Coupling of canine serotonin 5-HT(1B) and 5-HT(1D) receptor subtypes to the formation of inositol phosphates by dual interactions with endogenous G(i/o) and recombinant G(alpha15) proteins

Molecular cloning and expression of canine (ca) serotonin 5-HT(1B) and ca 5-HT(1D) receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT(1D) receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln(189) residue in the second extracellular loop of the ca 5-HT(1D) receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC(50) = 4.9 nM) the inositol phosphate formation at ca 5-HT(1D) receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 microM zolmitriptan) could be detected for the ca 5-HT(1B) receptor at a similar expression level. In contrast, both ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 microM zolmitriptan) upon co-expression with a mouse (m) G(alpha15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha15) protein-dependent ca 5-HT(1B) and ca 5-HT(1D) receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate betagamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha15) protein.     

Altered serotonin and dopamine metabolism in the CNS of serotonin 5-HT(1A) or 5-HT(1B) receptor knockout mice

Measurements of serotonin (5-HT), dopamine (DA), and noradrenaline, and of 5-HT and DA metabolites, were obtained by HPLC from 16 brain regions and the spinal cord of 5-HT(1A) or 5-HT(1B) knockout and wild-type mice of the 129/Sv strain. In 5-HT(1A) knockouts, 5-HT concentrations were unchanged throughout, but levels of 5-HT metabolites were higher than those of the wild type in dorsal/medial raphe nuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was taken as an indication of increased 5-HT turnover, reflecting an augmented basal activity of midbrain raphe neurons and consequent increase in their somatodendritic and axon terminal release of 5-HT. It provided a likely explanation for the increased anxious-like behavior observed in 5-HT(1A) knockout mice. Concomitant increases in DA content and/or DA turnover were interpreted as the result of a disinhibition of DA, whereas increases in noradrenaline concentration in some territories of projection of the locus coeruleus could reflect a diminished activity of its neurons. In 5-HT(1B) knockouts, 5-HT concentrations were lower than those of the wild type in nucleus accumbens, locus coeruleus, spinal cord, and probably also several other territories of 5-HT innervation. A decrease in DA, associated with increased DA turnover, was measured in nucleus accumbens. These changes in 5-HT and DA metabolism were consistent with the increased aggressiveness and the supersensitivity to cocaine reported in 5-HT(1B) knockout mice. Thus, markedly different alterations in CNS monoamine metabolism may contribute to the opposite behavioral phenotypes of these two knockouts.     

Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice

The role of serotonin (5-HT)1B receptors in the mechanism of action of selective serotonin re-uptake inhibitors (SSRI) was studied by using intracerebral in vivo microdialysis in conscious, freely moving wild-type and 5-HT1B receptor knockout (KO 5-HT1B) mice in order to compare the effects of chronic administration of paroxetine via osmotic minipumps (1 mg per kg per day for 14 days) on extracellular 5-HT levels ([5-HT]ext) in the medial prefrontal cortex and ventral hippocampus. Basal [5-HT]ext values in the medial prefrontal cortex and ventral hippocampus, approximately 20 h after removing the minipump, were not altered by chronic paroxetine treatment in both genotypes. On day 15, in the ventral hippocampus, an acute paroxetine challenge (1 mg/kg i.p.) induced a larger increase in [5-HT]ext in saline-pretreated mutant than in wild-type mice. This difference between the two genotypes in the effect of the paroxetine challenge persisted following chronic paroxetine treatment. Conversely, in the medial prefrontal cortex, the paroxetine challenge increased [5-HT]ext similarly in saline-pretreated mice of both genotypes. Such a challenge produced a further increase in cortical [5-HT]ext compared with that in saline-pretreated groups of both genotypes, but no differences were found between genotypes following chronic treatment. To avoid the interaction with raphe 5-HT1A autoreceptors, 1 micro m paroxetine was perfused locally through the dialysis probe implanted in the ventral hippocampus; similar increases in hippocampal [5-HT]ext were found in acutely or chronically treated wild-type mice. Systemic administration of the mixed 5-HT1B/1D receptor antagonist GR 127935 (4 mg/kg) in chronically treated wild-type mice potentiated the effect of a paroxetine challenge dose on [5-HT]ext in the ventral hippocampus, whereas systemic administration of the selective 5-HT1A receptor antagonist WAY 100635 did not. By using the zero net flux method of quantitative microdialysis in the medial prefrontal cortex and ventral hippocampus of wild-type and KO 5-HT1B mice, we found that basal [5-HT]ext and the extraction fraction of 5-HT were similar in the medial prefrontal cortex and ventral hippocampus of both genotypes, suggesting that no compensatory response to the constitutive deletion of the 5-HT1B receptor involving changes in 5-HT uptake capacity occurred in vivo. As steady-state brain concentrations of paroxetine at day 14 were similar in both genotypes, it is unlikely that differences in the effects of a paroxetine challenge on hippocampal [5-HT]ext are due to alterations of the drug's pharmacokinetic properties in mutants. These data suggest that there are differences between the ventral hippocampus and medial prefrontal cortex in activation of terminal 5-HT1B autoreceptors and their role in regulating dialysate 5-HT levels. These presynaptic receptors retain their capacity to limit 5-HT release mainly in the ventral hippocampus following chronic paroxetine treatment in mice.     

Biphasic alterations in serotonin-1B (5-HT1B) receptor function during abstinence from extended cocaine self-administration

Alterations in 5‐HT_1B receptor function during cocaine abstinence were evaluated in rats given either limited‐ or extended access (LA and EA, respectively) to cocaine self‐administration. The locomotor response to the 5‐HT_1B/1A agonist RU24969 was significantly reduced in cocaine‐experienced animals relative to cocaine‐naïve controls following 6 h of abstinence but became sensitized over the subsequent 14 days of abstinence. Both the early phase subsensitivity and later phase supersensivity to RU 24969‐induced activity were greater in EA versus LA animals. Intra‐nucleus accumbens administration of the 5‐HT_1B agonist CP 93, 129 produced significantly greater increases in dialysate dopamine levels in EA versus control animals following 14 days of abstinence. However, there was no difference between EA and cocaine‐naïve control animals in the augmentation of cocaine‐induced increases in nucleus accumbens DA produced by intra‐VTA CP 93, 129. Collectively these findings demonstrate that 5‐HT_1B receptor function is persistently altered by cocaine self‐administration.     

Molecular cloning and functional characterization of a human 5-HT1B serotonin receptor: a homologue of the rat 5-HT1B receptor with 5-HT1D-like pharmacological specificity.

We describe a genomic clone encoding the human 5-HT1B receptor. This apparently intronless gene encodes a 390 amino acid polypeptide homologous to the rat 5-HT1B serotonin receptor, with which it shares 93% amino acid sequence identity. Remarkably, [3H]5-hydroxytryptamine binding studies with transfected HeLa cells show that the human 5-HT1B receptor has a pharmacological profile that is markedly different from that of the corresponding rat receptor. Instead, human 5-HT1B drug specificity is highly similar to that of the human 5-HT1D receptor, with which it shares 59% amino acid sequence identity. The human 5-HT1B receptor, like the 5-HT1D receptor, can couple to Gi proteins. The presence of the threonine355 in the human receptor rather than an asparagine, as found in the corresponding rat gene product, may explain much of the marked pharmacological difference between the human and rat 5-HT1B receptors.     

