Malignant gliomas display altered pH regulation by NHE1 compared
with nontransformed astrocytes
Lee Anne
McLean1,
Jane
Roscoe2,
Nanna K.
Jørgensen1,
Fredric A.
Gorin2, and
Peter M.
Cala1
Departments of 1 Human Physiology and
2 Neurology, School of Medicine, University
of California, Davis, California 95616
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ABSTRACT |
Malignant
gliomas exhibit alkaline intracellular pH (pHi) and acidic
extracellular pH (pHe) compared with nontransformed
astrocytes, despite increased metabolic H+ production. The
acidic pHe limits the availability of
HCO
3, thereby reducing both passive
and dynamic HCO
3-dependent buffering.
This implies that gliomas are dependent upon dynamic HCO
3-independent H+
buffering pathways such as the type 1 Na+/H+
exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pHi
(pHi = 7.31-7.48) than normal astrocytes
(pHi = 6.98), and increased rates of recovery from
acidification, under nominally
CO2/HCO
3-free conditions.
Inhibition of NHE1 in the absence of
CO2/HCO
3 resulted in
pronounced acidification of gliomas, whereas normal astrocytes were
unaffected. When suspended in
CO2/HCO
3 medium astrocyte
pHi increased, yet glioma pHi unexpectedly
acidified, suggesting the presence of an
HCO
3-dependent acid loading
pathway. Nucleotide sequencing of NHE1 cDNA from the gliomas
demonstrated that genetic alterations were not responsible for this
altered NHE1 function. The data suggest that NHE1 activity is
significantly elevated in gliomas and may provide a useful target for
the development of tumor-selective therapies.
sodium/hydrogen antiport; tumor pH; astrocytomas; glioblastomas
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INTRODUCTION |
HIGHLY PROLIFERATING CANCER cells, such as malignant
gliomas, were first shown to generate increased levels of lactic acid by Warburg nearly 70 years ago (59). These tumors exhibit increased glucose uptake and hexokinase activity that increases glucose flux
through the glycolytic pathway (11, 33). Furthermore, tumor cells
tolerate the hypoxia associated with poor vascularization by
facilitated anaerobic glycolysis that is associated with an upregulation of membrane-bound lactate dehydrogenase (17).
Intracellular H+, increased as a result of lactic acid
dissociation, is actively transported out of the cell with resultant
acidification of the extracellular space. Microelectrode measurements
demonstrate average extracellular pH (pHe) values ranging
from 6.5-6.9 for tumors and values of 7.0-7.5 for normal
cells, with an average difference of ~0.5 pH units (19, 55, 60).
Despite their increased H+ production and the acidic
extracellular milieu, 31P-NMR spectroscopic studies of
gliomas have reported that the intracellular pH (pHi)
measured in situ is more alkaline (pH 7.12-7.24) than that of
normal brain (pH 6.99-7.05) (24, 45). This phenomenon of high
pHi with low pHe has been reported for many
types of transformed cells (49, 55, 60). Maintenance of an alkaline
pHi has been shown to be necessary for various mechanisms
involved in cellular proliferation because a number of intracellular
metabolic enzymes have alkaline pH optima. An important example is that
of phosphofructokinase (PFK), the rate limiting step of glycolysis. PFK
has a pH optimum in the range of 7.2 with decreasing activity as pH
becomes more acidic (14). Other key components of cellular
proliferation, such as protein, RNA, and DNA synthesis, are also
enhanced at alkaline pHi and inhibited under acidic
conditions (27). Thus it appears to be a general rule that higher
pHi corresponds with increased metabolic activity and cell proliferation.
To maintain an optimal pHi for these metabolic processes,
glial tumors must possess an efficient means of removing the excess H+ produced as a result of their increased
metabolism. Several of the dynamic pH regulatory mechanisms that have
been identified in astrocytes to date are illustrated in Fig.
1. These include the type 1 Na+/H+ exchanger (NHE1), the electrogenic
Na+-HCO
3 cotransporter,
the Na+-dependent and Na+-independent
Cl
/HCO
3 exchangers,
H+-lactate symport, and the vacuolar-type
H+-ATPase (see Fig. 1 legend for references). The acidic
extracellular environment of these malignant gliomas reduces external
HCO
3 concentrations so that there is
diminished H+ buffering provided by dynamic
HCO
3-dependent transporters and
reduced fixed buffer capacity provided by the CO2/HCO
3 system. Thus
whereas the glial tumor cells are highly reliant on dynamic,
HCO
3-independent H+
buffering mechanisms such as NHE1, normal astrocytes would have significant buffering capacity from
HCO
3-dependent pathways.

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Fig. 1.
Cultured mammalian astrocytes express a number of pH regulatory
mechanisms. Acid extrusion mechanisms include: 1,
Na+/H+ exchange (2, 4, 6, 32, 38, 48);
2, electrogenic
Na+-HCO 3 cotransport (1,
6, 48); 3, Na+-dependent
Cl /HCO 3 exchange
(48); 5, H+-lactate cotransport (57, 58); and
6, vacuolar-type H+-ATPase (36, 56). In addition,
an Na+-independent
Cl /HCO 3 exchanger
(4) has been shown to acidify astrocytes in response to
alkalinization (48).
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The present study evaluates the role of the
Na+/H+ exchanger in pH regulation of glioma
cells. Four different glioma cell lines exhibit a pHi that
is significantly elevated (0.3-0.5 pH units) above that of normal
astrocytes, under nominally
CO2/HCO
3-free conditions.
We demonstrate that this increased pHi is the result of
active acid extrusion by the type 1 Na+/H+
exchanger. In the presence of
CO2/HCO
3, astrocytes attain a slightly more alkaline pHi compared with that in
HEPES-buffered media whereas gliomas acidify. This is most likely due
to activation of the Na+-independent
Cl
/HCO
3 exchanger
in the gliomas that functions as an acid loading pathway in bicarbonate
media (Fig. 1). However, because the in situ external
HCO
3 concentration for the gliomas is
predicted to be minimal, the gliomas are primarily reliant on NHE1 for
regulation of pHi. The elevated steady-state pHi of these glial tumors is not the result of genetic
alterations of the NHE1 gene, because sequencing of NHE1 from each of
the gliomas studied revealed no differences compared with that
published for normal human NHE1. Furthermore, neither transcript nor
protein levels correlated with the observed values of pHi
in these cell lines. In conclusion, we have found that the alkaline
steady-state pHi measured in glioma cell lines is highly
dependent upon increased activity of NHE1.
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METHODS |
Established human glioma cell lines (U-118 and U-87) and the rat C6
glioma cell line were obtained from the American Tissue Culture
Collection (Rockville, MD). The U-251 human glioma cell line was
obtained from D. Littman with permission from J. Pontén (40).
