Modulation of Striatal GABA and Ach Release *Tmasako Kurokawa, *Ian P

Home , Phaclofen, Saclofen

Br. J. Pharmacol. (1994), 113, 43-48 (9" Macmillan Press Ltd, 1994 Inhibition by KF17837 of adenosine A2A receptor-mediated modulation of striatal GABA and ACh release *tMasako Kurokawa, *Ian P. Kirk, *Karen A. Kirkpatrick, tHiroshi Kase & '*Peter J. Richardson

*Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ and tPharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka-ken 411, Japan

1 The effect of the A2A adenosine receptor agonist, 2-p-(-2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]-y-aminobutyric acid ([3H]-GABA) from nerve terminals derived from the caudate-putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nM) inhibited the [3H]-GABA release evoked by 15 mM KCI but had no effect on that evoked by 30 mM KCl. 2 The ability of CGS 21680 (1 nM) to inhibit the release of [3H]-GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 gM), phaclofen (1001iM) and 2-hydroxysaclofen (100ZtM). Similarly the opioid receptor antagonist, naloxone (10 fiM), the adenosine Al receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 40 nM), and the cholinoceptor antagonists, mecamylamine (10lM) and atropine (100 nM) had no effect on this inhibition. 3 The ability of CGS 21680 (0.1 nM) to stimulate the release of [3H]-acetylcholine ([3H]-ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 iM), 2-hydroxysaclofen (10011M), phaclofen (100 AM), naloxone (1Op1M) and DPCPX (4nM). 4 The novel A2A receptor antagonist, (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837), blocked the CGS 21680 (1 nM)-induced inhibition of [3H]-GABA efflux with an EC5 of approximately 30 nM and also antagonized the CGS 21680 (0.1 nM)-induced stimulation of [3H]-ACh release with an EC50 of approximately 0.3 nM. 5 It is concluded that the A2A adenosine receptor is present on both GABAergic and cholinergic nerve terminals of the rat striatum and that in both the caudate-putamen and the globus pallidus this receptor inhibits [3H]-GABA release. No evidence was seen for a difference in the ligand binding sites of this receptor in the two groups of nerve terminals. Keywords: Acetylcholine release; GABA release; CGS 21680; A2A adenosine receptor; rat striatum; globus pallidus; KF17837

Introduction The adenosine A2A receptor has been reported to have a been observed in other areas of the brain (Barraco & Phillis; number of effects in the CNS, including the modulation of 1991; Simpson et al., 1992; Sebastiao & Ribeiro, 1992). Since transmitter release (Simpson et al., 1992; Kirkpatrick & the A2A receptor is present on the striato-pallidal neurones Richardson, 1993; Kirk & Richardson, 1994), the regulation which are overactive in Parkinson's disease (Mitchell et al., of dopamine D2 receptor affinity (Ferre et al., 1991a) and the 1990), and because the A2A receptor tends to oppose the control of motor behaviour (Green et al., 1982; Brown et al., influence of dopamine D2 receptors in vitro and in vivo, 1991; Barraco et al., 1993; Vellucci et al., 1993). This recep- (Brown et al., 1990; Ferre et al., 1991b; 1993; Vellucci et al., tor, whose mRNA has been localised by in situ hybridisation 1993; Schiffman & Vanderhaghen, 1993) there has been some to the GABAergic striato-pallidal medium-spiny neurones interest in the use of A2A receptor antagonists as therapies for (Schiffman et al., 1991), has been shown to inhibit y-amino Parkinsons disease (e.g. Ferre & Fuxe, 1992). It is therefore butyric acid (GABA) release from striatal nerve terminals important to characterize fully the effects of A2A receptor (Kirk & Richardson, 1993; 1994). There is also considerable stimulation. In this context it is interesting to note that in evidence for the presence of A2A receptors on striatal contrast to our results describing an inhibition of [3H]-GABA cholinergic nerve terminals (Brown et al., 1990; James & release by the A2A receptor, it has been reported that this Richardson, 1993; Kirk & Richardson, 1993; 1994; Kirkpat- receptor stimulates GABA release from slices of the globus rick & Richardson, 1993), despite the fact that in situ hy- pallidus (Mayfield et al., 1993). There are a number of possi- bridisation, using probes derived from the cloned A2A recep- ble explanations for these conflicting results, including the tor sequence (Maenhaut et al., 1990) failed to detect any possibility that the effects of A2A receptor stimulation may expression of A2A receptor mRNA in striatal cholinergic differ in the caudate-putamen and the globus pallidus. We neurones (Schiffman et al., 1991). Although this may have therefore set out to determine whether any significant been because the levels of mRNA expression in these cells difference can be observed in the effect of the A2A receptor on was below the limit of detection, it is also possible that the GABA release in these two areas of the striatum. Since there receptors present on the two nerve terminal populations are is also a discrepancy between the localization of the A2A significantly different in their primary sequences. receptor mRNA and its observed effects on the striatal The density of the A2A receptor is between 4 and 10 fold cholinergic nerve terminal, we have also further characterized greater in the striatum than other regions of the CNS (Bruns the effects on acetylcholine (ACh) release. In particular we et al., 1987), even though some A2A like receptor effects have have checked that they were not simply a consequence of an inhibition of endogenous GABA release, which could have reduced the inhibitory action of endogenous GABA on Author for correspondence. labelled ACh release. 44 M. KUROKAWA et al.

