Inhibition of glutamine transport into mitochondria protects astrocytes from ammonia toxicity

GLIA 55:801–809 (2007)

Inhibition of Glutamine Transport into Mitochondria Protects Astrocytes from Ammonia Toxicity V. B. R. PICHILI,1 K. V. RAMA RAO,1 A. R. JAYAKUMAR,1 AND M. D. NORENBERG1–3* 1 Department of Pathology, University of Miami School of Medicine, Miami, Florida 2 Veterans Affairs Medical Center, University of Miami School of Medicine, Miami, Florida 3 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida

KEY WORDS ammonia; astrocytes; ATP; free radicals; glutamine; hepatic encephalopathy; histidine; mitochondrial permeability transition; phosphate-activated glutaminase

ABSTRACT Hepatic encephalopathy (HE) is a major neurological complication that occurs in the setting of severe liver failure. Ammonia is a key neurotoxin implicated in this condition, and astrocytes are the principal neural cells histopathologically and functionally affected. Although the mechanism by which ammonia causes astrocyte dysfunction is incompletely understood, glutamine, a by-product of ammonia metabolism, has been strongly implicated in many of the deleterious effects of ammonia on astrocytes. Inhibiting mitochondrial glutamine hydrolysis in astrocytes mitigates many of the toxic effects of ammonia, suggesting the involvement of mitochondrial glutamine metabolism in the mechanism of ammonia neurotoxicity. To determine whether mitochondria are indeed the organelle where glutamine exerts its toxic effects, we examined the effect of L-histidine, an inhibitor of mitochondrial glutamine transport, on ammonia-mediated astrocyte defects. Treatment of cultured astrocytes with L-histidine completely blocked or significantly attenuated ammonia-induced reactive oxygen species production, cell swelling, mitochondrial permeability transition, and loss of ATP. These findings implicate mitochondrial glutamine transport in the mechanism of ammonia neurotoxicity. V 2007 Wiley-Liss, Inc. C

INTRODUCTION Liver failure represents the fourth leading cause of death in the United States, and approximately 75% of the patients with severe liver disease develop neurological abnormalities collectively referred to as hepatic encephalopathy (HE) (Lockwood, 1992). Chronic HE, the more common form of this condition, is usually a consequence of alcoholic cirrhosis and presents with changes in personality, altered mood, declining intellectual capacity, and abnormal muscle tone (Jones and Weissenborn, 1997). Acute HE, a complication of acute liver failure (ALF), also known as fulminant hepatic failure, generally occurs in previously healthy individuals as a result of viral hepatitis, acetaminophen toxicity, and exposure to various hepatotoxins, and presents with the abrupt onset of delirium, seizures, and coma. The principal neuropathological finding is severe brain edema, which C 2007 V

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leads to increased intracranial pressure and brain herniation. Acute HE is associated with a high mortality rate (Capocaccia and Angelico, 1991), and currently there is no effective treatment for the brain edema in ALF other than an emergency liver transplantation (Bismuth et al., 1988; Hoofnagle et al., 1995). Although the molecular basis underlying HE remains unclear, clinical, pathological, and neurochemical evidence suggests that elevated blood and brain ammonia levels are critical in its pathogenesis (Butterworth, 2002). Ammonia neurotoxicity involves altered energy metabolism, abnormal neurotransmitter function, electrophysiological derangements, and changes in ionic homeostasis (Albrecht and Jones, 1999). More recent studies have implicated oxidative stress and the mitochondrial permeability transition (MPT) in the mechanism of ammonia neurotoxicity (Norenberg et al., 2004; Rama Rao et al., 2003a). Several lines of evidence indicate that the pathogenesis of both acute and chronic HE primarily represent a ‘‘gliopathy’’ wherein astrocytes are the principal cells affected (Norenberg, 1998). Astrocytic changes are dominant in the histopathology of HE, with Alzheimer type II astrocytic changes found in chronic HE and cell swelling occurring in acute HE (Norenberg, 1981). Such astrocyte involvement is likely due to the fact that ammonia is mainly metabolized to glutamine, a process catalyzed by glutamine synthetase, an enzyme primarily localized in astrocytes (Norenberg and Martinez-Hernandez, 1979). Accordingly, levels of glutamine in brain and cerebrospinal fluid (CSF) have been shown to be highly elevated in both acute and chronic HE (Hourani et al., 1971; McConnell et al., 1995; Record et al., 1976). Because brain and CSF glutamine levels are elevated in HE, the possibility that glutamine might be toxic to the CNS has long been considered. In support of this view, methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, has been shown to diminish many of Grant sponsors: NIH; Department of Veterans Affairs, American Association for the Study of Liver Disease, American Liver Foundation; Grant number: DK063311. *Correspondence to: Michael D. Norenberg, Department of Pathology, University of Miami School of Medicine, PO Box 016960, Miami, FL 33101, USA. E-mail: [email protected] Received 11 December 2006; Revised 27 January 2007; Accepted 6 February 2007 DOI 10.1002/glia.20499 Published online 13 March 2007 in Wiley InterScience (www.interscience. wiley.com).

