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

通过对荧光标记的鞘脂进行成像和分析，揭示细胞外 1-磷酸鞘氨醇在体内和体外的分布和代谢途径

• 批准号: 92820999
• 负责人: Professor Dr. Markus Gräler, Ph.D.
• 金额: --
• 依托单位: Sektion Experimentelle Sepsisforschung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/92820999?language=en
• 关键词: Uncovering; routes; distribution; metabolism; extracellular

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.

血液中的 1-磷酸鞘氨醇 (S1P) 由红细胞储存，并诱导释放到血浆中，作为 S1P 受体的细胞外刺激物。根据初步数据，我们假设血液是 S1P 的主要来源，S1P 分布到淋巴组织、外周组织和淋巴液中。为了确定参与 S1P 分布的途径，将使用荧光标记的鞘脂来追踪 S1P 在体内和体外的释放和分布。 S1P从红细胞到血浆以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中以及通过流式细胞术在共培养实验中进行监测。缺乏鞘氨醇磷酸化酶鞘氨醇激酶 2 和 S1P 降解酶 S1P 裂解酶的小鼠将被用作受体小鼠，以确定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裂解酶缺陷小鼠组织中S1P积累的途径和来源。为了揭示细胞外 S1P 的代谢命运，将使用高效液相色谱 (HPLC) 中的荧光检测结合三重四极杆质谱 (Q-Trap) 来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在体外通过共聚焦激光扫描显微镜分析荧光标记的鞘脂的亚细胞定位。我们假设亚细胞鞘脂定位的差异将掺入的细胞外和内源鞘脂分开，并决定它们的命运，无论它们是分泌、储存、运输还是降解。