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S32504 [(+)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine] displayed marked affinity for cloned, human (h)D(3) receptors (pK(i), 8.1) at which, in total G-protein ([(35)S]GTPgammaS binding, guanosine-5'-O-(3-[(35)S]thio)-triphosphate), Galpha(i3) (antibody capture/scintillation proximity), and mitogen-activated protein kinase (immunoblot) activation procedures, it behaved as an agonist: pEC(50) values, 8.7, 8.6, and 8.5, respectively. These actions were blocked by haloperidol and the selective D(3) receptor antagonist S33084 [(3aR,9bS)-N-[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl]-(4-phenyl) benzamide)]. S32504 showed lower potency at hD(2S) and hD(2L) receptors in [(35)S]GTPgammaS binding (pEC(50) values, 6.4 and 6.7) and antibody capture/scintillation proximity (hD(2L), pEC(50), 6.6) procedures. However, reflecting signal amplification, it potently stimulated hD(2L) receptor-coupled mitogen-activated protein kinase (pEC(50), 8.6). These actions were blocked by haloperidol and the selective D(2) receptor antagonist L741,626 [4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol]. The affinity of S32504 for hD(4) receptors was low (5.3) and negligible for hD(1) and hD(5) receptors (pK(i), <5.0). S32504 showed weak agonist properties at serotonin(1A) ([(35)S]GTPgammaS binding, pEC(50), 5.0) and serotonin(2A) (G(q), pEC(50), 5.2) receptors and low affinity for other (>50) sites. In anesthetized rats, S32504 (0.0025-0.01 mg/kg, i.v.) suppressed electrical activity of ventrotegmental dopaminergic neurons. Correspondingly, S32504 (0.0025-0.63 mg/kg, s.c.) potently reduced dialysis levels (and synthesis) of dopamine in striatum, nucleus accumbens, and frontal cortex of freely moving rats, actions blocked by haloperidol and L741,626 but not by S33084. In contrast, S32504 only weakly inhibited serotonergic transmission and failed to affect noradrenergic transmission. Actions of S32504 were expressed stereospecifically versus its less active enantiomer S32601 [(-)-trans-3,4,4a,5,6,10b-hexahydro-9-carbomoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine]. Although the D(3)/D(2) agonist and antiparkinsonian agent ropinirole mimicked the profile of S32504, it was less potent. In conclusion, S32504 is a potent and selective agonist at dopamine D(3) and D(2) receptors.

Unilateral 6-hydroxydopamine (6-OHDA) lesions of substantia nigra pars compacta (SNPC) neurons in rats induce behavioural hypersensitivity to dopaminergic agonists. However, the role of specific dopamine receptors is unclear, and potential alterations in their transduction mechanisms remain to be evaluated. The present study addressed these issues employing the dopaminergic agonist, quinelorane, which efficaciously stimulated G-protein activation (as assessed by [35S]GTPgammaS binding) at cloned hD2 (and hD3) receptors. At rat striatal membranes, dopamine stimulated [35S]GTPgammaS binding by 1.9-fold over basal, but its actions were only partially reversed by the selective D2/D3 receptor antagonist, raclopride, indicating the involvement of other receptor subtypes. In contrast, quinelorane-induced stimulation (48% of the effect of dopamine) was abolished by raclopride, and by the D2 receptor antagonist, L741,626. Further, novel antagonists selective for D3 and D4 receptors, S33084 and S18126, respectively, blocked the actions of quinelorane at concentrations corresponding to their affinities for D2 receptors. Quinelorane potently induced contralateral rotation in unilaterally 6-OHDA-lesioned rats, an effect abolished by raclopride and L741,626, but not by D3 and D4 receptor-selective doses of S33084 and S18126, respectively. In functional ([35S]GTPgammaS) autoradiography experiments, quinelorane stimulated G-protein activation in caudate putamen and, to a lesser extent, in nucleus accumbens and cingulate cortex of naive rats. In unilaterally SNPC-lesioned rats, quinelorane-induced G-protein activation in the caudate putamen on the non-lesioned side was similar to that seen in naive animals (approximately 50% stimulation), but significantly greater on the lesioned side (approximately 80%). This increase was both pharmacologically and regionally specific since it was reversed by raclopride, and was not observed in nucleus accumbens or cingulate cortex. In conclusion, the present data indicate that, in rat striatum, the actions of quinelorane are mediated primarily by D2 receptors, and suggest that behavioural hypersensitivity to this agonist, induced by unilateral SNPC lesions, is associated with an increase in D2, but not D3 or D4, receptor-mediated G-protein activation.

