Characterization of [3H]Paroxetine Binding in Rat Brain
Characterization of [3H]Paroxetine Binding in Rat Brain
复制标题
[3H]帕罗西汀在大鼠脑中结合的表征
作者：
J. Marcusson;M. Bergstrom;K. Eriksson;S. Ross
翻译后摘要：5-羟色胺（5-HT，5-羟色胺）摄取抑制剂[3 H]帕罗西汀大鼠皮质匀浆的结合已被表征。检查了组织浓度的影响，在总体积为1，600 μl的情况下，以0.75 mg湿重组织/ml优化结合，表观解离常数（KD）为0.03-0.05 nM。5-HT、西酞普兰、诺齐美定和地昔帕明的竞争实验显示，符合单位点结合模型的置换结合比例较高（90%）。氟西汀和丙咪嗪显示，除了高亲和力（纳摩尔）位点外，还有一个低亲和力（微摩尔）位点，约占可置换结合的10%。5-HT是唯一结合的活性神经递质，而5-羟色胺S1和S2拮抗剂麦角新碱对结合没有影响，这一事实强调了[3 H]帕罗西汀结合的特异性。蛋白酶处理后，5-HT-和氟西汀敏感的[3 H]帕罗西汀结合完全消除，表明结合位点具有蛋白质性质。5-HT（100 μM）敏感性[3 H]帕罗西汀结合的饱和研究也与单位点结合模型一致，并且该结合被5-HT和丙咪嗪竞争性抑制。5-HT-敏感性[3 H]帕罗西汀和[3 H]丙咪嗪结合的结合位点数（Bmax）相同，表明放射性配体结合相同的位点。对氯苯丙胺的损伤实验导致额叶和顶叶皮质中的结合变得不可检测，纹状体和下丘脑中的结合减少>60%，表明5-HT末端的选择性定位。总之，这些发现表明[3 H]帕罗西汀特异性和选择性地标记大鼠脑中5-HT摄取的底物识别位点。
Abstract: The binding of the 5‐hydroxytryptamine (5‐HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 μl, the binding was optimized with an apparent dissociation constant (KD) of 0.03–0.05 nM. Competition experiments with 5‐HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single‐site binding model. Fluoxetine and imipramine revealed, in addition to a high‐affinity (nanomolar) site, also a low‐affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5‐HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5‐HT‐ and fluoxetine‐sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5‐HT (100 μM) sensitive [3H]paroxetine binding were also consistent with a single‐site binding model, and the binding was competitively inhibited by 5‐HT and imipramine. The number of binding sites (Bmax) for 5‐HT‐sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p‐chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a >60% reduction in the striatum and hypothalamus, indicating a selective localization on 5‐HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5‐HT uptake in rat brain.