Impairment of signal transduction in response to stimulation of the naturally occurring Pro279Leu variant of the h5-HT7(a) receptor

The aim of this study, performed in stably transfected HEK293 cells, was to investigate whether expression of the naturally occurring Pro279Leu variant of the h5-HT7(a) receptor (located in the third intracellular loop) is associated with changes in the pharmacological properties and/or second messenger formation compared to the wild-type receptor. Radioligand binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulation of [3H]cAMP formation in whole cells evoked by 5-HT receptor agonists were determined. Maximum binding (B(max)) to, and affinity (K(D)) of [3H]5-CT for, the variant receptor and the wild-type receptor were equal. All agonists and antagonists investigated exhibited no differences in affinity between the variant receptor and the wild-type receptor. However, the intrinsic activity of the 5-HT receptor agonists 5-HT, 5-CT, RU24969 and 8-OH-DPAT in stimulating [3H]cAMP accumulation in the cells expressing the Pro279Leu variant was almost abolished and their potency was 2.9-4.3-fold lower. Despite its affinity for both receptor isoforms, sumatriptan did not stimulate the accumulation of cAMP. In individuals expressing the Pro279Leu variant of the h5-HT7(a) receptor, a considerable attenuation of its function may be predicted. This may have relevance for the action of new classes of drugs which affect circadian rhythm or psychiatric diseases, such as schizophrenia.     

Inactivation of 5-HT(1A) receptors in hippocampal and cortical homogenates

5-HT(1A) receptor function can be assessed in rat hippocampal and cortical membrane preparations as agonist-stimulated [35S]GTPgammaS binding. Membranes were preincubated in vitro with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT]-stimulated [35S]GTPgammaS binding and [3H]8-OH-DPAT binding assays were used to assess 5-HT(1A) receptor function and density, respectively. EEDQ decreased both R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding in hippocampal and cortical membranes. The E(max) but not the EC(50) of R(+)-8-OH-DPAT to stimulate [35S]GTPgammaS binding was decreased by EEDQ in both preparations. Additionally, the IC(50) for EEDQ to reduce R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding was the same for both brain regions in both assays. In contrast to EEDQ alone, agonist-stimulated [35S]GTPgammaS binding was not reduced in hippocampal membranes preincubated with EEDQ and the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl- cyclohexanecarboxamide maleate (WAY 100,635), suggesting that EEDQ acts directly on the receptor. Due to parallel reductions in receptor density and maximal functional response, it is concluded that there is little or no reserve for 5-HT(1A) receptor coupling to G(alpha) in these preparations. In addition, the sensitivity of hippocampal and cortical 5-HT(1A) receptors to inactivation by EEDQ in vitro is the same.     

5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

1. It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT(1A) receptors. 2. Saturation studies using [(3)H]-8-OH-DPAT and [(3)H]-MPPF revealed a single, high affinity site (K(D)approximately 1 nM) in HEK293 cells expressing human 5-HT(1A) receptors and rat cortex. In recombinant cells, [(3)H]-MPPF labelled 3 - 4 fold more sites than [(3)H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [(3)H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT(1A) receptors but did not bind to native tissue 5-HT(1A) receptors. These data suggest that, in transfected HEK293 cells, human 5-HT(1A) receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. 3. Receptor agonists inhibited [(3)H]-MPPF binding from recombinant 5-HT(1A) receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [(3)H]-8-OH-DPAT and [(3)H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [(3)H]-MPPF and [(3)H]-8-OH-DPAT in a monophasic manner. 4. Functional evaluation of compounds, using [(35)S]-GTPgammaS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. 5. [(3)H]-8-OH-DPAT : [(3)H]-MPPF pK(i) difference correlated well with functional intrinsic activity (r=0.86) as did [(3)H]-8-OH-DPAT : [(3)H]-spiperone pK(i) difference with functional intrinsic activity (r=0.96). 6. Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT(1A) receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT(1A) receptors in which only the high agonist affinity state was detectable.     

