Implantation of a slow release corticosterone pellet induces long-term alterations in serotonergic neurochemistry in the rat brain.

Many studies point to an involvement of deficits in the serotonergic nervous system and hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis function with depression. Indeed early life stress, involving HPA axis activation, may predispose susceptible individuals to develop depression in later life. This study investigates the effects of elevating the neuroendocrine stress hormone, corticosterone, for 1 week in adolescent rats on markers of serotonergic neurone function at adulthood. Slow release corticosterone pellets were implanted for 7 days and various serotonergic parameters, as well as plasma corticosterone levels, were measured on day 7 or on day 28 (21 days following removal of the pellet). The corticosterone implant attenuated weight gain and reduced adrenal weights compared to that in control rats implanted with a cholesterol pellet. After 7 days, with the implant still in place, the diurnal variation in plasma corticosterone was reduced so that the level was approximately at that of the evening peak throughout the day. Twenty-one days after removal of the implant, the diurnal variation in plasma corticosterone returned. Corticosterone treatment decreased [3H] 8-hydroxy-2-(di-n-propylamino)tetralin binding to the 5-hydroxytryptamine1A receptor in the cortex but not in the hippocampus. Corticosterone treatment also enhanced the circadian rhythm observed in 5-hydroxyindoleacetic acid level and the ratio of 5-hydroxyindoleacetic acid to the 5-hydroxytryptamine in the frontal cortex. Despite corticosterone pellet removal 21 days earlier, there was a persistent decrease in whole body and adrenal weight, cortical 5-hydroxytryptamine1A receptor binding and an alteration in the diurnal variation in the 5-hydroxytryptamine "turnover" in the frontal cortex.

[3H]-SB-269970 radiolabels 5-HT7 receptors in rodent, pig and primate brain tissues.

The selective 5-HT7 receptor antagonist radioligand, [3H]-SB-269970, has been reported to radiolabel the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cortex (thomas et al, 2000). Saturation analysis of [3H]-SB-269970 binding to mouse forebrain, rat cortex, pig cortex, marmoset cortex and human thalamus membranes was consistent with labelling a homogenous population of binding sites in each tissue. K(D) values for [3H]-SB-269970 binding in these tissues ranged from 0.9 to 2.3 nM, being similar to those reported for the human cloned and guinea pig cortex 5-HT7 receptors (1.3 and 1.7 nM, respectively). Bmax values for [3H]-SB-269970 binding to the mouse, rat, pig, marmoset and human brain membranes were 20, 30, 31, 14 and 68 fmoles x mg x protein(-1), respectively. For each species the profile of inhibition of [3H]-SB-269970 binding, using a number of 5-HT7 receptor agonists and antagonists, correlated well with that reported for the human cloned 5-HT7(a) receptor (correlation coefficients were 0.95, 0.94, 0.92, 0.95, 0.97 versus the mouse, rat, pig, marmoset and human tissues, respectively). In conclusion, [3H]-SB-269970 has been shown to radiolabel 5-HT7 receptors in rodent, pig and primate brain and represents a valuable tool with which to further characterise the distribution and function of 5-HT7 receptors in native tissues and elucidate their potential role in disease states.

Ligands for the investigation of 5-HT autoreceptor function.

The existence of multiple 5-HT autoreceptors in the central nervous system is now firmly established and they have been pharmacologically identified as belonging to the 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptor subtypes. In addition, 5-HT(1F), 5-HT(5A), and 5-HT(7) receptors remain as potential candidates for additional autoreceptors. The emergence of selective ligands, such as SB-224289 (5-HT(1B) receptor antagonist), BRL 15572 (5-HT(1D) receptor antagonist), GR 127935 (a mixed 5-HT(1B/1D) receptor antagonist), LY 334370 (5-HT(1F) receptor agonist), and SB-269970 (5-HT(7) receptor antagonist), has aided the characterisation of 5-HT autoreceptors and has highlighted the complexity of mechanisms which modulate the release of 5-HT.

