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.     

Discovery and characterization of [H]8-OH-DPAT binding to HeLaS3 cells

Some G protein-coupled receptors (GPCRs) have functional links to cancer biology, yet the manifestation of GPCRs in tumor types is little studied to date. Using a battery of radioligand binding assays, we sought to characterize GPCR recognition binding sites on HeLaS3 tumor cells. High levels of binding of the selective serotonin 5-HT_1A receptor agonist [³H]8-OH-DPAT were observed in these cells. Saturation and homologous competition experiments indicated that [³H]8-OH-DPAT bound different populations of high- and low-affinity sites. In competition experiments, several serotonergic compounds displaced [³H]8-OH-DPAT binding with low potency from its high-affinity binding sites, suggesting that low-affinity binding is the predominant mode of binding. A variety of drugs targeting different classes of receptors did not affect [³H]8-OH-DPAT binding. These observations may help elucidate the pathophysiological and functional relevance of 5-HT receptors in tumor cells and link GPCRs and tumorigenic mechanisms to pharmacological and chemotherapeutic paradigms.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

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)     

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.     

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.     

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

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

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

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

Pharmacological and Biochemical Characterization of Rat Hippocampal 5-Hydroxytryptamine1A Receptors Solubilized by 3–[3–(Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate (CHAPS)

Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r = 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5'-guanylylimidodiphosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (approximately 60 kilodaltons) and one G protein (approximately 80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5-HT1A receptors.     

Differentiation of pre- and postsynaptic high affinity serotonin receptor binding sites using physico-chemical parameters and modifying agents

The two³H-labeled agonists [³H]8-hydroxy-2-(di-n-propylamino) tetralin ([³H]8-OH-DPAT) and [³H]serotonin ([³H]5-HT) have been used to examine the effects of physico-chemical parameters and modulatory agents on the high affinity 5-HT receptor binding sites in various regions of the rat central nervous system. Sites labeled by [³H]8-OH-DPAT and [³H]5-HT were differentially sensitive to changes in incubation demperature and pH, such that the optimal interaction of [³H]8-OH-DPAT with specific sites in the striatum was at 30°C and pH 7.4, whereas [³H]5-HT sites in the same region were most easily labeled at 2–23°C and pH 8.2. Micromolar concentrations of Mn²⁺ enhanced [³H]5-HT binding but inhibited markedly [³H]8-OH-DPAT binding to striatal membranes. In contrast, both [³H]5-HT and [³H]8-OH-DPAT binding were incerased by the cation in hippocampal membranes. Conversely, GTP reduced the binding of either ligand in the hippocampus but affected only [³H]5-HT binding in the striatum. Furthermore, N-ethylmaleimide inhibited equally [³H]5-HT and [³H]8-OH-DPAT binding to hippocampal membranes, but was markedly less potent against [³H]8-OH-DPAT binding to striatal     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

Alterations in 5-HT(1A) receptors and adenylyl cyclase response by trazodone in regions of rat brain

