N-terminal domains in mouse and human 5-hydroxytryptamine3A receptors confer partial agonist and antagonist properties to benzylidene analogs of anabaseine

The present study tested the hypothesis that mouse and human 5-hydroxytryptamine3A (5-HT3A) receptors may be differentially modulated by benzylidene analogs of anabaseine (BA) and that these analogs may be useful in assessing residues involved in receptor gating. Mouse and human wild-type and mouse and human chimeric 5-HT3A receptors expressed in Xenopus oocytes were evaluated with the two-electrode voltage clamp technique. Our previous studies demonstrated that 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA) is an antagonist at the mouse wild-type 5-HT3A receptor, but that its metabolites 3-(2-hydroxy, 4-methoxybenzylidene)-anabaseine (2-OHMBA), 3-(2-methoxy, 4-hydroxybenzylidene)-anabaseine (4-OHMBA), and 3-(2,4-dihydroxybenzylidene)-anabaseine (2,4-DiOHBA) are partial agonists (J Pharmacol Exp Ther, 299: 1112-1117, 2001). In the human wild-type (HWT) 5-HT3A receptor, none of the BA compounds possessed partial agonist activity. BA compounds antagonized 1.5 microM 5-HT-mediated (EC50) responses in the HWT 5-HT3A receptor with a rank order of potency (IC50 in muM) of 2-OHMBA (1.5 +/- 0.1) > DMXBA (3.1 +/- 0.2) > 4-OHMBA (7.4 +/- 0.5) > 2,4-DiOHBA (12.8 +/- 0.7). In mouse receptor chimeras containing N-terminal human receptor orthologs, 2-OHMBA inhibited 5-HT-mediated (EC50) currents with IC50 values of 2.0 +/- 0.08 and 3.0 +/- 0.13 microM, respectively. In human receptor chimeras containing N-terminal mouse receptor orthologs, 2-OHMBA displayed partial agonist activities with EC50 values of 1.3 +/- 0.15 and 5.0 +/- 0.4 microM; efficacies were 43 and 57%, respectively. Thus, amino acids present in the distal one-third of the N terminus of mouse and human 5-HT3A receptors are necessary and sufficient to confer partial agonist or antagonist properties of 2-OHMBA.     

Varenicline is a potent partial agonist at α6β2* nicotinic acetylcholine receptors in rat and monkey striatum

Extensive evidence indicates that varenicline reduces nicotine craving and withdrawal symptoms by modulating dopaminergic function at α4β2* nicotinic acetylcholine receptors (nAChRs) (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex). More recent data suggest that α6β2* nAChRs also regulate dopamine release and mediate nicotine reinforcement. The present experiments were therefore done to test the effect of varenicline on α6β2* nAChRs and their function, because its interaction with this subtype is currently unclear. Receptor competition studies showed that varenicline inhibited α6β2* nAChR binding (K(i) = 0.12 nM) as potently as α4β2* nAChR binding (K(i) = 0.14 nM) in rat striatal sections and with ～20-fold greater affinity than nicotine. Functionally, varenicline was more potent in stimulating α6β2* versus α4β2* nAChR-mediated [(3)H]dopamine release from rat striatal synaptosomes with EC(50) values of 0.007 and 0.086 μM, respectively. However, it acted as a partial agonist on α6β2* and α4β2* nAChR-mediated [(3)H]dopamine release with maximal efficacies of 49 and 24%, respectively, compared with nicotine. We also evaluated varenicline's action in striatum of monkeys, a useful animal model for comparison with humans. Varenicline again potently inhibited monkey striatal α6β2* (K(i) = 0.13 nM) and α4β2* (K(i) = 0.19 nM) nAChRs in competition studies. Functionally, it potently stimulated both α6β2* (EC(50) = 0.014 μM) and α4β2* (EC(50) = 0.029 μM) nAChR-mediated [(3)H]dopamine release from monkey striatal synaptosomes, again acting as a partial agonist relative to nicotine at both subtypes. These data suggest that the ability of varenicline to interact at α6β2* nAChRs may contribute to its efficacy as a smoking cessation aid.     

