Pharmacological characterization and visualization of the glial serotonin transporter
Introduction
The neuronal serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a member of a family of 12 transmembrane, Na+- and Cl−-dependent neurotransmitter transporters that aids in the termination of 5-HTergic neurotransmission by the re-uptake of 5-HT into presynaptic terminals from which it was released (Amara and Kuhar, 1993, Nelson, 1998). An understanding of the cellular mechanisms involved in regulating the expression, as well as activity, of SERT is important, since activation or inactivation of these mechanisms may be related to the pathogenesis of psychiatric disorders associated with altered 5-HTergic neurotransmission. SERTs are molecular targets for many clinically relevant drugs including cocaine, 3,4-methylenedioxymethamphetamine (ecstasy), tricyclic antidepressants and selective serotonin re-uptake inhibitors (SSRIs) (Marcusson and Ross, 1990, Rudnick and Wall, 1992). The elevation of the synaptic availability of 5-HT resulting from the blockade of SERT function may contribute to the therapeutic effect of many antidepressant drugs in depressive disorders. A SERT polypeptide cDNA has been isolated from rats (Blakely et al., 1991, Hoffman et al., 1991), mouse (Chang et al., 1996), guinea-pig (Chen et al., 1998) and humans (Lesch et al., 1993a, Lesch et al., 1993b, Ramamoorthy et al., 1993). The deduced amino acid sequences of these SERTs encode 630 amino acid polypeptides with 12 putative transmembrane spanning domains, cytoplasmic N- and C-termini and N-linked glycosylation sites within the large extracellular loop between transmembrane segments 3 and 4. Recently, the direct visualization of neuronal SERT proteins expressed in transfected and native membranes using SERT peptide and fusion protein-directed antibodies has been achieved (Tate and Blakely, 1994, Ovalle et al., 1995, Qian et al., 1995, Zhou et al., 1996). Various apparent molecular masses have been reported for SERT. Partial purification of SERT from human blood platelets by citalopram-derived affinity chromatography revealed two protein bands of 55 and 78 kDa (Biessen et al., 1990), whereas the rat midbrain and cortex transporter was identified as a 76-kDa polypeptide (Qian et al., 1995). In other experiments, the SERT from rat brains was shown to be 92, 74 and 64 kDa (Zhou et al., 1996). SERT contains two putative N-glycosylation sites in the second extracellular loop. These studies suggest that the difference in SERT mobility may be due to various glycosylation forms of the transporter protein, although the functional significance of such modifications is unclear.
Astrocytes also contain transport systems that are capable of removing various neurotransmitters, such as dopamine (DA), norepinephrine (NE), 5-HT, γ-aminobutyric acid and glutamate from the synaptic cleft by transporters present in the plasma membrane. Brain astrocytes are located around synapses and at the periphery of capillaries, so their uptake systems are likely to be important for regulating the neuronal environment. Primary astrocyte cultures derived from the cerebral cortex and other rat brain regions exhibit high-affinity, Na+-dependent and fluoxetine-sensitive 5-HT uptake (Katz and Kimelberg, 1985, Kimelberg and Katz, 1986, Amundson et al., 1992, Bel et al., 1997). [3H]5-HT uptake in brain slices prepared from astrogliotic hippocampus pretreated with kainic acid was twice that in control slices, suggesting the uptake of 5-HT by glia (Anderson et al., 1992). Further indirect evidence of 5-HT uptake into glial cells has recently been published by Bel et al. (1997), who demonstrated that tissue 5-HT recovery was unchanged after destruction of 5-HT nerve endings and axons with the selective toxin 5,7-dihydroxytryptamine. Their results strongly suggested the functional uptake of 5-HT by astrocytes. Since monoamine oxidase (MAO) and catechol-O-methyl-transferase (COMT) are present in astroglial cells (Pelton et al., 1981, Hansson and Sellstrom, 1983, Fitzgerald et al., 1990), uptake and subsequent metabolism by glial cells of neuronally released 5-HT may be of functional importance. Nonetheless, despite the physiological and clinical importance of glial SERT, the molecular properties of this transport system remain to be elucidated.
In the present study, we used cultured astrocytes derived from rat frontal cortex as a suitable in vitro model to investigate cellular aspects of 5-HT uptake. We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins in cultured rat astrocytes using specific site-directed monoclonal antibodies. Furthermore, we addressed the expression of SERT mRNA in cultured rat astrocytes.
Section snippets
Materials
Sprague–Dawley rats from Charles River (Tokyo, Japan) were used throughout the in vitro experiments. They were exposed to an artificial light–dark cycle (7:00–19:00 h light) with food and water available ad libitum. Tissue culture media, kanamycin, media supplements and fetal calf serum (FCS) were purchased from Gibco BRL (Gaithersburg, MD). Cells were grown on plastic dishes, plates or flasks (Falcon, Becton Dickinson, NJ). Dispase I (neutral protease; grade I), DNase I and a Neural Cell
Inhibitory effects of various monoamine uptake inhibitors on [3H]5-hydroxytryptamine uptake into cultured rat astrocytes and cortical synaptosomes
We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cortical synaptosomes (Fig. 1) and cultured rat astrocytes (Fig. 2). The Ki values for the inhibition of [3H]5-HT uptake were calculated from the corresponding inhibition curves and are given in Table 1. Tricyclic antidepressants (clomipramine and imipramine) as well as SSRIs (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In
Discussion
There have been many reports on 5-HT uptake in primary astrocyte cultures (Katz and Kimelberg, 1985, Kimelberg and Katz, 1986, Amundson et al., 1992, Anderson et al., 1992, Kimelberg et al., 1992, Dave and Kimelberg, 1994, Hösli and Hösli, 1995), and MAO-A and -B are present in astroglial cells (Levitt et al., 1982, Thorpe et al., 1987, Westlund et al., 1988, Fitzgerald et al., 1990). These reports support the notion that the uptake and subsequent metabolism by glial cells of neuronally
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