Comparison of DABA and GABA Transport Into Plasma Membrane Vesicles Derived from Synaptosomes

Home , Valinomycin

Jourtiol of Neurochemisiry 0022-3042/8 110201 -05441S02.0010 36(2):544-550. February. Raven Press, New York Q 1981 International Society for Neurochemistry

Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes

Robert Roskoski, Jr.

Depurrrnrnr of Biocliemistrj. Louisiana State University Medicrtl Center. Neil, Orleans, Louisiunu 701 12, 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 neuronal elements and inhibits GABA uptake. Localization of [:1H]DABA by auto- radiography has been used to identify neurons with the GABA high-affinity transport system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive ['HJGABA and ['HIDABA transport. For each. transport requires external sodium, is abolished by ionophores that dissipate the Na' gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor (K, = 0.6 mM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experiments, using enzymatic-fluorometric techniques, it was shown that the artificially imposed ion gradients drive net chemical transport of GABA into the vesicles. Key Words: GABA-DABA-Synaptosomes-Membrane vesicles- Transport. Roskoski R., Jr. Comparison of DABA and GABA transport into plasma membrane vesicles derived from synaptosomes. J. Neurochern. 36, 544-550 (1981).

GABA (y-aminobutyric acid) is a postulated neu- neurotransmitter action by uptake (Iversen, 1971). rotransmitter in the vertebrate CNS (Krnjevic, Levi and Raiteri (1974) and Simon et al. (1974) re- 1970). Nerve terminal preparations (synaptosomes) ported that the high-affinity synaptosomal transport accumulate GABA by high-affinity (low K,) and system mediates the exchange of endogenous and low-affinity (high K,) transport systems (Weinstein external GABA, but little net transport. We demon- et al., 1965; Martin, 1973; Levi and Raiteri, 1974). strated that synaptosomes take up net amounts of Two similar classes of transport systems occur for GABA, glutamate, and aspartate by the high- other neuroactive amino acids (Iversen and John- affinity system (Ryan and Roskoski, 1977; Ros- ston, 1971; Logan and Snyder, 1972). The low-af- koski, 197th; 1979); exchange of each amino acid finity transport systems are associated with both also occurs. We suggested that high-affinity trans- neuroactive and inactive amino acids (Logan and port is reversible and that the direction of movement Snyder, 1972). The high-affinity systems, which are may be related to the bioenergetics and mechanism Na+- and temperature-dependent are associated of uptake. with the neuroactive amino acids, including GABA. Iversen and Johnston (1971) demonstrated that It has been suggested that the high-affinity systems 0.1 mM-DABA (2,4-diaminobutyric acid) inhibits are specifically involved with the termination of [3H]GABA accumulation in brain slices. Simon and

Received January 30, 1980; accepted July 25. 1980. 2.4-Diaminobutyric acid: Mes. 2-(N-Morpholino) ethanesul- Abbreviations used: GABA. y-Arninobutyric acid; DABA, fonic acid.

544 DABA AND GABA TRANSPORT INTO PLASMA MEMBRANE VESICLES 545

Martin (1973) studied the transport of both GABA with 2 ml of 0.20 M-NaCI. After drying (5 min. IIO"), and DABA into rat brain synaptosomes. They radioactivity was measured by liquid scintillation spec- showed that DABA is a competitive inhibitor of trometry, using Budget-Solve (Research Products Inter- 13H]GABA uptake. Using low-specific-activity national Corp.) as scintillant with an efficiency of 35%. To measure background radioactivity. the ice-cold 0.2 hi- ["CIDABA, they showed that its uptake was par- NaCl was added before the membrane fraction; filtration tially inhibited by GABA. They pointed out that and washing were then performed immediately. as de- there may be a population of synaptosomes active scribed. in DABA, but not GABA, uptake. Kelly and Dick Each measurement was performed in duplicate: the (1975) reported that 13H]DABA is taken up by agreement was within 1057, or the measurement was re- neuronal elements, but not in glia in rat cerebellum. peated. There is a two- to threefold variation in rates and These investigators emphasized the similarity of extent from one membrane preparation to another based distribution of [3H]DABA and ["IGABA. on protein. Values from a given membrane preparation, Several investigators have prepared membrane however, varied only 10-15%. Each experiment was vesicles which take up substrates in response to ar- performed with at least three membrane preparations and tificially imposed ion gradients. Kanner (1978), for similar results were obtained. example, prepared fractions from synaptosomal membranes active in GABA transport. In the pres- Transport Measurement in Synaptosomes ent study the characteristics of [3H]DABA and [3H]GABA transport into these vesicles were com- These experiments were performed by the method prepared. Although there are many similarities, sub- viously outlined for choline uptake (Roskoski, 19786). stantive differences exist, which raise the possibil- Transport was terminated by addition of 4 ml of ice-cold 0.9% NaCl followed by filtration through GFiA filters; the ity that DABA is transported, at least in part, by a filters were washed twice with two additional 4-ml por- non-GABA transport system. tions.