Herein, we evaluate the interaction of the alpha(2)-AR antagonist, yohimbine, as compared to fluparoxan, at multiple monoaminergic receptors and examine their roles in the modulation of adrenergic, dopaminergic and serotonergic transmission in freely-moving rats. Yohimbine displays marked affinity at human (h)alpha(2A)-, halpha(2B)- and halpha(2C)-ARs, significant affinity for h5-HT(1A), h5-HT(1B), h5-HT(1D), and hD(2) receptors and weak affinity for hD(3) receptors. In [(35)S]GTPgammaS binding protocols, yohimbine exerts antagonist actions at halpha(2A)-AR, h5-HT(1B), h5-HT(1D), and hD(2) sites, yet partial agonist actions at h5-HT(1A) sites. In vivo, agonist actions of yohimbine at 5-HT(1A) sites are revealed by WAY100,635-reversible induction of hypothermia in the rat. In guinea pigs, antagonist actions of yohimbine at 5-HT(1B) receptors are revealed by blockade of hypothermia evoked by the 5-HT(1B) agonist, GR46,611. In distinction to yohimbine, fluparoxan shows only modest partial agonist actions at h5-HT(1A) sites versus marked antagonist actions at halpha(2)-ARs. While fluparoxan selectively enhances hippocampal noradrenaline (NAD) turnover, yohimbine also enhances striatal dopamine (DA) turnover and suppresses striatal turnover of 5-HT. Further, yohimbine decreases firing of serotonergic neurones in raphe nuclei, an action reversed by WAY100,635. Fluparoxan increases extracellular levels of DA and NAD, but not 5-HT, in frontal cortex. In analogy, yohimbine enhances FCX levels of DA and NAD, yet suppresses those of 5-HT, the latter effect being antagonized by WAY100,635. The induction by fluoxetine of FCX levels of 5-HT, DA, and NAD is potentiated by fluparoxan. Yohimbine likewise facilitates the influence of fluoxetine upon DA and NAD levels, but not those of 5-HT. In conclusion, the alpha(2)-AR antagonist properties of yohimbine increase DA and NAD levels both alone and in association with fluoxetine. However, in contrast to the selective alpha(2)-AR antagonist, fluparoxan, the 5-HT(1A) agonist actions of yohimbine suppress 5-HT levels alone and underlie its inability to augment the influence of fluoxetine upon 5-HT levels., (Copyright 2000 Wiley-Liss, Inc.)

