Malignant gliomas exhibit alkaline intracellular pH (pH(i)) and acidic extracellular pH (pH(e)) compared with nontransformed astrocytes, despite increased metabolic H(+) production. The acidic pH(e) 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 pH(i) (pH(i) = 7.31-7.48) than normal astrocytes (pH(i) = 6.98), and increased rates of recovery from acidification, under nominally CO(2)/HCO(-)(3)-free conditions. Inhibition of NHE1 in the absence of CO(2)/HCO(-)(3) resulted in pronounced acidification of gliomas, whereas normal astrocytes were unaffected. When suspended in CO(2)/HCO(-)(3) medium astrocyte pH(i) increased, yet glioma pH(i) 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.

Previously, we demonstrated that malignant glioma cell lines have increased intracellular pH (pH i ) as a result of increased activities of the type I sodium/hydrogen exchanger (NHE1). This alkalotic pH i of 7.2 to 7.4 is favorable for augmented glycolysis, DNA synthesis, and cell cycle progression. Conversely, reductions in pH i have been associated with reduced rates of proliferation in transformed cell types. The effects of reducing pH i directly and by NHE1 inhibition on human malignant glioma cells were systematically compared with those on primary rat astrocytes. Neither cariporide, nor direct acidification to pH i 6.9 altered the proliferative rates or viabilities of human U87 or U118 malignant glioma cell lines. However, amiloride significantly impaired glioma cell proliferation and viability while not affecting astrocytes at concentrations (500 M) that exceeded its inhibition of NHE1 in glioma cells (IC 50 ϭ 17 M). Preventing a reduction of pH i did not alter the drug's antiproliferative and cytotoxic effects on glioma cells. These findings indicated that amiloride's cytotoxic effects on glioma cells are independent of its ability to inhibit NHE1 or to reduce intracellular pH i . The amiloride derivative 2,4 dichlorobenzamil (DCB) inhibits the sodium-calcium exchanger (NCX) and was both antiproliferative and cytotoxic to glioma cells at low doses (20 M).preferentially blocks sodiumdependent calcium influx by NCX (reverse mode) and was nontoxic to glioma cells. It is proposed that DCB (20 M) and amiloride (500 M) impair calcium efflux by NCX, leading to elevations of intracellular calcium that initiate a morphologically necrotic, predominantly caspase-independent glioma cell death.High-grade malignant gliomas are the most common, lethal primary brain tumor in adults (Legler, 1999). Median survival is approximately 9 to 12 months after diagnosis (Stewart, 2002), and the tumors are usually refractory to aggressive multimodal therapy (Brandes et al., 1999). Glioma cells exhibit increased glycolytic fluxes associated with elevated lactate/pyruvate ratios (Miccoli et al., 1996) and would be expected to have an acidotic intracellular pH (pH i ). However, several 31 P spectroscopic studies measured intracellular human glioma pH in situ and reported alkaline values (pH 7.12-7.24) compared with surrounding brain (pH 6.99 -7.05) (Hubesch et al., 1990;Rutter et al., 1995). The alkaline pH is more optimal for obligate tumor glycolysis, DNA synthesis, and cell cycle progression (Hasuda et al., 1994;Miccoli et al., 1996;Katabi et al., 1999). In addition, a reduction in pH i has been associated with reduced rates of proliferation and growth arrest in transformed cell types (Musgrove et al., 1987;Rotin et al., 1989;Horvat et al., 1992).Our earlier investigation of four human and rat malignant glioma cell lines revealed an alkaline intracellular pH (pH i of 7.31-7.48) compared with primary rat astrocytes (pH i of 6.98 Ϯ 0.01) (McLean et al., 2000). Increased activity of the type 1 Na ϩ -H ϩ exchanger (NHE1) in gl...

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