Glutamate Uptake Into Synaptic Vesicles: Competitive Inhibition by Bromocriptine

Jvurnul of Neurochrmistry Raven Press, Ltd., New York 0 1989 International Society for Neurochemistry

Glutamate Uptake into Synaptic Vesicles: Competitive Inhibition by Bromocriptine

*Martha D. Carlson, $Phillip E. Kish, and *"fTetsufumi Ueda

The Departmenls of *Pharmacology and ?Psychiatry and the $Menla( Health Research Institute, The University of Michigan, Ann Arbor, Michigan, U.S.A.

Abstract: The ATP-dependent uptake of L-glutamate into vesicular glutamate uptake was not mimicked by agents synaptic vesicles has been well characterized, implicating a known to interact with dopamine and serotonin receptors. key role for synaptic vesicles in glutarnatergic neurotrans- Kinetic data suggest that bromocriptine competes with glu- mission. In the present study, we provide evidence that ve- tarnate for the glutamate binding site on the glutamate trans- sicular glutamate uptake is selectively inhibited by the pep- locator. It is proposed that this inhibitor could be useful as tide-containing halogenated ergot bromocriptine. It is the a prototype probe in identifying and characterizing the ve- most potent inhibitor of the agents tested; the ICso was de- sicular glutamate translocator, as well as in developing a more termined to be 22 pM. The uptake was also inhibited by specific inhibitor of the transport system. Key Words: Glu- other ergopeptines such as ergotamine and ergocristine, but tamate-Synaptic vesicle-ATP-dependent uptake-Er- with less potency. Ergots devoid of the peptide moiety, how- gots-Competitive inhibition-Glutamate translocator. ever, such as ergonovine, Iergotrile, and methysergide, had Carlson M. D. et al. Glutamate uptake into synaptic vesicles: little or no effect. Although bromocriptine is known to elicit Competitive inhibition by bromocriptine. J. Neirrochem. 53, doparninergic and serotonergic effects, its inhibitory effect on 1889-1894 (1989).

It is now widely accepted that glutamate functions (Storm-Mathisen et al., 1983). The vesicular glutamate as a major excitatory neurotransmitter in the vertebrate uptake is driven by an electrochemical proton gradient, CNS (for reviews see Watkins and Evans, 1981; Cot- which is generated by a proton pump Mg-ATPase, and man et al., 198 1 ; Fonnum, 1984; Ueda, 1986; Cotman stimulated by physiologically relevant concentrations et al., 1987; Nicholls, 1989) and at the neuromuscular ofchloride (Naito and Ueda, 1985; Maycox et al., 1988; junction of arthropods (Usherwood, 198 1). The neu- Shioi et al., 1989). The high degree of specificity and rotransmitter glutamate is released from nerve termi- the unique properties of the vesicular glutamate uptake nals on their depolarization in a calcium-dependent system have led to the proposal that whether or not manner (Bradford et al., 1973; Reubi and Cuknod, glutamate is utilized as a neurotransmitter is deter- 1979; Cotman et al., 198 1 ; Levi et al., 1982). Nicholls mined by the presence or absence of the glutamate- and Sihra (1986) have provided evidence that a non- specific vesicular translocator (Ueda, 1986). Recent cytosolic pool of glutamate is rapidly released in a Ca2+- studies by Fischer-Bovenkerk et al. (1988) suggest that dependent manner, suggesting a role for synaptic ves- the vesicular glutamate system is present in cerebellar icles in glutamate synaptic transmission. Glutamate granule cells, which are considered to be glutamatergic, has been shown to be specifically taken up into highly but not in GABAergic Purkinje cells. Moreover, such purified, isolated synaptic vesicles in an ATP-depen- a vesicular uptake system has been shown to develop dent manner (Naito and Ueda, 1983, 1985; Maycox in close parallel with the time course of synaptogenesis et al., 1988), in accord with the immunocytochemical (ash et al., 1989~).These lines of evidence all point evidence that glutamate is concentrated in synaptic to the importance of the vesicular glutamate uptake vesicles in certain nerve endings that are distinct from system in glutamate synaptic transmission. In this y-aminobutyric acid (GABA)-rich nerve terminals study, we have begun to investigate pharmacological

Received January 30, 1989; revised manuscript received May 16, Abbreviations used: GABA, y-aminobutyric acid; RCSV. rat crude 1989; accepted May 17, 1989. synaptic vesicles. Address correspondence and reprint requests to Dr. T. Ueda at Mental Health Research Institute, The University of Michigan, 205 Washtenaw Place, Ann Arbor, MI 48109, U.S.A.