Chloroamphetamine did not prevent the effects of chronic antidepressants on 5-hydroxytryptamine inhibition of forskolin-stimulated adenylate cyclase in rat hippocampus.

Administration of p-chloroamphetamine (PCA, 10 mg/kg i.p. on two occasions) to rats resulted in a severe depletion of [3H]paroxetine binding sites, a measure of presynaptic serotonergic terminals, in both cortex and hippocampus, but did not affect [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT) binding or 5-hydroxytryptamine (5-HT)-induced inhibition of forskolin-stimulated adenylate cyclase in hippocampal membranes. Administration of either imipramine (15 mg/kg i.p. for 2 weeks) or lithium (0.2% for 2 weeks) to PCA-treated rats did not affect [3H]8-OH-DPAT binding but reduced the degree of inhibition of forskolin-stimulated adenylate cyclase by 5-HT in hippocampal membranes. It is concluded that the effects of imipramine and Li+ on 5-HT1A receptor-mediated responses in the hippocampus are exerted postsynaptically, possibly at a level distal to the receptor.     

Differential modulation by GTPgammaS of agonist and inverse agonist binding to h5-HT(1A) receptors revealed by [3H]-WAY100,635

1. The interaction of serotonergic ligands at human (h) 5-HT(1A) receptors expressed in Chinese hamster ovary cells was examined with the selective 'neutral' 5-HT(1A) antagonist [(3)H]-WAY100,635. Its binding was saturable (K(D)=0.056 nM) with a B(max) (3.65 pmol mg(-1)) significantly higher than that of two other selective 5-HT(1A) radioligands: the partial agonist, [(3)H]-S15535 (2.77 pmol mg(-1)) and the agonist, [(3)H]-8-OH-DPAT (2.02 pmol mg(-1)). 2. The influence of GTPgammaS (100 microM) on the binding affinity of 15 serotonergic agonists, partial agonists, antagonists and inverse agonists was investigated in competition binding experiments with [(3)H]-WAY100,635. 3. Agonists, including 5-HT, 8-OH-DPAT and buspirone, displayed biphasic isotherms which shifted to the right in the presence of GTPgammaS. In contrast, isotherms of the inverse agonists, methiothepin, (+)butaclamol and spiperone, were shifted to the left in the presence of GTPgammaS. Unlabelled WAY100,635 was the only ligand that was unaffected by GTPgammaS, consistent with 'neutral' antagonist properties. 4. The magnitude of affinity changes induced by GTPgammaS for 13 ligands was highly correlated (r = 0.98) with their efficacy (positive and negative) previously determined by [(35)S]GTPgammaS binding. 5. In contrast, the napthylpiperazine derivative and high efficacy agonist, S14506, displayed only a modest GTPgammaS shift, in accordance with previous indications of 'atypical' binding properties of this ligand. A further full agonist, S14671, which is chemically closely-related to S14506, also displayed a minimal GTPgammaS shift, underpinning this observation. 6. In conclusion, [(3)H]-WAY100,635 constitutes a useful neutral antagonist radioligand for the characterization of drug actions at h5-HT(1A) receptors. GTPgammaS-induced affinity changes of agonist and inverse agonist competition isotherms generally correlate well with ligand efficacy, with the notable exception of two chemically-similar agents, S14506 and S14671, which are efficacious agonists, yet relatively insensitive to h5-HT(1A) receptor/G-protein coupling changes.     

