serotonin transporter (5-HTT) research


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(Updated 6/23/04)

Sitte, Harald H., Hiptmair, Birgit, Zwach, Julia, Pifl, Christian, Singer, Ernst A., Scholze, Petra
Quantitative Analysis of Inward and Outward Transport Rates in Cells Stably Expressing the Cloned Human Serotonin Transporter: Inconsistencies with the Hypothesis of Facilitated Exchange Diffusion
Mol Pharmacol 2001 59: 1129-1137
"Quantitative aspects of inward and outward transport of substrates by the human plasmalemmal serotonin transporter (hSERT) were investigated. Uptake and superfusion experiments were performed on human embryonic kidney 293 cells permanently expressing the hSERT using [(3)H]serotonin (5-HT) and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) as substrates. Saturation analyses rendered K(m) values of 0.60 and 17.0 microM for the uptake of [(3)H]5-HT and [(3)H]MPP(+), respectively. Kinetic analysis of outward transport was performed by prelabeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 microM). Apparent K(m) values for PCA induced transport were 564 microM and about 7 mM intracellular [(3)H]5-HT and [(3)H]MPP(+), respectively. Lowering the extracellular Na(+) concentrations in uptake and superfusion experiments revealed differential effects on substrate transport: at 10 mM Na(+) the K(m) value for [(3)H]5-HT uptake increased approximately 5-fold and the V(max) value remained unchanged. The K(m) value for [(3)H]MPP(+) uptake also increased, but the V(max) value was reduced by 50%. When efflux was studied at saturating prelabeling conditions of both substrates, PCA as well as unlabeled 5-HT and MPP(+) (all substances at saturating concentrations) induced the same efflux at 10 mM and 120 mM Na(+). Thus, notwithstanding a 50% reduction in the V(max) value of transport into the cell, MPP(+) was still able to induce maximal outward transport of either substrate. Thus, hSERT-mediated inward and outward transport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion." [Full Text]

Adams, Scott V., DeFelice, Louis J.
Ionic Currents in the Human Serotonin Transporter Reveal Inconsistencies in the Alternating Access Hypothesis
Biophys. J. 2003 85: 1548-1559
"We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore." [Abstract]

Kocabas AM, Rudnick G, Kilic F.
Functional consequences of homo- but not hetero-oligomerization between transporters for the biogenic amine neurotransmitters.
J Neurochem. 2003 Jun;85(6):1513-20.
"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." [Abstract]

Deniz Ozaslan, Sophie Wang, Billow A. Ahmed, Arif M. Kocabas, John C. McCastlain, Anca Bene, and Fusun Kilic
Glycosyl Modification Facilitates Homo- and Hetero-oligomerization of the Serotonin Transporter: A SPECIFIC ROLE FOR SIALIC ACID RESIDUES
J. Biol. Chem. 278: 43991-44000.
"The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec4 cells) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions." [Full Text]

Sammanda Ramamoorthy, Elena Giovanetti, Yan Qian, and Randy D. Blakely
Phosphorylation and Regulation of Antidepressant-sensitive Serotonin Transporters
J. Biol. Chem. 273: 2458-2466, January 1998. [Full Text]

Masson, J., Sagne, C., Hamon, M., Mestikawy, S. El
Neurotransmitter Transporters in the Central Nervous System
Pharmacol Rev 1999 51: 439-464 [Full Text]

Pineyro, Graciela, Blier, Pierre
Autoregulation of Serotonin Neurons: Role in Antidepressant Drug Action
Pharmacol Rev 1999 51: 533-591 [Full Text]

Qian, Yan, Galli, Aurelio, Ramamoorthy, Sammanda, Risso, Stefania, DeFelice, Louis J., Blakely, Randy D.
Protein Kinase C Activation Regulates Human Serotonin Transporters in HEK-293 Cells via Altered Cell Surface Expression
J. Neurosci. 1997 17: 45-57 [Full Text]

Yoel Smicun, Scott D. Campbell, Marisa A. Chen, Howard Gu, and Gary Rudnick
J. Biol. Chem. 274: 36058-36064, December 1999. [Full Text]

Andreas Androutsellis-Theotokis, Farshid Ghassemi, and Gary Rudnick
A Conformationally Sensitive Residue on the Cytoplasmic Surface of Serotonin Transporter
J. Biol. Chem. 276: 45933-45938, December 2001. [Full Text]

Christopher G. Tate, Erik Whiteley, and Michael J. Betenbaugh
Molecular Chaperones Stimulate the Functional Expression of the Cocaine-sensitive Serotonin Transporter
J. Biol. Chem. 274: 17551-17558.
"The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by the N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP." [Full Text]

Murphy, Dennis L., Lerner, Alicja, Rudnick, Gary, Lesch, Klaus-Peter
Serotonin Transporter: Gene, Genetic Disorders, and Pharmacogenetics
Mol. Interv. 2004 4: 109-123
"The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via mod-ulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette’s syndrome have detected signals in the chromosome 17q region where SERT is locat-ed. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs)." [Abstract]

Tafet GE, Idoyaga-Vargas VP, Abulafia DP, Calandria JM, Roffman SS, Chiovetta A, Shinitzky M.
Correlation between cortisol level and serotonin uptake in patients with chronic stress and depression.
Cogn Affect Behav Neurosci 2001 Dec;1(4):388-93
"In a recent study (Tafet, Toister-Achituv, & Shinitzky, 2001), we demonstrated that cortisol induces an increase in the expression of the gene coding for the serotonin transporter, associated with a subsequent elevation in the uptake of serotonin. This stimulatory effect, produced upon incubation with cortisol in vitro, was observed in peripheral blood lymphocytes from normal subjects. In the present work we investigated the cortisol-induced increase in serotonin uptake in lymphocytes from hypercortisolemic patients, including subjects with major depressive disorder (n = 8), and subjects with generalized anxiety disorder (n = 12), in comparison with a control group of normal healthy subjects (n = 8). A significant increase in serotonin uptake (+37% + 14, M + SD) was observed in the control group, whereas neither the generalized anxiety disorder nor the major depression group exhibited changes in serotonin uptake upon incubation with cortisol. It is likely that under chronic stress or depression, the capacity for increase in serotonin transporter has reached its limit due to the chronically elevated blood cortisol level. The physiological and diagnostic implications of this observation are discussed." [Abstract]

Jayanthi D. Ramamoorthy, Sammanda Ramamoorthy, Andreas Papapetropoulos, John D. Catravas, Frederick H. Leibach, and Vadivel Ganapathy
Cyclic AMP-independent Up-regulation of the Human Serotonin Transporter by Staurosporine in Choriocarcinoma Cells
J. Biol. Chem. 270: 17189-17195, 1995.
"Treatment of confluent cultures of JAR human placental choriocarcinoma cells with staurosporine caused a marked stimulation of serotonin transport activity in these cells. The stimulatory effect was noticeable at nanomolar concentrations of staurosporine, and a treatment time of > 4 h was required for staurosporine to elicit the effect. At 40 nM and with a treatment time of 16 h, the stimulation of the transport activity was 3.5-6.0-fold. None of the several other protein kinase inhibitors tested had similar effect except KT 5720, a protein kinase A inhibitor, which showed a small but significant (approximately 1.4-fold) stimulatory effect at a concentration of 5 microM. Blockade of RNA synthesis and protein synthesis in the cells prevented completely the stimulation of the transport activity induced by staurosporine. The stimulation was observed not only in intact cells but also in plasma membrane vesicles prepared from staurosporine-treated cells. The stimulation was accompanied by a 5-7-fold increase in the steady state levels of the transporter-specific mRNAs, by a 7-fold increase in the maximal velocity of the transport process, and by a 6-fold increase in the transporter density in the plasma membrane. Even though both staurosporine and cholera toxin had similar effects on the serotonin transport activity in these cells, the effect was not additive when the cells were treated with both reagents together. While treatment of the cells with cholera toxin markedly elevated intracellular levels of cAMP, staurosporine did not have any effect on the cellular levels of this cyclic nucleotide. It is concluded that staurosporine up-regulates the serotonin transport activity in JAR cells by increasing the steady state levels of the serotonin transporter mRNA and by the consequent increase in the transporter density in the plasma membrane and that the process involves a cAMP-independent signaling pathway." [Full Text]

