Dopamine D4-like binding sites labeled by [3H]nemonapride include substantial serotonin 5-HT2A receptors in primate cerebral cortex

Dopamine D4-like binding sites are abundant in human cerebral cortex as detected by [3H]nemonapride. The extremely low density of D4 mRNA in human cerebral cortex is inconsistent with the high amount of D4-like binding sites. To investigate the nature of the D4-like receptors, [3H]nemonapride binding sites in the nonhuman primate cerebral cortex were characterized. Although [3H]nemonapride binding sites were D4-like, displaceable by clozapine but not raclopride, [3H]nemonapride binding was not displaced by selective D4 antagonists but was displaced by the selective 5-HT2A antagonist MDL100907. Using [3H]ketanserin as a 5-HT2A ligand, nemonapride showed high affinity for monkey (Ki = 10.4 nM) and cloned human (Ki = 9.4 nM) 5-HT2A receptors, while its affinity for rat receptors was lower (Ki = 140 nM). The present study demonstrates that cerebral cortical D4-like binding sites labeled by [3H]nemonapride in nonhuman primates consist of a very small portion of D4, but a substantial portion of 5-HT2A receptors. The unexpectedly high affinity of nemonapride for primate 5-HT2A receptor suggests reconsidering previous data from other studies using [3H]nemonapride, particularly those on D4-like receptors.     

Thyrotropin releasing hormone increases 5-hydroxytryptamine1 receptors in the limbic brain of the rat

The effect of TRH on 5-HT1 receptors in the rat brain was investigated. A crude membrane preparation was incubated at 37 degrees C for 15 min with or without TRH prior to [3H]5-HT binding assay. TRH at 100 nM increased the number of 5-HT1 receptors significantly (approximately 20%) in the limbic forebrain and the hippocampus without altering their affinity. As this concentration of TRH is close to its dissociation constant (2 nM and 51 nM in the limbic forebrain, 11 nM in the hippocampus), this effect is probably of physiological relevance. This finding seems to support a pharmacological finding of others that the anti-convulsion effect of TRH may be related to increased serotonergic transmission.     

[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.     

Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain

Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of [3H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [(3)H]8-OH-DPAT and other serotonergic ligands

[(3)H]8-OH-DPAT is a selective ligand for labeling 5-HT(1A) receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [(3)H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [(125)I]RTI-55 and [(3)H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [(3)H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [(3)H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [(125)I]cyanopindolol, [(3)H]ketanserin/[(3)H]mesulergine, [(3)H]GR-65630, [(3)H]GR-113808 and [(3)H]LSD) that specifically labeled 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4) and 5-HT(5-7) receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Characterization of a Novel Serotonin Receptor Subtype (5-HTls) in Rat CNS: Interaction with a GTP Binding Protein

Three pharmacologically distinct high-affinity [3H]serotonin ([3H]5-HT) binding sites were identified in spinal cord synaptosomes. [3H]5-HT competition studies using selective 5-HT1A receptor ligands indicated that approximately 25% of high-affinity synaptosomal [3H]5-HT binding was inhibited by 5-HT1A-selective compounds, an estimate consistent with [3H](+-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) saturation experiments in which 5-HT1A receptors were directly labeled. [3H]5-HT competition studies using high-affinity 5-HT1B compounds performed in the presence of 100 nM 8-OH-DPAT (to block 5-HT1A receptors) indicated that approximately 26% of all specific, high-affinity [3H]5-HT binding to spinal cord synaptosomes was to 5-HT1B receptors. [3H]5-HT competition studies performed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (to block 5-HT1A and 5-HT1B receptors, respectively) indicated that the remaining 49% of [3H]5-HT binding did not possess the pharmacologic profile previous reported for 5-HT1C, 5-HT1D, 5-HT1E, 5-HT2, or 5-HT3 receptors. This residual 49% of [3H]5-HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (subsequently referred to as "5-HT1S") displayed high affinity and saturability (KD = 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 microM GTP or the nonhydrolyzable form of GTP, 5'-guanylylimidodiphosphate, inhibited [3H]5-HT binding to 5-HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Serotonin Receptors Recognized by Anti-Idiotypic Antibodies

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.     

