plasma daba & gaba

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Jourtiol of Neurochemis i ry36(2):544-550. February. Raven Press, New YorkQ 1981 International Society fo r Neurochemistry0022-3042/8110201 05441S02.0010

Comparison of DABA and GABA Transport into PlasmaMembrane Vesicles Derived from Synaptosomes

Robert Roskoski, Jr.Depurrrnrnr of Biocl iemistr j . Louisiana State Universi ty Medicrtl Cen ter . Nei l , Orleans, Louisiunu 7011 2 , U . S . A

Abstract: Transport of GABA by a high-affinity transport system ( K , =M ) is thought to terminate the action of this postulated neurotransmitter. 2,4-Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronalelements and inhibits GABA uptake. Localization of [:1H]DABA by auto -radiography has been used to identify neuro ns with the GA BA high-affinity trans-port system . After reconstitution of lysed synaptoso mal fractions in potassiumsalts, transfer of these membrane vesicles to sodium salts produces sodiumand potassium ion gradients which drive [ 'HJGABA and [ 'HIDABA trans-port. For each. transport requires external sodium, is abolished by ionoph oresthat dissipate the Na' gradient, and is enhan ced by conditions which make theintravesicular electromotive force more negative. Some characteristics of th etransport of these substances, however, differ. Fo r example, external chlorideis required for GABA, but not DAB A, transpo rt. Internal potassium is requiredfor DABA, but not GABA, transport. DABA is a competitive inhibitor ( K , =0.6 mM) of GABA transpo rt into membrane vesicle and synap tosomes. GA BA ,however, is a feeble inhibitor of DABA uptake into the membrane vesicles.Thes e differences suggest that the two substan ces are transported by differentmechanisms and possibly by different carriers. In addition to these experi-ments, using enzy matic-fluorometric techniques, it was show n that the artifi-cially imposed ion gradients drive net chemical transport of GABA into thevesicles. Key Words: GABA-DABA-Synaptosomes-Membrane vesicles-Transport . Roskoski R., r. Comparison of DABA and GABA transport intoplasma membrane vesicles derived from synaptosomes. J . Neurochern. 36,544-550 (1981).

GAB A (y-aminobutyric acid) is a postulated neu-rotransmitter in the ver tebra te C N S (Krnjevic,1970). Nerve terminal preparations (synaptosomes)accumulate GABA by high-affinity (low K,) andlow-affinity (high K,) trans port s ystem s (Weinsteinet al., 1965; Ma rtin, 1973; Lev i and Ra iteri, 1974).Two similar classes of transport systems occ ur forother neuroactive amino acids (Iversen and John-ston, 1971; Logan and Snyder, 1972). The low-af-finity transport systems are associated with bothneuroactive and inactive amino acids (Logan andSnyder, 1972). The high-affinity system s, w hich areNa+ - and tem perature-dependent are asso ciatedwith the neuroactive amino acids, including GAB A.It has been suggested tha t the high-affinity systemsare specifically involved with the termination of

neurotransmitter action by uptake (Iversen, 1971).Levi and Raiteri (1974) and Simon et al. (1974) re-ported that th e high-affinity synaptosomal transportsystem mediates the exchange of endogenous andexternal GABA, but little net transport. We demon-strated that synaptosomes take up net amounts ofGABA, glutamate , and aspar ta te by the h igh-affinity system (Ryan and Roskoski, 1977; Ros-koski, 197th; 1979); exchan ge of each am ino acidalso occurs. We suggested that high-affinity trans-port is reversible and that the direction of movementmay be related to the bioenergetics and mechanismof uptake.Iversen and Johnston (1971) demonstrated that0.1 mM-DABA (2,4-diaminobutyric acid) inhibits[3H]GABA accumulation in brain slices. Simon and

Received January 30, 1980; accepted July 25 . 1980.Abbreviations used: GABA. y-Arninobutyric acid; DABA,

2.4-Diaminobutyric acid: Me s. 2-(N-Morphol ino) ethanesul-fonic acid.