The novel, potential anxiolytic, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine), is an agonist and antagonist (weak partial agonist) at pre- and postsynaptic serotonin (5-HT)1A receptors, respectively. Herein, we characterized its influence on dialysate levels of 5-HT, dopamine (DA) and NAD simultaneously determined in single samples of the frontal cortex (FCX) of freely moving rats, and compared its activity in several other models of potential antidepressant (AD) properties with those of the 5-HT reuptake inhibitor (SSRI), fluoxetine. S 15535 displayed high affinity at cloned human (h) 5-HT1A receptors (Ki = 0.7 nM) and >250-fold lower affinity at cloned hD2 (400 nM), hD3 (248 nM) and h alpha2A-adrenergic (AR) (190 nM) receptors. S 15535 (0.08-5.0 mg/kg s.c.) markedly and dose-dependently suppressed dialysate levels of 5-HT in the FCX, nucleus accumbens and striatum of freely moving rats, whereas fluoxetine (10.0 mg/kg s.c.) elevated levels of 5-HT in each structure. In contrast to 5-HT, dialysate levels of DA and NAD in the FCX were dose-dependently increased by S 15535, and this effect was mimicked by fluoxetine. The influence of S 15535 and fluoxetine on FCX levels of DA was regionally specific inasmuch as dialysate levels of DA in the accumbens and striatum were not modified. The selective 5-HT1A antagonist, WAY 100,635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (0.16) transiently elicited a slight increase in cortical levels of 5-HT, an action opposite to that of S 15535. Further, in the presence of WAY 100,635 (0.16), the influence of S 15535 (0.63) on cortical levels of 5-HT, DA and NAD was markedly attenuated. Upon chronic administration of S 15535 or fluoxetine (10.0 mg/kg s.c. daily for 14 days, in each case), there was no significant alteration in the density of beta-AR receptors in the FCX. However, in contrast to fluoxetine, S 15535 elicited a significant (25%) decrease in the density (Bmax) of 5-HT2A receptors labeled by [3H]ketanserin in the cortex; there was no alteration in Kd. In a learned helplessness paradigm in rats, S 15535 (0.63-40.0 mg/kg p.o.) markedly reduced escape deficits on each of three consecutive days of testing. Fluoxetine (2.0-8.0 mg/kg i.p.) was also active in each session, but presented a biphasic dose-response curve. Finally, under the conditions used, neither S 15535 (0.63-10.0) nor fluoxetine (0.63-10.0) decreased immobility time in the forced swim test. In conclusion, S 15535 is a selective ligand of cloned, h5-HT1A receptors. Its agonist actions at 5-HT1A autoreceptors underlie its ability to decrease extracellular levels of 5-HT in the FCX, and likely contribute to the increase in extracellular levels of DA and NAD evoked by S 15535 in this structure. Further, S 15535 is active in several other, although not all, models of potential AD activity. Thus, although S 15535 is under development as an anxiolytic agent, a further characterization of its putative AD actions would be of interest.

In freely moving rats, the novel, selective dopamine (DA) D3 receptor agonist PD 128,907 dose-dependently [effective dose (ED25) = 0.07 mg/kg, s.c.] reduced dialysate levels of DA in the frontal cortex, a structure innervated by the ventral tegmental area (VTA). This action of PD 128,907 (0.16 mg/kg, s.c.) was abolished by a selective DA D3 receptor antagonist S 14297 (1.25 mg/kg, s.c.), which alone did not modify levels of DA. In contrast to S 14297, its inactive distomer, S 17777, did not modify the actions of PD 128,907. In addition, PD 128,907 dose-dependently and potently inhibited the firing rate of VTA-localized neurons in anesthetized rats (ED50 = 0.001 mg/kg, i.v.). S 14297, but not S 17777, completely reversed the actions of PD 128,907 (0.005 mg/kg, i.v.) with a 50% inhibitory dose of 0.03 mg/kg, i.v. and did not itself significantly modify the firing rate. In conclusion, these data provide the first direct evidence that DA D3 (auto) receptors modulate (inhibit) the release of DA in the frontal cortex.