WAY-163909 [(7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole], a novel 5-hydroxytryptamine 2C receptor-selective agonist with anorectic activity

The pharmacological profile of WAY-163909 [(7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole], a novel 5-hydroxytryptamine (HT)(2C) (serotonin) receptor-selective agonist is presented. WAY-163909 displaced [(125)I]2,5-dimethoxy-4-iodoamphetamine binding from human 5-HT(2C) receptor sites, in Chinese hamster ovary (CHO) cell membranes, with a K(i) value of 10.5 +/- 1.1 nM. Binding affinities determined for the human 5-HT(2A) and 5-HT(2B) receptor subtypes were 212 and 485 nM, respectively. In functional studies, WAY-163909 stimulated the mobilization of intracellular calcium in CHO cells stably expressing the human 5-HT(2C) receptor with an EC(50) value of 8 nM, and E(max) relative to 5-HT of 90%. WAY-163909 failed to stimulate calcium mobilization in cells expressing the human 5-HT(2A) receptor subtype (EC(50) > 10muM) and was a 5-HT(2B) receptor partial agonist (EC(50) 185 nM, E(max) 40%). WAY-163909 exhibited negligible affinity (<50% inhibition at 1 muM) for other receptor sites examined, including human 5-HT(1A), D2, and D3 receptors, and the 5-HT transporter binding site in rat cortical membranes. WAY-163909 exhibited weak affinity for the human D4 (245 nM) and 5-HT(7) (343 nM) receptor subtypes and the alpha1 binding site in rat cortical membranes (665 nM). WAY-163909 produced a dose-dependent reduction in food intake in normal Sprague-Dawley rats (ED(50) = 2.93 mg/kg), an effect blocked by a 5-HT(2C) receptor antagonist but not by a 5-HT(2A) or 5-HT(2B) receptor antagonist. In addition, WAY-163909 decreased food intake in obese Zucker rats and diet-induced obese mice with ED(50) values of 1.4 and 5.19 mg/kg i.p., respectively, consistent with the potential utility of 5-HT(2C) receptor agonists as anti-obesity agents.     

Characterization of vabicaserin (SCA-136), a selective 5-hydroxytryptamine 2C receptor agonist

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor subtype has received considerable attention as a target for drug discovery, having been implicated in a wide variety of disorders. Here, we describe the in vitro pharmacological profile of the novel 5-HT(2C) receptor-selective agonist vabicaserin [(-)-4,5,6,7,9,9a,10,11,12,12a-decahydrocyclopenta[c] [1,4]diazepino[6,7,1-ij]quinoline hydrochloride] (SCA-136), including a comprehensive strategy to assess 5-HT(2B) receptor selectivity using diverse preparations and assays of receptor activation. Vabicaserin displaced (125)I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT(2C) receptor sites in Chinese hamster ovary cell membranes with a K(i) value of 3 nM and was >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT(2B) receptor subtype using [(3)H]5HT was 14 nM. Vabicaserin was a potent and full agonist (EC(50), 8 nM; E(max), 100%) in stimulating 5-HT(2C) receptor-coupled calcium mobilization and exhibited 5-HT(2A) receptor antagonism and 5-HT(2B) antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. In rat stomach fundus and human colonic longitudinal muscle endogenously expressing 5-HT(2B) receptors, vabicaserin failed to induce a 5-HT(2B) receptor-dependent contraction and produced a rightward shift of the 5-HT and α-methyl-5-HT concentration-response curves in these preparations, respectively, consistent with 5-HT(2B) competitive antagonism. Likewise, vabicaserin failed to induce a 5-HT(2B) receptor-mediated contraction in arteries from deoxycorticosterone acetate-salt-treated rats, a model of hypersensitized 5-HT(2B) receptor function, and produced a rightward shift in the 5-HT-induced response that was consistent with 5-HT(2B) receptor antagonism. In summary, vabicaserin is a novel, potent, and selective 5-HT(2C) receptor agonist.     

General anesthetic-induced channel gating enhancement of 5-hydroxytryptamine type 3 receptors depends on receptor subunit composition

5-Hydroxytryptamine (serotonin) (5-HT) type 3 (5-HT(3)) receptors are members of an anesthetic-sensitive superfamily of Cys-loop ligand-gated ion channels that can be formed as homomeric 5-HT(3A) or heteromeric 5-HT(3AB) receptors. When the efficacious agonist 5-HT is used, the inhaled anesthetics halothane and chloroform (at clinically relevant concentrations) significantly reduce the agonist EC(50) for 5-HT(3A) receptors but not for 5-HT(3AB) receptors. In the present study, we used dopamine (DA), a highly inefficacious agonist for 5-HT(3) receptors, to determine whether the difference in sensitivity between 5-HT(3A) and 5-HT(3AB) receptors to the potentiating effects of halothane and chloroform is due to differential modulation of agonist affinity, channel gating, or both. Using the two-electrode voltage-clamp technique with 5-HT(3A) and 5-HT(3AB) receptors expressed in Xenopus oocytes, we found that chloroform and halothane enhanced currents evoked by receptor-saturating concentrations of DA for both receptor subtypes in a concentration-dependent manner but that the magnitude of enhancement was substantially greater for 5-HT(3A) receptors than for 5-HT(3AB) receptors. Isoflurane induced only a small enhancement of currents evoked by receptor-saturating concentrations of DA for 5-HT(3A) receptors and no enhancement for 5-HT(3AB) receptors. For both receptor subtypes, none of the three test anesthetics significantly altered the agonist EC(50) for DA, implying that these anesthetics do not affect agonist binding affinity. Our results show that chloroform, halothane, and (to a much lesser degree) isoflurane enhance channel gating for 5-HT(3A) receptors and that the incorporation of 5-HT(3B) subunits to produce heteromeric 5-HT(3AB) receptors markedly attenuates the ability of these anesthetics to enhance channel gating.     