1889 1890 M. D. CARLSON ET AL. means by which the vesicular glutamate transport sys- lipore HAWfilters (25 mm, 0.45 pm). The incubation tubes tem can be modulated. We provide evidence suggesting were washed with the KCl solution three times, and the filters that the peptide-containing halogenated ergot bro- were then washed four more times. Radioactivity retained mocriptine may serve as a prototype agent for devel- on the filters (dpm) was determined in ACS (Amersham) by oping a more specific, potent regulator of the vesicular using a liquid scintillation spectrophotometer (Beckman LS 9000). The amounts of radioactive glutamate nonspecifically glutamate transport system. bound to the filters were determined as follows: the prewarmed [3H]glutamate/ATP mixture was mixed with 2.0 ml MATERIALS AND METHODS of KCI solution and filtered; the filters were then washed with Materials the KCI solution five more times. The radioactivity retained on the filters (400-900 dpm) was subtracted from all of the L-Glutamic acid (potassium salt), ATP, bromocriptine, a- ergocriptine, ergonovine, and ergotamine tartrate were pur- values presented in the uptake experiments described here. chased from Sigma. Ergocristine methanesulfonate and di- The means 2 SEM of triplicate determinations were pre- hydroergocriptine (Sandoz Pharmaceuticals) were generously sented. All glutamate uptake values presented represent ATP- provided by Dr. Edward Domino, Department of Pharma- dependent uptake activity, which was defined as the activity cology, the University of Michigan. Huphenazine was a gift observed in the presence of ATP minus the activity observed from E. R. Squibb and Sons, Princeton. NJ, U.S.A.; chlor- in its absence, unless otherwise indicated. Synaptic vesicle promazine was from Smith, Kline, and French, Philadelphia. protein was determined according to Lowry et al. (195 1) with PA, U.S.A.; and lergotrile was from Eli Lilly, Indianapolis, bovine serum albumin used as standard. IN, U.S.A. Methysergide, trifluoperazine. sulpiride. lysergic acid diethylamide, and bromolysergc acid diethylamide were RESULTS provided by Dr. James Woods, Department of Pharmacology. the University of Michigan. ~-[2,3-~H]Glutamicacid (36 Ci/ Effects of ergots on vesicular glutamate uptake mmol), 4-arnin0-n-[2,3-~H]butyric acid (50 Ci/mmol), and Figure 1 A and B shows the results of an experiment [2-3H]glycine(I9 Ci/mmol) were obtained from Amersham. in which a number of derivatives of ergot alkaloids were tested for their effects on the ATP-dependent up- Preparation of synaptic vesicles take of glutamate into RCSV. Bromocriptine, an ergot Rat crude synaptic vesicles (RCSV) were prepared as de- with dopamimetic properties (Fuxe et al., 1978; Ke- scribed by Kish and Ueda (1989). In brief, rat cerebrum was babian and Calne, 1979), in micromolar concentra- homogenized in solution A (0.32 M sucrose, 0.5 mM calcium acetate, 1 mM magnesium acetate, and 1 mM NaHC03) and tions, caused a marked inhibition of ATP-dependent centrifuged at 12,100 g,,, for 20 min (10,000 rpm, Sorvall glutamate uptake into RCSV. Bromocriptine was the SS-34 rotor). The pellet was lysed for 45 rnin in 6 mM Tris- most potent inhibitor of glutamate uptake of the var- maleate (pH 8.1) and centrifuged at 43,500 g,,, for 15 rnin ious ergot derivatives tested, with an ICS0of 22 + 6 (1 9,000 rpm, Sorvall SS-34 rotor). The supernatant was cen- pM. ICS0values (the concentration of inhibitor that trifuged at 200,000 g,,, for 50 rnin (47,000 rpm, Beckman inhibited 50% of the ATP-dependent glutamate uptake TI 50 rotor) to pellet the synaptic vesicles. This crude vesicle activity) were obtained by log-logit transformation of preparation, which was resuspended in solution €3 (0.32 M the data in Fig. 1A and B. In other experiments not sucrose, 1 mM dithiothreitol, 1 mM NaHC03), was used in shown here, however, the potent dopamine agonist most of the experiments to be described here, except where apomorphine (100 pM) did not inhibit glutamate up- otherwise indicated. Although there was some ATP-independent glutamate uptake activity in the RCSV preparation, it take. Moreover, inhibition of glutamate uptake was represented only about 10% of the ATP-dependent uptake not observed with any of the dopamine antagonists activity. tested (at concentrations up to 500 pUn4): trifluoperazine, fluphenazine, chlorpromazine, and sulpiride (data Assay for vesicular glutamate uptake not shown). In addition, this inhibitory effect on glu- Vesicular glutamate uptake was assayed essentially as de- tamate uptake by bromocriptine was not mimicked by scribed previously by Naito and Ueda (1983, 1985). The any of the serotonergic agonists or antagonists tested standard incubation mixture for the glutamate uptake con- (at pM): lysergic acid diethylamide, bromolysergic tained, in a final volume of 100 pl, 50 fig of RCSV, 5 mM 500 Tris-maleate (pH 7.4), 4 mM MgS04,0.25 M sucrose, 2 mM acid diethylamide, and methysergide (not shown). L-aspartate, 0 or 2 mM ATP neutralized with Tris-base, 4 These observations suggest that the inhibitory effect of mM KCI, and 50 pM potassium ~-['H]glutamate (0.4 Ci/ bromocriptine is not mediated through dopamine or mmol). Aspartate was included to block any glutamate uptake serotonin receptor mechanisms. into resealed plasma membrane vesicles, and to block any Bromocriptine is structurally classified as a peptide binding to glutamate receptors occurring from any plasma alkaloid or ergopeptine, containing a peptide moiety membrane contamination in the crude synaptic vesicle prep- attached to the ergoline ring structure (Rall and Schlei- aration used in these experiments. Synaptic vesicles (10 pl fer, 1980). To determine whether the inhibitory effect containing approximately 50 pug of protein) and the Tris/ is attributable ergoline ring sucrose medium (70 pl) were preincubated at 30°C for 5 rnin of bromocriptine to the and then mixed with 20 pl of a prewarmed glutamate/ATP structure, the peptide moiety, or to both, the various mixture including the KCI and [3H]glutamate. The mixture ergot compounds were tested for their ability to inhibit was incubated for an additional 1.5 min. The glutamate up- the ATP-dependent glutamate uptake. As shown in take was terminated by the addition of 2.0 ml of ice-cold Fig. I, those ergots that possess peptide moieties, e.g., 0.15 M KCI, followed by immediate filtration through Mil- ergotamine (ICS0 = 30 * 2 pM), a-ergocriptine

