Ammonia Induces the MitochondrialPermeability Transition in Primary Culturesof Rat Astrocytes

G. Bai,1 K.V. Rama Rao,1 Ch.R.K. Murthy,2 K.S. Panickar,1 A.R. Jayakumar,1

and M.D. Norenberg1*1Veterans Affairs Medical Center, Departments of Pathology, Biochemistry, and Molecular Biology, Universityof Miami School of Medicine, Miami, Florida2Department of Animal Sciences, University of Hyderabad, Hyderabad, India

Ammonia is a toxin that has been strongly implicated inthe pathogenesis of hepatic encephalopathy (HE), andthe astrocyte appears to be the principal target of am-monia toxicity. The specific neurochemical mechanismsunderlying HE, however, remain elusive. One of the sug-gested mechanisms for ammonia toxicity is impairedcellular bioenergetics. Because there is evidence that themitochondrial permeability transition (MPT) is associatedwith mitochondrial dysfunction, we determined whetherthe MPT might be involved in the bioenergetic alterationsrelated to ammonia toxicity. Accordingly, we examinedthe mitochondrial membrane potential (Dcm) in culturedastrocytes and neurons using laser-scanning confocalmicroscopy after loading the cells with the voltage-sensitive dye JC-1. We found that ammonia induced adissipation of the Dcm in a time- and concentration-dependent manner. These findings were supported byflow cytometry using the voltage-sensitive dye tetram-ethylrhodamine ethyl ester (TMRE). Cyclosporin A, a spe-cific inhibitor of the MPT, completely blocked theammonia-induced dissipation of the Dcm. We also foundan increase in the mitochondrial permeability to2-deoxyglucose in astrocytes that had been exposed to5 mM NH4Cl, further supporting the concept that ammo-nia induces the MPT in these cells. Pretreatment withmethionine sulfoximine, an inhibitor of glutamine syn-thetase, blocked the ammonia-induced collapse of Dcm,suggesting a role of glutamine in this process. Over a24-hr period, ammonia had no effect on the Dcm incultured neurons. Collectively, our data indicate that am-monia induces the MPT in cultured astrocytes, whichmay be a factor in the mitochondrial dysfunction associ-ated with HE and other hyperammonemic states. J. Neu-rosci. Res. 66:981–991, 2001. © 2001 Wiley-Liss, Inc.

Key words: ammonia; hepatic encephalopathy; mito-chondrial permeability transition; cyclosporin A; astro-cytes; glutamine

Hepatic encephalopathy (HE) is a complex neuro-psychiatric syndrome resulting from severe liver failure

(see Jones and Weissenborn, 1997, for review). Thepathogenesis of this condition is not completely under-stood. The dominant view over many decades has beenthat gut-derived nitrogenous toxins are not detoxified bythe diseased liver or are not extracted by the liver becauseof vascular (portal-systemic) shunts that commonly occurin chronic liver disease. Ammonia is the most implicatedtoxin in HE and is found to be elevated in patients withHE (Conn, 1993).

A disturbance in brain bioenergetics has been sug-gested as an important factor in ammonia neurotoxicity(Cooper and Plum, 1987; Hawkins and Mans, 1989; Raoand Norenberg, 2001). Proposed mechanisms for impairedbioenergetics include inhibition of a-ketoglutarate dehy-drogenase (Lai and Cooper, 1986); diversion of glutamateto glutamine synthesis thereby depriving the astrocyte ofan energy source (McKenna et al., 1996); stimulation ofNa1,K1-ATPase resulting in depletion of ATP (Ratna-kumari and Murthy, 1989; Kosenko et al., 1996); impairedoxidation of pyruvate and glutamate (Ratnakumari et al.,1992); and a disturbance in the malate-aspartate shuttle(Hindfelt et al., 1977; Murthy and Hertz, 1988; Faff-Michalak and Albrecht, 1991). Evidence of ammonia-induced defects in bioenergetics in astrocytes has also beendescribed (Fitzpatrick et al., 1988; Murthy and Hertz,1988; Haghighat and McCandless, 1997; Haghighat et al.,2000).

The mitochondrial permeability transition (MPT)has been recently implicated as a factor in impaired mito-chondrial function. The MPT was initially described byHaworth and Hunter (1979) and Hunter and Haworth(1979a,b) as a sudden increase in the permeability of the

Contract grant sponsor: Department of Veterans Affairs; Contract grantsponsor: NIH; Contract grant number: NS34951.

Drs. Bai and Rama Rao contributed equally to this article and are consid-ered co-first authors.

