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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

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.


    METHODS
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INTRODUCTION
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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 (beta T) equals the sum of the intrinsic buffer capacity (beta i) and the buffering provided by CO2/HCO-3 (beta HCO-3). The beta i was determined over the investigated range of pHi studied by perfusing cells with progressively decreasing concentrations of NH+4-HR, such that beta i = Delta NH+4/Delta 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 beta 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).

                              
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Table 1.   Primers used for amplification of NHE1 from human glioma cell lines

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 gamma -actin probe labeled with 32P-dATP under similar conditions as for NHE1. Ratios of NHE1 to gamma -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 beta -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.


    RESULTS
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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.

Values of beta 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 Delta [NH+4]i was calculated based on the observed Delta 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 Delta [NH+4]i was equivalent to Delta [H+]i, with resulting beta i = Delta [H+]i/Delta pHi. beta 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 (beta i) for cultured astrocytes and glioma cell lines is dependent on pHi. A: example of experimental protocol used to determine beta 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: beta 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 beta i at steady-state (inset) is significantly lower in gliomas due to their elevated pHi.

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 beta i by Delta pHi/Delta 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.

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.

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.

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 beta T because the measured values of beta 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 beta 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 gamma -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta i, as demonstrated by the similar linear relationship of beta i vs. pHi for both astrocytes and gliomas (Fig. 3B). In fact, the higher pHi of the gliomas results in decreased beta 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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ABSTRACT
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RESULTS
DISCUSSION
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