Glioma cells were maintained in DMEM (Cellgro) supplemented with 5%
fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 mg/ml
streptomycin (Cellgro) at 37°C, 95% humidity, and 5% CO2. Primary cultures of rat astrocytes were isolated from
the cerebral cortices of neonatal rats (0-1 day old) as described by Hertz et al. (22). Primary cultures were maintained in Eagle's MEM
(GIBCO) supplemented with 10% fetal bovine serum (Hyclone), 1×
antibiotic/antimycotic, 1× vitamins, and 1× amino acids
(GIBCO) at 37°C, 95% humidity, and 5% CO2. Samples of
primary rat astrocytic cultures were routinely stained with an antibody
against glial fibrillary acidic protein (GFAP), with 90-95% of
cells demonstrating GFAP immunoreactivity. For measurements of
pHi, cells were grown on glass coverslips coated with rat
tail collagen type I (Collaborative Biomedical Products).
Measurement of pHi.
pHi was measured using the fluorescent ratio dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM
(BCECF-AM; Molecular Probes), as previously described (30). Cells on
coverslips were loaded with 0.5-1.5 µM of BCECF-AM in
HEPES-buffered Ringer (HR) solution for 30 min at 37°C, 0%
CO2. Coverslips were washed three times in HR, incubated in
HR for 30 min at 37°C, 0% CO2, and then transferred to
polystyrene cuvettes that permitted continuous perfusion of solution.
Cells were maintained at 37°C in the spectrophotometer (F-2000,
Hitachi Instruments) and BCECF fluorescence was measured at an emission
wavelength of 535 nm, using optimized excitation wavelengths of 507 and
440 nm. Autofluorescence, in the absence of dye, was measured from a
coverslip of cells grown in the same dish during perfusion with each of
the solutions utilized during the experiment. Particular care was taken
to correct for background at the pH-insensitive wavelength (440 nm) due
to considerable fluorescence emitted by amiloride and DIDS at this
wavelength. Calibration of BCECF was performed using
high-K+ solutions of known pHe in conjunction
with 10 µM nigericin (Sigma). Complete calibration curves were
constructed over the pH range of 6.2-8.2, with the
F507/F440 ratio normalized to the ratio
measured at either pH 7.0 (astrocytes) or 7.4 (gliomas). A single
calibration point was then measured at the end of each experiment as
described by Boyarsky et al. (3).
Determination of buffer capacity.
The total intracellular buffer capacity (
T) equals the
sum of the intrinsic buffer capacity (
i) and the
buffering provided by
CO2/HCO
3
(
HCO
3). The
i was determined over the investigated range of
pHi studied by perfusing cells with progressively
decreasing concentrations of NH+4-HR, such
that
i =
NH+4/
pHi (3). These
solutions were nominally HCO
3-free and
Na+-free to inhibit dynamic acid extrusion mechanisms.
Buffering by CO2/HCO
3 was
determined as described by Roos and Boron (42), where
HCO
3 = 2.3 × [HCO
3]i, and
[HCO
3]i = S × PCO2 × 10(pHi - pK), with S = 0.0314 (solubility of CO2 in cell water),
PCO2 = 40 mmHg, and pK = 6.12.
Solutions.
The standard HR was composed of the following, in mM: 125 NaCl, 5.5 KCl, 24 HEPES, 1 MgCl2, 0.5 CaCl2, 5 glucose,
and 10 NaOH, adjusted to pH 7.4 at 37°C with NaOH or HCl.
Bicarbonate-buffered Ringer solution contained the following, in mM:
111 NaCl, 5.5 KCl, 24 NaHCO3, 1 MgCl2, 0.5 CaCl2, and 5 glucose, maintained at pH 7.4 at 37°C by
continuous bubbling with humidified 5% CO2-95% air.
High-K+ calibration solutions contained, in mM: 130 KCl, 24 HEPES, 1 MgCl2, 0.5 CaCl2, 10 N-methyl-D-glucamine (NMDG)-Cl, and 5 glucose, using NMDG-OH or HCl to adjust pH. For Na+-free solutions,
Na+ was substituted with an equimolar amount of the
impermeant cation. In
NH3/NH+4-containing solutions,
NaCl was replaced with NH4Cl at a ratio of 1:2
milliequivalents. In Cl
-free
CO2/HCO
3-buffered
media, Cl
was replaced by gluconate, and total
calcium was increased to achieve free Ca2+ of 0.5 mM, as
measured with an ion-selective electrode. Solutions for astrocytes and
high-grade gliomas were identical with the exception of an additional
5.5 mM glucose added to solutions for cultured astrocytes. Amiloride
(Sigma) was dissolved in DMSO and HOE-694 (a generous gift from H. Lang, Hoechst AG, Germany) dissolved in distilled H2O to
stock concentrations of 500 mM and 20 mM, respectively, and diluted to
final concentrations in solution as indicated. DIDS (Molecular Probes)
was dissolved directly into final solutions at a concentration of 500 µM.
Sequencing of NHE transcripts in gliomas.
Total RNA was isolated from nearly confluent U-87, U-118, and U-251
human glioblastoma cell lines (8). cDNA was generated from 3 µg of U-251 total RNA, and 1 µg of U-87 and U-118
poly(A)+ RNA using Superscript II (GIBCO) according to
manufacturer's instructions, then diluted to 1 ml final volume with
Tris-EDTA (TE, pH 8.0). Primers (F1 and R1) were added to an aliquot of the reaction mix (0.25%) to amplify a transmembrane spanning region (amino acids 230-399 of human NHE1) that is highly conserved among all of the mammalian NHE isoforms (see Table
1). Adjacent regions of NHE that are
5' and 3' to this transmembrane domain were also amplified
using primer pairs F2, R2, and F3, R3, respectively (Table 1). These
cDNA regions were subcloned into either pUC 18/19 or Bluescript SK+
vectors, and double-strand sequencing was performed either manually or
by using an automated fluorescent dideoxynucleotide sequencer (Applied
Biosystem, Gene Sequencing Lab, University of California, Davis, CA).
Nucleotide and deduced amino acid sequences were compared with
sequences obtained from GenBank and with other published NHE sequences
(16, 47, 50).
RNA blots.
Poly(A)+ mRNA was isolated from primary rat astrocytes, C6
rat glioma, and three human glioma cell lines (U-87, U-118, and U-251)
at or near confluence using oligo(dT) cellulose (46). RNA concentration
was determined spectrophotometrically and total RNA (5 µg) was
size-fractionated electrophoretically through a 1.2% agarose gel
containing 2.2 M formaldehyde (29). RNA was transferred by capillary
action to a Duralon-ultraviolet nylon membrane (Stratagene) in
20× SSC (0.3 M sodium citrate, 3 M NaCl, pH 7.0).
