Na+/H+ exchange subtype 1 inhibition during ... - Wiley Online Library

Journal of Neurochemistry, 2002, 80, 36±44

Na+/H+ exchange subtype 1 inhibition during extracellular acidi®cation and hypoxia in glioma cells Kristine Glunde,*,1 Heiko DuÈûmann, Hans-Paul Juretschkeà and Dieter Leibfritz* Departments of *Chemistry and Biophysics, University of Bremen, Bremen, Germany àAventis Pharma Deutschland GmbH, In Vivo NMR Laboratory, Frankfurt/Main, Germany

Abstract Lactacidosis is a common feature of ischaemic brain tissue, but its role in ischaemic neuropathology is still not fully understood. Na+/H+ exchange, a mechanism involved in the regulation of intracellular pH (pHi), is activated by low pHi. The role of Na+/H+ exchange subtype 1 was investigated during extracellular acidi®cation and subsequent pH recovery in the absence and presence of (4-isopropyl-3-methylsulphonylbenzoyl)-guanidine methanesulfonate (HOE642, Cariporid), a new selective and powerful inhibitor of the Na+/H+ exchanger subtype 1 (NHE-1). It was compared for normoxia and hypoxia in two glioma cell lines (C6 and F98). pHi was monitored by ¯uorescence spectroscopy using the intracellularly trapped pH-

sensitive dye 2¢,7¢-bis(carboxyethyl)-5(6)-carboxy¯uorescein (BCECF). Alterations in glial cell metabolism were characterized using high-resolution 1H, 13C and 31P NMR spectroscopy of perchloric acid extracts. NHE-1 contributed to glial pH regulation, especially at pathologically low pHi values. NHE-1 inhibition with HOE642 during acidi®cation caused exacerbated metabolic disorders which were prolonged during extracellular pH recovery. However, NHE-1 inhibition during hypoxia protected the energy state of glial cells. Keywords: ¯uorescence, hypoxia, intracellular pH, Na+/ H+ exchange, NMR. J. Neurochem. (2002) 80, 36±44.

Cerebral ischaemia causes intra- and extracellular lactacidosis (for reviews, see SiesjoÈ et al. 1993; Tombaugh and Sapolsky 1993; Hossmann 1994) owing to anaerobic glycolysis (Pasteur effect) and the release of acid equivalents due to hydrolysis of ATP (Roos and Boron 1981; SiesjoÈ and Wieloch 1985). Recent investigations on neuronal tissue culture and an animal model of focal ischaemia suggested that mild acidosis or blockage of Na+/H+ exchange may protect ischaemic brain tissue (Simon et al. 1993; Vornov et al. 1996) possibly by suppressing the pH-sensitive injury mechanisms or by preventing sodium entry due to Na+/H+ exchange inhibition. However, the role of acidosis during cerebral ischaemia is controversial. Intense acidosis is linked to enhanced tissue damage, based mainly on pre-ischaemic hyperglycaemia, which aggravates the damage caused by a subsequent period of transient ischaemia (Nedergaard and Diemer 1987; SiesjoÈ 1988; SiesjoÈ et al. 1993). Furthermore, lactacidosis induces cell swelling and cell damage in glial cells (Kempski et al. 1988; Staub et al. 1996). This is of particular interest because astroglial cells control the pH of interstitial fluid (Deitmer 1992) and thereby influence neuronal activity (Chesler 1990). To date, six systems have

been identified that regulate glial cell pHi (Volk et al. 1998). pHi is controlled by the Na+/H+ exchanger existing as at least five isozymes; by three different bicarbonate transporters, e.g. Na+-dependent Cl±/HCO3±-countertransport, Na+-independent

