and differentiation. In astrocytes, intracellular Ca2+ modulates the proliferative effects of UTP, EGF, and brain-derived neurotrophic factor, among others (6, 25, 36), whereas migrating human astrocytoma cells exhibit complex Ca2+ oscillations depending on the prevailing growth conditions (24). A major component of intracellular Ca2+ homeostasis is the endoplasmic reticulum (ER), which stores micromolar levels of Ca2+. Central to the regulation of [Ca2+]i are the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which pump Ca2+ against its gradient into the ER, thus maintaining a low cytosolic [Ca2+] in the face of a continuous basal outward leak of Ca2+ into the cytosol. Specific inhibition of SERCA activity with thapsigargin (TG) results in ER Ca2+ depletion, and consequent opening of store-operated calcium channels (SOCC) located in the plasma membrane. Plasma membrane SOCCs elevate cytosolic [Ca2+] and under physiological conditions permit refilling of the ER Ca2+ pool. The magnitude of the increase in [Ca2+]i evoked by TG is thus dependent on the size of the TG-sensitive ER Ca2+ stores, the leakiness of the ER membrane for Ca2+, and the activity of the SOCC.

Several studies have demonstrated that changes in the magnitude of the ER Ca2+ store directly influence cell behavior. Depletion of the ER pool arrests proliferation in C6 glioma cells (5), whereas in human fibroblasts and human embryonic kidney-293 cells, inhibition of SERCA by TG prolonged the cell cycle and prevented the cells progressing into S phase (28, 32). ER-mediated Ca2+-release pathways are also involved in apoptosis in many cell types. Because the micromolar Ca2+ content of the ER is essential for protein synthesis, folding, and trafficking, depletion of ER Ca2+ content induces an unfolded protein response, an integrated intracellular signaling pathway that transmits information about protein folding status in the ER to the nucleus and cytoplasm, and leads to activation of adaptive mechanisms essential for cell survival (13, 23).

A second tightly regulated intracellular ionic parameter is the concentration of H+ ions. The role of intracellular pH (pHi) in proliferation and tumor formation has been investigated for many years (see Ref. 27 for review). Tumors generate an excessive amount of acidic metabolites such as lactate, and thus their microenviroment is acidic due to increased proton efflux (9). However, the pHi of tumor cells is neutral to alkaline, enhancing cellular processes such as DNA, RNA, and protein synthesis, which are involved in proliferation and attenuating apoptosis (18). Consequently, an alkaline pHi observed in human glioma cells could contribute to tumor proliferation (19, 20). It was recently demonstrated that in glioblastoma cells pHi is regulated predominantly by the Na+/H+ exchanger (NHE), whereas pHi regulation occurs through HCO-dependent mechanisms in normal astrocytes (20). Among the regulators of cellular pH, the activity of NHE has been most clearly demonstrated to have a significant influence on apoptosis and proliferation (see Ref. 27 for review). In addition, the upregulation of NHE activity was also found to be crucial in tumor formation (26). Furthermore, SOCC activity and pHi have mutual effects. It has been reported that administration of SERCA inhibitors affected pHi (15), whereas capacitative Ca2+ entry was substantially altered by pHi in platelets (10).

In the present study, we have used fluorescent dyes to examine the role that the SERCA pump plays in Ca2+ homeostasis and pHi in human glioma cell lines and primary cultures of human GBMs and normal human astrocytes. Inhibition of SERCA function with TG revealed a significant divergence in intracellular Ca2+ homeostasis and pH regulation between the normal and tumor cell types and it also affected the proliferation of the tumor cells. The ability of GBM cells to maintain a higher cytosolic and ER [Ca2+], a more alkaline pHi, and an increased plasma membrane permeability to Ca2+, may facilitate the increased proliferation and migration characteristic of the glioblastoma cell phenotype.


    METHODS
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 ABSTRACT
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Cell culture. The human glioblastoma cell lines (SK-MG-1, U87-MG, D54-MG, U251-MG, and U373-MG), primary cultures of normal human astrocytes (generated from tissue removed from surgery for intractable epilepsy), and human glioblastoma cells were obtained from the Brain Tumor Tissue Bank at University of Alabama at Birmingham. The cells were cultured in DMEM/F-12 (1:1) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cultures were maintained at 37°C in a 5% CO2-95% air atmosphere. Cell lines between passages 25 and 39, and primary cultures between passages 2 and 7 were used. For fluorescence measurements, cells were plated on uncoated glass coverslips in 60-mm cell culture dishes 24–48 h before experiments.