A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5-Hydroxytryptamine-1A Sites in Rat Brain

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

Identification of 5-Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex

5-Hydroxytryptamine1A (5-HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5-HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P-40, and bound effectively to 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP)-coupled Affi-Gel 10 in a time-dependent manner. PAPP was shown previously to be a selective ligand for the 5-HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5-HT or 1 microM [3H]1-[2-(4-azidophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine ([3H]p-azido-PAPP). [3H]p-Azido-PAPP is a selective photoaffinity labeling probe for the 5-HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p-azido-PAPP into these two proteins decreased significantly when the solubilized fraction was preincubated with excess 5-HT or PAPP (saturating all 5-HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5-HT1A receptor protein. The isoelectric points of the photolabeled 5-HT1A receptor proteins were 6.0 and 6.5.     

Lack of Usefulness of DN-1417 for Characterization of a CNS Receptor for Thyrotropin-Releasing Hormone

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [H]8-OH-DPAT and other serotonergic ligands

[³H]8-OH-DPAT is a selective ligand for labeling 5-HT_1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [³H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [¹²⁵I]RTI-55 and [³H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [³H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [³H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [¹²⁵I]cyanopindolol, [³H]ketanserin/[³H]mesulergine, [³H]GR-65630, [³H]GR-113808 and [³H]LSD) that specifically labeled 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄ and 5-HT_5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Dopaminergic Receptors in Bovine Retina and Their Interaction with Thyrotropin-Releasing Hormone

A superfusion technique was employed to study the release of [3H]dopamine from isolated bovine retina. Only K+-stimulated release was observed from both light- and dark-adapted retina; release by other stimuli was from dark-adapted retina only. Light-evoked release of [3H]dopamine from dark-adapted retina was blocked by thyrotropin-releasing hormone (TRH), which has previously been identified as a retinal neuropeptide. TRH itself released small amounts of [3H]dopamine from dark-adapted retina. These results are interpreted as indicating that TRH acts as a modulator of dopaminergic activity in retina through the agency of presynaptic autoreceptors. Evidence of the existence of a feedback inhibition system, probably mediated by dopaminergic autoreceptors, was found by the inclusion of sulpiride, a dopaminergic D2 receptor antagonist in the perfusate, which, in a stereoselective manner, enhanced spontaneous and light-evoked release of [3H]dopamine. On the other hand, dopamine (1 microM) reduced these effects. TRH did not affect the high-affinity uptake system for dopamine in retina; this, then, could not account for the effects on release. Radioligand binding showed a specific, saturable high-affinity binding system for [3H]TRH, with an apparent KD of 2.2 nM and a Bmax of 23 fmol/mg protein in bovine retinal membranes. Displacement experiments showed that specific [3H]TRH binding was displaced in the nanomolar range by spiperone and in the micromolar range by dopamine, whereas L-(--)-sulpiride was virtually inactive in displacing [3H]TRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine receptors in rat brain membranes: characterization of high affinity binding of [3H]-2-chloroadenosine

1. [3H]-2-chloroadenosine has been found to be a suitable ligand for the study of adenosine receptors in rat brain synaptic membranes. 2. Binding sites labelled by [3H]-2-chloroadenosine had a high affinity with a KD value of 23.5 nM. 3. Binding is heat sensitive, pH dependent and probably involves protein molecules. 4. The IC50 values for 2-chloroadenosine, adenosine, L-N6-phenylisopropyladenosine and D-N6-phenylisopropyladenosine, N6-cyclohexyladenosine and adenosine-5'-N-ethyl-carboxamide inhibition of [3H]2-chloroadenosine binding are in good agreement with the values obtained in studies of the ability of these compounds to inhibit adenylate cyclase, suggesting that [3H]-2-chloroadenosine binding sites reported here are comparable to the adenosine A1 receptor site. 5. There are regional differences in [3H]-2-chloroadenosine binding to brain membranes. 6. This difference is probably due to the discrepancies in the number of binding sites, and is probably not caused by changing affinities of receptors to the ligand.     