36

Received January 25, 2001; revised manuscript received September 18, 2001; accepted September 22, 2001. Address correspondence and reprints requests to D. Leibfritz, Department of Chemistry, University of Bremen, NW 2, FB 2, PO Box 330440, 28334 Bremen, Germany. E-mail: [email protected] 1 The present address of Kristine Glunde is Johns Hopkins University, School of Medicine, Radiology Department ± NMR Research, 217 Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205, USA. Abbreviations used: AM, acetoxymethylester; BCECF, 2¢,7¢bis(carboxyethyl)-5(6)-carboxyfluorescein; Cr, creatine; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; Gro-3-P, glycerol-3-phosphate; Gro-3-P-DH, glycerol 3-phosphate dehydrogenase; HOE642, (4-isopropyl-3-methylsulphonyl-benzoyl)-guanidine methanesulfonate; KHB, Krebs±Henseleit buffer; NHE-1, Na+/H+ exchange subtype 1; NOE, nuclear Overhauser effect; PCA, perchloric acid; PCr, phosphocreatine; PFK, phosphofructokinase; pHe, extracellular pH; pHi, intracellular pH; TCA, tricarboxylic acid; TSP, (trimethylsilyl)propionic-2,2,3,3,d4-acid.

Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 36±44

Na+/H+ exchange inhibition in glial cells 37

Other chemicals were pure analytical grade and obtained from Merck (Darmstadt, Germany).

Fig. 1 Structure of 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE642; Cariporid).

Cl±/HCO3±-exchanger and Na+/HCO3±-cotransport; and furthermore by carbonic anhydrase and a proton-translocating H+-ATPase. Among these Na+/H+ exchange seems to be the predominant mechanism by which glial cells extrude acid equivalents at pathologically low pHi values as shown with the Na+/H+ exchange inhibitor amiloride (Cragoe et al. 1967; FloÈgel et al. 1994). However, recent studies concerned with selectivity and pharmacology indicated that amiloride nonspecifically inhibits most plasma membrane Na+ transport systems, e.g. Na+ channel, Na+/Ca2+ exchanger and all subtypes of the Na+/H+ exchanger (Kleyman and Cragoe 1988; Counillon and PouysseÂgur 1995). (4-Isopropyl-3-methylsulphonyl-benzoyl)-guanidine methanesulfonate (HOE642; Cariporid; Fig. 1) has been characterized as a new selective, powerful and tolerable inhibitor of Na+/H+ exchange subtype 1 (NHE-1) (Scholz et al. 1995). In this study, this compound was used to investigate how NHE-1 is involved in glial pHi homeostasis. pHi was monitored in immobilized viable cells using the fluorescence 2¢,7¢-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) ratio technique (Rink et al. 1982). Alterations in glial cell metabolism during extracellular acidosis and subsequent pHi recovery under normoxia and hypoxia were followed up using 1H, 13C and 31P NMR spectra of perchloric acid extracts from F98 and C6 glioma cells. Possible mechanisms relating the role of NHE-1 in glial pHi regulation to cellular metabolism are being discussed. Clinical implications arise from beneficial effects of NHE-1 inhibition on glial energy metabolism during hypoxia.

Materials and methods Materials Rat C6 glioma cells (Benda et al. 1968) were purchased from ICN Biomedicals (Meckenheim, Germany), F98 glioma cells (Ko et al. 1980a,b) were donated by K.-A. Hossmann (Max-Planck-Institut fuÈr Neurologische Forschung, Germany). Dulbecco's modi®ed Eagle's medium (DMEM), fetal calf serum (FCS), phosphate-buffered saline (PBS), trypsin/EDTA and penicillin/streptomycin were obtained from Gibco (Eggenstein, Germany). Macroporous gelatine microcarriers CultispherTM±G were purchased from Percell Biolytica (Lund, Sweden). HOE642 was a gift of Hoechst Marion Roussel (Frankfurt, Germany). BCECF and Nigericin were purchased from Sigma (Deisenhofen, Germany), deuterated solvents and EDTA from Merck (Darmstadt, Germany) and [1-13C]glucose from Cambridge Isotope Laboratories (Promochem; Wesel, Germany).