Intracellular Ca2+ and pH measurements. pHi and [Ca2+]i were measured using BCECF and fura-2, respectively. For measurement of [Ca2+]i, 50 µg of fura-2 AM was dissolved in DMSO containing 15% (wt/vol) Pluronic F-127. Five microliters of this stock solution were added to 4 ml of cell culture medium. The cells were loaded with 12.5 µM fura-2 AM in this prepared culture medium at 37°C for 1.5 h. The final concentration of 15% Pluronic acid F-127 was <0.02% (wt/vol). For pH experiments, 50 µg of BCECF AM was dissolved in DMSO and 5 µl of this stock solution were added to 4 ml of cell culture medium. The cells were loaded in this culture medium containing 15.2 µM BCECF AM at 37°C for 10 min. In double-loading experiments, cells were loaded first with fura-2 as described above, washed with Krebs buffer composed of (in mM) 130 NaCl, 2.4 K2HPO4, 1 CaSO4, 1 MgSO4, 10 D-glucose, and 10 HEPES, pH 7.4, for 10 min, followed by loading of the cell with BCECF (see above) for 3 min. Finally, the cells were rinsed with Krebs buffer for 10 min.

With the use of a cuvette-based fluorescent spectrofluorimeter equipped with dual-excitation and a single-emission monochromators (Deltascan-1, Photon Technology International, Lawrenceville, NJ), the fluorescent intensity of fura-2 was measured at excitation wavelengths of 340 and 380 nm with 2.5-nm bandwidth and at emission wavelength of 510-nm with 4-nm bandwidth. The pH experiments were performed at 490- and 440-nm excitation wavelengths and 530-nm emission wavelength with the same bandwidths. One 455-nm long-pass filter was applied in front of the photomultiplier tube at the emission side to reduce background fluorescence, which results from scattered excitation light. Raw 340/380 ratio values were converted into [Ca2+] using the following equation (12):

(1)
where Kd is the dissociation constant for fura-2 (taken as 224 nM), R is the raw 340/380 fluorescence ratio, Rmin is the 340/380 ratio in the absence of Ca2+ using 10 mM EGTA, Rmax is the 340/380 ratio in the presence of 10 mM Ca2+, and Sf2/Sb2 is the ratio of the fura-2 fluorescence signal at 380 nm in the absence of Ca2+ (Ca2+-free fura-2) and in the presence of saturating [Ca2+] (Ca2+-bound fura-2). The calibration procedure was performed in the presence of 10 µM ionomycin to equilibrate intra- and extracellular Ca2+.

To convert raw 490/440 ratios to pHi values, we applied the nigericin-high-KCl calibration method (8). Briefly, we used a series of solutions with pH values of 8.0, 7.5, 7.0, 6.5, and we equilibrated pHi and extracellular pH using 10 µM nigericin, a K+/H+ antiporter in the presence of 120 mM KCl and 10 mM NaCl. The calibration curve was generated by plotting the acquired raw ratios against the known pH values. The equation of the derived calibration curve was used for the transformation of measured ratio values to pHi. All measurements were background corrected and were performed in Krebs buffer at room temperature and pH 7.4.

The double-loading experiments with fura-2 and BCECF were performed on a cuvette-based fluorescent spectrofluorometer (model QM-8, Photon Technology International) equipped with a Delta RAM V, which allowed us to switch between excitation wavelengths in milliseconds. The bandwidths were adjusted to 2 and 3 nm at the excitation and emission side, respectively. A 455-nm long-pass filter at the emission side was also used. The specimen was alternately illuminated at fura-2 (340 and 380 nm) and BCECF (490 and 440 nm) excitation wavelengths controlled by the computer. Emission wavelengths (510 nm for fura-2 and 530 nm for BCECF) were adjusted in accordance with the excitation wavelengths. The temporal difference in data collection of BCECF and fura-2 signals via the computer-operated, alternate wavelength switching ensured that no significant cross-talk could exist between BCECF and fura-2 signals. In addition, the excitation spectra for fura-2 and BCECF are well separated, permitting these double-loading experiments.

Mn2+-quenching technique. We used Mn2+ to measure exclusively Ca2+ entry through the plasma membrane. This divalent cation is transported by several plasmalemmal Ca2+ entry pathways, including SOCCs, and it quenches the activity of fura-2. On the basis of these features, we assessed the rate of Ca2+ entry through the plasma membrane measuring the rate of decrease of fura-2 intensity at its isosbestic point where fura-2 fluorescence is insensitive to Ca2+ changes. For our experimental setup, the isosbestic point was measured to be 359 nm.