Kekuda, Ramesh, Leibach, Frederick H., Furesz, Todd C., Smith, Carl H., Ganapathy, Vadivel
Polarized Distribution of Interleukin-1 Receptors and Their Role in Regulation of Serotonin Transporter in Placenta
J Pharmacol Exp Ther 2000 292: 1032-1041
"We investigated the expression of interleukin-1 (IL-1) receptors and their involvement in the regulation of the serotonin transporter gene expression in human placenta. IL-1beta is an activator of the serotonin transporter gene expression in JAR human placental choriocarcinoma cells as demonstrated by an increase in the steady-state levels of the transporter mRNA and in serotonin transport activity. This activation is blocked by IL-1 receptor antagonist. Genistein also blocks the effect of IL-1beta, indicating involvement of tyrosine phosphorylation in the process. Treatment of JAR cells with IL-1beta activates mitogen-activated protein kinases and nuclear factor-kappaB. The nuclear factor-kappaB that is responsive to IL-1beta in these cells is the p65 homodimer. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that JAR cells and human placenta express type I and type II IL-1 receptors. The binding sites for [125]I-IL-1beta are localized predominantly in the maternal-facing brush border membrane of the syncytiotrophoblast. These results show that IL-1 in the maternal circulation is likely to play a critical role in the regulation of the serotonin transporter gene expression in the placenta."
[Full Text]

Persico, Antonio M., Mengual, Elisa, Moessner, Rainald, Hall, Scott F., Revay, Randal S., Sora, Ichiro, Arellano, Jon, DeFelipe, Javier, Gimenez-Amaya, Jose Manuel, Conciatori, Monica, Marino, Ramona, Baldi, Alfonso, Cabib, Simona, Pascucci, Tiziana, Uhl, George R., Murphy, Dennis L., Lesch, K. Peter, Keller, Flavio
Barrel Pattern Formation Requires Serotonin Uptake by Thalamocortical Afferents, and Not Vesicular Monoamine Release
J. Neurosci. 2001 21: 6862-6873 [Full Text]

Upton, A. L., Salichon, N., Lebrand, C., Ravary, A., Blakely, R., Seif, I., Gaspar, P.
Excess of Serotonin (5-HT) Alters the Segregation of Ispilateral and Contralateral Retinal Projections in Monoamine Oxidase A Knock-Out Mice: Possible Role of 5-HT Uptake in Retinal Ganglion Cells During Development
J. Neurosci. 1999 19: 7007-7024
"In this paper we show that in MAOA-KO mice, elevated levels of brain 5-HT during the first 2 weeks of postnatal development prevent the ipsilateral and contralateral retinal projections from segregating into eye-specific areas in their target structures. Furthermore, we show that in normal and MAOA-KO mice SERT, VMAT2, and 5-HT1B are jointly expressed by a subpopulation of developing RGCs during the period of axonal remodeling. We propose that 5-HT could, via these molecules, influence retinofugal pathways and thereby help in sculpting their adult pattern of connections." [Full Text]

Salichon, Nathalie, Gaspar, Patricia, Upton, A. Louise, Picaud, Sandrine, Hanoun, Naima, Hamon, Michel, De Maeyer, Edward, Murphy, Dennis L., Mossner, Rainald, Lesch, Klaus Peter, Hen, Rene, Seif, Isabelle
Excessive Activation of Serotonin (5-HT) 1B Receptors Disrupts the Formation of Sensory Maps in Monoamine Oxidase A and 5-HT Transporter Knock-Out Mice
J. Neurosci. 2001 21: 884-896 [Full Text]

Yura A, Kiuchi Y, Uchikawa T, Uchida J, Yamazaki K, Oguchi K.
Possible involvement of calmodulin-dependent kinases in Ca(2+)-dependent enhancement of [3H]5-hydroxytryptamine uptake in rat cortex.
Brain Res 1996 Oct 28;738(1):96-102
"Effects of Ca2+ on [3H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca2+ in Krebs-Ringer medium and the uptake at 0.3-5 mM Ca2+ was 2.4-2.7 times greater than that observed in the absence of Ca2+. The maximal increase at the concentration of 1 mM Ca2+ was achieved after 2 min preincubation. Ca(2+)-dependent enhancement of the [3H]5-HT uptake reflected an increase in Vmax of the uptake process. However, Kd and Bmax values for [3H]paroxetine were not significantly changed in the presence of 1 mM Ca2+ compared with Ca(2+)-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca(2+)-free after preincubation with 1 mM Ca2+. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca(2+)-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazin e (KN-62, an inhibitor of Ca2+ /calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca(2+)-channel blockers suppressed enhancement of the uptake. These results indicate that Ca(2+)-dependent enhancement of [3H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake." [Abstract]

Ase, Ariel R., Reader, Tomas A., Hen, Rene, Riad, Mustapha, Descarries, Laurent
Regional changes in density of serotonin transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the 5-HT1B knockout.
J Neurochem 2001 78: 619-630 [Abstract]

S Ramamoorthy, DR Cool, VB Mahesh, FH Leibach, HE Melikian, RD Blakely, and V Ganapathy
Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding
J. Biol. Chem. 268: 21626-21631, October 1993.
"Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4- fold) serotonin transport activity in these cells. Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation. Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA. Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected. Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity. Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels." [Abstract/Full Text]

Roxanne A. Vaughan, Robin A. Huff, George R. Uhl, and Michael J. Kuhar
Protein Kinase C-mediated Phosphorylation and Functional Regulation of Dopamine Transporters in Striatal Synaptosomes
J. Biol. Chem. 272: 15541-15546, 1997.
"Dopamine transporters (DATs) are members of a family of Na+- and Cl-dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 µM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, ()-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4-phorbol 12,13-didecanoate at 10 µM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity." [Full Text]

Carvelli, Lucia, Moron, Jose A, Kahlig, Kristopher M, Ferrer, Jasmine V, Sen, Namita, Lechleiter, James D, Leeb-Lundberg, L. M. Fredrik, Merrill, Gerald, Lafer, Eileen M, Ballou, Lisa M, Shippenberg, Toni S, Javitch, Jonathan A, Lin, Richard Z, Galli, Aurelio
PI 3-kinase regulation of dopamine uptake
J Neurochem 2002 81: 859-869
"The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3 H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [3 H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [3 H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs." [Abstract]

Miller, Dennis K., Sumithran, Sangeetha P., Dwoskin, Linda P.
Bupropion Inhibits Nicotine-Evoked [3H]Overflow from Rat Striatal Slices Preloaded with [3H]Dopamine and from Rat Hippocampal Slices Preloaded with [3H]Norepinephrine
J Pharmacol Exp Ther 2002 302: 1113-1122
"Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent." [Abstract]

Miller, Dennis K., Wong, Erik H. F., Chesnut, M. Dathan, Dwoskin, Linda P.
Reboxetine: Functional Inhibition of Monoamine Transporters and Nicotinic Acetylcholine Receptors
J Pharmacol Exp Ther 2002 302: 687-695
"The present study determined whether repeated administration of the antidepressant and selective norepinephrine (NE) uptake inhibitor reboxetine resulted in an adaptive modification of the function of the NE transporters (NETs), serotonin (5-HT) transporters, or dopamine (DA) transporters. Because antidepressants may be effective tobacco smoking cessation agents and because antidepressants have recently been shown to interact with nicotinic acetylcholine receptors (nAChRs), the interaction of reboxetine with nAChRs was also evaluated. Repeated administration of reboxetine (10 mg/kg i.p., twice daily for 14 days) did not alter the potency or selectivity of reboxetine inhibition of [3H]NE, [3H]DA, or [3H]5-HT uptake into striatal or hippocampal synaptosomes (IC50 values = 8.5 nM, 89 µM, and 6.9 µM, respectively). In a separate series of experiments, reboxetine did not inhibit (Ki > 1 µM) [3H]methyllycaconitine, [3H]cytisine, or [3H]epibatidine binding to rat whole brain membranes. However, at concentrations that did not exhibit intrinsic activity, reboxetine potently inhibited (IC50 value = 7.29 nM) nicotine-evoked [3H]NE overflow from superfused hippocampal slices via a noncompetitive mechanism. In the latter experiments, the involvement of NET was eliminated by inclusion of desipramine (10 µM) in the superfusion buffer. Reboxetine also inhibited (IC50 value = 650 nM) nicotine-evoked 86Rb+ efflux at reboxetine concentrations that did not exhibit intrinsic activity in this assay. Thus, in addition to inhibition of NET function, reboxetine inhibits nAChR function, suggesting that it may have potential as a smoking cessation agent."