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the toxic effects of ammonia, including a reduction in seizures and death (Warren and Schenker, 1964), inhibition of brain edema (Blei et al., 1994; Takahashi et al., 1991), attenuation of astrocyte swelling (Norenberg and Bender, 1994; Willard-Mack et al., 1996), and recovery of altered cerebral metabolic rate (Hawkins et al., 1993). For review see (Albrecht and Norenberg, 2006). Although the effects of MSO indirectly implicate glutamine in the abnormalities associated with HE/hyperammonemia, recent studies have shown that glutamine can directly exert toxic effects on cultured astrocytes by increasing reactive oxygen species (ROS) production as well as by inducing the MPT (Jayakumar et al., 2004; Rama Rao et al., 2003b). Such effects of glutamine were either completely or significantly inhibited by 6-diazo-5oxo-L-norleucine (DON), an inhibitor of mitochondrial phosphate-activated glutaminase (PAG), suggesting that the hydrolysis of glutamine in astrocytic mitochondria, and the resulting generation of high levels of ammonia in that organelle, represents a key mechanism of ammonia neurotoxicity (Jayakumar et al., 2006; Rama Rao et al., 2005a). To further test the view that mitochondria are indeed the critical organelles in which glutamine initiates its toxic action, this study examined the effect of L-histidine, an inhibitor of mitochondrial glutamine transport (Albrecht et al., 2000), on ammonia-induced toxic effects in cultured astrocytes. Our study demonstrates that inhibition of glutamine transport by histidine blocks or attenuates the deleterious effects of ammonia, including free radical production, induction of the MPT, astrocyte swelling, and loss of ATP.

MATERIALS AND METHODS Astrocyte Cultures Primary cultures of astrocytes were prepared as described by Ducis et al. (1990). Briefly, dissociated cells from the cerebral cortices of 1 to 2-day-old rats were plated in 35 mm dishes at a density of 0.5 3 106 in Dulbecco’s Modified Eagle Medium (DMEM). After 2 weeks, cells were maintained with dibutyryl cAMP and 3 to 5week-old cells were used for the experiments. Cultures consisted of 95–99% astrocytes based on immunohistochemistry for glial fibrillary acidic protein and glutaAbbreviations

ALF ATP CNS CsA DCFDA DMEM DON HE MPT MSO OMG PAG ROS TMRE

acute liver failure adenosine-50-triphosphate central nervous system cyclosporin A dichlorofluorescein diacetate Dulbecco’s modified Eagle medium 6-diazo-5-oxo-L-norleucine hepatic encephalopathy mitochondrial permeability transition methionine sulfoximine O-methyl-glucose phosphate-activated glutaminase reactive oxygen species tetramethylrhodamine ethyl ester.

GLIA DOI 10.1002/glia

mine synthetase. For each experiment, the required number of plates was selected randomly from at least two different seeding batches.