Cannabinoid activation of recombinant and endogenous vanilloid receptors.

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

SB-243213; a selective 5-HT2C receptor inverse agonist with improved anxiolytic profile: lack of tolerance and withdrawal anxiety.

SB-243213 (5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline hydrochloride) is a new, selective 5-hydroxytryptamine (5-HT)2C receptor inverse agonist. SB-243213 has high affinity for the human 5-HT2C receptor (pK(i) 9.37) and greater than a 100-fold selectivity over a wide range of neurotransmitter receptors, enzymes and ion channels. In in vitro functional studies, SB-243213 acted as an inverse agonist at the human 5-HT2C receptor with a pK(b) of 9.8. In in vivo studies, SB-243213 was a potent inhibitor of central 5-HT2C receptor-mediated function in rats, blocking meta-chlorophenylpiperazine-induced hypolocomotion with an ID50 of 1.1 mg/kg p.o. and a long duration of action (>8 h). In rats, SB-243213 exhibited anxiolytic-like activity in both the social interaction and Geller-Seifter conflict tests. Importantly, unlike diazepam, chronic administration of SB-243213 did not result in the development of either tolerance to the anxiolytic-like effects or withdrawal anxiogenesis. Furthermore, in rodents, SB-243213 did not affect seizure threshold, did not increase body weight or induce catalepsy, but attenuated the haloperidol-induced catalepsy. SB-243213 did not affect amphetamine-, MK-801- or phencyclidine-induced hyperactivity. In conclusion, SB-243213 may possess an improved anxiolytic profile compared to benzodiazepines. SB-243213 also modulates dopaminergic transmission, lacks pro-psychotic properties and may have utility in the treatment of schizophrenia and motor disorders.

SB-272183, a selective 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptor antagonist in native tissue.

A novel compound, SB-272183 (5-Chloro-2, 3-dihydro-6-[4-methylpiperazin-1-yl]-1[4-pyridin-4-yl]napth-1-ylaminocarbonyl]-1H-indole), has been shown to have high affinity for human 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors with pK(i) values of 8.0, 8.1 and 8.7 respectively and is at least 30 fold selective over a range of other receptors. [(35)S]-GTPgammaS binding studies showed that SB-272183 acts as a partial agonist at human recombinant 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors with intrinsic activities of 0.4, 0.4 and 0.8 respectively, compared to 5-HT. SB-272183 inhibited 5-HT-induced stimulation of [(35)S]-GTPgammaS binding at human 5-HT(1A) and 5-HT(1B) receptors to give pA(2) values of 8.2 and 8.5 respectively. However, from [(35)S]-GTPgammaS autoradiographic studies in rat and human dorsal raphe nucleus, SB-272183 did not display intrinsic activity up to 10 microM but did block 5-HT-induced stimulation of [(35)S]-GTPgammaS binding. From electrophysiological studies in rat raphe slices in vitro, SB-272183 did not effect cell firing rate up to 1 microM but was able to attenuate (+)8-OH-DPAT-induced inhibition of cell firing to give an apparent pK(b) of 7.1. SB-272183 potentiated electrically-stimulated [(3)H]-5-HT release from rat and guinea-pig cortical slices at 100 and 1000 nM, similar to results previously obtained with the 5-HT(1B) and 5-HT(1D) receptor antagonist, GR127935. Fast cyclic voltammetry studies in rat dorsal raphe nucleus showed that SB-272183 could block sumatriptan-induced inhibition of 5-HT efflux, with an apparent pK(b) of 7.2, but did not effect basal efflux up to 1 microM. These studies show that, in vitro, SB-272183 acts as an antagonist at native tissue 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors.

Low pH modulation of recombinant vanilloid receptors and perivascular capsaicin-sensitive sensory neurotransmission.