Complexities of measuring antagonist potency at P2X(7) receptor orthologs

The ability of P2 antagonists to affect agonist-stimulated fluorescent dye accumulation in cells expressing human, rat, or mouse P2X(7) receptors was examined. Several compounds, including pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), which was previously thought to be a weak P2X(7) receptor antagonist, possessed high potency (nanomolar IC(50)) at human and rat P2X(7) receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5'-phosphate (P5P), and periodate-oxidized ATP (OxATP) exhibiting 20- to 500-fold higher potency for human than for mouse P2X(7) receptors. HMA (5-(N,N-hexamethylene)amiloride) was also selective for human over rat P2X(7) receptors but potentiated responses at mouse P2X(7) receptors. Coomassie Brilliant Blue G (CBB) was a nonselective antagonist with high potency at mouse P2X(7) receptors (IC(50) approximately 100 nM). All compounds were noncompetitive antagonists, and potency could only be quantified by measuring IC(50) values. These values were similar when determined against EC(50) concentrations of ATP or 2'- and 3'-O-4(-benzoylbenzoyl)-ATP and, for most compounds, only slightly (3- to 5-fold) affected by agonist concentration. However, IC(50) values for KN62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) and suramin, varied up to 25-fold depending upon agonist concentration. Furthermore, IC(50) values for KN62 and OxATP were 10-fold lower at 22 degrees C than at 37 degrees C, whereas IC(50) values for PPADS, P5P, suramin, and OxATP were up to 20-fold lower in NaCl than in sucrose buffer. Potency estimates for CBB and PPADS decreased 5-fold in the presence of bovine serum albumin, possibly due to protein binding. Given the species differences, and the effects of assay conditions on antagonist potency, caution must be exercised when interpreting results obtained with the available antagonists.     

Characterization of the serotonin receptor mediating contraction in the mouse thoracic aorta and signal pathway coupling

The purpose of this study was to characterize pharmacologically the 5-HT receptor(s) mediating contraction in the mouse aorta and the pathways these receptors are coupled with to mediate contraction. We hypothesized that a 5-HT2A receptor, as in the rat, mediates contraction by activating L-type calcium channels, phospholipase C (PLC), and tyrosine kinase(s). Endothelium-denuded aortic strips were placed in a tissue bath for measurement of isometric contractile force. 5-HT, the 5-HT2A receptor agonist alpha-methyl-5-HT, and partial 5-HT2A receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (+/--DOI) caused the most potent and efficacious contraction. The 5-HT(1E/1F) receptor agonist BRL 54443 also induced contraction (-log EC(50) = 6.52); however, the 5-HT2A receptor antagonist ketanserin antagonized this contraction. Our hypothesis was further supported by the finding that antagonists with affinity for the 5-HT2A receptor, ketanserin, 1-(1-naphthyl)piperazine, spiperone, and LY53857, reduced 5-HT-induced contraction. A correlation of 0.927 was found between literature-derived compound binding affinities for the agonists and antagonists at the 5-HT2A receptor of the rat and the data generated in our experiments (-log EC(50) and pK(B) values). The L-type calcium channel blockers nifedipine and nitrendipine, PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, and tyrosine kinase inhibitors genistein and PD 098,059 all shifted and/or reduced maximum contraction to 5-HT. We conclude that contraction to 5-HT in the mouse aorta is mediated primarily by a 5-HT2A receptor and is coupled to L-type calcium channels, PLC, and tyrosine kinases.     