The membrane was baked in a vacuum oven at 80°C for 30 min and then
cross-linked by ultraviolet irradiation (Stratalinker 1800, Stratagene).
Hybridization conditions for RNA blot analyses.
Purified DNA probes were radiolabeled with 32P-dCTP using
random priming (RTS RadPrime DNA labeling system, GIBCO) and
unincorporated nucleotides removed by passage over a G-50 Sephadex
column. The specific activities of the DNA probes were between 1 and 3 × 109 cpm/µg DNA. Blots were first hybridized with
a 0.5-kb probe that contained the highly conserved transmembrane
spanning region encompassing amino acids 230-399 of human NHE1
using high stringency hybridization and moderately stringent wash
conditions (29). Briefly, RNA blots were prehybridized and hybridized
at 42°C in 40% (vol/vol) formamide, 5× Denhardt's reagent,
6× SSPE (0.9 M NaCl, 50 mM NaHPO4, 5 mM EDTA, pH
7.4), and 0.5% SDS. Blots were washed for 15 min at 25°C in
2× SSC, and for an additional 15 min at 42°C in 0.2× SSC, 0.2% SDS.
Quantitation of steady-state transcript levels.
Normalization of RNA blots was performed using a 1.5-kb human
-actin
probe labeled with 32P-dATP under similar conditions as for
NHE1. Ratios of NHE1 to
-actin signal were used to quantitatively
compare relative amounts of mRNA between normal rat astrocytes and rat
C6 gliomas, and among the human tumor cell lines. There was no attempt
to quantitatively compare mRNA levels between rat and human tissues.
Autoradiographic bands were digitized from the RNA blots using a
PhosphorImager and ImageQuant software (Molecular Dynamics). Digitized
autoradiographs were imported into Adobe Photoshop 4.0 for sizing and
labeling without manipulation of relative band intensities (Adobe).
Immunoblots.
The membrane-enriched protein fractions of all cell lines were isolated
as follows. Cells were rinsed twice in PBS, then scraped from the dish
in a rinse solution composed of 1× PBS, 2 mM EDTA (pH 8.0), 2 mM
phenylmethylsulfonyl fluoride, 4.2 µM leupeptin, 1.5 µM pepstatin
A, and 2 mM
-mercaptoethanol (Sigma). Large debris was reduced by
passing through a 23-gauge needle and the cell suspensions were
centrifuged at 4,000 g for 10 min at 4°C. The pelleted
cells were resuspended in 1 ml rinse solution and lysed by sonication
for 1 min while on ice. The lysates were then centrifuged at 12,000 g for 10 min at 4°C to remove large cytosolic debris. The
supernatants were subjected to ultracentrifugation at 150,000 g
for 45 min at 4°C, and the resulting pellets were resuspended in
50-100 µl of rinse solution plus 0.1% SDS. Protein concentration was determined by the Bradford method. This protein fraction (50 µg) was loaded on a 7.5% polyacrylamide-SDS gel, size-fractionated by electrophoresis, and transferred to a
nitrocellulose membrane. Blots were put in blocking buffer (1×
PBS, 0.5% Tween 20, 3% nonfat dry milk) for 1 h, then incubated for 1 h with a 1:2,000 dilution of a rabbit polyclonal antibody to actin
(Sigma). Immunoblots were rinsed three times in wash buffer (1×
PBS, 0.5% Tween 20), then incubated at 4°C for 12-16 h with a
mouse monoclonal antibody to NHE1 (1:2,000 dilution) (10). Secondary
antibodies were incubated sequentially for 1 h each with horseradish
peroxidase (HRP)-conjugated goat anti-rabbit antibody at 1:4,000
followed by HRP-conjugated goat anti-mouse antibody at 1:20,000, with
three 5 min rinses in wash buffer before and after each secondary
antibody incubation.
Antibody complexes were visualized by enhanced chemiluminescence using
X-ray film after incubation with Super Signal Ultra (Pierce). Films
were scanned by a Molecular Dynamics laser densitometer for
quantification. The relative amounts of NHE1 protein were normalized to
actin for each sample. These ratios of NHE1 to actin were used to
compare relative amounts of NHE1 between normal rat astrocytes and rat
C6 glioma cells, with a separate comparison of NHE1 protein levels
compared among the human tumor cell lines.
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RESULTS |
Steady-state pHi and buffer capacity in the absence of
CO2/HCO
3.
Steady-state pHi was determined in the absence of
CO2/HCO
3 for primary
cultures of cortical rat astrocytes and four established glioma cell
lines, three of human origin (U-118, U-87, and U-251) and one rat (C6)
cell line (Fig. 2). The primary astrocytes
maintained an average steady-state pHi of 6.98 ± 0.01 in
HEPES-buffered media, similar to previous reports for cultured
astrocytes (4, 6, 12, 38). In contrast, the rat and human glioma cell
lines exhibited significantly more alkaline steady-state
pHi than astrocytes, with values ranging from 7.31 ± 0.02 for U-87 gliomas to 7.48 ± 0.01 for the U-118 glioma cell line. Our
measured pHi of 7.38 ± 0.02 for the C6 glioma cell line
was nearly identical to that reported by Shrode and Putnam under
similar conditions (48).

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Fig. 2.
Steady-state intracellular pH (pHi) of cultured cortical
astrocytes and several established glioma cell lines. pHi
was measured using fluorescent ratio dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM in
HEPES-buffered media under nominally
HCO 3-free conditions. Values are means ± SE. Number of individual experiments is in parentheses.
* Significant difference compared with astrocytes (P < 0.001), as determined by Student's t-test.
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Values of
i were determined for the primary astrocytes
and two of the glioma cell lines (42). An example of a typical buffer capacity experiment, performed on primary astrocytes, is illustrated in
Fig. 3A. Cells were progressively
perfused with decreasing concentrations of
NH3/NH+4-containing solution, and
the change in pHi measured for each change in
[NH+4]i was
determined. These experiments were performed in Na+- and
HCO
3-free media to prevent active acid extrusion. The
[NH+4]i was
calculated based on the observed
pHi that occurred as a
result of changing the external [NH4Cl]. The
value of [NH+4]i was
determined from [NH3]i, pK,
and pH, with [NH3]i assumed to be
equal to [NH3]o. It was further
assumed that
[NH+4]i was
equivalent to
[H+]i, with
resulting
i =
[H+]i/
pHi.
i was found to vary with pHi in a linear
fashion (Fig. 3B), similar to values previously reported for
cultured astrocytes (2). A similar linear relationship between buffer
capacity and pH was observed for both the gliomas and the
nontransformed astrocytes. However, the higher steady-state
pHi of the glioma cells places them at a lower point on the
buffer capacity curve (Fig. 3B, inset), accounting for
their diminished buffer capacity compared with normal astrocytes.