*Correspondence to: Michael D. Norenberg, MD, Department of Pathol-ogy (D-33), PO Box 016960, University of Miami School of Medicine,Miami, FL 33101. E-mail: mnorenbe@med.miami.edu

Received 13 July 2001; Accepted 6 August 2001

Journal of Neuroscience Research 66:981–991 (2001)

© 2001 Wiley-Liss, Inc.

inner mitochondrial membrane resulting from an overloadof Ca21. This calcium sensitivity is further enhanced byoxidative stress, redox state of various thiol groups, oxi-dative state of pyridine nucleotides, nitric oxide, alkalinepH and other factors (see Zoratti and Szabo, 1995 forreview). The MPT results from the opening of a perme-ability transition pore (PTP) located in the inner mito-chondrial membrane, thereby increasing its permeabilityto protons, ions and other solutes #1,500 Da. This in-creased permeability leads to a collapse of the inner mito-chondrial membrane potential (Dcm) that is created by thepumping of protons by the electron transport chain. Lossof the Dcm leads to colloid osmotic swelling of the mito-chondrial matrix (Gunter and Pfeiffer, 1990), redistribu-tion of metabolites (Ca21, Mg21, glutathione, NADPH)across the inner membrane, defective oxidative phosphor-ylation, cessation of ATP synthesis, and the generation ofROS; the latter may act to further aggravate the MPT.These mitochondrial changes may initiate a cascade ofevents culminating in cell death (necrosis or apoptosis)(Bernardi et al., 1998; Kroemer and Reed, 2000). TheMPT is characteristically inhibited by the immunosup-pressant cyclosporin A (CsA). The MPT has been impli-cated in the pathogenesis of a number of neurologicaldisorders, including ischemia (Uchino et al., 1995; Fol-bergrova et al., 1997), excitotoxicity (White and Reyn-olds, 1996), hypoglycemia (Friberg et al., 1998), trauma(Okonkwo et al., 1999; Scheff and Sullivan, 1999), Reyesyndrome (Trost and Lemasters, 1997) and Parkinson dis-ease (Cassarino et al., 1999; Berman and Hastings, 1999).

Astrocytes represent the primary target of neurotox-icity in HE/hyperammonemia (HA) (Norenberg, 1998).The effects of ammonia on astrocytes in culture have beenextensively studied and used as an in vitro model of HE(Norenberg, 1998). Exposure of cultured astrocytes toammonia mimics many of the changes observed in HE,including Alzheimer type II changes, cell swelling, alteredprotein phosphorylation, decreased cyclic AMP levels, anddecreased ability to take up glutamate, GABA, potassiumand myo-inositol (Norenberg, 1998). In this study weexamined the effects of ammonia on the Dcm, and onmitochondrial permeability in cultured astrocytes andneurons. Our results demonstrate that ammonia inducesthe MPT in astrocytes but not in neurons, and suggest thatglutamine may be involved in this process.

MATERIALS AND METHODS

Materials

5,59,6,69-Tetrachloro-1,19,3,39-tetraethyl-benzimidazolo-carbocyanine iodide (JC-1) and tetramethylrhodamine ethyl es-ter (TMRE) were obtained from Molecular Probes (Eugene,OR). Opti-MEM1 was purchased from GibcoBRL. All otherchemicals were from Sigma Chemical Co. (St. Louis, MO), andwere of analytical grade.

Cell Cultures

Primary cultures of astrocytes were prepared as describedby Ducis et al. (1990). Briefly, dissociated cells from cerebral

cortices of 1–2-day-old rats were plated in 35 mm dishes at adensity of 0.5 3 106 in Dulbecco modified Eagle medium(DMEM). After 2 weeks, the cells were maintained with dibu-tyryl cAMP. Cultures consisted of 95–99% astrocytes based onimmunohistochemistry of glial fibrillary acidic protein and glu-tamine synthetase. Three- to 5-week-old cells were used forexperiments. For each experiment, 7–8 culture plates wereselected randomly from at least two different seeding batches.

Neuronal cell cultures from cerebral cortex of rat fetuses(gestational age 16–18 days) were prepared by a method similarto that described by Schousboe et al. (1989). Cytosine arabino-side (10 mM) was added to the culture medium 48 hr afterseeding to prevent proliferation of astrocytes. The cultures con-sisted of at least 90% neurons as determined by immunohisto-chemical staining for neurofilament protein; the remaining cellswere predominantly astrocytes as shown by immunohistochem-ical staining for GFAP. Experiments were carried out on cul-tures 10–12 days old.

Cell cultures were treated with variable concentrations ofammonium chloride (1–10 mM) (referred hereafter as ammonia;the terms NH4

1 and ammonia are used interchangeably in thisarticle) for indicated time periods; however, in most studies weused 5 mM ammonia because this level has been identified inbrains of animals with HE (Swain et al., 1992). Ammoniatreatment was maintained throughout the experimental period,including the assay period. The external pH was monitored anddid not significantly change upon ammonia treatment. Controlcultures were treated with equal amounts of NaCl. In someexperiments, cells were pretreated with 3 mM methionine sul-foximine (MSO) 30 min before the addition of 5 mM NH4Cl.When cyclosporin A (CsA) was used, it was always added15 min before the addition of NH4Cl. CsA, FCCP and FK506were dissolved in 100% ethanol; the final concentration ofethanol was 0.1%; control cultures received an equivalentamount of ethanol.

