Filtration disrupts synaptosomes during radiochemical analysis of serotonin uptake: comparison with chronoamperometry in SERT knockout mice.
Filtration disrupts synaptosomes during radiochemical analysis of serotonin uptake: comparison with chronoamperometry in SERT knockout mice.
复制标题
在血清素摄取的放射化学分析过程中，过滤会破坏突触体：与 SERT 敲除小鼠中的计时电流分析法进行比较。
DOI：
10.1016/j.jneumeth.2005.12.017
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Andrews,AnneM
中科院分区：
文献类型：
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作者：
Perez,XiomaraA;Bianco,LauraE;Andrews,AnneM
放射化学方法未能揭示缺乏血清素转运蛋白（SERT）基因的一种功能性拷贝的小鼠中突触体血清素摄取的减少。相比之下，通过计时电流分析法测定的 SERT+/- 小鼠突触体的摄取率显示出与基因相关的减少。我们重新审视了 SERT 敲除小鼠中的 [3H]5-HT 摄取，以确定孵育缓冲液中包含 O2 对通过该方法获得的动力学参数的影响。在从额叶皮层和纹状体制备的含氧突触体中，与 SERT+/+ 小鼠相比，SERT+/- 小鼠的放射性标记 5-HT 摄取量分别减少了 25% 和 35%。然而，即使在存在 O2 的情况下，SERT+/- 和 SERT+/+ 小鼠脑干中的 [3H]5-HT 摄取也没有检测到差异，而计时安培法测定的减少了 60%。此外，虽然 O2 的加入适度增加了 [3H]5-HT 的摄取速率，但在 O2 存在的情况下通过计时电流分析法测定的速率比放射化学测定的速率高 40 倍。我们提供的证据表明，放射化学方法中使用的过滤过程会导致运输的 5-HT 大量损失，从而导致表观吸收率降低。这些发现解释了放射化学方法在确定生物学上重要的摄取变化（例如 SERT+/- 和 SERT+/+ 小鼠之间发生的变化以及对 O2 的反应）时相对不敏感。
Radiochemical methods have failed to reveal decreases in synaptosomal serotonin uptake in mice lacking one functional copy of the serotonin transporter (SERT) gene. By contrast, uptake rates determined by chronoamperometry in synaptosomes from SERT+/− mice show gene-related reductions. We revisited [3H]5-HT uptake in SERT knockout mice to determine the effects of inclusion of O2in the incubation buffer on the kinetic parameters obtained by this method. In oxygenated synaptosomes prepared from frontal cortex and striatum, modest 25 and 35% reductions in radiolabeled 5-HT uptake were detected in SERT+/− versus SERT+/+ mice. However, even in the presence of O2, no differences in [3H]5-HT uptake were detected between SERT+/− and SERT+/+ mice in brain stem in contrast to 60% reductions determined by chronoamperometry. Moreover, while inclusion of O2modestly increased the rates of [3H]5-HT uptake, rates determined by chronoamperometry in the presence of O2were 40-fold greater than those determined radiochemically. We present evidence that the filtration process used in the radiochemical method leads to substantial loss of transported 5-HT resulting in lower apparent uptake rates. These findings explain the relative insensitivity of radiochemical methods for determining biologically important alterations in uptake such as those occurring between SERT+/− and SERT+/+ mice and in response to O2.