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D A B A A N D G A B A T R A N S P O R T I NT O P L A S M A M E M B R A N E V E SI CL E S 545

Martin (1973) studied the transport of both GABAand DABA into rat brain synaptosomes. Theyshowed that DABA is a competitive inhibitor of13H]GABA uptake . Using low -specif ic-activi ty["CIDABA, they showed that its uptake was par-tially inhibited by GABA. They pointed out thatthere may be a population of synaptosomes activein DABA, but not GABA, uptake. Kelly and Dick(1975) repor ted tha t 13H]DABA is taken up byneuronal elements, but not in glia in rat cerebellum.These investigators emphasized the similarity ofdistribution of [3H ]DABA and ["IGABA.Several investigators have prepared membranevesicles which take up su bstrates in response to ar-tificially imposed ion gradients. Kann er (1978), forexample, prepared fractions from synaptosomalmembranes active in GABA transport. In the pres-ent study th e chara cteristic s of [3H]DABA and[3H]GABA transport into these vesicles w ere com-pared. Although there are many similarities, sub-stantive differences exist, which raise the possibil-i ty that DABA is transported, at least in part, by anon-GABA transport system.

MA T E R IA L S A N D ME T H O D SPreparation of Membrane Fractions

Rat cortical synaptoso mes were prepared from maleSprague-Dawley ra t s (125-200 g) by the me thod ofHaycock e t al. (1978). The synapto somes were lysed andthe plasma membran e fract ion was prepared by the pro-cedure of Kanner (1978). The lysed membrane fract ionwas stored in 200-pl aliquots [about 5 mgiml protein de-termined by the pro cedure of Low ry et a l . (1951)l inliquid N,.

Transpor t Assay sTransport m easurements w ere carr ied out by the meth-od s of Rudnick (1977) and Kann er (1978). wi th minormodif icat ions . Un less otherwise noted, the thawed mem-brane fract ion was reconst i tuted in 4 volumes of 100m w K C I . 10 m w N a C I , a n d 9 0 m M - c h o l i n e c h l o r i d ebuffered with 5 mal-Mes-Tris (pH 7.0) at a concentrat ionof about 1 mgiml protein for 10 min at 37". After centrifu-

gation (27.000 x gfor 10min). the mem branes were resus-pended (about 1 mgiml protein) in fresh solution of thesame composi t ion by vortex mixing. To measure t rans-port , a 20-pl port ion was t ransferred to 180 p1 of t ransportsolution consisting of 100 m w N a C I p l us 100 mM-cholinechloride buffered with 5 m w M e s - T r i s (p H 7.0). This pro-cedure genera t es Na* (Naa , ,u ,c , r , ,> N a + l n s l d r ) n d K '(K- ,",,,,,, >K',,, ,,,, ,r ) gradients . The external t ransport so-lution also contained ["HIGABA (26,600 c.p.m.ipmol) orL-["H]DABA (6850 c .p.m.ipm ol) to give a f inal concen-tration of 0.14 pxi . After incubation fo r the specified timeat ambient temperature (22-24") , t ransport was termi-nated by addition of 2 ml of ice-cold 0.20 M-NaCI in 5mxi-Mes-Tris (p H 7.0) and f i l t ra t ion through 25-mm-diameter W hatman GFiA glass-f iber discs . Th e f i l t ra t ionra t e was 2 ml per 2-4 s. The f i l ters were washed twice

with 2 ml of 0.20 M-NaCI. After drying ( 5 min. IIO"),radioactivity was measured by liquid scintil lation spec-t rometry, us ing Budget-Solve (Research Products Inter-national C orp .) as scintil lant with a n efficiency of 35%. Tomeasure background radioactivity. the ice-cold 0.2 hi-NaCl w as added before the mem brane fract ion; f i lt rat ionand washing were then performed imm ediately. as de-scribed.Each measurement was performed in duplicate: theagreement was wi thin 1057, or the measurement was re-peated. There is a two- to threefold variation in rates andextent from one membrane preparat ion to another basedon protein. Values from a given membrane preparat ion,however , va r i ed on ly 10-15%. Each exper iment wasperformed with a t leas t three membran e preparat ions ands imilar resul ts were obtained.Transpor t Measurement in Synaptosomes

These experim ents were performed by the method pre-viously outlined fo r choline uptak e (Rosko ski, 19786).Transp ort was terminated by addi t ion of 4 ml of ice-cold0.9% N aCl followed by fil tration thro ugh GF iA filters; thefilters were washed twice with two additional 4-ml por-t ions .