MATERIALS AND METHODS Marerials Preparation Membrane Fractions of Labeled GABA and DABA were obtained from New Rat cortical synaptosomes were prepared from male England Nuclear Corp. and Amersham. respectively. Sprague-Dawley rats (125-200 g) by the method of Nigericin and monensin were gifts from Eli Lilly and Co. Haycock et al. (1978). The synaptosomes were lysed and Other drugs and compounds were purchased from Sigma the plasma membrane fraction was prepared by the pro- Chemical Co. cedure of Kanner (1978). The lysed membrane fraction was stored in 200-pl aliquots [about 5 mgiml protein determined by the procedure of Lowry et al. (1951)l in RESULTS liquid N,. General Characteristics of DABA and GABA Transport Transport Assays Using 0.14 FM labeled substrate, the rates of Transport measurements were carried out by the meth- DABA and GABA transport into reconstituted ods of Rudnick (1977) and Kanner (1978). with minor vesicles are linear for about 30 s (Fig. 1). GABA modifications. Unless otherwise noted, the thawed mem- uptake exhibits a characteristic overshoot. With 1.O brane fraction was reconstituted in 4 volumes of 100 mM substrate under the same experimental condi- mwKCI. 10 mwNaCI, and 90 mM-choline chloride tions, there is appreciable DABA uptake (Fig. 1). buffered with 5 mal-Mes-Tris (pH 7.0) at a concentration Maximal uptake of DABA occurs by 1 and 4 min, of about 1 mgiml protein for 10 min at 37". After centrifugation (27.000 x gfor 10 min). the membranes were resus- respectively, with the low and high substrate con- pended (about 1 mgiml protein) in fresh solution of the centrations. Although GABA transport can be same composition by vortex mixing. To measure trans- demonstrated with higher protein concentrations, it port, a 20-pl portion was transferred to 180 p1 of transport is not readily detected under these conditions with a solution consisting of 100 mwNaCI plus 100 mM-choline 1 .O mM concentration because of the relatively low chloride buffered with 5 mwMes-Tris (pH 7.0). This pro- specific radioactivity. The time course of transport cedure generates Na* (Naa,,u,c,r,,> Na+lnsldr)and K' of GABA into synaptosomes differs from that into (K- ,",,,,,, > K',,, ,,,, r,r) gradients. The external transport so- vesicles (Fig. 1). Uptake plateaus after 10 min in lution also contained ["HIGABA (26,600 c.p.m.ipmol) or synaptosomes and after 2 min in the membrane L-["H]DABA (6850 c.p.m.ipmol) to give a final concen- vesicles. The former, but not the latter, possesses tration of 0.14 pxi. After incubation for the specified time at ambient temperature (22-24"), transport was termi- the metabolic machinery to maintain ion gradients. nated by addition of 2 ml of ice-cold 0.20 M-NaCI in 5 Dissipation of the artificially imposed ion gradients mxi-Mes-Tris (pH 7.0) and filtration through 25-mm- occurs rather rapidly. diameter Whatman GFiA glass-fiber discs. The filtration Experiments were next performed to determine rate was 2 ml per 2-4 s. The filters were washed twice the ionic requirements for transport. Both DABA