Serotonin agonists inhibit synaptic potentials in the rat locus ceruleus in vitro via 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors

Intracellular recordings were made from rat locus ceruleus neurons in the slice preparation in vitro. Depolarizing synaptic potentials (DSP)2 elicited by electrical stimulation were typically 10 to 15 mV in amplitude and 200 msec in duration. Superfusion with 5-hydroxytryptamine (5-HT, serotonin) or the 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT), produced an inhibition of the DSP. The maximal inhibition was 55 +/- 2% (mean +/- S.E.M.). The EC50 for 5-CT was 60 nM, whereas for 5-HT it was 12 microM. Cocaine (10 microM) shifted the 5-HT concentration-response curve to the left and the EC50 to 320 nM. 8-Hydroxy-2-(di-n-propylamino)tetralin, a selective 5-HT1A receptor ligand, also inhibited the DSP, but only produced about 65% of the maximal 5-CT or 5-HT response (EC50 = 50 nM). A relatively selective 5-HT1B ligand (65-fold 5-HT1B greater than 5-HT1A), 1-(m-trifluoromethyl-phenyl)-piperazine, acted as a full agonist (EC50 = 110 nM). None of these compounds had any effects on the membrane properties of the cell at the doses tested. The response to 8-hydroxy-2-(di-n-propylamino) tetralin was antagonized by pretreatment with the 5-HT1A antagonist spiperone (1 microM). The estimated KD for spiperone was 16 nM. At this same concentration, however, there was no effect on the 5-CT-induced inhibition. The antagonist 4-(3-ter-butyl-amino-2-hydroxy-propoxyl)-indol-2-carbonic acid isopropyl ester (LM 21-009, 100 nM) was found to be a partial agonist producing a 26 +/- 4% inhibition of the DSP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin1c receptor reserve in choroid plexus masks receptor subsensitivity

This paper tests the hypothesis that spare serotonin 5-HT1c receptors are present in the rat choroid plexus and explores the possible influence of such sites on the adaptive regulation of the 5-HT1c receptor. The consequences of partial receptor inactivation were compared for the natural agonist 5-HT and the putative partial agonists trifluoromethylphenylpiperazine (TFMPP) and (+)-lysergic acid diethylamide (LSD). These studies showed approximately 50% reserve of 5-HT1c receptors in the rat choroid plexus. The calculated KA for 5-HT obtained by partial irreversible inactivation was 36 nM. Phenoxybenzamine reduced the maximum response elicited by TFMPP and LSD, without shifting the EC50 values, consistent with the interpretation that TFMPP and LSD are partial agonist at the 5-HT1c receptor in rat choroid plexus. The KA of TFMPP and LSD was 0.16 microM and 9 nM, respectively. Quantitative analysis of percentage of receptor occupancy vs. percentage of maximum response showed that 5-HT occupied only 70% of the receptors to give a maximum response, whereas a linear relationship between percentage of occupancy and response was found for TFMPP. These differences had functional consequences as demonstrated in studies of regulation of the 5-HT1c receptor. Chronic administration of the 5-HT agonist quipazine produced a 32% loss of 5-HT1c binding sites in the choroid plexus, with no change in the 5-HT-induced phosphoinositide hydrolysis response. This dissociation between binding and function is likely explained by the receptor reserve that exists for the 5-HT1c receptors. Consistent with this interpretation, the TFMPP-induced phosphoinositide hydrolysis signal was reduced to the same extent as the loss of binding sites. These results show that the 5-HT1c receptor in the choroid plexus adapts predictably to chronic receptor activation and suggest the possibility that the paradoxical regulation that has been described for other 5-HT receptors might be explained partially by the unrecognized existence of receptor reserve.     

Benzylidene analogs of anabaseine display partial agonist and antagonist properties at the mouse 5-hydroxytryptamine(3A) receptor.

The nicotinic receptor drug candidate, 3-(2,4-dimethoxybenzylidene)-anabaseine (also known as GTS-21; DMXBA), its hydroxy metabolites, and some related analogs were evaluated with the two-electrode voltage-clamp technique in mouse 5-hydroxytryptamine (5-HT)(3A) receptors expressed in Xenopus oocytes. Although DMXBA lacked partial agonist activity, its hydroxy-benzylidene metabolites and related analogs were partial agonists, displaying the following rank order of potency (EC(50)) and apparent efficacy: 5-HT, 0.9 +/- 0.06 microM (100% efficacy) > 3-(2-hydroxy,4-methoxybenzylidene)-anabaseine (2-OH-MBA), 2.0 +/- 0.3 microM (63% efficacy) > 3-(2,4-dihydroxybenzylidene)-anabaseine, 2.6 +/- 0.3 microM (63% efficacy) > 3-(2-methoxy,4-hydroxybenzylidene)-anabaseine, 17.2 +/- 1.0 microM (30% efficacy). To examine the influence of a benzylidene ring hydroxy substituent, the agonist actions of the three possible monohydroxy isomers were examined. The rank order of potency, based on EC(50) determinations, and apparent efficacy was: 3-(2-hydroxybenzylidene)-anabaseine, 20.3 +/- 2.6 microM (63% efficacy) > 3-(4-hydroxybenzylidene)-anabaseine, 32.3 +/- 5.9 microM (14% efficacy) > 3-(3-hydroxybenzylidene)-anabaseine (3-OH-BA) (no agonist activity). Both DMXBA and 3-OH-BA antagonized 5-HT-mediated currents, with IC(50) values of 15.7 +/- 0.9 and 27.5 +/- 4.7 microM, respectively. DMXBA demonstrated both competitive and noncompetitive forms of antagonism over the range of concentrations tested. These results suggest that a hydroxy substituent at the 2' position of the benzene ring is necessary and sufficient for partial agonist activity; substitution at the 4' position with a hydroxy or methoxy group further enhances agonist potency. Because 2-OH-MBA is a primary metabolite of DMXBA, it may contribute to the physiological, biochemical, and behavioral effects of the parent compound when administered in vivo.     

