Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes
Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes
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DOI：
10.1194/jlr.d071068
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发表时间：
2016-11-01
影响因子：
6.5
通讯作者：
Nishi, Tsuyoshi
中科院分区：
文献类型：
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作者：
Kobayashi, Naoki;Otsuka, Masato;Nishi, Tsuyoshi
1-磷酸鞘氨醇 (S1P) 存在于血浆中，通过从胸腺和次级淋巴组织募集淋巴细胞，充当免疫系统中关键的细胞间信号传递者。血浆S1P浓度由红细胞供给S1P来维持。之前，我们表明红细胞中 S1P 的释放是由 ATP 依赖性转运蛋白介导的。在本研究中，我们尝试建立一种快速可靠的方法，通过使用荧光 S1P 类似物 7-硝基-2-1,3-苯并恶二唑-4-基 (NBD) 标记的 S1P 来测量红细胞中的 S1P 转运活性。 NBD-S1P 以时间依赖性方式从红细胞中释放。接触格列本脲后，NBD-S1P 的释放减少，格列本脲是红细胞中 S1P 转运蛋白的抑制剂。此外，红细胞中NBD-S1P和S1P的释放分别受到细胞内S1P和NBD-S1P的竞争性抑制。这些结果表明红细胞S1P转运蛋白输出NBD-S1P。我们优化了样品制备条件和脂质提取，以提高测定的灵敏度。此外，我们通过将测定缓冲液中 BSA 的浓度降低至 0.1%，成功测量了 NBD-S1P 的释放，无需进行脂质提取。该方法可用于使用传统荧光计高通量筛选 S1P 转运蛋白抑制剂。
Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.