Glutamine Transport in Mitochondria For mitochondrial isolation, astrocyte cultures were grown on 100 mm culture dishes at a density of 5 3 106 cells/ plate, and 3–4 plates were pooled in each experiment. Mitochondria were isolated from cultured astrocytes by the method of Almeida and Medina (1998). Cultures were washed three times with ice-cold PBS and cells were harvested into 0.32 M sucrose, homogenized using a motor driven Potter-Elvehjem homogenizer, and subjected to low-speed centrifugation (2,500 rpm) for 8 min. The supernatant was then centrifuged at 14,000 rpm for 30 min. The pellet was washed 2–3 times with ice-cold 0.32 M sucrose and recentrifuged. The final pellet was used for transport studies. To compare the purity (enrichment) of mitochondria used in the study, activities of citrate synthase were measured in both homogenate and mitochondrial fractions employing the method of Shepherd and Garland (1969). Mitochondrial transport of [14C]-glutamine was studied following the method of Roberg et al. (1999). Briefly, to the reaction mixture containing 130 mM KCl, 5 mM MgSO4, 20 mM sucrose, 20 mM HEPES (pH 7.4), 100 lM glutamine, and 0.5 lCi [14C]-glutamine, with or without 50–1,000 lM histidine, 100 lg of mitochondria were added and incubated at 37°C for 3 min. Transport was terminated by rapid filtration of samples under vacuum. Samples were washed four times with a large amount of ice-cold reaction buffer, filters were dried overnight and the radioactivity retained in the filters was determined using a Packard-Tricarb liquid scintillation counter. Nonspecific transport was determined by incubating samples at 0°C. The specific transport of glutamine was estimated by subtracting the nonspecific transport from the total transport.

Measurement of Free Radicals Free radical production was determined by the method of Thorburne and Jurlink (1996) employing dichlorofluorescein diacetate (DCFDA), as modified by Jayakumar et al. (2004). Results were expressed as fluorescence units/ mg protein.

Measurement of the Mitochondrial Membrane Potential (DWm) The DWm was measured as described by Rao and Norenberg (2004). The potentiometric fluorescent dye, TMRE, was loaded at a final concentration of 50 nM for 20 min. Cells were excited at 488 nm, and the fluorescence emission was recorded at 590 nm. Images were taken with a Nikon Diaphot inverted fluorescent microscope equipped

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with multivariant fluorescent filters. Fluorescent intensities were analyzed using Sigma Scan Pro; total number of pixels were quantified on a grey scale (0–255) and the average pixel (fluorescent intensity) value in each image was obtained and expressed as the mean 6 SEM of the total fluorescence intensity from at least 15 random image fields in each group.

Cell Volume Determination Astrocyte cell volume was determined by measuring the intracellular water space by the method of Kletzien et al. (1975), as modified for cell cultures by Kimelberg (1987) and Norenberg et al. (1991). 1 mM 3-OMG and 0.5 lCi/mL [3H]-3-OMG were added to the cultures 12 h prior to the volume assay. At the end of the incubation period, culture medium was aspirated and an aliquot was saved for radioactivity determination. The cells were rapidly washed six times with ice-cold buffer containing 229 mM sucrose, 1 mM Tris, and 0.5 mM calcium nitrate (pH 7.4). Phloretin (0.1 mM) was dissolved in absolute alcohol and added to the buffer. Cells were harvested into 0.5 mL of 1 N sodium hydroxide, and the radioactivity in the cell extract was counted using a scintillation spectrometer (Packard Tricarb, Meriden, CT). The remaining cell extract was used for protein estimation with the Bio-Rad bicinchoninic acid kit (Pierce). The amount of radioactivity in the cell extract was transformed to intracellular water space as described by Kletzien et al. (1975) and expressed as microliter/milligram protein.

Measurement of ATP At the end of treatment, cultured astrocytes were quickly washed with ice-cold PBS, rapidly frozen and immediately extracted into 12% TCA and centrifuged at 12,000 rpm The supernatant was used for the estimation of ATP using the Sigma Diagnostic kit (Cat no. 366UV) based on the method of Adams (1963). This method measures the decrease in absorbance because of the oxidation of NADH in enzyme-coupled reactions. Briefly, to the reaction mixture containing phosphoglyceric acid buffer (18 mM), NADH (1 mg/mL) and the TCA supernatant, glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase were added in a 2:1 ratio, and the oxidation of NADH monitored spectrophotometrically at 340 nm.