Cell growth curves. Cells were seeded at a density of 2.5 x 105 cells/dish into 11 uncoated 60-mm cell culture dishes (24 h). One day later (0 h), cells in one dish were harvested and stained with 0.2% trypan blue. Viable cells (excluding trypan blue) were counted with the use of a hemocytometer. The other 10 dishes were divided into two groups (5 for 24 h and 5 for 48 h) and were randomly selected and treated with the appropriate drugs. At 24 and 48 h, cells in 5–5 dishes (24- and 48-h group) were harvested, stained, and counted as described above. The culture medium with the appropriate drugs was replenished for the 48-h group after 24 h. This experimental procedure was performed three times per cell line.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. Cells were counted with the use of a hemocytometer and resuspended in phenol red-free medium. Next, the cells were seeded at various densities (from 0 to 5 x 103 cells/well) into 96-well plates in 100-µl aliquots. After 24 h of incubation (0 h), cells were treated with the appropriate drugs. At 24 h, the culture medium was replenished. One day later (48 h), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay was performed, following the manufacturer's instructions. Briefly, a 12 mM stock solution of MTT was made by dissolving 5 mg of MTT in sterile PBS. The cell culture medium was removed and replaced with 100 µl of fresh phenol red-free medium mixed with 10 µl of MTT stock solution. The cells were incubated in this medium at 37°C for 4 h. This was followed by the addition of 100 µl of 0.1 gm/ml SDS dissolved in 0.01 N HCl. After 4 h of incubation, the absorbance was measured at 570 nm with the use of a microplate reader (model 550, Bio-Rad Laboratories, Hercules, CA). The absorbance of the wells containing no cells was subtracted as background.

Materials. BCECF AM and fura-2 AM were purchased from TEFLabs (Austin, TX). TG, 2-APB, ionomycin, and nigericin were obtained from Calbiochem (La Jolla, CA). Amiloride and 5-(N,N-hexamethylene)-amiloride were purchased from Sigma (St. Louis, MO). Cell culture medium and reagents were bought from GIBCO-Invitrogen (Carlsbad, CA). Vybrant MTT Cell Proliferation Assay Kit was purchased from Molecular Probes/Invitrogen (Eugene, OR).

Statistics. Data are expressed as means ± SD. Statistical significance was tested using ANOVA or unpaired Student's t-test at P < 0.05.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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TG-evoked changes of [Ca2+]i. We initially measured [Ca2+]i under basal conditions. Resting [Ca2+]i was found to be significantly higher in SK-MG-1 and U87 MG cells (113.6 ± 23 nM, n = 17, and 115.2 ± 34.8 nM, n = 16) compared with primary human astrocytes (92.6 ± 14.3 nM, n = 23) (see traces in Fig. 1 and summarized data in Fig. 2A). The basal free cytosolic Ca2+ was not elevated in primary human GBM cells (83.3 ± 19.0 nM, n = 13; Fig. 2A). In nominally Ca2+-free buffer, the basal [Ca2+]i was markedly reduced in SK-MG-1 cells and primary astrocytes but not in U87 MG and primary GBM cells (Fig. 2A). The addition of 50 µM 2-APB, an SOCC blocker, caused a small increase in [Ca2+]i, which then fell by 14.7 ± 5.2 nM below the basal level in SK-MG-1 cells. However, in primary human GBM cells, U87 MG cells, and normal astrocytes, 2-APB caused a small increase in basal [Ca2+]i (by 4.6 ± 2.6, 2.2 ± 1.5, and 6.9 ± 2.8 nM, respectively, n = 6, in each cell type, data not shown). Administration of 100 nM TG, which is an effective and (at this concentration) a specific sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) blocker, evoked a significantly larger and faster elevation of cytosolic free Ca2+ in human primary GBM, SK-MG-1, and U87 MG cells than in normal primary astrocytes (Fig. 1, A and B, for representative traces, and see Fig. 2, B and C, for summarized data). The peak of the [Ca2+]i rise was followed by a prolonged plateau phase in all cell types, but this phase was maintained at a much higher level in the tumor cells (Fig. 1, A and B). Removal of extracellular Ca2+ or administration of 50 µM 2-APB after exposure of TG, resulted in a rapid return of the [Ca2+]i to basal or slightly below basal values (Fig. 1, A and B). The effect of 2-APB was reversible (Fig. 1B). The effect of calcium removal was also reversible (data not shown). In nominally Ca2+-free buffer or in 2-APB pretreated cells, the large TG-induced [Ca2+]i increase was markedly diminished in all cell types (Fig. 1, C and D, as representative traces; Fig. 2, B and C, for summarized data). The same effects were observed in normal astrocytes (Figs. 1, C and D, and 2, B and C). However, the increase in cytosolic Ca2+ in nominally Ca2+-free buffer was significantly smaller in the primary normal astrocytes than in the SK-MG-1 or primary human GBM cells without any difference in the rate of the [Ca2+]i increase (Fig. 2, B and C). DMSO alone did not change basal [Ca2+]i or alter the TG-evoked Ca2+ response (data not shown).