The effect of low pH on capsaicin-sensitive sensory neurotransmission in the rat isolated mesenteric arterial bed and at recombinant (rVR1) vanilloid receptors was investigated. Mesenteric sensory neurogenic vasorelaxation elicited by electrical field stimulation was reversibly inhibited by lowering pH from 7.4 to 6.9 and 6.3. Capsaicin-induced vasorelaxation was not different at pH 6.9, but was attenuated at pH 6.3. Vasorelaxation to calcitonin gene-related peptide, the principal sensory motor neurotransmitter in rat mesenteric arteries, was not different at pH 6.9 or pH 6.3. In rVR1-transfected HEK293 cells, acidic conditions enhanced the affinities of capsaicin and capsazepine at rVR1, but did not affect the potency of carbachol at endogenous muscarinic receptors. Following inactivation of endogenous acid-sensitive ion channels, lowering pH (6.0-4.5) directly increased [Ca2+]i in rVR1-HEK293 cells (EC50 5.5). This response was abolished by 1 microM capsazepine. In conclusion, a decrease in pH (to 6.9 and 6.3) enhances the affinity of capsaicin at rVR1, but inhibits sensory neurotransmission in the rat mesenteric arterial bed. This likely explains why there is no evidence of an enhancement of sensitivity to capsaicin at endogenous vanilloid receptors, as observed with rVR1. When pH is reduced still further (6.0-5.5) there is direct activation of rVR1.

Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT4 receptor in the central nervous system by TaqMan real time RT-PCR.

5-HT4 receptors mediate several physiological effects of 5-HT, particularly in the central nervous system (CNS), heart and gut. Recently, several C-terminal splice variants of the human 5-HT4 (h5-HT4) receptor have been described, namely h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g). Previous tissue distribution data suggest some degree of specificity in the mRNA expression patterns of the different h5-HT4 receptor splice variants. However, comparison of the mRNA expression profiles of these splice variants is difficult due to the non-quantitative methods used, and in addition, there is very limited data on the expression of each splice variant in human CNS subregions. In the present study we used a single technique, TaqMan real time quantitative RT-PCR, to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. Using a primer/probe set that amplified all 5-HT4 splice variants (5-HT4pan), the highest CNS expression of 5-HT4 receptor mRNA was observed in basal ganglia, amygdala and hippocampus, consistent with previous studies. h5-HT4(a), h5-HT4(b), h5-HT4(c) and h5-HT4(g) were predominantly expressed in various CNS tissues, compared to most peripheral tissues, but there were differences in expression levels and distribution patterns of each variant. The distribution profile and expression levels observed for the 5-HT4(b) splice variant were virtually identical to that obtained with the 5-HT4pan primer/probe set, whilst the other splice variants were expressed at much lower levels and with different expression patterns obtained with both 5-HT4(b) and 5-HT4pan primer/probe sets. Highest levels of 5-HT4(g) were observed in the hypothalamus and cortex, whilst the 5-HT4(a) variant was highest in the amygdala. 5-HT4(c) expression was highest in the pituitary gland whilst 5-HT4(d) mRNA was only detected in the small intestine at very low levels and not in the CNS. In conclusion, we have shown quantitative differences in the mRNA distribution profiles of the 5-HT4 receptor C-terminal splice variants in human CNS subregions as well as peripheral tissues. In addition, our data suggests that the h5-HT4(b) variant is the most predominant form of the 5-HT4 receptor in humans.

The effect of SB-269970, a 5-HT(7) receptor antagonist, on 5-HT release from serotonergic terminals and cell bodies.