Quantitative Autoradiography of [3H]Indalpine Binding Sites in the Rat Brain: I. Pharmacological Characterization

The binding of [3H]indalpine (4-[2-(3-indolyl)]ethyl piperidine) to slide-mounted sections of rat brain has been characterized. This 5-hydroxytryptamine (5-HT) uptake blocker binds to sections with high affinity (KD approximately 1 nM). The binding is saturable, and can be displaced by the addition of clomipramine (1 microM). Other drugs inhibiting the uptake of 5-HT also have the capacity to inhibit the binding of [3H]indalpine. A significant correlation (r = 0.86) was found between the capacity of these compounds to inhibit the uptake of 5-HT and their potencies as inhibitors of [3H]indalpine binding. Binding was Na+ - and Cl- -dependent and was inhibited competitively by 5-HT. Furthermore, electrolytic lesions of the dorsal raphe or medial forebrain bundle, which cause a degeneration of 5-HT cell bodies and fibers, respectively, resulted in a 30-40% reduction in the binding of [3H]indalpine. [3H]Indalpine binds to the 5-HT uptake recognition sites in a different manner from imipramine-like compounds.     

(125I)iodo-zacopride: new ligand for the study by autoradiography of central 5-HT3 receptors

This paper describes the synthesis and the pharmacological characteristics of the first radioiodinated ligand of central 5-HT3 receptors: [125I]iodo-zacopride. Specific sites having a high affinity (Kd = 4.3 nM) for [125I]iodo-zacopride have been found in membranes from the rat entorhinal cortex. In addition, a highly significant correlation (r = 0.995) existed between the Ki of several 5-HT-related drugs for displacing both [125I]iodo-zacopride from its specific binding sites, and [3H]zacopride from 5-HT3 receptors. Finally, [125I]iodo-zacopride was successfully used for the autoradiographic mapping of 5-HT3 receptors in the rat central nervous system.     

High-affinity agonist binding correlates with efficacy (intrinsic activity) at the human serotonin 5-HT2A and 5-HT2C receptors: evidence favoring the ternary complex and two-state models of agonist action

Many modern models of receptor-G protein function assume that there is a direct relationship between high-affinity agonist binding and efficacy. The validity of this assumption has been recently questioned for the serotonin 5-HT2A receptor. We examined the intrinsic activities of various ligands in activating phosphoinositide hydrolysis and measured their respective binding affinities to the high- and low-affinity states of the 5-HT2C (VNV isoform) and 5-HT(2A) receptors. Ligand binding affinities for the high-affinity state of the receptors were determined using 1-(4-[125I]iodo-2,5-dimethoxyphenyl)2-aminopropane, whereas [3H]mesulergine and N-[3H]methylspiperone were used, in the presence of excess guanine nucleotide [guanosine 5'-O-(3-thiotriphosphate)], to define binding to the low-affinity state of the 5-HT2C and 5-HT2A receptors, respectively. Antagonists labeled the high- and low-affinity states of each receptor with comparable affinities. Previously identified inverse agonists of the 5-HT2C receptor behaved as silent antagonists in our systems even when the receptor was overexpressed at a relatively high density. In contrast, the ability of agonists to bind differentially to the high- and low-affinity states of the 5-HT2A and 5-HT2C receptors was highly correlated (r2 = 0.86 and 0.96, respectively) with their intrinsic activities. These data suggest that high-affinity agonist states can account for agonist efficacy at human 5-HT2A or 5-HT2C receptors without the need for considering additional transition or active states of the receptor-ligand complex. The procedure described herein may expedite drug discovery efforts by predicting intrinsic activities of ligands solely from ligand binding assays.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.