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Fig. 1. Representative tracings showing the effect of 100 nM thapsigargin (TG) on intracellular [Ca2+] ([Ca2+]i) in SK-MG-1 glioma cells and in human (Hu) primary (Pr) astrocytes using the Ca2+-sensitive fluorescent dye fura-2. The effect of TG in the presence of 1 mM external Ca2+, followed by removal of extracellular Ca2+ or administration of 50 µM 2-APB, a store-operated calcium channel (SOCC) blocker is shown in A and B, respectively. C and D: effect of 100 nM TG in nominally extracellular Ca2+-free buffer or in the presence of 50 µM 2-APB, respectively.

 


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Fig. 2. Summarized data from fura-2 experiments. A: baseline [Ca2+]i. B and C: magnitude and the rate of the Ca2+ response evoked by 100 nM TG, respectively. Each group contains at least 6 separate experiments. *P < 005 compared with the corresponding group of normal astrocytes; #P < 005 compared with the control group of each cell type.

 
Effect of TG on rate of plasmalemmal Ca2+ entry assessed by Mn2+ quenching technique. With the use of the Mn2+ quenching technique, we found that basal Ca2+ entry through the plasma membrane was significantly higher in all five tested GBM cell lines but not in primary human GBM cells compared with the normal astrocytes (Fig. 3, AC). Basal Mn2+ entry was significantly reduced by 2-APB in all cell types (Fig. 3C). When TG was added, Ca2+ entry was increased slightly in normal astrocytes, but in primary GBM cells and GBM cell lines it increased markedly (Fig. 3, C and D). In every case, 2-APB completely abolished the effect of TG (Fig. 3, C and D). In the human primary astrocytes, SK-MG-1, and U87-MG cell lines (the cells have been tested), the inhibitory effect of 2-APB on TG-induced Mn2+ entry was found to be reversible (n = 4, data not shown).



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Fig. 3. Effect of TG and 2-APB on Ca2+ entry pathways in human glioblastoma multiforme (GBM) cells and in normal human astrocytes. A and B: representative traces of experiments with SK-MG-1 glioma cells and human primary astrocytes, respectively; 250 µM MnCl2, the SOCC blocker 2-APB (50 µM), and TG (100 nM) were added as indicated by the arrows. C: summary data showing the rate of fluorescence quenching under control (basal), in the presence of 50 µM 2-APB and/or 100 nM TG. D: 2-APB abolishes the TG stimulation of Mn2+ entry in every cell type. Data are means of at least 5 separate experiments. *P < 005 compared with the corresponding group of normal astrocytes.

 
Effect of TG on pHi. In accordance with previously published findings (20), resting pHi in SK-MG-1, U87-MG, and primary human GBM cells was markedly higher than in normal astrocytes (7.58 ± 0.11, n = 53; 7.36 ± 0.1, n = 19; 7.18 ± 0.07, n = 23; and 7.09 ± 0.1, n = 16, respectively), and it was not affected by removal of extracellular Ca2+ (Figs. 4 and 5A). However, basal pHi was not as high in the primary GBM cells as in the GBM cell lines (Fig. 5A). The addition of 50 µM 2-APB significantly reduced basal pHi (–0.19 ± 0.07 pH units, n = 17; –0.09 ± 0.01, n = 5; –0.17 ± 0.01 and –0.14 ± 0.05 pH units, n = 6) in SK-MG-1, U87 MG cells, primary human GBM cells, and astrocytes, respectively. DMSO alone did not exert any effect on basal pH or on TG-evoked pH changes (data not shown). In normal astrocytes, 100 nM TG slowly decreased the pHi, which stabilized at a lower level. By contrast, in the tumor cells, TG evoked a rapid acidification, with a significantly larger change in pHi than was observed in normal astrocytes (Figs. 4, A and B, and 5, B and C). However, because the pH scale is not linear, changes in pH cannot be compared if the starting pH is different. Nonetheless, changes in intracellular free [H+] are comparable. The increase of intracellular [H+] in response to TG was found not to be different in all cell types. This acidification was temporary and pHi returned to the resting level in ~30 min (Fig. 4, A and B). The addition of 2-APB further reduced the pHi in normal astrocytes, whereas it resulted in only a transient acidification in SK-MG-1 cells (Fig. 4A). When extracellular Ca2+ was removed after TG treatment, pHi started to increase slowly in normal astrocytes and quickly in SK-MG-1 cells (Fig. 4B). Pretreatment of the cells with 2-APB diminished the effect of TG on intracellular [H+] in all tumor cells and in primary astrocytes (Figs. 4C and 5, B and C). In nominally Ca2+-free buffer, the intracellular acidification in response to TG was inhibited (Figs. 4D, and 5, B and C). Readdition of Ca2+ evoked the same responses as were observed in all cell types when TG was added in the presence of external Ca2+.



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Fig. 4. Representative tracings showing the effect of 100 nM TG on pH in SK-MG-1 glioma cells and human primary astrocytes assessed by BCECF fluorescence. A and B: in normal Krebs solution, followed by removal of extracellular calcium or addition of 50 µM 2-APB. C: in the presence of 2-APB. D: in nominally Ca2+-free buffer.

