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 pre- warmed [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-indepen- dent 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): trifluopera- zine, 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