Bauman, Andrea L., Apparsundaram, Subbu, Ramamoorthy, Sammanda, Wadzinski, Brian E., Vaughan, Roxanne A., Blakely, Randy D.
Cocaine and Antidepressant-Sensitive Biogenic Amine Transporters Exist in Regulated Complexes with Protein Phosphatase 2A
J. Neurosci. 2000 20: 7571-7578
"Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration." [Full Text]

Francis, Michael M., Vazquez, Raymond W., Papke, Roger L., Oswald, Robert E.
Subtype-Selective Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Cocaine Is Determined by the alpha 4 and beta 4 Subunits
Mol Pharmacol 2000 58: 109-119 [Full Text]

Prasad PD, Leibach FH, Mahesh VB, Ganapathy V.
Human placenta as a target organ for cocaine action: interaction of cocaine with the placental serotonin transporter.
Placenta 1994 Apr;15(3):267-78
"When equilibrium interaction was allowed, cocaine inhibited the function of the transporter with a Ki of 0.09 microM. It is concluded that cocaine and its analog RTI-55 are potent inhibitors of the function of the serotonin transporter that is expressed in the normal human placenta and in cultured human placental choriocarcinoma cells. Since the reported values for cocaine concentration in the blood of cocaine users are several-fold higher than the inhibition constant for cocaine, the present study strongly suggests that the function of the placental serotonin transporter may be severely impaired by maternal use of cocaine during pregnancy. These findings may be relevant to fetal and placental complications of cocaine abuse during pregnancy." [Abstract]

Ramamoorthy JD, Ramamoorthy S, Leibach FH, Ganapathy V.
Human placental monoamine transporters as targets for amphetamines.
Am J Obstet Gynecol 1995 Dec;173(6):1782-7
"The results show that the norepinephrine transporter and, to a lesser extent, the serotonin transporter are cellular targets in the human placenta for the abusable drugs amphetamine and methamphetamine." [Abstract]

Haughey, Heather M., Fleckenstein, Annette E., Metzger, Ryan R., Hanson, Glen R.
The Effects of Methamphetamine on Serotonin Transporter Activity: Role of Dopamine and Hyperthermia
J Neurochem 2000 75: 1608-1617 [Abstract]

Brown, Pierre, Molliver, Mark E.
Dual Serotonin (5-HT) Projections to the Nucleus Accumbens Core and Shell: Relation of the 5-HT Transporter to Amphetamine-Induced Neurotoxicity
J. Neurosci. 2000 20: 1952-1963 [Full Text]

Gobbi, M., Moia, M., Pirona, L., Ceglia, I., Reyes-Parada, M., Scorza, C., Mennini, T.
p -Methylthioamphetamine and 1-(m -chlorophenyl)piperazine, two non-neurotoxic 5-HT releasers in vivo, differ from neurotoxic amphetamine derivatives in their mode of action at 5-HT nerve endings in vitro.
J Neurochem 2002 82: 1435-1443 [Abstract]

Quick MW.
Regulating the conducting states of a mammalian serotonin transporter.
Neuron. 2003 Oct 30; 40(3): 537-49.
"Serotonin transporters (SERTs), sites of psychostimulant action, display multiple conducting states in expression systems. These include a substrate-independent transient conductance, two separate substrate-independent leak conductances associated with Na(+) and H(+), and a substrate-dependent conductance of variable stoichiometry, which exceeds that predicted from electroneutral substrate transport. The present data show that the SNARE protein syntaxin 1A binds the N-terminal tail of SERT, and this interaction regulates two SERT-conducting states. First, substrate-induced currents are absent because Na(+) flux becomes strictly coupled to 5HT transport. Second, Na(+)-mediated leak currents are eliminated. These two SERT-conducting states are present endogenously in thalamocortical neurons, act to depolarize the membrane potential, and are modulated by molecules that disrupt SERT and syntaxin 1A interactions. These data show that protein interactions govern SERT activity and suggest that both cell excitability and psychostimulant-mediated effects will be dependent upon the state of association among SERT and its interacting partners." [Abstract]

Joel W. Schwartz, Randy D. Blakely, and Louis J. DeFelice
J. Biol. Chem. 278: 9768-9777.
"Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP+ (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP+ accumulation is Na+-, Cl-, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP+ competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na+ and Cl, binding is independent of Na+ and actually increases in low Cl. We further demonstrate that ASP+ interacts with transporters not only in transfected cells but in cultured neurons. ASP+ is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery."

Androutsellis-Theotokis, Andreas, Rudnick, Gary
Accessibility and Conformational Coupling in Serotonin Transporter Predicted Internal Domains
J. Neurosci. 2002 22: 8370-8378
"The intracellular topology of serotonin transporter (SERT) was examined using mutants containing single cysteine residues in the predicted cytoplasmic domain of the protein. Cysteine residues in each predicted cytoplasmic domain, including the NH2 and COOH termini and the five predicted internal loops, reacted with methanethiosulfonate (MTS) reagents only when the plasma membrane was permeabilized with digitonin or in membrane preparations but not in intact cells. The reaction was monitored by inactivation of high-affinity binding activity and by incorporation of biotin groups into the protein. Of the seven endogenous cysteine residues predicted to lie in the cytoplasmic domain, modification of only Cys-357 in the third internal loop (IL3) led to loss of activity. Cys-15 in the NH2 terminus and Cys-622 in the COOH terminus also reacted with MTS reagents. Modification of cysteine residues inserted at positions 137 in IL1, 277 in IL2, and 441 in IL4 also led to inactivation, and at positions 157 in IL1 and 532 in IL5, cysteine was modified without an effect on binding activity. These results are in agreement with the originally proposed topology for SERT and argue against an alternative topology proposed for the closely related GABA and glycine transporters. The reactivity of many of the cytoplasmic cysteine residues studied was influenced by ion and ligand binding, suggesting that the internal domains of SERT participate in conformational changes during neurotransmitter transport." [Abstract]

Fusun Kilic, and Gary Rudnick
Oligomerization of serotonin transporter and its functional consequences
PNAS 97: 3106-3111; published online before print as 10.1073/pnas.060408997
"Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer." [Full Text]

Kilic, Fusun, Murphy, Dennis L., Rudnick, Gary
A Human Serotonin Transporter Mutation Causes Constitutive Activation of Transport Activity
Mol Pharmacol 2003 64: 440-446
"A rarely occurring variant of human serotonin transporter (hSERT) was tested for its functional consequences in HeLa and COS-7 cells. The variant, in which Ile-425 is converted to Val, was significantly different from wild type with respect to its catalytic properties. In both cell types, rates of serotonin (5-HT) transport were higher for the I425V variant. Both an increase in Vmax and a decrease in KM caused this increase in rate. The increase in Vmax was not accounted for by increases in transporter expression or in the distribution of transporter between the cell surface and intracellular pools. The decrease in KM was accompanied by a decrease in the KD for binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane. In both HeLa and COS-7 cells, the nitric oxide donor S-nitroso-N-acetylpenicillamine increased the activity of wild-type hSERT to that of the variant but did not change the activity of the I425V variant. This stimulation was prevented by the presence of oxyhemoglobin, which quenches nitric oxide, and by an inhibitor of guanylyl cyclase." [Abstract]

KJ Miller, and BJ Hoffman
Adenosine A3 receptors regulate serotonin transport via nitric oxide and cGMP
J. Biol. Chem. 269: 27351-27356, November 1994.
"Many antidepressants inhibit 5-hydroxytryptamine (5HT) transport resulting in increased 5HT levels in the synapse. However, physiological regulation of neurotransmitter uptake has not been demonstrated. We have examined the effect of receptor-activated second messengers on the 5HT transporter in rat basophilic leukemia cells (RBL 2H3). Here, we show that activation of an A3 adenosine receptor results in an increase of 5HT uptake in RBL cells, due to an increase in maximum velocity (Vmax). The A3 adenosine receptor-stimulated increase in transport is blocked by inhibitors of nitric oxide synthase and by a cGMP-dependent kinase inhibitor. In fact, compounds that generate nitric oxide (NO) and the cGMP analog 8-bromo-cGMP mimicked the effect of A3 receptor stimulation, suggesting that the elevation in transport occurs through the generation of the gaseous second messenger NO and a subsequent elevation in cGMP. Additionally, the 5HT transporter is differentially regulated by second messengers since direct activation of protein kinase C by phorbol esters decreases 5HT uptake by decreasing Vmax. Our results suggest that the changes in transport are due to a direct modification of the 5HT transporter, possibly by phosphorylation, which appears to alter the rate at which transport occurs. As the 5HT transporter in RBL cells is identical to that in neurons, our results suggest that analogous mechanisms may operate in the brain." [Abstract/Full Text]

I. Scott Ramsey, and Louis J. DeFelice
Serotonin Transporter Function and Pharmacology Are Sensitive to Expression Level. EVIDENCE FOR AN ENDOGENOUS REGULATORY FACTOR
J. Biol. Chem. 277: 14475-14482, April 2002.
"We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires ~5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity."

