- - -

The FASEBJournal · Research Communication

Oral L-glutamine increases GABA levels in striatal tissueand extracellular fluid

Lei Wang,* Timothy J. Maher,*,t and Richard J. Wurtman*,1*Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge,Massachusetts, USA; andtDepartment of Pharmaceutical Sciences, Massachusett., College ofPharmacy and Health Sciences, Boston. Massachusetts, USA

ABSTRACT We explored the possibility that circulat-ing glutamine affects y-aminobutyric acid (GABA) lev-els in rat striatal tissue and GABA concentrations in

striatal extracellular fluid (ECF). Striatal microdialy-sates, each collected over a 20 min interval, wereobtained after no treatment, oral L-glutamine (0.5g/kg), or glutamine followed by NMDA (administeredvia the microdialysis probe). GABA concentrationswere measured by HPLC using a stable OPA/sulfiteprecolumn derivatization and an electrochemical detec-tion method. L-Glutamine administration significantlyincreased ECF GABA concentrations by 30%, and en-hanced the response evoked by NMDA alone (70%) to120% over baseline (all P<0.05). Striatal GABA levelsincreased significantly 2.5 h after oral L-glutamine (e.g.,from 1.76 :t 0.04 J.1mol/g in vehicle-treated rats to2.00 :t 0.15 J.1mol/g in those receiving 2.0 g/kg ofglutamine). Striatal glutamine levels also increased sig-nificantly, but not those of glutamate. These datasuggest that GABA synthesis in, and release from, ratstriatum may be regulated in part by circulating glu-tamine. Hence, glutamine administration may provide auseful adjunct for treating disorders (e.g., anxiety,seizures) when enhanced GABAergic transmission isdesired. Moreover, the elevation in plasma and brainglutamine associated with hepatic failure may, by in-creasing brain GABA release, produce some of themanifestations of hepatic encephalopathy.-Wang, L.,Maher, T.J., Wurtman, R.J. Oral L-glutamine increasesGABA levels in striatal tissue and extracellular fluid.

FASEBJ. 21, 1227-1232 (2007)

Key Words: microdialysi.r' glutamate' neurotran.rmiUer pre-Cltnor' hepatic encePhalopathy

A NUMBEROF NEUROTRANSMITTERSexhibit the prop-erty of having their rates of synthesis and/or releasedepend primarily on brain levels of their amino acidprecursors (1). Thus, tyrosine levels can control cate-cholamine synthesis in and release fi'om neurons thatare firing frequently (2, 3); serotonin's synthesis andrelease vary with brain tryptophan levels (4, 5). Thesynthesis of brain glutamate and -y-aminobutyric acid(GABA), the major central nervous system (CNS) exci-tatory and inhibitory neurotransmitters, respectively,are both linked to a substrate cycle involving glutamine

0892-6638/0710021-1227 (0 FASEB

metabolism (6) in neurons and astroglial cells. Circu-lating glutamate is unable to cross the blood-brainbarrier (BBB) (7) whereas glutamine can be trans-ported into the brain by facilitated diffusion, a Na +-independent process (8) in competition with otherneutral amino acids, or by a Na +-dependent process(mainly N-system transport) (9, 10), and brain gluta-mate serves as the precursor for GABA synthesis in vivo(11). The enzyme glutamate decarboxylase (GAD),which catalyzes the conversion of glutamate to GABA,has a high K,n for glutamate (2-5 mM) compared withusual glutamate levels in GABAergic neurons (1.6 mM)(12), and thus is likely unsaturated with its substrateunder normal conditions (13).

The eflects of providing supplemental glutamine onGABA synthesis and levels have been studied previouslyin vitro. In cultured rat pancreatic beta cells, cellularGABA content and release increased dose-dependentlywith increasing concentrations of glutamine (0.25-2mM) in the medium (14); in synaptosomes preparedfrom substantia nigra, GABA synthesis was stimulated3.1-fold when 500 fJ.M glutamine was added to theincubation medium (15); and in brain slices, the rate ofGABA synthesis, estimated in the presence of the GABAtransaminase (GABA:r) inhibitor gabaculine (20 fJ.M),was increased by 50% with 500 fJ.M glutamine (16).Furthermore, inhibition of glutamine transport candeplete glutamate and GABA in brain slices (17), andnuclear magnetic resonance studies have been inter-preted as showing that glutamine is the major precur-sor for GABA synthesis in rat in vivo (18). Apparentlyno information is available, however, about whether

exogenous glutamine administration might affect brainGABAlevels or release in vivo.