Laser Scanning Confocal Microscopy

At the end of the treatment period, culture medium wasremoved and the cells were loaded with JC-1 for 20 min. JC-1was first dissolved in DMSO (1 mg/ml) and added to theOpti-MEM1 medium (final concentration of JC-1 was1 mg/ml). After the loading period, the cells were rinsed withPBS and examined with a laser-scanning confocal microscope(Multiprobe 2001, Molecular Dynamics, Sunnyvale, CA). Cellswere excited at 488 nm and the fluorescence emission wasrecorded at 530 and 590 nm. Fluorescence level was coded witha gray scale representing pixel intensity of 0–255, and the imageswere collected at 1,024 3 1,024 pixels. Semiquantitation of redand green fluorescence intensity was carried out in 10–16 ran-domly selected areas by an unbiased observer using the ImageSpace 3.10 software. Quantitation was expressed as the mean ofred/green fluorescence ratios, compared to control that was setat 100%. The results were statistically analyzed using the pairedStudent’s t-test, where P , 0.05 was considered significant.

At high mitochondrial membrane potentials, JC-1 accu-mulates sufficiently in the mitochondria to form aggregates(J-aggregates) that fluoresce red (emission 590 nm). At lowermitochondrial potentials, less dye enters mitochondria resultingin monomers that fluoresce green (emission 530 nm) (Smiley et

982 Bai et al.

al., 1991). The 590 nm/530 nm ratios generated with JC-1 canfurther enhance the sensitivity of changes in the Dcm anddecrease the variability that may result from differences in dyeloading and changes in mitochondrial size, shape and density,and number of cells among samples (Smiley et al., 1991; Salvioliet al., 1997; Bedner et al., 1999). The ratioing property of JC-1also makes it suitable for semiquantitative measurements ofchanges in the Dcm (Reers et al., 1991). In preliminary studies,we employed JC-1 concentrations ranging from 0.1–2.0 mMand found the 1.5 mM concentration (1 mg/ml) to be optimal asthis concentration resulted in consistent and reliable degree ofred/green emission fluorescence ratios. Additionally, we carriedout cytotoxicity studies and found that 3 hr treatment of astro-cyte cultures with JC-1 (1 mg/ml) did not result in cytopathicchanges or LDH release, and mitochondrial function was notcompromised as determined by the MTT assay (Shearman et al.,1995). We also observed that the fluorescent properties of thedye were not affected by ammonia (5 mM), or by changes in pHfrom 6.5 to 8.0 (data not shown). Also, treatment of culturedastrocytes with 5 mM ammonia for 30 min did not influence dyeloading compared to controls as measured by red/green fluo-rescence. Care was also taken to avoid photobleaching of thedye by exposing the cells for only a short period of time duringconfocal measurements as well as by using the Opti-MEM1medium.

Determination of the Dcm was also carried out usingTMRE (150 nM). At the end of the experimental period,culture medium was removed and the cells were loaded withTMRE for 30 min in regular growth media (in the presence orabsence of NH4Cl). After dye loading, the cells were rinsed andexamined by confocal microscopy as was done with JC-1,except that the TMRE fluorescence was measured at 590 nm.Semiquantitation of fluorescence intensity was carried out aswith JC-1.

Flow Cytometry

Controls and NH4Cl treated astrocytes were incubated for30 min in medium (DMEM/10% horse serum) containingTMRE (150 nM). Cells were then rinsed with PBS and sub-jected to trypsinization (0.25% trypsin in Hanks balanced saltsolution without Ca21 or Mg21). Trypsinization was stoppedwith DMEM containing 10% FBS. Cells were suspended bygentle trituration and collected by centrifugation (5,000 rpm for1 min at room temperature).

The flow cytometric procedure was carried out similar tothat described by Johnson et al. (2000). Briefly, cells were rinsedwith PBS and resuspended into 0.5 ml each of FACS buffer(PBS, 1% BSA, 0.01% sodium azide and 15 nM TMRE). TheDcm was analyzed by using a FACScan flow cytometer (BectonDickinson) equipped with 488 nm argon laser. A minimum of104 cells per sample were analyzed through a FL3-H channel.Identical results were observed when the cells were loaded withTMRE after trypsinization. Data were acquired in list mode andanalyzed with a CellQuest software.

Permeabilization of Astrocytes

Permeabilization of astrocytes was carried out followingthe methods of Huang and Philbert (1995). To permeabilize thecells, the medium was aspirated and cells were washed three

times with ice-cold PBS. To each culture dish, 0.25 ml ofice-cold isolation buffer (250 mM mannitol, 2.5 mM EDTA and17 mM 3-[N-morpholino]propane sulfonic acid, MOPS; pH7.4) was added and the culture plate was kept on ice for 5 min,harvested in the same medium, and an ice-cold solution ofdigitonin was added to a final concentration of 0.1 mg/ml. Afterexactly 1 min at 0°C, permeabilized cells were centrifuged at14,000 3 g for 1 min. The supernatant was discarded and thepellet was washed twice in a large volume (;2 ml) of ice-coldmedium and resuspended in isolation buffer (50 ml/plate). Theprotein content was determined by the BioRad bicinchoninicacid method. All operations were carried out at 2–4°C. Thepurity of the preparations was assessed by measuring the activ-ities of LDH and citrate synthase (Bergmeyer, 1974), whereasthe integrity was assessed by measuring the activities of mono-amine oxidase (Yu and Hertz, 1982) and citrate synthase, whichare mitochondrial outer membrane and matrix markers, respec-tively.

