In vitro receptor specificity of the 5HT1A selective phenylpiperazine, LY165163

LY165163, a ligand reported to be selective for the 5HT1A subtype of serotonin receptor, was examined for its ability to interact with 5HT2 receptors in the rat jugular vein and alpha-receptors in the rat aorta. In these smooth muscle preparations, no agonist activity of LY165163 occurred in concentrations up to 10(-5) M. However, LY165163 was an antagonist of serotonin-induced contractions in the jugular vein and of norepinephrine-induced contractions in the rat aorta. The dissociation constant calculated for LY165163 at 5HT2 receptors in the rat jugular vein was 10(-8) M and at alpha-receptors in the rat aorta was 2 X 10(-7) M. Thus, LY165163 is a relatively potent antagonist at vascular 5HT2 sites and possesses appreciable affinity at alpha-receptors. Based on these data, the multiple receptor interactions of LY165163 must be taken into consideration when utilizing this agent as a probe for the 5HT1A subtype of serotonin receptor.     

mCPP but not TFMPP is an antagonist at cardiac 5HT3 receptors.

The prototypic arylpiperazines, meta-chlorophenylpiperazine (mCPP), meta-trifluoromethylphenylpiperazine (TFMPP) and quipazine are widely studied serotonergic ligands with nonselective effects at 5HT1 and 5HT2 receptor subtypes. The present study was designed to compare the affinities of these arylipiperazines at 5HT3 receptors, and to determine agonist or antagonist activity at 5HT3 receptors. Quipazine showed high affinity at brain 5HT3 receptors (IC50 = 4.4 nM) and was a potent agonist of the von Bezold-Jarisch reflex in anesthetized rats, a response mediated by cardiac 5HT3 receptors. In concentrations that activated 5HT3 receptors, quipazine also antagonized serotonin-induced bradycardia in anesthetized rats. Taken together, these data suggest that quipazine is an agonist/antagonist with high affinity at 5HT3 receptors in both brain and cardiac tissue. Although mCPP also showed relatively high affinity at brain 5HT3 receptors (IC50 = 61.4 nM), it did not activate the von Bezold-Jarisch reflex; instead, mCPP potently antagonized serotonin-induced bradycardia. Thus, mCPP acts as an antagonist at 5HT3 receptors in the periphery. Although both quipazine and mCPP possessed relatively high affinity at brain 5HT3 receptors, TFMPP did not bind appreciably to 5HT3 receptors in brain (IC50 = 2373 nM) and neither activated nor inhibited cardiac 5HT3 receptors. That TFMPP did not interact with 5HT3 receptors, whereas quipazine and mCPP did, is in marked contrast to the similar effects of all three arylpiperazines at other serotonin receptors. The selectivity of TFMPP for 5HT1 and 5HT2 receptors (i.e., its minimal affinity for 5HT3 receptors) suggests that this arylpiperazine may be a preferred ligand relative to mCPP when studying 5HT1 or 5HT2 receptor mediated responses.     

Quantitative structure-activity relationship study on tetrahydro-beta-carboline antagonists of the serotonin 2B (5HT2B) contractile receptor in the rat stomach fundus

The antagonist actions of three sub-series of tetrahydro-beta-carbolines at the serotonin 2B (5HT2B) contractile receptor in the rat stomach fundus are analyzed in relation to the physicochemical properties of the molecules. Significant correlations are obtained between the 5HT2B receptor antagonist affinity and the hydrophobic, steric, electronic, hydrogen bond acceptor and some indicator variables of substituents. Based on these findings, the mode of actions of these congeneric series and future strategy to synthesize more potential compounds are discussed.     