Subunit-dependent modulation of the 5-hydroxytryptamine type 3 receptor open-close equilibrium by n-alcohols

5-Hydroxytryptamine (5-HT, serotonin) type 3 (5-HT(3)) receptors belong to the alcohol-sensitive superfamily of Cys-loop ligand-gated ion channels, and they are thought to play an important role in alcoholism. Alcohols with small molecular volumes increase the amplitude of currents evoked by low 5-HT concentrations and shift the 5-HT concentration-response curve for 5-HT(3) receptor activation leftward, indicative of increased receptor sensitivity to agonist. This action is significantly smaller when currents are mediated by heteromeric 5-HT(3AB) receptors compared with homomeric 5-HT(3A) receptors. In this study, we used the highly inefficacious 5-HT(3) receptor agonist dopamine to determine whether this difference between 5-HT(3A) and 5-HT(3AB) receptors reflects differential alcohol modulation of agonist binding affinity or channel gating efficacy. Human recombinant 5-HT(3A) and 5-HT(3AB) receptors were expressed in Xenopus oocytes, and currents were measured in the absence and presence of alcohols using the two-electrode voltage-clamp technique. Modulation by alcohols of peak currents elicited by maximally activating concentrations of dopamine was alcohol concentration-dependent. Potentiation by smaller alcohols was consistently significantly greater in 5-HT(3A) than in 5-HT(3AB) receptors, whereas inhibition by larger alcohols was not. A representative small (butanol) and large (octanol) alcohol failed to alter the EC(50) value for channel activation by dopamine. We conclude that the presence of the 5-HT(3B) subunit in 5-HT(3AB) receptors significantly reduces the enhancement of gating efficacy by small alcohols without altering the inhibitory actions of large alcohols.     

Real-time intracellular Ca2+ mobilization by travoprost acid,bimatoprost, unoprostone,and other analogs via endogenous mouse,rat, and cloned human FP prostaglandin receptors

The ability of a number of prostaglandin F 2 alpha (PGF 2 alpha) analogs to mobilize intracellular Ca2+[Ca2+]iand to compete for [3H]PGF 2 alpha binding to prostaglandin F 2 alpha receptors (FP) was evaluated. Radioligand binding studies measuring displacement of [3H]PGF 2 alpha by a variety of FP prostaglandin analogs yielded the following rank order of affinities: travoprost acid [(+)-16-m-trifluorophenoxy tetranor PGF 2 alpha; (+)-fluprostenol] > bimatoprost acid (17-phenyl-trinor PGF 2 alpha) > unoprostone (13,14-dihydro-15-keto-20-ethyl PGF 2 alpha) = bimatoprost (17-phenyl-trinor PGF 2 alpha ethyl amide) > or = Lumigan (bimatoprost ophthalmic solution). In FP functional studies, travoprost acid (EC50= 17.5-37 nM, n = 13), bimatoprost acid (EC50= 23.3-49.0 nM, n = 6-12), unoprostone (EC50= 306-1270 nM, n = 4-8), bimatoprost (EC50= 3070- 3940 nM, n = 4-9), and Lumigan (EC50= 1470-3190 nM, n = 5-9) concentration dependently stimulated [Ca2+]imobilization via the rat (A7r5 cells), mouse (3T3 cells), and cloned human ocular FP prostanoid receptors. The rank order of potency of these compounds at the FP receptor of the three species was similar and in good agreement with the determined binding affinities. The agonist effects of these compounds were concentration dependently blocked by the FP receptor-selective antagonist, AL-8810 (11beta-fluoro-15-epi-15-indanyl-tetranor PGF 2 alpha) (Ki= 0.6-1.3 microM). These studies have demonstrated that bimatoprost, unoprostone, and bimatoprost acid possess direct agonist activities at the rat, mouse, and human FP prostanoid receptor and that travoprost acid is the most potent of the synthetic FP prostaglandin analogs tested.     