Plasma and brain glutamine levels can be augmentedby exogenous glutamine. Giving preteml infants glu-tamine-supplemented parenteral nutrition (0.4 g/kg/day of glutamine) for IS days increases their plasmaglutamine levels by 50% without adverse effects (19).Giving rats L-glutamine intraperitioneally (2g/kg) in-creased brain and serum glutamine levels by 50% and300%, respectively, without affecting those of glutamate

I Correspondence: 46-5023B, MIT, 43 Vassar St., Cam-bridge, MA 02139, USA. E-mail: dick@mit.edu

doi: 1O.1096/fj.06-7495com

1227

---

or aspartate (20). In the current study, we determinedwhether brain GABA levels and GABA concentrations

in ECF under conditions of spontaneous or evokedrelease can be increased by orally administering 1.-glutamine.

MATERIALS AND METHODS

Drugs and chemicals

Standards for GABA, glutamate, and L-glutamine were pur-chased from Sigma Chemicals (St. Louis, MO, USA). Q-Ph thai aldehyde (OPA) was purchased from Pierce Biotech-nology (Rockford, II.. USA). Sodium sulfite, N-methyl-u-aspartic acid (NMDA). perchloric acid (PCA) , sodium bordte,GABase (a mixture of GABA-T and succinic semialdehydedehydrogenase). GAD. and ~-mercaptoethanol were pur-chased from Sigma Chemicals.

Animals

Sixty-six male Sprague-Dawley rats (3 months old, -250 g)were purchased from the Charles River Laboratories (Wil-mington, MA, USA). The animals were exposed to a 12 hlight/dark cycle (7 AM to 7 PM). A standard rodent diet(LabDiet"" 5POO), which lacks glutamine. and water wereprovided ad libitum. All experimental procedures were ap-proved by the Massachusetts Institute of Technology Commit-tee on Animal Care.

Surgery and microdialysis

Thirty-two rats (4 groups) were utilized to measure ECFGABA concentrations using in vivo microdialysis. Rats re-ceived vehicle (saline, 2.5 ml/kg; groups I and 2) or 1.-glutamine (0.5 g/kg; groups 3 and 4) by gavage, then half ofthem (groups 2 and 4) funher received NMDA [500 j.lM inanificial cerebrospinal fluid (aCSF)] via retrodialysis. L-Glu-tamine was mixed in saline to form a suspension (4 g [0 10 mlsaline): this \\-'asdivided into daily doses and stored at-80°C.

Stereotaxic surgeries were carried out I wk prior to treat-ments, using ClIvtA/ll guide cannula aimed at the rightstriatum [AP= +0.5, ML=-3.0, DV=3.3 mm; according to theatlas of Paxinos and Watson (21»), under ketamine andxylazine (80 and 10 mg/kg i.p., respectively) anesthesia.

Microdialysis was perfOlmed on freely moving rats kept ina circular Plexiglas bowl on a rotating platform, obviating theneed for a liquid swivel. Each rat was dialyzed once using anew CMA/ll probe (4 mm membrdne length). ACSF con-taining. in mM: NaCl, 121; NaHCO~, 25; KCI, 3.5; CaCI\!, 1.2:MgCI\!, 1.2; NaH2PO", 1.0, was mixed with 95% O2 and 5%C02 for 15 min. This gas mixture was used to bring the finalpH close to 7.4. ACSF was then perfused through the probescontinuously at a rate of 1.5 j.ll/rnin. Samples collectedduring the first 2 h of microdialysis were discarded; subse-quent collections over the next 4 h were for 20 min intervals.The first three samples weT'e collected 10 establish baselineECF GA13A concentrations (estimated by actual GABA con-centrdtions in the dialysates; data were not corrected forprobe recoveries). Vehicle or L-glutamine were administeredby gavage, and three samples were collected to study 1.-glutamine's effects on ECF GABA concentrations under con-ditions of spontaneous GABA release; then striala were per-fused with aCSF or aCSF containing 500 j.lM NMDA, and sixadditional samples were collected to study changes in ECF

1228 Vol. 21 April 2007

--

GAl~A concentrations when GABA release was being evoked.This procedure yielded a total of 12 samples from each rat (3baseline + 3 samples under spontaneous conditions+6 evokedsamples with or witholll NMDA=12). All experiments werecarried out in a quiet cubicle. The acthities of the rats weremonitored for reference but not for data analysis. All sampleswere collected on crushed ice. then immediately fi'ozen andkept at -80°C until HPLC analysis.

Sample preparation for plasma and striatal levels ofglutamate, glutamine, GABA

Thirty-four rats (4 groups) were used to measure plasma andstriatal glutamate, glutamine, and GABA levels after 1.-glu-tamine administration. Rats received vehicle (saline; 2.5 ml/kg) or l.-glutamine (0.5, 1.0, and 2.0 g/kg). Preliminarymicrodialysis experiments showed that maximal GA13A re-lease evoked by NMDA occurs 2 to 3 h after l.-glutamineadministralion; hence, in this study rats were anesthetized(with ketamine), sacrificed, and decapitated 2.5 h afterdosing. Trunk blood and brain samples were then obtained.