Quantitative Structure-Activity Relationship Study on Tetrahydro-β-carboline Antagonists of the Serotonin 2B (5HT 2B) Contractile Receptor in the Rat Stomach Fundus

The antagonist actions of three sub-series of tetrahydro- β -carbolines at the serotonin 2B (5HT 2B) contractile receptor in the rat stomach fundus are analyzed in relation to the physicochemical properties of the molecules. Significant correlations are obtained between the 5HT 2B receptor antagonist affinity and the hydrophobic, steric, electronic, hydrogen bond acceptor and some indicator variables of substituents. Based on these findings, the mode of actions of these congeneric series and future strategy to synthesize more potential compounds are discussed.     

5-HT-1a and 5-HT-1b selectivity of two phenylpiperazine derivatives: evidence for 5-HT-1b heterogeneity

The ability of m-trifluoromethylphenylpiperazine (TFMPP) and an N-substituted derivative, LY 165163 (p-NH2-PE-TFMPP), to discriminate 5-HT-1 binding sites labelled by [3H]5-HT is compared in rat corpus striatum and rat cortex. TFMPP displays at least a 30 fold selectivity for the 5-HT-1b subtype. Furthermore, TFMPP reveals heterogeneity within the 5-HT-1b binding sites. TFMPP displays a Kd of 6 nM for approximately two-thirds of the 5-HT-1b binding sites and a Kd of 273 nM for the remaining one third of the 5-HT-1b sites. p-NH2-PE-TFMPP, on the other hand, discriminates the 5-HT-1 sites in a manner similar to spiperone, displaying a 110 fold selectivity for the 5-HT-1a sites. p-NH2-PE-TFMPP displays a Kd of about 3 nM for the 5-HT-1a sites. p-NH2-PE-TFMPP does not discriminate subtypes within the 5-HT-1b binding sites. The significance of the selectivity of these two compounds as well as structurally related compounds is discussed.     

5HT7 receptors in the rodent suprachiasmatic nucleus

Serotonergic modulation of circadian rhythms in rodent model preparations has received considerable attention over the past decade. Investigators have also been trying to determine which of the many serotonin receptor subtypes may be mediating the effects of serotonin in the suprachiasmatic nucleus, the location of the biological clock that generates the circadian rhythms. A single study in 1993 using the in vitro rat hypothalamic slice preparation suggested that serotonergic modulation of circadian rhythms at the level of the suprachiasmatic nucleus was acting via the newly discovered 5HT7 receptor subtype. Since that initial claim, serotonin modulation of circadian rhythms at the level of the suprachiasmatic nucleus has generally been attributed to 5HT7 receptor activation. However, when trying to cite relevant literature in support of 5HT7 involvement, it becomes evident that attributing rhythm-related serotonin activity in the suprachiasmatic nucleus to 5HT7 receptors may be somewhat premature. There are issues related to pharmacological specificity, species-specific results, and significant knowledge gaps that necessitate a careful review of the literature to make a judgment as to whether 5HT7 receptors are responsible for serotonergic activity in the rodent suprachiasmatic nucleus. In addition, there is sufficient data available at present to make an initial determination as to the degree of 5HT7 receptor involvement at any level in the generation or modulation of circadian rhythms in rodent species.     

The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5. When expressed in Cos-7 cells and NIH-3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5-HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.     

5HT2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition

Contractile responses to serotonin were examined $\text{in}$$\text{vitro}$ in the longitudinal portal vein to determine whether such responses were mediated by the interaction of serotonin with 5HT₁ receptors (those that preferentially bind [³H]serotonin) or 5HT₂ receptors (those that preferentially bind [³H]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the affinity for serotonin receptors in the rat portal vein and the ability to bind to 5HT₂, but no 5HT₁ receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED₅₀ value) relative to contractions produced by the non-cumulative addition of serotonin. Affinities of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentration-response curves compared to affinities obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT₂ receptor affinities of antagonists in the rat portal vein are best determined by the shift of non-cumulative responses to serotonin.     