An antagonistic 5-HT receptor system in the auricles of the systemic heart complex of Sepia officinalis L. (Cephalopoda) shows 5-HT1 and 5-HT4 subtype properties

In pharmacological bioassays on isolated ring-shaped auricle preparations of Sepia officinalis, the action of the specific 5-hydroxytryptamine (5-HT) agonists 8-OH-DPAT (5-HT1a), CP-93129 (5-HT1b), TFMPP (5-HT1b) and RS-67333 (5-HT4) on these autonomously contractile compartments was studied. 8-OH-DPAT and CP-93129 induced mainly positive effects on frequency and tone on the isotonically suspended auricles. The positive effect of 8-OH-DPAT on frequency was blocked by the specific 5-HT1a antagonist NAN-190. The 5-HT1b agonist TFMPP caused similar effects on tone and a positive impact on the auricular amplitude. The highly specific 5-HT4 agonist RS-67333 induced an effect opposite to the action of 5-HT1 agonists inducing mainly negative effects on frequency, amplitude and tone, causing a diastolic standstill at a concentration of 10(-6) M. These negative effects were blocked by the adenylyl cyclase inhibitor SQ-22,536 in the absence of a diastolic standstill. The opposing action of 5-HT1 and 5-HT4 agonists on auricular contractile activity suggests that an antagonistic 5-HT-receptor system exists within the auricular myocardial cells of S. officinalis, probably consisting of 5-HT1- and 5-HT4-like subtypes. The results also suggest that adenylyl cyclase acts as the intracellular target enzyme of both signal transduction mechanisms.     

The 5-HT(1A) receptor agonist 8-OH-DPAT prevents prefrontocortical glutamate and serotonin release in response to blockade of cortical NMDA receptors

We studied the role of 5-HT(1A) receptors in controlling the release of glutamate (GLU) in the medial prefrontal cortex (mPFC) of conscious rats with the in vivo microdialysis technique. The effect of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin infused in the prefrontal cortex was examined under basal conditions and on the rise of extracellular GLU (+106%) induced by co-infusion of the competitive N-methyl-d-aspartate receptor antagonist 3-[(R)-2-carboxypiperazin-4yl]-propyl-1-phosphonic acid (CPP). 8-OH-DPAT (0.3 and 3 microm) had no effect on basal extracellular GLU, but the higher concentration completely abolished the rise of extracellular GLU induced by CPP. CPP also increased extracellular serotonin (5-HT) in the mPFC (+50%) and this effect was antagonized by 3 microm 8-OH-DPAT which, by itself, had no effect on basal 5-HT release. The effects of 8-OH-DPAT on extracellular GLU and 5-HT were reversed by the 5-HT(1A) receptor antagonist WAY100 635 (100 microm), indicating a selective involvement of 5-HT(1A) receptors. WAY100 635 had no effect by itself. These results show that the stimulation of cortical 5-HT(1A) receptors prevents the CPP-evoked rise of extracellular GLU and 5-HT and suggest that these effects may contribute to the ability of intracortical 8-OH-DPAT to counteract cognitive deficits caused by the blockade of NMDA receptors.     

SB-656104-A: a novel 5-HT(7) receptor antagonist with improved in vivo properties

A focused SAR study around the previously reported selective 5-HT(7) receptor antagonist, SB-269970-A has resulted in the identification of a structurally related analogue having an improved pharmacokinetic profile. Replacement of the phenolic group in SB-269970-A with an indole moiety, and replacement of the piperidinyl 4-methyl group with a heterocyclic ring system proved to be the key changes leading to the identification of SB-656104-A.     

Immunological properties of trehalose dimycolate (cord factor) and other mycolic acid-containing glycolipids--a review

Mycolic acids are characteristic fatty acids of Mycobacteria and are responsible for the wax-like consistence of these microorganisms. Decades of research revealed that mycolic acid-containing glycolipids, in particular trehalose-6,6'-dimycolate (TDM, cord factor) as their best-studied representative, exert a number of immunomodifying effects. They are able to stimulate innate, early adaptive and both humoral and cellular adaptive immunity. Most functions can be associated with their ability to induce a wide range of chemokines (MCP-1, MIP-1alpha, IL-8) and cytokines (e.g., IL-12, IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10). This review tries to link well-known properties of mycolic acid-containing glycolipids, e.g., stimulation of cellular and humoral immunity, granuloma formation and anti-tumor activity, with recent findings in molecular immunology and to give an outlook on potential practical applications.     

