Regulation of hippocampal gonadotropin releasing hormone (GnRH) receptor mRNA and GnRH-stimulated inositol phosphate production by gonadal steroid hormones

Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS-42358, [3H]granisetron, or 1-(m-[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS-42358, and [3H]RS-25259, but not [3H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT2A receptor selective antagonist [3H]MDL100,907 ((+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [3H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd = 0.2-0.3 nM), and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [3H]MDL100,907 was compared to the autoradiographical patterns obtained with [3H]Ketanserin, [3H]Mesulergine, and [3H]RP62203 (N-[3-[4-(4-fluorophenyl)piperazin-1-y1]propyl]-1,8-naphtalenes ultam) and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [3H]MDL100,907 labelled a single population of sites (5-HT2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [3H]MDL100,907-labelled receptors and 5-HT2A mRNA further supports the selectivity of this radioligand for 5-HT2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [3H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT2A receptors.     

3H]MDL 100,907 labels 5-HT2A serotonin receptors selectively in primate brain

The selective antagonist for the 5-HT2A serotonin receptor MDL 100,907, recently characterized autoradiographically in rat brain, has been characterized as a radioligand for the visualization of this receptor in human and monkey brain. In both species [3H]MDL 100,907 binding to brain sections was saturable, had sub-nanomolar affinity (Kd = 0.14-0.19 nM in human brain; Kd= 0.16-0.19 nM in monkey brain) and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL 100,907-labeled receptors: MDL 100,907 > spiperone > ketanserin > mesulergine). The autoradiographical signal obtained with [3H]MDL 100,907 was compared to the signal obtained with [3H]ketanserin, [3H]RP62203 and [3H]mesulergine in both species, and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization in monkey brain. At variance with the other radioligands, [3H]MDL 100,907 showed a single population of binding sites with extremely low levels of non-specific binding. As expected, mesulergine showed low affinity for [3H]MDL 100,907-labeled receptors and the autoradiographic pattern shown by [3H]mesulergine confirmed the lack of labeling of the 5-HT2A receptor by this radioligand in primate brain. The similarity of the distribution of [3H]MDL 100,907-labeled receptors and 5-HT2A mRNA in monkey brain, supports the selectivity of this radioligand for 5-HT2A receptors and suggests a somatodendritic localization of these receptors. The present results confirm [3H]MDL 100,907 as the radioligand of choice at present for the autoradiographic visualization of 5-HT2A receptors in mammalian brain including post-mortem human brain.     

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to adenylate cyclase stimulation

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) (h5-HT7(a)) receptor isoform was performed using stably transfected LM(tk-) cells. Expression levels of the h5-HT7(a) receptor determined from saturation studies using either a labeled agonist ([3H]5-HT) or antagonist ([3H]LSD) were very similar (Bmax = 160-190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]i) with an EC50 value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine >/= 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC50 values) to stimulate [cAMP]i were more than 25-fold lower relative to their respective binding affinities (Ki values) obtained in [3H]5-HT competition assays. In contrast, antagonist potencies (Kb values) to block 5-HT-stimulated [cAMP]i were in close agreement with their corresponding Ki values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]i, indicating that the 5-HT7(a) subtype directly interacts with Galphas protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT7 receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT7 receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT ([3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 microM) displayed a pharmacological profile similar to the recombinant 5-HT7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [3H]5-CT binding to residual, possibly, 5-HT1A sites. Competition for this [3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT7 receptor. Saturation studies also indicated that (+/-)-pindolol (10 microM)/WAY 100635 (100 nM)-insensitive [3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (Bmax=33.2+/-0.7 fmol mg(-1) protein, pKd=8.78+/-0.05, mean+/-S.E.M., n=3). The development of this 5-HT7 receptor binding assay will aid investigation of the rat native 5-HT7 receptor.     

2-aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived substituted benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned human dopamine D2A and D3 receptors, and for [3H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (12a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM) as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.     

Alniditan, a new 5-hydroxytryptamine1D agonist and migraine-abortive agent: ligand-binding properties of human 5-hydroxytryptamine1D alpha, human 5-hydroxytryptamine1D beta, and calf 5-hydroxytryptamine1D receptors investigated with [3H]5-hydroxytryptamine and [3H]alniditan

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.     