MarerialsLabe led GABA and DABA were ob ta ined f rom NewEngland Nuc lea r Corp. and Amersham. re spec t ive ly .Nigericin and mon ensin were gifts from Eli Lilly and Co.Other drugs and compou nds were purchased f rom S igmaChemical Co.

R E S U L T SG e n e ra l C h a ra c t e r i s t ic s of DABA andGABA Transp or t

Using 0.14 FM labeled substrate, the rates ofDABA and GABA t ranspor t in to recons t i tu tedvesicles are linear for about 30 s (Fig. 1) . GABAuptake exhibits a characteristic ov ershoot. With 1OmM subs trate und er the sam e experim ental condi-tions, there is appreciable DABA uptake (Fig. 1).Maximal uptake of DABA occurs by 1 and 4 min,respectively, with the low and high substrate con-cen t ra t ions . A l though GABA t ranspor t can bedemonstrated with higher protein concentrations, itis not readily detected under these conditions w ith a1 O mM con cen tratio n beca use of the relatively lowspecific radioactivity. The time course of transportof GABA into synaptosomes differs from that intovesicles (Fig. 1). Uptake plateaus after 10 min insynaptosomes and after 2 min in the membranevesicles. The former, but not the latter, possessesthe metabolic machinery to maintain ion gradients.Dissipation of the artificially imposed ion gradientsoccurs rather rapidly.Experiments were next performed to determinethe ionic requirements for transport. Both DABA

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546 R . R O S K O S KI

8

150lime, mnTime, mn

FIG. 1. Time course of DABA and GABA uptake. Trans-port into membrane vesicles and synaptosomes was mea-sured by procedures given in Materials and Methods. (A)GABA (A-A) and DABA(0-0)ransport into membranevesicles using 0.14 ~ L Mabeled substrate concentrations.(B)DABA transport into membrane vesicles using a 1.0mM concentration. (C) GABA (0-0)nd DABA (0-0)transport into synaptosomes using 0.14 p~ radioactivesubstrate. The values given are the mean of duplicate de-terminations. Similar results were obtained with four dif-ferent preparations.

lime, min

an d GABA transport are N a+-depen dent (Table 1 ) .Substitution of Li+ or choline for Na+ decreasedapparent uptake by 98%. Since GABA transport isalso dependent on external C1- (K ann er, 1978), itsrole in DABA transport was examined. In contrastto GABA, DABA transport is not dependent on thepresence of external C1-. Using external sodiumphosphate under conditions where GABA transportis less than 1% of that in the presence of sodiumchloride, DABA transport is 76% of that obtainedwith NaCl. This suggests that the mechanism ofDABA transport differs from that of GABA, or thatit is transported in part by a different ca m er system .The presence of external S CN - increa ses the rateof DABA an d GABA transport by about 30%. Thisanion, which permeates membranes more readilythan C1-, is expected to produce a more negativeintravesicular electromotive force. T he thiocyanateenhancement o f t r anspor t of b o t h s u b s t a n c e ssuggests that their transport is electrogenic in na-ture. To substantiate this hypothesis, experimentswere also performed with valinomycin, a compoundthat specif ica l ly enhances the t ranspor t of K+across membranes. By transporting K+ down its

concentration gradient (from inside to outside)valinomycin also produces a more negative intra-vesicular electromotive force. Consonant with thepostulated electrogenic nature of transport, valino-mycin increases the rate ofDABA an d GABA trans-port by 90% (Table 2).TABLE 1. Role of th e ionic composit ion of t h eexternal so lut ion o n D A B A mid G A B A t ransport

Uptake. pmoVmg proteinExternal solution DABA GABANaCl 3.61 1.92LiCl 0.11 0.01ChCI 0.08 0.00Sodium phosphate 2.14 0.01Na S CN 4.14 2.54

The synaptosome membrane vesicles were reconstituted in100 ~ M - K C ~nd 100 mM-choline chloride as described in Mate-r ials and Methods. The external solution contained 0.14 p ~ -labeled DABA or G A B A , the specified salt (100 mM), 100 mM-choline chloride, and 2 . 5 pM-valinomycin. Incubations werecarr ied ou t for 30 s at 24". Similar results were obtained in fourother m embrane preparations.