5-HT(1A) receptor mutant mice exhibit enhanced tonic, stress-induced and fluoxetine-induced serotonergic neurotransmission

Mutant mice that lack serotonin(1A) receptors exhibit enhanced anxiety-related behaviors, a phenotype that is hypothesized to result from impaired autoinhibitory control of midbrain serotonergic neuronal firing. Here we examined the impact of serotonin(1A) receptor deletion on forebrain serotonin neurotransmission using in vivo microdialysis in the frontal cortex and ventral hippocampus of serotonin(1A) receptor mutant and wild-type mice. Baseline dialysate serotonin levels were significantly elevated in mutant animals as compared with wild-types both in frontal cortex (mutant = 0.44 +/- 0.05 n M; wild-type = 0.28 +/- 0.03 n M) and hippocampus (mutant = 0.46 +/- 0.07 n M; wild-type = 0.27 +/- 0.04 n M). A stressor known to elicit enhanced anxiety-like behaviors in serotonin(1A) receptor mutants increased dialysate 5-HT levels in the frontal cortex of mutant mice by 144% while producing no alteration in cortical 5-HT in wild-type mice. There was no phenotypic difference in the effect of this stressor on serotonin levels in the hippocampus. Fluoxetine produced significantly greater increases in dialysate 5-HT content in serotonin(1A) receptor mutants as compared with wild-types, with two- and three-fold greater responses being observed in the hippocampus and frontal cortex, respectively. This phenotypic effect was mimicked in wild-types by pretreatment with the serotonin(1A) antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (p-MPPI). These results indicate that deletion of central serotonin(1A) receptors results in a tonic disinhibition of central serotonin neurotransmission, with a greater dysregulation of serotonin release in the frontal cortex than ventral hippocampus under conditions of stress or increased interstitial serotonin levels.     

Quantitative assay of 5-HT(1A) serotonin receptor gene expression in the brain

Serotonin 5-HT(1A) receptors participate in the regulation of many kinds of behavior and are implicated in the mechanism of action of anxiolitics and antidepressants. The investigation of 5-HT(1A) receptor gene expression is complicated by low concentration of the receptor mRNA. Our method of quantification of the receptor gene expression in brain structures includes estimation of the concentration of genomic DNA contamination, the number of cDNA copies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)--one of the "housekeeping genes", and the number of cDNA copies of 5-HT(1A) receptor in the sample. To evaluate the number of cDNA copies of the receptor and GAPDH, the fluorescence intensity of PCR-product was calibrated using genomic DNA-standard of a known concentration. The intensity of 5-HT(1A) receptor gene expression was corrected by genomic DNA contamination and was evaluated as a number of copies of 5-HT(1A) receptor cDNA per 100 copies of GAPDH cDNA. Using this method an increase of 5-HT(1A) receptor gene expression in the frontal cortex and amygdala in monoamine oxidase A knockout mice was shown.     

5-HT(1B) receptor-mediated regulation of serotonin clearance in rat hippocampus in vivo