J. Neiirochem.. Vd.36. No. 2, 1981 546 R. ROSKOSKI

5 10 15 lime, min Time, min

FIG. 1. Time course of DABA and GABA uptake. Trans- port into membrane vesicles and synaptosomes was measured by procedures given in Materials and Methods. (A) GABA (A-A) and DABA (0-0)transport into membrane vesicles using 0.14 ~LMlabeled substrate concentrations. (B) DABA transport into membrane vesicles using a 1.0 mM concentration. (C) GABA (0-0)and DABA (0-0) transport into synaptosomes using 0.1 4 p~ radioactive substrate. The values given are the mean of duplicate determinations. Similar results were obtained with four different preparations.

lime, min

and GABA transport are Na+-dependent (Table 1). concentration gradient (from inside to outside) Substitution of Li+ or choline for Na+ decreased valinomycin also produces a more negative intra- apparent uptake by 98%. Since GABA transport is vesicular electromotive force. Consonant with the also dependent on external C1- (Kanner, 1978), its postulated electrogenic nature of transport, valino- role in DABA transport was examined. In contrast mycin increases the rate of DABA and GABA trans- to GABA, DABA transport is not dependent on the port by 90% (Table 2). presence of external C1-. Using external sodium phosphate under conditions where GABA transport is less than 1% of that in the presence of sodium TABLE 1. Role of the ionic composition of the chloride, DABA transport is 76% of that obtained external solution on DABA mid GABA transport with NaCl. This suggests that the mechanism of Uptake. pmoVmg protein DABA transport differs from that of GABA, or that it is transported in part by a different camer system. External solution DABA GABA The presence of external SCN- increases the rate NaCl 3.61 1.92 of DABA and GABA transport by about 30%. This LiCl 0.11 0.01 anion, which permeates membranes more readily ChCI 0.08 0.00 than C1-, is expected to produce a more negative Sodium phosphate 2.14 0.01 intravesicular electromotive force. The thiocyanate NaSCN 4.14 2.54 enhancement of transport of both substances The synaptosome membrane vesicles were reconstituted in suggests that their transport is electrogenic in na- 100 ~M-KC~and 100 mM-choline chloride as described in Mate- ture. To substantiate this hypothesis, experiments rials and Methods. The external solution contained 0.14 p~- labeled DABA or GABA, the specified salt (100 mM), 100 mM- were also performed with valinomycin, a compound choline chloride, and 2.5 pM-valinomycin. Incubations were that specifically enhances the transport of K+ carried out for 30 s at 24". Similar results were obtained in four across membranes. By transporting K+ down its other membrane preparations.

J. Neurochem.. Vol. 36. No. 2, 1981 DABA AND GABA TRANSPORT INTO PLASMA MEMBRANE VESICLES 547

TABLE 2. Ejfict of iotiophores on the This finding, in conjunction with the different spe- rlite of DABA transport cificities in the external anion, suggests that there are substantive differences in the mechanism of Uptake, pmoYmg protein DABA and GABA transport. Addition DABA GABA