J Neurorhem., Vol. 53,No. 6, 1989 GLUT,4MA TE UPTAKE INHIBITION BY BROMOCRIPTINE 1891

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04/'. '.....' ".,...I ' ...,..I ' "..J 10-6 10-5 10-4 10-3 ERGOT (M) ERGOT (M) FIG. 1. Effects of various concentrations of ergots on ATP-dependent glutamate uptake into xsynaptic vesicles. A: Glutamate uptake was determined in the presence of various concentrations of bromocriptine (O),a-ergocriptine (A),ergotamine (m), and dihydroergocriptine (A). B: Glutarnate uptake was determined in the presence of various concentrations of bromocriptine (@), ergocristine (U), ergonovine (0),and lergotrile (+), as described in Materials and Methods. [3H]Glutamate uptake activity is expressed as percent of the activity in the absence of test agent (control). The glutamate uptake in the control was 11.5 +- 0.24 pmol/l.5 min. All of the ergots except ergonovine and lergotrile were dissolved in 50% or 75% (for a-ergocriptine only) ethanol to make a stock solution of 10 mM. Ethanol alone at a concentration of 0.5% (the concentration used for M ergot), or less, had no effect on glutamate uptake. Ethanol at 2.5%, the concentration used for 500 pM ergot, lowered the glutamate uptake 42%. This control value was used to determine the effect of this concentration of water- insoluble ergot. The data on bromocriptine and the other ergots are representative of five and three individual experiments, respectively. The various ergots inhibited the ATP-independent uptake 5-1 0% only at the two highest concentrations of ergot tested (100 and 500 pM).