The 5-hydroxytryptamine (5-HT; serotonin) transporter (5-HTT) is important in terminating serotonergic neurotransmission and is a primary target for many psychotherapeutic drugs. Study of the regulation of 5-HTT activity is therefore important in understanding the control of serotonergic neurotransmission. Using high-speed chronoamperometry, we have demonstrated that local application of 5-HT(1B) antagonists into the CA3 region of the hippocampus prolongs the clearance of 5-HT from extracellular fluid (ECF). In the present study, we demonstrate that the 5-HT(1B) antagonist cyanopindolol does not produce this effect by increasing release of endogenous 5-HT or by directly binding to the 5-HTT. Dose-response studies showed that the potency of cyanopindolol to inhibit clearance of 5-HT was equivalent to that of the selective 5-HT reuptake inhibitor fluvoxamine. Local application of the 5-HT(1A) antagonist WAY 100635 did not alter 5-HT clearance, suggesting that the effect of cyanopindolol to prolong clearance is not via a mechanism involving 5-HT(1A) receptors. Finally, the effect of low doses of cyanopindolol and fluvoxamine to inhibit clearance of 5-HT from ECF was additive. These data are consistent with the hypothesis that activation of terminal 5-HT(1B) autoreceptors increases 5-HTT activity.     

Anti-depressant-like activity of a novel serotonin type-3 (5-HT3) receptor antagonist in rodent models of depression

N-Cyclohexyl-3-methoxyquinoxalin-2-carboxamide (QCM-13), a novel 5-HT3 antagonist identified from a series of compounds with higher pA2 (7.6) and good log P (2.91) value was screened in rodent models of depression such as forced swim test (FST), tail suspension test (TST), interaction studies with standard anti-depressants and confirmatory studies such as reversal of parthenolide induced depression and reserpine induced hypothermia. In FST (2 and 4 mg/kg) and TST (2 and 4 mg/kg), QCM-13 significantly reduced the duration of immobility in mice without affecting the base line locomotion. QCM-13 (2 and 4 mg/kg) was also found to have significant interaction with standard anti-depressants (fluoxetine and bupropion in FST and TST respectively). Further, reversal of parthenolide induced depression in mice and reserpine induced hypothermia in rat models indicate the serotonergic influence of QCM-13 for anti-depressant potential.     

Ketanserin, an antagonist of 5-HT2A receptor of serotonin, inhibits testosterone secretion by rat Leydig cells in vitro.

The effect of ketanserin, an antagonist of 5-HT2A receptor of serotonin, added to the culture medium, on basal and LH-stimulated testosterone secretion was studied in primary cultures of adolescent rat Leydig cells. Ketanserin decreased the basal secretion of testosterone but showed an insignificant influence on the LH-stimulated process. It can be concluded that ketanserin may affect the testosterone-secreting cells by an indirect action at the vascular level as well as directly at the level of Leydig cells, at least in adolescent rats, leading to down-regulation of the basal testosterone secretion.     

Association analysis of the catechol-o-methyltransferase (COMT), serotonin transporter (5-HTT) and serotonin 2A receptor (5HT2A) gene polymorphisms with obsessive-compulsive disorder

Family and twin studies have supported a strong genetic factor in the etiology of obsessive-compulsive disorder (OCD), although the precise mechanism of inheritance is unclear. Clinical and pharmacological studies have implicated the serotonergic and dopaminergic systems in disease pathogenesis. In this cross-sectional study, we have examined the allelic and genotypic frequencies of a Val-158-Met substitution in the COMT gene, a 44-base pair (bp) length variation in the regulatory region of the serotonin transporter gene ( 5-HTTLPR ) and the T102C and C516T variants in the serotonin receptor type 2A ( 5HT2A ) gene in 79 OCD patients and 202 control subjects. There were no observed differences in the frequencies of allele and genotype between patients and control groups for the COMT , the 5HTTLPR and the T102C 5HT2A gene polymorphisms. In contrast, a statistically significant difference between OCD patients and controls was observed on the genotypic distribution (χ² = 16.7, 2df, P  = 0.0002) and on the allelic frequencies (χ² = 15.8, 1df, P  = 0.00007) for the C516T 5HT2A gene polymorphism. The results suggest that the C516T variant of the 5HT2A gene may be one of the genetic risk factors for OCD in our sample. However, further studies using larger samples and family based methods are recommended to confirm these findings.     