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Fig. 3.
Intrinsic buffer capacity ( i) for cultured astrocytes
and glioma cell lines is dependent on pHi. A:
example of experimental protocol used to determine i for
cultured astrocytes. Cells were sequentially perfused with decreasing
concentrations of NH+4 in
Na+-free, HEPES-buffered media and change in
pHi was determined for each step change in
NH+4. B: i plotted as
a function of pHi for cultured astrocytes and two glioma
cell lines (U-118 and U-87). Although similar trend is seen for each of
the cell types, it is important to note that i at
steady-state (inset) is significantly lower in gliomas
due to their elevated pHi.
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Response to acidification in the absence of
CO2/HCO
3.
To study active pH regulation, cells were acidified using the
NH+4-prepulse method (42) and recovery from the acid load was evaluated. H+ flux rates
(JH+: mM
H+/min) were determined by multiplying
i by
pHi/
t measured during the initial linear portion of
recovery (Fig. 4). U-118 gliomas displayed
rapid recovery from the acid load (Fig. 4A) that was dependent
on external Na+ and inhibited by amiloride, an NHE1
inhibitor, in a dose-dependent manner. Primary astrocytes also
recovered from acidification in a Na+-dependent,
amiloride-sensitive manner (Fig. 4B), although recovery rates
for astrocytes were significantly lower than those of the gliomas. A
plot of JH+ as
a function of pHi for astrocytes and U-118 gliomas, under
nominally CO2/HCO
3-free conditions, is displayed in Fig. 5. It is
clear that at any given pHi, the glioma cells display
significantly greater flux rates in response to acidification than do
the nontransformed astrocytes. Furthermore, the glioma cells have an
apparent steady-state pHi (point where net
JH+ approaches
0) that is ~0.4 pH units more alkaline than that of normal
astrocytes. This is consistent with the measured steady-state
pHi values (see Fig. 2) and indicates the principal role
played by the Na+/H+ exchanger in nominally
CO2/HCO
3-free media.

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Fig. 4.
Acidification of (A) U-118 gliomas and (B) cultured
astrocytes by ammonium prepulse in nominally
HCO 3-free media. Cells were perfused
with 10 mM NH4-HEPES-buffered Ringer solution (HR) for 5 min, followed by washout in NH4-free and
Na+-free media. Recovery from acidification was dependent
on presence of external Na+ and was inhibited by amiloride
(Amil) in a reversible, dose-dependent manner. Maximum rates of
H+ efflux were determined during the initial linear portion
of recovery.
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Fig. 5.
Comparison of H+ efflux rates for U-118 gliomas and
cultured astrocytes under nominally
CO2/HCO 3-free conditions.
Maximal rates of flux
(JH+; mM
H+ per min) were determined during the initial linear
portion of recovery from an acid load and plotted as a function of
pHi. Glial tumor cells exhibit more robust H+
efflux at any given pHi compared with astrocytes and
approach a more alkaline pHi at steady state.
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Dose-response experiments with established NHE1 inhibitors were
performed to confirm that NHE1 was responsible for this
Na+-dependent pH regulation. Dose-response curves for both
primary astrocytes and U-118 gliomas (Fig.
6) were constructed from data collected
during experiments such as those shown in Fig. 4. The maximum rate of
recovery after acidification was determined in the presence and absence
of varying concentrations of inhibitors. Calculated IC50
values were 6.6 and 16.6 µM for astrocytes and U-118 gliomas,
respectively. These values are slightly higher than those reported for
rat (1.6 µM) and human (3 µM) NHE1 (9, 35). However, the studies
cited were performed in the nominal absence of external
Na+, which is a known competitive inhibitor of amiloride,
whereas our studies were performed using physiological concentrations (135 mM) of external Na+. Previous work from our laboratory
demonstrated that the IC50 for amiloride in physiologic
Na+-containing media increased approximately fivefold
compared with that in nominally Na+-free media (30). With
the use of the more potent and specific inhibitor for the
NHE1 isoform, HOE-694, a lower IC50 value (0.34 µM) was
measured in studies of U-118 gliomas (Fig. 6B), consistent with
other reports on this compound (9). These pharmacological data provide
further evidence that the Na+/H+ exchanger
involved in pH regulation in these gliomas is the NHE1 isoform.

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Fig. 6.
Recovery from acidification in nominally
HCO 3-free media can be blocked by
inhibitors of type 1 Na+/H+ exchanger (NHE1).
A: amiloride dose-response curves for cultured astrocytes and U-118
gliomas. Maximal rates of H+ efflux
(JH+; mM
H+ per min) following an acid load were determined over
increasing concentrations of amiloride. B: comparison of
dose-response curves for amiloride and HOE-694 for U-118 gliomas. Data
were normalized as a percentage of maximal
JH+ in absence of
inhibitor.
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Acidification due to removal of external
Na+ or inhibition of NHE1.
In the absence of
CO2/HCO
3, regulation of
glial cell pHi by
HCO
3-dependent pathways (Fig. 1) would
be expected to be minimal because the apparent Km
for HCO
3 is in the range of 6-10
mM (5, 7). If NHE1 is the predominant pH regulatory pathway under these
conditions, then inhibition of NHE1 will result in intracellular
acidification. Furthermore, the rate of acidification would reflect the
metabolic rate of H+ production. U-118 cells exhibited
rapid, sustained acidification when net Na+/H+
exchange was inhibited by suspension in Na+-free media
(Fig. 7A), which was completely
reversed upon reintroduction of Na+ to the perfusate.
Addition of 1 mM amiloride to Na+-free media did not alter
this response to any significant degree, indicating that the
acidification in Na+-free media is not due to the exchanger
running in the reverse direction (data not shown). As expected, the
U-118 gliomas also displayed rapid, reversible acidification when
exposed to 1 mM amiloride in the presence of external Na+
(Fig. 7C).

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Fig. 7.
U-118 gliomas and astrocytes respond differently to removal of external
Na+ or inhibition of NHE1 under nominally
HCO 3-free conditions. A: U-118
gliomas exhibit rapid, sustained acidification upon Na+
removal, which is completely reversed upon reintroduction of
Na+ to perfusate. B: in contrast, astrocytes
behaved irregularly in absence of external Na+. Although
removal of external Na+ generally caused an initial slight
acidification, prolonged exposure to Na+-free media often
resulted in a gradual alkalinization, as shown here. Inset
shows typical time scale shown by other investigators who report
acidification upon removal of Na+ from media (2, 4).
C: exposure to 1 mM amiloride significantly acidifies U-118
gliomas, whereas normal astrocytes (D) were relatively
unaffected by NHE1 inhibition.
