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

血液中的鞘氨醇1-磷酸（S1 P）由红细胞储存，并可诱导释放到血浆中，在血浆中作为S1 P受体的细胞外刺激物。基于初步数据，我们假设血液是S1 P的主要来源，其分布于淋巴和外周组织以及淋巴。为了确定参与S1 P分布的途径，将使用荧光标记的鞘脂来跟踪S1 P在体内和体外的释放和分布。将通过共聚焦激光扫描显微镜在组织切片中和通过流式细胞术在共培养实验中监测S1 P从红细胞释放和分布到血浆和周围细胞和组织中。将鞘氨醇磷酸化酶鞘氨醇激酶2和S1 P降解酶S1 P裂解酶缺陷的小鼠用作受体小鼠，以确定去磷酸化和再磷酸化对血液传播的S1 P的组织分布的作用，并确定S1 P裂解酶缺陷小鼠组织中积累S1 P的途径和来源。为了揭示细胞外S1 P的代谢命运，将使用高效液相色谱法（HPLC）中的荧光检测结合三重四极杆质谱法（Q-Trap）鉴定和分析荧光标记的鞘脂代谢物。将使用组织细胞系和原代细胞，通过共聚焦激光扫描显微镜在体外分析荧光标记鞘脂的亚细胞定位。我们假设，在亚细胞鞘脂定位的差异分离纳入细胞外和内源性鞘脂，并决定他们的命运，无论是分泌，储存，运输，或降解。

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.