Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.     

Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.     

Design, synthesis, and biological activity of N6-substituted-4'-thioadenosines at the human A3 adenosine receptor

A large series of N6-substituted-4'-thioadenosines were synthesized starting from D-gulonic-gamma-lactone, and structure-activity relationships were studied at the human A3 and other subtypes of adenosine receptors (ARs). 2-Chloro-substituted and 2-H analogues were compared. 2-Chloro-N6-methyl-4'-thioadenosine 19b was a highly potent and selective agonist (Ki=0.8+/-0.1 nM in binding) at the A3AR, and displayed the same relative efficacy in receptor activation as a known full agonist, Cl-IB-MECA. Most of N6-substituted-4'-thioadenosines were less potent in binding than the corresponding N6-substituted-adenosines or N6-substituted-4'-thioadenosine-5'-uronamides. N6-(3-Iodobenzyl) derivative 19g was demonstrated to be an A3AR-selective partial agonist displaying a Ki value of 3.2 nM.     

The binding of SCH 39166 and SCH 23390 to 5-HT1C receptors in porcine choroid plexus

SCH 39166 is a novel benzonaphthazepine, which has been characterized as a potent and selective D1 antagonist. Recently, its D1 selective benzazepine predecessor, SCH 23390, has been shown to bind to 5-HT1C binding sites in the choroid plexus. Therefore, the present studies were undertaken to determine if SCH 39166 has any measurable affinity for 5-HT1C binding sites. Our results indicate that SCH 39166 exhibited poor affinity for the 5-HT1C receptor, with a Ki of 1327 nM. In contrast, SCH 23390 inhibited [3H]-mesulergine binding to 5-HT1C receptors with a Ki of 30 nM. The non-selective 5-HT antagonist, methysergide, inhibited binding with a Ki of 2.4 nM. Finally, studies with the stereoisomers of SCH 39166 and SCH 23390 demonstrated that stereoselectivity at the 5-HT1C site is significantly less than for the D1 site.     

[3H]Spiroxatrine: A 5-HT1A Radioligand with Agonist Binding Properties

Spiroxatrine has been reported to be a 5-HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5-HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5-HT1A antagonist radioligand. [3H]8-Hydroxydipropylaminotetralin [( 3H]8-OH-DPAT), a well-characterized 5-HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8-OH-DPAT binding produced KD values of 0.9 nM and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r = 0.98; p less than 0.001) exists between Ki values obtained for a series of drugs in competing for [3H]-spiroxatrine and [3H]8-OH-DPAT binding. Of special interest was the observation that 5-HT1A agonists such as serotonin, 8-OH-DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8-OH-DPAT-labelled 5-HT1A receptors. [3H]Spiroxatrine and [3H]8-OH-DPAT binding to 5-HT1A receptors was inhibited by guanosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration-dependent manner whereas adenosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of ATP) had no effect. The similarities in the 5-HT1A receptor radiolabelling properties of [3H]spiroxatrine and [3H]8-OH-DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has "agonist-like" binding properties in its interaction with the 5-HT1A receptor.     

7-Arylpiperazinylalkyl and 7-tetrahydroisoquinolinylalkyl derivatives of 8-alkoxy-purine-2,6-dione and some of their purine-2,6,8-trione analogs as 5-HT(1A), 5-HT(2A), and 5-HT(7) serotonin receptor ligands

On the basis of our earlier studies with the serotonin receptor ligands in the group of 1,3-dimethyl-3,7-dihydropurine-2,6-dione derivatives, a series of new arylpiperazinylalkyl and tetrahydroisoquinolinylalkyl analogs of 8-alkoxy-1,3-dimethyl-3,7-dihydropurine-2,6-dione (10-25) and 1,3-dimethyl-7,9-dihydro-3H-purine-2,6,8-trione (26-30) were synthesized and their 5-HT(1A), 5-HT(2A), and 5-HT(7) receptor affinities were determined. The new compounds 17, 18, 20, and 21 were found to be highly active 5-HT(1A) receptor ligands (K(i)=11-19nM) with diversified affinity for 5-HT(2A) receptors (K(i)=15-253nM). Compounds 12, 13, 15, and 19 were moderately potent 5-HT(2A) ligands (K(i)=23-57nM), whereas 17, 18, 24, and 25 showed distinct affinity for 5-HT(7) receptors (K(i)=51-83nM). Purine-2,6,8-triones showed weak affinities for 5-HT(1A) and 5-HT(7) receptors; among them, 27 and 29 were classified as 5-HT(2A) receptor ligands. The selected compounds 17 and 21 were pharmacologically evaluated to determine their functional activities at pre-(hypothermia in mice) and post-(lower lip retraction in rats) synaptic 5-HT(1A) receptors. Compound 17 showed features of a potential agonist of pre- and post-synaptic 5-HT(1A) receptors, whereas 21 was classified as a potential, weak partial agonist of postsynaptic sites. Last of all, the most interesting compound 17 tested in behavioral models showed potential anxiolytic and antidepressant activities.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