Striata were quickly dissected on a chilled dissection board,homogenized on ice (50 mg of tissue with ] ml 400 mMHCI01, 50 IJ.MEDTA), and neutralized with 100 mM boratebuffer (1:10). The homogenates were then centrifuged(14,000 rpm, 15 min, 4°C) and filtered with Ultrafree-MCcentrifugal filter units (Millipore, Billerica, MA, USA; 14,000rpm, 1 min, 4°C). Glutamate, glutamine, and GABA concen-trations were detetmined by HPLC as described below.

GABA analysis

GABA concentrations in dialysates and striatal homogenateswere detelmined using a precolumn OPA/sulfite denvatiza-tion method. according to Smith and Sharp (22). This HPLCelectrochemical detection sYStem consisted of a Shimadzu

lOA HPLC pump, an Allt~ch 580 aULOsampler, an ESACoulochem II 5100A detector, and an ESA 50148 microdialy-sis cell (E.=+150 mV, E\!=+600 mV, E arc\=650 mY).Twenty microliter samples were automaticallY' mixed \\ith 10j.ll OPA/sulflte working (klivalizing reagent; 20 min later, 25j.ll of this mixtm'e was injected. The flow rate of the mobilephase (100 mM Na2HP04; ]0% acetonitrille) was 0.6 milmin. The column (ESA MD 150, 3x150 mm, 3j.lm) was keptat 40°C. Samples were injected using an Alltech 580 autosat1l-pIer, then data were collected and analyzed using an AII-Chrom workstation (Alltech, Deetiield, IL, USA).

Stock solutions of the OPA/sulfite-derivatizing "eagentwere prepared by dissolving 22 mg OPA in 0.5 ml of absolllleethanol. To this was added 0,5 ml of 1M sodium sulfite,followed by 9 ml of 100 mM sodium borate buffer adjusted topH lOA with sodium hydmxide. The working OPA/sulfitesolution was prepared by diluting 50 j.ll of OPA/sulfite stocksolution with 5 ml of deionized water.

We validated the GA13A peak in the dialysate using OPAlsul/ite dcrivalization of samples pretreated with GABase,which destroyed the GABA. We were unable to validate theglutamate peak by convening the glutamate to GABA withGAD, then treating it \\ith OPA/sulfite. Hence, glutamateand glutamine were measured using the OPA/~-mercapto-ethanol method as described below.

Glutamate and glutamine analysis

Glutamate and glutamine were measured as their OPA/~-mercaptoethanol derivatives according to Donzanti andYamamoto (23). HPLC conditions were similar to those used

The FASEBJournal WANG ET AL.

- ------ -- --

for GABA detenninations, except for the following: E~ of theESA 5014B microdial)'sis ceIl was set at + 530 mY, and the

mobile phase consisted of 100 mM Na2HPO'j at pH 6.75 with38% methanol. Twenty microliter samples were automaticaIlymixed with 10 f.ll of the OPA/j3-mercaptoethanol workingderivatizing reagenr; 25 f.ll of this mixture was injected afterwaiting for 2 min.

Stock solutions of the OPA/I3-mercaptoethanol derivatiz-ing reagent were prepared by dissolving 27 rng OPA in I IIIIof methanol. Five microliters of j3-mercaptoethanol and 9 mlborate hulTer (\00 mM, pH 9.3) were then added. Theworking OPA/j3-mcrcaptoethanol solution was prepared bydiluting I ml of OPA/j3-mercaptoethanol stOck solution with3 ml of borate buffer.

Data analysis

Statistical analyses wel'e carried out using SPSS 12.0. Data arerepresenred as means :t SEs. Student's t t.est (2-t.ailed), I-way.and 2-way ANOVA wit.h repeated measurements were used toassess the statistical significance of effects. Tukey !}()J! ho(analyses wer'e used when appropriate. The significance levelwas set at P < (1.05.

RESULTS

Effects of oral L-glutamine on striatal ECF GABAconcentrations measured by microdialysis

Striatal baseline GABA concentrations in microdialy-sates did not differ (F3. 2~)=0.05; P>0.05) among thefour groups of rats prior to oral administration ofvehicle or L-glutamine. Average values were 21 ::!:1 nM(43.~~::!:2.1pg/20 fl.l).