None 1.37 0.62 Characteristics of Inhibition of DABA and Valinomycin (2.5 pm) 2.60 1.14 GABA Transport Monensin (5 pgirnl) 0.02 0.01 Nigericin (2.5 phi) 0.01 0.01 A Lineweaver-Burk kinetic analysis of the trans- Gramicidin D (1.25 pgiml) 0.57 0.29 port of these two components was performed. The synaptosome membrane fraction was reconstituted as de- DABA is a linearly competitive inhibitor of GABA scribed in Materials and Methods. The external solution con- transport into membrane vesicles (Fig. 2). The K, tained 100 mM-NaCI. 100 mM-choline chloride and 0.14 p~ for GABA ranges between 3 and 6 FM. The Ki of labeled substrate. The ionophores were dissolved in 95% ethanol DABA for GABA transport into membrane vesicles and 2-4 portions were added to 180 pl of the transport solution prior to the addition of 20 pl of vesicles. Incubations were for is 0.6 mM. Similar results were obtained when the 15 s at 24"; the means of duplicate determinations are given. Ki of DABA for GABA transport into synapto- Similar results were obtained with two other membrane prep- somes was measured (Fig. 3). In both experiments, arations. there was no preincubation of the fractions with DABA and the results are consistent with competi- To further demonstrate the role of ion gradients in tive inhibitory patterns. The greater V,,, for the supplying the energy for transport, the role of synaptosome preparation may reflect, in part, the ionophores on uptake was determined. Monensin, more physiological ion gradients (150 mM-Na+ ver- which abolishes the Na+ gradient (Harold et al., sus 100 mM-Na+) used in the former case. 1974), decreases the rate of DABA transport by In performing similar experiments on the inhibi- 99% (Table 2). Nigericin, which also abolishes the tion of DABA uptake by GABA, different results Na+ gradient (Pressman et al., 1967), similarly de- were obtained. In the first place, the apparent creases DABA uptake. In addition, gramicidin D, DABA K, for transport into synaptosomes (Fig. 4) an ionophore with broad specificity (Mueller and Rudin, 1967), decreases the rate of transport by 53% (Table 2). These studies support the notion that the ion gradients provide the driving force for the up- / take of DABA and GABA into the membrane vesicles. Experiments were performed to determine the specificity of the internal cation on DABA and GABA transport. Although internal K+ and Rb+ are optimal 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 support DABA uptake into the membrane vesicles.

TABLE 3. Internal cation requiremetilts for DABA and GABA transport

Uptake, pmoVmg protein

Cation DABA GABA

K- 4.31 1.92 Rb' 4.24 1.97 0.5 1 Tris 0.00 0.94 Li' 0.02 1.21

The membrane vesicles were reconstituted in 100 mM concentrations of the specified chloride salt, 100 mM-choline chloride, FIG. 2. DABA inhibition of GABA transport into membrane and 5 mM-Mes-Tris as described in Materials and Methods. The vesicles. After reconstitution of the vesicles, the rate of GABA external solution contained 100 mM-NaCI, 100 mM-choline chlo- transport was measured during a 10-s incubation (24") as ride, 5 mM-Mes-Tris, and 0.14 PM of the labeled amino acid (no described in Materials and Methods. (0-O),Control; valinomycin). Incubations were performed for 30 s (24"), and the (A-A), 0.8 mu-DABA; (0-O), 1.6 mM-DABA. The K, was reactions were terminated as described in Materials and Methods. determined by the equation: slope = (1 + [IYK,) (Plowman, Similar results were obtained with four other membrane prepara- 1972). Similar results were obtained in three other membrane tions. preparations.

J. Neurochem.. Vol. 36. No. 2, 1981 548 R. ROSKOSKl

is Na+- and temperature-dependent (Table 4). An- other unsuspected finding was that GABA is a very weak inhibitor of DABA transport (Table 5). Very high concentrations of GABA (40 mM) are required to produce a 50% decrease in DABA transport, using concentrations ranging from to M. Similar results were also found with synaptosomes. Since DABA seems to be transported by a system other than the GABA transport system, the effect of other amino acids on its transport was examined. Of the various classes of amino acids tested, alanine inhibits DABA transport by 33% (Table 6). Other structural classes of amino acids, including leucine, lysine, glutamic acid, phenylalanine, and glycine are without effect. DABA was the only amino acid that substantially decreased [3H]GABA uptake.