Statistical Analysis Data are represented as mean 6 SEM. The data were analyzed by Neuman-Keuls multiple test. Each experiment was repeated at least three times with five plates in each group.

Fig. 1. Inhibition of glutamine transport into mitochondria by histidine. Histidine (50, 100, and 1,000 lM) significantly blocked glutamine transport into mitochondria. *P < 0.01 versus glutamine. The rate of transport of glutamine in control mitochondria is 6 6 0.2 nmol/mg protein/min. Gln, glutamine; His, histidine.

RESULTS Effect of Histidine on Mitochondrial Glutamine Transport Albrecht et al. (2000) had previously investigated the inhibitory potency of several amino acids on mitochondrial glutamine transport and found that, of all the amino acids tested, histidine inhibited glutamine transport with the greatest potency (>60% in nonsynaptic mitochondria). As there is no information available on the effect of histidine on mitochondria derived from cultured astrocytes, mitochondria were isolated from cultured astrocytes and the effect of histidine on glutamine transport examined. First, the purity of isolated mitochondria was determined by assaying the activity of citrate synthase, which showed a 4-fold enrichment in the mitochondrial fraction as compared with crude homogenates of astrocytes (117 nmol/mg protein/min in mitochondria vs. 29.5 nmol/mg protein/min in crude homogenates). We then examined the effect of different concentrations of histidine (50–1,000 lM) on the transport of glutamine (100 lM) into mitochondria isolated from cultured astrocytes. In control mitochondria, the rate of glutamine (100 lM) transport was found to be
6 nmol/ mg protein/min. In the presence of histidine at 50, 100, and 1,000 lM, glutamine transport was inhibited significantly by 29 (P < 0.05), 32, and 49% (P < 0.05), respectively (Fig. 1). The percent change between different concentrations (50–1,000 lM) of histidine on glutamine transport was not statistically significant. These data are similar to an earlier report demonstrating the inhibitory GLIA DOI 10.1002/glia

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5 mM ammonia showed a 40% loss of TMRE fluorescence, consistent with the dissipation of the DWm, and such loss was completely blocked by 1 lM CsA, indicating that ammonia induces the MPT. A 10 min pretreatment of cultures with L-histidine (50–1,000 lM) significantly blocked the ammonia-induced MPT to a variable degree. Lower concentrations of histidine (50 and 100 lM) completely blocked the ammonia-induced dissipation of DWm, whereas, surprisingly, a higher concentration (1 mM) was less effective (45–50%, P < 0.05) in protecting against the ammonia-induced MPT (see Fig. 4). At all the concentrations used, histidine alone had no effect on TMRE fluorescence (data not shown).

Effect of Histidine on Ammonia-Induced Astrocyte Swelling

Fig. 2. Effect of L-histidine on ammonia-induced reactive oxygen species (ROS) production in astrocytes. Astrocytes exposed to ammonia showed a significant increase in ROS production, and such increase was completely blocked by 1 mM histidine, while 100 lM caused a 49% blockade. *P < 0.05 versus control. **P < 0.05 versus NH4. C, control; NH4, NH4Cl (5 mM).

effects of histidine on glutamine transport in nonsynaptic mitochondria (Albrecht et al., 2000). Effect of Histidine on Ammonia-Induced Free Radical Generation Treatment of astrocytes with ammonia (5 mM NH4Cl) resulted in a 100% increase in free radical production, which is consistent with our earlier studies (Murthy et al., 2001). The concentration of ammonia used in the present study is pathophysiologically relevant, since such levels of ammonia are observed in brain in animal models of ALF (Swain et al., 1992). To determine whether histidine blocks free radical production, cultures were pretreated with 100 lM and 1 mM histidine. One micromolar histidine completely blocked free radical production while with 100 lM a 49% (P < 0.05) reduction was observed (Fig. 2). At both 100 lM and 1 mM concentrations, histidine alone had no effect on ROS content (data not shown).

