Zhu Chong-Bin, Steiner Jennifer A, Munn Jaclyn L, Daws Lynette C, Hewlett William A, Blakely Randy D
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
J Pharmacol Exp Ther. 2007 Jul;322(1):332-40. doi: 10.1124/jpet.107.121665. Epub 2007 Apr 25.
The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A(3) adenosine receptors (A(3)AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A(3)ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A(3)AR agonist N(6)-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A(3)AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A(3)AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport V(max) with no significant change in K(m). As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK(5)H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A(3)ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A(3)ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.
突触血清素（5-羟色胺，5-HT）的失活主要是通过突触前、对抗抑郁药敏感的5-羟色胺转运体（SERT，SLC6A4）的作用来实现的。最近的研究表明，包括蛋白激酶C和G（PKC和PKG）以及p38丝裂原活化蛋白激酶（MAPK）在内的多种丝氨酸/苏氨酸激酶以及丝氨酸/苏氨酸磷酸酶PP2A介导了SERT的翻译后调控。针对这些信号通路以控制SERT表面表达和/或催化速率的特定表面受体的研究较少。我们之前使用大鼠嗜碱性白血病2H3细胞系（RBL-2H3）证实，A3腺苷受体（A3AR）的激活通过PKG和p38 MAPK刺激SERT活性（Zhu等人，2004a）。A3AR是否在中枢神经系统（CNS）中调节SERT尚不清楚。在此我们报告，A3AR激动剂N6-(3-碘苄基)-N-甲基-5'-氨基甲酰腺苷（IB-MECA）能快速（10分钟）且选择性地刺激小鼠中脑、海马体和皮质突触体中的5-HT转运。选择性A3AR拮抗剂3-乙基-5-苄基-2-甲基-苯乙炔基-6-苯基-1,4(±)二氢吡啶-3,5-二羧酸酯（MRS1191）可阻断IB-MECA诱导的5-HT摄取刺激，且在从A3AR基因敲除小鼠制备的突触体中不存在这种刺激。动力学分析表明，IB-MECA诱导5-HT转运Vmax增加，而Km无显著变化。与在RBL-2H3细胞中一样，用PKG拮抗剂N-[2-(甲氨基)乙基]-5-异喹啉磺酰胺（H8）和DT-2（YGRKKRRQRRRPPLRK(5)H）以及p38 MAPK抑制剂SB203580 [4-(4-氟苯基)-2-(4-甲亚磺酰基苯基)-5-(4-吡啶基)-1H-咪唑]预孵育可阻断IB-MECA对突触体5-HT摄取的刺激。在麻醉大鼠海马体中的计时电流法研究支持A3AR在体内SERT调节中的作用。总之，这些结果确定了中枢神经系统A3AR在调节SERT介导的5-HT转运方面一种新的、区域特异性作用，这可能与5-HT相关脑疾病的病因和/或治疗有关。
