[<sup>3</sup>H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Whitaker, Patricia M.; Vint, Carley K.; Morirn, Rosemarie

doi:10.1111/j.1471-4159.1983.tb00827.x

Cited by 32 publications

(10 citation statements)

References 16 publications

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“…This system is characterized by ligand specificity, Na+ and temperature dependence, antagonizability by known blockers (nortriptyline, imipramine, and desipramine), and saturable and high-affinity uptake kinetics. In particular, the Km and Vma values are well within the range of such parameters measured for 5-HT-uptake systems in the central nervous system and blood platelets (6)(7)(8)(9)(10)(11)(12)(13).…”

Section: Discussionsupporting

confidence: 66%

“…Although the physiology and pharmacology of a number of neurotransmitter transport systems including that for serotonin have been extensively studied (6)(7)(8)(9)(10)(11)(12)(13), little is known about the biochemistry and molecular biology of any of these systems. With the hope of attaining such information, we report here the use of gene transfer (14)(15)(16)(17) in the establishment and identification of a clonal mouse fibroblast cell line, L-S1, that stably expresses a presumptively human highaffinity uptake system for serotonin (5-HT).…”

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confidence: 99%

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Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Chang¹,

Frnka²,

Chen³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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By transfecting mouse fibroblast L-M cells with human genomic DNA, we have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-[3H]HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (ii) both Nal and temperature dependences; (iv) kinetic saturability; and (v) high affinity for 5-HT (Km = 0.39 + 0. 10 jIM; V.. = 2.14 ± 0.55 pmol/min per mg of protein). This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, we postulate that the observed 5-HT-uptake system resulted from the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neurotransmitteruptake systems as well as substrates for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.Inactivation of neurotransmitters, following their stimulated release into the synaptic cleft, is a key regulatory process in neurotransmission. For biogenic amine and amino acid neurotransmitters, the inactivation process is achieved mainly via transmitter-specific high-affinity transport systems present in the presynaptic neuron and/or surrounding glial cells (1-6). These transport mechanisms are also of significant clinical interest because some of their inhibitors are therapeutic agents for certain neurological disorders.Although the physiology and pharmacology of a number of neurotransmitter transport systems including that for serotonin have been extensively studied (6-13), little is known about the biochemistry and molecular biology of any of these systems. With the hope of attaining such information, we report here the use of gene transfer (14-17) in the establishment and identification of a clonal mouse fibroblast cell line, L-S1, that stably expresses a presumptively human highaffinity uptake system for serotonin (5-HT). MATERIALS AND METHODSTransfection of L-M Fibroblasts. Mouse L-M fibroblast cells (ATCC CCL 1.2) were plated in 100-mm culture dishes so that by the time of transfection the cell number would be about 106. Human genomic DNA and pSV2-neo, a plasmid bearing neomycin-resistance as a selectable marker (18), were cotransfected essentially by the method of calcium phosphate precipitation (19). After transfection, the cells were trypsinized and plated into 96-well plates. Selection of resistance to G418 (GIBCO; 400 tkg/ml) was maintained until transfectant colonies (typically three to five per well) were well established. Each well was then assayed for 5-[3H]HT uptake activities.Identificatio...

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Section: Discussionsupporting

confidence: 66%

mentioning

confidence: 99%

Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Chang¹,

Frnka²,

Chen³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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“…A strong accumulation of GABA has already been demonstrated in cultured normal astrocytes of brain cortex of newborn mice, and a quantitative correlation between GABA and sodium transport has been established . Uptake of 5-HT by glial cells has also been reported and a Na+-sensitive component of this influx has been shown (Katz and Kimelberg 1985;Suddith et al, 1978;Whittaker et al, 1983). Similarly, glial uptake of NA has also been demonstrated (Kimelberg and Pelton 1983;Kimelberg, 1986;Pelton et al, 1981) but its saturability and Na+ dependency has been questioned (Hansson, 1985;Hertz and Peterson, 1988).…”

Section: Discussionmentioning

confidence: 96%

Uptake, metabolism, and release of [³H]‐histamine by glial cells in primary cultures of chicken cerebral hemispheres