Native rat hippocampal 5-HT1A receptors show constitutive activity

Previous studies have shown that human 5-hydroxytryptamine (5-HT)1A receptors stably expressed in transfected cell lines show constitutive G-protein activity, as revealed by the inhibitory effect of inverse agonists, such as spiperone, on basal guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding. In the present study, we evaluated the constitutive activity of native rat 5-HT1A receptors in hippocampal membranes. Using anti-Galphao-antibody capture coupled to scintillation proximity assay under low sodium (30 mM) conditions, we observed high basal [35S]GTPgammaS binding to Galphao subunits (defined as 100%). Under these conditions, 5-HT and the prototypic selective 5-HT1A agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] both stimulated [35S]GTPgammaS binding to Galphao to a similar extent, raising binding to approximately 130% of basal with pEC50 values of 7.91 and 7.87, respectively. The 5-HT1A-selective neutral antagonist [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with pKb values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its pKi value at r5-HT1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [35S]GTPgammaS binding to Galphao in a concentration-dependent manner to 90% of basal with pIC50 values of 7.37 and 7.98, respectively. The inhibition of basal [35S]GTPgammaS binding induced by maximally effective concentrations of spiperone (10 microM) or methiothepin (1 microM) was antagonized by WAY100,635 in a concentration-dependent manner (pKb, 9.52 and 8.87, respectively), thus indicating that this inverse agonism was mediated by 5-HT1A receptors. These data provide the first demonstration that native rat serotonin 5-HT1A receptors can exhibit constitutive activity in vitro.     

Pharmacological properties of the naturally occurring Phe-124-Cys variant of the human 5-HT1B receptor: changes in ligand binding, G-protein coupling and second messenger formation

The aim of this study was to analyse whether substitution of phenylalanine in position 124 of the human (h) 5-HT1B receptor by cysteine, a naturally occurring variant of this receptor, modifies not only ligand binding, but also G-protein coupling and second messenger formation. Stably transfected rat C6 glioma cells, which express either the h5-HT1B variant receptor (VR) or the wild-type receptor (WTR) were used. In saturation experiments with [3H]5-carboxamidotryptamine ([3H]5-CT), the maximum binding (Bmax) of the VR amounted to only 60% of that to WTR. In competition experiments with 1 nM [3H]5-CT, the following 5-HT receptor ligands exhibited a higher affinity for the mutant receptor than for the WTR: L-694,247, 5-CT, 5-HT, sumatriptan (agonists listed at decreasing order of potency) and SB-224289 (a selective h5-HT1B receptor inverse agonist with competitive antagonistic properties). In contrast, the mixed 5-HT1B/1D receptor antagonist GR-127935 exhibited equal affinity for both isoforms. The efficacy of L-694,247, 5-CT, 5-HT and sumatriptan in stimulating [35S]GTPgammaS binding (a measure of G protein coupling) to membranes of cells expressing the VR was approximately 50-65% lower compared to membranes of cells expressing the WTR, but their potency was 2.8-3.6-fold higher. SB-224289, which decreased [35S]GTPgammaS binding when given alone, but not GR-127935, was more potent in antagonizing the stimulatory effect of 5-CT on [35S]GTPgammaS binding to membranes expressing the VR compared to membranes expressing the WTR. In whole cells expressing the VR, 5-CT and sumatriptan inhibited the forskolin-stimulated cAMP accumulation 3.2-fold more potently than in cells expressing the WTR. In conclusion, our data suggest that the Phe-124-Cys mutation modifies the pharmacological properties of the h5-HT1B receptor and may account for pharmacogenetic differences in the action of h5-HT1B receptor ligands. Thus, the sumatriptan-induced vasospasm which occurs at low incidence as a side-effect in migraine therapy may be related to the expression of the (124-Cys)h5-HT1B receptor in patients with additional pathogenetic factors such as coronary heart disease.     

5-HT1A receptor-mediated G protein activation assessed by [35S]GTPgammaS binding in rat cerebral cortex

To date, 5-hydroxytryptamine1A (5-HT1A) receptor-mediated functional assays (adenylyl cyclase inhibition, high-affinity GTPase activity and [35S]guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding) have been performed mainly in hippocampal membranes. In the current study, 5-HT-stimulated G protein activation assays were carried out in rat cerebral cortical membranes. High-affinity GTPase activity was stimulated by 5-HT, but not by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). By contrast, 5-HT- and 8-OH-DPAT-stimulated [35S]GTPgammaS binding displayed sufficient dynamic range enough to warrant further pharmacological analysis. Under standard conditions, which were determined precisely in terms of the concentrations of GDP, MgCl2 and NaCl, the profile of 5-HT-stimulated [35S]GTPgammaS binding investigated using a series of 5-HT receptor agonists and antagonists clearly indicated the involvement of the 5-HT1A receptor subtype. There appeared to be no evidence supporting the presence of regional heterogeneity in coupling efficiency between 5-HT1A and G proteins in the hippocampus or cortex. This method is a useful tool for investigating functional coupling between postsynaptic 5-HT1A receptors and G proteins in cerebral cortical membranes.     