Astrocyte cell volume was determined after exposure of cultures to NH4Cl (5 mM) for 24 h. The intracellular space in the control groups was found to be 5.7 6 0.13 lL/ mg protein. Addition of ammonia to the cultures increased cell volume by 44% (Fig. 5). The effect of histidine on ammonia-induced astrocyte swelling was examined by treating cells with different concentrations of histidine (50–1,000 lM), 10 min before exposure to ammonia. At all concentrations, histidine significantly (60%, P < 0.01) blocked ammonia-induced astrocyte swelling (Fig. 5). Histidine alone had no effect on cell volume (data not shown). Ammonia-induced astrocyte swelling has been shown to be attenuated by MSO an inhibitor of glutamine synthesis (Willard-Mack et al., 1996). Since in the present study histidine only partially inhibited such swelling (Fig. 5), we examined whether a combination of MSO and histidine had additive effects on ammonia-induced astrocyte swelling. Such an additive effect, if present, would support the concept that glutamine formation, as well as its transport into mitochondria, are key factors in the mechanism of ammonia-induced astrocyte swelling. Accordingly, cultures were treated with MSO (3 mM) and histidine (1 mM), alone and in combination, and cell volume was determined. MSO blocked ammonia-induced astrocyte swelling by 67% (P < 0.05), while histidine inhibited cell volume by 60% (P < 0.05). The combination of MSO and histidine blocked such swelling by 100% (P < 0.01) (Fig. 6). Effect of Histidine on ATP levels

Effect of Histidine on the Ammonia-Induced MPT Treatment of astrocyte cultures with ammonia resulted in a significant induction of the MPT, as shown by a cyclosporin A (CsA)-sensitive dissipation of the mitochondrial inner membrane potential (DWm) employing the potentiometric fluorescent dye TMRE. These data are consistent with our earlier studies demonstrating induction of the MPT by ammonia (Bai et al., 2001). As shown in Figs. 3 and 4, astrocyte cultures treated with GLIA DOI 10.1002/glia

Opening of the MPT pore leads to a loss of the inner membrane potential and decreased oxidative phosphorylation, ultimately resulting in depletion of ATP (Bernardi et al., 1998). Accordingly, levels of ATP were determined in cultured astrocytes exposed to ammonia. The ATP content in control astrocyte cultures was found to be 60 nmol/mg protein. Ammonia caused a 27% decrease in cellular ATP levels, which was completely blocked by 100 and 1,000 lM histidine (Fig. 7). Treatment of cultures with CsA (1 lM)

Fig. 3. Effect of histidine on the ammonia-induced MPT. Upper Panel: Representative TMRE fluorescence images showing the effect of ammonia on the dissipation of the inner mitochondrial membrane potential (DWm) in cultured astrocytes. Cultures were treated with 5 mM NH4Cl for 24 h. Control (C) astrocytes show bright TMRE fluorescence indicating polarized mitochondria. Astrocytes treated with ammonia

(NH4) show a significant loss of TMRE fluorescence indicating a dissipation of the DWm. Pretreatment of cultures with cyclosporin A (CsA, 1 lM) completely blocked the ammonia-induced dissipation of (DWm), indicating induction of the MPT by ammonia. Lower Panel: Treatment of cultured astrocytes with 100 lM L-histidine (His) 10 min prior to the exposure of cells to ammonia completely blocked the ammonia-induced MPT.

Fig. 4. Quantification of TMRE fluorescent intensities. Cultured astrocytes were exposed to various concentrations of histidine (50 lM–1 mM) and the fluorescence intensity of each image was quantified as described under ‘‘Experimental Procedures’’. Control fluorescence was set at 100%. Values are mean 6 S.E.M of total fluorescence intensities of at least 15 different random images captured in each group. Experiments were repeated using cultures derived from at least two different seedings. *P < 0.05 versus control. C, control; NH4, NH4Cl (5 mM); CsA, cyclosporin A (1 lM).

Fig. 5. Effect of different concentrations of histidine on ammoniainduced astrocyte swelling. Cultures exposed to 5 mM NH4Cl (NH4) showed an increased astrocyte swelling after 24 h when compared with control (C). Histidine both at lower (50 and 100 lM), as well as higher (1 mM) significantly reduced ammonia-induced astrocyte swelling. *P < 0.05 versus control. **P < 0.05 versus NH4.