Calcium-Dependent Mitochondrial PermeabilityChange

Mitochondrial permeability was assessed by determiningchanges in permeability to 2-deoxy-glucose-6-phosphate (2-DG-6-P) using an adaptation of the in vivo method of Kerr etal. (1999). Briefly, control as well as ammonia-treated astrocytecultures were incubated with 1 mM 2-deoxyglucose dissolved inmedium containing 0.5 mCi [3H]-2-DG for 1 hr at 37°C. At theend of the incubation, an aliquot of medium from each cultureplate was collected for radioactivity determination, the plateswere washed three times with ice-cold PBS, and then processedfor digitalization as described above. Permeabilized cells, com-pletely devoid of cytosol, were resuspended in a small volume ofisolation buffer, and an aliquot of the cell suspension was trans-ferred to scintillation vials for radioactivity determination,whereas the rest of the sample was retained for citrate synthaseassay. Results were expressed as the amount of 2-DG-P inpermeabilized cells after normalization to citrate synthase activ-ity. To establish the validity of this method, we incubated thecells with t-butyl-hydroperoxide (TBH; 250 mM; 30 min) andphenylarsine oxide (PAO; 10 mM agents that are known toinduce the MPT (Nieminen et al., 1995; Zoratti and Szabo,1995).

LDH Assay

LDH activity in the media and cell supernatant was mea-sured spectrophotometrically after oxidation of NADH in thepresence of pyruvate (Wroblewsky and LaDue, 1955).

Statistical Analysis

Data are presented as means 6 SD. The data were ana-lyzed by a paired Student’s t-test (Sigma Stat). Where necessary,the data was also subjected to one-way analysis of variance(ANOVA) followed by Dunnett or Newman-Keuls post-hoctests.

RESULTSEffect of Ammonia on the Dcm

Laser-scanning confocal microscopy was carried outusing the potentiometric dye JC-1. Control cultures dis-

Ammonia-Induced Permeability Transition 983

played uniform red fluorescence throughout the soma andprocesses indicating a polarized state (Fig. 1A). As shownin Figure 1B,E, addition of 5 mM NH4Cl (24 hr) induceda dissipation of the Dcm in cultured astrocytes comparedto their respective controls (Fig. 1A,D). FCCP (3 mM,20 min) completely collapsed Dcm and served as a positivecontrol (Fig. 1A inset).

The changes produced by ammonia on the Dcmwere time- and concentration-dependent (Fig. 2). With5 mM NH4Cl, a change in the JC-1 fluorescence patternwas first noted at 4 hr as the extent of green fluorescenceincreased and red fluorescence diminished. With 1 mMNH4Cl, very little effect was observed even after 24 hr,whereas with 10 mM NH4Cl, the change in fluorescencepattern commenced earlier, i.e., at 2 hr (data not shown).Prolonged treatment of astrocytes with 5 mM NH4Cl (48and 72 hr) resulted in a greater magnitude of green fluo-rescence with a concomitant decrease in red fluorescence.It should be noted that ammonia treatment did not resultin cell death as determined morphologically and by LDHrelease.

Quantitative analyses of the fluorescence intensitieswere carried out by determining the change in the ratio of

red/green fluorescence intensities in 5 mM ammonia-treated astrocytes and were compared to untreated controlcells, which was set at 100% (Fig. 2). The quantitation offluorescence intensity correlated well with the visual anal-ysis of fluorescence changes.

To confirm whether the ammonia-induced dissipa-tion of the Dcm was a result of the MPT, we examined theeffects of CsA, a specific inhibitor of MPT (Crompton etal., 1988). At 1 mM, CsA displayed only a slight attenua-tion of the ammonia-induced Dcm collapse, whereashigher concentrations of CsA (2.5 and 5 mM) almostcompletely blocked the ammonia effect (Figs. 1A–C, 3).CsA by itself had no effect on the Dcm at the testedconcentrations (data not shown).

CsA has also been reported to act as an inhibitor ofthe Ca21/calmodulin-dependent protein phosphatase,calcineurin (Halloran, 1996). To rule out the possibilitythat the effects of CsA might be due to calcineurin inhi-bition, we examined the effects of FK506 (generous giftfrom Dr. Ihor Bekersky, Fujisawa Health Care Inc.), acalcineurin inhibitor (Liu et al., 1991) that has no influ-ence on the MPT (Schweizer et al., 1993). FK506 (1 mM)did not block the ammonia-induced collapse of the Dcm

Fig. 1. Laser scanning confocal mi-croscopic images of JC-1 fluores-cence of ammonia-treated cultures(5 mM; 24 hr). Red fluorescence in-dicates a polarized state whereasgreen fluorescence indicates a depo-larized state. A–C: Effect of CsA onammonia-induced collapse of theDcm. A: Control astrocytes. B:NH4Cl treatment showing a signifi-cant degree of depolarization. C:Cells treated with NH4Cl 1 CsA(5 mM) are similar to control. FCCP(3 mM, 20 min) induced a total col-lapse of the Dcm (inset in A). D–F:Effects of MSO on ammonia-induced MPT. D: Control astrocytes.E: NH4Cl treatment. F: NH4Cl 1MSO (3 mM). MSO completelyblocked the ammonia-induced depo-larization. G–I: Effect of ammoniaon neurons. G: Control neurons. H:Neurons treated with 5 mM NH4Clshowing no effect. I: Neurons treatedwith 10 mM showing minimal depo-larization. Scale bar 5 100 mm.