Recently a novel A2A receptor antagonist, (E)-8-(3,4-dime- any one run, thus enabling a second set of release ratios to thoxystyryl)-1 ,3-dipropyl-7-methylxanthine (KF17837), has be calculated. been synthesized which shows much greater selectivity for this receptor than those previously available (Nonaka et al., [3H]-acetylcholine release assays 1994). This therefore could provide an opportunity for deter- mining whether the adenosine receptor(s) responsible for Two methods were used to assess the effect of A2A receptor modulating both ACh and GABA release are of the A2A stimulation on the release of [3H]-ACh. In the perfusion subtype, and whether or not they can be distinguished by this method a similar system to that described above was used ligand. except 1.OIM [3H]-choline was used to label the terminals and physostigmine (100 tIM) was included in the loading buffer (pH 7.4) which had the following composition (mM): Methods NaCI 125, KCI 4.75, MgCl2 1.4, CaC12 2.0, HEPES 20.0 and glucose 10.0 and the synaptosomes incubated for 30 min at Subcellular fractionation 37°C. The identity of the 3H label released by elevated KCI concentrations in this system has been shown to be greater Nerve terminals were prepared from the striatum of adult than 75% ACh (Kirk & Richardson, 1994). In the second Wistar rats of either sex. In most experiments the whole method, after a 2 min preincubation at 37'C the release of striatum, including both caudate-putamen and globus pal- ACh was evoked from the nerve terminals by the addition of lidus, was used but in experiments where the differences 75tiM veratridine in the presence of 100pM physostigmine. between the two areas of the striatum were being inves- After a further 2 min the reaction was stopped by centrifuga- tigated, great care was taken to dissect each area free of the tion at 10,000 g for 2 min at 0'C. The release of [3H]-ACh other. After dissection the tissue was homogenized in 0.32 M into the supernatant was then measured by the choline kinase sucrose, 10 mM HEPES (pH 7.4) with a motor driven (640 extraction method (Pittel et al., 1990), as modified by Kirk- r.p.m.), loose fitting Teflon-glass homogenizer. After centri- patrick & Richardson (1993). fugation (1,000 g, 10 min, 4'C) the terminals were purified on a Percoll gradient (Verhage et al., 1989) and resuspended in a Drugs and chemicals balanced salt solution (pH 7.4) of the following composition (mM): NaCl 126.4, KCI 3.6, NaH2PO4 0.4, NaHCO3 5, HEPES [3H]-GABA (60 Ci mmol ') and [3H]-choline (75 Ci mmol ') 20, MgCl2 1, glucose 10 and CaCl22. After centrifugation were obtained from Amersham International. Phaclofen, 2- (15,000 g, 20 min, 4C) the nerve terminals were stored on ice hydroxysaclofen, naloxone, physostigmine, bicuculline, atro- for up to 3 h prior to use. pine, mecamylamine, nipecotic acid, Percoll, amino-oxyacetic acid and adenosine deaminase were all from Sigma Chemi- [3H]-GABA release assays cals. 2-p-(-2-Carboxyethyl)phenethylamino 5'-N-ethylcarboxamidoadenosine (CGS 21680) and 8-cyclopentyl-1,3-dipropyl- The nerve terminals were resuspended in the balanced salt xanthine (DPCPX) were from Research Biochemicals Incor- solution containing 0.1 mM aminoxyacetic acid to give a final porated. (E)- 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl) protein concentration of 0.5-1.5 mg ml-' and then incubated xanthine (KF17837) was from Kyowa Hakko Kogyo Co. at 37°C for 10 min, after which [3H]-GABA was added to Ltd., Shinzuoka-Ken, Japan. DPCPX and KF17837 were give a final concentration of 0.0 14 14M (1 pCi ml-') in the dissolved in dimethylsulphoxide, the final concentration of presence of 0.1 JiM unlabelled GABA. After incubation at this solvent being 0.02%. KF17837 is stereoselective, the E 37°C for 60 min the nerve terminals were diluted 1:4 with isomer exhibiting a higher affinity for the A2A receptor than perfusion buffer of the following composition (mM): NaCl the Z isomer. Exposure of the E isomer to visible light results 125, KCl 3, NaH2PO4 1, NaHCO3 22, MgCl2 1.3, glucose 10, in a photo-isomerization which finally results in a stable CaCI2 1.3 containing 0.05 mM aminooxyacetic acid; 1 u ml-' mixture of the two isomers. Such a mixture was used in these adenosine deaminase and 1 JiM nipecotic acid. The terminals experiments, in which the ratio of the E to Z isomers was 2:8 were then drawn into perfusion chambers containing What- (Nonaka et al., 1994). All other chemicals were of the highest man GF/B filters where they were superfused at 37°C at a available purity and were obtained from BDH Chemicals. rate between 0.4 and 0.5 ml min-' and constantly aerated with 95% 02/5% CO2. A series of 8 parallel perfusion Statistics chambers were used. Control and test conditions were performed in duplicate, thereby allowing a maximum of three Statistical analysis of the normalized data (derived from each drug concentrations plus controls to be tested with any one individual determination) was evaluated using either a one set of stimuli. After an equilibration period of 36 min, sam- way analysis of variance (ANOVA) followed by a Dunnett's ples were collected at 2 min intervals and counted for test, when making multiple comparisons from the same set of radioactivity. Release of [3H]-GABA was evoked by inclusion data with control values. Alternatively, Student's t test was of elevated KCI (15 mM) for 90s in the perfusion buffer. used for comparing individual treatments with their respec- Isotonicity was maintained by a corresponding reduction in tive controls. In both cases a probability of P<0.05 was the NaCl concentration. The evoked release for each stimulus accepted as denoting a statistically significant difference. was expressed as a percentage of the total amount of radiolabel present in the synaptosomes at the point at which release was evoked. Evoked release was calculated by sub- Results