SPECIFICITY IN ENDOTHELIAL CELL CALCIUM SIGNALING

内皮细胞钙信号的特异性。内皮细胞接收并整合与血流控制（即剪切应力和血管活性介质）、炎症和生长/修复相关的各种刺激。内皮细胞对这些刺激的不适当或有缺陷的反应可导致各种血管病变。来自所有这些刺激的信号转导部分依赖于细胞内钙的动员。在大血管内皮细胞中，细胞内钙池由两种肌（内皮细胞）浆钙ATP酶（SERCA）管理;独特表达的SERCA 3和普遍存在的SERCA 2b泵。缺乏SERCA 3的小鼠（SERCA 3敲除）出生时具有完整的血管系统。然而，这些小鼠的动脉瘤对血管扩张剂乙酰胆碱没有反应。此外，SERCA 3缺陷的主动脉内皮细胞不产生钙瞬变响应乙酰胆碱。即使SERCA 2b存在，SERCA 3敲除小鼠中存在的内皮细胞活性缺陷也会发生。这些观察结果导致了这样的假设，即内皮细胞利用不同的细胞内钙池，如这两个SERCA泵所定义的，来介导对不同类型的刺激的反应。本项目的目标是通过完成四个特定目标来验证这一假设，即通过证明SERCA 3维持的钙池参与内皮细胞应答的一个子集，并且该钙池与内皮细胞内SERCA 2b管理的钙池分离。具体目标1和2将表征SERCA 3和SERCA 2b管理的钙在钙瞬变产生中的分布和空间组织，并且这些小鼠内皮细胞中由四类血管刺激（剪切应力、血管活性、炎症和血管修复）诱导的内皮细胞反应将为这些研究提供实验基础。在两只小鼠的细胞和血管之间观察到的选择性差异可归因于缺乏由SERCA 3建立的钙储存，并表明依赖于该钙库的那些过程。这些目标的完成将提供进一步了解钙介导的内皮细胞信号转导的特异性是如何建立的，从而为针对治疗血管疾病的特异性疗法提供可能的途径。

Specificity in Endothelial Cell Calcium Signaling. The endothelial receives and integrates a variety of stimuli related to blood flow control (i.e. shear stress and vasoactive mediators), inflammation, and growth/repair. Inappropriate or defective endothelial cell responses to these stimuli can lead to a variety of vascular pathologies. Transduction of signals from all of these stimuli relies, in part, on mobilization of intracellular calcium. In large vessel endothelial cells, intracellular calcium pools are managed by two sarco (endothelial cell) plasmic calcium ATPases (SERCA); the uniquely expressed SERCA3 and the ubiquitous SERCA2b pumps. Mice lacking SERCA3 (SERCA3 knockout) are born viable with intact vasculature. However, aortas from these mice fail to respond to the vasodilator acetylcholine. Furthermore, SERCA3-deficient aortic endothelial cells do not generate a calcium transient in response to acetylcholine. The defects in endothelial cell activity present in the SERCA3 knockout mouse occur even though SERCA2b is present. These observations have lead to the hypothesis that endothelial cells utilize distinct intracellular calcium pools, as defined by these two SERCA pumps, to mediate responses to different types of stimuli. The goal of this project is to test this hypothesis, through completion of four specific aims, by demonstrating that the calcium pool maintained by SERCA3 participates in a subset of endothelial cell responses and that this calcium pool is segregated from the SERCA2b-managed pool within the endothelial cell. Specific aims 1 and 2 will characterize the distribution and the spatial organization of the SERCA3- and SERCA2b- managed calcium in the generation of calcium transients and endothelial cell responses induced by four classes of vascular stimuli (shear stress, vasoactivity, inflammation, and vascular repair) in endothelial cells from these mice will provide the experimental basis for these studies. Selective differences observed between cells and vessels of the two mice can be attributed to the absence of calcium stores established by SERCA3 and indicate those processes relying on this calcium pool. Completion of these aims will provide further insight into how specificity in calcium-mediated endothelial cell signal transduction is established and thus provide possible avenues for specific therapies directed at treating vascular disease.