Ramamoorthy, Sammanda, Blakely, Randy D.
Phosphorylation and Sequestration of Serotonin Transporters Differentially Modulated by Psychostimulants
Science 1999 285: 763-766
"Many psychotropic drugs interfere with the reuptake of dopamine, norepinephrine, and serotonin. Transport capacity is regulated by kinase-linked pathways, particularly those involving protein kinase C (PKC), resulting in transporter phosphorylation and sequestration. Phosphorylation and sequestration of the serotonin transporter (SERT) were substantially impacted by ligand occupancy. Ligands that can permeate the transporter, such as serotonin or the amphetamines, prevented PKC-dependent SERT phosphorylation. Nontransported SERT antagonists such as cocaine and antidepressants were permissive for SERT phosphorylation but blocked serotonin effects. PKC-dependent SERT sequestration was also blocked by serotonin. These findings reveal activity-dependent modulation of neurotransmitter reuptake and identify previously unknown consequences of amphetamine, cocaine, and antidepressant action." [Full Text]

Whitworth, Terri L., Herndon, Laura C., Quick, Michael W.
Psychostimulants Differentially Regulate Serotonin Transporter Expression in Thalamocortical Neurons
J. Neurosci. 2002 22: 192-
"5-HT transporters (SERTs) are transiently expressed in thalamocortical neurons during development, permitting these glutamatergic neurons to co-release 5-HT as a "borrowed" transmitter. The high level of SERT expression in these neurons is likely important in the serotonergic modulation of neocortical circuits and provides a system for examining endogenous SERT regulation. We tested the hypothesis that developmental expression of SERT in thalamocortical neurons is regulated by psychostimulants that are agonists and antagonists of SERT. Cultured thalamocortical neurons from embryonic day 18 rats were examined for SERT expression until P15. In untreated cultures, SERT protein levels peaked at postnatal day 3 (P3) and were absent by P10. Chronic treatment with SERT substrates (5-HT, 3,4-methylenedioxymethamphetamine) increased both peak SERT protein levels (fourfold) and the time course of SERT expression. SERT substrates also shifted the relative functional expression of SERT by redistributing intracellular SERT protein to the plasma membrane. The subcellular redistribution was prevented by PKC activators. SERT antagonists (e.g., fluoxetine, cocaine) reduced total SERT expression levels and the time course of SERT expression. These data (1) show that endogenous SERT is differentially regulated by 5-HT and psychostimulants, (2) indicate that SERT modulation occurs via changes in both total SERT protein levels and subcellular redistribution of the transporter, and (3) suggest that some of the actions of drugs of abuse in neocortical development may be attributable to alterations in SERT expression and concomitant changes in 5-HT signaling." [Full Text]

G Rudnick, and SC Wall
The Molecular Mechanism of "Ecstasy" [3,4-Methylenedioxy-Methamphetamine (MDMA)]: Serotonin Transporters are Targets for MDMA-Induced Serotonin Release
PNAS 89: 1817-1821, 1992.
"MDMA ("ecstasy") has been widely reported as a drug of abuse and as a neurotoxin. This report describes the mechanism of MDMA action at serotonin transporters from plasma membranes and secretory vesicles. MDMA stimulates serotonin efflux from both types of membrane vesicle. In plasma membrane vesicles isolated from human platelets, MDMA inhibits serotonin transport and [3H]imipramine binding by direct interaction with the Na+-dependent serotonin transporter. MDMA stimulates radiolabel efflux from plasma membrane vesicles preloaded with [3H]serotonin in a stereospecific, Na+-dependent, and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, which contain the vesicular biogenic amine transporter, MDMA inhibits ATP-dependent [3H]serotonin accumulation and stimulates efflux of previously accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due to dissipation of the transmembrane pH difference generated by ATP hydrolysis and to direct interaction with the vesicular amine transporter." [Abstract/Full Text]

Fletcher PJ, Korth KM, Robinson SR, Baker GB.
Multiple 5-HT receptors are involved in the effects of acute MDMA treatment: studies on locomotor activity and responding for conditioned reinforcement.
Psychopharmacology (Berl) 2002 Jul;162(3):282-91
"AbstractRATIONALE. Responding for conditioned reinforcement is increased by the dopamine releasing agent amphetamine, but reduced by drugs that enhance serotonin (5-HT) function. The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) releases both monoamines.OBJECTIVES. The primary purpose of this study was to examine the effects of MDMA on responding for conditioned reinforcement as well as on locomotor activity. The roles of several 5-HT receptor sub-types in mediating these behavioural effects of MDMA were also examined.METHODS. Locomotion was measured in photocell activity monitors. For conditioned reinforcement experiments thirsty rats learned to associate a conditioned stimulus (CS) with water in operant chambers. Subsequently, two response levers were available; responding on one lever delivered the CS, while responding on the second lever had no consequences. Drug effects on this operant response were measured.RESULTS. MDMA dose-dependently increased locomotion but reduced responding for conditioned reinforcement. This latter effect differs from that induced by amphetamine, which potentiates conditioned reinforcement responding. The stimulant effect of MDMA was attenuated by GR127935 and ketanserin, indicating facilitatory roles of 5-HT(1B) and 5-HT(2A) receptors in mediating this effect. The 5-HT(2C) antagonist SB242084 enhanced the stimulant effect of MDMA. Only SB242084 attenuated the suppressant effect of MDMA on responding for conditioned reinforcement.CONCLUSIONS. The results show that 5-HT(2A) and 5-HT(1B/1D) receptors play a facilitatory role in mediating the stimulant effect of MDMA, whereas 5-HT(2C) receptors are inhibitory. Activation of 5-HT(2C) receptors also contributes to the deficit in operant responding. Multiple 5-HT receptor sub-types appear to contribute to the behavioural effects of MDMA." [Abstract]

Bengel, Dietmar, Murphy, Dennis L., Andrews, Anne M., Wichems, Christine H., Feltner, Douglas, Heils, Armin, Mossner, Rainald, Westphal, Heiner, Lesch, Klaus-Peter
Altered Brain Serotonin Homeostasis and Locomotor Insensitivity to 3,4-Methylenedioxymethamphetamine ("Ecstasy") in Serotonin Transporter-Deficient Mice
Mol Pharmacol 1998 53: 649-655 [Full Text]

Byrne SE, Rothschild AJ.
Loss of antidepressant efficacy during maintenance therapy: possible mechanisms and treatments.
J Clin Psychiatry 1998 Jun;59(6):279-88
"BACKGROUND: Many patients with unipolar depression experience a return of depressive symptoms while taking a constant maintenance dose of an antidepressant. METHOD: All cited studies were found using computerized literature searches of the MEDLINE database since 1966. RESULTS: The return of depressive symptoms during maintenance antidepressant treatment has occurred in 9% to 57% of patients in published trials. Possible explanations include loss of placebo effect, pharmacologic tolerance, increase in disease severity, change in disease pathogenesis, the accumulation of a detrimental metabolite, unrecognized rapid cycling, and prophylactic inefficacy. CONCLUSION: Although several strategies have been proposed to overcome the loss of antidepressant efficacy, double-blind controlled studies are needed to ascertain the optimal strategy for this perplexing clinical problem." [Abstract]

Fryer, John D, Lukas, Ronald J
Antidepressants Noncompetitively Inhibit Nicotinic Acetylcholine Receptor Function
J Neurochem 1999 72: 1117-1124
"Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression." [Abstract]

Hennings, Esteban C. P., Kiss, Janos P., Oliveira, Karine De, Toth, Peter T., Vizi, E. Sylvester
Nicotinic Acetylcholine Receptor Antagonistic Activity of Monoamine Uptake Blockers in Rat Hippocampal Slices
J Neurochem 1999 73: 1043-1050
"The aim of our study was to investigate the effect of different monoamine uptake blockers on the nicotine-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices. We found that desipramine (DMI), nisoxetine, cocaine, citalopram, and nomifensine inhibit the nicotine-evoked release of [3H]NA with an IC50 of 0.36, 0.59, 0.81, 0.93, and 1.84 µM, respectively. These IC50 values showed no correlation with the inhibitory effect (Ki) of monoamine uptake blockers on the neuronal NA transporter (r = 0.17, slope = 0.02), indicating that the NA uptake system is not involved in the process. In whole-cell patch clamp experiments neither drug blocked Na+ currents at 1 µM in sympathetic neurons from rat superior cervical ganglia, and only DMI produced a pronounced inhibition (52% decrease) at 10 µM. Comparison of the effect of DMI and tetrodotoxin (TTX) on the electrical stimulation- and nicotine-evoked release of [3H]NA showed that DMI, in contrast to TTX, inhibits only the nicotine-induced response, indicating that the target of DMI is not the Na+ channel. Our data suggest that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the nicotinic acetylcholine receptors in the CNS. Because these compounds are widely used in the therapy of depressed patients, our findings may have great importance in the evaluation of their clinical effects." [Abstract]

XM Guan and WJ McBride
Fluoxetine increases the extracellular levels of serotonin in the nucleus accumbens.
Brain Res Bull, Jul 1988; 21(1): 43-6.
"The effects of an IP injection of the monoamine uptake inhibitor fluoxetine on the extracellular concentration of serotonin (5-HT), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens of awake and freely moving rats were examined using a push-pull perfusion technique. Baseline values of 5-HT, 5-HIAA, DA, DOPAC and HVA in the perfusates were approximately 0.07, 13, 0.8, 49 and 12 pmol/hr, respectively. The IP administration of 5 and 10 mg/kg fluoxetine dose-dependently elevated the amounts of 5-HT 3- and 13-fold, respectively, in the push-pull perfusate, with the maximum reached within one hour after drug administration. Moreover, 10 mg/kg fluoxetine also significantly decreased the levels of 5-HIAA in the perfusate as much as 50% within 2-3 hours. On the other hand, no significant effect of 5 or 10 mg/kg fluoxetine was observed on the contents of DA, DOPAC and HVA in the push-pull perfusates. The data indicate that fluoxetine, in accord with its role as a 5-HT uptake inhibitor, increases the physiologically active pool of 5-HT in the nucleus accumbens under in vivo conditions."