tracting basal release from total release for each stimulus. Modulation of release was assessed by changes in the ratio In order to determine if there was a difference in the effect of of the 3H-label released between two stimuli spaced 12 min adenosine A2A receptor stimulation in two areas of the apart (i.e. S2/S1 ratio). The individual test ratios were then striatum, the effect of the A2A selective agonist, CGS 21680 expressed as a percentage of the mean ratio in the control (Jarvis et al., 1989; Lupica et al., 1990) on [3H]-GABA perfusions in order to normalize the data. Experimental con- release was measured separately in nerve terminals derived trols were run in parallel, i.e. both stimuli contained elevated from the caudate-putamen and the globus pallidus. CGS KCI alone, while test experiments included putative modu- 21680, 1 nM, was used as this concentration of the A2A lators in the second stimulation. Agonists were added during agonist has previously been shown to have a maximal effect the depolarizing stimulus only, whereas antagonists were per- on [3H]-GABA release from nerve terminals derived from the fused 6min prior to, and during depolarization. Using this whole striatum (Kirk & Richardson, 1994). Figure 1 shows system it was possible to carry out a total of four stimuli on that CGS 21680 (1 nM) inhibited the release of [3H]-GABA A2A MODULATION OF STRIATAL TRANSMIMTER RELEASE 45 from both areas of the rat striatum when the stimulus was (1 nM) inhibited the efflux of [3H]-GABA by only 8.9 ± 2.0% 15 mM KCl but not when the efflux of [3H]-GABA was (n = 4) in the globus pallidus (Figure la) and by 8.0 ± 0.5% evoked by 30 mM KCI (Figure la,b). In the globus pallidus (n = 3) in the caudate-putamen (Figure lb). Since A2A recep- the inhibition of [3H]-GABA release was 22.2 ± 3.8% (n = 4, tor stimulation had similar effects in both areas of the Figure la) while the same concentration of CGS 21680 striatum subsequent experiments were performed using nerve reduced the efflux of tritiated GABA from the caudate- terminals derived from the whole striatum. putamen by 23.1 ± 3.2% (n = 11, Figure lb). Figure 1 also Table 1 illustrates that neither the GABAA antagonist, shows that the selective A2A antagonist, KF17837, greatly bicuculline (10 gM) nor the GABAB antagonist, 2-hydroxy- impaired the CGS 21680-mediated inhibition of [3H]-GABA saclofen (100 gM), had any effect on the potassium-evoked release. In the presence of KF17387 (100 nM), CGS 21680 release, nor on the ability of CGS 21680 (1 nM) to inhibit evoked [3H]-GABA release. Similarly, the potassium-evoked release of [3H]-GABA, and its inhibition by CGS 21680, were a unaffected by the presence of the cholinoceptor (muscarinic 30 and nicotinic) antagonists atropine (100 nM) and mecamylamine (1OIM). Since the striato-pallidal neurone bearing A2A receptors also express Met-enkephalin (Schiffman et al., 1991), the effect of the opioid receptor antagonist, naloxone < 20- (10 gM), on the inhibition of [3H]-GABA release was also assessed, as was the Al receptor antagonist, DPCPX (40 nM). Once again no effect was observed. None of the antagonists T 0- on00 used had a significant effect on the basal efflux of [3H]-GABA 01 (data not shown). * 1 0 ' Table 2 demonstrates that the ability of CGS 21680 (0.1 nM) to enhance the release of [3H]-ACh from rat striatal .C synaptosomes is also unimpaired by the presence of GABA, opioid or Al adenosine receptor antagonists. The concentration of CGS 21680 (0.1 nM) was chosen because it was 0 previously shown to affect the evoked efflux of [3H]-ACh b maximally (Kirkpatrick & Richardson, 1993). The antago- 30- nists used were: bicuculline (10 IM), 2-hydroxysaclofen (100 sM), phaclofen (100 AM) and DPCPX (4 nM). None of the antagonists used had any effect on the evoked release of [3H]-ACh (see Table 2) nor on the basal efflux (data not m shown). Y 20 In a previous study, we reported that increasing concentra- IE-.- tions of CGS 21680 enhanced the veratridine-evoked efflux of a,() %I- cn [3H]-ACh (Kirkpatrick & Richardson, 1993) but in these O n experiments [3H]-ACh release was measured by a batch o00a) analysis method. When using this particular method it is .10- more likely that the continued presence of endogenous -C released neurotransmitter(s) could affect the release of other (labelled) transmitters. Therefore, we investigated the effect of GABA receptor antagonists and naloxone on the ability of CGS 21680 to increase the veratridine (75 AM)-evoked release of [3H]-ACh. Table 2 shows that under these conditions Figure 1 A2A receptor-mediated inhibition of [3H]-GABA release CGS 21680 (0.1 nM) enhanced the release of [3H]-ACh by from nerve terminals of either (a) the globus pallidus or (b) caudate- 22.7 ± 6.4% (n = 3). This augmentation was unaffected by putamen. Nerve terminals were perfused as described and the release the presence of the GABA receptor antagonists, bicuculline of [3H]-GABA evoked by 15 mm KCl (open columns), 15 mm KCI (10 tM) and phaclofen (100 PM), and naloxone (10 rM). No with 100 nm KF17837, (hatched columns) or 30mM KCl (solid effects on either the veratridine-evoked release of [3H]-ACh columns); all in the presence of I nM CGS 21680. The results are (see Table 2) nor the basal efflux were seen with any of the expressed as % inhibition of the control release of [3H]-GABA, i.e. antagonists used. that evoked by elevated KCI in the absence of CGS 21680, and are means ± s.e.mean of 3 experiments, each experiment containing trip- Having established that there are adenosine A2A-like recep- licate determinations. *Indicates significantly different from the tors on both cholinergic and GABAergic nerve terminals, we inhibition of release induced in the presence of 15 mM KCI assessed further the ability of the novel A2A antagonist, (P < 0.05). KF17837, to inhibit the modulation of transmitter release.