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The response of the cultured astrocytes to Na+-free
HEPES-buffered media differed from that of the gliomas (Fig.
7B). The astrocytes exhibited an initial, slight acidification
that lasted for several minutes (Fig. 7B, inset),
similar to reports by other investigators (2, 48). However, the
majority of the astrocytes in our study gradually alkalinized
back to, or above, the initial steady-state pHi if
maintained in HEPES-buffered Na+-free media for a prolonged
period. In addition, exposure to 1 mM amiloride produced only minimal
acidification of the primary astrocytes (Fig. 7D), consistent
with the results in Na+-free media. Taken together, these
data suggest that U-118 gliomas rely primarily on the
Na+/H+ exchanger to maintain their alkaline
pHi set point, whereas nontransformed astrocytes are able
to regulate pHi even in the absence of NHE activity, under
nominally CO2/HCO
3-free conditions.
Effect of
CO2/HCO
3 on
pHi.
Having established the differences in pH regulation in the
CO2/HCO
3-free media, the
next step was to determine whether pH regulation differed between
astrocytes and gliomas in the presence of
CO2/HCO
3. When exposed to CO2/HCO
3-buffered media,
nontransformed astrocytes initially acidify as CO2 entering
the cell forms carbonic acid and dissociates to H+ and
HCO
3. This anticipated acidification
phase was followed by alkalinization to a pHi slightly in
excess of that measured in HEPES-buffered media (Fig.
8A). The measured rate of this
alkalinization for the astrocytes was 3.65 ± 0.27 mM/min (n = 22) and was similar to other measurements for primary rat astrocytes
(2). U-118 gliomas also acidified when CO2 was added to the
media, to approximately the same pH as that of astrocytes (see Fig.
8A). Unexpectedly, the glioma cells demonstrated limited recovery from the CO2-induced acidification, with a new
resting pHi which was significantly more acidic than that
in HEPES-buffered media. In addition, the rate of this alkalinization
was significantly slower for gliomas (1.02 ± 0.14 mM/min; n = 14) compared with the nontransformed astrocytes (P < 0.001).
These differences in rates of alkalinization were not due to
differences in
T because the measured values of
T were nearly identical for both the astrocytes (34.3 ± 0.8 mM/pH) and gliomas (34.9 ± 0.9 mM/pH). A comparison of
steady-state pHi in the absence and presence of
CO2/HCO
3 for astrocytes
and U-118 gliomas is shown in Fig. 8B. Values of pHi in HEPES-buffered media were significantly more
alkaline for U-118 gliomas and more acidic for astrocytes than those
measured in
CO2/HCO
3-buffered
media. In addition, the pHi values of astrocytes were
significantly more acidic than the corresponding
pHi measured in U-118 gliomas in HEPES-buffered media yet more alkaline in
HCO
3-buffered media.

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Fig. 8.
Effect of CO2/HCO 3 on
pHi for astrocytes and U-118 gliomas. A: after
stabilization in HR, cells were perfused with
CO2/HCO 3-buffered media.
Rapid initial acidification due to CO2 entering cells was
followed by recovery to a new steady-state pHi.
After stabilization of pHi in
CO2/HCO 3, cells were
acidified by perfusion with
CO2/HCO 3 media containing
10 mM NH4Cl. Removal of NH+4, in
presence of CO2/HCO 3,
resulted in rapid recovery for both astrocytes and gliomas back to
CO2/HCO 3 steady-state
pHi values. Removal of
CO2/HCO 3 caused rapid
initial alkalinization, followed by recovery back to previous
steady-state pHi values in HEPES-buffered media. B:
comparison of steady-state pHi values for astrocytes and
U-118 gliomas in absence and presence of
CO2/HCO 3. Values of
pHi in HEPES-buffered media are significantly different
from those in
CO2/HCO 3-buffered media
for both cell types (P < 0.001), and pHi of
astrocytes differs significantly from U-118 gliomas in both
HEPES-buffered (P < 0.001) and
CO2/HCO 3-buffered
(P < 0.05) media. C: comparison of rates of recovery
from NH+4-induced acidification for U-118
gliomas and astrocytes. Values of
JH+ were
determined during initial linear portion of recovery from acid load,
using only data from cells that were acidified to a similar degree
(pHi = 6.5-6.6). Rates of recovery were significantly
greater for U-118 gliomas in HEPES-buffered media compared with
astrocytes (P < 0.001), whereas there was no significant
difference between gliomas and astrocytes in
CO2/HCO 3-buffered media.
In addition, rates of recovery were reduced in gliomas in presence of
CO2/HCO 3 compared with
that in HEPES (P < 0.01), whereas recovery was enhanced for
astrocytes in CO2/HCO 3
compared with HEPES (P < 0.01).
|
|
Acidification by NH+4 prepulse in
CO2/HCO
3 media generated
similar pH regulatory responses by both the astrocytes and the gliomas,
with mean recovery rates of 6.12 ± 1.09 mM/min (n = 3) and
8.29 ± 0.63 mM/min (n = 3), respectively (Fig.
8C). The astrocytes displayed a significantly enhanced response
to the acid load compared with that in HEPES-buffered media
(Fig. 8C), with nearly a fivefold increase in recovery rate. Interestingly, the rate of recovery from
NH+4-induced acidification for the gliomas
was significantly decreased (~30%) in the presence of
CO2/HCO
3 compared with
that in the HEPES-buffered media (Fig. 8C). Because pH
regulatory H+ flux is a graded function (both in rate and
magnitude) of pH disturbance, only those experiments in which the
astrocytes and gliomas were acidified to the same extent
(pHi = 6.5-6.6), in both HEPES-buffered and
CO2/HCO
3-buffered media, were used to determine the above flux rates. In
CO2/HCO
3 media, the rates
of recovery from acidification for both astrocytes and gliomas could be
reduced by either amiloride or DIDS, indicating that both
Na+/H+ exchange and
HCO
3-dependent pH regulatory
mechanisms are involved (data not shown). Lastly, washout of
CO2/HCO
3 resulted in an
initial, rapid alkalinization as CO2 left the cell, followed by a gradual return to HEPES-buffered steady-state
pHi values for both cell types. These results suggest that
in the presence of
CO2/HCO
3, the
HCO
3-dependent pH regulatory
mechanisms are more dominant than the
Na+/H+ exchanger in the primary astrocytes.
However, the contribution of
HCO
3-dependent pathways in glioma pH
regulation appears to be more complex.
Effect of Cl
on steady-state pHi in
the presence of
CO2/HCO
3.