1. The presence of 5-HT(7) receptor mRNA and protein in 5-HT neurons suggests that this receptor may act as a 5-HT autoreceptor. In this study, the effect of the 5-HT(7) receptor antagonist, SB-269970 ((R)-1-[3-hydroxy phenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine), was investigated on 5-HT release in the guinea-pig and rat cortex and the rat dorsal raphe nucleus (DRN), using the techniques of in vitro [(3)H]-5-HT release or fast cyclic voltammetry, respectively. 2. Cortical slices were loaded with [(3)H]-5-HT and release was evoked by electrical stimulation. 5-CT inhibited the evoked release of [(3)H]-5-HT in a concentration-dependent manner. SB-269970 had no significant effect on [(3)H]-5-HT release while the 5-HT(1B) receptor antagonist, SB-224289 significantly potentiated [(3)H]-5-HT release. In addition, SB-269970 was unable to attenuate the 5-CT-induced inhibition of release while SB-224289 produced a rightward shift of the 5-CT response, generating estimated pK(B) values of 7.8 and 7.6 at the guinea-pig and rat terminal 5-HT autoreceptors respectively. 3. Rat DRN slices were electrically stimulated and the evoked 5-HT efflux detected by voltammetric analysis. 8-OH-DPAT inhibited evoked 5-HT efflux and was fully reversed by WAY 100635. SB-269970 had no effect on either 5-HT efflux per se or 8-OH-DPAT-induced inhibition of 5-HT efflux. In addition, 5-CT inhibited 5-HT efflux in a concentration-dependent manner. SB-269970 was unable to attenuate the 5-CT-induced inhibition of 5-HT efflux. 4. In conclusion, we were unable to provide evidence to suggest a 5-HT autoreceptor role for 5-HT(7) receptors. However, investigations with more selective 5-HT(7) receptor agonists are needed to confirm the data reported here.

Pharmacological characterisation of human 5-HT2 receptor subtypes.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

SB-236057-A: a selective 5-HT1B receptor inverse agonist.

5-HT1B autoreceptors are involved in the control of extracellular 5-HT levels from both the terminal and cell body regions of serotonergic neurons. In this manuscript we review the pharmacological and pharmacokinetic data available for the selective and potent 5-HT1B receptor inverse agonist, SB-236057-A (1'-ethyl-5-(2'-methyl-4'-(5-methyl-1,3,4-oxadiazolyl-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro (furo[2,3-f]indole-3,4'-piperidine) hydrochloride). SB 236057-A has been shown to have high affinity for human 5-HT1B receptors (pK(i) = 8.2) and displays 80 or more fold selectivity for the human 5-HT1B receptor over other 5-HT receptors and a range of additional receptors, ion channels and enzymes. In functional studies at human 5-HT1B receptors SB-236057-A displayed inverse agonism (pA(2) = 8.9) using [(35)S]GTPgammaS binding, and silent antagonism (pA(2) = 9.2) using cAMP accumulation. SB-236057-A also acted as an antagonist at the 5-HT terminal autoreceptor as measured by [3H]5-HT release from electrically stimulated guinea pig and human cortical slices. In the guinea pig, pharmacokinetic analysis demonstrated that SB-236057-A was bioavailable and according to in vivo pharmacodynamic assays it enters brain and has a long duration of action. Importantly no side effect liability was evident at relevant doses from anxiogenic, cardiovascular, sedative or migraine viewpoints. In vivo microdialysis studies demonstrated that SB-236057-A is an antagonist in the guinea pig cortex but has no effect on extracellular 5-HT levels per se. In contrast, SB-236057-A increased extracellular 5-HT levels in the guinea pig dentate gyrus. This increase in 5-HT release was comparable to that observed after 14 days of paroxetine administration. SB-236057-A has been a useful tool in confirming that, in either guinea pigs or humans, the terminal 5-HT autoreceptor is of the 5-HT1B subtype. It appears that acute 5-HT1B receptor blockade, by virtue of increased 5-HT release in the dentate gyrus, may provide a rapidly acting antidepressant.

Cloning and characterization of human NTT5 and v7-3: two orphan transporters of the Na+/Cl- -dependent neurotransmitter transporter gene family.

Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.

1-[2-[(Heteroaryloxy)heteroaryl]carbamoyl]indolines: novel and selective 5-HT2C receptor inverse agonists with potential as antidepressant/anxiolytic agents.

Bisaryl ethers have been identified with excellent 5-HT2C affinity and selectivity over both 5-HT2A and 5-HT2B receptors. Compounds such as 11, 27 and 38 have potent oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function and their potential as novel non-sedating anxiolytic and antidepressants is under investigation.