Net Transport of GABA into Membrane Vesicles In addition to characterizing the requirements of FIG. 3. DABA inhibition of GABA transport in rat cortical synaptosomes. Transport into synaptosomes was performed DABA and GABA transport into membrane vesi- as described previously (Roskoski, 1978b), except that the cles, an experiment was performed to determine concentration of [3H]GABA was varied from 1 to 10 p~.Incu- whether the ion gradients would mediate net uptake bations were carried out for 10 s at 24". (O--O), Control; of GABA. In all studies previously performed on (A-A), 0.6 mM-DABA; (0-0).1.2 mM-DABA. The data represent the mean of duplicate determinations. Similar results substrate transport into membrane vesicles, mea- were obtained with three different synaptosome preparations. surements of radioactive uptake, and not chemical uptake, have been performed. As with synaptosome transport studies, it is possible that exchange of and membrane vesicles (not shown) is 2.5 mM. The intravesicular and external substrate may occur data are not consistent with appreciable transport (Levi and Raiteri, 1974). The uptake of GABA into by a high-affinity transport system. When mi- reconstituted membrane vesicles was measured cromolar concentrations of [3H]DABA were used, both by chemical-enzymatic and radioactive deter- the extrapolated K, was greater than 1 mM. Using a minations. The artificially imposed ion gradients wide range of WBA concentrations, best fits are mediate net GABA uptake measured chemically; an obtained with single K, of 2.5 mM. Transport of 1.0 equivalent amount of transport occurred, as deter- ~M-[~H]DABAinto membrane vesicles, moreover, mined by radioactive uptake (Table 7). Monensin,

FIG. 4. Determination of the K, for DABA transport into synaptosomes. Transport was measured as described in Fig. 3, except that the concentration of [3H]DABA was varied; incubations were performed for 10 s at 24". Similar results were obtained with three different synaptosome preparations.

J. Neurochem.. Yo/.36, No. 2, 1981 DABA AND GABA TRANSPORT INTO PLASMA MEMBRANE VESICLES 549

TABLE 4. Sodiuni tiriti ti~tliperrirriredeperidetice TABLE 6. Ejyecf of'utnirto ticid3 ott DABA frritispori of DABA !ratisport [ 'HIDABA uptake Temperature DABA uptake Addition (1 mM) (pmoVmg protein) Medium ("C) (nmoVmg protein) None 3.1 2 0.15 NaCl 24 48.9 Alanine 2.0 2 0.12'1 NaCl 0 0.2 Leucine 3.3 2 0.14 ChCl 24 0.3 Lysine 3.2 t 0.14 LiCl 24 0.1 Phen ylalanine 2.9 5 0.13 Glycine 3.2 -c 0.16 Membrane vesicles were prepared and reconstituted, and DABA 1.0 * 0.11" transport was measured as described in Materials and Methods. P-Alanine 3.1 t 0.14 Incubations were performed for 30 s using 1.0 mM labeled ["HI- DABA in medium containing 100 mhr-choline chloride plus 100 Membranes were prepared and reconstituted, and transport mxr of the specified salt. Means of duplicate determinations are was measured as described in Materials and Methods. Portions given. Similar results Were obtained in two other membrane (10 pl) of unlabeled amino acid were added to the external trans- preparations. port solution to give the final specified concentration. The means t S.E.M. of quadruplicate determinations are given. Incubations were performed for 30 s at 24" with a [ 'HIDABA concentration which dissipates the Na+ gradient, inhibits chemical of 0.14 p~.Similar results were obtained with two other membrane preparations. transport and uptake of radioactive GABA. Trans- " p < 0.01. port is also abolished by omitting