Chronic administration of venlafaxine fails to attenuate 5-HT1A receptor function at the level of receptor-G protein interaction

In this study venlafaxine was administered to rats at a low, moderate or high dose; for comparison, the selective serotonin reuptake inhibitor (SSRI) sertraline and the tricyclic antidepressant (TCA) amitriptyline were also included. We evaluated, using quantitative autoradiography, the effect of these antidepressant treatments on [35S]GTPgammaS binding stimulated by the 5-HT1A receptor agonist 8-OH-DPAT, a measure of the capacity of 5-HT1A receptors to activate G proteins. Chronic administration of amitriptyline resulted in a marked increase in 5-HT1A receptor-stimulated [35S]GTPgammaS binding in the hippocampus which was accompanied by an increase in 5-HT1A receptor number. 5-HT1A receptor-stimulated [35S]GTPgammaS binding in the hippocampus was also increased by chronic treatment with the highest dose of venlafaxine; 5-HT1A receptor number, however, was not significantly altered. In serotonergic cell body areas (i.e. dorsal and median raphe nuclei), 5-HT1A receptor-stimulated [35S]GTPgammaS binding was not altered by chronic administration of amitriptyline, sertraline or venlafaxine. Chronic TCA treatment does not desensitize somatodendritic 5-HT1A autoreceptor function. However, the lack of effect of chronic sertraline treatment on 5-HT1A receptor-stimulated [35S]GTPgammaS binding is in contrast to what has been observed previously following chronic administration of the SSRI fluoxetine, and suggests that different SSRIs may regulate somatodendritic 5-HT1A autoreceptor function differently depending on their pharmacology. Our data also suggest that the desensitization of somatodendritic 5-HT1A autoreceptors observed in electrophysiological studies following chronic venlafaxine administration is not at the level of receptor-G protein interaction. The hypothermic response in vivo to acute injection of 8-OH-DPAT was significantly attenuated following chronic treatment with venlafaxine or sertraline, but not amitriptyline.     

Regulation of the avidity of ternary complexes containing the human 5-HT(1A) receptor by mutation of a receptor contact site on the interacting G protein alpha subunit

1 Fusion proteins were constructed between the human 5-HT(1A) receptor and pertussis toxin-resistant forms of both G(i1)alpha and G(o1)alpha mutated at residue(351) from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. 2 Increasing concentrations of GDP inhibited binding of the agonist [(3)H]-8-OH-DPAT but not the antagonist [(3)H]-MPPF to each construct. 3 The IC(50) for GDP was greater for constructs containing isoleucine at residue(351) of the G proteins compared to those with glycine at this position. 4 The G protein antagonist suramin had similar effects to GDP on the binding of [(3)H]-8-OH-DPAT. 5 The proportion of 5-HT(1A) receptor binding sites detected by [(3)H]-MPPF that displayed high affinity for 8-OH-DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue(351). 6 The 5-HT(1A) receptor displayed similar avidity of interaction with G(i1)alpha and G(o1)alpha. 7 These results indicate that a higher avidity ternary complex is formed between 8-OH-DPAT, the 5-HT(1A) receptor and G proteins when isoleucine rather than glycine is located at residue(351) of the interacting G protein.     

Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists.