= 57 k 10 pM), dihydroergocriptine (IC50= 73 & 3 absence of ATP might represent glutamate binding to pM), and ergocristine (IC5,, = 83 * 9 pM), were all nonvesicular membrane proteins and/or to enzymes effective but less potent than bromocriptine in inhib- involved in the metabolism of glutamate. In an effort iting glutamate uptake. In contrast, those ergots that to determine whether bromocriptine interacts with the lack peptide moieties, such as lergotrile (ICsO> 500 glutamate translocator component, kinetic experiments pM) and ergonovine (I& > 1 mM), had little or no were carried out in the absence and presence of 10 and effect. The presence of the bromine moiety also appears 20 uM bromocriptine. As shown in Fig. 3, the V,,, of to enhance- the potency of the inhibitory kffect on glutamate uptake, because a-ergocriptine, which is iden- 40 tical to bromocriptine except that it lacks the bromine moiety, has a reduced potency. Therefore, it appears that both the peptide and bromine moieties attached w 30 to the ergoline ring structure are important in causing Y- a .E a potent and selective inhibition of ATP-dependent +2 glutamate uptake. 5:

Nature of the bromocriptine inhibition ak? 20 Figure 2 shows a time course of glutamate uptake IS in the absence and presence of bromocriptine, following 5:a$ the standard 5-min preincubation time with the ergot. 0- Bromocriptine showed its inhibitory effect throughout 10 the incubation times tested; however, the effect de- creased in magnitude with increasing incubation time. A preincubation time of 30 s is sufficient to achieve a maximal inhibitory effect by a given concentration of bromocriptine (data not shown), suggesting that full TIME (min) interaction between bromocriptine and a component FIG. 2. Time course of vesicular glutamate uptake in the presence of the glutamate uptake system occurs within 30 s. or absence of bromocriptine. Synaptic vesicles were preincubated Figure 2 also shows that bromocriptine inhibited at 30°C for 5 rnin in the absence (0,0) or presence (m, 0)of 100 glutamate uptake but had no signifi- pM bromocriptine. A mixture of [3H]glutamate with ATP (0,m) or without ATP (0,0) was then added, and glutamate uptake was cant effect On ATP-independent uptake. The allowed to occur for the various times indicated. The data are (approximately 2 pmo1/50 pg protein) obtained in the representative of two individual experiments.