The effects of L-glutamine (0.5 g/kg) on striatal ECFGABA concentrations, estimated by it,>concentrationsin dialysates, arc shown in Fig. IA (spontaneous releasecondition), and B (spontaneous release, followed byevoked release with NMDA). Each data point repre-sents the sample collected during the preceding 20min. During the first hour of collection, GABA baselineconcen trations in each rat were established, pooled,and set as 100%. At the start of the second hour,

glutamine or vehicle was administered orally. Two-wayANOVA with repeated measurements (glutamine orvehicle as between-subject factors and time as within-subject factors) revealed that glutamine increased ECFGABA concentrations compared with those after vehi-cle WI.!J=6.02, P<0.05, Fig. IA). The maximal increase(by 30% over baseline, P<0.05) occurred around thestart of the 3rd hour (i.e., 1 h after rats receivedglutamine; Fig. IA). NMDA alone (500 fJ.M in aCSF,perfused continuously during the 3rd and 4th hours)significantly increased ECF GABA concentrations, caus-ing a maximal 70% rise (P<O.OI; Fig. IB). Amonganimals that had also received glutamine, the NMDAincreased ECF GABA concentrations, causing a maxi-mal 120% rise (P<O.O1; Fig. IlJ). I.-Glutamine treat-ment increased ECF GABA concentrations under

NMDA-stimulated conditions (t=2.68, P<0.05, Fig. IB).

GLUTAMINE INCREASES BRAIN GABA RELEASE

Changes in ECF GABA concentrations (8 value)between baseline samples during the first hour ofcollection and those thereafter are presented in Fig. 1C(spontaneous conditions; data collected during the firsthour were omitted) and Fig. ID (evoked conditions;data collected during the first 2 h were omitted).Horizontal axes indicate time (hours) of the microdi-alysis experiments. Data represent the average 8 valuesper hour obtained by pooling the three 20 min intervalsamples; thus each data point represent'> the samplecollected during the preceding hour. At the start of thesecond hour, vehicle or glutamine were administered(Fig. 1C); NMDA was perfused during the 3rd and 4thhours to rats that had or had not received glutamine(Fig. ID). Two-way ANOVA using time (hours) andgroups (glutamine or vehicle) as the factors revealed asignificant effect of glutamine on ECF GABA concen-trations under both spontaneous (F1.41=5.8, P<0.05)and evoked conditions (FI.29=4.3, P<O.05).

Effects of oral L-glutamine on striatal glutamate,glutamine, and GABA tissue levels

The effecL,>of oral L-glutamine on striatal glutamate,glutamine, and GABA tissue levels are shown in Table1. In vehicle-treated animals, striatal glutamate, glu-tamine, and GABA tissue levels were 11.25 ::!: 0.19

fl.mol/g, 5.60 ::!:0.11 fl.mol/g, and 1.76 ::!:0.04 fl.mol/g(wet weight of tissue), respectively. Oral L-glutamineadministration (0.5 g/kg) significantly increased stria-tal glutamine by 5% (F:!.2!J=6.0, P<O.OI) and GABAlevels by 11% (F3,:!7=3.5, P<0.05) without affectingthose of glutamate (F2.29=1.4, P>0.(5) (I-way AN OVAwith L-glutamine dose as the factor). The effect of 1.0g/kg glutamine (8%) on striatal GABA was not signif-icant, but the effect of 2.0 g/kg (14%) was.

Effects of oral L-glutamine on plasma glutamate andglutamine levels

The effects of oral I.-glutamine on plasma glutamateand glutamine concentrations are shown in Table 1. Invehicle-treated animals, plasma glutamate and glu-tamine concentrations were 116.9 ::!: 5.4 fl.M and722.2 ::!: 38.5 fl.M. Oral I.-glutamine administrationsignificantly incrt'ased dose-dependently plasma glu-tamine concentrations obtained after 2.5 h (F.) "I =4.6,P<0.05) without affecting those of gl~It.'unate(1'2,21=1.1, P>0.05) (I-way AN OVA with I.-glutaminedose as the factor).

DISCUSSION

These data demonstrate that giving animals L-glu-tamine, an amino acid precursor for brain glutamateand GABA synthesis, by gavage increases their striataltissue levels and ECF concentrations of GABA (Table 1;Fig. 1), as measured by postmortem assays and in vivomicrodialysis. The enhancement of ECF GABA occurs

1229

- ---- - --

B5tJtl -.

VHI , 1\~1f):\. liLN + !\MDi\~5n ..

:m . (;I.J\ VI'II

~~o. .. .

100.

511~ t\MDi\:,OU 1'\1

1\ '.'¥

(.lJ L~U j S~) :~n I:Zd 1 1N'

Tim\: (min)

Dl~n h nkl'd

rJ VEIl prctreah:d

. Iii 1\ prelreat,..!L:~

WH

:- --1i !

iI j r-.",- I ~

.<

:'41

?;'

.. !