The serotonin receptor SER-1 (5HT2ce) contributes to the regulation of locomotion in Caenorhabditis elegans

Serotonin (5-hydroxytryptamine: 5HT) is an important neuroactive substance in the model roundworm, Caenorhabditis elegans. Aside from having effects in feeding and egg-laying, 5HT inhibits motility and also modulates several locomotory behaviors, notably food-induced slowing and foraging. Recent evidence showed that a serotonergic 5HT2-like receptor named SER-1 (also known as 5HT2ce) was responsible for the effect of 5HT on egg-laying. Here we confirm this observation and show that SER-1 also plays an important role in locomotion. A mutant lacking SER-1 was found to be highly resistant to exogenous 5HT in the absence of food and this resistant phenotype was rescued by reintroducing the SER-1 gene in a mutant background. Pharmacological studies showed that the same antagonists that blocked the activity of recombinant SER-1 in vitro also inhibited the effect of 5HT on motility, suggesting the same receptor was responsible for both effects. When tested for locomotory behaviors, the SER-1 mutant was found to be moderately defective in food-induced slowing. In addition, the mutant changed direction more frequently than the wildtype when searching for food, suggesting that SER-1 may play a role in navigational control during foraging. Both these effects required the presence of MOD-1, a 5HT gated chloride channel, and the results indicate that SER-1 and MOD-1 modulate these behaviors through a common pathway. On the basis of expression analysis of a ser-1::GFP translational fusion, SER-1 is prominently located in central, integrating neurons of the head ganglia (RIA and RIC) but not the body wall musculature. The evidence suggests that SER-1 controls locomotion through indirect modulation of neuromuscular circuits and has effects both on speed and direction of movement.     

Selective 5-HT1B receptor agonist reduces serotonin synthesis following acute, and not chronic, drug administration: results of an autoradiographic study

The effects of acute and chronic administration of the serotonin (5-HT)1B agonist CP-93,129, on 5-HT synthesis rates were evaluated using the alpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method. In the acute treatment study, CP-93,129 (7 mg/kg) was injected intraperitoneally 30 min before the alpha-MTrp injection (30 microCi over 2 min). A single dose of CP-93,129 caused a significant increase in the synthesis in the median raphe nucleus (MR) without a significant influence on the dorsal raphe nucleus (DR). There was a reduction in 5-HT synthesis in almost all of the projection areas. In the chronic treatment study, CP-93,129 was administered continuously (7 mg/kg/day) for 14 days using an osmotic minipump implanted subcutaneously. The chronic treatment with CP-93,129 did not produce a significant change in 5-HT synthesis in the raphe nuclei nor in the nerve terminal structures, except for the medial frontal bundle and the visual and sensory-motor cortices. The unaltered 5-HT synthesis rates in the chronic treatment study probably reflect a normalization of the synthesis as a result of the desensitization of 5-HT1B autoreceptors and/or heteroreceptors.     

Synthesis and activity of functionalizable derivatives of the serotonin (5-HT) 5-HT2A receptor (5-HT2AR) antagonist M100907

The approach of tethering together two known receptor ligands, to be used as molecular probes for the study of G protein-coupled receptor (GPCR) systems, has proven to be a valuable approach. Selective ligands that possess functionality that can be used to link to other ligands, are useful in the development of novel antagonists and agonists. Such molecules can also be attached to reporter molecules, such as fluorophores, for the study of GPCR dimerization and its role in signaling. The highly selective serotonin (5-HT) 5-HT_2A receptor (5-HT_2AR) antagonist M100907 (volinanserin) is of clinical interest in the treatment of neurological and mental health disorders. Here, we synthesized the most active (+)-M100907 enantiomer as well as a series of derivatives that possessed either an alkyne or an azide. The triazole resulting from the dipolar cycloaddition of these groups did not interfere with the ability of the bivalent ligand to act as an antagonist. Thus, we have synthesized a number of compounds which will prove useful in elucidating the role of the 5-HT_2AR in the central nervous system.