Uncovering the routes of distribution and metabolism of extracellular sphingosine 1-phosphate in vivo and in vitro by imaging and analyzing fluorescently-labelled sphingolipids

血液中的1-磷酸鞘氨醇(S1P)由红细胞储存，并被诱导释放到血浆中，在血浆中作为S1P受体的细胞外刺激。根据初步数据，我们假设血液是S1P的主要来源，S1P分布在淋巴、外周组织和淋巴中。为了确定参与S1P分布的途径，将使用荧光标记的鞘脂来跟踪S1P在体内和体外的释放和分布。S1P从红细胞到血浆及周围细胞和组织的释放和分布将通过共聚焦激光扫描显微镜在组织切片上和在共培养实验中通过流式细胞仪进行监测。缺乏鞘氨醇磷酸化酶鞘氨醇激酶2和S1P降解酶S1P裂解酶的小鼠将作为受体小鼠，以确定S1P去磷酸化和再磷酸化对血源性S1P组织分布的作用，并确定S1P在S1P裂解酶缺陷小鼠组织中积累的途径和来源。为了揭示细胞外S1P的代谢去向，荧光标记的鞘磷脂代谢物将通过高效液相色谱和三重四极杆质谱仪(Q-Trap)相结合的荧光检测进行鉴定和分析。荧光标记的鞘磷脂的亚细胞定位将使用组织细胞系和原代细胞在体外用激光共聚焦扫描显微镜进行分析。我们假设，亚细胞鞘脂定位的不同将结合的细胞外和内源性鞘脂分开，并决定它们的命运，无论它们是被分泌、储存、运输还是降解。

Sphingosine 1-phosphate (S1P) in blood is stored by erythrocytes, and it is inducibly released into plasma, where it acts as an extracellular stimulus for S1P-receptors. Based on preliminary data we hypothesize that blood serves as a major source for S1P, which is distributed to lymphoid and peripheral tissues and lymph. In order to define the pathways that are involved in the distribution of S1P, fluorescently-labelled sphingolipids will be used to track the release and distribution of S1P in vivo and in vitro. The release and distribution of S1P from erythrocytes into plasma and surrounding cells and tissues will be monitored in tissue sections by confocal laser scanning microscopy and in co-culture experiments by flow cytometry. Mice deficient for the sphingosine-phosphorylating enzyme sphingosine kinase 2 and for the S1P-degrading enzyme S1P-lyase will be used as recipient mice to determine the role of de- and re-phosphorylation for tissue distribution of blood-borne S1P, and for determining the routes and origin of accumulating S1P in tissues of S1P-lyase deficient mice. To uncover the metabolic fate of extracellular S1P, fluorescently-labelled sphingolipid metabolites will be identified and analyzed using fluorescence detection in high performance liquid chromatography (HPLC) combined with tripple-quadrupole mass spectrometry (Q-Trap). The subcellular localization of fluorescently-labelled sphingolipids will be analyzed by confocal laser scanning microscopy in vitro using tissue cell lines and primary cells. We hypothesize that differences in subcellular sphingolipid localization separate incorporated extracellular and endogenous sphingolipids, and determine their fate whether they are secreted, stored, transported, or degraded.