Characterization of Adenosine Receptors in the PC 12 Pheochromocytoma Cell Line Using Radioligand Binding: Evidence for A-2 Selectivity

Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6-cyclohexyladenosine [( 3H]CHA), [3H]cyclopentyladenosine [( 3H]CPA), and [3H]5'-N-ethylcarboxamido adenosine [( 3H]NECA) to membranes prepared from PC12 cells showed that the A-1-selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve-fitting programs. However, [3H]NECA, a nonselective A-1/A-2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD = 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM), a finding suggesting involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC12 cell membranes was stereo-selective, with the R isomer of N6-phenylisopropyladenosine (PIA) being approximately 12 times more active than S-PIA. The A-1-selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki = 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA greater than or equal to 2-chloroadenosine greater than CHA greater than or equal to 5'-N-methylcarboxamido adenosine greater than or equal to R-PIA greater than CPA greater than S-PIA. Binding was also displaced by the marine adenosine agonist 1-methylisoguanosine and by a series of xanthine antagonists with the activity order being 1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 8-phenyltheophylline greater than 8-p-sulfophenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the alpha 1-adrenergic receptor subtype in a smooth muscle cell line

We have identified in the DDT1 smooth muscle cell line a [3H]dihydroergocryptine-binding site having the characteristics of an alpha 1-adrenergic receptor. Specific binding of [3H]dihydroergocryptine to DDT1 cells grown either in monolayer or suspension culture was reversible, saturable, and of high affinity, and the binding site demonstrated stereoselectivity. [3H]Dihydroergocryptine dissociation constants of 1.4 +/- 0.2 nM and 1.4 +/- 0.3 nM were observed for suspension and monolayer cells, respectively. However, the concentration of binding sites in suspension-cultured cells (65,100 +/- 8,300 sites/cell) was significantly greater (p less than 0.001) than that found in monolayer cells (27,900 +/- 4,300 sites/cell). The order of agonist competition for the binding site was epinephrine (Ki = 0.92 +/- 0.32 microM) greater than or equal to norepinephrine (Ki = 2.2 +/- 1.0 microM) greater than isoproterenol (Ki = 137 +/- 17 microM), consistent with an alpha-adrenergic interaction. Results of competition experiments with specific antagonists prazosin (alpha 1-selective) or yohimbine (alpha 2-selective) and a computer modeling technique indicated that the alpha-adrenergic receptor of the DDT1 cell was predominantly (greater than 95%) the alpha 1-subtype.     

The influence of modifications in imide fragment structure on 5-HT(1A) and 5-HT(7) receptor affinity and in vivo pharmacological properties of some new 1-(m-trifluoromethylphenyl)piperazines

New, flexible (7, 9, 11 and 13) and rigid (8, 10, 12 and 14) imides with a 1-(m-trifluorophenyl)piperazine fragment and a tetramethylene or a 1e,4e-cyclohexylene spacer, respectively, showed very high affinity (K(i)=0.3-34 nM) and agonistic in vivo activity for 5-HT(1A) receptors. Flexible new compounds and the previously described 5 also bound to 5-HT(7) receptors (K(i)=21-134 nM). Selected glutarimide derivatives, that is, the most potent postsynaptic 5-HT(1A) receptor agonist rigid compound 8 and its flexible analogue 7, as well as the previously described full agonist-rigid compound 6 and the partial agonist-its flexible counterpart 5 exhibited moderate affinity for alpha(1)-adrenoceptors (K(i)=85 - 268 nM), but were practically devoid of any affinity for dopamine D(2) sites. Those glutarimides demonstrated anxiolytic- (5 and 7) and antidepressant-like (5, 6 and 8) activity in the four-plate and the swim tests in mice, respectively; at the same time, however, they inhibited the locomotor activity of mice. The antidepressant-like effect of 8 was significantly stronger than that induced by imipramine used as a reference antidepressant.     

Characterization of the 5-Hydroxytryptamine Receptor Modulating the Release of 5-[3H]Hydroxytryptamine in Slices of the Human Neocortex

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

The influence of substitution at aromatic part of 1,2,3,4-tetrahydroisoquinoline on in vitro and in vivo 5-HT(1A)/5-HT(2A) receptor activities of its 1-adamantoyloaminoalkyl derivatives.