SL65.0155, a novel 5-hydroxytryptamine(4) receptor partial agonist with potent cognition-enhancing properties

SL65.0155 [5-(8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-3-[1-(2-phenyl ethyl)-4-piperidinyl]-1,3,4-oxadiazol-2(3H)-one monohydrochloride] is a novel benzodioxanoxadiazolone compound with high affinity for human 5-hydroxytryptamine (5-HT)(4) receptors (K(i) of 0.6 nM) and good selectivity (greater than 100-fold for all other receptors tested). In cells expressing the 5-HT(4(b)) and 5-HT(4(e)) splice variants, SL65.0155 acted as a partial agonist, stimulating cAMP production with a maximal effect of 40 to 50% of serotonin. However, in the rat esophagus preparation, SL65.0155 acted as a 5-HT(4) antagonist with a pK(b) of 8.81. In addition, SL65.0155 potently improved performance in several tests of learning and memory. In the object recognition task, it improved retention at 24 h when administered i.p. or p.o. (0.001-0.1 mg/kg). This effect was antagonized by the 5-HT(4) antagonist SDZ 205,557, itself without effect, demonstrating that the promnesic effects of SL65.0155 are mediated by 5-HT(4) agonism. SL65.0155 also reversed the cognitive deficits of aged rats in the linear maze task and the scopolamine-induced deficit of mice in the water maze task. Furthermore, the combined administration of an inactive dose of SL65.0155 with the cholinesterase inhibitor rivastigmine resulted in a significant promnesic effect, suggesting a synergistic interaction. SL65.0155 was devoid of unwanted cardiovascular, gastrointestinal, or central nervous system effects with doses up to more than 100-fold higher than those active in the cognitive tests. These results characterize SL65.0155 as a novel promnesic agent acting via 5-HT(4) receptors, with an excellent preclinical profile. Its broad range of activity in cognitive tests and synergism with cholinesterase inhibitors suggest that SL65.0155 represents a promising new agent for the treatment of dementia.     

Serotonin-induced reduction of the calcium-dependent plateau in frog dorsal root ganglion cells is blocked by serotonergic agents acting at 5-hydroxytryptamine1A sites

Intracellular recordings from the dorsal root ganglion cells of adult frogs in the presence of tetraethylammonium display action potentials with a prominent calcium-dependent plateau. These action potentials can be altered by serotonergic agents in one of two ways. The superfusion of 5-HT (0.1-1 microM) usually produces a dose-dependent reduction of the action potential duration, whereas 8-hydroxy dipropylaminotetralin (8-OH-DPAT) (10-50 microM) produces a dose-dependent increase in duration. A series of 5-HT antagonists were tested for their ability to block either the 5-HT or the 8-OH-DPAT effect. The antagonists were chosen for their reported selectivity in distinguishing receptors of the 5-HT1A, 5-HT2 and 5-HT3 subtypes. The antagonists' action on 5-HT narrowing [blockade by methiothepin, spiperone and spiroxitrine, but not by ketanserin or 3-tropyl-indole-3-carboxylate (ICS 205-930)] suggests that this response is mediated by 5-HT1A receptors. The widening effect produced by 8-OH-DPAT (a putative 5-HT1A agonist) was not blocked by any antagonist tested. At lower concentrations (0.1-2.5 microM) 8-OH-DPAT exhibited no agonist actions, but antagonized the 5-HT-induced narrowing. These results suggest the 5-HT receptors mediating 5-HT action potential narrowing in these cells are of the 5-HT1A subtype, but that they differ from the 5-HT1A receptors described in other tissues in which 8-OH-DPAT is an agonist or a partial agonist.     

Characterization of the molecular fragment that is responsible for agonism of pergolide at serotonin 5-Hydroxytryptamine2B and 5-Hydroxytryptamine2A receptors