Biguanide derivatives: agonist pharmacology at 5-hydroxytryptamine type 3 receptors in vitro

The effects of 24 biguanide and four guanidine derivatives on 5-hydroxytryptamine (5-HT)3 receptors in N1E-115 neuroblastoma cells were examined using radioligand binding and whole-cell voltage-clamp techniques. Displacement of the selective 5-HT3 receptor antagonist [3H]BRL 43694 by phenylbiguanide (PBG) derivatives revealed Ki values ranging from 3.4 x 10(-4) to 4.4 x 10(-10) M. The rank order of potency of agonists was 2,3,5-trichloro-PBG > 2,3-dichloro-PBG = 2,5-dichloro-PBG = 3,5-dichloro-PBG > 3,4-dichloro-PBG = 3-chloro-PBG > 2-chloro-PBG = 4-chloro-PBG = 2-methyl-PBG = 2,4-difluoro-PBG > PBG = 2-trifluoro-5-chloro-PBG > 4-fluoro-PBG = 3-trifluoromethyl-PBG > 4-nitro-PBG = 1,5-bis-4-chloro-PBG = 3,5-ditrifluoromethyl-PBG > 4-ethoxy-PBG > 4-sulfonic acid-PBG. All of the benzylbiguanides and indanylbiguanide were inactive on [3H]BRL 43694 binding or displaced it only weakly. The four guanidine derivatives were quite inactive. In the PBG series, all antagonist competition curves were steep (pseudo-Hill coefficients ranging from 1.05 to 1.58), monophasic, and best fit with a one-site model. Among PBG derivatives, the chlorinated compounds exhibited a good degree of selectivity for 5-HT3 receptors versus other 5-HT receptor subtypes and other neurotransmitter binding sites. Electrophysiological studies showed that the PBG derivatives tested produced rapid inward currents, at a holding potential of -65 mV, that showed rapid desensitization. The current induced by the 2,3,5-trichloro-PBG derivative was inhibited by the specific 5-HT3 receptor antagonist ICS 205-930 but was unaffected by the 5-HT2 receptor antagonist ketanserin. Analysis of concentration-response curves for the PBG derivatives gave EC50 values ranging from 2.2 x 10(-5) to 2.7 x 10(-8) M and Hill slopes ranging from 1.02 to 2.10. The rank order of potency was similar to that obtained from the binding data, and a good correlation was found between Ki and EC50 values. It is concluded that the triple-chloro substitution yielded a compound that is 30-fold more potent than 3-chloro-PBG and approximately 10-fold more potent than dichloro-PBG derivatives, making 2,3,5-trichloro-PBG the most potent 5-HT3 agonist described thus far.     

Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes

The existence of endogenous compounds interacting with the serotonergic system was previously postulated. In the present work, rat brain tissues were extracted by acidic and organic procedures. The resulting extract was tested for its capacity to interact with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to 5-HT1 receptors. Compounds responsible for the observed inhibitory activities were isolated and purified by high pressure liquid chromatography. A tetrapeptide corresponding to a novel amino acid sequence Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of [3H]5-HT to 5-HT1 receptors at low concentration (IC50 = 10(-10) M). This effect corresponds to a specific interaction at 5-HT1B receptors since LSAL does not significantly affect other neurotransmitter bindings. LSAL appears heterogeneously distributed throughout the brain (hippocampus > cerebellum > striatum > brain stem) and in peripheral tissues (kidney > lung > stomach > blood > liver > spleen). Two other peptides, Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected [3H]5-HT binding compared with LSAL. They presumably represent degradation products of the functional peptide LSAL. The fact that LSAL interacts specifically with 5-HT1B receptors that inhibit the release of neurotransmitters and particularly that of 5-HT itself suggests that this peptide may be involved in mechanisms controlling 5-HT neurotransmission and, accordingly, may play an important role in pathophysiological functions related to 5-HT activity.     

Autoradiographic mapping of 5-HT1B- and 5-HT1D receptors in human brain using [3H]alniditan, a new radioligand