The density of 5-hydroxytryptamine (5-HT)1B receptors and their coupling to the inhibition of cAMP accumulation were investigated in opossum kidney cells maintained in culture. The density and properties of the receptor were determined using [125I] iodocyanopindolol as the radioligand. The pharmacological specificity of the binding site was consistent with that expected for a 5-HT1B receptor. Serotonin inhibited forskolin-stimulated cAMP accumulation with an EC50 of 4-8 nM. Compounds known to show selectivity at the 5-HT1B receptor, such as trifluoromethyl-phenylpiperazine and CGS-12066B, also inhibited forskolin-stimulated cAMP accumulation, acting as full agonists with efficacies comparable to that of serotonin. Other beta-adrenergic receptor antagonists, including (-)-pindolol and (-)-alprenolol, bound to the receptor with high affinity and acted as partial agonists capable of inhibiting forskolin-stimulated cAMP accumulation. Exposure of cells to 5-HT resulted in a time- and dose-dependent decrease in the density of 5-HT1B receptors that was not accompanied by a change in the Kd of the binding site for [125I] iodocyanopindolol. A maximum decrease of 60% in the number of 5-HT1B receptors was evident after a 16-hr treatment with 1 microM 5-HT. Concomitant with the observed decrease in the density of receptors was a marked increase in the EC50 for 5-HT-mediated inhibition of forskolin-stimulated cAMP accumulation. The EC50 was increased 4-5-fold after a 16-hr exposure to 1 microM 5-HT, and the maximal level of inhibition was markedly decreased. Whereas pretreatment with moderate concentrations of 5-HT (100-300 nM) for 16 hr produced significant decreases in the density of 5-HT1B receptors and increases in the EC50 for inhibition of forskolin-stimulated cAMP formation, there was little change in the maximal level of inhibition that could be attained. Such a combination of changes could be explained by the presence of "spare" 5-HT1B receptors on these cells.     

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes.

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.     

Pindolol antagonises G-protein activation at both pre- and postsynaptic serotonin 5-HT1A receptors: a [35S]GTPγS autoradiography study

The arylalkylamine, pindolol, may potentiate the clinical actions of antidepressant agents. Although it is thought to act via blockade of 5-HT1A autoreceptors, its efficacy at these sites remains controversial. Herein, we evaluated the actions of pindolol at 5-HT1A autoreceptors and specific populations of postsynaptic 5-HT1A receptors employing [35S]GTPgammaS autoradiography, a measure of receptor-mediated G-protein activation. Both 8-OH-DPAT (1 microM) and 5-HT (10 microM) elicited a pronounced increase in [35S]GTPyS binding in the dorsal raphe nucleus, which contains serotonergic cell bodies bearing 5-HT1A autoreceptors. Pindolol abolished their actions. In the dentate gyrus, lateral septum and entorhinal cortex, structures enriched in postsynaptic 5-HT1A receptors, 8-OH-DPAT (1 microM) and 5-HT (10 microM) also elicited a marked increase in [35S]GTPgammaS binding which was likewise blocked by pindolol. The antagonism of 5-HT-induced [35S]GTPgammaS labelling in the dentate gyrus was shown to be concentration-dependent, yielding a pIC50 of 5.82. Pindolol did not, itself, affect [35S]GTPgammaS binding in any brain region examined. In conclusion, these data suggest that, as characterised by [35S]GTPgammaS autoradiography, and compared with 5-HT and 8-OH-DPAT, pindolol possesses low efficacy at both pre- and postsynaptic 5-HT1A receptors.     

Region-specific changes in 5-HT(1A) receptor-activated G-proteins in rat brain following chronic buspirone