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Fig. 6. Combined effects of MSO and histidine on ammonia-induced astrocyte swelling. Cultures exposed to 5 mM NH4Cl (NH4) showed an increase in astrocyte swelling after 24 h when compared with control (C). Both MSO (3 mM) and histidine (1 mM) significantly blocked ammonia-induced astrocyte swelling, where as a combination of MSO and histidine completely blocked such swelling. *P < 0.05 versus control. **P < 0.05 versus MSO and histidine.

also completely blocked the ammonia-mediated ATP loss (Fig. 7), implicating the MPT in the loss of ATP. Histidine alone had no effect on ATP levels (data not shown).

DISCUSSION This study demonstrates that L-histidine at various concentrations (50–1,000 lM) inhibits glutamine transport into mitochondria isolated from cultured astrocytes. Histidine at these concentrations significantly blocked or attenuated ammonia-induced free radical production, induction of the MPT, and loss of ATP. These findings are consistent with the view that glutamine transport into mitochondria represents a critical component in the mechanism of ammonia-mediated astrocyte dysfunction. Histidine also significantly blocked ammonia-induced astrocyte swelling by 60%, suggesting the involvement of mitochondrial glutamine transport in the astrocyte swelling produced by ammonia. Such blockade of swelling by histidine was further enhanced by MSO, resulting in a complete inhibition of astrocyte swelling following ammonia treatment. These data on the combined effects of MSO and histidine support the concept that glutamine production, as well as its transport into mitochondria, represent key factors in the mechanism of ammonia-induced astrocyte swelling. Glutamine has been implicated in many of the deleterious effects of ammonia in the setting of HE. Inhibition GLIA DOI 10.1002/glia

Fig. 7. Effect of L-histidine on the ammonia-induced ATP depletion in cultured astrocytes. Astrocytes exposed to 5 mM NH4Cl (NH4) showed a significant decrease in ATP levels when compared with controls (C). Treatment of cultures with cyclosporin A (CsA 1 lM) significantly increased ATP levels above the controls. Histidine (100 lM–1 mM), treatment completely inhibited the loss of ATP by ammonia. *P < 0.05 versus control. **P < 0.01 versus NH4. ATP levels in control cultures are 60 nmol/mg protein.

of glutamine synthetase activity by MSO mitigates many of the ammonia-mediated abnormalities in brain. It reduces the number of seizures, and delays the onset of death in animal models of hyperammonemia (Warren and Schenker, 1964); normalizes the decreased glucose utilization in a rat model of HE (Hawkins and Jessy, 1991), and restores altered vascular CO2 responsiveness (Takahashi et al., 1992). MSO has also been shown to block cytotoxic edema in experimental models of HE and hyperammonemia (Blei et al., 1994; Takahashi et al., 1991; Willard-Mack et al., 1996). Likewise, MSO blocks ammonia-induced free radical production (Murthy et al., 2001) as well as induction of the MPT (Bai et al., 2001) in ammonia-treated cultured astrocytes. These findings implicate glutamine, albeit indirectly, in the mechanism of ammonia neurotoxicity. Glutamine has recently been shown to directly exert toxic effects on astrocytes in culture, including the production of free radicals (Jayakumar et al., 2004) and induction of the MPT (Rama Rao et al., 2003b). The mechanism by which glutamine exerts these effects is not completely understood. However, one might predict that mitochondria represent critical loci, as mitochondria are a major site of free radical production and the organelle where the permeability transition occurs. Most of the ammonia (>98%) in brain is metabolized to glutamine with an extremely rapid turnover rate of t1/2 < 3 s (Cooper et al., 1979). This process occurs in the smooth endoplasmic reticulum and is catalyzed by glutamine syn-