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D A B A A N D G A B A T R A N S P O R T I N TO P L A S M A M E M B R A N E V E SI C LE S 547T A B L E 2. Ejfict of iotiophores on th erlite of DAB A t ransport

Uptake, pmoYmg proteinAddition DABA GABA

None 1.37 0.62Valinomycin (2.5 p m ) 2.60 1.14Monensin (5 pgirnl) 0.02 0.01Nigericin (2.5 p h i ) 0.01 0.01Gramicidin D (1.25 pgiml) 0.57 0.29The synaptosome m embrane fraction was reconstituted as de-scribed in Materials and Methods. The external solution con-tained 100 mM-NaCI. 100 mM-choline chloride and 0.14 p~labeled substrate. The ionoph ores were dissolved in 95% ethanolan d 2 - 4 portions were added to 180pl of the transport solutionprior to the addition of 20 pl of vesicles. Incubations were for

15 s at 24"; the means of duplicate determinations are given.Similar results were obtained with two other membrane prep-arations.

To further demonstrate the role of ion gradients insupplying the energy for transport, the role ofionophores on uptake was determined. Monensin,which abolishes the Na+ gradient (Harold et al.,1974), decreases the rate of DABA transport by99% (Table 2). Nigericin, which also abolishes theNa+ gradient (Pressman et al., 1967), similarly de-creases DABA uptake. In addition, gramicidin D ,an ionophore with broad specificity (Mueller andRudin, 1967), decreases the rate of transport by 53 %(Table 2). These studies support the notion that theion gradients provide the driving force for the up-take of DABA and GABA into the membrane vesi-cles.

Experiments were performed to determine thespecificity of the internal cation on DABA andGABA transport. Although internal K+ and Rb+ areoptimal for GABA, internal Tris or Li+ also sup-ports substantial uptake (Table 3) (Kanner, 1978).Internal Tris or Li+,on the other hand, fails to sup-port DABA uptake into the membrane vesicles.

TABLE 3. Internal cation requiremetilts forDABA an d G AB A t ran spor tUptake, pmoVmg protein

Cation DABA GABAK- 4 .31 1.92Rb ' 4.24 1.97Tris 0.00 0.94Li' 0.02 1.21

The membrane vesicles were reconstituted in 100mM concen-trations of the specified chloride salt, 100 mM-choline chloride ,an d 5 mM-Mes-Tris as described in Materials and Metho ds. Th eexternal solution contained 100mM-NaCI, 100 mM-choline chlo-ride, 5 mM-Mes-Tris, and 0.14 P M of the labeled amino acid (novalinomycin). Incubations were performed for 30 s (24"),and thereactions were terminated as described in Materials and M ethods.Similar results were obtained with four other membrane prepara-tions.

This finding, in conjunction with the different spe-cificities in the external anion, suggests that thereare substantive differences in the mechanism ofDABA and GABA transport.

Characteristics of Inhibition of DABA an dGABA TransportA Lineweaver-Burk kinetic analysis of the trans-

port of these two components was performed.DABA is a linearly competitive inhibitor of GABAtransport into membrane vesicles (Fig. 2). The K,for GABA ranges between 3 and 6 F M . The K i ofDABA for GABA transport into membrane vesiclesis 0.6 mM. Similar results were obtained when theKi of D A B A fo r GABA transport into synapto-somes was measured (Fig. 3). In both experiments,there was no preincubation of the fractions withDABA and the results are consistent with competi-tive inhibitory patterns. The greater V,, , for thesynaptosome preparation may reflect, in part, themore physiological ion gradients (150 mM-Na+ ver-sus 100 mM-Na+) used in the former case.

In performing similar experiments on the inhibi-tion of DABA uptake by GABA, different resultswere obtained. In the first place, the apparentDABA K , fo r transport into synaptosomes (Fig. 4)

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0. 5 1

FIG. 2. DABA inhibition of GABA transpor t in to membraneves ic les . After recons t i tu t ion of the ves ic les , th e ra te of GABAt r a n s p o rt w a s m e a s u r e d d u r i ng a 10-s incuba t ion (24") a sd e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . (0-O), o n t r o l ;(A-A), 0.8 mu-DABA; (0-O), 1.6 mM-DABA. The K , w a sd e te r min e d b y th e e q u a t io n : s lo p e = (1 + [IYK,) ( P lo wma n ,1972). Simila r r esu l ts wer e obta ined in three o ther me mb raneprepara t ions .