 


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Fig. 5. Summary data showing the effect of 100 nM TG and 50 µM 2-APB on changes in pHi in human primary astrocytes and SK-MG-1 cells. A: baseline pH values in each cell type. B and C: summarized data of the pH changes and the cytosolic free [H+] changes induced by TG. Data are the means of at least 6 separate experiments. Note that statistical tests were only used to compare changes in [H+]i because pH difference ({Delta}pH) values are not comparable with different starting pHs, as the pH scale is logarithmic. *P < 005 compared with the appropriate group of the human primary astrocytes; #P < 005 compared with the control group of each cell type.

 
Next, we wanted to explore whether NHE is involved in the pHi recovery in response to TG-induced acidification. First, we investigated the effects of withdrawal of extracellular Na+ and addition of amiloride in SK-MG-1 cells. We found that the recovery phase was reduced by 10 µM amiloride and totally abolished by 100 µM amiloride, which blocks NHE in the micromolar range (Fig. 6, B and C). In contrast, amiloride had no significant effect on resting pH (Fig. 6, A and C). When extracellular Na+ was completely removed and isosmotically replaced by N-methyl-D-glucamine, resting pHi was slightly decreased (7.37 ± 0.06, n = 8; Fig. 6B). In Na+-free buffer, TG evoked an increase in [H+]i of 176 ± 44.9 nM that was ~10-fold greater compared with [H+]i changes in the presence of 130 mM [Na+]i (Fig. 6D). In addition, no recovery was observed (Fig. 6B).



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Fig. 6. The Na+ dependency and amiloride sensitivity of the recovery of intracellular pH (pHi) after the TG-evoked intracellular acidosis in SK-MG-1 cells. A: representative tracing shows the effect of amiloride at various concentrations on basal pHi in SK-MG-1 cells. B: representative tracings of the effect of TG in the presence and absence of extracellular sodium as well as the effect of amiloride on the recovery of pHi after TG-induced acidosis in the presence of extracellular Na+. C: summary data show the effect of amiloride on pHi without or with TG pretreatment. D: comparison of the magnitude of TG-evoked intracellular acidosis in the presence and absence of extracellular sodium. Data are the means of at least 5 separate experiments. *P < 005 compared with the appropriate control group.

 
Next, we tested the effect of pretreatment of amiloride and 5-(N,N-hexamethylene)-amiloride (HMA; a more NHE-specific agent) on the TG-induced pHi changes in SK-MG-1, U87 MG, and primary human GBM cells. We found that 100 µM amiloride and 500 nM HMA increased [H+]i significantly only in U87 MG, and primary human GBM cells, but not in SK-MG-1 cells (Fig. 8, A and B). There was no difference between the effects of these two drugs. In all three tumor cells (SK-MG-1, U87 MG, and primary human GBM cells), both amiloride and HMA abolished the recovery of the acidification evoked by TG (Fig. 7, AC). In addition, the change in [H+]i in response to TG was significantly increased by both of these agents in all investigated cell types, except in the case of amiloride in U87 MG cells (Figs. 8, C and D).



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Fig. 8. The role of the Na+/H+ exchanger in pHi recovery after TG-evoked acidification. A and B: effect of 500 nM HMA and 100 µM amiloride on cytoplasmic [H+] in different GBM cells represented as change of [H+] and of the basal pHi, respectively. The effect of TG on cytoplasmic [H+] concentration (C) and pHi(D) is shown under control conditions and in the presence of 500 nM HMA or 100 µM amiloride. Note that statistical tests were only used to compare changes in [H+] because {Delta}pH values are not comparable because pH scale is pH is measured as a logarithmic scale. Data are the means of at least 4 experiments. *P < 005 compared with the appropriate control group.

 


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Fig. 7. The role of the Na+/H+ exchanger in the pHi recovery after TG-evoked acidification. Representative traces show the effect of pretreatment of 100 µM amiloride or 500 nM 5-(N,N-hexamethylene)-amiloride (HMA) on the recovery phase, following TG-evoked acidification in SK-MG-1 (A), U87-MG (B), and human primary GBM cells (C). TG was added at 100 s, as indicated.

 
Finally, we measured [Ca2+]i and pHi simultaneously to explore the connection between these parameters. We found in all cell types that the pHi started to drop only when [Ca2+]i was already elevated (Fig. 9). The time delay between the start of the [Ca2+]i rise and the decline in pHi was markedly longer in the normal astrocytes compared with SK-MG-1 or U87-MG cells (Fig. 9).



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Fig. 9. Time course of TG-sensitive changes in pHi and [Ca2+]i in SK-MG-1, U87 MG cells, and human primary astrocytes using BCECF-AM and fura-2 AM double-loaded cells. {Delta}t represents the difference in time between the time points when [Ca2+]i and pHi started to deflect from baseline, respectively. *P < 005 compared with normal astrocytes.