Further structure-activity relationship (SAR) studies with the 1,2,3,4-tetrahydroisoquinoline (THIQ) class of 5-HT(1A) ligands led to the synthesis of new 1-adamantoyloaminoalkyl derivatives. The impact of substituent variations in the aromatic part of THIQ moiety on 5-HT(1A) and 5-HT(2A) receptor affinities, as well as in vivo functional properties of the investigated compounds were discussed. It was found that those modifications reduced the binding affinity for 5-HT(1A) receptors (in comparison with unsubstituted THIQ derivatives); however, the majority of new compounds still remained potent 5-HT(1A) ligands (K(i)=4.9-46 nM) and most of them showed features of partial agonists of postsynaptic 5-HT(1A) receptors. At the same time, their 5-HT(2A) receptor affinity was slightly increased (K(i)=40-1475 nM), which resulted in a loss of 5-HT(2A)/5-HT(1A) selectivity. 5-Br,8-OCH3 derivative-the most potent, mixed 5-HT(1A)/5-HT(2A) ligand-produced activation of presynaptic 5-HT(1A) receptors and showed properties of a 5-HT(2A) receptor antagonist.     

Synthesis, receptor binding and functional studies of mesoridazine stereoisomers

The four stereoisomers of mesoridazine were synthesized and evaluated in D2, 5-HT1A, 5-HT2A, 5-HT2C, D1, and D3 receptor binding and functional assays. Two isomers demonstrated potent D2 receptor binding (Ki < 3 nM) and functional antagonism (IC50 < or = 10 nM) activities. These two isomers also showed moderate affinity for the 5-HT2A and D3 receptors. A third isomer was devoid of significant D2 receptor binding, but did have moderate affinity for the 5-HT2A and D3 receptors. The fourth isomer demonstrated poor affinity for all the receptors tested. Most significantly, the stereochemistry of the sulfoxide moiety played a dominant role in the observed structure-activity relationship (SAR).     

Characterization of a [3H]-5-hydroxytryptamine binding site in rabbit caudate nucleus that differs from the 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D subtypes

[3H]5-HT binding sites were analyzed in membranes prepared from the rabbit caudate nucleus (CN). [3H]5-HT labeled both 5-HT1A and 5-HT1C recognition sites, defined by nanomolar affinity for 8-OH-DPAT and mesulergine respectively; however, these represented only a fraction of total specific [3H]5-HT binding. Saturation experiments of [3H]5-HT binding in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine to block 5-HT1A and 5-HT1C sites revealed that non-5-HT1A/non-5-HT1C sites represented about 60% of the total 5-HT1 sites and that they exhibited saturable, high affinity, and homogeneous binding. The pharmacological profile of the non-5-HT1A/non-5-HT1C sites (designated 5-HT1R) also differed from that of 5-HT1B and 5-HT2 sites, but was similar to that of the 5-HT1D site. However, significant differences existed between the 5-HT1D and 5-HT1R sites for their Ki values for spiperone, spirilene (an analog of spiperone), metergoline, and methiothepin. The study of modulatory agents (calcium and GTP) also showed differences between the 5-HT1R and 5-HT1D sites. For example, the effects of GTP on agonist binding to the 5-HT1R sites were less than on the 5-HT1D sites in bovine caudate. In addition, calcium enhanced the effects of GTP on the 5-HT1R sites, whereas calcium inhibited the GTP effect on the 5-HT1D sites. The present findings demonstrate the presence of a high-affinity [3H]5-HT binding site in rabbit CN, designated 5-HT1R, that is different from previously defined 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT2 sites.     

Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]

In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.     

Detection of a Novel Serotonin Receptor Subtype (5-HT1E) in Human Brain: Interaction with a GTP-Binding Protein

[3H]Serotonin (5-hydroxytryptamine, [3H]5-HT) was used as a radioligand probe of brain 5-HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 microM pindolol (to block 5-HT1A and 5-HT1B receptors), and 100 nM mesulergine (to block 5-HT1C and 5-HT2 receptors). One of these sites demonstrated high affinity for 5-carboxyamidotryptamine (5-CT) and ergotamine, consistent with the known pharmacology of the 5-HT1D receptor; the second site demonstrated low affinity for 5-CT and ergotamine. Computer-assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5-CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5-CT and ergotamine, respectively) for 45% of the sites. To investigate the non-5-HT1D component of the binding, 100 nM 5-CT (to block 5-HT1A, 5-HT1B, and 5-HT1D receptors) was coincubated with [3H]5-HT, membranes, and mesulergine. The remaining [3H]5-HT binding (hereafter referred to as "5-HT1E") displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5-CT and ergotamine displayed the highest selectivity for the 5-HT1D site versus the 5-HT1E site.(ABSTRACT TRUNCATED AT 250 WORDS)     

[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.