1-[2-[(Heteroarylmethoxy)aryl]carbamoyl]indolines are selective and orally active 5-HT2C receptor inverse agonists.

Bisarylmethoxyethers have been identified with nanomolar 5-HT2C affinity and selectivity over both 5-HT2A and 5-HT2B receptors. Compounds such as 1, 2, 8, 12, 14 and 18 have potent oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function and their therapeutic potential is currently under further investigation.

The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C).

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

The effect of SB-236057-A, a selective 5-HT1B receptor inverse agonist, on in vivo extracellular 5-HT levels in the freely-moving guinea-pig.

5-HT1B autoreceptors are involved in the control of extracellular 5-HT levels from both the terminal and cell body regions of serotonergic neurones. In this study we report on the effect of a selective and potent 5-HT1B receptor inverse agonist, SB-236057-A (1'-ethyl-5-(2'-methyl-4'-(5-methyl- 1,3,4-oxadiazolyl-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydros piro [furo[2,3-f]indole-3,4' -piperidine] hydrochloride), on extracellular 5-HT levels in the cortex and dentate gyrus of the freely-moving guinea-pig, using the technique of in vivo microdialysis. SB-236057-A had ca. 23% bioavailability following oral drug administration. In vivo hypothermia pharmacodynamic assays demonstrated it was brain penetrant with a duration of action in excess of 18 h. SB-236057-A (0.75 mg/kg p.o.) increased extracellular 5-HT levels in the dentate gyrus to a maximum of 167+/-7% of basal but had no effect in the frontal cortex. However, a small increase in cortical 5-HT levels (117+11% of basal) was evident at 2.5 mg/kg p.o. In addition, SB-236057-A (0.75 mg/kg and 2.5 mg/kg p.o.) antagonised the sumatriptan-induced inhibition of extracellular 5-HT levels in the guinea-pig frontal cortex. These differences were attributed to MRN-innervated regions (e.g. dentate gyrus) being more responsive to 5-HT1B receptor-mediated negative feedback than DRN-innervated regions (e.g. frontal cortex). In the dentate gyrus, the increase in 5-HT release induced by SB-236057-A (0.75 mg/kg p.o.) was comparable to that after 14 days of paroxetine (10 mg/kg p.o.) administration, reaching a maximum of 183+/-13% of basal. These data suggest that acute 5-HT1B receptor blockade, by virtue of increased 5-HT release in the dentate gyrus, may provide a rapidly acting antidepressant.

Characterization of [(125)I]-SB-258585 binding to human recombinant and native 5-HT(6) receptors in rat, pig and human brain tissue.

SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzen esulphonamide) is a high affinity ligand at 5-HT(6) receptors. It displays over 100 fold selectivity for the 5-HT(6) receptor over all other 5-HT receptors tested so far. SB-258585 has been radiolabelled, to high specific activity, for its characterization as a 5-HT(6) receptor selective radioligand. [(125)I]-SB-258585 bound, with high affinity, to a single population of receptors in a cell line expressing human recombinant 5-HT(6) receptors. Kinetic and saturation binding experiments gave pK(D) values of 9.01+/-0.09 and 9.09+/-0.02, respectively. In membranes derived from rat or pig striatum and human caudate putamen, [(125)I]-SB-258585 labelled a single site with high levels (>60%) of specific binding. Saturation analysis revealed pK(D) values of 8.56+/-0.07 for rat, 8.60+/-0.10 for pig and 8.90+/-0.02 for human. B(max) values for the tissues ranged from 173+/-23 and 181+/-25 fmol mg(-1) protein in rat and pig striatum, respectively, to 215+/-41 fmol mg(-1) protein in human caudate putamen. The pK(i) rank order of potency for a number of compounds, determined in competition binding assays with [(125)I]-SB-258585, at human caudate putamen membranes was: SB-271046>SB-258585>SB-214111>methiothepin>clozapine>5-Me-OT>5-HT>Ro 04-6790>mianserin>ritanserin=amitriptyline>5-CT>mesulergine. Similar profiles were obtained from pig and rat striatal membranes and recombinant 5-HT(6) receptors; data from the latter correlated well with [(3)H]-LSD binding. Thus, [(125)I]-SB-258585 is a high affinity, selective radioligand which can be used to label both recombinant and native 5-HT(6) receptors and will facilitate further characterization of this receptor subtype in animal and human tissues.