Cabrera-Vera, Theresa M., Garcia, Francisca, Pinto, Wilfred, Battaglia, George
Effect of Prenatal Fluoxetine (Prozac) Exposure on Brain Serotonin Neurons in Prepubescent and Adult Male Rat Offspring
J Pharmacol Exp Ther 1997 280: 138-145
"The present study examines the consequences of prenatal fluoxetine exposure on brain serotonin [5-hydroxytryptamine (5-HT)] neurons in male offspring. Pregnant rats were administered either saline or fluoxetine (10 mg/kg s.c.) daily from gestational day 13 through gestational day 20. The biochemical status of brain 5-HT neurons was assessed in prepubescent and adult offspring by measuring 1) the 5-HT and 5-hydroxyindoleacetic acid content, 2) the density of [3H]paroxetine-labeled 5-HT uptake sites and 3) the ability of the 5-HT-releasing drug p-chloroamphetamine to reduce 5-HT content. Biochemical parameters were assessed in the frontal cortex, hypothalamus, hippocampus, striatum and midbrain. Comparative effects on dopamine and norepinephrine content in selected regions were also determined. Prenatal exposure to fluoxetine significantly reduced (28%) 5-HT content in the frontal cortex of prepubescent but not adult male offspring. In contrast, in adult progeny prenatal fluoxetine exposure produced a significant decrease only in midbrain 5-HT content (28%). In addition, p-chloroamphetamine markedly reduced 5-HT content in all brain regions examined, but the ability of p-chloroamphetamine to reduce 5-HT content was significantly attenuated only in the midbrain of adult progeny prenatally exposed to fluoxetine. No significant differences were observed between control and fluoxetine-exposed progeny with respect to brain 5-hydroxyindoleacetic acid content, the 5-hydroxyindoleacetic acid/5-HT ratio or the density of 5-HT uptake sites, regardless of the brain region examined or the age of the offspring. These data provide additional evidence that prenatal exposure to fluoxetine can produce limited, rather than global, changes in brain 5-HT neurons in male rat offspring and that the effects observed are region-specific and age-dependent. The potential functional consequences and clinical implications of these alterations in brain 5-HT systems remain to be elucidated." [Full Text]

Thomas, Dierk, Gut, Bernd, Wendt-Nordahl, Gunnar, Kiehn, Johann
The Antidepressant Drug Fluoxetine Is an Inhibitor of Human Ether-A-Go-Go-Related Gene (HERG) Potassium Channels
J Pharmacol Exp Ther 2002 300: 543-548
"Fluoxetine is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Although this group of antidepressant drugs is generally believed to cause fewer proarrhythmic side effects compared with tricyclic antidepressants, serious concerns have been raised by case reports of tachycardia and syncopes associated with fluoxetine treatment. To determine the electrophysiological basis for the arrhythmogenic potential of fluoxetine, we investigated the effects of this drug on cloned human ether-a-go-go-related gene (HERG) potassium channels heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. We found that fluoxetine blocked HERG channels with an IC50 value of 3.1 µM. Inhibition occurred fast to open channels with very slow unbinding kinetics. Analysis of the voltage dependence of block revealed loss of inhibition at membrane potentials greater than 40 mV, indicating that channel inactivation prevented block by fluoxetine. No pronounced changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or inactivation time constant could be observed, and block was not frequency-dependent. This is the first study demonstrating that HERG potassium channels are blocked by the selective serotonin reuptake inhibitor fluoxetine. We conclude that HERG current inhibition might be an explanation for the arrhythmogenic side effects of this drug."

J. García-Colunga, J. N. Awad, and R. Miledi
Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac)
PNAS 94: 2041-2044, March 1997.
"Fluoxetine (Prozac), a widely used antidepressant, is said to exert its medicinal effects almost exclusively by blocking the serotonin uptake systems. The present study shows that both muscle and neuronal nicotinic acetylcholine receptors are blocked, in a noncompetitive and voltage-dependent way, by fluoxetine, which also increases the rate of desensitization of the nicotinic receptors. Because these receptors are very widely distributed in the both central and peripheral nervous systems, the blocking action of fluoxetine on nicotinic receptors may play an important role in its antidepressant and other therapeutical effects. Our findings will help to understand the mode of action of fluoxetine, and they may also help to develop more specific medicinal drugs."
[Full Text]

Blakely, Randy D.
Physiological Genomics of Antidepressant Targets: Keeping the Periphery in Mind
J. Neurosci. 2001 21: 8319-8323
"The plasma membrane transporters that clear extracellular serotonin (5-HT) and norepinephrine (NE), serotonin transporters (SERTs) and NE transporters (NETs), have received considerable attention over the past four decades because of their roles in amine neurotransmitter inactivation. In addition, they interact with many centrally active drugs, including multiple classes of antidepressants such as the serotonin-selective reuptake inhibitors, typified by fluoxetine (Prozac), and the more recently developed norepinephrine-selective transporter antagonists, such as reboxetine. The therapeutic utility of these agents supports biogenic amine theories of affective disorders and raises the question as to whether SERT and NET exhibit a functional genetic variation that could influence risk for behavioral disorders. Although evidence exists that a promoter polymorphism in SERT may influence behavioral states, this contention is not without complexity and its mechanism of action remains poorly understood. The identification of coding variants of NETs and SERTs would offer important opportunities to connect genotype to phenotype. However, given the limited frequency of transporter coding variations evident to date in general population surveys or in psychiatric genetic studies, the identification of informative functional variants of transporters will likely require refined phenotypes. In this regard, NET and SERT play critical roles in cardiovascular and gastrointestinal physiology, respectively. This perspective reviews recent human and mouse studies that suggest how peripheral autonomic phenotypes, linked to genetic disruption of NET and SERT function, can aid in the phenotypic segregation needed for advanced theories of biogenic amine dysfunction and pharmacogenetics." [Abstract]

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Recent Serotonin Transporter Research

1) Brown GW, Ban M, Craig TK, Harris TO, Herbert J, Uher R
Depress Anxiety. 2012 Jul 27;
BACKGROUND: Key questions about the interaction between the serotonin transporter length polymorphism (5-HTTLPR) and stress in the etiology of depression remain unresolved. We test the hypotheses that the interaction is restricted to childhood maltreatment (as opposed to stressful events in adulthood), and leads to chronic depressive episodes (as opposed to any onset of depression), using gold-standard assessments of childhood maltreatment, severe life events, chronic depression, and new depressive onsets. METHOD: In a risk-enriched sample of 273 unrelated women, childhood maltreatment was retrospectively assessed with the Childhood Experience of Care and Abuse (CECA) interview and 5-HTTLPR was genotyped. A subset of 220 women was followed prospectively for 12 months with life events assessed with the Life Events and Difficulties (LEDS) interview. Any chronic episode of depression (12 months or longer) during adulthood and onset of a major depressive episode during a 12-month follow-up were established with the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) interview. RESULTS: The short alleles of 5-HTTLPR moderated the relationship between childhood maltreatment and chronic depression in adulthood, reflected in a significant gene-environment interaction (RD = 0.226, 95% CI: 0.076-0.376, P = .0032). 5-HTTLPR did not moderate the effects of either childhood maltreatment or severe life events on new depressive onsets. CONCLUSIONS: The short variant of the serotonin transporter gene specifically sensitizes to the effect of early-life experience of abuse or neglect on whether an adult depressive episode takes a chronic course. This interaction may be responsible for a substantial proportion of cases of chronic depression in the general population. Depression and Anxiety 00:1-9, 2012. © 2012 Wiley Periodicals, Inc. [PubMed Citation] [Order full text from Infotrieve]