Cell culture C6 or F98 glioma cells were cultivated in 10- or 15-cm culture dishes in a humidi®ed atmosphere of 10% CO2 in air at 37°C in DMEM, supplemented with 5% FCS and penicillin/streptomycin (100 units/mL). Inoculation was performed at a density of 1 ´ 104 cells/mL, and the medium was routinely changed 3 days later. Con¯uence was reached within 5 days. For pHi measurements, C6 cells were grown on macroporous gelatine microcarriers (van Wezel 1983; Nilsson 1988). Approximately 107 cells, 3 mL gelatine microcarriers, and 50 mL DMEM, supplemented with 5% FCS and penicillin/streptomycin (100 units/ mL) were transferred into a silicon-coated stirring ¯ask. This mixture was alternately stirred for 3 min at 40 r.p.m. and then kept at rest for 30 min in a humidi®ed atmosphere of 10% CO2 in air at 37°C, for a total of 4 h. Cells then became adherent to the microcarriers, and additional 75 mL DMEM, supplemented with 5% FCS and penicillin/streptomycin (100 units/mL) was added with continuous stirring at 32 r.p.m. Three days later, cell density was 2 ´ 107 cells per mL microcarrier as determined after each ¯uorescence measurement by release of cells from microcarriers using dispase (2.4 U/mL in Puck's solution). pHi measurements in viable cells with BCECF For ¯uorescence measurements 200-lL microcarriers coated with C6 cells were transferred into a thermostatisized quartz cuvette (37°C), which is part of a perfusion system driven by a peristaltic pump at a rate of 0.5 mL HEPES-buffered saline containing 5 mM glucose per min. pHi was measured using the pH-sensitive ¯uorescent dye BCECF. C6 cells were perfused with 2 lM BCECF-acetoxymethylester (BCECF-AM) for 10 min. The dye signal was monitored continuously during the loading procedure and subsequent washing period, when extracellular BCECF-AM diffused into the cells and was completely hydrolysed to BCECF by cytosolic esterases until signals remained constant (20±30 min). pHi was recorded using a home-built ¯uorescence spectrometer. BCECF was alternately excited at 440 and 500 nm by a 150-W xenon arc lamp and a monochromator with maximal bandwidth of 3 nm. The ¯uorescence light emitted orthogonal to the excitation at 535 nm (535 nm interference ®lter with 10 nm bandwidth) was detected using a photomultiplier. During each 30-s measurement interval cells were kept dark for 20 s in order to prevent the dye photobleaching. The signals of the photomultiplier were collected using a 12-bit A/D converter on a personal computer with self-programmed BASIC software which also controlled the monochromator, shutter and photomultiplier. The ¯uorescence excitation ratio R F(500 nm)/F(440 nm) was calculated from raw data after subtraction of scattering which was measured before dye incubation in each experiment. The calibration following each experiment was done with the high [K+]ex (130 mM) and Nigericin (10 lM) method (Thomas et al. 1979). Extracellular pH values were calibrated over the range of pH 6.0±8.0. The pHi values were then calculated according to the Henderson-Hasselbalch equation pHi pK + log[(R ) Rmin)/ (Rmax ) R)].