Na+ from the external medium. Reconstitution in potassium phosphate and 220 mM-NaCI were chosen to maximize specified artificially imposed ion gradients, and not the extent of transport in this experiment. from experiments performed with synaptosomes. Although GABA transport exhibits an uptake system with high affinity ( M), DABA transport DISCUSSION exhibits a low-affinity transport system. DABA is a The use of membrane vesicles has allowed a linearly competitive inhibitor of GABA transport comparison of the transport properties of GABA into reconstituted membrane fractions and synapto- and DABA. There are several similarities in the somes. The latter finding is in agreement with pre- two systems. Transport of each is Na+- and tem- vious studies of Simon and Martin (1973). On the perature-dependent. Transport is abolished by other hand, GABA is a feeble inhibitor of DABA ionophores which dissipate the Na+ gradient. Up- take of each is also electrogenic, being enhanced by SCN- and valinomycin. On the other hand, there TABLE 7. Net trunsport o,f GABA irifo t?ienibrurie are several differences in the properties of transport vrsicles dri\.eti by irtti grridierits of these two substances. Internal K+ (or Rb+), for Chemical Radioactive example, is required for DABA, but not GABA, Transport transport transport transport. External CIF is required for GABA, but solution (nmoUmg) (nmoVmg) not DABA, transport. These differences suggest NaCl (220 mM) 602 669 that DABA is transported by a carrier different from NaCl (220 mhf) plus that of GABA. These characteristics can be ascer- monensin (5 pglml) I74 190 tained by reconstitution of membrane vesicles with KPi (150 mM) 167 184 Membrane vesicles were reconstituted in 0. 15 hi-potassium phosphate (pH 6.8) as described in Materials and Methods. After TABLE 5. CABA itiliibirioti of DABA trurisporf centrifugation (15,000 x g, I0 min). these were resuspended in the same solution with a protein concentration of I5 mglml. Por- DABA uptake tions (100 pl) were transferred to 1.4 ml of the specified solution [GABA] (pmoUmg protein) containing 10 ~M-[~H]GABA(10.4 c.p.m.!pmol). After 3 min at ambient temperature, the suspensions were centrifuged for 1 min None 1.20 at 13,000 x g in an Eppendorf Microfuge. After aspiration of the 20 pM 1.18 supernatant. ice-cold 220 mht-NaC1 (I ml) was gently added to 0.67 mv 1.04 the tube and aspirated to decrease the extravesicular [,"H]GABA. 2.0 mv 0.94 After adding 250 pl of distilled water. the solution was heated to 20 mv 0.88 90°C to denature any metabolizing enzymes, and the pellet was 40 mv 0.63 dispersed by brief sonication with a Kontes micro-ultrasonic cell disrupter. After centrifugation for 15 min at 13,000 x g. duplicate Membrane vesicles were prepared and reconstituted and 100-pl portions were taken for the fluorometric determination of transport was measured as described in Materials and Methods. GABA as previously described (Roskoski. 1978u), using a modi- Portions of GABA were added to the external transport solution fication of the procedure of Graham and Aprison (1966). Dupli- to give the final specified concentration. Incubations were per- cate 15-pI portions were taken for radioactive determination by formed for 15 s at 24" with ["IDABA concentration of 0.14 p~. liquid scintillation spectrometry. The values represent the means Similar results were obtained in three different membrane prep- of duplicate samples. Similar results were obtained in two other arations. membrane preparations.

