Pharmacological characterization and visualization of the glial serotonin transporter

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Abstract

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine)>NE uptake inhibitors (nisoxetine and desipramine)=cocaine>DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 (P<0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.

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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