Relationship between glycine transporter 1 inhibition as measured with positron emission tomography and changes in cognitive performances in nonhuman primates.

Several lines of evidence suggest that schizophrenia is associated with deficits in glutamatergic transmission at the N-methyl-d-aspartate (NMDA) receptors. Glycine is a NMDA receptor co-agonist, and extracellular levels of glycine are regulated in the forebrain by the glycine type-1 transporters (GlyT-1). GlyT-1 inhibitors elevate extracellular glycine and thus potentiate NMDA transmission. This mechanism represents a promising new avenue for the treatment of schizophrenia. Here, the recently introduced positron emission tomography radiotracer [11C]GSK931145 was used to quantify the relationship between occupancy of GlyT-1 by a GlyT-1 inhibitor, Org 25935, and its impact on spatial working memory performances in rhesus monkeys. The effect of Org 25935 on working memory was assessed both in control conditions and during a state of relative NMDA hypofunction induced by ketamine administration, at a dose selected for each animal to reduce task performance by about 50%. Under control conditions, Org 25935 had no effect on working memory at GlyT-1 occupancies lower than 75% and significantly impaired working memory at occupancies higher than 75%. Under ketamine conditions, Org 25935 reversed the deficit in working memory induced by ketamine and did so optimally in the 40-70% GlyT-1 occupancy range. The results confirm the efficacy of this mechanism to correct working memory deficits associated with NMDA hypofunction. These data also suggest the existence of an inverted-U dose-response curve in the potential therapeutic effect of this class of compounds.

Identification and evaluation of [11C]GSK931145 as a novel ligand for imaging the type 1 glycine transporter with positron emission tomography.

The type-1 glycine transporter (GlyT1) is an important target for the development of new medications for schizophrenia. A specific and selective positron emission tomography (PET) GlyT1 ligand would facilitate drug development studies to determine whether a drug reaches this target and help establish suitable doses for clinical trials. This article describes the evaluation of three candidate GlyT1 PET radioligands (GSK931145, GSK565710, and GSK991022) selected from a library of compounds based on favorable physicochemical and pharmacological properties. Each candidate was successfully labeled using [(11)C]methyl iodide or [(11)C]methyl triflate and administered to a pig pre- and postadministration with a pharmacological dose of a GlyT1 inhibitor to determine their suitability as PET ligands in the porcine brain in vivo. All three candidate ligands were analyzed quantitatively with compartment analyses employing a plasma input function. [(11)C]GSK931145 showed good brain penetration and a heterogeneous distribution in agreement with reported GlyT1 localization. Following pretreatment with GSK565710, uptake of [(11)C]GSK931145 was reduced to homogeneous levels. Although [(11)C]GSK565710 also showed good brain penetration and a heterogeneous distribution, the apparent level of specific binding was reduced compared to [(11)C]GSK931145. In contrast, [(11)C]GSK991022 showed a much lower brain penetration and resultant signal following pretreatment with GSK565710. Based on these findings [(11)C]GSK931145 was identified as the most promising ligand for imaging GlyT1 in the porcine brain, possessing good brain penetration, specific signal, and reversible kinetics. [(11)C]GSK931145 is now being progressed into higher species.

Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1.

BACKGROUND AND PURPOSE: M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtype-selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M(1) mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. EXPERIMENTAL APPROACH: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. KEY RESULTS: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. CONCLUSIONS AND IMPLICATIONS: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M1 mAChR.

Pharmacological assessment of the role of the glycine transporter GlyT-1 in mediating high-affinity glycine uptake by rat cerebral cortex and cerebellum synaptosomes.