.I.Neurochem.. Vol. 36. No. 2, 1981 550 R. ROSKOSKI transport by the membrane vesicle system (Table Iversen L. L. (1971) Role of transmitter uptake mechanisms in 5). This argues that DABA is transported by a non- synaptic transmission. Br. J. Pharmucol. 41, 571 -591. lversen L. L. and Johnston G. A. R. (1971) GABA uptake in rat GABA carrier. This occurs at both low and high central nervous system: Comparison of uptake in slices and concentrations of [3H]DABA, and further raises the homogenates and the effects of some inhibitors. J. possibility, first hypothesized by Simon and Martin Neurochem. 18, 1939-1950. (1973), that DABA is taken up into elements that Kanner B. I. (1978) Active transport of y-aminobutyric acid by membrane vesicles isolated from rat brain. Biochemisrry 17, fail to take up GABA. 1207-1211. These results, obtained in experiments performed Kelly J. S. and Dick F. (1975) Differential labeling of glial cells with synaptosomes and membrane vesicles, differ and GABA-inhibitory interneurons and nerve terminals fol- from experiments performed with brain slices lowing the microinjection of Lp-3H]Alanine, ["IDABA and (Weitsch-Dick et al., 1978). These investigators re- ['HIGABA into single folia of the cerebellum. Cold Spring Harb. Sym. Qutirt. Biol. 40, 93-106. ported that D,L-DABA has a K, of 20.7 p~,which Krnjevic K. (1970) Glutamate and y-aminobutyrate in brain. occurs in the high-affinity range. They show, Nature 250, 735-737. moreover, that a concentration of 17 p~ in GABA Levi G. and Raiteri M. (1973) Detectability of low and high affin- inhibits D,L-DABAtransport (lo-* M) by 50%. As in ity uptake systems for GABA and glutamate in rat brain slices and synaptosomes. Life Sci. 12, 81 -88. the present studies, transport was Na+-dependent. Levi G. and Raiteri M. (1974) Exchange of neurotransmitter With the more intact brain slices, DABA transport amino acid at nerve endings can simulate high affinity up- was linear for 20 min. The differences in those take. Nature 250, 735 -737. studies and the present experiments seem most Logan W. J. and Snyder S. H. (1972) High affinity uptake sys- likely to be due to the differences in transport into tems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissues. Brain Kes. 42, slices and membrane fractions. In the former case, 413-431. diffusion barriers exist for the exogeneously applied Lowry 0. H., Rosebrough N. J., Farr A. L., and Randall R. J. substance. The size of the slices, for example, also (1951) Protein measurement with the Folin phenol reagent. affects the kinetic parameters of uptake (Riddall et J. Biol. Chem. 193, 265-275. al., 1976). Levi and Raiteri (1973), moreover, Martin D. L. (1973) Kinetics of the sodium-dependent transport of gamma-aminobutyric acid by synaptosomes. J. Neuro- suggested that transport in large slices may be into chem. 21, 345-356. nonsynaptosomal elements, which may be selec- Mueller P. and Rudin D. 0. (1967) Development of K+-Na' dis- tively destroyed in the preparation of synapto- crimination in experimental bimolecular lipid membranes by somes. In the present experiments, the difference in macrocyclic antibiotics. Biochem. Biophys. Res. Commun. 26, 398-404. chloride and potassium requirements provides the Plowman K. (1972) Enzyme Kinetics. p. 58, McGraw-Hill Book best evidence for the transport of DABA and Company, New York. GABA by different carriers. Differences in the ki- Pressman B. C., Hams E. J., Jagger W. S., and Johnston J. H. netic parameters between brain slices and synapto- (1967) Antibiotic-mediated transport of alkali ions across some or membrane preparations seem most likely to lipid barriers. Proc. Narl. Acad. Sci. USA 58, 1949-1976. Riddall D. R., Leach M. J., and Davison A. N. (1976) Neuro- be related to differences in substrate diffusion and transmitter uptake into slices of rat cerebral cortex in vitro: metabolic activity of the respective preparations. Effect of slice size. J. Neurochem. 27, 835-839. The present experiments, and those of Simon and Roskoski R., Jr. (1978~)Net uptake of L-glutamate and GABA Martin (1973), raise the possibility that DABA may by high affinity synaptosomal transport systems. J. Neurorhem. 31, 493-498. be taken up into cellular elements which lack the Roskoski R., Jr. (197%) Acceleration of choline uptake after high-affinity GABA transport system, and point to depolarization-induced acetylcholine release in rat cortical the need for additional controls in localization of synaptosomes. J. Neurochem. 30, 1357- 1361. DABA and GABA uptake by radioautography. Roskoski R., Jr. (1979) Net uptake of aspartate by a high-affinity rat cortical synaptosomal transport system. Brain Res. 160, 85-93. Rudnick G. (1977) Active transport of 5-hydroxytryptamine by ACKNOWLEDGMENT plasma membrane vesicles isolated from human platelets. J. Biol. Chem. 252, 2170-2174. This work was supported by USPHS Grant NS Ryan L. D. and Roskoski R., Jr. (1977) Net uptake of y-amino- 15994. butyric acid by a high affinity synaptosomal transport system. J. Pharmacol. Exp. Ther. 200, 285-291. Simon J. R. and Martin D. L. (1973) The effects of L-2.4 diaminobutyric acid on the uptake of gamma amino butyric REFERENCES acid by a synaptosomal fraction from rat brain. Arch. Biochem. Biophys. 157, 348-355. Graham L. T., Jr. and Aprison M. H. (1966) Flurometric deter- Simon J. R., Martin D. L., and Kroll M. (1974) Sodium- mination of aspartate, glutamate & y-aminobutyrate in nerve dependent efflux and exchange of GABA in synaptosomes. tissue using enzymic methods. Anal. Biochem. 15,487-497. J. Neurochem. 23, 981 -991. Harold F. M., Altendorf K. H., and Hirata H. 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J. Neurochem.. Vul. 36%No. 2, 1981