38 K. Glunde et al.

Incubation, perchloric acid extraction and protein determination Experiments were performed after the cells had reached con¯uence. Approximately 108 cells were washed twice with 5 mL PBS per dish and then incubated with Krebs±Henseleit (KHB) buffer or bicarbonate-free HEPES buffer containing 5 mM [1-13C]glucose at 37°C. For KHB, different extracellular pH values were set by means of bicarbonate buffers and the CO2 content in the air. An extracellular pH (pHe) of 7.4 was adjusted with 26 mM NaHCO3 and 5% CO2/95% air, pHe 6.4 with 6 mM NaHCO3 and 20% CO2/ 80% air. The pH values of the HEPES buffers were adjusted with HCl or NaOH. Extracellular acidi®cation was maintained for 2 h. Subsequently, buffers were exchanged and pH recovery with pHe 7.4 lasted 10 or 20 min. Cells were then extracted. Hypoxia was imposed by gassing the used KHB (pH 7.4) and the incubation chamber with 95% N2/5% CO2 in a humidi®ed atmosphere at 37°C. The incubation buffer contained 5 mM [1-13C]glucose. These conditions were maintained for 2 h. After removal of the experimental medium the cells were immediately washed twice with 5 mL ice-cold isotonic saline, frozen in liquid nitrogen and extracted with 2 ´ 2 mL 12% perchloric acid (PCA). The suspension was centrifuged and the aqueous layer removed, neutralized and lyophilized. The pellet remaining after PCA extraction was resuspended in 3 mL H2O. The protein content was quanti®ed from the pellet using a slightly modi®ed Biuret reaction (Goa 1953; FloÈgel et al. 1995). Visual absorption was recorded at 540 nm. Protein concentrations were calculated using bovine serum albumin as a standard. NMR measurements PCA cell extracts were dissolved in 0.5 mL D2O, centrifuged and the pH standardized to 7 using deuterated sodium hydroxide (NaOD) or hydrochloric acid (DCl). After measuring 1H- and 13 C NMR spectra, 100 mmol/L EDTA were added to the sample to complex divalent cations, the pH was readjusted to 7 and 31P NMR spectra were acquired. NMR spectra were recorded on Bruker AMX 360 and AM 360 NMR spectrometers operating at frequencies of 360 MHz for 1H, 145.7 MHz for 31P and 90.5 MHz for 13C measurements. 13 C NMR spectra were measured with a 5-mm 1H/13C dual probe, 20 000 accumulations, ¯ip angle 27°, repetition time 2.5 s, composite pulse decoupling with WALTZ-16, spectral width 20 833 Hz, data size 16 K, zero ®lling to 32 K, exponential weighting with a 2-Hz line broadening. Chemical shifts were referenced to the C3 signal of lactate at 21.3 p.p.m. Fully relaxed 1H NMR spectra were recorded for absolute quanti®cation of the metabolites with a 5-mm HX probe, 500 accumulations, ¯ip angle 25°, repetition time 15 s, spectral width 3600 Hz, data size 16 K, zero ®lling to 32 K. The lactate resonance at 1.33 p.p.m. was used as internal reference for chemical shifts of metabolites. Absolute quanti®cation of metabolites in fully relaxed 1H NMR spectra were performed using 59 lM (trimethylsilyl)propionic-2,2,3,3,d4-acid (TSP) as an external standard. The concentrations of all metabolites were calculated with respect to the protein content ([nmol]/[mg protein]) obtained as described above. 31 P NMR spectra were recorded with a 5-mm HX probe, 5000 accumulations, ¯ip angle 80°, repetition time 3.5 s, spectral width

5155 Hz, composite pulse decoupling with WALTZ-16. Chemical shifts were referenced to phosphocreatine at )2.33 p.p.m. The 13C enrichment was calculated after subtraction of natural abundance 13C contribution of a metabolite based on the known myo-inositol concentration as an internal standard. The 13C NMR spectra were corrected for the nuclear Overhauser effect (NOE) by comparison with a standard mixture of amino acids measured under the same recording conditions. Fractional enrichments were calculated according to Badar-Goffer and Bachelard (1991). HOE642 concentration HOE642 concentrations of 10 or 100 lM were used for the NMR or ¯uorescence spectroscopy experiments, respectively. It has been shown that HOE642 exhibits neither acute nor chronic toxicity in the concentration range 1 lM to 1 mM HOE642 for C6 and F98 glioma cells (Glunde 2000). HOE642 inhibits NHE-1 with a half inhibitory concentration (IC50) between 0.1 and 1 lM depending on the cell type (Scholz et al. 1995). Because the aim of this study was to investigate NHE-1 related mechanisms of glial pHi regulation and metabolism, HOE642 was used at relatively high concentrations to observe maximal effects. The applied HOE642 doses were higher than its IC50 for NHE-1, but much lower than its toxicity limit. HOE642 (10 lM) was used to prepare PCA extracts measured by multinuclear NMR spectroscopy in order to provoke sizeable effects within an incubation time of 2 h to allow for the relatively insensitive NMR spectroscopy. The experiments described in Ômetabolic effects of extracellular acidi®cationÕ were carried out with 10 lM and 1 mM HOE642. No signi®cant differences in metabolite levels or fractional 13C enrichments were detected between experiment with these two different concentrations. For ¯uorescence measurements of pHi in living cells, 100 lM HOE642 was used in the perfusion buffer to achieve complete blockage of NHE-1 with respect to the ¯ow rate of 0.5 mL/min. Data analysis All results are presented as mean SD values. Differences between results were tested by Student's t-test. A probability of p < 0.05 was considered signi®cant.