Cardiac-valve regurgitation observed in Parkinson patients treated with the ergoline dopamine receptor agonist 8beta-methylthiomethyl-6-propylergoline (pergolide) has been associated with the agonist efficacy of the drug at 5-hydroxytryptamine(2B) (5-HT(2B)) receptors. 5-HT(2A) receptors may also play a role in pergolide-induced cardiac-valve regurgitation. We studied the pharmacological profile of pergolide and eight derivatives in porcine vascular rings endowed with 5-HT(2B) and 5-HT(2A) receptors to detect the molecular fragment of the pergolide molecule that may be responsible for agonism at these receptors. Pergolide derivatives showed a different substitution pattern at N(6), and the side chain at C(8) was modified by replacement of the sulfur against an oxygen atom. We demonstrate that the potent agonism of pergolide both at 5-HT(2B) and 5-HT(2A) receptors is retained when the N(6) propyl substituent is replaced by ethyl. However, agonism can be converted into antagonism if N(6) propyl is replaced by methyl. The N(6)-unsubstituted derivative was a low efficacy 5-HT(2B) receptor partial agonist and a 5-HT(2A) receptor antagonist. This pharmacological pattern was also applicable for pergolide congeners with an oxygen in the side chain at C(8). 6-Methylpergolide retained agonist efficacy and potency compared with pergolide at human (h) D(2LONG(L)) and hD(2SHORT(S)) receptors as determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Based on the ability of pergolide to produce potent agonism at 5-HT(2B) receptors and the failure of 6-methylpergolide to act as an agonist but as a potent antagonist, we conclude that the N(6) propyl substituent of pergolide is crucial for 5-HT(2B) receptor agonism and thus a determinant of valvular regurgitation.     

Pharmacological and behavioral profile of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel 5-hydroxytryptamine(2A) receptor inverse agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of [(3)H]ketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.     

Triptan-induced contractile (5-HT1B receptor) responses in human cerebral and coronary arteries: relationship to clinical effect

Triptans are agonists at 5-HT1B and 5-HT1D (where 5-HT is 5-hydroxytryptamine; serotonin) receptors and cause vasoconstriction of isolated blood vessels. The aim of the present study was to determine vasoconstrictor potency (EC50) of triptans in human coronary and cerebral arteries and to examine whether there was any relationship with the maximal plasma concentrations (Cmax; nM) of the drugs achieved following oral administration of clinically relevant doses to man using values reported in the literature. We also examined the expression of 5-HT1B receptors in atherosclerotic and normal coronary arteries. The vasocontractile responses to sumatriptan, rizatriptan or eletriptan were characterized by in vitro pharmacology. The ratio of Cmax/EC50 was calculated. 5-HT1B and 5-HT1D receptors were visualized by immunohistochemical techniques in coronary arteries. Sumatriptan, rizatriptan and eletriptan were powerful vasoconstrictors in cerebral artery. The rank order of agonist potency was eletriptan=rizatriptan=sumatriptan. In the coronary artery, the triptans were weaker vasoconstrictors. The rank order of potency was similar. In cerebral artery the ratio of Cmax/EC50 was not significantly different from unity, indicating a relationship between these two parameters. In general for the coronary artery, the ratios were significantly less than unity, indicating no direct relationship. Immunohistochemistry showed expression of 5-HT1B receptors in the medial layer, but did not reveal any obvious difference in 5-HT1B receptor expression between normal and atherosclerotic coronary arteries. The results support the notion that triptans are selective vasoconstrictors of cerebral arteries over coronary arteries and that there is a relationship between vasoconstrictor potency in cerebral arteries and clinically relevant plasma levels.     

5-Hydroxytryptamine(1A) receptor-stimulated [(35)S]GTPgammaS binding in rat brain: absence of regional differences in coupling efficiency

In hippocampal membranes, the selective 5-hydroxytryptamine (5-HT(1A)) receptor agonists 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and N,N-dipropyl-5-carboxamidotryptamine (N,N-DP-5-CT) stimulated guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) binding by 130 to 140%; binding stimulated by nonselective agonists (5-HT and 5-CT) was approximately 30% greater. However, the selective 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohex anecarboxamide (WAY100,635) completely abolished the increases produced by 8-OH-DPAT and N,N-DP-5-CT but only eliminated 70% of that elicited by 5-CT. The rank potency order of the tested agonists was identical with their rank order of affinity for 5-HT(1A) receptors [5-CT congruent with N,N-DP-5-CT > R-(+)-8-OH-DPAT > 5-HT > ipsapirone]. Racemic 8-OH-DPAT and the partial agonist ipsapirone exhibited lower intrinsic activity than R-(+)-8-OH-DPAT. R-(+)-8-OH-DPAT also stimulated [(35)S]GTPgammaS binding in cortex, but not in striatum, which lacks 5-HT(1A) receptors. Partial irreversible inactivation of 5-HT(1A) receptors, in vitro with phenoxybenzamine (0.3 or 1 microM) or in vivo with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1 mg/kg), reduced the maximal response produced by R-(+)-8-OH-DPAT but did not alter its EC(50). In autoradiographic sections, R-(+)-8-OH-DPAT stimulated [(35)S]GTPgammaS binding in 5-HT(1A) receptor-rich regions (dorsal hippocampus, 123%; lateral septum, 111%; midhippocampus, 110%; dorsal raphe nucleus, 83%; medial prefrontal cortex, approximately 60%). The EC(50) of R-(+)-8-OH-DPAT did not vary significantly among brain regions (46-96 nM). Partial irreversible blockade of 5-HT(1A) receptors in brain sections (phenoxybenzamine, 10 microM) reduced the maximal response without altering the EC(50) in both the hippocampus and dorsal raphe. Despite prior evidence that dorsal raphe somatodendritic 5-HT(1A) autoreceptors exhibit high receptor/effector coupling efficiency (receptor reserve) compared with postsynaptic receptors in hippocampus, there was no evidence of a difference at the level of receptor/G protein coupling.     