2) Fisher HL, Cohen-Woods S, Hosang GM, Korszun A, Owen M, Craddock N, Craig IW, Farmer AE, McGuffin P, Uher R
Interaction between specific forms of childhood maltreatment and the serotonin transporter gene (5-HTT) in recurrent depressive disorder.
J Affect Disord. 2012 Jul 25;
BACKGROUND: There is inconsistent evidence of interaction between stressful events and a serotonin transporter promoter polymorphism (5-HTTLPR) in depression. Recent studies have indicated that the moderating effect of 5-HTTLPR may be strongest when adverse experiences have occurred in childhood and the depressive symptoms persist over time. However, it is unknown whether this gene-environment interaction is present for recurrent depressive disorder and different forms of maltreatment. Therefore, patients with recurrent clinically diagnosed depression and controls screened for the absence of depression were utilised to examine the moderating effect of 5-HTTLPR on associations between specific forms of childhood adversity and recurrent depression. METHOD: A sample of 227 recurrent unipolar depression cases and 228 never psychiatrically ill controls completed the Childhood Trauma Questionnaire to assess exposure to sexual, physical and emotional abuse, physical and emotional neglect in childhood. DNA extracted from blood or cheek swabs was genotyped for the short (s) and long (l) alleles of 5-HTTLPR. RESULTS: All forms of childhood maltreatment were reported as more severe by cases than controls. There was no direct association between 5-HTTLPR and depression. Significant interactions with additive and recessive 5-HTTLPR genetic models were found for overall severity of maltreatment, sexual abuse and to a lesser degree for physical neglect, but not other maltreatment types. LIMITATIONS: The cross-sectional design limits causal inference. Retrospective report of childhood adversity may have reduced the accuracy of the findings. CONCLUSIONS: This study provides support for the role of interplay between 5-HTTLPR and a specific early environmental risk in recurrent depressive disorder. [PubMed Citation] [Order full text from Infotrieve]

3) Spiller R, Lam C
An Update on Post-infectious Irritable Bowel Syndrome: Role of Genetics, Immune Activation, Serotonin and Altered Microbiome.
J Neurogastroenterol Motil. 2012 Jul;18(3):258-68.
The literature on post-infectious irritable bowel syndrome (IBS) is reviewed with special emphasis on recent new data. Further accounts of this phenomenon continue to be reported following a range of infections including giardiasis as well as viral and bacterial gastroenteritis. Risk factors such as severity of initial illness, female gender together with adverse psychological factors have been confirmed. Recent evidence of a genetic predisposition needs replication. Animal studies suggest activation of mast cells and inflammation driven impairment of serotonin transporter may be important, which are findings supported by some recent human studies in IBS with diarrhoea. Experimentally induced inflammation leads to damage and remodelling of enteric nerves. Similar changes have been reported in IBS patients with increase in nerves expressing transient receptor potential cation channel V1. While changes in microbiota are very likely this area has yet to be explored using modern techniques. Since the prognosis is for slow improvement, treatments should currently target the key symptoms of diarrhoea and abdominal pain. Future therapies aimed at correcting underlying mechanisms including immune activation and serotonin excess are currently being explored and may provide better treatments in the future. [PubMed Citation] [Order full text from Infotrieve]

4) Priess-Groben HA, Hyde JS
5-HTTLPR X Stress in Adolescent Depression: Moderation by MAOA and Gender.
J Abnorm Child Psychol. 2012 Jul 27;
Depression surges in adolescence, especially among girls. Most evidence indicates that the short allele of a polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) interacts with stress to influence the onset of depression. This effect appears to be less robust in adolescents, particularly among boys, and may be moderated by other genetic polymorphisms. Seeking to explain the adolescent gender difference in depression, this study examined the effects of 5-HTTLPR (rs25531), the monoamine oxidase A-upstream variable number tandem repeat (MAOA-uVNTR), and negative life events (NLE). A community-based longitudinal sample of 309 adolescents reported depressive symptoms and NLE at ages 11, 13, and 15. 5-HTTLPR and MAOA-uVNTR genotypes were ascertained via buccal swabs. A significant four-way interaction of 5-HTTLPR, MAOA-uVNTR, NLE at age 13, and gender predicted depressive symptoms at age 15. Girls were most likely to exhibit elevated depressive symptoms when experiencing NLE if they possessed low-expression MAOA-uVNTR alleles and short 5-HTTLPR alleles, whereas low-expression MAOA-uVNTR alleles but long 5-HTTLPR alleles were implicated in boys. The results indicate that the commonly reported 5-HTTLPR by stress interaction for depression may be limited to individuals with low-expression MAOA-uVNTR alleles. These data also provide new evidence that the short allele of 5-HTTLPR confers susceptibility to stress differently for females compared with males. [PubMed Citation] [Order full text from Infotrieve]

5) Rylands AJ, Hinz R, Jones M, Holmes SE, Feldmann M, Brown G, McMahon AW, Talbot PS
Pre- and Postsynaptic Serotonergic Differences in Males with Extreme Levels of Impulsive Aggression Without Callous Unemotional Traits: A Positron Emission Tomography Study Using (11)C-DASB and (11)C-MDL100907.
Biol Psychiatry. 2012 Jul 24;
BACKGROUND: Impulsive aggression (IA) in adults is associated with brain serotonin (5-HT) system abnormalities and is more common following childhood adversity. Within aggressive behavior, IA and callous unemotional (CU) traits are core components of differentiable factors with opposing 5-HT abnormalities. We aimed to investigate 5-HT abnormalities in IA and potential correlations with severity of childhood adversity while controlling for confounding 5-HT effects of high CU traits and mental disorders. METHODS: Healthy male subjects (mean age 34 ± 9 years) without high CU traits were recruited with IA ratings in the high (n = 14) and low (n = 13) population extremes. Serotonin transporter (SERT) and 5-HT(2A) receptor availability was measured in multiple brain regions using positron emission tomography with (11)C-DASB and (11)C-MDL100907, respectively, and compared between high-IA and low-IA groups. Correlations were measured between SERT and 5-HT(2A) receptor availability, impulsivity and aggression, and childhood adversity. RESULTS: Compared with the low-IA group, SERT were significantly higher in brainstem regions in the high-IA group (by 29.0% ± 11.4%) and modestly lower across cortical regions (by 11.1% ± 6.0%), whereas 5-HT(2A) receptors were also modestly lower (by 8.6% ± 4.0%). Across all subjects, brainstem SERT were significantly positively correlated with impulsivity, aggression, and childhood trauma ratings. Within the high-IA group, higher brainstem SERT was most strongly predicted by severity of childhood trauma (r = .76 in midbrain). CONCLUSIONS: Pre-and postsynaptic 5-HT differences are present in men with high levels of IA and are strongly suggestive of a persisting effect of childhood adversity on serotonergic neurodevelopment and emotional-behavioral control. [PubMed Citation] [Order full text from Infotrieve]

6) Pascale E, Lucarelli M, Passarelli F, Butler RH, Tamellini A, Addessi E, Visalberghi E, Manciocco A, Vitale A, Laviola G
Monomorphic Region of the Serotonin Transporter Promoter Gene in New World Monkeys.
Am J Primatol. 2012 Jul 25;
Genetic variation in the human serotonin system has long been studied because of its functional consequences and links to various neuropsychiatric and behavior-related disorders. Among non-human primates, the common marmosets (Callithrix jacchus) and tufted capuchins monkeys (Cebus apella) are becoming increasingly used as models to study the effects of genes, environments, and their interaction on physiology and complex behavior. In order to investigate the independent functions of and potential interactions between serotonin-related genes, anxiety and neuropsychiatric disorders, we analyzed the presence and variability of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) in marmoset and capuchin monkeys. By PCR and using heterologous primers from the human sequence, we amplified and then sequenced the corresponding 5-HTT region in marmosets and capuchins. The resulting data revealed the presence of a tandem repeat sequence similar to that described in humans, but unlike humans and other Old World primates, no variable length alleles were detected in these New World monkeys, suggesting that if serotonin transporter is involved in modulating behavior in these animals it does so through different molecular mechanisms. Am. J. Primatol. 00:1-6, 2012. © 2012 Wiley Periodicals, Inc. [PubMed Citation] [Order full text from Infotrieve]

7) Payer DE, Nurmi EL, Wilson SA, McCracken JT, London ED
Effects of methamphetamine abuse and serotonin transporter gene variants on aggression and emotion-processing neurocircuitry.
Transl Psychiatry. 2012 Feb 21;2:e80.
Individuals who abuse methamphetamine (MA) exhibit heightened aggression, but the neurobiological underpinnings are poorly understood. As variability in the serotonin transporter (SERT) gene can influence aggression, this study assessed possible contributions of this gene to MA-related aggression. In all, 53 MA-dependent and 47 control participants provided self-reports of aggression, and underwent functional magnetic resonance imaging while viewing pictures of faces. Participants were genotyped at two functional polymorphic loci in the SERT gene: the SERT-linked polymorphic region (SERT-LPR) and the intron 2 variable number tandem repeat polymorphism (STin2 VNTR); participants were then classified as having high or low risk for aggression according to individual SERT risk allele combinations. Comparison of SERT risk allele loads between groups showed no difference between MA-dependent and control participants. Comparison of self-report scores showed greater aggression in MA-dependent than control participants, and in high genetic risk than low-risk participants. Signal change in the amygdala was lower in high genetic risk than low-risk participants, but showed no main effect of MA abuse; however, signal change correlated negatively with MA use measures. Whole-brain differences in activation were observed between MA-dependent and control groups in the occipital and prefrontal cortex, and between genetic high- and low-risk groups in the occipital, fusiform, supramarginal and prefrontal cortex, with effects overlapping in a small region in the right ventrolateral prefrontal cortex. The findings suggest that the investigated SERT risk allele loads are comparable between MA-dependent and healthy individuals, and that MA and genetic risk influence aggression independently, with minimal overlap in associated neural substrates. [PubMed Citation] [Order full text from Infotrieve]