Results

Intracellular pH The steady-state pHi was 7.14 0.04 (n 8; Fig. 2) during perfusion at an extracellular pH (pHe) of 7.4 with HEPESbuffered medium. Lowering the pHe to 6.4 decreased pHi slowly to 6.55 0.04 (n 4; Fig. 2a, Table 1). Derivation of the data showed that the maximum absolute rate of d(pHi)/ dt )0.060 [min)1] was reached 3 min after onset of pHe 6.4 (Fig. 3a, Table 1). The rate then slowly returned to zero and the new steady-state pHi was reached after 20 min. Reconstitution of pHe 7.4 recovered pHi to the control steady-state value. pHi was restored with a maximum rate d(pHi)/dt +0.174 [min)1] 3 min after reconstitution of control conditions (Fig. 3a, Table 1). Perfusion with HEPES buffer at pHe 7.4 in the presence of 100 lM HOE642 reduced pHi to 6.99 0.05 (n 4;



Fig. 2 Time course of intracellular pHi during extracellular acidi®cation and subsequent pHe recovery without (a, h) and with (b, s) additional inhibition of Na+/H+ exchange subtype 1 by HOE642. The values are means SD (each n 4).

Fig. 2b). On switching to pHe 6.4 in the presence of the NHE-1 inhibitor pHi decreased more quickly to 6.40 0.05 (Fig. 2b, Table 1) with a more pronounced maximum rate d(pHi)/dt )0.145 [min)1] reached earlier, 1 min after acidification (Fig. 3b). The equilibration process was finished after 10 min. Reperfusion with pHe 7.4 and 100 lM HOE642 restored pHi to control values. The maximum rate of this increase was lower d(pHi)/dt +0.092 [min)1], which was reached 3 min after restoration (Fig. 3b, Table 1). The final control perfusion with HEPES pH 7.4 led to the control pHi of 7.14 0.05 (n 4; Fig. 2b). Metabolic effects of extracellular acidi®cation Two hours of extracellular acidification decreased the [PCr]/ [Cr] ratio significantly to 67 8% of control. On subsequent

Fig. 3 Differential quotient d(pHi)/dt of the means (each n 4) of intracellular pHi per time during extracellular acidi®cation and subsequent pHe recovery without (a) and with (b) additional inhibition of Na+/H+ exchange subtype 1 by HOE642.

reconstitution of pHe 7.4, PCr recovered to control values within 20 min (n 6; Fig. 4). However, the presence of 10 lM as well as 1 mM HOE642 caused an even more pronounced decrease in the [PCr]/[Cr] ratio to 21 5% of control. It remained at this low level during extracellular pH recovery (n 6; Fig. 4). Nucleoside triphosphate levels remained constant. The corresponding 13C NMR spectra of the glioma cell lines showed strongly decreased cytosolic concentrations of labelled metabolites produced from [1-13C]glucose after the period of extracellular acidification. Enrichments of Ala C3, Lac C3, and Glu C4 decreased to less than half of the control values. Concomitant with the pH recovery the 13C enrichment was restored in all metabolites within 20 min (n 6; Figs 5 and 6). Upon extracellular acidification, absolute quantification of metabolite concentrations from 31P and 13C NMR spectra

Table 1 Summary of results: intracellular pHi and its derivation per time during extracellular acidi®cation and pHe recovery in the absence and presence of the inhibition of Na+/H+ exchange subtype 1 by HOE642 in C6 glioma cells.

Experimental condition

Intracellular pH pHe 7.4*

Control 7.14 0.04 100 lM HOE642 6.99 0.05

pHe 6.4*

Extracellular acidi®cation (d(pHi)/dt)min [1/min]***

at time [min]

pHe recovery equil. time [min]

(d(pHi)/dt)max [1/min]***

at time [min]

equil. time [min]

6.55 0.04 6.40 0.05

)0.060 )0.145

30.5 28.0

20 10

+ 0.174 + 0.092

91.5 91.5

10 20

The pHi values are means SD (n 4), for calculation see Materials and methods. *p < 0.05, ***p < 0.001 vs. control. Minima and maxima are taken from the derivation curve (see Fig. 3).