Table 1 The effects of various antagonists on the evoked release of [3H]-GABA from rat striatal synaptosomes in the presence and absence of CGS 21680 (1 nM) Antagonists Antagonists plus alone CGS 21680 None 100.0 ± 3.0 (14) 75.5 ± 3.2 (14) Bicuculline (10IM) plus 2-hydroxysaclofen (100 nM) 100.0 ± 4.G (4) 75.9 ± 7.2 (4)* Atropine (100 nM) plus mecamylamine (10 gM) 97.4 ± 8.3 (3) 73.0 ± 3.7 (3)* Naloxone (10 gM) 93.6 ± 6.6 (4) 79.8 ± 5.0 (4)* DPCPX (40 nM) 92.1 ± 1.3 (3) 67.7 ± 6.4 (3)* Results are expressed as a percentage of the release observed in the controls (i.e. 15 mm KC1 alone) and are means ± s.e.mean from the number of experiments indicated in parentheses, each experiment containing duplicate determinations. In the control experiment the S2/S I ratios were 1.180 ± 0.033. *Indicates significantly different from antagonists alone. 46 M. KUROKAWA et al.

Table 2 The effect of various antagonists on the 15 mM KCI-evokeda and veratridine (75 pM)-evokedb release of [3H]-ACh from rat striatal synaptosomes, in the presence and absence of CGS 21680 (0.1 nM) Antagonists Antagonists plus alone CGS 21680 None' 100.0±2.7 (6) 155 ± 8.1 (6) Bicuculline (10 juM) plus 2-hydroxysaclofen (100liM)4 113.3 ± 7.8 (3) 146.7 ± 0.3 (3)* DPCPX (4 nM)4 98.2 ± 4.6 (3) 153.4 ± 6.2 (3)* Noneb 100.0 ± 3.3 (3) 122.7 ± 6.4 (3) Bicuculline (10 fiM) plus phaclofen (100 jM)b 100.0± 11.8 (3) 121.9 ± 0.3 (3)* Naloxone (1O jLM)b 100.0 ± 2.6 (3) 118.7 ± 6.3 (3)* Results are expressed as a percentage of the release observed in the controls (i.e. 15 mm KCl alone or veratridine alone) and are means ± s.e.mean from the number of experiments indicated in parentheses, each experiment containing duplicate determinations. In the perfusion experiments the S2/SI ratios were 1.090 ± 0.036. *Indicates significantly different from antagonists alone (P <0.05).

