Fast calcium responses along arteriolar endothelium in vivo

描述（由申请人提供）：我们实验室中心的长期研究目标是确定协调单个内皮细胞（EC）和平滑肌细胞（SMC）沿着微血管调节血液流向活性组织的信号事件。我们的工作假设是，血流的局部控制代表了微血管阻力网络中构成小动脉壁的细胞之间的协调活动。乙酰胆碱（ACh）刺激启动沿内皮细胞在细胞间传播的信号，从而启动SMC松弛，从而产生血管舒张。我们的实验室已经证明，ACh启动双向Ca2+波，以-0.1毫米/秒的速度传播几百微米的距离，刺激一氧化氮的释放，促进血管舒张。我的初步实验揭示了一种新型的“快速钙反应（Fast Calcium Response， FOR）”，它的传播速度要快得多（毫米每秒），距离也远得多，但只在血液流动的方向上。本文描述的项目重点是了解这种新颖的FOR是如何沿着容器启动和传播的。实验在体内进行，使用麻醉转基因小鼠表达Ca2+指示蛋白（GCaMP2）特异性靶向动脉内皮细胞。FOR的发生和传播机制尚不清楚。目的1将通过检测多种内皮依赖性血管扩张剂（如乙酰胆碱、缓激肽、P物质和ATP）来确定FOR是如何启动的。我将确定血管扩张激动剂进入血流及其沿血流路径的对流是否可以解释FOR。或者，一种次级物质可以作为对这些激动剂的反应而产生，从而触发FOR。我将用微栓塞来控制小动脉网络中的血流分布，并通过微灌注特定的拮抗剂来解决这个问题。目标2将确定与血液运动相关的FOR实际上是如何传播的。通过引入血流荧光示踪剂（标记微球和红细胞），我将确定FOR与血流速度和分布之间的关系。解决FCR的性质将为考虑这种以前未被识别的信号通路如何在体内调节提供新的见解。反过来，这种独特的理解可以应用于开发新的治疗方法来治疗诸如糖尿病、高血压和缺血等病理生理状况，在这些疾病中，微血管EC和SMC的功能完整性受到损害。我的总体目标是将我的研究成果应用于了解和治疗心血管疾病。

DESCRIPTION (provided by applicant): The long term research goals of our laboratory center on defining the signaling events that coordinate the activity of individual endothelial cells (EC) and smooth muscle cells (SMC) along microvessels that regulate blood flow to active tissue. Our working hypothesis is that the local control of blood flow represents a coordinated activity among the cells that comprise the walls of arterioles in microvascular resistance networks. Stimulating with acetylcholine (ACh) initiates signals that propagate from cell to cell along the endothelium to initiate SMC relaxation and thereby produce vasodilation. Our laboratory has shown that ACh initiates a bi-directional Ca2+ wave that propagates at -0.1 millimeter per second over distances of several hundred micrometers and stimulates the release of nitric oxide to promote vasodilation. My preliminary experiments have revealed a novel 'Fast Calcium Response (FOR)' that travels much more rapidly (millimeters per second) and for far greater distances but only in the direction of blood flow. The project described herein is focused on understanding how this novel FOR is initiated and propagated along a vessel. Experiments are performed in vivo using anesthetized transgenic mice expressing a Ca2+ indicator protein (GCaMP2) targeted specifically to arteriolar endothelial cells. The mechanism of FOR initiation and propagation are unknown. Aim 1 will determine how the FOR is initiated by testing a variety of endothelium-dependent vasodilators (e.g. ACh, bradykinin, substance P, and ATP). I will determine whether entry of the vasodilator agonist into the blood stream and its convection along the flow path can explain the FOR. Alternatively, a secondary substance may be produced in response to such agonists that in turn triggers the FOR. I will resolve this question using microoclusion to control blood flow distribution in arteriolar networks and by microperfusing defined segments with specific antagonists. Aim 2 will determine how the FOR is actually propagated in relation to the movement of blood. By introducing fluorescent tracers of blood flow (labeled microspheres and red blood cells), I will determine the relationship between FOR and the velocity and distribution of blood flow. Resolving the nature of the FCR will provide new insight for considering how this previously unrecognized signaling pathway is regulated in vivo. In turn, this unique understanding may be applied to developing new therapeutic approaches for treating such pathophysiological conditions such as diabetes, hypertension and ischemia, where, the functional integrity of microvascular EC and SMC are compromised. My overall goal is to apply the findings of my research to understanding and treating cardiovascular disease.