40 K. Glunde et al.

Fig. 4 Representative sections of 1H NMR spectra from control, extracellular acidi®cation (2 h) and subsequent pHe recovery (10 and 20 min) of F98 glioma cell extracts (ai) and the same experimental set-up with addition of 10 lM or 1 mM HOE642 (aii). Absolute contents of PCr and Cr were quanti®ed as [PCr]/[Cr] ratio and related to the control value to give percentage of control (b). The values are means SD (each n 6). ns, Not signi®cant p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Assignments: PCr, phosphocreatine; Cr, creatine.

Fig. 5 Representative sections of 13C NMR spectra from control, extracellular acidi®cation (2 h) and subsequent pHe recovery (10 and 20 min) of F98 glioma cell extracts. Assignments: Glc, glucose (a- and b-anomer); Ins, myo-inositol; Gro, glyceryl-derivatives; Gro-3-P, glycerol-3-phosphate; Glu, glutamate; Lac, lactate; Ala, alanine.



Fig. 6 Fractional 13C enrichment changes of alanine C3, lactate C3 and glutamate C4 and cytosolic concentrations of glycerol-3-phosphate and glyceryl derivatives upon extracellular acidi®cation (2 h) and subsequent pHe recovery (10 and 20 min) of F98 glioma cell extracts compared to control conditions. The values are means SD (each n 6). ns, Not signi®cant p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

showed a strong intensity reduction of C1 of glycerophosphocholine and glycerophosphoethanolamine (Glyceryl-C1) to 55 9% of the control level, and of glycerol-3-phosphate (Gro-3-P) to 47 12%, respectively. The subsequent recovery of these metabolites was also completed within 20 min, at which Glyceryl-C1 was increased transiently to 132 12% (n 6; Figs 5 and 6). The metabolic results described above were obtained from experiments carried out both in KHB or bicarbonate-free HEPES buffer. Metabolic effects of hypoxia After 2 h of hypoxia, the [PCr]/[Cr] ratio was reduced to 45 6% of control, whereas cytosolic inorganic phosphate was increased to 142 10% of control (n 6; Fig. 7). In the presence of 10 lM HOE642 during oxygen deprivation, the [PCr]/[Cr] ratio decreased to 87 8% of control only. This is comparable with the control incubation with

Fig. 7 Changes in [PCr]/[Cr] ratio and inorganic phosphate in the presence of 10 lM HOE642 (2 h, light bars), during hypoxia (2 h, grey bars) and during hypoxia plus presence of 10 lM HOE642 (2 h, black bars) of F98 glioma cell extracts compared with control conditions without HOE642. The values are means SD (each n 6). ns, Not signi®cant p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Fig. 8 Changes in cytosolic concentrations of lactate C3, alanine C3 and glycerol-3-phosphate (a) and 13C enrichments of lactate C3 and alanine C3 (b) in the presence of 10 lM HOE642 (2 h, light bars), during hypoxia (2 h, grey bars) and during hypoxia plus presence of 10 lM HOE642 (2 h, black bars) of F98 glioma cell extracts compared to control. The values are means SD (each n 6). ns, Not signi®cant p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

HOE642, but without hypoxia, which was 85 7% (n 6; Fig. 7). Hypoxia increased the cytosolic lactate concentration slightly to 112 5% of control (n 6; Fig. 8a). 13C enrichment at lactate C3 was increased to 133 7% (n 6; Fig. 8b). 13C and 1H NMR spectra of the incubation media indicated an enormous export of labelled lactate, which was increased to 263 8% of the fractional enrichment of the control medium (n 6; data not shown). Alanine was also markedly increased to 151 7% of the control concentration (n 6; Fig. 8a) and its enrichment at C3 was doubled (n 6; Fig. 8b). Glycerol-3-phosphate was increased twofold during oxygen deprivation (n 6; Fig. 8a). With respect to these metabolites, additional incubation with 10 lM HOE642 had no significant effects during hypoxia (n 6; Fig. 8). Discussion

Recent investigations characterized HOE642 (Fig. 1) as an efficient and very specific inhibitor of NHE-1 (Scholz et al. 1995). In contrast, amiloride has been shown to nonspecifically inhibit all Na+/H+ exchanger isoforms, the Na+