5-Hydroxytryptamine(1A) (5-HT(1A)) receptors, which activate inhibitory G-proteins, are implicated in psychiatric disorders including anxiety and depression. Studies suggest that chronic 5-HT(1A) receptor agonist administration alters 5-HT(1A) receptor function, but the effect of chronic treatment on 5-HT(1A) receptor-activated G-proteins is unclear. In this study, agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding was examined following chronic administration of buspirone. Brains were processed for [35S]GTPgammaS autoradiography using R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) for 5-HT(1A) receptors or baclofen for GABA(B) receptors. Net 8-OH-DPAT-stimulated [35S]GTPgammaS binding was decreased by 25-30% in the septum and dorsal raphe nucleus of buspirone-treated animals. No significant changes in 8-OH-DPAT-stimulated [35S]GTPgammaS binding were found in the prefrontal, entorhinal or cingulate cortices or hippocampus in buspirone-treated rats. GABA(B) receptor-stimulated [35S]GTPgammaS binding was increased by 25% in the hippocampus, with no significant changes in any other region examined. These results demonstrate region-specific alterations in 5-HT(1A) and GABA(B) receptor-activated G-proteins following chronic buspirone treatment, which may contribute to the clinical effects of this drug.     

Pharmacology of polymorphic variants of the human 5-HT1A receptor

The 5-HT1A receptor is a critical mediator of serotonergic (5-HT) function. We have identified 13 potential single nucleotide polymorphisms resulting in amino acid changes throughout the human 5-HT1A receptor. The pharmacological profiles of these 13 polymorphic variants were then characterized using a high-throughput assay based on ligand-dependent transformation of NIH/3T3 cells. The majority of the polymorphic variants displayed wild-type pharmacological profiles in response to a panel of well-established agonists at the 5-HT1A receptor. However, the A50V polymorphic variant, which had an alanine to valine substitution in transmembrane 1, exhibited a loss of detectable response to 5-HT. Interestingly, all other agonists tested, including buspirone, lisuride, and (+)8-OH-DPAT, exhibited efficacies similar to that of the wild-type receptor. The competitive antagonist, methiothepin, also displayed a 19-fold decrease in potency at the A50V variant receptor. However, both 5-HT and methiothepin were able to compete for [3H]WAY-100635 binding to the A50V variant with affinities similar to the wild-type receptor. Moreover, the Bmax of [3H]WAY-100635 binding was 14-fold lower for the A50V variant than for the wild-type receptor. Thus, the A50V receptor variant exhibited ligand-specific functional alterations in addition to lower expression levels. These data suggest a previously unappreciated role for transmembrane 1 in mediating 5-HT response at the 5-HT1A receptor. Furthermore, individuals that potentially harbor the A50V polymorphism might display aberrant affective behaviors and altered responses to drugs targeting the 5-HT1A receptor.     

Lack of apparent receptor reserve at postsynaptic 5-hydroxytryptamine1A receptors negatively coupled to adenylyl cyclase activity in rat hippocampal membranes.

Previous studies have demonstrated the existence of a large receptor reserve for agonists at somatodendritic 5-hydroxytryptamine1A (5-HT1A) serotonin receptors in the raphe nuclei of the rat. 5-HT1A agonists with anxiolytic properties (e.g., buspirone, gepirone, and ipsapirone) display full intrinsic activity at these receptors but are partial agonists at postsynaptic 5-HT1A receptors, which suggests that the latter sites may be devoid of a receptor reserve. In the present studies, this was directly determined by examining the relationship between receptor occupancy and response at postsynaptic 5-HT1A receptors, in rat hippocampus, mediating the inhibition of forskolin-stimulated adenylyl cyclase activity, using the method of partial irreversible receptor inactivation. Rats were treated with vehicle or the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), and 24 hr later hippocampi were removed for saturation analysis of [3H]8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) binding to 5-HT1A receptors or for adenylyl cyclase assays. EEDQ (1 and 6 mg/kg) dose-dependently reduced the maximal number of [3H]8-OH-DPAT binding sites by 68.5 and 80%, respectively, without altering the Kd. Concentration-response curves were generated for inhibition of forskolin-stimulated adenylyl cyclase activity by 5-HT and the selective 5-HT1A agonist N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT). EEDQ treatment dose-dependently reduced the maximal inhibitory effect of 5-HT [percentage of inhibition: control, 23.6; EEDQ (1 mg/kg), 13.4; EEDQ (6 mg/kg), 8.9], without altering either the slope factor (1.01) or the EC50 (96.4 nM). Analogous results were obtained with DP-5-CT [percentage of maximal inhibition: control, 24.1; EEDQ (1 mg/kg), 15.2; EEDQ (6 mg/kg), 10.7), again without changes in slope factor (0.89) or EC50 (9.9 nM). Analysis of double-reciprocal plots of equieffective concentrations of agonist, followed by calculation of fractional receptor occupancy, revealed a linear relationship between receptor occupancy and response for both 5-HT and DP-5-CT (i.e., an absence of receptor reserve). The receptor specificity of the effect of EEDQ was demonstrated in two ways. First, it was shown that pretreatment of rats with the selective 5-HT1A partial agonist BMY 7378 (10 mg/kg) before EEDQ afforded substantial protection (about 75%) against loss of the inhibitory effect of DP-5-CT on forskolin-stimulated adenylyl cyclase activity. Second, EEDQ did not alter the inhibition of forskolin-stimulated adenylyl cyclase activity induced by the adenosine A1 receptor agonist phenylisopropyladenosine (PIA).(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site