Figure 2 demonstrates that increasing concentrations of a KF17837 (0.1-100 nM) inhibited A2A receptor-mediated 401 modulation of [3H]-ACh and [3H]-GABA release. Figure 2a illustrates that in these experiments CGS 21680 (1 nM) alone decreased the release of [3H]-GABA by 30.1 ± 3.2% (n = 8) I = and that this effect was antagonized in a dose-dependent c 30 77 manner by increasing concentrations of KF17837; the IC50 U_ being approximately 30 nM with a maximal inhibition of the I' effect of CGS 21680 occurring at 100 nM. This maximal con- C,- 20-00 centration of KFI7837 alone had no significant effect on C cn 20 either the evoked (an inhibition of 5.7 ± 8.2%, n = 4) or O a) basal release of [3H]-GABA. The effects of increasing concen- * trations of KFI7837 on [3H]-ACh release, evoked by 15 mM potassium in perfusion experiments, are shown in Figure 2b. 10. In the absence of KF17837, CGS 21680 (0.1 nM) increased the efflux of [3H]-ACh by 55.5 ± 6.9% (n = 4). KF17837 (0.1-1O nM) inhibited the action of CGS 21680 in a dose- 0 dependent manner with an apparent IC50 of approximately 0 1 301(nM 0.3 nM and its effects appeared maximal at 1 nM (Figure 2b). [KF 17837] (nm) In the absence of CGS 21680, KF17837 (10 nM) had no effect b on the evoked (an inhibition of 5.8 ± 2.7%, n = 3) or basal 70- (data not shown) release of [3H]-ACh. 60 Discussion - 50' The A'A receptor-mediated inhibition of labelled GABA release from striatal nerve terminals has already been des- M -40 cribed, and shown to be due to a reduction in calcium- ".a) dependent release (Kirk & Richardson, 1993; 1994). The C 0) 30 experiments described in this paper were undertaken in order E to clarify the effects of the adenosine A2A receptor on transmitter release in the striatum. The recent that this E 20 report ._, receptor stimulated the release of GABA from globus pal- (n lidus slices (Mayfield et al., 1993), contradicted our previous observations that stimulation of A2A receptors inhibited the release of [3H]-GABA (Kirk & Richardson, 1993; 1994). This could have been because the overwhelming majority of the 0 0.1 0.3 1 10 100 terminals in our preparation were derived from the caudate- [KF 178371 (nM) putamen and were not derived from the striato-pallidal neurones, or that this receptor stimulated release at the Figure 2 A2A receptor-mediated inhibition of striatal [3H]-GABA axonal terminals of these neurones in the globus pallidus but and [3H]-ACh release by KF17837. Striatal nerve terminals were inhibited release at the recurrent collaterals in the caudate- perfused as described, and the release of [3H]-GABA (a) or [3H]-ACh putamen. The results in Figure 1 clearly demonstrate that (b) evoked by 15 mM KCI, in the presence of either 1 nm (a) or A2A receptor stimulation inhibits [3H]-GABA release from 0.1 nM (b) CGS 21680 and various concentrations of KF17837. The nerve terminals derived from both the caudate-putamen and results are expressed as % inhibition (GABA) or % stimulation the globus pallidus, and that this inhibition is antagonized in (ACh) of the control release (i.e. that evoked by 15 mM KCI alone) and are means ± s.e.mean of experiments each containing duplicate both regions by the novel A2A antagonist, KF17837. There determinations. *Indicates significantly different from the modulation are a number of differences between our methodology and of release observed in the absence of KF17837 (P<0.05). that of Mayfield et al. (1993) including the use of high potassium concentrations rather than electrical stimulation. Although it is possible that their results are a consequence of an inhibition of GABA release by the A2A receptor, resulting Mayfield et al. (1993) permitted the observation of two A2A in a reduction of an endogenous tonic (GABA mediated) receptor mediated effects, inhibition as well as stimulation. inhibition of [3H]-GABA release, the methodology used by Since, under the stimulation conditions used in this paper A2A MODULATION OF STRIATAL TRANSMITTER RELEASE 47