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548 R . R O S K O S Kl

FIG. 3. DABA inhibi t ion of GABA t ransport in rat cort icalsynaptosomes. Transpor t in to synaptosomes was per for medas described previously (Roskoski , 1978b), except that theconcent ra tion o f [3H]GABA was var ied f rom 1to 10 p ~ .ncu-bat ions were carried out for 10 s at 24". (O--O), C on t ro l ;(A-A), 0.6 mM-DABA;(0-0)..2 mM-DABA. The d ata repre-sent the mean o f d up l i ca te determinat ions . S imi lar resu l t swere obta ined w i th three d i f fe ren t synaptosome preparat ions.

and mem brane vesicles (not shown) is 2.5 mM. Thedata are not consistent with appreciable transportby a high-affinity transport system. When mi-cromolar concentrations of [3H]DABA were used,the extrapolated K , was greater than 1 mM. Using awide range of W B A concentrations, best fits areobtained with single K , of 2.5 mM. Tra nspo rt of 1.0~ M - [ ~ H ] D A B Anto membrane vesicles, moreover,

is Na +- and temperature-dependent (Table 4). An-other unsuspected finding was that GABA is a veryweak inhibitor of DABA transport (Table 5) . Veryhigh concentrations of GABA (40 m M ) are requiredto produce a 50% decrease in DABA transport,using concentration s ranging from to M .Similar results were also found with synaptosomes.Since DABA seems to be transported by a systemother than the GABA transport system , the effect ofother amino acids on its transport was examined. Ofthe various classes of amino acids tested, alanineinhibits DABA transport by 33% (Table 6 ) . Otherstructu ral classes of amino ac ids, including leucine,lysine, glutamic acid, phenylalanine, and glycineare without effect. DABA was the only amino acidthat substantially decreased [3H]GABA uptake.

N et Transport of G A B A in toMem brane Vesic lesIn addition to characterizing the requirements ofDABA and GABA transport into membrane vesi-cles, an experiment was performed to determinewhether the ion gradients would mediate net uptakeof GABA. In all studies previously performed onsubstrate transport into membrane vesicles, mea-surements of radioactive uptake, and not chemicaluptake, have been performed. As with synaptosometransport studies, i t is possible that exchange ofintravesicular and external substrate may occur(Levi and R aiteri, 1974). The uptake of GABA into

reconstituted membrane vesicles was measuredboth by chemical-enzymatic and radioactive deter-minations. The artificially imposed ion gradientsmediate net GABA uptake measured chemically; anequivalent amount of transport o ccurred, a s deter-mined by radioactive uptake (Table 7) . Monensin,

FIG. 4. D et e rmi na t i on of th e K , fo rD ABA t ranspo r t i n t o synap t osomes .Transport was measured as describedin Fig. 3, except that the conc ent ra t ionof [3H]DABA was var ied; incubat ionswere per form ed for 10 s at 24". Similarresu l t s were obta ined w i th three d i f -ferent synaptosome preparat ions.

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D A B A A N D G A B A T R A N S P O R T I N T O P L A S M A M E M B R A N E V E SI C LE S 549T A B L E 4. Sodiuni tiriti t i~tliperrirriredeperidetice

of D A B A ! r a t i s p o r tTemperature DABA uptakeMedium ("C) (nmoVmg protein)

NaCl 24NaCl 0ChCl 24LiCl 2448 .90 . 20. 30.1

Membrane vesicles were prepared and reconstituted, andtransport was measured as described in Materials and Methods.Incubations were performed for 30 s using 1.0mM labeled ["HI-DABA in medium containing 100 mhr-choline chloride plus 100mxr of the specified salt. Means of duplicate determinations aregiven. Similar results Were obtained in two other membranepreparations.

which dissipates the Na+ gradient, inhibits chemicaltransport and uptake of radioactive GABA. Trans-port is also abolished by omitting Na+ from the ex-ternal medium. Reconstitution in potassium phos-phate and 220 mM-NaCI were chosen to maximizethe extent of transport in this experiment.