8) Singh YS, Altieri SC, Gilman TL, Michael HM, Tomlinson ID, Rosenthal SJ, Swain GM, Murphey-Corb MA, Ferrell RE, Andrews AM
Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells.
Transl Psychiatry. 2012 Feb 21;2:e77.
The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans. [PubMed Citation] [Order full text from Infotrieve]

9) Agren T, Furmark T, Eriksson E, Fredrikson M
Human fear reconsolidation and allelic differences in serotonergic and dopaminergic genes.
Transl Psychiatry. 2012 Feb 21;2:e76.
Fear memory persistence, central for the development and maintenance of anxiety disorders, is partially genetically controlled. Recently, consolidation and reconsolidation processes have been reported to affect fear memory stability and integrity. This study explored the impact of reconsolidation processes and genetic make-up on fear reacquisition by manipulating reconsolidation, using extinction performed outside or inside a reconsolidation interval. Reacquisition measured by skin conductance responses was stronger in individuals that extinguished outside (6?h) than inside (10?min) the reconsolidation interval. However, the effect was predominantly present in val/val homozygotes of the functional val158met polymorphism of the catechol O-methyltransferase (COMT) enzyme and in short-allele carriers of the serotonin-transporter length 5-HTTLPR polymorphism. These results demonstrate that reconsolidation of human fear memory is influenced by dopamine and serotonin-related genes. [PubMed Citation] [Order full text from Infotrieve]

10) Surguladze SA, Radua J, El-Hage W, Gohier B, Sato JR, Kronhaus DM, Proitsi P, Powell J, Phillips ML
Interaction of catechol O-methyltransferase and serotonin transporter genes modulates effective connectivity in a facial emotion-processing circuitry.
Transl Psychiatry. 2012 Feb 21;2:e70.
Imaging genetic studies showed exaggerated blood oxygenation level-dependent response in limbic structures in carriers of low activity alleles of serotonin transporter-linked promoter region (5-HTTLPR) as well as catechol O-methyltransferase (COMT) genes. This was suggested to underlie the vulnerability to mood disorders. To better understand the mechanisms of vulnerability, it is important to investigate the genetic modulation of frontal-limbic connectivity that underlies emotional regulation and control. In this study, we have examined the interaction of 5-HTTLPR and COMT genetic markers on effective connectivity within neural circuitry for emotional facial expressions. A total of 91 healthy Caucasian adults underwent functional magnetic resonance imaging experiments with a task presenting dynamic emotional facial expressions of fear, sadness, happiness and anger. The effective connectivity within the facial processing circuitry was assessed with Granger causality method. We have demonstrated that in fear processing condition, an interaction between 5-HTTLPR (S) and COMT (met) low activity alleles was associated with reduced reciprocal connectivity within the circuitry including bilateral fusiform/inferior occipital regions, right superior temporal gyrus/superior temporal sulcus, bilateral inferior/middle prefrontal cortex and right amygdala. We suggest that the epistatic effect of reduced effective connectivity may underlie an inefficient emotion regulation that places these individuals at greater risk for depressive disorders. [PubMed Citation] [Order full text from Infotrieve]

11) Yan C, Xin-Guang L, Hua-Hong W, Jun-Xia L, Yi-Xuan L
Effect of the 5-HT4 receptor and serotonin transporter on visceral hypersensitivity in rats.
Braz J Med Biol Res. 2012 Jul 26;
[PubMed Citation] [Order full text from Infotrieve]

12) Kozaka T, Uno I, Kitamura Y, Miwa D, Ogawa K, Shiba K
Syntheses and in vitro evaluation of decalinvesamicol analogues as potential imaging probes for vesicular acetylcholine transporter (VAChT).
Bioorg Med Chem. 2012 Jun 29;
A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and ? receptors (?-1, ?-2) were evaluated by in vitro binding assays using rat cerebral or liver membranes. OIDV and OBDV showed greater binding affinity to VAChT (K(i)=20.5±5.6 and 13.8±1.2nM, respectively) than did vesamicol (K(i)=33.9±18.1nM) with low affinity to ? receptors. A saturation binding assay in rat cerebral membranes revealed that [(125)I]OIDV had a single high affinity binding site with a K(d) value of 1.73nM and a B(max) value of 164.4fmol/mg protein. [(125)I]OIDV revealed little competition with inhibitors, which possessed specific affinity to each ? (?-1 and ?-2), serotonin (5-HT(1A) and 5-HT(2A)), noradrenaline, and muscarinic acetylcholine receptors. In addition, BBB penetration of [(125)I]OIDV was verified in in vivo. The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging probes with high affinity and selectivity. [PubMed Citation] [Order full text from Infotrieve]

13) Raby KL, Cicchetti D, Carlson EA, Cutuli JJ, Englund MM, Egeland B
Genetic and Caregiving-Based Contributions to Infant Attachment: Unique Associations With Distress Reactivity and Attachment Security.
Psychol Sci. 2012 Jul 24;
In the longitudinal study reported here, we examined genetic and caregiving-based contributions to individual differences in infant attachment classifications. For 154 mother-infant pairs, we rated mothers' responsiveness to their 6-month-old infants during naturalistic interactions and classified infants' attachment organization at 12 and 18 months using the Strange Situation procedure. These infants were later genotyped with respect to the serotonin-transporter-linked polymorphic region (5-HTTLPR). Maternal responsiveness uniquely predicted infants' attachment security. Infants' 5-HTTLPR variation uniquely predicted their subtype of attachment security at 12 months and their subtype of attachment insecurity at 12 and 18 months. The short allele for 5-HTTLPR was associated with attachment classifications characterized by higher emotional distress. These findings suggest that 5-HTTLPR variation contributes to infants' emotional reactivity and that the degree to which caregivers are responsive influences how effectively infants use their caregivers for emotion regulation. Theoretical implications for the study of genetic and caregiving influences are discussed. [PubMed Citation] [Order full text from Infotrieve]

14) Thomas JR, Gedeon PC, Grant BJ, Madura JD
LeuT Conformational Sampling Utilizing Accelerated Molecular Dynamics and Principal Component Analysis.
Biophys J. 2012 Jul 3;103(1):L1-3.
Monoamine transporters (MATs) function by coupling ion gradients to the transport of dopamine, norepinephrine, or serotonin. Despite their importance in regulating neurotransmission, the exact conformational mechanism by which MATs function remains elusive. To this end, we have performed seven 250 ns accelerated molecular dynamics simulations of the leucine transporter, a model for neurotransmitter MATs. By varying the presence of binding-pocket leucine substrate and sodium ions, we have sampled plausible conformational states representative of the substrate transport cycle. The resulting trajectories were analyzed using principal component analysis of transmembrane helices 1b and 6a. This analysis revealed seven unique structures: two of the obtained conformations are similar to the currently published crystallographic structures, one conformation is similar to a proposed open inward structure, and four conformations represent novel structures of potential importance to the transport cycle. Further analysis reveals that the presence of binding-pocket sodium ions is necessary to stabilize the locked-occluded and open-inward conformations. [PubMed Citation] [Order full text from Infotrieve]

15) Lanzenberger R, Kranz GS, Haeusler D, Akimova E, Savli M, Hahn A, Mitterhauser M, Spindelegger C, Philippe C, Fink M, Wadsak W, Karanikas G, Kasper S
Prediction of SSRI treatment response in major depression based on serotonin transporter interplay between median raphe nucleus and projection areas.
Neuroimage. 2012 Jul 22;
Recent mathematical models suggest restored serotonergic burst-firing to underlie the antidepressant effect of selective serotonin reuptake inhibitors (SSRI), resulting from down-regulated serotonin transporters (SERT) in terminal regions. This mechanism possibly depends on the interregional balance between SERTs in the raphe nuclei and in terminal regions before treatment. To evaluate these hypotheses on a systems level in humans in vivo, we investigated SERT availability and occupancy longitudinally in patients with major depressive disorder using positron emission tomography (PET) and the radioligand [(11)C]DASB. Measurements were performed before and after a single oral dose, as well as after three weeks (mean 24.73±3.3days) of continuous oral treatment with either escitalopram (10mg/day) or citalopram (20mg/day). Data were analyzed using voxel-wise linear regression and ANOVA to evaluate SERT binding, occupancy and binding ratios (SERT binding of the entire brain compared to SERT binding in the dorsal and median raphe nuclei) in relation to treatment outcome. Regression analysis revealed that treatment response was predicted by pre-treatment SERT binding ratios, i.e., SERT binding in key regions of depression including bilateral habenula, amygdala-hippocampus complex and subgenual cingulate cortex in relation to SERT binding in the median but not dorsal raphe nucleus (p<0.05 FDR-corrected). Similar results were observed in the direct comparison of responders and non-responders. Our data provide a first proof-of-concept for recent modeling studies and further underlie the importance of the habenula and subgenual cingulate cortex in the etiology of and recovery from major depression. These findings may indicate a promising molecular predictor of treatment response and stimulate new treatment approaches based on regional differences in SERT binding. [PubMed Citation] [Order full text from Infotrieve]