[3H]alniditan, a new potent non-indole serotonin 5-HT1B/1D agonist, was used as a radioligand to characterize 5-HT1B and 5-HT1D receptor (previously termed 5-HT1D beta and 5-HT1D alpha) in various regions of the human brain. Quantitative receptor autoradiography was applied for high anatomical resolution and sensitivity. Highest densities of 5-HT1B/1D receptors were found in the substantia nigra and in the globus pallidus. High to moderate densities were measured in the caudate nucleus, putamen, nucleus accumbens, central gray and hippocampal formation. Very low densities were detected in various cortical regions. In the cerebellum no [3H]alniditan binding was detected. Selective 5-HT1B receptor labeling was achieved using [3H]alniditan in the presence of 300 nM of ketanserin (sufficient to block 5-HT1D receptor labeling). The identity of the 5-HT1B binding sites under these conditions was corroborated by the pIC50 of sumatriptan, which corresponded to its affinity for cloned human 5-HT1B receptors expressed in cells. Surprisingly, the distribution of selective 5-HT1B receptor labeling was completely identical to the distribution of labeling of 5-HT1B + 5-HT1D receptors. The present data indicate that [3H]alniditan is a suitable radioligand for measuring 5-HT1B/1D receptor in the human brain and that the 5-HT1B binding sites are predominant in the presently investigated regions of the human brain.     

Dilated fallopian tubes: MR imaging characteristics

A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.     

Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719

1. The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2. The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]ben zene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT > 5-HT > 8-OH-DPAT) was the same in functional and binding studies. 4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2+/-0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6. It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.     

Carrier-dependent and Ca(2+)-dependent 5-HT and dopamine release induced by (+)-amphetamine, 3,4-methylendioxymethamphetamine, p-chloroamphetamine and (+)-fenfluramine

1. The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the 3H neurotransmitters. 2. Their rank order of potency for [3H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCA > or = MDMA > or = (+)-Fen > > (+)-Amph). Similarly, their rank order as [3H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph > > pCA = MDMA > > (+)-Fen). We also confirmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine). 3. [3H]-5HT and/or [3H]-dopamine release induced by all these compounds was partially (31-80%), but significantly Ca(2+)-dependent. Lack of extracellular Ca2+ did not alter uptake mechanisms nor did it modify the carrier-dependent dopamine-induced [3H]-dopamine release. (+)-Amph-induced [3H]-dopamine release and pCA- and MDMA-induced [3H]-5-HT release were significantly inhibited by omega-agatoxin-IVA, a specific blocker of P-type voltage-operated Ca(2+)-channels, similar to the previous results on (+)-Fen-induced [3H]-5-HT release. 4. Methiothepin inhibited the Ca(2+)-dependent component of (+)-Amph-induced [3H]-dopamine release with high potency (70 nM), as previously found with (+)-Fen-induced [3H]-5-HT release. The inhibitory effect of methiothepin was not due to its effects as a transporter inhibitor or Ca(2+)-channel blocker and is unlikely to be due to its antagonist properties on 5-HT1/2, dopamine or any other extracellular receptor. 5. These results indicate that the release induced by these compounds is both 'carrier-mediated' and Ca(2+)-dependent (possibly exocytotic-like), with the specific carrier allowing the amphetamines to enter the synaptosome. The Ca(2+)-dependent release is mediated by Ca(2+)-influx (mainly through P-type Ca(2+)-channels), possibly triggered by the drug interacting with an unknown intracellular target, affected by methiothepin, common to both 5-HT and dopamine synaptosomes.     

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.     

Atypical in vitro and in vivo binding of [3H]S-14506 to brain 5-HT1A receptors

The tritiated derivative of the potent 5-HT1A receptor agonist S-14506 ?1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)pipera zine? was tested for its capacity to selectively label the serotonin 5-HT1A receptors both in vitro in the rat and the mouse brain, and in vivo in the mouse. In vitro studies showed that the pharmacological profile and the distribution of [3H]S-14506 specific binding sites (Kd = 0.15 nM) in different brain regions matched perfectly those of the prototypical 5-HT1A receptor ligand [3H]8-OH-DPAT. However, in the three regions examined (hippocampus, septum, cerebral cortex), the density of [3H]S-14506 specific binding sites was significantly higher (+66-90%) than that found with [3H]8-OH-DPAT. Whereas the specific binding of [3H]8-OH-DPAT was markedly reduced by GTP and Gpp(NH)p and increased by Mn2+, that of [3H]S-14506 was essentially unaffected by these compounds. In addition, the alkylating agent N-ethylmaleimide was much less potent to inhibit the specific binding of [3H]S-14506 than that of [3H]8-OH-DPAT. Measurement of in vivo accumulation of tritium one hour after i.v. injection of [3H]S-14506 to mice revealed marked regional differences, with about 2.5 times more radioactivity in the hippocampus than in the cerebellum. Pretreatment with 5-HT1A receptor ligands prevented tritium accumulation in the hippocampus but not in the cerebellum. Autoradiograms from brain sections of injected mice confirmed the specific in vivo labeling of 5-HT1A receptors by [3H]S-14506, therefore suggesting further developments with derivatives of this molecule for positron emission tomography in vivo in man.     