 
Effect of TG and 2-APB on cell proliferation. When we tested the effect of TG and 2-APB on cell proliferation rates of five different human GBM cell lines, we found that TG slowed down the proliferation at 24 h and clearly reduced the number of viable cells after 48 h in each cell line (Fig. 10). In three of the five cell lines (U87-MG, D54, and U373-MG), 50 µM 2-APB also clearly reduced cell proliferation after 48 h. In the remaining two cell lines (SK-MG-1 and U-251), the effect of 2-APB was not as evident, although in each experimental series, 2-APB reduced the proliferation rate to a small extent. Nevertheless, 100 µM 2-APB clearly reduced proliferation rate in SK-MG-1 cells (data not shown). Vehicle alone (DMSO) did not exert any effect on cell proliferation, whereas primary human astrocytes exhibited nearly no proliferation in the 48-h time period (data not shown).



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Fig. 10. Effect of the depletion of the endoplasmic reticulum (ER) calcium content and of the SOCC on cell proliferation of GBM cells. Cell growth curves show the effect of TG and/or 2-APB on cell proliferation rates of 5 different GBM cell lines: D54 (A), U251 (B), U373 (C), SK-MG-1 (C), and U87 (D). Each point at 24 and 48 h represents the mean ± SD of 3 data.

 
Effect of TG on cell viability of SK-MG-1 cells. To further investigate the effect of TG and 2-APB on glioma cells, we used the MTT cell viability assay. This assay assesses the number of viable cells by measuring the mitochondrial-dependent reduction of water-soluble tetrazolium into insoluble formazan product. The measured absorbance is directly proportional to the number of the viable cells. This assay is more sensitive and it is quantitative compared with the trypan blue assay. First, we optimized the starting cell concentrations to obtain an appropriate working range for SK-MG-1 cells, over the 48-h drug treatment period. We found that 100 nM and 1 µM TG inhibited the reduction of the MTT reagent, consistent with a decrease in cell viability under these conditions. In addition, 50 µM 2-APB also reduced the number of viable cells, while increasing the effect of TG (Fig. 11, B and C). At a concentration of 100 µM, 2-APB almost completely abolished the MTT reduction rate in the presence or absence of TG (Fig. 11, B and C).



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Fig. 11. Effect of TG and 2-APB on cell proliferation of SK-MG-1 cells. With the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. A: dose-dependent effect of TG on cell proliferation rate at different cell-plating densities. B and C: effect of 2-APB pretreatment (50 and 100 µM concentrations) shown on the TG-evoked inhibition of cell proliferation of the SK-MG-1 cells at 2,500 and 5,000 seeded cell number, respectively. Each bar represents the mean ± SD of 3 separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is one of the major regulators of intracellular Ca2+ homeostasis. It helps to keep resting cytosolic Ca2+ at a low level, and it maintains the high resting luminal [Ca2+] in the ER. Several agonists increase cytosolic Ca2+ either by release of Ca2+ from intracellular stores via the inositol 1,4,5-trisphosphate receptor or ryanodine receptor and/or by increasing Ca2+ influx through the plasma membrane. SERCA is one of the three known major Ca2+ extrusion mechanisms. Besides the Ca2+-ATPase in the plasma membrane and the plasmalemmal Na+/Ca2+ exchanger, SERCA is crucial to the maintenance of the low cytosolic [Ca2+] and the ER Ca2+ store. A combination of SERCA activity and basal Ca2+ leakage from the ER determines the magnitude of the ER Ca2+ store. The level of the ER Ca2+ store is also the primary regulatory signal for store-operated Ca2+ channel (SOCC) in the plasma membrane. If ER [Ca2+] decreases, the SOCC will be activated and it will increase cytosolic Ca2+, which will in turn be pumped into the ER by SERCA. In nonexcitable cells, SOCC is the major calcium entry pathway. Because cytosolic and ER Ca2+ are crucial regulators of such essential cell functions as apoptosis, cell cycle control, and migration, a permanent disturbance in cellular Ca2+ homeostasis could initiate or promote tumor formation. Consequently, we tested whether the blockade of SERCA activity by the selective inhibitor TG (35) revealed any differences in the subsequent Ca2+ response between glioblastoma cells and human primary astrocytes.