984 Bai et al.

(Fig. 3), confirming that the effect of CsA was due to itsMPT inhibiting effect.

In addition to JC-1, we also used the potentiometricdye TMRE to detect changes in the Dcm by confocalmicroscopy. Treatment of cultured astrocytes with 5 mM

NH4Cl for 24 hr gave a similar attenuation of the Dcm aswas observed with JC-1 (;45% reduction) (data notshown).

The ammonia-induced collapse of Dcm was alsodemonstrated by flow cytometry using TMRE (Fig. 4).Treatment of astrocytes with 5 mM ammonium chloridefor 24 and 48 hr caused a leftward shift in the spectralcurve (curves A–C), indicating that fewer cells retainedTMRE in their mitochondria (i.e., depolarized). Again,CsA blocked the ammonia-induced collapse of the Dcm.Treatment with FCCP (3 mM, 20 min) caused a markedspectral shift to the left (curve E). Unexpectedly, CsAalone at 5 mM caused a small degree of depolarization thathad not been observed in the confocal studies. The reasonfor such differences is not known. It is possible that certainprocedures, such as the trypsinization, employed to disso-ciate astrocytes for FACScan analysis might have alteredastrocyte properties and predisposed these cells to a smalldegree of depolarization when exposed to CsA. A repre-sentative histogram of the flow cytometric data is shown inFigure 4.

Cultures of cortical neurons (10–12-day-old) wereexamined for their sensitivity to ammonia-inducedchanges in the Dcm. Twenty-four hour treatment with5 mM NH4Cl had no effect on the Dcm whereas treat-ment with 10 mM NH4Cl only minimally reduced theDcm (Fig. 1G–I).

Some of the deleterious effects of ammonia havebeen attributed to glutamine (Brusilow and Traystman,1986; Takahashi et al., 1991; Hawkins et al., 1993), theproduct of ammonia detoxification that could potentiallymediate the changes in the Dcm. We therefore examinedthe effect of methionine sulfoximine (MSO), an inhibitor

Fig. 2. Time-dependence of ammonia (5 mM)-induced dissipation ofthe Dcm in astrocytes. Quantitation is expressed as mean red/greenfluorescence ratios and compared to untreated controls that were set at100%. *P , 0.05 vs. control.

Fig. 3. Quantitation of CsA, FK506, and MSO effects on ammonia-induced dissipation of the Dcm in astrocytes. Cells were treated withNH4Cl (5 mM) 6 CsA (1, 2.5, and 5 mM), FK506 (1 mM) and MSO(3 mM) for 24 hr. Quantification of the fluorescent intensities wascarried out, and the ratio of red/green were compared to untreatedcontrols that were set at 100%. *P , 0.05 vs. controls; †P , 0.05 vs.NH4

1.

Fig. 4. Effect of 48 hr treatment with 5 mM NH4Cl on the Dcm asdetermined by flow cytometry. Data is plotted as cell counts vs. TMREfluorescence (as measured in FL3-Height). Ammonia results in a spec-tral shift to the left indicating mitochondrial depolarization. This effectwas blocked by CsA (5 mM). Similar results were obtained after 24 hrtreatment with ammonia. Treatment with 3 mM FCCP for 20 mincaused a marked spectral shift to the left. A: Control. B: 5 mM NH4Cl.C: 5 mM CsA. D: NH4Cl 1 CsA. E: FCCP.

Ammonia-Induced Permeability Transition 985

of glutamine synthetase (GS), on the ammonia-inducedcollapse of Dcm. Treatment of cells with 3 mM MSO for30 min before a 24 hr treatment with 5 mM NH4Clblocked the ammonia-induced collapse of Dcm in astro-cytes (Fig. 1D–F). This finding suggests that the accumu-lation of glutamine or the process of glutamine synthesismay mediate some of the toxic effects of ammonia. Quan-titative analysis of the MSO effects on ammonia-inducedcollapse of Dcm is shown in Figure 3.

Characterization of Permeabilized AstrocytesPermeabilized cell preparations were used in lieu of

isolated mitochondria, because the latter requires the gen-eration of sufficient mitochondria from a large number ofcultured cells, and the procedure for isolating the mito-chondria from cultured cells is cumbersome. We used theprocedure of Huang and Philbert (1995) for preparingpermeabilized cells from control as well as NH4Cl treatedastrocytes. This method requires very few cells (1 million)and yields sufficient material for several biochemical andenzymatic assays. Exposing the cells to very low concen-trations of digitonin (0.001%) for a brief period of time(1 min) solubilizes mostly the plasma membrane withouthaving any direct effect on mitochondria. This results in apermeabilized cell preparation from which the solublecytosolic proteins have been removed to a very greatextent and provides a ready access of substrates to mito-chondrial membranes. The mitochondria in these prepa-rations are metabolically intact, and have been extensivelyused for MPT related studies including protection byBcl-2 (Murphy et al., 1996; Kowaltowski et al., 2000),calcium-dependent and independent pathways of theMPT (Kristal and Dubinsky, 1997), and apoptosis-relatedactivities (Fiskum et al., 2000).