(i.e. elevated potassium concentrations), any effect arising are the same or different. The concentrations of the from receptor-mediated modulation of potassium channels antagonists and agonists affecting the release of ACh and would be negated (McMahon & Nicholls, 1991), it may be GABA are different in the two systems, while the relative that the stimulation of GABA release observed by Mayfield potencies of the agonists CGS 21680, N-ethylcarboxamido- et al. (1993) was a consequence of A2A receptor-mediated adenosine and R-phenylisopropyladenosine are similar. Inter- inhibition of such a channel. estingly the antagonist KF17837 appeared to be more The results in this paper provide yet more evidence that effective in blocking the modulation of ACh release than that the A2A receptor is present on striatal cholinergic nerve ter- of GABA, although it would be necessary to determine the minals. The inability of the GABA receptor and cholinocep- pA2 value of KF17837 at the A2A receptor on the two nerve tor (and other) antagonists to affect A2A receptor-mediated terminals in order to determine whether or not these are modulation of transmitter release clearly demonstrates that indeed two different receptors. the effect of adenosine agonists on acetylcholine release is not In summary KF17837, the most A2A-selective antagonist a consequence of a secondary effect mediated by changes in synthesized to date, is a potent inhibitor of CGS 21680- GABA release in our experimental system. Given the absence mediated modulation of both striatal GABA and ACh of detectable A2A receptor mRNA in cholinergic neurones release. Under the conditions described in this paper the A2A and since A2A agonists inhibit the release of GABA but receptor inhibits potassium-evoked release of GABA in both stimulate that of ACh (Kirkpatrick & Richardson, 1993; the caudate-putamen and globus pallidus, while stimulating Kirk & Richardson, 1994), it is possible that more than one the release of ACh. type of A2A receptor exits. Indeed, two A2A-like binding sites have been reported in human brain (James et al., 1992) and This work was funded by the Medical Research Council and the Sir rat brain (Johansson et al., 1993). It is not however possible Jules Thorne Charitable Trust. I.P.K. is the recipient of a Glaxo to determine from the data presented whether the ligand Group Research Training Award. We are grateful to Kyowa Hakko binding sites of the receptors on these two nerve terminals Kogyo Co. Ltd. for the supply of KF17837.

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