Expression of 5HT-1A and 5HT-1B receptor genes in brains of Wistar-Zagreb 5HT rats

By selective breeding, two sublines of rats with high or low activity of platelet serotonin (5HT) transporter (5HTt) have been developed (Wistar-Zagreb 5HT rats). Previous studies demonstrated significant differences between the sublines in the expression of platelet 5HTt at the level of both, mRNA and protein. Pharmacological studies showed marked alterations in brain 5HTt function, indicating differences in central serotonin homeostasis, although analysis of regional brain 5HTt gene expression did not show analogous differences. In this study, we searched for possible changes in the expression of the two central 5HT receptor subtypes: 5HT-1A and 5HT-1B, both participating in the regulation of brain 5HT transmission. Semi-quantitative RT-PCR, with three different housekeeping genes as internal standards, showed no differences in the levels of 5HT-receptor expression between the sublines. Results suggest that constitutional alteration of 5HT homeostasis, induced by selective breeding for the extremes of platelet 5HTt activity, did not cause measurable changes in the expression of central 5HT-1A (hippocampus) and 5HT-1B (striatum) receptors in the mentioned rat sublines under physiological conditions.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Structural features of the ligand-binding domain of the serotonin 5HT3 receptor

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation.     

Evidence for hyperdensity of 5HT1B binding sites in the substantia nigra of the rat after 5,7-dihydroxytryptamine intraventricular injection

High affinity serotonin (5HT) binding sites have been found highly concentrated in the substantia nigra (SN) of the rat brain in each classical anatomical subdivisions of this structure, SN reticulata (SNR), SN lateralis (SNL), SN compacta (SNC). In all of the anatomical samples examined along the posteroanterior brain axis (at 200 um intervals), they corresponded to 5HT1B binding sites. The analysis of their distribution performed in rats 15 days after 5,7-DHT intraventricular injection has revealed : (1) the post-synaptic localization of these 5HT1B sites ; (2) the selective increase in their density at the level of SNR. This increase was found heterogeneously distributed inside the SNR and clearly differentiated in external and internal portions of this structure. This hyperdensity in 5HT1B sites in the SNR likely explains the functional hypersensitivity previously demonstrated by local injection of exogenous 5HT into the SN and systemic administration of RU 24969, a preferential 5HT1B agonist.     

Effect of LY53857, a selective 5HT2 receptor antagonist, on 5HT-induced increases in cutaneous vascular permeability in rats

Both serotonin and histamine increased cutaneous vascular permeability in rats; however, serotonin was approximately 100-fold more potent than histamine. LY53857 (0.1 and 1.0 mg/kg, i.p.), a selective 5HT2 receptor antagonist, blocked serotonin- but not histamine-induced increases in cutaneous vascular permeability. the alpha 1 receptor antagonist, prazosin, did not significantly affect increases in vascular permeability produced by serotonin. These data extend previous studies with LY53857 by further documenting its selectivity as a 5HT2 receptor antagonist. In addition, these results with a selective 5HT2 receptor antagonist provide evidence that 5HT2 receptor activation may be the predominant mechanism associated with vascular permeability changes induced by serotonin.     

Inhibition of serotonin-amplified human platelet aggregation by ketanserin, ritanserin, and the ergoline 5HT2 receptor antagonists-LY53857, sergolexole, and LY237733

Human platelets are known to possess 5HT2 receptors which, when activated, amplify the aggregation response produced by other aggregating agents. Several 5HT2 receptor antagonists, including ketanserin and ritanserin, are known to antagonize serotonin-mediated aggregation of human platelets. In the present study, we document the ability of three ergoline 5HT2 receptor antagonists, LY53857, sergolexole, and LY237733, to antagonize the serotonergic component of the human platelet aggregation response. Potencies of the ergoline esters (LY53857 and sergolexole) and the ergoline amide (LY237733) to inhibit serotonin-amplified platelet aggregation responses were similar to the potencies of ketanserin and ritanserin under the conditions of our study. Furthermore, all five 5HT2 receptor antagonists were capable of fully inhibiting the serotonergic component of the platelet aggregation response. In contrast to these potent ergoline esters and amides, 1-isopropyl dihydrolysergic acid (up to 10(-5)M), a putative metabolite of the ergoline esters, was ineffective under these in vitro conditions. These data are consistent with the high potency of these ergolines as antagonists of 5HT2 receptors and further support the involvement of 5HT2 receptors on human platelets in the amplifying response to serotonin.     