Two distinct types of glycine transporter, GlyT-1 and GlyT-2, have been characterised. GlyT-1 and GlyT-2 are known to be differentially expressed amongst CNS areas, but direct functional evidence for their relative contributions to high-affinity glycine uptake by brain tissues is lacking. In the present study, we have used the selective GlyT-1 inhibitor N[3-(4"-fluorophenyl)-3-(4"-phenylphenoxy)propyl]sarcosine (NFPS) to investigate the role of GlyT-1 in mediating glycine uptake. HEK293 cells expressing human GlyT-1c or GlyT-2 showed high levels of Na(+)-dependent glycine uptake, with K(m) values of 117+/-13 and 200+/-22 microM, respectively. NFPS potently inhibited uptake in GlyT-1c cells (IC(50) value 0.22+/-0.03 microM), being around 500-fold more potent than glycine or sarcosine, but had no effect on uptake in GlyT-2 cells (IC(50) >10 microM). Efflux of pre-loaded [3H]-glycine from GlyT-1c cells was increased by glycine or sarcosine, whereas NFPS had no effect on its own but blocked the effects of glycine or sarcosine. These results confirm that NFPS is a potent, selective and non-transportable GlyT-1 inhibitor. Rat cortex and cerebellum synaptosomes also showed a high-affinity Na(+)-dependent component of glycine uptake, with affinities similar to those observed for uptake in GlyT-1c or GlyT-2 cells. In cortex synaptosomes, NFPS and sarcosine produced the same maximal inhibition of uptake as glycine itself. However, in cerebellum synaptosomes, the maximal inhibition produced by NFPS and sarcosine was only half that produced by glycine. In both tissues NFPS was around 1000-fold more potent than glycine or sarcosine. Overall, our findings indicate that high-affinity glycine uptake in cerebral cortex occurs predominantly via GlyT-1. However, in cerebellum, only a part of the high-affinity uptake is mediated by GlyT-1, with the remaining NFPS-insensitive component most likely mediated by GlyT-2.

Characterization of the binding of [(125)I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor.

GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K:(D:) values of 0.026+/-0.006 and 0.57+/-0.14 nM respectively, and B(max) values of 3010+/-400 and 8570+/-2240 fmol mg protein(-1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K:(D:) of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.

Solubilisation of a novel anticonvulsant binding site from pig cortical membranes.

The present study describes the solubilisation of the novel anticonvulsant, SB-204269, binding site from pig cortical membranes. Throughout the study the binding of a close analogue of this compound, [125I]-SB-217644 (trans 6- Acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b ]pyran-3R-ol) was used to monitor the success of the solubilisation procedure. [125I]-SB-217644 was an ideal mechanistic tool for quantifying the binding to this novel anticonvulsant site, with a high specific activity and affinity (K(D) of 3 nmol/l). Optimum conditions for the solubilisation of this anticonvulsant binding site were investigated using a multifactorial experimental design to assess a large number of variables. Detergent type, detergent-protein ratio, absence of Mg2+ and temperature were deemed to be important factors. However, the increases observed in binding site specific activity were minimal compared with those achieved for yields. Maximum percentage yields of binding activity (25%) were achieved with a low concentration of the zwitterionic detergent, CHAPS, in the presence of a low protein concentration. This yield was further enhanced on combining mixtures of detergents. The highest recovery (37%) was achieved with a 50:50 (v:v; 1.5 x critical micelle concentration) mixture of the ionic detergent, sodium cholate, and the non-ionic detergent, MEGA-10. In summary, we report the successful solubilisation of a novel anticonvulsant binding site, identified by its selective affinity for SB-204269 and its analogues. The recovery of nearly 40% of the target binding sites from the starting material should provide a good starting point for the purification of this protein.

Evaluation of a series of anticonvulsant 1,2,3,4-tetrahydroisoquinolinyl-benzamides.

SAR studies around a series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides, identified by high-throughput screening at the novel SB-204269 binding site, have provided compounds such as 13, 29-33 with high affinity and excellent anticonvulsant activity in animal models.

Use of the radioligand [(125)I]-SB-217644 in the characterisation and solubilisation of the novel binding site for the anticonvulsant SB-204269 in rat brain membranes and cell lines.

SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol) shows anticonvulsant activity in a range of animal seizure models, with a high therapeutic index and a lack of side-effects. We have previously reported the characterisation of a novel binding site for [(3)H]-SB-204269 in rat forebrain, which has a unique profile unrelated to other known anticonvulsant sites of action. We now describe the use of a [(125)I]-labelled form of SB-217644 (trans-6-acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol), an analogue of SB-204269, for studies on this novel binding site. In rat forebrain membranes, [(125)I]-SB-217644 shows a similar binding profile to that of [(3)H]-SB-204269, with a maximum specific binding capacity (B(max)) of 286+/-12 fmol/mg protein, but has twenty-fold higher affinity (K(d) value 1.7+/-0.1 nM). The high affinity and high specific activity of [(125)I]-SB-217644 allowed it to be used for detection and characterisation of the detergent-solubilised form of the binding site. Specific [(125)I]-SB-217644 binding to cholate-solubilised rat cerebellum showed a K(d) value of 2.7+/-0.3 nM and a B(max) value of 55+/-11 fmol/mg protein, with a 7.3+/-0.3% yield of solubilised binding sites. [(125)I]-SB-217644 was also used in whole-cell binding assays for investigation of the properties of the novel binding site in a range of cell lines. Both rat brain neuronal and glial primary cultures and several CNS-related cell lines were found to have levels of specific [(125)I]-SB-217644 binding similar to those present in rat forebrain membranes. The solubilisation of this novel binding site, and the ability to quantify and characterise it in solubilised tissues and whole cells using [(125)I]-SB217644, will allow further studies towards the ultimate identification of the molecular target of SB-204269.