DISCUSSIONThe use of membrane vesicles has allowed acomparison of the transport properties of GABAand DABA. There are several similarities in the

two systems. Transport of each is Na+- and tem-perature-dependent. Transport is abolished byionophores which dissipate the Na+ gradient. Up-take of each is also electrogenic, being enhanced bySCN- and valinomycin. On the other hand, thereare several differences in the properties of transportof these two substances. Internal K + (o r Rb+), forexample, is required for D A B A , but not GABA,transport. External CIF is required for G A B A , butnot D A B A , transport. These differences suggestthat DABA is transported by a carrier different fromthat of GABA. These characteristics can be ascer-tained by reconstitution of membrane vesicles with

TABLE 5 . C A B A itiliibiriotiof DABA trurisporf

[GABA] DABA uptake(pmoUmg protein)None20 pM0.67 m v2.0 m v20 mv40 m v

1.201.181.040 .940 .880 .63Membrane ves ic les were prepared and recons t i tu ted andtransport was measured as described in Materials and Methods.Portions of GABA were added to the external transport solutionto give the final specified concentration. Incubations were per-formed for 15 s a t 24" with ["IDABA conc entra tion of 0.14 p ~ .Similar results were obtained in three different membrane prep-arations.

T A B L E 6. Ejyecf of'utnirto ticid3 o t t D A B A frri t ispori[ HIDABA uptakeAddition ( 1 mM) (pmoVmg prote in)

NoneAlanineLeucineLysinePhen ylalanineGlycineDABAP-Alanine

3. 1 2 0.152 .0 2 0.12'13 .3 2 0 .143 .2 t 0.142 .9 5 0 .133. 2 -c 0 .161.0 * 0.11"3.1 t 0 .14

Membranes were prepared and reconstituted, and transportwas measured as described in Materials and Methods. Portions(10 pl) of unlabeled amino acid were added to the external trans-port solution to give the final specified concentration. T he meanst S . E . M . of quadruplicate determinations are given. Incubationswere performed for 30 s at 24" with a [ HIDABA concentrationof 0.14 p ~ .imilar results were obtained with two other mem-brane preparations."p


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550 R . ROSKOSKItransport by the membrane vesicle system (Table5) . This argues that DABA is transported by a non-GABA carrier. This occurs at both low and highconcentrations of [3H]DA BA , and further raises thepossibility, first hypothesized by Simon and Martin(1973), that DABA is taken up into elements thatfail to take up GABA.These results, obtained in experimen ts performedwith synaptosomes and membrane vesicles, differf rom exper imen ts pe r fo rmed wi th b ra in s l i ces(Weitsch-Dick et al ., 1978). These investigators re-ported that D ,L-DAB A has a K , of 20.7 p ~ ,hichoccurs in the h igh-aff in i ty range. They show,moreover, that a concentration of 17 p~ in GABAinhibits D,L-DA BA ransp ort ( lo-* M) by 50%.As inthe present studies, transport was Na+-dependent.With the more intact brain slices, DABA transportwas linear for 20 min. The differences in thosestudies and the present exp erim ents seem mostlikely to be due to the differences in transport intoslices and mem brane fractions. In the former case,diffusion barriers ex ist for the exoge neously appliedsubstance. Th e size of the slices, for exam ple, alsoaffects the kinetic parameters of uptake (Riddall eta l . , 1976). Levi and Rai ter i (1973) , moreover ,suggested that transport in large slices may be intononsynaptosomal elements, which may be selec-tively destroyed in the preparation of synapto-somes. In the present experime nts, the difference inchloride and potassium requirements provides thebes t ev idence fo r the t r anspor t o f DABA andGABA by different carriers. Differences in the ki-netic parameters between brain slices and synapto-some or mem brane preparations seem most likely tobe related to differences in substrate diffusion andmetabolic activity of the respective preparations.The present experiments, and those of Simon andMartin (1973), raise the possibility that DABA maybe taken up into cellular elements which lack thehigh-affinity GABA transpo rt system, and point tothe need for additional controls in localization ofDABA and GABA uptake by radioautography.

ACKNOWLEDGMENTThis work was supported by USPHS Grant NS15994.

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transmitters f rom cortical synaptosomes: Emux or secre-tion? J . Neurochem. 30, 1113 - 1125.

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