\ ., ~

1\\10.'\(,U, 1\\11),\

Time (Hours)

Figure 1. Effect of oral glutamine on ECF GABA concenlrations, The effect~ of L~htamine (0.5 g/kg) on striatal ECF GABAconcentrations estimated by it~ concentnuions in dialysates arc shown in panel A (spontaneous release condition) and panel B(sp011laneous release, followed by evoked release with NMDA). Each data point represents the sample collected during thepreceding 20 min. During the first hour of collection, GABA baseline concentrdtiollS in each rat were established, pooled, andset as 100%. At the start of the second hour, glutamine or vehicle was administered orally. Glutamine increased ECF GABAconcentrations during the next 2 h compared with those after vehicle (A). The maximal increase (by 30%, P<O.(5) occurredaround the start of the 3rd hour (i.e., 1 h after rats received glutamine; panel A). NMDA alone (500 f.l.Min aCSF, perfusedcontinuously during the 3rd and 4th hours) significantly increased ECF GABA concentrations, causing a maximal 70% rise(P<O.OI, panel B). Among animals that had also received glutamine, the NMDA dose increased ECF GABA concentrations,causing a maximal 120% \;se (P<O.01. panel B). Changes in ECF GABA concentrations (0. value) between baseline samplesduring the first hour of collection and those thereaftet' are presented in panel C (spontaneous conditions; data collected duringthe first hour were omitted) and panel D (evoked conditions; data collected during the first 2 h were omined). Horizontal axesindicate time (hours) of the microdial)'sis experiments. Data represent the average 0 values per hOllT obtained b)' pooling thethree 20 Olin intetval samples; thus each data point represents the sample collected during the preceding hour. At the start ofthe second hour. vehicle or glutamine were administered (C); NMDA was perfused during the 3rd and 4th hours to rat~ .hathad or had not received glutamine (D). Two-way ANOVA using time (hours) and groups (glutamine or vehicle) as the fa, torsrevealed a significant effect of glutamine on EeF GABA concenrrations under both spontaneous and evoked conditions. Dataare given as means:!: st:. *p < 0.05 lIS. the groups of rats received vehicle with/without NMDA (unpaired Student's test,2-tailed). GLN: L-glutamine; VEH: vehicle.

under both spontaneous and evoked (by NMDA) con-ditions. Oral l.-glutamine also dose-dependently in-creases plasma and striatal gltttamine levels withoutaffecting those of glutamate.

Measuring GABA is challenging given its low concen-tration in brain microdialysates. The common HPLC-based method for amino acid analysis, which utilizes aprecolumn derivatization of OPA and f3-mercaptoetha-nol, forms an unstable derivative with GABA, with ahalf~life of only several minutes (23). Hence, we usedthe OPA/sulfite derivatization procedure, which pro-

vides a sensitive and robust method to measure GABA

concentrations in both brain tissue and microcjalysissamples.

Oral l.-glutamine administration most likdy in-creased the synthesis and level of GABA by increasingthe saturation of GAD, in GABAergic neurol'S, withglutamate. We hypothesize that some of the cil' ulatingglutamine crossed the BBB, and entered GABAergicneurons, where it was deaminated by phosphate-acti-vatedglutaminase (PAG) to glutamate, then dl carboxy-lated by GAD to GABA.

Brain (~mol/g wel weight of tissue)

TABLE 1. Effect oJ L-glutamine on brain and plllJ7/1a leueLJ oJ gLUlll71tl1ll',glutamine and CABAn

Plasma (~M)

TreatmentvehicleGLN (0.5 g/kg)GLN (1.0 g/kg)GLN (2.0 g/kg)

Glut.'1mate11.25:!: 0.19 (11)11.20 :!: 0.23 (9)10.81 :t 0.20 (10)

Glutamine5.60 :!: 0.11 (11)5.88 :!: 0.11 (9)*6.09 :!: 0.08 (10) 1

GABA1.76 :!: 0.04 (11)1.96 :!: 0.05 (9)*1.91 ::+:0.07 (8)2.00 :!: 0.15 (4)*

Glut.'1mate116.9 :!: 5.4 (9)115.0 :!: 6.2 (8)105.6 :t 3.9 (7)

Glut.'1mine722.2 :!: 38.5 (9)865.3 :!: 46.1 (6)*887.0 :t 44.9 (9)*

"Rats (250 g) were omlly administered various doses of L-glutalllille or vehicle. All d,'ugs were given 2.5 I, before sacrifice. Data are givenas means:!: st: GLN: L-glutamine. :-.lumber of determinations is shown in parentheses. *P < 0.05. ditTers from vehicle group. : P < 0.0 I. differsfrom vehicle group.

