Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
J Biol Chem. 2011 Mar 25;286(12):10712-24. doi: 10.1074/jbc.M110.167056. Epub 2011 Jan 18.
The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y(1) and P2Y(13) because the effects are partially reversed by the specific antagonists N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate and pyridoxal-5'-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y(12) receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca(2+) mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y(1) receptor. Sensitivity to 2-methylthioadenosine 5'-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.
钠氯协同甘氨酸神经递质转运体（GLYT）控制甘氨酸介导突触中甘氨酸的可用性。主要的神经胶质 GLYT1 是甘氨酸能和谷氨酸能途径中甘氨酸水平的关键调节剂，而神经元 GLYT2 参与从抑制性突触间隙中回收突触甘氨酸。在这项研究中，我们报告说，在大鼠脑干/脊髓原代神经元培养物和成年大鼠突触体中，用 2-甲基硫腺苷 5'-二磷酸刺激 P2Y 嘌呤能受体，通过旁分泌调节抑制 GLYT2 并刺激 GLYT1。这些作用主要是由 ADP 偏好型 P2Y(1) 和 P2Y(13) 亚型介导的，因为特定的拮抗剂 N(6)-甲基-2'-脱氧腺苷-3'，5'-双磷酸和吡哆醛-5'-磷酸-6-偶氮（2-氯-5-硝基苯基）-2,4-二磺酸盐部分逆转了这些作用，而苏拉明则完全阻断了这些作用。P2Y(12) 受体也参与了 GLYT1 的刺激。我们使用药理学方法和 siRNA 介导的蛋白敲低方法阐明了 GLYT 调节的分子机制。调节通过涉及磷脂酶 C 激活、肌醇 1,4,5-三磷酸生成、细胞内 Ca(2+)动员、蛋白激酶 C 刺激、一氧化氮形成、环鸟苷单磷酸生成和蛋白激酶 G-I（PKG-I）激活的信号级联进行。GLYT1 和 GLYT2 在脑源性制剂以及表达重组转运体和 P2Y(1) 受体的异源系统中对 NO/cGMP/PKG-I 的敏感性不同。用小干扰 RNA（siRNA）介导的一氧化氮合酶敲低，消除了 GLYT1 和 GLYT2 对 2-甲基硫腺苷 5'-二磷酸的敏感性。我们的数据可能有助于确定 GLYTs 在伤害感受和疼痛敏化中的作用。