Excised outside-out patches from HEK293 cells stably transfected with the human (h) 5-HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5-HT-induced current using the patch clamp technique. In addition, binding studies with radioligands for 5-HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5-HT-induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): 9-THC, WIN55,212-2, anandamide, JWH-015 and CP55940. The WIN55,212-2-induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212-3, an enantiomer of WIN55,212-2, did not affect the 5-HT-induced current. WIN55,212-2 did not change the EC50 value of 5-HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]-SR141716A and the CB1/CB2 receptor ligand [3H]-CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5-HT3A receptor cDNA, WIN55,212-2, CP55940, anandamide and SR141716A did not affect [3H]-GR65630 binding, but 5-HT caused a concentration dependent-inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5-HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5-HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.     

The cloned human 5-HT7 receptor splice variants: a comparative characterization of their pharmacology, function and distribution

Serotonin (5-hydroxytryptamine, 5-HT) receptor pre-mRNA is alternatively spliced in human tissue to produce three splice variants, h5-HT7(a), h5-HT7(b) and h5-HT7(d), which differ only in their carboxyl terminal tails. Using membranes from transiently and stably transfected HEK293 cells expressing the three recombinant h5-HT7 splice variants we compared their pharmacological profiles and ability to activate adenylyl cyclase. Using PCR on cDNA derived from various human tissues, the 5-HT7(a) and 5-HT7(b) splice variants were detected in every tissue examined. The h5-HT7(d) splice variant was detected in 13 of 16 tissues examined, with predominant expression in the heart, small intestine, colon, ovary and testis. All three h5-HT7 splice variants displayed high affinity binding for [3H]5-HT (pKd=8.8-8.9) in the presence and absence of 100 microM GTP and had similar binding affinities for all 17 ligands evaluated. In HEK293 cells expressing similar, high levels of receptor (approximately 10,000 fmol/mg protein), 5-CT (5-carboxamidotryptamine), 5-MeOT (5-methoxytryptamine) and 5-HT were full agonists while 8-OH-DPAT ((2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin) was a partial agonist with relative efficacy of approximately 0.8. Even at this high receptor level, EC50 values for stimulation of adenylyl cyclase were 10- to 50-fold higher than the Kd values, indicating a lack of spare receptors. No significant differences in coupling to adenylyl cyclase were observed between the three splice variants over a wide range of receptor expression levels. For antagonists, binding affinities determined by displacement of [3H]5-HT binding and by competitive inhibition of 5-HT-stimulated adenylyl cyclase activity were essentially identical amongst the splice variants. These studies indicate that the three human splice variants are pharmacologically indistinguishable and that modifications of the carboxyl tail do not influence coupling to adenylyl cyclase.     

