S33005, a novel ligand at both serotonin and norepinephrine transporters: I. Receptor binding, electrophysiological, and neurochemical profile in comparison with venlafaxine, reboxetine, citalopram, and clomipramine (original) (raw)
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The serotonin transporter from human brain: Purification and partial characterization
Neurochemistry International, 1996
The serotonin (5-HT) transporter from hhman striatum was solubilized by digitonin and purified by affinity chromatography. The native protein-detergent complex had a molecular mass of 205 kDa, as estimated by gel-exclusion chromatography of the eluates obtained from affinity chromatography. The purified 5-HT transporter migrated as a single band of 67 kDa in SDS-PAGE. To clarify the spatial relationships between the binding sites of the tricyclic antidepressants, as [3H]-imipramine, and of the selective serotonin reuptake inhibitors, as [3H]-paroxetine, on the 5-HT transporter, both radioligands were used to label it in the purification steps.
Journal of Pharmacology and Experimental Therapeutics, 2005
Previous studies identified partial inhibitors of serotonin (5-HT) transporter and dopamine transporter binding. We report here on a partial inhibitor of 5-HT transporter (SERT) binding identified among a group of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine analogs (4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-1-(2-trifluoromethyl-benzyl)-piperidine; TB-1-099). Membranes were prepared from rat brains or human embryonic kidney cells expressing the cloned human dopamine (hDAT), serotonin (hSERT), and norepinephrine (hNET) transporters. -(4Ј-125 Iodophenyl)tropan-2-carboxylic acid methyl ester ([ 125 I]RTI-55) binding and other assays followed published procedures. Using rat brain membranes, TB-1-099 weakly inhibited DAT binding (K i ϭ 439 nM), was inactive at NET binding ([ 3 H]nisoxetine), and partially inhibited SERT binding with an extrapolated plateau ("A" value) of 20%. Similarly, TB-1-099 partially inhibited [ 125 I]RTI-55 binding to hSERT with an extrapolated plateau (A value) of 14%. Upon examining the effect of increasing concentrations of TB-1-099 on the apparent K d and B max of [ 125 I]RTI-55 binding to hSERT, we found that TB-1-099 decreased the B max in a dose-dependent manner and affected the apparent K d in a manner well described by a sigmoid dose-response curve. TB-1-099 increased the K d but not to the magnitude expected for a competitive inhibitor. In rat brain synaptosomes, TB-1-099 noncompetitively inhibited [ 3 H]5-HT, but not [ 3 H]dopamine, uptake. Dissociation experiments indicated that TB-1-099 promoted the rapid dissociation of a small component of [ 125 I]RTI-55 binding to hSERT. Association experiments demonstrated that TB-1-099 slowed [ 125 I]RTI-55 binding to hSERT in a manner unlike that of the competitive inhibitor indatraline. Viewed collectively, these results support the hypothesis that TB-1-099 allosterically modulates hSERT binding and function. The serotonin (5-HT) transporter (SERT) is a member of the twelve transmembrane-spanning transporter family that transports 5-HT across cell membranes in a sodium-dependent manner (Amara and Kuhar, 1993; Uhl and Johnson, 1994). SERT, in the central nervous system, is an important target for a wide range of medications used to treat a variety of psychiatric conditions such as anxiety, depression, and obsessive-compulsive disorder (Gorman and Kent, 1999; Zohar and Westenberg, 2000). Drugs that interact with transporters generally interact with this protein in two distinct ways. Reuptake inhibitors bind to transporter proteins but are not transported. These drugs elevate extracellular concentrations of transmitter by blocking transporter-mediated uptake of transmitters from the synapse. Substrate-type releasers bind to transporter proteins and are subsequently transported into the cytoplasm of nerve terminals. Releasers elevate extracellular Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
Neuroscience, 2007
Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT ؊/؊ ) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT ؊/؊ rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in nonserotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis. transporter; SERT Ϫ/Ϫ , homozygous serotonin transporter knockout rat; SERT ϩ/Ϫ , heterozygous serotonin transporter knockout rat; SERT ϩ/ϩ , wildtype littermate of heterozygous serotonin transporter knockout and homozygous serotonin transporter knockout rats; SSRI, selective serotonin reuptake inhibitor; TPH, tryptophan hydroxylase; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HTP, 5-hydroxytryptophan.
Neuropsychopharmacology, 2013
Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [ 3 H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (45-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [ 3 H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.
Serotonin Receptor and Transporter Ligands - Current Status
Current Medicinal Chemistry, 2001
The serotonin (5-HT) receptor system has 14 different subtypes classified by pharmacology and function. Many ligands are widely used for therapeutic and diagnostic purposes in some severe human diseases. Most of the ligands that are specific for each 5-HT receptor have distinctive chemical structures with regard to pharmacophore elements including 4-arylpiperazine, benzimidazole, benzamide, chroman, aminopyridazine, tetralin, and polycycles. However, their affinity and selectivity for 5-HT, dopamine and α1 receptors depend on their substituents and linker spacers. 5-HT transporter inhibitors have also been developed as potential antidepressants. In contrast to classical tricyclic compounds, newly developed secondary amine derivatives such as paroxetine and tetralin show high binding affinity and selectivity. Radioisotope-labeled ligands have also been developed, including [carbonyl-11 C]WAY 100635 for 5-HT 1A receptor, [ 11 C or 18 F]ketanserine derivatives for 5-HT 2 receptor, [ 125 I]DAIZAC for 5-HT 3 receptor, and [ 123 I]IDAM for 5-HT transporter, and these are accumulated in brain regions that are rich in the respective receptors. This review summarizes the recent development of 5-HT receptor-and transporter-specific ligands and their pharmacological properties on the basis of their chemical structures.