J. Neurochem.. Vol. 53, No. 6, 1989 1892 M. D. CARLSON ET AL. glutamate uptake was determined to be 0.26 k 0.02 glycine in comparison with the effect on vesicular glu- nmol/min in both the absence and presence of 10 or tamate uptake. As shown in Table 1, the inhibitory 20 pM bromocriptine. Corresponding K, values were effect of bromocriptine is greater on the glutamate up- 2.05 k 0.04, 4.41 * 0.06, and 6.10 f 0.09 mM, re- take system than on the GABA and glycine uptake spectively. Thus, the inhibitory action of bromocriptine systems. There was some inhibition by bromocriptine is reflected in a change (increase) in the K, for gluta- of the ATP-independent uptake activity; the inhibition mate, and not in changes in the V,,, of the uptake of glutamate uptake in the cerebral synaptic vesicles system. These results suggest that the nature of the in- and spinal cord vesicles was 2 and 8%, respectively, hibitory effect on glutamate uptake by bromocriptine and the inhibition for both GABA and glycine uptake is competitive with respect to glutamate. was 8%. These results suggest that the inhibitory action Other experiments not presented in this study failed of bromocriptine is selective for the ATP-dependent to support the possibility that bromocriptine inhibits glutamate uptake system. glutamate uptake by inhibiting the Mg-ATPase hydrolysis activity in the synaptic vesicle. Bromocriptine ( 1- DISCUSSION 100 pM)exhibited no effect on the Mg-ATPase activity. Moreover, the degree of the bromocriptine inhibition In this study, we have demonstrated that bromo- was not affected by varying the concentration of ATP criptine selectively inhibits the ATP-dependent gluta- from 0.05 to 2 mM. These studies lend further support mate uptake into synaptic vesicles. The kinetic data to the notion that the glutamate translocator is the site shown in Fig. 3 indicate that the nature of bromocrip- of action of bromocriptine. tine’s inhibitory action on uptake is competitive, with Effect of bromocriptine on ATP-dependent vesicular respect to glutamate, in that the V,,, of glutamate up- uptake of GABA and glycine take is unchanged, whereas the K, for glutamate is The amino acids GABA and glycine are also taken increased. These results suggest that bromocriptine is up into synaptic vesicles in an ATP-dependent manner acting at the glutamate translocator site of the synaptic vesicle, thereby preventing the uptake glutamate into (Kish et al., 1987, 1988, 19896; Fykse and Fonnum, of 1988; Hell et a]., 1988). These uptake systems are dis- synaptic vesicles. Bromocriptine’s competitive interaction at the glu- tinct from each other and from the glutamate uptake system (ash et al., 19896). In an effort to substantiate tamate translocator site is substantiated further by the selective action of bromocriptine on the glutamate studies that demonstrated that increasing the concen- translocator, we have examined the effect of the ergot tration of glutamate attenuated bromocriptine’s ability to inhibit the vesicular glutamate uptake (data not pre- on the ATP-dependent vesicular uptake of GABA and sented). The competitive inhibition of glutamate uptake by bromocriptine also appears to be reversible, because removal of bromocriptine by centrifugation 6o 1 followed by incubation and subsequent centrifugation 50 - of the synaptic vesicles in the presence of high concentrations of glutamate (e.g., 50 mM) reversed the inhibitory effect of bromocriptine (not shown). This sup- ports the view that the bromocriptine interaction with .-C . E the glutamate translocator is via noncovalent bonding. Other experiments not presented in this study sug- gested that bromocriptine does not inhibit the Mg- ATPase hydrolysis activity in the synaptic vesicle. In addition, using the fluorescent probes Oxonol VI and Acridine Orange, which are indicators of membrane potential (Russell, 1984) and pH gradients (Xie et al., 19831, respectively, we observed that bromocriptine had no detectable effect on the membrane potential or 0 0.5 10 15 2.0 pH gradient across the synaptic vesicle membrane (re- l/(L-GLU) (mM” ) sults not shown). Although bromocnptine is known to mimic some FIG. 3. Effect of bromocriptine on the kinetic parameters of the of the dopaminergic and serotonergic effects, this study ATP-dependent vesicular uptake of glutamate. The vesicular glutamate uptake was determined at various concentrations of glu- indicates that the inhibitory effect on vesicular gluta- tamate (0.5-5 rnM) in the absence (0)or presence of 10 pM bro- mate uptake is not attributable to its dopaminergic and mocriptine (m)or 20 pM bromocriptine (A),as described in Materials serotonergic properties. The data in Fig. 1 demonstrate and Methods. Specific activities of [3H]glutamate were 0.04 Ci/ that the tripeptide moiety of bromocriptine is crucial mmol for 0.5 mM; 0.027 Cipnmol for 0.75 mM; and 0.05,0.025, 0.013, and 0.01 Ci/rnmol for 1, 2, 4, and 5 rnM glutamate, re- in achieving potent inhibition of glutamate uptake. spectively. The data are representative of three individual experi- Thus, ergonovine, which is devoid of the peptide ments. Intercepts were determined by linear regression analysis. moiety, exhibited no effect even at 1 mM, the highest

J. Neurochem , Vol. 53. No. 6, 1989 GLUTAMA TE UPTAKE INHIBITION BY BROMOCRIPTINE 1893

TABLE 1. Efect of bromocriptine on ATP-dependent amino acid neurotransmitter uptake into synaptic vesicles

ATP-dependent uptake (dpm/5 min) Source of Amino acid Inhibition synaptic vesicles neurotransmitters - Bromocriptine + Bromocriptine (%)

Cerebrum Glutamate 82,825 i 1,542 21,565 f 373 74 GABA 3,145 k 305 2,284 _i 307 27 Spinal cord Glutamate 79,216 f 1,294 28,947 +- 860 63 Glycine 13,032 f 932 9,322 k 601 28

In the experiments in the upper half, crude synaptic vesicles were prepared from rat cerebrum as described by ffish and Ueda (1 989) and purified further by discontinuous sucrose-densitygradient (0.4,0.6, and 0.8 Msucrose) centrifugation at 141,400 g,, for 120 min (28,000 rpm, SW 28 rotor), and 100 pg of vesicle protein were used per assay. In those in the lower half, synaptic vesicles were purified from rat spinal cord by Percoll-density gradient centrifugation as described by Kish et al., (19896), and 150 pg of vesicle protein were used per assay. Vesicular amino acid uptake was assayed as described previously by Kish et al. (19896), using 150 FM ~-[~H]glutamate,[3H]GABA, or ['Hlglycine (each 0.13 Ci/ mmol), using 10 mM ATP in the absence or presence of 100 pM bromocriptine (in 0.5% ethanol) in the standard incubation medium described for glutamate uptake in Materials and Methods, except that 0.5% ethanol was included in the control and the assay volume was 200 pl. Uptake was allowed to occur for 5 min at 30°C. The data are representative of two separate experiments; the variation in the percent inhibition from this experiment was 5- 14%.