It was surprising that U-118 gliomas attained a more acidic
steady-state pHi (Fig. 8B) and exhibited decreased
recovery from acidification (Fig. 8C) in the presence of
CO2/HCO
3 compared with
that in HEPES-buffered media. It was anticipated that external
CO2/HCO
3 would enhance
intracellular alkalinization by both increased buffer capacity (from
HCO
3) and from the
contribution of either the
Na+-HCO
3 cotransporter or
the Na+-dependent
Cl
/HCO
3 exchanger,
both of which function as net acid extruders (see Fig. 1). This
certainly appeared to be the case for the cultured astrocytes, which
alkalinized by an average of 0.1 pH units in the presence of
HCO
3 (Fig. 8B), as shown by
other investigators (2, 12, 48). This raised the question as to what
other HCO
3-dependent pathway might
account for intracellular acidification of gliomas.
One explanation could be that the gliomas express a robust
Na+-independent
Cl
/HCO
3 exchanger
which functions as an acid loader. Previous reports have demonstrated
that astrocytes possess an Na+-independent
Cl
/HCO
3 exchanger
that is apparently active only when pHi is more alkaline
than normal steady-state pHi (31, 48). The function of a
potential Na+-independent
Cl
/HCO
3 exchanger
in gliomas was examined by exposing them to
CO2/HCO
3 media in which
external Cl
was replaced with gluconate (Fig.
9). This chloride-substituted media would
inhibit a functional
Cl
/HCO
3 exchanger
without affecting NHE1 and
Na+-HCO
3 cotransport (see
Fig. 1). Consistent with the prediction of a functional
Cl
/HCO
3 exchanger,
the gliomas recovered from CO2-induced acidification more
rapidly and attained a significantly more alkaline steady-state
pHi in the absence of Cl
(Fig.
9B) than that observed in Cl
-containing
media (Fig. 9A). By contrast, in astrocytes neither the
steady-state pHi nor the rate of alkalinization after the CO2-induced acidification were significantly affected by
the absence of Cl
in
CO2/HCO
3 media (data not
shown). These data suggest that compared with astrocytes, gliomas
display significantly higher steady-state pHi in the
absence of CO2/HCO
3 due to
increased activity of NHE1, whereas in the presence of CO2/HCO
3, the activity of
the Cl
/HCO
3
exchanger results in significant acidification.

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Fig. 9.
Recovery of U-118 gliomas from CO2-induced acidification is
enhanced by removal of extracellular Cl . A:
in presence of external Cl , exposure to
CO2/HCO 3 results in rapid
acidification followed by recovery to a more acidic steady-state
pHi compared with that in HEPES-buffered media.
B: in Cl -free
CO2/HCO 3-buffered media,
gliomas regain a new steady-state pHi value similar to that
in HEPES-buffered media, whereas addition of Cl back
to CO2/HCO 3 media results
in rapid acidification.
|
|
Mutational analysis of the type 1 Na+/H+
exchanger in human glioma cell lines.
The preceding data demonstrate that the Na+/H+
exchanger is primarily responsible for the elevated steady-state
pHi of malignant gliomas under nominally
CO2/HCO
3-free conditions. Although the inhibitor data (Fig. 6) fit the pharmacological profile of
the type 1 Na+/H+ exchanger (NHE1), it does not
exclude the possibility that the gliomas express a mutated version of
the NHE1 isoform. Given that there are multiple genetic mutations
reported in glioblastomas (54), one possibility is that mutations of
the NHE1 gene are responsible for their elevated steady-state
pHi.
This genetic hypothesis was directly examined by sequencing the NHE
cDNA that was amplified and cloned from three human glioma cell lines
(U-87, U-118, and U-251) that demonstrated elevated steady-state
pHi. Comparison of nucleotide sequences of these human NHE1
cDNAs to the nucleotide sequences reported for the wild-type exchanger
revealed that one clone out of four isolated from the U-87 cell line
contained cDNA that encoded for an NHE1 protein with an
NH2-terminal deletion. There were no protein differences encoded by the three other NHE1 cDNAs from this cell line, and neither
RNA blots nor immunoblots detected truncated transcripts or proteins
for NHE1, indicating the instability of this aberrant transcript.
Results for the other two cell lines (U-118 and U-251) indicated no
differences of amino acid sequences compared with that of normal human NHE1.
Expression of NHE1.
Results of RNA blot analyses of various cell lines indicated that
similar levels of NHE1 mRNA (relative to
-actin) were expressed for
primary rat astrocytes and the rat C6 glioma cell line, whereas transcript levels of NHE1 in the three human glioma cell lines (U-118,
U-87, and U-251) varied over a fourfold range (data not shown).
Overall, there was no direct correspondence of levels of mRNA
transcript expression with measured values of steady-state pHi. Because there was no correspondence between transcript
levels and increased pHi, we then examined if NHE1 protein
levels corresponded with measurements of the elevated steady-state
pHi. Immunoblot analyses of primary rat astrocytes, rat C6
glioma, and the three human glioma cell lines are shown in Fig.
10A, along with relative amounts
of NHE1 protein normalized to levels of actin (Fig. 10B). Comparison of the two rat cell lines reveals that NHE1 protein expression is increased in the C6 tumor line compared with normal astrocytes. In addition, each of the three human gliomas express abundant levels of NHE1 protein compared with actin, with the U-118
gliomas expressing the largest amount. However, comparisons with the
measured steady-state pHi values (Fig. 2) again reveal no
direct correlation of pHi with levels of protein
expression. These data indicate that the elevated steady-state
pHi of the gliomas cannot simply be attributed to increased
levels of expression of the NHE1 protein, or due to pretranslational
alterations of transcripts, but rather that some posttranslational
modification must be involved.

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Fig. 10.
Immunoblot of membrane-enriched protein fractions from primary rat
astrocytes (1°RA), rat C6 gliomas, and three human glioma cell
lines (U-87, U-118, and U-251). A: protein fractions (50 µg
each) were separated by SDS-PAGE, transferred to nitrocellulose, and
sequentially probed with antibodies to actin and to NHE1. B:
normalization of NHE1 signal to levels of actin.
|
|
 |
DISCUSSION |
Steady-state pHi.
In this study, human and rat malignant gliomas were found to exhibit
significantly higher steady-state pHi (0.3-0.5 pH
units) compared with normal astrocytes (pHi = 6.98 ± 0.01), under nominally CO2/HCO
3-free conditions
(Fig. 2). This variation was not due to differences in
i, as demonstrated by the similar linear relationship of
i vs. pHi for both astrocytes and gliomas (Fig. 3B). In fact, the higher pHi of the gliomas
results in decreased
i, compared with primary
astrocytes, as a result of their respective positions on the composite
buffer titration curve (see Fig. 3B, inset). The effect
of CO2/HCO
3 on
steady-state pHi also differed between gliomas and primary
astrocytes (Fig. 8). Whereas the astrocytes attained a more alkaline
pHi that was consistent with previous reports (2, 12, 48),
the acidification of the gliomas was unexpected, particularly because
the addition of CO2/HCO
3
permits both passive and dynamic HCO
3-dependent buffering. These U-118
glioma data are contrary to a study of C6 gliomas by Shrode and Putnam, where a slightly more alkaline steady-state pHi was
reported in the presence of
CO2/HCO
3 compared with
that in HEPES-buffered media (48). Instead, our data indicate that U-118 gliomas likely possess a bicarbonate-dependent acid loading mechanism (see below).