Toward selective drug development for the human 5-hydroxytryptamine 1E receptor: a comparison of 5-hydroxytryptamine 1E and 1F receptor structure-affinity relationships

The 5-hydroxytryptamine (5-HT) 1E receptor is highly expressed in the human frontal cortex and hippocampus, and this distribution suggests the function of 5-HT(1E) receptors might be linked to memory. To test this hypothesis, behavioral experiments are needed. Because rats and mice lack a 5-HT(1E) receptor gene, knockout strategies cannot be used to elucidate this receptor's functions. Thus, selective pharmacological tools must be developed. The tryptamine-related agonist BRL54443 [5-hydroxy-3-(1-methylpiperidin-4-yl)-1H-indole] is one of the few agents that binds 5-HT(1E) receptors with high affinity and some selectively; unfortunately, it binds equally well to 5-HT(1F) receptors (K(i) ≈ 1 nM). The differences between tryptamine binding requirements of these two receptor populations have never been extensively explored; this must be done to guide the design of analogs with greater selectivity for 5-HT(1E) receptors versus 5-HT(1F) receptors. Previously, we determined the receptor binding affinities of a large series of tryptamine analogs at the 5-HT(1E) receptor; we now examine the affinities of this same series of compounds at 5-HT(1F) receptors. The affinities of these compounds at 5-HT(1E) and 5-HT(1F) receptors were found to be highly correlated (r = 0.81). All high-affinity compounds were full agonists at both receptor populations. We identified 5-N-butyryloxy-N,N-dimethyltryptamine as a novel 5-HT(1F) receptor agonist with >60-fold selectivity versus 5-HT(1E) receptors. There is significant overlap between 5-HT(1E) and 5-HT(1F) receptor orthosteric binding properties; thus, identification of 5-HT(1E)-selective orthosteric ligands will be difficult. The insights generated from this study will inform future drug development and molecular modeling studies for both 5-HT(1E) and 5-HT(1F) receptors.     

Pharmacological evidence for a functional serotonin-2B receptor in a human uterine smooth muscle cell line

The present study investigated the serotonin-induced increase in phosphoinositide hydrolysis and mobilization of intracellular Ca2+ ([Ca2+]i) in human uterine smooth muscle cells (HUSMCs) to identify the serotonergic receptor positively coupled to phospholipase C in these cells. In phosphoinositide (PI) assays, serotonin (5-HT) and alpha-methyl-5-HT were potent, full agonists (EC50 = 20 and 4.1 nM, respectively), whereas the phenylethylamine, R-(-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride, was less active (EC50 = 63 nM). Proposed 5-HT2B-selective agonists, BW-723C86 [alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride] and (+)-norfenfluramine, exhibited strong agonist potency and efficacy comparable with 5-HT (EC50 = 18 and 33 nM, respectively) and approximately 15-fold more potency than (-)-norfenfluramine (EC50 = 500 nM). 5-HT2C receptor agonists m-chlorophenylpiperazine and MK-212 [6-chloro-2-(1-piperaxinyl)pyrazine] were weak agonists in these cells, with potencies of 110 and 880 nM, respectively. A similar rank order of potency was observed in [Ca2+]i mobilization assays (r = 0.9, p < 0.005) in the HUSMC and with contraction of rat stomach fundus strips that contain a 5-HT2B receptor (r = 0.9, p < 0.001). Antagonist studies revealed that a 5-HT2B-selective antagonist, RS-127445 [2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine] (Ki = 0.13 nM), was significantly more effective at inhibiting 5-HT-induced activity than a 5-HT2A antagonist, M-100907 (R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol]) (Ki= 914 nM) and the 5-HT2C antagonists RS-102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride) (Ki = 2.5 microM) and SB-242084 (6-chloro-5-methyl-1-[6-92-methylpyridin-3-yloxy) pyridine-3-ylcarbamoyl] indoline) (Ki = 42.4 nM) in the HUSMC PI turnover assays. Taken together, these studies strongly suggest the presence of a functionally active 5-HT2B receptor subtype in HUSMCs. The physiological role of this receptor in these cells remains to be defined.