concentration tested, whereas all the ergots containing tine is a selective transport inhibitor with reasonable the peptide moiety tested are capable of inhibiting the potency, it is also possible that it could serve as a pro- glutamate uptake at much lower concentrations. The totype probe in identifying and characterizing the glu- bromine moiety appears to enhance the inhibitory po- tamate translocator in a purified synaptic vesicle prep- tency. aration. Consistent with the notion that bromocriptine interacts with the vesicular glutamate translocator is ev- Acknowledgment: This work was supported by grants from idence that the halogenated ergot selectively inhibited the National Science Foundation (BNS 8509679) and the the glutamate uptake. Thus, GABA and glycine uptakes National Institutes of Health (NS 26884). were less affected (Table 1). In addition to its interaction with the vesicular glutamate uptake system, bromocriptine has been reported to inhibit the high-affinity REFERENCES glutamate reuptake system in striatal membranes (Nieoullon et al., 1982). However, in contrast to its Bradford H. F., Bennett G. W., and Thomas A. J. (1973) &polarizing stimuli and the release of physiologically active amino acids from effect on the vesicular uptake system, the inhibition suspensions of mammalian synaptosomes. J. Neurochem. 21, appeared to be achieved through activation of D2-like 495-505. dopamine receptors located on the presynaptic corti- Cotman C. W., Foster A. C., and Lanthorn T. H. (198 I) An overview costriatal glutamatergic nerve endings. The authors of glutamate as a neurotransmitter, in Glutamate as u Neziro- transmitter (Di Chiara G. and Gessa G. L., eds), pp. 1-27. Raven proposed that nigrostriatal dopaminergic input may Press, New York. regulate corticostriatal glutamatergic activity. We have Cotman C. W., Monaghan D. T., Ottersen 0. P., and Storm-Mathisen also observed the inhibitory effect of bromocriptine on J. (1987) Anatomical organization of excitatory amino acid re- the sodium-dependent glutamate uptake into synap- ceptors and their pathways. Trends Neurosci. 10, 273-279. Fischer-Bovenkerk C., Kish P. E., and Ueda T. (1988) ATP-dependent tosomes; in our experiments, 20 pLM bromocriptine in- glutamate uptake into synaptic vesicles from cerebellar mutant hibited about 30% of the glutamate uptake into syn- mice. J. Nrurochem. 51, 1054-1059. aptosomes in the presence of 50 pA4 glutamate. The Fonnum F. (1984) Glutamate: a neurotransmitter in mammalian glutamate reuptake system located on synaptosomal brain. J. Neurochem. 42, 1-1 1. plasma membranes has quite different characteristics Fuxe K., Bredholm B. B., Ogren S.-O., Agnati L. F., Hokfelt T., and Gustafsson J. A. (1978) Ergot drugs and central monoaminergic from the vesicular glutamate uptake system, particu- mechanisms: a histochemical, biochemical, and behavioral larly with respect to its ability to recognize asparate analysis. Fed. Proc. 37, 2 I8 1-2 I9 1. (Logan and Snyder, 1972; Naito and Ueda, 1985). The Fykse E. M. and Fonnum F. (1988) Uptake of y-aminobutyric acid inhibitory effects on these two types of glutamate by a synaptic vesicle fraction isolated from rat brain. J. Nrrtro- transport systems, one in the synaptic vesicle mem- chem. 50, 1237-1242. Hell J. W., Maycox P. R., Stadler H., and Jahn R. (1988) Uptake of brane and the other in the presynaptic plasma mem- GABA by rat brain synaptic vesicles isolated by a new procedure. brane, raises the possibility that in vivo short exposure EMBO J. 7,3023-3029. to bromocriptine could prolong or potentiate the glu- Kebabian J. W. and Calne D. B. (1979) Multiple receptors for do- tamate-evoked excitation by inhibiting the reuptake of pamine. Nature 277, 93-96. fish P. E. and Ueda T. (1 989) Glutamate accumulation into synaptic glutamate from the synaptic cleft. Longer exposures vesicles, in Methods in Enzymology, Vol. 174 (Fleischer S. and could lead to hypoglutamatergic transmission by de- Fleischer B., eds). Academic Press, New York (in press). pleting vesicular glutamate pools. Because bromocrip- Kish P. E., Fischer-Bovenkerk C., and Ueda T. (1987) Garnma-ami-

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