Selective Serotonin Transporter Inhibitor Research Articles

Functional consequences of homo- but not hetero-oligomerization between transporters for the biogenic amine neurotransmitters.

Before this study, the human norepinephrine transporter (hNET) was the only member of the biogenic amine neurotransmitter transporter family that had not been demonstrated to be a functional homo-oligomer. Here, using two forms of the transporter, I155C and hNET-myc, with distinct antigenicity and inhibitor sensitivity, we demonstrated that hNET exists as a homo-oligomer. hNET I155C is a functional mutant and is sensitive to inactivation by the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate, while hNET-myc is resistant to inactivation by this reagent. Coimmunoprecipitation of these two forms demonstrated that a physical interaction exists between norepinephrine transporter monomers. Further characterization of this physical interaction has revealed that the activity of norepinephrine transporters depends on interactions between monomers. Because norepinephrine transporters and serotonin transporters are the only two members of the neurotransmitter transporter family endogenously expressed in the cell membrane of the same cells, placental syncytiotrophoblasts, we tested the ability of norepinephrine transporters and serotonin transporters to associate and function in a hetero-oligomeric form. Similarly, coexpression of hNET-myc with serotonin transporter-FLAG showed a physical interaction in coimmunoprecipitation assays. However, coexpression of serotonin and norepinephrine transporters did not sensitize norepinephrine transporter activity to inhibition by citalopram, a selective serotonin transport inhibitor. Thus, the norepinephrine transporter-serotonin transporter physical association did not produce functional consequences. Based on this, we propose that the transporters for biogenic amine neurotransmitters interact functionally in homo- but not hetero-oligomeric forms.

Synthesis, radiolabeling and preliminary biological evaluation of radiolabeled 5-methyl-6-nitroquipazine, a potential radioligand for the serotonin transporter

5-Methyl-6-nitroquipazine, a novel analogue of the potent and selective serotonin transporter inhibitor 6-nitroquipazine was synthesized and radiolabeled with tritium and the positron emitter carbon-11. [ 3H]5-methyl-6-nitroquipazine was found to have a K d=51±7 pM. The high affinity and the facile labeling of [ 11C]5-methyl-6-nitroquipazine makes it a promising radioligand for visualization of the serotonin transporter with positron emission tomography.

Synthesis of [11C]2?-carbomethoxy-3?-(3?-iodo-4?-methyl, -ethyl and isopropyl phenyl)nortropane as potential radiotracers for examination of the serotonin transporter with positron emission tomography

Research on depression and anxiety disorders would benefit from the development of suitable radioligands for PET-imaging of the serotonin transporter. Three cocaine analogues, 2β-carbomethoxy-3β-(3′-iodo-4′-methyl, -ethyl and isopropyl phenyl)nortropane (LBT-14, EINT and LBT-44), were prepared in a three-step synthesis by 1-4 addition of the appropriate Grignard reagent to anhydroecgonine methyl ester as first step. Iodination of the phenyl ring was accomplished with a mixture of yellow mercuric oxide, perchloric acid and acetic acid followed by a solution of iodine in dichloromethane. N-desmethylation was performed by using 2,2,2-trichloroethylchloroformiate followed by treatment of zinc in acetic acid. Acidic hydrolysis of the ester functions gave the carboxylic acid analogues of LBT-14, EINT and LBT-44. The precursors were labelled with 11C using [11C]methyl iodide or [11C] methyl triflate in dimethyl formamide (DMF) and tetrabutyl ammonium hydroxide (TBAH) as base. [11C]LBT-14, [11C]EINT and [11C]LBT-44 were all examined in Cynomolgus monkey with PET. All three compounds entered the monkey brain to a high degree (5∽–10% of injected dose). There was a marked uptake of radioactivity in the thalamus and the brain stem, regions known to contain serotinin transporters. Pre-treatment with the selective serotonin transporter inhibitor citalopram had minor effect on the binding ratios, suggesting that none of the three examined radioligands are preferable to the previously examined non-iodinated 4′-isopropenyl analogue [11C]RTI-357 for the study of the serotonin reuptake system with PET. Copyright © 2000 John Wiley & Sons, Ltd.

The Role of External Loop Regions in Serotonin Transport: LOOP SCANNING MUTAGENESIS OF THE SEROTONIN TRANSPORTER EXTERNAL DOMAIN

Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na(+). In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter.

Interactions of fluoxetine with metabolism of dopamine and serotonin in rat brain regions

Given evidence of inhibitory effects of serotonin on dopaminergic neurotransmission, a series of experiments sought neurochemical evidence of interactions between the selective serotonin transport inhibitor fluoxetine and the metabolism of dopamine (DA) or serotonin (5-HT) in regions of rat brain that might account for extrapyramidal side-effects associated with clinical use of fluoxetine. There were significant inhibitory effects of acute or repeated fluoxetin treatment on the turnover of 5-HT (accumulation of 5-hydroxytryptophan, or ratio of [5-hydroxyindoleacetic acid]/[5-HT]) in striatum, nucleus accumbens and frontal cerebral cortex, but only minor effects on metabolism of DA (accumulation of dihydroxyphenylalanine, or [homovanillio acid]/[DA] ratio), even at high doses or with repeated treatment, and no significant inhibition of the DA metabolism-increasing actions of haloperidol.