Regulation of Endothelial Barrier through the Compartmentation of Cyclic AMP

通过环 AMP 的区室调节内皮屏障

基本信息

批准号：
7407872
负责人：
Sarah Louise Sayner
金额：
$ 5.13万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-01 至 2011-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The pulmonary microvascular endothelium forms a barrier limiting the movement of blood components into the underlying tissue. Barrier disruption leads to increased permeability that contributes to multiple pathologies including sepsis and acute respiratory distress syndrome. Cell-cell adhesions critically maintain this barrier and in turn are governed by intracellular cAMP concentrations. However, cAMP signaling specificity is encoded in the spatial and temporal fluctuations of this second messenger. Endogenous transmembrane adenylyl cyclase (AC) 6 synthesizes an endothelial barrier protective near-membrane cAMP-pool which is targeted to physiologically relevant effectors by phosphodiesterase (PDE) 4 confined to the membrane by spectrin. In contrast, the pathogenic bacteria Pseudomonas aeruginosa inserts a soluble AC, ExoY, into pulmonary microvascular endothelial cells (PMVECs) and generates a cytosolic cAMP pool that disrupts the barrier. Thus, this proposal tests the overall Hypothesis that endogenous transmembrane ACs generate a membrane cAMP pool does not saturate the cytosolic compartment while soluble ACs generate a cytosolic cAMP pool that does not reach the plasma membrane. Specific aims test the related hypotheses that (1) endogenous transmembrane ACs generate a membrane-to-cytosol cAMP gradient while soluble ACs disrupt this gradient and (2) plasma membrane localized PDE4(D4) activity prevents the spread of cAMP away from the plasma membrane domain. cAMP fluctuations in live PMVECs in response to endogenous- or ExoY-AC activity will be determined using electrophysiological techniques to detect near-membrane cAMP concentrations while the extent of second messenger diffusion will be demonstrated through FRET studies. Completion of these studies will give a clearer understanding of how endogenous ACs are barrier protective while bacterial toxins, such as ExoY of P. aeruginosa, disrupt the endothelial barrier.

描述（由申请人提供）：肺微血管内皮形成屏障，限制血液成分移动到下面的组织中。屏障破坏导致渗透性增加，导致多种病理，包括败血症和急性呼吸窘迫综合征。细胞与细胞之间的粘附至关重要地维持了这一屏障，并且反过来又受到细胞内 cAMP 浓度的控制。然而，cAMP 信号传导特异性被编码在第二信使的空间和时间波动中。内源性跨膜腺苷酸环化酶 (AC) 6 合成内皮屏障保护性近膜 cAMP 库，通过血影蛋白限制在膜上的磷酸二酯酶 (PDE) 4 靶向生理相关效应器。相比之下，致病菌铜绿假单胞菌将可溶性 AC（ExoY）插入肺微血管内皮细胞 (PMVEC) 中，并产生胞质 cAMP 池，破坏屏障。因此，该提案测试了总体假设，即内源性跨膜 AC 生成的膜 cAMP 池不会使胞质室饱和，而可溶性 AC 生成不会到达质膜的胞质 cAMP 池。具体目标测试了以下相关假设：(1) 内源性跨膜 AC 产生膜到细胞溶质的 cAMP 梯度，而可溶性 AC 则破坏该梯度；(2) 质膜局部 PDE4(D4) 活性阻止 cAMP 远离质膜扩散领域。将使用电生理学技术检测近膜 cAMP 浓度来确定活 PMVEC 响应内源性或 ExoY-AC 活性的 cAMP 波动，同时将通过 FRET 研究证明第二信使扩散的程度。完成这些研究将使人们更清楚地了解内源性 ACs 如何发挥屏障保护作用，而细菌毒素（例如铜绿假单胞菌的 ExoY）会破坏内皮屏障。