The in vivo effect of trazodone on the density of [(3)H]5-HT binding sites and 5-HT(1A) receptors and adenylyl cyclase (AC) response was studied in regions of rat brain. The chronic administration of trazodone (10 mg/Kg body wt, 40 days) resulted in a significant downregulation of [(3)H]5-HT binding sites and 5-HT(1A) receptors in cortex and hippocampus. Trazodone significantly (p < 0.0001) decreased the density of [(3)H]5-HT binding sites in cortex (42.6 +/- 3.6 fmol/mg protein, 65%) and hippocampus (12.6 +/- 1.6 fmol/mg protein, 87%) when compared to control values of 121.9 +/- 5.4 and 99.3 +/- 7.5 fmol/mg protein in these regions, respectively. Similarly there was a significant (p < 0.0001) decrease in the density of 5-HT(1A) receptors in both cortex (7.2 +/- 0.5 fmol/mg protein, 70%) and hippocampus (6.3 +/- 1.2 fmol/mg protein, 79%) when compared to control values of 24.2 +/- 2.1 and 30.6 +/- 3.7 fmol/mg protein, in these regions respectively. However, the affinity of [(3)H]5-HT to 5-HT binding sites (1.83 +/- 0.26 nM, p < 0.0001) and [(3)H]8-OH-DPAT to 5-HT(1A) receptors (0.60 +/- 0.06 nM, p < 0.05) was significantly decreased only in cortex when compared to the control K(d) values of 0.88 +/- 0.04 nM and 0.47 +/- 0.02 nM in these regions, respectively.The basal AC activity did not alter in treated rats, where as, the inhibition of forskolin-stimulated AC activity by 5-HT (10 microM) was significantly (p < 0.0001) decreased both in cortex (43%) and hippocampus (40%) when compared to control levels. In conclusion, chronic treatment with trazodone results in downregulation of 5-HT(1A) receptors in cortex and hippocampus along with concomitant increased AC response, suggesting the involvement of 5-HT(1A) receptor-mediated AC response in the mechanism of action of trazodone.     

WAY-100635 antagonist-induced plasticity of 5-HT receptors: regulatory differences between a stable cell line and an in vivo native system

We present evidence that the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY-100635), can induce receptor internalization in a human (h)5-HT(1A) receptor Chinese hamster ovary (CHO-K1) cell system. Exposure of h5-HT(1A) CHO cells to WAY-100635 decreased the cell-surface h5-HT(1A) receptor density in a way that was both time (24-72 h) and concentration (1-100 nm) dependent.[(3)H]WAY-100635 and [(3)H]8-hydroxy-dipropylaminotetralin ([(3)H]8-OH-DPAT) saturation analyses demonstrated a significant reduction (50-60%) in total h5-HT(1A) receptor number in the WAY-100635-treated (100 nm; 72 h) compared with control cells. In WAY-100635-treated cells, the 8-OH-DPAT-mediated inhibition of forskolin (FSK)-stimulated cAMP accumulation was right-shifted and the maximal inhibitory response of 8-OH-DPAT was impaired compared with control cells. Similar results were obtained for 8-OH-DPAT-mediated Ca(2+) mobilization after WAY-100635 treatment. h5-HT(1A) receptors labeled with [(3)H]WAY-100635, as well as [(3)H]4-(2'-Methoxy)-phenyl-1-[2'-(N-2'-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF), exhibited a time-dependent rate of cellular internalization that was blocked by endocytotic suppressors and was pertussis-toxin insensitive. In contrast, quantitative autoradiographic studies demonstrated that chronic treatment of rats with WAY-100635 for two weeks produced a region-specific increase in the 5-HT(1A) receptor density. In conclusion, prolonged exposure of an h5-HT(1A) cell-based system to the 5-HT(1A) antagonist, WAY-100635, induced a paradoxical internalization of cell surface receptor resulting in depressed functional activity. This suggests that an antagonist can influence 5-HT(1A) receptor recycling in vitro differently to in vivo regulatory conditions.     