GLUTAMINE TRANSPORT AND AMMONIA TOXICITY

thetase (Sellinger and Verster, 1962). The bulk (>75%) of glutamine derived from ammonia is then transported into mitochondria (Sonnewald et al., 1996; Yudkoff et al., 1988) by a specific carrier-mediated process (Minn, 1982; Steib et al., 1986) and then hydrolyzed by PAG, yielding ammonia and glutamate (Kvamme et al., 1982). Given the fact that glutamine levels are highly elevated in hyperammonemic conditions, and that ammonia has been shown to increase glutamine transport into mitochondria (Dolinska et al., 1996), such increase in mitochondrial glutamine following its hydrolysis may result in the generation of very high levels of ammonia in the mitochondrial compartment. Such high levels of ammonia confined to mitochondria we believe might be responsible for increased free radical production and the induction of the MPT in cultured astrocytes. Moreover, the localization of PAG on the outer side of inner mitochondrial membrane (Kvamme et al., 1982) would conceivably make glutamine immediately available for its hydrolysis once it enters mitochondria. Interestingly, studies by Zieminska et al. (2004) suggest that histidine blocks the transport of a distinct pool of glutamine that is preferentially available for its subsequent hydrolysis by PAG. In support of this role of mitochondria in glutamine toxicity, studies have shown that the PAG inhibitor DON, either completely or significantly, blocked free radical production and the MPT by ammonia in cultured astrocytes (Jayakumar et al., 2004; Rama Rao et al., 2005a). Another possible consequence of high glutamine levels in mitochondria is the excessive production of a-ketoglutaramate (KGM) (a transamination product of glutamine), mediated by glutamine aminotransferase K, an enzyme that is also localized in mitochondria (Cooper and Meister, 1974; Malherbe et al., 1995). It is noteworthy that increased levels of KGM have been found in rat brain, as well as in CSF of patients with hepatic coma (Vergara et al., 1973, 1974). Since these studies also suggest the involvement of glutamine in the mechanisms of ammonia neurotoxicity, the potential role of KGM in the deleterious effects of ammonia/glutamine on the CNS needs to be re-examined. While histidine is able to block mitochondrial glutamine transport, histidine may also inhibit plasma membrane glutamine transport into astrocytes (Chaudhry et al., 2002; Rae et al., 2003). Such inhibition may potentially deplete the cytosolic pool of glutamine, and hence its availability to mitochondria for hydrolysis. Whether this effect is relevant to hyperammonemic conditions, however, is uncertain, as in hyperammonemia essentially all of the glutamine is derived intracellularly in astrocytes, and there is likely little to no transport of glutamine into the intracellular compartment from the extracellular space. A few reports have commented that histidine has free radical scavenging properties, particularly for singlet oxygen species, which may possibly confound our findings. However, such free radical scavenging properties of histidine have been shown to occur only at very high concentrations (>25 mM) (Ishibashi et al., 1996; Obata et al., 2001; Zhai and Ashraf, 1995). Moreover, studies by Obata

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and Yamanaka (2001) showed that histidine at lower concentrations (1–10 mM) was unable to scavenge free radicals. Further, there is no documentation that histidine at lower concentrations, as employed in the present study can scavenge free radicals in tissues or cultured cells. It is therefore highly unlikely that the reduction in ROS levels by histidine observed in this study was due to its purported free radical scavenging property. It should also be mentioned that while the present study, along with other reports (Albrecht et al., 2000; Zieminska et al., 2000), indicate that histidine is a blocker of glutamine transport, the effect of 1 mM histidine in blocking glutamine transport was by only 49%, whereas it blocked ROS production by 100%. Although this discordance cannot be precisely explained, it does suggest that the amount of glutamine that enters mitochondria in the presence of histidine (1 mM) (
50%) is not sufficient to trigger ROS production. Oxidative stress has been implicated in ammonia neurotoxicity. For review, see (Norenberg et al., 2004), and one major consequence of oxidative stress is induction of the MPT (Castilho et al., 1995; Halestrap et al., 1997). In support of a role of oxidative stress in the induction of the MPT by ammonia, we earlier demonstrated that antioxidants significantly blocked the ammonia-induced MPT in cultured astrocytes (Rama Rao et al., 2005b). The present study showing the ability of histidine blocking ammonia-induced ROS production and the MPT are consistent with such a view. Although histidine was able to block both ROS production and the induction of the MPT by ammonia, such blockade was not in parallel, in that higher concentrations of histidine completely blocked ammonia-induced ROS production, while it only blocked the MPT by 50% (Fig. 4). Conversely, histidine at lower concentration (100 lM) completely protected against ammonia-induced MPT, whereas it blocked ROS production by only (49%). The reason for the mismatch regarding the effects of histidine is unknown. At the moment we can only speculate that histidine at higher concentrations might be triggering other events that potentially interfere with its ability to completely block the ammonia-induced MPT, while at the same time, a partial reduction of ROS by lower concentrations of histidine may be sufficient to completely block such MPT. Despite the lack of a linear response, the data are nonetheless consistent with the view that mitochondrial glutamine transport is central in the mechanism of ammonia neurotoxicity. It is known that the induction of the MPT results in a dissipation of the inner mitochondrial membrane potential, and such dissipation of the membrane potential can decrease the rate of oxidative phosphorylation that could eventually lead to a loss of ATP production. The induction of the MPT by ammonia may partially explain the disturbance in brain energy metabolism, including a reduction of a-ketoglutarate dehydrogenase activity, altered NAD/NADH ratios in both cytosolic and mitochondrial compartments, as well as a moderate loss of ATP caused by ammonia (For review see Rao and Norenberg, 2001). The present study demonstrates that GLIA DOI 10.1002/glia