Characterization of SB-271046: a potent, selective and orally active 5-HT(6) receptor antagonist.

SB-271046, potently displaced [(3)H]-LSD and [(125)I]-SB-258585 from human 5-HT(6) receptors recombinantly expressed in HeLa cells in vitro (pK(i) 8.92 and 9.09 respectively). SB-271046 also displaced [(125)I]-SB-258585 from human caudate putamen and rat and pig striatum membranes (pK(i) 8.81, 9.02 and 8.55 respectively). SB-271046 was over 200 fold selective for the 5-HT(6) receptor vs. 55 other receptors, binding sites and ion channels. In functional studies on human 5-HT(6) receptors SB-271046 competitively antagonized 5-HT-induced stimulation of adenylyl cyclase activity with a pA(2) of 8.71. SB-271046 produced an increase in seizure threshold over a wide-dose range in the rat maximal electroshock seizure threshold (MEST) test, with a minimum effective dose of < or =0.1 mg kg(-1) p.o. and maximum effect at 4 h post-dose. The level of anticonvulsant activity achieved correlated well with the blood concentrations of SB-271046 (EC(50) of 0.16 microM) and brain concentrations of 0.01-0.04 microM at C(max). These data, together with the observed anticonvulsant activity of other selective 5-HT(6) receptor antagonists, SB-258510 (10 mg kg(-1), 2-6 h pre-test) and Ro 04-6790 (1-30 mg kg(-1), 1 h pre-test), in the rat MEST test, suggest that the anticonvulsant properties of SB-271046 are likely to be mediated by 5-HT(6) receptors. Overall, these studies demonstrate that SB-271046 is a potent and selective 5-HT(6) receptor antagonist and is orally active in the rat MEST test. SB-271046 represents a valuable tool for evaluating the in vivo central function of 5-HT(6) receptors.

Acute and chronic administration of the selective D(3) receptor antagonist SB-277011-A alters activity of midbrain dopamine neurons in rats: an in vivo electrophysiological study.

This study examined the effect of acute and repeated p.o. administration of the selective D(3) receptor antagonist SmithKline Beecham (SB)-277011-A (1, 3, or 10 mg/kg) on the activity of spontaneously active midbrain dopamine (DA) neurons in anesthetized, male Sprague-Dawley rats. This was accomplished with the technique of in vivo extracellular single-unit recording. A single administration of either 3 or 10 mg/kg SB-277011-A produced a significant increase in the number of spontaneously active substantia nigra pars compacta (or A9) DA neurons compared with vehicle-treated (2% methylcellulose) animals. The 10-mg/kg dose of SB-277011-A produced a significant increase in the number of spontaneously active A10 DA neurons compared with vehicle-treated animals. The acute administration of SB-277011-A produced a significantly greater alteration in the firing pattern of spontaneously active A10 DA neurons, particularly at the 3- and 10-mg/kg doses, compared with vehicle-treated animals. The i.v. administration of SB-277011-A (0.01-1.28 mg/kg) did not significantly alter the firing rate or firing pattern of either A9 or A10 DA neurons. The repeated p.o. administration of 1, 3, or 10 mg/kg SB-277011-A once a day for 21 days produced a significant decrease in the number of spontaneously active A10 DA neurons. The repeated administration of SB-277011-A produced a greater effect on the firing pattern of spontaneously active A10 DA neurons, particularly at the 3-mg/kg dose, compared with A9 DA neurons. Overall, our results indicate that SB-277011-A alters the activity of midbrain DA neurons in rats.