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

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

Pharmacology of [3H]R(+)-7-OH-DPAT binding in the rat caudate-putamen

Dopamine D3 receptors may be involved in drug addiction and in disorders such as schizophrenia and Parkinson's disease. To determine the pharmacological properties of dopamine D3 receptors in the rat caudate-putamen, we have investigated R(+)-[3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]R(+)-7-OH-DPAT) binding to membrane preparations from the rat caudate-putamen. Kinetic analyses showed that [3H]R(+)-7-OH-DPAT binding reached equilibrium in approximately 1 h and that both association and dissociation curves were composed of at least two components. Likewise, saturation curves showed at least two binding components with a combined Bmax value of about 600 fmol/mg protein, which is three times higher than what is present in the subcortical limbic area. Competition curves were performed with agonists such as R(-)-propylnorapomorphine, dopamine, PD 128907, quinpirole, and bromocriptine, and antagonists such as haloperidol, raclopride, clozapine, GR 218231x, remoxipride, and U99194A. These experiments revealed that [3H]R(+)-7-OH-DPAT binding could be resolved into three specific binding sites (R1-R3) and one nonspecific binding site, with R1-R2 probably representing D3 receptor binding and the minor R3 representing D2 receptor binding. The low affinities of (+/-)-8-OH-DPAT and 1,3-di(2-tolyl)guanidine to inhibit [3H]R(+)-7-OH-DPAT binding indicate negligible involvement of 5-HT1A or sigma binding sites, respectively. The pharmacological profile of [3H]R(+)-7-OH-DPAT (2 nM) binding in the caudate-putamen was similar to that of dopamine on [125I]iodosulpride binding in the cerebellar lobule X, which contain D3 but not D2 receptors. Mg2+ increased and GTP and Na+ decreased the binding of [3H]R(+)-7-OH-DPAT, suggesting a coupling of endogenous D3 receptors to G proteins. Taken together, these results suggest that dopamine D3 receptors display multiple agonist binding states, and that D3 receptors are present in high concentrations in the rat caudate-putamen. These results may have implications for the physiological and pathological roles of dopamine D3 receptors in the brain.     

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.     

Nad-299 antagonises 5-HT-stimulated and spiperone-inhibited [35S]GTPgammaS binding in cloned 5-HT1A receptors

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxytryptamine1A A (5-HT1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-3H]-3,4-dihydro-2H-1-benzopyan-5-carboxamide ([3H]NAD-299) exhibited high affinity (Kd = 0.16 nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-3H]di-n-propylamino)tetralin ([3H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [35S]GTPgammaS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [35S]GTPgammaS binding 2.5-fold while spiperone and methiothepin inhibited [35S]GTPgammaS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [35S]GTPgammaS binding to basal levels. The KiL/KiH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (+/-)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [35S] GTPgammaS (r = 0.97).     

Effects of a Chronic Lithium Treatment on Cortical Serotonin Uptake Sites and 5-HT1A Receptors

The objectives of this study were to characterize the effects of a chronic lithium (Li⁺) treatment on serotonin (5-HT) uptake sites and on 5-HT_1A receptors, and to determine the eventual reversibility of the treatment. The experiments were carried out with membranes from rat cerebral cortex using 8-hydroxy-2-(propylamino)tetralin, or [³H]8-OH-DPAT, and [³H]citalopram to label 5-HT_1A receptors and 5-HT uptake sites, respectively. Endogenous levels of 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) were measured by high-performance liquid chromatography in the cingulate cortex. The saturation curves with [³H]8-OH-DPAT were always best fitted a two-site model. After a treatment with Li⁺ for 28 days, no alterations in the binding parameters of [³H]8-OH-DPAT to the high- and low-affinity binding sites could be documented. However, competition curves with 5-HT to inhibit [³H]8-OH-DPAT binding revealed a decreased proportion of sites with high affinity for the agonist, together with an increased density of sites with low affinity for 5-HT, suggesting an alteration in the coupling efficacy between 5-HT_1A receptors and their transduction systems. Saturation studies with [³H]citalopram showed an increase (>40%) in the density of 5-HT uptake sites after chronic Li⁺, suggesting a more efficient 5-HT uptake process for the treated animals, in accord with clinical observations. Although 5-HT contents in cingulate cortex remained unchanged after the treatment, 5-HIAA levels decreased (>30%), leading to a diminished (almost 50%) 5-HT turnover; and also reflecting a more efficient uptake in the treated rats, so that less 5-HT could be degraded by extracellular monoamine oxidase. All the effects revealed by [³H]8-OH-DPAT and [³H]citalopram were reversed following a recovery period of two days without Li⁺. Since symptoms of bipolar affective disorders may reappear if the chronic Li⁺ treatment is interrupted, the reversibility of the observed effects further supports the importance of central 5-HT synaptic transmission in the pathophysiology and treatment of human affective disorders.