Cloning, functional characterisation and population analysis of a variant form of the human glycine type 2 transporter.

Two forms of glycine transporter have been described to date, GlyT-1 and GlyT-2. The GlyT-2 form is expressed mainly in the spinal cord, brainstem and cerebellum. Here we describe the identification of a variant form of the human GlyT-2 (SC6), showing three amino acid changes to the previously reported protein. Population analysis identified the allele causing one of the polymorphisms, D463N, at 10% within the population with 3% being homozygous for the change. We also transfected our new variant into mammalian cells and compared it to the published cDNA, showing that the three amino acid changes present have no major effect on the biochemical properties of the transporter.

Identification of (-)-cis-6-acetyl-4S-(3-chloro-4-fluoro-benzoylamino)- 3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S-ol as a potential antimigraine agent.

Optimisation of novel cis- and trans-4-(substituted-amido)benzopyran-3-ol derivatives has led to the identification of SB-220453 20 with an in vivo pre-clinical CNS profile predictive of potential antimigraine activity.

Identification of a series of 1,2,3,4-tetrahydroisoquinolinyl-benzamides with potential anticonvulsant activity.

A series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides was identified by high-throughput screening at the novel SB-204269 binding site. SAR studies have provided compounds 4 and 14 with high affinity and good anticonvulsant activity in animal models.

Profile of SB-204269, a mechanistically novel anticonvulsant drug, in rat models of focal and generalized epileptic seizures.

1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1-30 mg Kg-1) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MEST)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg-1, p.o. 4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1-10 microM) that had no effect on normal synaptic activity and neuronal excitability. 5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg-1, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED50 in the MES test) for SB-204269 of > 31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8. At concentrations (> or = 10 microM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 microM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na+ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures.

Characterization of the binding of [3H]-SB-204269, a radiolabelled form of the new anticonvulsant SB-204269, to a novel binding site in rat brain membranes.

1. SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [3H]-SB-204269 in rat forebrain membranes. 2. Specific [3H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (KD) of 32 +/- 1 nM and a maximum binding capacity (Bmax) of 253 +/- 18 fmol mg-1 protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na+, K+, Ca2+ or Mg2+ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues. 3. Specific [3H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action. 4. None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [3H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na+ or K+ channels or various other receptors did not demonstrate any affinity for the binding site. 5. These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability.

The novel anticonvulsant SB 204269 binds to a stereospecific site in the mouse brain.

The novel compound SB 204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)- 3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in the mouse maximal electroshock seizure threshold test. The binding of [3H]SB 204269 to mouse forebrain membranes is saturable (Bmax 217 fmol/mg protein, Kd 32 nM) and stereospecific. The excellent anticonvulsant profile of SB 204269, combined with the identification of a unique binding site for the compound, suggest that it has potential clinical utility as a novel treatment for epilepsy.

SB 201823-A, a neuronal Ca2+ antagonist is neuroprotective in two models of cerebral ischaemia.

We have characterised the Ca2+ channel blocking properties of a new non-peptide Ca2+ channel antagonist, SB 201823-A, in cultures of rat sensory neurones. The IC50 for SB 201823-A against total Ca2+ current in sensory neurones was 4.9 microM. SB 201823-A showed little selectivity for sub-types of neuronal Ca2+ channel but was selective for Ca2+ channels over Na+ and K+ channels. Efficacy against other types of cation channel such as agonist gated channels was not assessed. SB 201823-A was neuroprotective in vivo when administered post-ischaemia in one focal and one global model of neuronal ischaemia. In the rat photothrombotic focal lesion model, SB 201823-A administered i.p. 10 min post-ischaemia resulted in a dramatic reduction in lesion volume. In the gerbil bilateral carotid artery occlusion global model, SB 201823-A dosed i.p. 30 min post-occlusion resulted in both histological and functional improvements when compared to vehicle treated animals. These data suggest that such novel neuronal Ca2+ channel antagonists may have potential in ameliorating both the pathological and functional consequences of stroke in man.