1230 Vol. 21 April 2007 The FASEBJournal WANG ET AL.

AI.I

Vi'll':Jz . liL1\::1<:J tfl2t;-J'. ::.."2 (;1 !\" III

II i .. .< . .::Q ,,'II..;I . .:'!....,

Q.

h

c-':J'" 1 Sp,'nl,mc,JU'<J,

0:'.J-;;:j... ";1

;;'"

.D IIt-o"

1"g'":.- III

:0

< " LI'"",c:<

"iJ u.. -h;

VIII

- -

Prcvious sludies and our curren I dala confirm lhal

exogenous L-glutamine can increase glutamine levels inthe brain (rer. ~O and Table 1). l:IC.Labeled tracerstudies have shown that dietary glutamine can beabsorbed unchanged into blood (24). L-[He] Glu-tamine perfusion studies in rats showed that plasmaglutamine can be taken up into the brain, mainly by the:'>Ja+-dependent N-system transport (9, 10). Under nor-mal conditions, the glutamine concentration in plasmais 0.5-0.8 mM (ref. 25 and Table 1), which is muchlower than the ~l (3.3 mM) of the N-system trans-porter (9); hence, this transporter in BBB is unsatur-ated by circulating glutamine. Since ECF concentra-tions of glutamine, measured by microdialysis ",ith azero-net-flux technique, are in the submillimolar range(0.38 mM) (26), most of the increase in brain glu-tamine is intr.lcellular, probably including GABA-form-ing neurons.

There appears to be a limited supply of glutamineavailable in GABAergic neurons to form glutamate.Brain contains mainly a kidney-type PAG (25), whichexhibits a ~, for glutamine in the r.lnge of 2-5 mM(25). Glutamine concentration in GABAergic neuronsis estimated to be -1.6 mM [20% of the whole brainglutamine concentrations (12)], which is lower thanthe 1\., of PAG. Moreover, given that glutamate levels inGABAergic neurons are much lower than that of gluta-matergic neurons (discussed below) and PAG has es-sentially the same activity in cultured GABAergic andglutamatergic neurons (27, 28), PAG probably does notreach its maximal activity in GABAergic neurons, since'apparently more glutamate can be formed in glutama-tergic neurons.

Increased glutamate availability in GABAergic neu-rons may regulate GABA formation and release. Brainglutamate levels are -10 f.1mol/g tissue (20), but thedistribution of glutamate is highly compartmentalized(29) and GABAergic neurons utilize a small precursorpool of glutamate «5% of the total amount; ref. 30) inwhich the glutamate concentration (-0.6 mM) appearsto be lower than the Km of GAD (0.2-1.2 mM) (13).Moreover, the r.lte of GABA syn thesis in brain is a smallfraction (3-17%) of total GAD activity (13), indicatingthat GAD also operates at only a small fraction of itsmaximal catalytic capacity. GABA, like glutamate, isunable to cross the BBB (31); hence, glutamine-in-duced increase in brain GABA levels <md release are

unlikely to be derived from circulating GABA.Our data suggest that increased circulating (32, 33)

and br.lin (33-36) glutamine levels leading to in-creased concentrations and release of brain GABA maybe involved in the hepatic encephalopathy of liverfailure and ammonia toxicity (37). GABAergic trans-mission has been implicated in the pathogenesis ofhepatic encephalopathy (38, 39), and antagonists ofGABA receptors can ameliorate manifestations of thisdisorder. Decreased glutamate levels have consistentlybeen observed in brain homogenates from rats withacute liver failure (40) and from patients who died ofhepatic coma (41).

GLUTAMINE INCREASES BRAIN GABA RELEASE

Deficienl GABA-m(~dial.ed brain nCllrolransmission

may be a factor in various diseases, including anxietydisorders (42), epilepsy (43), and schizophrenia (44).The ability of oral L-glutamine administration to in-crease brain GABA level and release in rats raises the

possibility that this compound might possess sometherapcntic benefits in tl1ese disorders. ~

The authors thank Carol Watkins, Meredith Albrecht, andMark Bohlke for advice and excellent assistance. These

studies were supported by grants from the National Institutesof Mental Health (#2 ROl MH028783-31) and from TheCenter for Brain Sciences and Metabolism Charitable Trust.

REFERENCES

1. Wurtman, R Joo Hefti, F., and Melamed, E. (1980) Precursorconu'ol of neurouansmiuer synthesis. Pharmacal. Rev. 32, 315-3~ .

2. Melamed, E., Hefti, F.. and Wurtman. R. J. (1980) Tyrosineadministration increases sniatal dopamine release in rats withpartial nigrostriatal lesions. ?roc. Nail. Amd. Sci. U. S. A. 77,4305-4309

3. Acwonh, I. N., During, M. .J., and Wurtman. R. J. (1988)Tvrosine: effects on catecholamine release. Brain Res. BulL 21,473-477

4. Fernstrom, J. D.. and Wurtman. R J. (1971) Brain serotonincontent: ph>'Siological dependence on plasma tryptophan levels.Scienc:e173, 149-152

5. Schaechter, J. D., and Wurtman, R.J. (1990) Serotonin release~-aries with blain tryptophan levels. Brain &.f. 532, 203-210

6. Bak. L. K., Schousboe, A.. and Waagepetersen. H. S. (2006) TheglUlamateiGABA-glutamine cycle: aspects of !lansport, neuro-transmitter homcostasis and ammonia transfer. J. Nt>1I1"Ochem.98,641-653