The importance of the Na+/H+ exchanger in
maintaining the alkaline steady-state pHi of the gliomas,
under nominally
CO2/HCO
3-free conditions,
is demonstrated in Fig. 7. U-118 gliomas exhibited rapid, sustained
acidification when placed in either Na+-free media (Fig.
7A) or in the presence of the NHE1 inhibitor amiloride (Fig.
7C). This acidification was fully reversible upon reintroduction of Na+ or washout of amiloride. By contrast,
nontransformed astrocytes were only mildly affected by amiloride (Fig.
7D) and were capable of maintaining steady-state
pHi even in the absence of external Na+ and
HCO
3 (Fig. 7B). These data
demonstrate the higher H+ production of the gliomas
compared with nontransformed astrocytes, as predicted by their
increased glycolytic metabolism. Furthermore, the ability of the
astrocytes to maintain their steady-state pHi in the
absence of both Na+ and
HCO
3 indicates the presence of other
H+ extruding pathways. Likely candidates are the vacuolar
type H+-ATPase (36, 56) or H+-lactate symport
(57, 58), both of which function as acid-extruding mechanisms and have
been reported in primary astrocytes (see Fig. 1). However, activity of
these Na+- and
HCO
3-independent mechanisms appears to be minimal in these primary astrocyte cultures, because negligible pH
recovery was seen after a substantial acidification in
Na+-free, HEPES-buffered media (see Fig. 4B). Yet,
given the modest acid production by the astrocytes, these
Na+- and HCO
3-independent
mechanisms appear to be adequate to maintain their normal steady-state
pHi under basal conditions. Taken together, these data
suggest that U-118 gliomas rely primarily on the
Na+/H+ exchanger to maintain their alkaline
pHi set point, whereas nontransformed astrocytes are able
to maintain a constant pHi even in the absence of NHE
activity, under nominally
CO2/HCO
3-free conditions.
pH recovery in response to acidification.
Active pH regulation in response to acidification under
CO2/HCO
3-free conditions
is highly dependent upon the Na+/H+ exchanger
for both malignant gliomas and astrocytes. This is best illustrated in
Fig. 4, where recovery from an NH+4-induced acidification depends upon the presence of external Na+ and
is inhibited by amiloride in a dose-dependent manner. Furthermore, the
activity of the Na+/H+ exchanger is
significantly greater in gliomas than astrocytes at any given
pHi (Fig. 5). However, recovery from an
NH+4-induced acidification is enhanced nearly
fivefold for astrocytes in the presence of
CO2/HCO
3, whereas it is
reduced nearly 30% in gliomas (Fig. 8C). These data suggest
that astrocytes utilize either the
Na+-HCO
3 cotransporter or
Na+-dependent
Cl
/HCO
3 exchanger
for HCO
3 influx, thus effectively
functioning as net H+ extruding pathways under these acidic
conditions (see Fig. 1). Recent studies by Boron and co-workers have
confirmed that rat hippocampal astrocytes primarily utilize the
electrogenic Na+-HCO
3
cotransporter for bicarbonate-dependent pH regulation (1, 2).
In contrast, the diminished pH recovery and more acidic steady-state
pHi of the gliomas in the presence of
CO2/HCO
3 (Fig. 8) suggests
that they possess a HCO
3-dependent acid loading mechanism that acts in opposition to the above
H+ extruding pathways (see Fig. 1). A likely candidate
would be the Na+-independent
Cl
/HCO
3 exchanger,
which transports intracellular HCO
3
out of the cell in exchange for equimolar extracellular
Cl
. To test this hypothesis, we exposed cells that
had been preincubated in HEPES-buffered media to
CO2/HCO
3-buffered media in
the presence (Fig. 9A) and absence (Fig. 9B) of
external Cl
. Gliomas in Cl
-free
media initially acidified in response to
CO2/HCO
3 and then rapidly
recovered to a new steady-state pHi that was ~0.05 pH
units more alkaline than that measured in HEPES-buffered media (Fig.
9B). The rate of pH recovery after CO2-induced
acidification for the gliomas was substantially reduced (~80%) by
DIDS, an anion exchange inhibitor, and to a lesser extent (~25%) by
amiloride in the absence of external Cl
(data not
shown). These data indicate that the DIDS-sensitive Na+-HCO
3 cotransporter and
the Na+/H+ exchanger are active during pH
regulation in the tumor cells in the absence of external
Cl
. It is also possible that the
Na+-dependent
Cl
/HCO
3 exchanger
could be activated if internal Cl
is not fully
depleted. In contrast, when astrocytes were similarly exposed to
Cl
-free media, neither the steady-state
pHi nor the rate of recovery from the
CO2-induced acidification was significantly affected compared with that seen in normal HCO
3
media (data not shown).
Thus there appears to be a functional
Cl
/HCO
3 exchanger
in U-118 gliomas that counteracts the acid efflux via NHE1 and
Na+-driven HCO
3 uptake in
24 mM HCO
3 media. As a consequence,
pHi is less alkaline than that measured in HEPES (nominally
HCO
3-free) media. This assumes that at
steady-state pH the
Cl
/HCO
3 exchanger
in high-HCO
3 media has a turnover rate
that approaches that of the acid extrusion pathways. Sustained
operation of this anion exchanger requires some relatively robust
mechanism for Cl
efflux/recycling to prevent
dissipation of the chloride electrochemical gradient that drives
HCO
3 transport by the
Cl
/HCO
3 exchanger.
In this regard, there are recent reports of glioma-specific, outwardly
rectified Cl
channels (53) which could function as a
means of recycling Cl
entering the cell via the
Cl
/HCO
3 exchanger,
in the presence of external HCO
3.
However, the in situ extracellular environment of the tumors is acidic
and would minimize the availability of external
HCO
3, so that the physiological role of this anion exchanger is unclear. Indeed, 31P-NMR
spectroscopy studies demonstrate that the pHi of gliomas in
vivo is significantly more alkaline than adjacent normal brain (18, 24,
45). This is consistent with our
HCO
3-free steady-state pHi
data and would further indicate that the
HCO
3-dependent pathways are not
significant in gliomas in vivo.