16) Mitjans M, Gastó C, Catalán R, Fañanás L, Arias B
Genetic variability in the endocannabinoid system and 12-week clinical response to citalopram treatment: the role of the CNR1, CNR2 and FAAH genes.
J Psychopharmacol. 2012 Jul 23;
First line treatment of major depression is based on selective serotonin re-uptake inhibitors (SSRIs) that enhance serotonergic neurotransmission by blocking the serotonin transporter. However, clinical response is a complex phenomenon in which other systems such as the endocannabinoid system could be involved. Given the evidence for the role of the endocannabinoid system in the pathogenesis of depression as well as in the mediation of antidepressant drug effects, the aim of this study was to analyze genetic variability in the endocannabinoid system genes (CNR1, CNR2 and FAAH genes) and their role in clinical response (at week 4) and remission (at week 12) in SSRI (citalopram) treatment in a sample of 154 depressive outpatients, all of Spanish origin. All patients were treated with citalopram and followed over 12 weeks. Severity of depressive symptomatology was evaluated by means of the 21-item Hamilton Depression Rating Score (HDRS). No differences were found in any of the genotype distributions according to response or remission. The longitudinal study showed that (i) the CNR1 rs1049353-GG genotype conferred a better response to citalopram treatment in the subgroup of male patients and (ii) G allele carriers (CNR2 rs2501431) presented higher HDRS scores in the follow-up than AA homozygous allele carriers. Our results seem to suggest the involvement of CNR1 and CNR2 genes in clinical responses to citalopram treatment. [PubMed Citation] [Order full text from Infotrieve]

17) Descarries L, Riad M
Effects of the antidepressant fluoxetine on the subcellular localization of 5-HT1A receptors and SERT.
Philos Trans R Soc Lond B Biol Sci. 2012 Sep 5;367(1601):2416-25.
Serotonin (5-HT) 5-HT(1A) autoreceptors (5-HT(1A)autoR) and the plasmalemmal 5-HT transporter (SERT) are key elements in the regulation of central 5-HT function and its responsiveness to antidepressant drugs. Previous immuno-electron microscopic studies in rats have demonstrated an internalization of 5-HT(1A)autoR upon acute administration of the selective agonist 8-OH-DPAT or the selective serotonin reuptake inhibitor antidepressant fluoxetine. Interestingly, it was subsequently shown in cats as well as in humans that this internalization is detectable by positron emission tomography (PET) imaging with the 5-HT(1A) radioligand [(18)F]MPPF. Further immunocytochemical studies also revealed that, after chronic fluoxetine treatment, the 5-HT(1A)autoR, although present in normal density on the plasma membrane of 5-HT cell bodies and dendrites, do not internalize when challenged with 8-OH-DPAT. Resensitization requires several weeks after discontinuation of the chronic fluoxetine treatment. In contrast, the SERT internalizes in both the cell bodies and axon terminals of 5-HT neurons after chronic but not acute fluoxetine treatment. Moreover, the total amount of SERT immunoreactivity is then reduced, suggesting that SERT is not only internalized, but also degraded in the course of the treatment. Ongoing and future investigations prompted by these finding are briefly outlined by way of conclusion. [PubMed Citation] [Order full text from Infotrieve]

18) Pinna M, Zompo MD
[Post partum depression: future perspectives].
Riv Psichiatr. 2012 May-Jun;47(3):195-9.
Aim. Post partum depression (PPD) is a psychiatric illness approximately affecting 10-20% of women after childbirth. The objective of this work is to update our knowledge of PPD giving particular emphasis to etiopathogenetic hypotheses. Methods. An accurate search of the literature on this topic was conducted using free dedicated websites such as PubMed. Results. The most recent studies reveal that PPD is a complex disease, whose pathogenesis is not yet clarified, determined by a mix of genetic, biological and environmental factors. Genetic studies have shown a possible involvement of polymorphisms of genes coding for serotonin transporter, 5HT2A and 5HT2C receptors, HMCN1 and METTL13 genes, D2 receptor and GABAA receptor (GABAAR). The involvement of these systems might provide an explanation of the relations among genetic alterations, hormonal fluctuations in the post partum, changes in neurotransmission and mood fluctuations typical of PPD. Discussion.The results obtained so far are not exhaustive. However, there is a substantial evidence showing that patients with PPD may have a high genetic vulnerability, although we have not been able yet to pinpoint a specific biological marker of the disease. Recent research is focusing on the ? subunit of GABAAR and the possible role of selective agonists of this subunit, such as gaboxadol, in the treatment of PPD. [PubMed Citation] [Order full text from Infotrieve]

19) Xu L, Tian H, Wang W, Ru S
Effects of monocrotophos pesticide on serotonin metabolism during early development in the sea urchin, Hemicentrotus pulcherrimus.
Environ Toxicol Pharmacol. 2012 Jul 6;34(2):537-547.
Organophosphate pesticides can interfere with the serotonergic nervous system and potentially lead to malformations and behavioral abnormalities during early development in sea urchin. To investigate the mechanism by which monocrotophos (MCP) pesticide disrupts the serotonergic nervous system, we evaluated its effects on serotonin metabolism. Fertilized embryos of sea urchin were incubated with 40% MCP pesticide at nominal concentrations of 0.01, 0.10 and 1.00mg/L, and the effects on tryptophan hydroxylase of Hemicentrotus pulcherrimus (HpTPH), serotonin reuptake transporter (SERT), monoamine oxidase (MAO), and serotonin levels were investigated. The results indicated that MCP pesticide disturbed the baseline pattern of HpTPH and SERT mRNA expression and MAO activity during early development in H. pulcherrimus. When serotonin should be quickly metabolized at 36-hpf stage, HpTPH and SERT expression was decreased and MAO activity was induced by MCP pesticide, leading to the impairment of serotonergic synaptic activity. But when serotonin should be metabolized at low levels during the other six stages, MCP pesticide induced HpTPH and SERT expression, resulting in the improvement of serotonergic synaptic activity. We concluded that this metabolic disturbance is one of the major mechanisms by which MCP pesticides affect the serotonergic nervous system and potentially contribute to various developmental abnormalities. [PubMed Citation] [Order full text from Infotrieve]

20) Ineichen C, Sigrist H, Spinelli S, Lesch KP, Sautter E, Seifritz E, Pryce C
Establishing a probabilistic reversal learning test in mice: Evidence for the processes mediating reward-stay and punishment-shift behaviour and for their modulation by serotonin.
Neuropharmacology. 2012 Jul 21;
Valid animal models of psychopathology need to include behavioural readouts informed by human findings. In the probabilistic reversal learning (PRL) task, human subjects are confronted with serial reversal of the contingency between two operant stimuli and reward/punishment and, superimposed on this, a low probability (0.2) of punished correct responses/rewarded incorrect responses. In depression, reward-stay and reversals completed are unaffected but response-shift following punished correct response trials, referred to as negative feedback sensitivity (NFS), is increased. The aims of this study were to: establish an operant spatial PRL test appropriate for mice; obtain evidence for the processes mediating reward-stay and punishment-shift responding; and assess effects thereon of genetically- and pharmacologically-altered serotonin (5-HT) function. The study was conducted with wildtype (WT) and heterozygous mutant (HET) mice from a 5-HT transporter (5-HTT) null mutant strain. Mice were mildly food deprived and reward was sugar pellet and punishment was 5-s time out. Mice exhibited high motivation and adaptive reversal performance. Increased probability of punished correct response (PCR) trials per session (p = 0.1, 0.2 or 0.3) led to monotonic decrease in reward-stay and reversals completed, suggesting accurate reward prediction. NFS differed from chance-level at p PCR = 0.1, suggesting accurate punishment prediction, whereas NFS was at chance-level at p = 0.2-0.3. At p PCR = 0.1, HET mice exhibited lower NFS than WT mice. The 5-HTT blocker escitalopram was studied acutely at p PCR = 0.2: a low dose (0.5-1.5 mg/kg) resulted in decreased NFS, increased reward-stay and increased reversals completed, and similarly in WT and HET mice. This study demonstrates that testing PRL in mice can provide evidence on the regulation of reward and punishment processing that is, albeit within certain limits, of relevance to human emotional-cognitive processing, its dysfunction and treatment. [PubMed Citation] [Order full text from Infotrieve]