42 K. Glunde et al.

channel and the Na+/Ca2+ exchanger (Kleyman and Cragoe 1988). Furthermore, amiloride accumulates within the cells because of its lipophilicity (Benos et al. 1983). This may affect the Na+/H+ exchange in various ways, such as by inhibition of intracellular proteins, e.g. protein kinases (Ralph et al. 1982; Besterman et al. 1985) and adenylate cyclase (Mahe et al. 1985), which may be involved in the 1 regulation of Na+/H+ exchange (Moolenaar et al. 1984; Reuss and Petersen 1985), or through increasing intracellular pH which may inactivate the Na+/H+ exchanger (Aronson et al. 1982). Bearing this in mind, earlier investigations based on amiloride (Sapirstein and Benos 1984; FloÈgel et al. 1994) are questionable with respect to the specific mechanism involved. HOE642, however, affects the Na+/H+ exchanger subtype 1 (NHE-1) only. This study using HOE642 clearly shows that NHE-1 exists in both investigated glioma cell lines. Data obtained by fluorescence spectroscopy of BCECFloaded viable, immobilized C6 glioma cells in HEPES-buffered media confirm an active pHi regulation at physiological pHe 7.4. This is proved by the decrease in pHi of 0.15 units after exposure to the Na+/H+ exchange subtype 1 inhibitor HOE642 at pHe 7.4. If active pHi regulation were absent, pHi would be adapted to the Donnan equilibrium, and should therefore be calculated according to Roos and Boron (1981) as 6.59 for C6 glioma cells assuming a temperature of 37°C, a pHe of 7.4 and a membrane potential of )50 mM (Strupp et al. 1993). Although our data show that the NHE-1 is involved in the regulation of pHi in bicarbonate-free media, its contribution to pHi control at physiological pHi values appears relatively low. This is in good agreement with 2 results of Counillon and PouysseÂgur (1995), who characterized the allosteric activation of Na+/H+ exchangers by intracellular acidification. Therefore, Na+/H+ exchange is negligible at physiological pHi, but as the pHi decreases, the antiporter is rapidly activated and reaches its maximum transport rate upon acidification by < 1 pH unit. Furthermore, the pHi dependence of the Na+/H+ exchanger activity varies according to cell type and membrane origin (Frelin et al. 1988). At a pathological pHe of 6.4, HOE642 clearly inhibits the rate of NHE-1 in C6 glioma cells as indicated by the increasing response time of pHi adaptation upon extracellular acidification in the presence of HOE642 (Fig. 3, Table 1). Furthermore, HOE642 slows down the rate of pHi recovery and doubles its equilibration time. This confirms the hypothesis of NHE-1 activation at high proton concentrations, as the strongest inhibition by HOE642 is manifest if pHi decreases below the physiological level. Therefore, NHE-1 of this glial cell line is an important part of pH regulation at pathologically low pHi values, that could occur during cerebral ischaemia. In contrast to former investigations performed with amiloride, our data do not support the hypothesis of a fully inactive Na+/H+ exchanger at physiological pHi in C6 cells (Jean et al. 1986). Rather, the