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treatment of cultured astrocytes with ammonia results in 27% loss of ATP and such loss was completely blocked by CsA (Fig. 6), confirming the involvement of the MPT in the ammonia-induced ATP depletion. Similarly, the present study shows that histidine (0.1 and 1 mM) completely restores the loss of ATP by ammonia (Fig. 6), suggesting that inhibition of glutamine transport into mitochondria attenuates the ammonia-induced loss of ATP in astrocytes, presumably by preventing the MPT. A major abnormality in acute HE and hyperammonemia is cytotoxic edema, principally because of astrocyte swelling (Blei and Larsen, 1999; Norenberg et al., 2005; Vaquero et al., 2003). Such edema has been strongly correlated with increased arterial ammonia concentrations (Clemmesen et al., 1999). Moreover, it has been reported that in patients with fulminant hepatic failure, extracellular brain glutamine levels strongly correlate with the severity of intracranial pressure (Tofteng et al., 2006). While the mechanisms responsible for brain edema/ astrocyte swelling by ammonia are not completely understood, a commonly held view is that the accumulation of glutamine, resulting from the ‘‘detoxification’’ of ammonia, leads to an osmotic shift of water into astrocytes (Brusilow, 2002). However, such an osmotic mechanism appears to be inconsistent with data showing a lack of temporal correlation of astrocyte glutamine concentration with the extent of swelling (Jayakumar et al., 2006), as well as with reports documenting the reduction of brain edema by various modalities, in the absence of a commensurate reduction in cerebral glutamine concentrations (Chatauret et al., 2003; Zwingmann and Butterworth, 2005; Zwingmann et al., 2004). The above findings, however, do not at all detract from the central role of glutamine in the mechanism of astrocyte swelling in hyperammonemic states. Rather than acting as an osmolyte, we have instead proposed that glutamine contributes to brain edema/astrocyte swelling by inducing oxidative stress, and the MPT, both of which have been implicated in the mechanism of ammonia-induced astrocyte swelling (Norenberg et al., 2005). Energy depletion, possibly resulting from the MPT, may additionally contribute to astrocyte swelling, as cellular energy is an essential requirement for cell volume regulation (Kimelberg, 2005; Olson and Evers, 1992; Olson et al., 1986). However, it is not known whether a modest loss of ATP (27%) following ammonia treatment is sufficient to bring about astrocyte swelling. In summary, our study demonstrates that L-histidine, known to inhibit mitochondrial glutamine transport, is able to block ammonia-induced astrocyte swelling, free radical production, the MPT, and ATP depletion. The effects of histidine appear to be mediated by its inhibition of glutamine transport into mitochondria, and are consistent with earlier findings showing that inhibition of mitochondrial glutamine hydrolysis protects against ROS production, the MPT and astrocyte swelling following ammonia and glutamine treatment. Ammonia-glutamine-mitochondrial interactions may provide novel therapeutic targets for counteracting ammonia neurotoxicity associated with HE and other hyperammonemic states. GLIA DOI 10.1002/glia

ACKNOWLEDGMENTS We thank K. Panneerselvam and A. Fernandez for the preparation of astrocyte cultures.

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GLIA DOI 10.1002/glia