Circannual variations in the binding of [3H]lysergic acid diethylamide to serotonin2A receptors and of [3H]paroxetine to serotonin uptake sites in platelets from healthy volunteers

BACKGROUND: Circannual variations occur in several serotonergic parameters, including platelet serotonin uptake and platelet [3H]imipramine binding. METHODS: Binding of [3H]lysergic acid diethylamide ([3H]LSD) to platelet serotonin (5-HT)2A receptors and binding of [3H]paroxetine to platelet serotonin uptake sites were studied longitudinally for 1 year in 12 healthy volunteers. RESULTS: For [3H]LSD, the number of binding sites (Bmax) showed no significant seasonal variation (two-way analysis of variance), although Bmax was significantly higher during the months October through February than during the months April through August (32.6 vs. 29.8 fmol/mg protein; p = .015). For [3H]paroxetine, Bmax showed a significant seasonal variation (p = .003) with maximum in August (1322 fmol/mg protein) and minimum in February (1168 fmol/mg protein). The affinity constant (Kd) showed a significant seasonal variation for [3H]LSD binding (p = .046), but not for [3H]paroxetine binding. The seasonal fluctuations in [3H]LSD binding and in paroxetine binding tended to be inversely correlated for Bmax (r = -.70; p = .08) and were significantly negatively correlated for Kd (r = -.88; p = .009). CONCLUSIONS: The present study demonstrates a seasonal effect on platelet serotonin uptake site binding and indicates a possible seasonal effect on 5-HT2A receptor binding. The results imply that circannual fluctuations should be taken into account when these platelet serotonin markers are studied.     

Agonist activity of antimigraine drugs at recombinant human 5-HT1A receptors: potential implications for prophylactic and acute therapy

The actions of several serotonergic ligands in use or under development for the treatment of migraine headaches were examined at recombinant human 5-HT1A receptors stably expressed in Chinese Hamster Ovary cells. Affinities (K(i)s) at this site were determined in competition binding experiments with [3H]-8-OH-DPAT ([3H](+/-)8-hydroxy-N,N-dipropylaminotetralin), whilst agonist efficacy was measured by stimulation of [35S]-GTP gamma S (guanylyl-5'-[gamma[35S]thio]-triphosphate) binding. Of the prophylactic antimigraine drugs tested, methysergide and lisuride behaved as efficacious agonists (Emax > or = 90% relative to 5-HT) whereas pitozifen and (-)propranolol acted as a partial agonist (60%) and an antagonist, respectively. This suggests that there is no correlation between agonism at 5-HT1A receptors and prophylactic antimigraine action. In contrast, serotonin, dihydroergotamine, sumatriptan, naratriptan and alniditan, which are effective in acute interruption of migraine attacks, each displayed high efficacy (Emax = 100, 100, 92.6, 79.3, 79.1% respectively) and marked affinity (Ki = 18.7, 0.6, 127, 26.4 and 3.0 nM respectively) at 5-HT1A receptors. EC50 values for agonist stimulation of [35S]-GTP gamma S binding correlated with respective Ki values at 5-HT1A receptors (r = 0.93) and the stimulation of [35S]-GTP gamma S binding by these compounds was antagonised by the selective 5-HT1A antagonist WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide; 100 nM). These data suggest that agonism at 5-HT1A receptors may be involved in some actions of drugs used in acute antimigraine therapy. In comparison with the above compounds, novel ligands targeted at 5-HT1B/1D receptors, such as GR125,743 (N-[4-methoxy-3-(4-methyl-piperazin-1-yl)phenyl] -3-methyl-4-(4-pyridyl)benzamide) and GR 127,935 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-2'-methyl-4'-(5-m ethyl-1, 2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide), only weakly activated [35S]-GTP gamma S binding (32.4 and 32.1% efficacy) and displayed moderate affinity at 5-HT1A receptors (Kis 53.1 and 49.8 nM) suggesting that they constitute useful tools to differentiate 5-HT1A and 5-HT1B/1D receptor-mediated actions. In conclusion, the present data indicates that several antimigraine agents exhibit marked 5-HT1A receptor activity and that although this is unlikely to be important for prophylactic action it may be relevant to the ancilliary properties of drugs used for acute migraine treatment.