WAY100635 prevents the changes induced by fluoxetine upon the 5-HT1A receptor functionality

5-HT1A receptor-mediated signalling in rat brain was evaluated after chronic administration (14 days; s.c.) of the selective serotonin reuptake inhibitor (SRRI) fluoxetine (10 mg/kg/day) alone, or in combination with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg/day). The density of 5-HT1A binding sites was unchanged following fluoxetine, WAY100635, or the combination of fluoxetine and WAY100635. However, the net stimulation of [35S]GTPgammaS binding induced by the 5-HT1A agonist 8-OH-DPAT was significantly attenuated in dorsal raphe nucleus (DRN), but not in hippocampus, after chronic fluoxetine. Moreover, depending of the area analysed, the basal binding of [35S]GTPgammaS was differentially affected by this treatment: increased in DRN and decreased in hippocampal dentate gyrus. Interestingly, the changes in [35S]GTPgammaS basal binding and on 5-HT1A receptors functionality were prevented by the concomitant administration of WAY100635. The inhibition of dorsal raphe firing by 8-OH-DPAT was also attenuated in fluoxetine-treated rats (ED50 = 2.12 +/- 0.32 microg/kg and 4.34 +/- 0.09 microg/kg, for vehicle and fluoxetine respectively), an effect which was also prevented by the concomitant administration of WAY100635 (ED50 = 2.10 +/- 0.58 microg/kg). Chronic administration of WAY100635 alone did not affect the 5-HT1A receptor-induced stimulation of [35S]GTPgammaS binding, nor the 8-OH-DPAT-induced inhibition of 5-HT neuron firing. These results demonstrate that the concomitant blockade of 5-HT1A receptors when administering fluoxetine prevents those adaptive changes of 5-HT1A receptor function associated with the chronic administration of this antidepressant. These findings could be relevant from the therapeutic point of view, and further support the potential benefit of treatments with a SSRI/5-HT1A receptor antagonist combination.     

Synthesis and molecular modeling of new 1-aryl-3-[4-arylpiperazin-1-yl]-1-propane derivatives with high affinity at the serotonin transporter and at 5-HT(1A) receptors

It has been proposed that 5-HT(1A) receptor antagonists augment the antidepressant efficacy of selective serotonin (5-HT) reuptake inhibitors. In a search toward new and efficient antidepressants, 1-(aryl)-3-[4-arylpiperazin-1-yl]-1-propane molecular hybrids were designed, synthesized, and evaluated for 5-HT reuptake inhibition and 5-HT(1A) receptor affinity. The design was based in coupling structural moieties related to inhibition of serotonin reuptake, such as benzo[b]thiophene derivatives to arylpiperazines, typical 5-HT(1A) receptor ligands. In binding studies, several compounds showed affinity at the 5-HT transporter and at 5-HT(1A) receptors. Molecular modeling studies predicted the pharmacophore elements required for high affinity binding and the features that enable to discriminate between agonist, partial agonist, or antagonist action at 5-HT(1A) receptors and 5-HT transporter inhibition. Solvent interactions in desolvation prior to the binding step along with enthalpy and enthropy compensations might be responsible to explain agonist, partial agonist, and antagonist character. Hydrogen-bonding capability seems to be important to break hydrogen interhelical hydrogen bonds or alternatively to form other bonds upon ligand binding. Partial agonists and antagonists are unable to do this as the full agonist, which interacts closely by long-range forces or directly. The compounds showing the higher affinity at both the 5-HT transporter (K(i) < 50 nM) and the 5-HT(1A) receptors (K(i) < 20 nM) were further explored for their ability to stimulate [(35)S]GTPgammaS binding or to antagonize 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT)-stimulated [(35)]GTPgammaS binding to rat hippocampal membranes, an index of agonist/antagonist action at 5-HT(1A) receptors, respectively. Compound 8g exhibited agonist activity (EC(50) = 30 nM) in this assay, whereas compounds 7g and 8h,i behaved as weak partial agonists and 7h-j and 8j,l antagonized the R(+)-8-OH-DPAT-stimulated GTPgammaS binding. Functional characterization was performed by measuring the antagonism to 8-OH-DPAT-induced hypothermia in mice.