ACS chemical neuroscience, 2016
Previous studies of transgenic mice carrying a single isoleucine to methionine substitution (I172M) in the serotonin transporter (SERT) demonstrated a loss of sensitivity to multiple antidepressants (ADs) at SERT. However, the ability of AD metabolites to antagonize SERT was not assessed. Here, we evaluated the selectivity and potency of these metabolites for inhibition of SERT in mouse brain-derived synaptosomes and blood platelets from wild-type (I172 mSERT) and the antidepressant-insensitive mouse M172 mSERT. The metabolites norfluoxetine and desmethylsertraline lost the selectivity demonstrated by the parent compounds for inhibition of wild-type mSERT over M172 mSERT, whereas desvenlafaxine and desmethylcitalopram retained selectivity. Furthermore, we show that the metabolite desmethylcitalopram accumulates in the brain and that the metabolites desmethylcitalopram, norfluoxetine, and desvenlafaxine inhibit serotonin uptake in wild-type mSERT at potencies similar to those of thei...
Going with the Flow: Trafficking-Dependent and -Independent Regulation of Serotonin Transport
Traffic, 2008
Antidepressant-, cocaine-and 3,4-methylenedioxymethamphetamine-sensitive serotonin (5hydroxytryptamine, 5-HT) transporters (SERTs) are expressed on presynaptic membranes of 5-HTsecreting neurons to provide efficient uptake of the biogenic amine after release. SERTs also support 5-HT transport across platelet, placental, gastrointestinal and pulmonary membranes and thus play a critical role in central nervous system and peripheral nervous system 5-HT signaling. SERTs are subject to multiple levels of posttranslational regulation that can rapidly alter 5-HT uptake and clearance rates. Specific cell surface receptors are now known to regulate SERT trafficking and/or catalytic function, with pathways activating protein kinase C, protein kinase G and p38 mitogenactivated protein kinase receiving the greatest attention. Remarkably, disease-associated mutations in SERT not only impact basal SERT activity but also selectively impact one or more SERT regulatory pathway(s). In this review, we describe both trafficking-dependent and traffickingindependent modes of SERT regulation and also the suspected roles played in regulation by SERTassociated proteins. Elucidation of the SERT 'regulome' provides important depth to our understanding of the likely origins of 5-HT-associated disorders and may help orient research to develop novel therapeutics.
European Journal of Pharmacology: Molecular Pharmacology, 1990
Citalopram binds with high affinity to a specific binding site located or, the serotonin (5-HT) transporter in 5-HT neurons. The binding affinity of [3H]citalopram was found to incre~c with increasing concentration of eitalopram. This may be a homotropic positive allosteric effect, thus indicating the presence of an allosteric binding site for citalopram. The molecular weight of the proteins containing the high-affinity binding sites for citalopram and paroxetine, as well as the allosteric binding site for eitalopram were determined by the irradiation method. The molecular weights of the three binding site proteins were found to be the same. suggesting that all three binding sites are located on the same protein molecule in the 5-HT transporter. The results support a hypothesis that the binding area for [3H]citalopram is located deeper in the transport channel than the [3H]paroxetine binding area. Thus the two high-affinity binding sites probably cover different, but overlapping, parts of the protein raolecule. The allostefic binding site may be located elsewhere on the protein where it induces conformational changes of the 5-HT transporter with the result that high-affinity bound figands get trapped in the transport channel, thereby explaining the increase in affinity.
Toxicology Letters, 2007
5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a synthetic orally active hallucinogenic tryptamine derivative, known also as Foxy or Foxy methoxy. However, few studies have examined its effects in vitro. In the present study, we investigated the actions of 5-MeO-DIPT against monoamine neurotransmitter transporters, including the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), using COS-7 cells heterologously expressing these transporters and rat brain synaptosomes. 5-MeO-DIPT specifically inhibited the uptake of [ 3 H]serotonin (5-HT) by the SERT-expressing COS-7 cells and rat striatal synaptosomes in a high affinity manner at concentrations similar to those for cocaine. The effect was reversible and competitive. 5-MeO-DIPT failed to stimulate reverse transport of [ 3 H]5-HT through SERT, while it prevented the releasing action of methamphetamine. 5-MeO-DIPT induced cell toxicity at high concentrations in COS-7 cells, and it was not influenced by the expression of SERT. These results demonstrated that 5-MeO-DIPT acts as a competitive SERT inhibitor and has an inability to cause reverse transport, underlying its serotonergic actions. (S. Kitayama). compound was placed on the U.S. DEA Schedule I list in 2004, and also has newly come under legal control as of 2005 in Japan. Clinically, it is believed to promote emotional expression, a talkative uninhibited state, and visual and auditory sensory distortion, but few clinical studies appear in the medical literature . The actions of 5-MeO-DIPT on the serotonergic system correspond to its pharmacology. However, there are few studies in the literature involving pharmacological investigations of 5-MeO-DIPT in animal models, or 0378-4274/$ -see front matter