We found that the resting cytosolic [Ca2+] was markedly higher in the glioblastoma cell lines but not in primary GBM cells compared with normal astrocytes. In nominally Ca2+-free buffer, the [Ca2+]i decreased only in human primary astrocytes and SK-MG-1 cells. 2-APB, a SOCC blocker, at least transiently elevated [Ca2+]i in all cell types. In addition, basal Ca2+ entry was significantly higher in all five investigated glioblastoma cell lines, although not in human primary GBM cells, and it was almost completely abolished by 2-APB in every cell type. The resting [Ca2+] has been reported to be even higher in other glioblastoma cell lines, such as U87-MG, 1321N1, and U373-MG (7, 16, 22). These results suggest that glioblastoma cells tend to have higher resting [Ca2+]i because the activity of the SOCC (and/or other 2-APB sensitive calcium entry pathways) is much higher than in normal astrocytes. Ca2+ entry into cells can also be modulated by cell membrane potential; however, the slightly depolarized membrane potential of glioblastoma cells would be predicted to reduce Ca2+ entry via the SOCC.

When SERCA activity was blocked with TG, the intracellular Ca2+ increased to a substantially higher level in SK-MG-1, U87 MG, and human primary GBM cells compared with normal astrocytes. This increase was diminished if extracellular Ca2+ was removed, and it was almost completely abolished in the presence of 2-APB in all cell types. It should be noticed that even with 2-APB pretreatment, the elevation of intracellular Ca2+ in response to TG was bigger in the tumor cells, suggesting that more Ca2+ is sequestered in the ER in the glioblastoma cells than in normal astrocytes. With the use of the Mn2+ quenching assay, we also observed a much larger 2-APB-sensitive increase in plasmalemmal Ca2+ entry in response to TG in the glioblastoma cells than in normal astrocytes. This, together with the Ca2+ data, confirmed that SOCC is activated by TG to a much greater extent in glioblastoma cells compared with normal astrocytes. There are, at least three possible explanations for this finding. First, there could be a large TG-resistant ER Ca2+ pool in normal astrocytes, and, consequently, SOCC would not be fully activated by TG. However, one recent study (37) has shown that the TG-resistant pool in U87 MG cells was much larger than that in both U251 MG glioblastoma cells and normal rat astrocytes. In our study, both U87 MG and U251 MG cells responded to TG with a large increase in plasma membrane Ca2+ influx, suggesting that the relative size of the TG-resistant pool cannot account for the increase in Ca2+ entry observed in tumor cells. Because the SOCC was significantly more activated even in U251 cells, this option is not very likely. A second possibility is that the Ca2+ level in the ER is higher in the GBM cells compared with normal astrocytes. This idea is supported by the data showing that in the presence of 2-APB or in nominally Ca2+-free buffer, the TG-induced increase in [Ca2+]i is higher in GBM cells than in normal primary astrocytes. Third, the activity and/or the number of SOCCs might be increased in tumor cell lines as opposed to normal astrocytes. This hypothesis is supported by our finding of a larger basal plasmalemmal Ca2+ entry in these cells and that the TG-induced changes in Ca2+ influx and cytosolic [Ca2+] were abolished with 2-APB. However, this might not be the case in human primary GBM cells. SOCC activity also seems to be related to the proliferative ability of the cell. In proliferating human pulmonary arterial myocytes, SOCC activity, and the transient receptor potential channel isoform 1 Ca2+ channel was reported to be upregulated compared with growth-arrested cells (11, 31). A similar study showed that upregulation of transient receptor potential channel isoform 6 and enhancement of SOCC activity were involved in PDGF-stimulated proliferation of pulmonary myocytes (39). In addition, intracellular alkalization was found to increase SOCC activity, whereas intracellular acidification exerted the opposite effect in platelets (10). Finally, an as-yet unidentified signal for the activation of the SOCC could be enhanced.

Our proliferation and viability assays showed that 2-APB reduced the number of viable glioblastoma cells. This effect of 2-APB is probably due to inhibition of the activity of SOCC, but other cellular effects, such as inhibition of inositol 1,4,5-trisphosphate receptors, the mitochondrial Na+/Ca2+ exchanger or SERCA activity, as well as enhancement of the nonspecific leak of Ca2+ from the ER pools cannot be excluded. Although these effects were observed mostly using higher concentrations of 2-APB (>100 µM) (13, 21), we found that even 50 µM 2-APB caused a transient Ca2+ increase and intracellular acidification. One explanation for these observations is that protonated 2-APB enters cells and increases the passive Ca2+ leakage from the ER as has been suggested by others (1, 3). In addition, we have also observed that although the depletion of ER Ca2+ stores triggered programmed cell death in glioblastoma cells, activation of SOCC was not required. In fact, our results show that activation of the SOCC reduced the effect of ER store depletion on viability. This phenomenon has been previously observed in androgen-sensitive prostate cancer cells, where inhibition of SOCCs facilitated TG-induced apoptosis (29). In these cells, SERCA 2B was also involved in the regulation of cell growth (17).