Preliminary studies were carried out to characterizethe extent of mitochondrial enrichment as well as theintegrity of permeabilized astrocytes. After digitization ofcultured astrocytes and subsequent centrifugation, the su-pernatant that presumably represents cytosol, and the pel-let containing mitochondria, were assessed for activities ofmarker enzymes. We found negligible contamination ofthe permeabilized cell pellet by cytosol as indicated by,1% of LDH activity in the pellet (data not shown). Wealso determined the activity of citrate synthase (CS), amitochondrial matrix marker, and found .95% activity inthe pellet fraction (120 6 10 nmol/min/mg protein)compared to the cytosol (6.5 6 1 nmol/min/mg protein).Likewise, the activity of monoamine oxidase (MAO), anouter mitochondrial membrane marker, was highly en-riched in the pellet (mitochondria) (0.137 6 0.02 nmol/min/mg protein) as compared to the supernatant (cytosol)fraction (0.006 6 0.001 nmol/min/mg protein), indicat-ing the integrity of the outer mitochondrial membrane, aswell as of the mitochondrial matrix. In preliminary studies,we also observed that respiratory control ratios (RCI)were in the range of 2.5–3.5. These results indicate thatthe permeabilized preparations are enriched with intactmitochondria.

Effect of Ammonia on Mitochondrial PermeabilityTo determine whether ammonia is capable of elic-

iting an alteration in mitochondrial permeability, we mea-sured the amount of [3H]-2-deoxyglucose-6-phosphate(2-DG-6-P) entrapped in mitochondria. When 2-deoxy-glucose (2-DG) enters the cell via the glucose transporter,it is phosphorylated by hexokinase to 2-DG-6-P that isnot further metabolized and is then trapped inside thecytoplasm. As the inner membrane of normal mitochon-dria is not permeable to 2-DG-6-P, very little label will befound in the mitochondria of normal cells. When theMPT occurs, however, 2-DG-6-P enters mitochondriathrough the PTP and equilibrates with the cytosolic pool.Addition of the Ca21 chelator EDTA (2.5 mM) in thepermeabilization buffer results in the closure of the PTPduring the isolation procedure leading to the entrapmentof 2-DG-6-P in mitochondria. This method has the ad-vantage of directly assessing the status of MPT in situ.

As a positive control for the permeability procedure,astrocytes were treated with TBH (250 mM; 30 min) orwith phenylarsine oxide (PAO, 10 mM; 30 min), and2-DG permeability was determined. Results indicated thattreatment with TBH showed a significant (112 6 10%P , 0.01) increase in 2-DG permeability that was blockedby treatment with CsA (Fig. 5). Similarly, PAO alsosignificantly increased 2-DG permeability (118 6 13%P , 0.01) that was sensitive to CsA.

Astrocytes were treated with 5 mM NH4Cl andchanges in 2-DG-6-P permeability were studied at differ-ent time points during NH4Cl treatment. As shown inFigure 6, 1 and 2 hr post-treatment with ammonia showedno significant changes in 2-DG-6-P permeability, whereas4 hr post-treatment caused a 50% increase (P , 0.05) inpermeability. After 12 hr there was a further increase(120%, P , 0.01) in 2-DG-6-P permeability that persistedup to 48 hr. NH4Cl (10 mM) treatment for 2 days in-creased the 2-DG-6-P permeability 2-fold (P , 0.01) ascompared to 5 mM NH4Cl treatment. The relatively highbackground values that were obtained in the controlgroups may be due to the slow MPT-independent entry of2-DG-6-P into a subpopulation of mitochondria (Kerr etal., 1999), or due to the retention of some 2-DG-6-P incell vesicles. Nevertheless, the background values we ob-tained in controls were always consistent with a ,5%variance. To confirm whether these changes could beblocked by CsA, we subjected astrocytes to parallel treat-ment with 5 mM NH4Cl, and CsA (1 mM or 5 mM CsA)for 48 hr. As shown in Figure 7, CsA at both 1 and 5 mMconcentrations completely normalized the ammonia-induced increase in 2-DG-6-P permeability.

Taken together, these data show that ammonia sig-nificantly increased the 2-DG-6-P permeability in a time-and concentration-dependent manner, indicating that theMPT occurred in the presence of ammonia. These resultsare consistent with the temporal- and concentration- de-pendent dissipation of the mitochondrial membrane po-tential that was observed in the presence of ammonia.