7. Pardridge, W. M. (1979) Regulation of amino acid availability tobrain: selective control mechanisms for glutamate. In GlutamicAcid: Atlvanas in Biochemistry' and PhYJiolQgy(Filer. L. J., Jr., ed)pp. 125-137, Raven Press, New York, NY

8. Smith, Q. R, Momma, S., Aoyagi, M.. and Rapoport. S. I. (1987)Kinetics of neutral amino acid transport across the blood-brainban'ier..f. Neurochem. 49, 1651-1658

9. Keep, R F.. and Xiang.J. (1995) N-system amino acid transportat the blood-CSF barrier. J. Neurochem. 65, 2571-2576

10. Ennis, S. R, Kawai, N., Ren, X. D., Abdelkarim, G. E., and Keep,R. F. (1998) Glutamine uptake at the blood-brain barrier ismediated by N-system transport. J. Neurochem. 71, 2565-2573

II. 'hite, H. L. (1981) Glutamate as a precursor of GABA in ratbrain and peripheral tissues. 1"101.Ceu Bioclll!1n.39, 253-259

12. Kanamori. K., and Ross, B. D. (2005) Suppression of glialglUtamine release to the extracellular fluid stUdied in vivo by:'\MR and microdialysis in h)'j)erammonemic rat brain. J. Nellr/jochenl. 94, 74-85

13. Martin, D. L., and Rimvall, K. (1993) Regulation of Ir.unma-aminobutyric acid s)'I1thesis in the brain. J. Neurochem. 60,395-407

14. Smismalls. A., Schuit, F.. and Pipeleers, D. (1997) Nutrientregulation of gamma-aminobUtytlc acid release from islel betacells. Di~tologia40, 1411-1415

15. Battaglioli. G., and Martin, D. L. (1996) Glutamine stimulatesGABA synthesis in synaptosomes but other putative astrocyte-to-neuron shuttle substrates do no!. NmTOJci. l..e/l. 209, 129-133

I Ii. Battaglioli. G., and Martin, D. L. (1991) GA!3J\ synthesis in brainslices is dependent on glutamine produced in astrocytes. Nt'lIro-chenL Res. 16, 151-156

17. Rae, c.. Hare, N., Bubb, W. A., McEwan, S. R., Bruer, A..

McQuillan,.J. A., Balear. V. J., Conigr.l.ve, A. D., and Broer. S.(2003) Inhibition of glutamine transport depletes glutamateand GABA neurotransmitter pools: further evidence for meta-bolic compartmentalization. J. Neurochem. 85, 503-514

18. Patel, A. B., Rothman. D. L., Cline, G. W.. and Behar, K. L.

(2001) Glutamine is the major precursor for GABA synthesis in

1231

rat. neocollex in vivo following acut.e GABA-mmsaminase inhi-bition. Brain 111'.1.919, 2U7-:!20

19. Lacey,]. M.. Crouch,.J. B., Benfell, K.. Ringer, S. A., Wilmore,C. K., Maguire, D.. and Wilmore. D. W. (1996) The e!Tects ofglutamine-supp]ement.ed parenterdl nutrilion in premature in-fants..I Parentcr. F:nll'ral. Nutr. 20, 74-80

20. Liebschutz,j.. Airoldi, L.. Bmwnstein, M.J.. Chinn. N. G., andWUrlman, R.]. (1977) Regional distribution of endogenous andparenteral glutamate, aspartate and glutamine in rat brain.Biochem. Pll(mnllCol. 26, 143-446

21. Paxinos, G.. and Watson, C. (2004) 771cRat BUlin in Stt'rcotuxirCnordiuflte.\' (5th Ed). Elsevier, San Diego

22. Smith, S., and Sharp, T. (]994) Measurement of GABA in rat.brain microdialysates nsing o-phthaldialdehyde-sulphite denva-lizalion and high-pel1ormance liquid chromatogmphy withelectrochemical detection..I Cll11mlalogr.B. 652, 228-233

23. Donzmiti, B. A., and Yamamoto, B. K. (1988) An improved andrapid HPLC EC method for the isocrdtic separation of aminoacid neurotransmitters from bmin tissue and microdialysispelfusates. Life St:i. 43, 913-922

24. Decheloue, P., Darmaun. D.. Rongier, M., Hecketsweiler, B.,Rigal, 0., and De~jeux,J. F. (1991) Absorption and metabolice!Tects of enter all}' administered glutamine in humans. Am.].PhYJiol. 260, G677-G682

25. Curthoys, X P., and Watford, M. (1995) Regulation of glutam-inase activity and glutamine metabolism. :htnu. Rr.u. Nutr. 15,133-159

26. Kanamori, K., and Ross. B. D. (2004) Quantitalive detennina-tion of extracellular glutamine concentration in rat brain, andits elevation in vivo b}' system A transport inhibitor. alpha-(methylamino) isobutyrate.]. Neurochcnl. 90, 203-210