Huszti

Rimanóczy

Juhász

et al. 1990

Glia

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Labelled histamine was taken up into cultured glial cells of chick embryonic brain by a system with high affinity for histamine and diffusion. The active uptake, occurring at low concentrations of the amine, was Na+ dependent and gave an apparent Km of 0.24 microM and a Vmax of 0.31 pmol x mg protein-1 x min-1. The uptake was completely blocked by desmethylimipramine (Ki = 2.5 microM) and partially by the histamine agonists and histamine-N-methyltransferase blockers 4-methylhistamine and 2-methylhistamine (I30 values obtained were 2 microM and 5 microM). Other psychoactive drugs were either ineffective (imipramine) or they showed moderate inhibitory effects (amitriptyline and cocaine). Ouabain (100 microM) inhibited uptake by approximately 50%. Diffusion occurred at high concentrations of the amine, was insensitive to extracellular Na+, and was proportional to histamine concentration up to 1 mM. [3H]-Histamine, taken up into the cells, was metabolized and/or released. The spontaneous efflux of the radioactivity measured after 10 min of exposure to [3H]-histamine (when most of it was still unmetabolized), was moderately Ca++ dependent, accelerated by both reduced concentrations of extracellular Na+ and enhanced concentrations of K+ and inhibited by desmethylimipramine. After prolonged (60 min) incubation, histamine metabolites detected in the cells presented 78% of the chromatogram radioactivity and consisted of N tau-methylhistamine and N tau-methylimidazole acetic acid. These results indicate that at low nM concentrations, histamine is taken up and metabolized by (and released from) glial cells by an Na(+)-dependent system, and the intracellular metabolism seems to serve an increased uptake of the amine.

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“…Reith et al (1983) demonstrated that the highaffinity binding of [3H]imipramine is related to the neuronal uptake system for 5-HT but inhibition of the low-affinity binding did not correlate with the inhibition of the neuronal uptake of 5-HT. Whitaker et al (1983) suggested that part of the [3H]imipramine binding occurs on astroglial cells and is not related to 5-HT transport. It is possible that the low-affinity, sodium-independent site in different brain regions could be the glial cell site.…”

Section: Discussionmentioning

confidence: 99%

High‐ and Low‐Affinity Binding of [³H]Imipramine in Rat Hypothalamus

Moret

Briley

1986

Journal of Neurochemistry

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In the rat hypothalamus [3H]imipramine binding is inhibited by tricyclic and nontricyclic antidepressant drugs in a complex manner, with biphasic curves and Hill coefficients less than 1.0. 5-Hydroxytryptamine (serotonin) inhibited with high affinity a decreasing proportion of the [3H]imipramine binding sites as the [3H]imipramine concentration was raised. In the absence of sodium ions, IC50 values for the inhibition by tricyclic and nontricyclic antidepressants were increased by approximately 1,000-fold, and the inhibition curves became classically monophasic with Hill coefficients close to 1.0. These data are interpreted as suggesting that [3H]imipramine binds to two independent sites in the rat hypothalamus. One site is sodium-dependent with a high affinity for the drugs tested; the other is sodium-independent and has a low affinity for these drugs.

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[³H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Cited by 32 publications

References 16 publications

Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Uptake, metabolism, and release of [³H]‐histamine by glial cells in primary cultures of chicken cerebral hemispheres

High‐ and Low‐Affinity Binding of [³H]Imipramine in Rat Hypothalamus

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[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Cited by 32 publications

References 16 publications

Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Uptake, metabolism, and release of [3H]‐histamine by glial cells in primary cultures of chicken cerebral hemispheres

High‐ and Low‐Affinity Binding of [3H]Imipramine in Rat Hypothalamus

Contact Info

Product

Resources

About

[³H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Uptake, metabolism, and release of [³H]‐histamine by glial cells in primary cultures of chicken cerebral hemispheres

High‐ and Low‐Affinity Binding of [³H]Imipramine in Rat Hypothalamus