986 Bai et al.

DISCUSSIONThis study has shown that ammonia induces a col-

lapse of the mitochondrial inner membrane potential thatis associated with enhanced mitochondrial permeability.These effects were abrogated by CsA, supporting the viewthat ammonia induces the MPT in cultured astrocytes.Similar treatment of cultured neurons failed to produce adissipation of the mitochondrial inner membrane poten-tial.

A significant feature of the MPT is the sudden in-crease in the permeability of certain compounds across theinner mitochondrial membrane in a Ca21-sensitive man-ner (Haworth and Hunter, 1979). In the present study, weemployed the 2-DG-6-P entrapment method as a measureof the MPT, and observed that ammonia caused a time-and concentration-dependent increase in 2-DG-6-P per-meability (Fig. 6). This effect was completely blocked by

CsA (Fig. 7) further supporting the concept that ammoniainduces the MPT.

Our findings are consistent with reports of disturbedbioenergetics in HE/HA (see Introduction), and with anumber of studies showing morphologic changes in mi-tochondria in HE/HA, principally swelling of the matrixand intracristal space (Martinez, 1968; Norenberg, 1977;Drewes and Leino, 1985; Gregorios et al., 1985). Theseobservations further support the concept that anammonia-induced dysfunction in astrocytes (gliopathy) isa key aspect in the pathogenesis of HE/HA (Norenberg,1981).

The mechanism by which ammonia induces theMPT remains to be defined. Although it is possible thatammonia directly affects the transition pore, the timecourse of changes in the Dcm and in mitochondrial per-meability suggests an indirect effect. It is of interest that anumber of factors believed to be operative in HE/HAhave been shown to induce the MPT in other systems.Free radicals are perhaps the most important inducers ofthe MPT and oxidative stress is an evolving concept in thepathogenesis of HE/ammonia neurotoxicity (Kosenko etal., 1999). There is evidence of lipid peroxidation incultured astrocytes after ammonia treatment (Murphy etal., 1992). Additionally, Alzheimer type II astrocytes arecharacterized by excessive amounts of lipofuscin (Noren-

Fig. 5. Effect of t-butyl-hydroperoxide (TBH) and phenylarsine oxide(PAO) on the mitochondrial permeability as measured by [3H]-2-deoxyglucose-6-phosphate (2-DG-6-P) entrapment. Astrocytes wereincubated with TBH (250 mM) as well as PAO (10 mM) with andwithout CsA (5 mM) for 30 min and 2-DG-6-P permeability wasdetermined as described in Materials and Methods. Values are expressedas % dpm of 2-DG-6-P/U citrate synthase. The dpm values in thecontrol group is set at 100% and the values are mean 6 SEM of 3–5individual cultures in each group taken from two different seedings.*Significant from control (P , 0.01); †significant from TBH (P ,0.01); ††significant from PAO (P . 0.01).

Fig. 6. Effects of 5 mM NH4Cl treatment at different time points onthe mitochondrial permeability as measured by [3H]-2-deoxyglucose-6-phosphate (2-DG-6-P) entrapment. Values are expressed as % dpm of2-DG-6-P/U citrate synthase. The dpm values in the control group isset as 100% and the values are mean 6 SE 7–8 individual cultures ineach group, taken from 3–4 different seedings. *P , 0.01 vs. control;†P , 0.01 vs. 4 hr.

Ammonia-Induced Permeability Transition 987

berg, 1981), a cytologic feature of lipid peroxidation(Brunk, 1989). Recently, we have shown that ammonia iscapable of generating free radicals in cultured astrocytes(Murthy et al., 2001). Studies showing the beneficial effectof free radical scavengers in experimental HE/HA (Guer-rini, 1994; Bruck et al., 1999) and in patients with fulmi-nant hepatic failure (Wendon et al., 1994) further supporta role of free radicals in HE. Preliminary studies in ourlaboratory have shown that the spin trapping agenta-phenyl-N-butyl nitron (PBN) partially blocked theammonia-induced collapse in the Dcm.

Nitric oxide (NO) is an inducer of the MPT (Scarlettet al., 1996). Nitric oxide synthase (NOS) activity hasbeen shown to be elevated in experimental models of HE(Rao et al., 1995; Norenberg and Itzhak, 1995), andincreased NO production in brain was reported inportacaval-shunted rats after ammonia infusions (Master etal., 1999). Our study on ammonia-induced increase inastrocyte uptake of arginine, the precursor for NO, isconsistent with these observations (Hazell and Norenberg,1998). Furthermore, the NOS inhibitor, nitroarginine, hasbeen shown to attenuate ammonia toxicity (Kosenko etal., 1995).

Changes in mitochondrial respiration secondary toammonia are also relevant to the issue of the MPT, and

defective mitochondrial respiration may be a factor in theammonia-induction of MPT. Thus far, the effect of am-monia on mitochondrial respiration in cultured astrocyteshas not been reported. An in vivo study by Kosenko et al.(1996) reported, however, that isolated mitochondriafrom a rat model of acute hyperammonemia had reducedstate III respiration and a subsequent decrease in the re-spiratory control index (RCI). Other studies employingcongenital hyperammonemic sparse-fur mouse model in-dicated a progressive decrease in the activity and expres-sion of cytochrome C oxidase and complex IV of themitochondrial respiratory chain in different brain regions(Rao and Qureshi, 1997), as well as reduced activities ofother respiratory complexes in synaptic and non-synapticmitochondria (Qureshi et al., 1998).