Proposed causes of altered steady-state pHi in gliomas.
Our data suggest that the gliomas exhibit elevated steady-state
pHi due to increased activity of the
Na+/H+ exchanger, under nominally
CO2/HCO
3-free conditions. This increased NHE activity could be due to either pre- or
posttranslational modification of the exchanger. Data from our study
preclude the notion of pretranslational NHE modification by several
lines of evidence. First, functional studies using known inhibitors of NHE reveal a pharmacological profile that is consistent with that of
the normal mammalian NHE1 isoform (Fig. 6). Second, analyses by both
Northern and Western (Fig. 10) blot indicate that NHE1 is the primary
isoform expressed in these cells. Furthermore, steady-state NHE
transcript and protein levels in the gliomas do not directly correspond
with their increased pHi set point. Last, and most
important, nucleotide sequence data indicate that the NHE isoform
expressed in the glioma cells is identical to that published for normal
human NHE1, thus mutations of the exchanger are not responsible for its
increased activity in these cells.
The most plausible explanation for the elevated pHi in
glial tumors would be that there is upregulation of the signaling
pathways that activate NHE1. In a review by Grinstein and Rothstein, it was shown that an alkaline shift of the pHi-dependence
curve of ~0.2-0.3 pH units occurs when NHE1 is activated by
growth factors, phorbol esters, or hypertonic stimulation (20). This
alkaline shift in pH dependence of NHE1 activity for gliomas vs.
astrocytes is consistent with our observations in this study (see Fig.
5). In addition, studies by other investigators provide circumstantial evidence favoring this view. For example, phorbol esters are known to
stimulate protein kinase C (PKC), which in turn would
stimulate NHE1 activity. Also, growth factors such as epidermal growth
factor and platelet-derived growth factor activate tyrosine kinases, which could indirectly affect both PKC and NHE1 activity. Finally, both
epidermal growth factor receptor (15, 25) and PKC (39) upregulation
have been implicated in glioma pathogenesis, either of which could
manifest itself by an alkaline shift of the pH-dependence curve.
Elevated pHi and tumorigenicity.
There is compelling evidence that the Na+/H+
exchanger plays an important role in cellular proliferation (see Ref.
21 for review). In fact, mutated tumor cell lines that lack a
functional exchanger have been shown to lose, or severely reduce, their
ability to develop solid tumors when implanted in immune-deficient mice (26, 43). There is additional evidence that elevating pHi alone can result in tumorigenicity. Perona and Serrano demonstrated that mammalian fibroblasts transfected with a yeast
H+-ATPase exhibited a more alkaline pHi and
acquired tumorigenic characteristics, whereas cells transfected with an
inactive form of the H+-ATPase did not (37). Therefore, it
would appear that maintaining an alkaline pH set point by increasing
activity of acid extruding mechanisms, such as NHE1 or proton pumps,
gives cells a proliferative advantage.
Although the elevated pHi is one important element in
cellular proliferation and tumor growth, there is increasing evidence that maintenance of the acidic extracellular environment contributes to
tumor growth and invasion as well. A hallmark for malignant gliomas is
their invasive capacity, which is dependent upon the breakdown of the
surrounding extracellular matrix by a number of secreted proteolytic
enzymes (13, 52). These enzymes include cathepsins, metalloproteinases,
and serine proteases, all of which show enhanced activity at acidic pH
(28). Studies on glioblastoma cells have demonstrated that increased
invasiveness of the tumors correlates with increased levels of
cathepsin B expression, implicating an important role for these
proteases in glioma invasion (41). Additional studies on glioma cell
lines have implicated a 72-kDa matrix metalloprotease (MMP-2) as
another key player in tumor invasion, and that MMP-2 activity could be
reduced by inhibition of PKC (52). Thus it appears that increased acid
extrusion provides a twofold advantage to tumor cells: the alkaline
pHi favors metabolic processes associated with cellular
proliferation, whereas the acidic extracellular environment, which is a
consequence of H+ extrusion, enhances the invasive capacity
of the transformed cells. This suggests that compounds that prevent the
active acid extrusion from tumor cells, resulting in decreased
pHi and increased pHe, would be important tools
for effective tumor-specific chemotherapy.
In conclusion, we have shown that malignant gliomas exhibit altered
H+ metabolism compared with normal astrocytes and that
their elevated steady-state pHi is likely due to increased
activation of the type 1 Na+/H+ exchanger,
under nominally
CO2/HCO
3-free conditions. Because tumors in situ typically have a more acidic extracellular environment, this would result in decreased external
[HCO
3] with concomitant
reduction in total buffer capacity as well as reduced activity of
HCO
3-dependent transporters. This
implies that the tumor cells would be largely reliant on the dynamic
NHE1-mediated H+ buffering to maintain an alkaline
steady-state pHi despite their high levels of
H+ production. Inhibition of NHE1 in the tumors results in
significant cytosolic acidification (and presumably extracellular
alkalinization), thus precluding the conditions necessary for continued
cellular proliferation. By contrast, normal astrocytes are not affected by NHE1 inhibition because of their higher fixed buffer capacity (both
intrinsic and HCO
3 dependent) and the availability of dynamic HCO
3-dependent
pH regulatory mechanisms due to their more alkaline pHe
(see Fig. 7). In fact, several investigators have demonstrated that
NHE1 inhibitors can be utilized to selectively kill tumors in vivo (23,
34, 44, 51). We are currently employing in vivo NMR spectroscopy to evaluate the effects of NHE inhibitors on the growth of gliomas implanted in rat brains.
 |
ACKNOWLEDGEMENTS |
P. M. Cala thanks Lazaro J. Mandel, his mentor, colleague, and
friend. Laz was always a source of inspiration and good cheer. Laz's
untimely death due to a glioma served as the inspiration for this work.
It is our hope that our efforts honor his memory and contribute to a
rational and effective approach to therapy. The authors also thank
Elizabeth Nguyen for excellent technical assistance, Dr. Dan
Biemesderfer (Yale University) for the anti-NHE1 antibody (MAb 4E9),
and Dr. Hans-J. Lang of Hoechst, Germany, for providing the HOE-694 compound.
 |
FOOTNOTES |
This research was supported by National Heart, Lung, and Blood
Institute (NHLBI) Grant HL-21179 (to P. M. Cala) and a University of
California-Davis Health System Award (to F. A. Gorin). L. A. McLean was
partially supported by NHLBI Predoctoral Training Grant HL-07682 (to J. Longhurst).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. M. Cala,
Dept. of Human Physiology, Univ. of California, One Shields Ave.,
Davis, CA 95616 (E-mail: pmcala{at}ucdavis.edu).
Received 16 August 1999; accepted in final form 18 October 1999.
 |
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