Investigations of the roles of dihydropyridine and omega-conotoxin-sensitive calcium channels in mediating depolarisation-evoked endogenous dopamine release from striatal slices.

The relative roles of L- and N-type voltage-sensitive calcium channels (VSCC) in mediating endogenous dopamine release have been investigated by examining the effects of the dihydropyridine (DHP) agonist BAY K 8644 and the antagonist PN 200-110, as well as the VSCC-blocking peptide omega-conotoxin GVIA, on depolarisation-evoked dopamine release from superfused rat striatal slices. Dopamine release evoked by electrical field stimulation was virtually unaffected by either of the DHP drugs, but release evoked by raising the K+ concentration to 25 mmol/l was significantly increased by BAY K 8644 and reduced stereospecifically by PN 200-110. Quantitative differences between electrically-evoked and K+-evoked dopamine release with respect to their dependence on extracellular calcium concentration were also observed, with electrically-evoked release requiring higher calcium concentrations. The adenylate cyclase activator forskolin itself increased dopamine release, but did not appear to influence the effectiveness of either DHP drug in altering dopamine release. In contrast to the relatively small effects of the DHP drugs, omega-conotoxin produced a major reduction in electrically-evoked dopamine release as well as a substantial decrease in K+-evoked release. Since omega-conotoxin is thought to block both L- and N-type neuronal VSCC whereas DHP drugs affect only L-type VSCC, these findings suggest that electrically-evoked dopamine release is mediated mainly by calcium influx through N-type VSCC, accounting for the reported lack of effect of many organic calcium antagonists on this process. In contrast, K+-evoked dopamine release appears to involve both L- and N-type VSCC, and can occur at lower extracellular calcium concentrations.

Endogenous dopamine release from rat striatal slices and its regulation by D-2 autoreceptors: effects of uptake inhibitors and synthesis inhibition.

The effects of various dopaminergic drugs on the spontaneous and veratrine-stimulated release of endogenous dopamine (DA) from superfused rat striatal slices have been examined using a high-sensitivity HPLC system. The DA uptake inhibitor nomifensine greatly increased both veratrine-stimulated and spontaneous DA release, whilst the effects of the more potent and selective inhibitor GBR 12921 were much smaller. The DA agonists pergolide and LY 171555 reduced both spontaneous and veratrine-stimulated DA release; conversely, the D-2 selective antagonist l-sulpiride stereospecifically increased spontaneous and veratrine-stimulated release, and blocked the effects of pergolide and LY 171555. Inhibition of DA synthesis did not directly influence the actions of either pergolide or sulpiride. These studies indicate that nomifensine may have a DA-releasing action in addition to its uptake blocking action, the regulation of endogenous DA release by D-2 autoreceptors shows properties similar to those reported previously for radiolabelled DA release, with the novel finding that spontaneous release is also regulated, the autoreceptors do not appear to selectively influence newly synthesised DA release.

Comparison between radiolabelled and endogenous dopamine release from rat striatal slices: effects of electrical field stimulation and regulation by D2-autoreceptors.

Direct comparisons have been made between the release of radiolabelled and endogenous dopamine from superfused rat striatal slices prelabelled with 3H-dopamine. Both spontaneous release and release evoked by electrical field stimulation (3 Hz, 2 min) were measured using a high-sensitivity HPLC system with electrochemical (coulometric) detection, plus scintillation counting of chromatographically separated superfusate fractions. Two periods of electrical stimulation released similar amounts of endogenous dopamine, but the second stimulation released much less 3H-dopamine than did the first, although the levels of spontaneous release immediately before the two stimuli were similar. Substantial increases in endogenous 3,4-dihydroxy phenyl acetic acid (DOPAC) release but only minor increases in 3H-DOPAC release occurred following the two stimuli. The dopamine agonist pergolide (1 microM) reduced the electrically-stimulated release of both 3H-dopamine and endogenous dopamine to a similar extent, whilst the D2-selective antagonist sulpiride (1 microM) produced large increases in both 3H-dopamine and endogenous dopamine electrically-stimulated release. In addition, spontaneous release of both 3H-dopamine and endogenous dopamine were decreased by pergolide and increased by sulpiride. Co-addition of sulpiride and pergolide produced lesser increases than those seen with sulpiride alone. These studies indicate that, despite major differences between 3H-dopamine and endogenous dopamine release in response to various stimuli, their regulation by D2-autoreceptors appears similar; a novel finding being the modulation of spontaneous 3H-dopamine release by autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)