Using [125I]-iodosulpride as a radioligand, the novel naphthofurane, (+/-)-S 11566 [(+/-)-[7-(N,N-dipropylamino)-5,6,7,8-tetra-hydro- naphtho(2,3b)dihydro,2,3-furane]) showed a marked preference for human, recombinant D3 as compared with D2 receptors stably transfected into Chinese hamster ovary cells (Kis = 24/529 nM). This activity resided in its (+)-eutomer, (+)-S 14297 (13/297 nM) as compared with its (-)-distomer, (-)-S 17777 (406/3544 nM). In contrast, (+)-AJ 76 manifested only a mild 2-fold preference for D3 sites (70/154 nM), whereas haloperidol and six additional antagonists showed a mild (2-7-fold) preference for D2 sites. As concerns agonists, (+)-7-OH-DPAT, (+/-)-CGS 15855A, quinelorane, (-)-quinpirole and N-0434 displayed a preference (6-40-fold) for D3 receptors, whereas piribedil showed a slight, 2-fold, preference for D2 sites (243/126 nM). (+)-S 14297 showed low (> 1.0 microM) affinity at rat D1 and D2 sites and at cloned, human D4 and D5 receptors and only low affinity (145 to > 10,000 nM) at all other sites examined. In vivo, administered s.c., (+)-7-OH-DPAT, CGS 15855A, quinelorane, (-)-quinpirole and N-0434 potently evoked hypothermia. Across all (8) agonists tested, potency correlated significantly with affinity at D3 sites (r = .84, P < .001) but not D2 sites (r = .50, P > .05). (+)-S 14297 (0.16-1.25 mg/kg, s.c.) blocked the induction of hypothermia by (+)-7-OH-DPAT, CGS 15855A and (-)-quinpirole, but not by the alpha 2-adrenergic agonist, clonidine, without influencing core temperature alone. In contrast, (-)-S 17777 (10.0 mg/kg, s.c.) was only partially active. Across all (9) antagonists, potency for inhibition of (+)-7-OH-DPAT-induced hypothermia correlated more strongly with affinity at D3 (r = .96, P < .001) than D2 (r = .75, P < .02) sites. Whereas haloperidol and the other antagonists provoked prolactin secretion and elicited catalepsy, (+)-S 14297 and (+/-)-S 11566 at doses of up to 10.0 and 40.0 mg/kg, s.c., respectively, were not significantly effective (P > .05). Across all antagonists, potency for eliciting prolactin secretion and catalepsy correlated better with affinity at D2 (r = .95 and .96) than D3 (r = .76 and .91) sites. In conclusion, these data demonstrate that the novel naphtofurane, (+)-S 14297, is a selective ligand (antagonist) at dopamine D3 receptors and suggest that their activation mediates hypothermia in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

In Chinese hamster ovary cells stably transfected with recombinant rat dopamine D2 or D3 receptors, the naphthofurane antagonist, (+)-S 14297 [(+)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro- naphtho(2,3b)dihydro,2,3-furane]], displayed a pronounced preference for dopamine D3 versus D2 receptors: Ki values = 13 and 365 nM, respectively. In contrast, its distomer, (-)-S 17777, showed low affinity (296 versus 3403 nM). The aminotetralin agonist, (+)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin), also showed high affinity at dopamine D3 (1.8 nM) versus D2 (96 nM) receptors while its (-)-isomer showed low affinity (71 and 1461 nM). In freely moving rats, (+)-7-OH-DPAT (0.16 mg/kg s.c.)-but not (-)-7-OH-DPAT-decreased dialysate levels of dopamine in the nucleus accumbens. (+)-S 14297 (1.25 mg/kg s.c.) markedly inhibited the action of (+)-7-OH-DPAT without influencing dopamine levels alone. Further, this action was stereospecific in that (-)-S 17777 (20.0 mg/kg s.c.) was inactive. In conclusion, data obtained with the novel, selective dopamine D3 receptor antagonist, (+)-S 14297 suggest that dopamine D3 autoreceptors modulate the release of dopamine from mesolimbic dopaminergic neurones.

The selective dopamine D3 receptor agonist, 7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and the novel naphthofurane, S 14297 ((+)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro- naphtho(2,3b)dihydro,2,3-furane]), bound with high affinity and selectivity to recombinant, human dopamine D3 versus D2 receptors stably transfected into Chinese hamster ovary cells: Ki values = 2 versus 103 nM for 7-OH-DPAT and 13 versus 297 nM for S 14297. In contrast, the putative dopamine D3 receptor antagonist, AJ 76 (cis-(+)-5-methoxy-1-methyl-2-(n- propylamino)tetralin), displayed low affinity and selectivity for dopamine D3 versus D2 sites (70 versus 154 nM). 7-OH-DPAT (0.01-0.16 mg/kg s.c.) provoked hypothermia in rats, an action abolished by S 14297 (0.04-0.63 mg/kg s.c.) and, less potently, by AJ 76 (0.16-2.5 mg/kg s.c.). S 14297 (20.0 mg/kg s.c.) did not modify prolactin secretion. These data suggest that dopamine D3 receptors mediate hypothermia in the rat and that S 14297 acts as a selective antagonist at these sites.