Another potential factor in ammonia neurotoxicity isalkalinization of intracellular pH ([pH]i). Alkaline pH in-duces the MPT (Haworth and Hunter, 1979; Bernardi etal., 1992; Petronilli et al., 1993) and is known to aggravateHE, presumably due to the generation of the more gas-eous, and more permeable NH3 (Plum and Hindfelt,1976). It should be noted, however, that the literature on[pH]i in astrocytes after ammonia treatment shows con-flicting results. Ammonia alkalinizes cultured rat astrocytes(Boyarsky et al., 1993; Busch et al., 1996), as in other cells(Roos and Boron, 1981), and astrocytes from portacaval-shunted rats show increased [pH]i (Swain et al., 1991).Nagaraja and Brookes (1998), however, have recentlyreported that ammonia acidifies astrocytes derived frommice but not in rats. Because the potential cellular entry ofNH4

1 is presumably occurring via K1 channels (Nagarajaand Brookes, 1998), it is possible that pH discrepanciesmay be due to the unusually high number of K1 channelsfound in mouse astrocytes as compared to rat astrocytes(Walz and Kimelberg, 1985).

Elevated cellular and mitochondrial Ca21 is a criticalevent in the production of the MPT (Petronilli et al.,1993), although the MPT may occur in the absence ofelevated Ca21 levels (Huser et al., 1998). The precise roleof calcium in the ammonia induction of the MPT remainsto be defined. Little is currently known about the effects ofammonia on Ca21 homeostasis in astrocytes. Ammonia oralkalosis may lead to increased intracellular Ca21 in cul-tured neurons (Mironov and Lux, 1993; Hoyt and Reyn-olds, 1998), arterial smooth muscle (Chen and Rembold,1995; Siskind et al., 1989), platelets (Siffert et al., 1990),and HT29 cells (Nitschke et al., 1996; Benning et al.,1996). Kosenko et al., (2000) have shown that acuteammonia toxicity in vivo leads to an increase in mito-chondrial Ca21 accumulation, and recent unpublishedobservations in our laboratory have shown that ammoniaelevated intracellular Ca21 levels in cultured astrocytes.

Our study also showed that MSO inhibited theammonia-induced collapse of the Dcm suggesting thatglutamine may be involved in its formation. This findingis consistent with a recent report showing that glutaminewas capable of inducing swelling of isolated brain mito-chondria (Ziemiska et al., 2000). The latter study also

Fig. 7. Effect of CsA (1 and 5 mM) on the ammonia-induced increasein mitochondrial permeability as measured by [3H]-2-deoxyglucose-6-phosphate (2-DG-6-P) entrapment. Values are expressed as % dpm of2-DG-6-P/U citrate synthase. The dpm values in the control group isset at 100% and the values are mean 6 SE 7–8 individual cultures ineach group, taken from 3–4 different seedings. *P , 0.001 vs. control;†P , 0.001 NH41 alone.

988 Bai et al.

showed that acute (20 min) treatment with high concen-trations of NH4Cl failed to show such mitochondrialswelling. These findings can be explained by the fact thatisolated mitochondria cannot generate glutamine fromammonia as glutamine synthetase is only found in thesmooth endoplasmic reticulum (Sellinger and de BalbianVerster, 1962).

For some years, many of the detrimental effects ofammonia, such as depressed glucose utilization (Hawkinsand Jessy, 1991), altered CNS metabolism (Hawkins et al.,1993), vascular CO2 responsiveness (Takahashi et al.,1992), edema (Blei et al., 1994), and astrocyte swelling(Norenberg and Bender, 1994; Willard-Mack et al., 1996;Takahashi et al., 1991) have been attributed to glutaminerather than ammonia per se. Our findings with MSO areconsistent with the potential deleterious role of glutaminein ammonia neurotoxicity.

In summary, we have shown that ammonia inducesa dissipation of the Dcm in cultured astrocytes, but not incultured neurons. Ammonia also enhanced mitochondrialpermeability in cultured astrocytes. The effects of ammo-nia on the Dcm and on mitochondrial permeability wereblocked by cyclosporin A indicating that the MPT wasinduced by ammonia treatment. The effect of ammonia onthe Dcm was also blocked by MSO suggesting that dissi-pation of the Dcm may be a consequence of glutamineproduction. The induction of the MPT in astrocytes mayrepresent a fundamental aspect of the pathogenesis of HEand other hyperammonemic conditions.

ACKNOWLEDGMENTSThe confocal microscopic data was obtained at the

Core Facility, Sylvester Cancer Center, University of Mi-ami School of Medicine. We are grateful to Weiyun Sunfor preparation of cell cultures, to Drs. Youwei Chen andLilly Bourguignon for assistance in the use of the confocalmicroscope, and to Enrique Cepero and James Phillips forassistance with the flow cytometer. The helpful discussionswith Drs. Martin A. Philbert and Andrew P. Halestrap aregratefully acknowledged.

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