Pharmacological characterization and visualization of the glial serotonin transporter
Pharmacological characterization and visualization of the glial serotonin transporter
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DOI：
10.1016/s0197-0186(01)00010-9
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发表时间：
2001-07-01
影响因子：
4.2
通讯作者：
Matsumiya, T
中科院分区：
文献类型：
--
作者：
Inazu, M;Takeda, H;Matsumiya, T
星形胶质细胞含有能够通过存在于质膜中的转运体从突触间隙移除各种神经递质的转运系统。胶质细胞5-羟色胺转运体（SERT）在5-羟色胺（5-HT）的重摄取中起重要作用。我们研究了大鼠皮质突触体和培养的大鼠星形胶质细胞的5-HT摄取的药理学特征，以及使用特异性定点单克隆抗体（MoAb）的胶质SERT蛋白的免疫检测。此外，使用逆转录-聚合酶链反应（RT-PCR）的方法，我们解决了SERT mRNA在培养的大鼠星形胶质细胞的表达。我们研究了各种单胺摄取抑制剂对[H-3]5-HT摄取到培养的星形胶质细胞和皮质突触体的抑制作用。三环类抗抑郁药（氯丙咪嗪和丙咪嗪）以及选择性5-羟色胺再摄取抑制剂（氟伏沙明，氟西汀和zimelidine）是非常有效的抑制剂[H-3]5-HT摄取在这两种制剂。与此相反，NE摄取抑制剂（尼索西汀和地昔帕明）和可卡因的抑制作用弱于5-HT摄取抑制剂。此外，多巴胺（DA）摄取抑制剂（诺米芬辛和GBR-12935）表现出低微摩尔范围内的Ki值。抑制效力的顺序为5-HT摄取抑制剂（氯丙咪嗪、氟伏沙明、氟西汀、丙咪嗪和齐美利定）> NE摄取抑制剂（尼索西汀和地昔帕明）=可卡因> DA摄取抑制剂（诺米芬辛和GBR-12935）。两种制剂对各种单胺摄取抑制剂的抑制作用顺序无差异。各种单胺摄取抑制剂对[H-3]5-HT进入培养的星形胶质细胞和皮质突触体的抑制效力的相关分析产生高度显著的相关系数为0.9893（P < 0.0001）。在培养的星形胶质细胞中使用抗SERT MoAb进行免疫细胞化学染色显示，质膜以及细胞内、核周区室（可能是内质网或高尔基体膜）显示出相当高水平的免疫反应性。用抗SERT单克隆抗体对皮质星形胶质细胞和突触体的提取物进行免疫印迹。SDS-PAGE/Western印迹表明，抗SERT单克隆抗体识别两种制剂中的120和73 kDa的两个条带。RT-PCR结果显示，培养的星形胶质细胞表达克隆的SERT蛋白的mRNA，其特征在于神经元SERT。这些药理学实验表明，这种摄取过程通过神经胶质SERT发生，这与神经元SERT非常相似。此外，目前的数据还表明，神经元SERT的mRNA和蛋白质的存在下，建立在培养的大鼠星形胶质细胞，和SERT的多肽部分星形胶质细胞和额叶皮质可能是相同的基因产物。(C)2001爱思唯尔科技有限公司版权所有。
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 [H-3]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 [H-3]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 [H-3]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. (C) 2001 Elsevier Science Ltd. All rights reserved.