The clinical relevance of enhanced SOCC activity in tumors has recently been reported. A beneficial effect of NSAIDs in reducing the initiation and the proliferation of colorectal tumors has been reported by several clinical trials and epidemiological investigations (33, 34). Inhibition of store-operated calcium influx seems to play a role in the antiproliferative effect of NSAIDs (38). In addition, myeloid leukemia cells have also been shown to exhibit a high sensitivity to SOCC blockade (30). Our finding that the viability of glioblastoma cells is significantly affected by the activity of the SOCC, suggests that the inhibition of SOCC might be useful for future therapeutic approaches.

We also found that the resting pHi was significantly higher in glioblastoma cells using a CO2/HCO-free buffer, a finding that is consistent with previous observations (14, 20). However, unlike the tumor cell lines, the pHi of primary GBM cells was very similar to the values obtained by NMR measurements in human patients. It is well known that intracellular alkalization promotes proliferation compared with acidification, which plays a role in apoptosis. Although we were able to reduce pHi either with 100 µM amiloride or 500 nM HMA in human primary GBM and in U87 MG cells, neither agent had any significant effect on SK-MG-1 cells. Furthermore, removal of extracellular Na+ in SK-MG-1 cells did not reduce pHi. In addition, in U87 MG cells, pHi did not drop to the level found in primary astrocytes after administration of amiloride or HMA. Similarly, in primary GBM cells, pHi remained slightly higher compared with normal astrocytes. These data suggest that in U87 MG and primary GBM cells, another Na+- and HCO-independent H+ extrusion mechanism in addition to NHE could be responsible for the alkaline resting pHi. In SK-MG-1 cells, the activity or the number of NHE transporters under control conditions might also be reduced.

The TG-induced pHi change is a cascade of consecutive events. Data from our double-loading experiments, together with the data from pHi measurements showing the dependence of the intracellular acidification on extracellular Ca2+ and Ca2+ influx via SOCC, clearly show that in both cell types, the primary response was elevation of [Ca2+]i, followed by intracellular acidification. In addition, in primary astrocytes, in which the Ca2+ response was clearly slower and smaller, the time interval between the start of the Ca2+ response and the start of the pHi response was substantially longer. We also found that intracellular acidification was only transient in tumor cells, whereas the recovery phase was clearly sensitive to amiloride and HMA. Furthermore, when NHE activity was not blocked, the change in intracellular proton concentration was the same that we observed in tumor cells. However, removal of extracellular Na+ in SK-MG-1 cells or inhibition of NHE in every investigated tumor cell not only abolished the recovery phase, but also revealed that the elevation of the intracellular proton concentration is markedly increased in response to TG in tumor cells. These findings suggest that a relatively small increase in intracellular Ca2+ is required to activate H+ entry (which could be reached with TG even in normal astrocytes), and that this proton entry is proportional to the elevation in [Ca2+]i. These observations confirm that NHE in the plasma membrane is activated in glioblastoma cells to recover the pHi. The possible inhibition of epithelial Na+ channels (ENaCs) by amiloride cannot be the explanation for the pHi recovery, because ENaC inhibition would result in a decrease in [Na+]i, followed by accelerated recovery due to enhanced NHE activity. In addition, 1 µM amiloride, which is an effective inhibitory concentration for ENaC but not for NHE, had no effect on basal pHi or on the recovery phase after TG-induced acidification. This finding is consistent with the results published by McLean et al. (20), namely, that in glioblastomas, NHE activity is much more predominant in the regulation of pHi than in primary astrocytes, which was found to rely on HCO-dependent pHi regulatory mechanisms.

In summary, the resting [Ca2+]i and basal Ca2+ influx were found to be increased in gliobastoma cell lines compared with human primary astrocytes. In addition, the ER Ca2+ store was found to be increased in both GBM cell lines and in primary GBM cells. Furthermore, in the tumor cell lines and primary tumor cells, depletion of the ER Ca2+ store with TG evoked a much greater increase in SOCC activity than did the same maneuver in normal human astrocytes. pHi under normal conditions was alkaline in all tumor cells. However, TG evoked a larger increase in intracellular proton concentration in tumor cells, which was masked by the activation of NHE in the plasma membrane. Our study has revealed fundamental differences in intracellular Ca2+ homeostasis and confirmed the previously observed changes in pHi regulation in glioblastoma cells. Further studies are required to elucidate the underlying molecular differences to develop therapeutic approaches targeting Ca2+ regulatory components of Ca2+ homeostasis in glioblastoma cells.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This study was supported by National Institutes of Health Grant CA-101952, National Institute of Diabetes and Digestive and Kidney Diseases Grants 3202 and P50CA97247, and Hungarian Scientific Research Fund T037524.


    ACKNOWLEDGMENTS
 
The authors thank Melissa McCarthy (Department of Physiology and Biophysics) and Cathy Langford (Division of Neurosurgery) for excellent cell culture assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Fuller, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, Birmingham, AL 35294 (e-mail: fuller{at}physiology.uab.edu)

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.


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