Expression Cloning of a Serotonin Transporter: A New Way to Study Antidepressant Drugs

Hoffman, Beth J.

doi:10.1055/s-2007-1014268

Cited by 26 publications

(7 citation statements)

References 9 publications

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“…SERTs are expressed in several tissues, including brain, platelets, placenta, lung, and adrenal gland Hoffman, 1994), and the genetic elements required to deliver quantitatively appropriate, cell-specific expression are unknown but could involve sequences surrounding exons lA and/ or lB. Recently, a common polymorphism within an enhancer-richregion of the hSERT exon lA promoter was implicated in differential hSERT gene expression in vitro and in vivo (Lesch et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Alternative Splicing of the Human Serotonin Transporter Gene

Bradley

Blakely

1997

Journal of Neurochemistry

106

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To explore the structural basis for regulation of human serotonin transporter (hSERT) gene expression, we used primer extension and 5' rapid amplification of cDNA ends (5'RACE) techniques to estimate levels of and identify 5'-noncoding elements of hSERT mRNAs and genomic cloning to place these elements within the overall map of the hSERT gene. Primer extension on JAR cell mRNA suggested the presence of significant hSERT mRNA sequence upstream of the 5' end of our cloned hSERT cDNA. Using 5'RACE and reverse transcription-PCR (RT-PCR) methodologies, we cloned these sequences from brain and placenta and found this material to be composed of alternatively spliced exons using a previously reported noncoding exon (1A) and a novel 97-bp noncoding exon (1B). RT-PCR of JAR cell mRNA blotted with exon-specific oligonucleotides revealed both exons 1A and 1B to be regulated in a cholera toxin-dependent manner. To clarify the structure of the hSERT gene including exon 1B, we isolated and characterized genomic hSERT clones from Lambda Fix II and P1 artificial chromosome libraries. In agreement with previous findings, a single hSERT gene was identified that accounts for hybridizing bands on genomic Southern blots and was found to utilize 13 exons to encode the transporter's coding sequences along with the two noncoding 5' exons. Exon 1B was identified approximately 14 kb downstream of exon 1A in the hSERT gene and 737 bp upstream of exon 2, where the initiation site for translation is located. Exon 1B is surrounded by several elements potentially suitable for regulating serotonin transporter gene expression in vivo, including consensus sites for transcription factors AP-1, AP-2, CREB/ATF, and NF-kappaB. These data reveal additional complexity in hSERT gene structure and expression that may be relevant to regulated and compromised transporter expression in vivo.

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Section: Discussionmentioning

confidence: 99%

Alternative Splicing of the Human Serotonin Transporter Gene

Bradley

Blakely

1997

Journal of Neurochemistry

106

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“…This scheme is consistent with (i) glial uptake of 5-HT sensitive to SSRIs, as previously shown by Kimelberg and associates (see References) and further confirmed in the present study, and (ii) the inability of MAO-A and MAO-B inhibitors to increase extracellular 5-HT after a single administration of a drug of either class. after destruction of catecholaminergic terminals with 6-OHDA and the low affinity of the cloned catecholaminergic transporters for 5-HT (Hoffman, 1994) seem to rule out a quantitatively important contribution of catecholaminergic terminals to in vivo 5-HT uptake in 5,7-DHT-lesioned rats.…”

Section: Serotonergic Terminalmentioning

confidence: 96%

Antidepressant Drugs Inhibit a Gial 5‐Hydroxytryptamine Transporter in Rat Brain

Bel

Figueras

Vilaró

et al. 1997

Eur J of Neuroscience

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We assessed the role of glial cells in the uptake of serotonin (5‐hydroxytryptamine, 5‐HT). Primary cultures of rat and mouse cortical astrocytes took up and deaminated 5‐HT. The antidepressants citalopram, clomipramine, fluoxetine, fluvoxamine, paroxetine and sertraline inhibited this process. The presence of the mRNAs for the 5‐HT transporter and monoamine oxidase‐A (MOA‐A) was established in cultured astrocytes and in adult rat brain areas with (midbrain and brainstem) and without (frontal cortex) serotonergic cell bodies after reverse transcription‐polymerase chain reaction and hybridization with probes complementary to the cloned neuronal 5‐HT transporter and MAO‐A. To examine in vivo the role of astrocytes in the elimination of 5‐HT from the extracellular brain space, 5‐HT was perfused through dialysis probes implanted in the frontal cortex of conscious rats and its concentration was measured at the probe outlet. Tissue 5‐HT recovery was dose‐dependently inhibited by the concurrent perfusion of citalopram, fluoxetine and paroxetine, showing that it essentially measured uptake through the high‐affinity 5‐HT transporter. Rats lesioned with 5,7‐dihydroxytryptamine (5,7‐DHT; 88% reduction of tissue 5‐HT) displayed tissue 5‐HT recovery slightly higher than sham‐operated rats (55 ± 2 vs. 46 ± 3%, P < 0.001), a finding perhaps attributable to the astrogliosis induced by 5,7‐DHT denervation. Rats lesioned with 6‐hydroxydopamine showed tissue 5‐HT uptake similar to controls, suggesting negligible reuptake of 5‐HT by catecholaminergic terminals. These results are consistent with the presence of a glial component of 5‐HT uptake in the rodent brain, sensitive to antidepressants, which takes place through a 5‐HT transporter very similar or identical to that present in neurons.

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“…However, competition binding experiments revealed a rank order of affinities of transporter ligands that was characteristic of binding to NETs and uncharac- teristic for binding to serotonin or dopamine transporters (Hoffman et al, 1991;Pacholczyk et al, 1991;Hoffman, 1994;Cheetham et al, 1996). For example, the relative affinities of transporter ligands in the locus coeruleus, dorsal raphe, and median raphe were identical, i.e., desipramine Ͼ imipramine Ͼ citalopram.…”

Section: Although [mentioning

confidence: 99%

Pharmacology and Distribution of Norepinephrine Transporters in the Human Locus Coeruleus and Raphe Nuclei

et al. 1997

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The norepinephrine transporter (NET ) is a site of action for tricyclic antidepressant drugs and for drugs of abuse such as amphetamine and cocaine. In this study, the binding of [ 3 H]nisoxetine to NETs in the noradrenergic cell group, the locus coeruleus, and the serotonergic cell groups, the dorsal raphe nuclei, was measured autoradiographically in postmortem human brain. 3 H]nisoxetine binding to NETs in monoaminergic nuclei was assessed by measuring the inhibition of its binding by desipramine, imipramine, or citalopram. The order of affinities of these drugs was identical in the locus coeruleus and dorsal and median raphe and was characteristic of binding to NETs (desipramine Ͼ imipramine Ͼ citalopram). Thus, high levels of NETs and an uneven distribution of NETs occur in the locus coeruleus as well as in the dorsal raphe nuclei of the human.

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Expression Cloning of a Serotonin Transporter: A New Way to Study Antidepressant Drugs

Cited by 26 publications

References 9 publications

Alternative Splicing of the Human Serotonin Transporter Gene

Alternative Splicing of the Human Serotonin Transporter Gene

Antidepressant Drugs Inhibit a Gial 5‐Hydroxytryptamine Transporter in Rat Brain

Pharmacology and Distribution of Norepinephrine Transporters in the Human Locus Coeruleus and Raphe Nuclei

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