observations in the presence of HOE642 show that NHE-1 contributes to glial pHi control in bicarbonate-free HEPESbuffered media under physiological, and in particular pathologically low, pH conditions. The metabolic findings support a reversible disorder of the energy state due to extracellular acidification in C6 and F98 glioma cells. This is indicated by the decrease in the [PCr/Cr] ratio following extracellular acidification, monitored by 1 H NMR spectroscopy of PCA extracts, and the subsequent restoration of PCr within the 20 min of pH recovery (Figs 4ai and b). In the presence of HOE642, however, the decrease in [PCr/Cr] ratio is greater during extracellular acidification and PCr is not restored within 20 min of pH recovery (Figs 4aii and b). This is remarkable, as the pH recovers in the presence of HOE642 to almost physiological pHi within 20 min (Table 1). 13C (Fig. 5) and 31P NMR spectra reveal similar reversible decreases in several metabolites derived from glycolysis and the TCA cycle, such as Lac, Ala, Gro-3-P, Glyceryl-C1 and Glu (Fig. 6). This means that glycolysis and the subsequent TCA cycle are significantly, but reversibly, hindered during extracellular acidification. It has been shown that phosphofructokinase (PFK, EC 2.7.1.11), the rate-limiting enzyme in glycolysis, is inactivated by decreasing pHi values (Fidelman et al. 1982). Consequently, the increase in intracellular acidification results in a shortage of acetyl-CoA and subsequent metabolites. Reversible metabolic inhibition upon extracellular acidification was present in both cases, HOE642 present or absent. Thus, the lack of PCr recovery after reconstitution of pHe 7.4 caused by HOE642 cannot be explained by the slower pHi recovery. Although, at first sight, there seems to be a correlation between the time course of pHi recovery and restoration of the high-energy phosphate PCr, which could be mediated by the re-upregulation of PFK. However, the lack of PCr recovery in the presence of HOE642 is in contrast to the slower, but nonetheless occurring pHi recovery and the reversibility of the metabolic inhibition in the presence of this inhibitor. Obviously, a mechanism other than metabolic inhibition via proton mediated inactivation of PFK is responsible for this phenomenon. Eventually, the pH-dependent creatine kinase reaction may account for this. Glial hypoxia in this cellular model increased both absolute cytosolic concentrations and 13C enrichments of Lac and Ala in C6 and F98 glioma cells, whether HOE642 is present or not (Fig. 8). In addition, large amounts of labelled Lac are exported into the extracellular space. The corresponding 31 P NMR spectra showed a strong increase in the cytosolic Gro-3-P concentration. The inhibition of oxidative phosphorylation under prolonged hypoxic conditions decreases the rate of pyruvate oxidation by the TCA cycle and thereby increases the NADH pool. Therefore, the alternative source of ATP production is anaerobic glycolysis. The resulting pyruvate and NADH accumulation is controlled by lactate dehydrogenase (EC 1.1.1.27), leading to increased cytosolic



lactate concentrations, which are responsible for lactacidosis. If the regeneration of NAD+ via lactate dehydrogenase is overcome, the NAD+-dependent glycerol 3-phosphate dehydrogenase (Gro-3-P-DH, EC 1.1.1.177, EC 1.1.1.8) reaction is used to maintain the cytoplasmic NADH/NAD+ redox state (Ben-Yoseph et al. 1993; FloÈgel et al. 1994). We also observed a significant increase in the alanine concentration and in de novo synthesis from the enlarged pyruvate pool during hypoxia, which has also been reported by Ben-Yoseph et al. (1993) in cerebral)cortex slices. This may be part of the secondary excitotoxic cell death in chronic ischaemia due to alanine-mediated exaggerated synthesis of the excitatory amino acid aspartate (Griffin et al. 1998). During hypoxia, HOE642 has no additional effects on these metabolic features typically observed in hypoxic glial cells (Fig. 8). The cellular energy charge in hypoxic glial cells, reflected by the [PCr/Cr] ratio, is strongly reduced (Fig. 7), although the ATP level is maintained by an increased pseudo-firstorder rate constant of the creatine kinase (EC 2.7.3.2) reaction, which regenerates ATP at the expense of PCr (Cox et al. 1988). Therefore, cytosolic inorganic phosphate is increased, reflecting the loss of PCr (Fig. 7). Interestingly, the inhibition of NHE-1 by addition of 10 lM HOE642 protects the energy state during hypoxia in glial cells because the [PCr/ Cr] ratio retains control values (Fig. 7). These data support the hypothesis that the inhibition of NHE-1 has protective effects during cerebral ischaemia as suggested previously (Tombaugh and Sapolsky 1993; Vornov et al. 1996). In summary, these results show that the NHE-1 is an important part of glial pHi regulation especially at pathologically low extracellular pH values. In addition, even at physiological pHe NHE-1 is involved in maintaining the pHi against the Donnan equilibrium to a relatively small, but sizeable extent. From a metabolic point of view, the pHi decrease leads to a disorder of the energy state and inhibition of glycolysis and their subsequent biochemical pathways. HOE64-mediated aggravation of this phenomenon is independent of the low pHi. Interestingly, inhibition of NHE-1 with HOE642 during hypoxia protects the energy state and has no additional effects on the bulk metabolism in glial cells during hypoxia, to the extent that it is measurable by NMR spectroscopy. Acknowledgement We thank Professor Dr A. Mayer-Heinricy for providing us with a home-built ¯uorescence spectrometer and for his expert assistance.

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