27. Larsson, O. M., DI'ejer,].. Kvamme, E., Svenneby, G.. Hertz, L.,and Schousboe. A. (1985) Ontogenetic development of gluta-mate and GABA metabolizing enzymes in cultured cerebralcortex interneurons and in cerebral cortex in vivo. Int..I Der'.NeuroJCi. 3, ] 77-185

28. Waagepetersen, H. S., Sonnewa]d, U., and Schousboe, A. (1999)The GABA paradox: multip]e roles as metabolite, neurotrans-mitter, and neurodi!Terenliative agent../. Neumche7ll. 73, 1335-1342

29. Storm-Mathisen, j., Leknes, A. K., Bore. A. T.. Vaaland, j. L.,Edminson, P., Haug. F. M.. and Ottersen, O. P. (1983) Firstvisuali7.ation of glmamate and GABA in neurones by immuno-cytochemistry. Nature 301,517-520

30. Patel, A. j., Johnson, A. L., and Balazs, R. (1974) Metaboliccomparnnentation of glutamate associated with the formationof g'.lmma-aminobutyrate. J. Neurocltem. 23, 1271-1279

31. Zeneroli, M. L., Iuliano, E., Ra(:agni, G., and Bardldi. M. (\982)Ml"tabolism and bmin uptake of g'.lmma-aminobutyric acid ingalactosamine-induced hepatic encephalopathy in rats.]. Nt'UTO-ch~n. 38, 1219-1222

1232 Vol. 21 April 2007 The FASEB Journal WANG ET AL.

32. Van l.eeuwen. P. A., Hong, R. W., Round~,j. D., Rodrick, M. L.,and Wilmore, D. (1991) Hepatic failure and coma afler liverresection is reversed by manipulation of gut contents: the role ofendotoxin. Surgt'lY 110, 169-174

33. D~jong, C. H., Kampman, M. T.. Dl"utz,:>I. E., and Soeters, P. B.(1992) Cerebral cortex ammonia and glutamine ml"tabolismduring liver insnfficil'ncy-innuccn hyperammonemia in the ral.J. Neurorhem. 59, 1071-1079

34. Weiser, :'vI., Riederer, I)., and Kleinberger. G. (1978) Humancerebral free amino acids in hepatic ('oma.]. Neural. Tromm.Sup/II. 14,95-102

3:3. Vogels, B. A., van Steynen, B., Maas, M. A.,.Torning. G. G., andChamuleau, R. A. (1997) The e!Tects of ammonia and ponal-systemic shun ling on brain metabolism. neurotransmission andintracmnial hypertension in hyperanunonaemia-induced en-cephalopathy. J. lIepatol. 26, 387-395

36. Funovics,.J. M., and Fischer,.J. E. (1978) Brain energy metabo-lism and alterations of transmiuer profiles in acme hepaticcoma.]. NeuraL TrallslII. Suppl. 14,61-67

37. Grippon, P., Le Poncin Lafitte, M., Boschat. M.. Wang, S., Faure,G., Dutertre, D., and Opolon, P. (1986) E\~dence for the J"Oleofammonia in the intracerebrdl transfl"r and metabolism of tl)'P-tophan. Hepatology 6, 682-686

38. Basile, A. S. (2002) Direct and indirect enhancement ofGABAergic neurotransmission by ammonia: implications for thepathogenesis of hyperammonemic syndromes. Neurocltem. /tIt.41, 115-122

39. Wysmyk, U., qja, S. S., Saransaari, P., and Albrecht, J. (1992)Enhanced GABA release in cerebral conical slices derived fJ"Om

rat~ with thioacetamide-induced hepatic encephalopathy. Neu-rochcnL ReJ. 17, 1187-1190

40. Hilgier, W., and Olson,]. E. (1994) Brain ion and amino acidcontents during edema developme11l in hepatic encephalopa-thy..! NeuTOchnn. 62, 197-201

41. Lavoie, j., Gigul"re, j. F., Layrargues. G. P.. and Butterworth,R. F. (1987) Amino add changes in autopsied brdin tissue fromcirrhotic patients \\~th hepalic encephalopathy.]. Nmroclteln. 49,692-697 .

42. Anagnostaras, S. G., Craske, M. G., and Fanselow, M. S. (1999)Anxiety: at the intersection of gl"nes and l"xpeIience. Nat.Neurosci. 2, 780-782

43. Koh]ing, R. (2002) GABAbecomes exciting. Science298, 1350-1351

44. Benes, F. M., McSparren,j.. Bird, E. D.. SanGiovanni,j. P., andVincent, S. L. (1991) Deficits in small interneurons in prefrontaland cingulatl" cortices of schizophrenic and schizoaffectivepatients. Arch. Gen.P.rychiatry48, 996-1001

Received for publication October 12, 2006.

Accepll'dfor publicationNovember21, 2006.