Clozapine: selective labeling of sites resembling 5HT6 serotonin receptors may reflect psychoactive profile.

BACKGROUND: Clozapine, the classic atypical neuroleptic, exerts therapeutic actions in schizophrenic patients unresponsive to most neuroleptics. Clozapine interacts with numerous neurotransmitter receptors, and selective actions at novel subtypes of dopamine and serotonin receptors have been proposed to explain clozapine''s unique psychotropic effects. To identify sites with which clozapine preferentially interacts in a therapeutic setting, we have characterized clozapine binding to brain membranes. MATERIALS AND METHODS: [3H]Clozapine binding was examined in rat brain membranes as well as cloned-expressed 5-HT6 serotonin receptors. RESULTS: [3H]Clozapine binds with low nanomolar affinity to two distinct sites. One reflects muscarinic receptors consistent with the drug''s anticholinergic actions. The drug competition profile of the second site most closely resembles 5HT6 serotonin receptors, though serotonin itself displays low affinity. [3H]Clozapine binding levels are similar in all brain regions examined with no concentration in the corpus striatum. CONCLUSIONS: Besides muscarinic receptors, clozapine primarily labels sites with properties resembling 5HT6 serotonin receptors. If this is also the site with which clozapine principally interacts in intact human brain, it may account for the unique beneficial actions of clozapine and other atypical neuroleptics, and provide a molecular target for developing new, safer, and more effective agents.     

Structural analogues of 5-OMe-BPAT: synthesis and interactions with dopamine D2, D3, and serotonin 5-HT1A receptors.

Several structural analogues of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1), a representative of a series of 2-aminotetralin-derived benzamides with potential atypical antipsychotic properties, were synthesized and evaluated for their ability to bind to dopamine D2A, D3, and serotonin 5-HT1A receptors in vitro. The structure affinity relationships revealed that the aromatic ring of the benzamide moiety of 1 contributes to the high affinities for all three receptor subtypes. Furthermore, 1 may interact with the dopamine D2 and D3 receptors through hydrogen bond formation with its carbonyl group. Investigation of the role of the amide hydrogen atom by amide N-alkylation was not conclusive, since conformational aspects may be responsible for the decreased dopaminergic affinities of the N'-alkylated analogues of 1. The effects of the amide modifications on the serotonin 5-HT1A receptor affinity were less pronounced, suggesting that the benzamidoethyl side-chain of 1 as a whole enhances the affinity for this receptor subtype probably through hydrophobic interactions with an accessory binding site. The structural requirements for the substituents at the basic nitrogen atom supported the hypothesis that the 2-aminotetralin moieties of the 2-aminotetralin-derived substituted benzamides may share the same binding sites as the 2-(N,N-di-n-propylamino)tetralins.     

R-(-)-N-alkyl-11-hydroxy-10-hydroxymethyl- and 10-methyl-aporphines as 5-HT1A receptor ligands

Several N-substituted-11-hydroxy-10-hydroxymethyl- and 11-hydroxy-10-methylaporphines were synthesized and their binding affinities at dopamine D(1) and D(2) receptors and serotonin 5-HT(1A) and 5-HT(2A) receptors in rat forebrain tissue were evaluated. Tested compounds displayed moderate to high affinity to 5-HT(1A) receptors but low affinity to D(1) and D(2) receptors. The most potent novel 5-HT(1A) agent was R-(-)-N-methyl-10-hydroxymethyl-11-hydroxyaporphine.