Quantitative Vasodilation Studies

描述（由申请人提供）：本研究提案描述了一系列旨在帮助确定循环系统中阻力血管（即小动脉和毛细血管）控制血管张力的确切机制的实验。具体而言，该建议将有助于确定红细胞（RBC）在控制肺血管阻力中的作用。当穿过微血管床时，例如在肺中，RBC经受机械变形。先前的研究结果表明，RBC是肺中一氧化氮（NO）合成所需的，并且RBC通过微米大小的孔或管道导致ATP的释放，ATP是内皮细胞NO合成的已知刺激物。在这个建议中，我们描述了一系列的研究，旨在量化的速率和持续时间的ATP从红细胞释放，因为他们穿越微孔通道制造的聚二甲基硅氧烷（PDMS）芯片与内径的阻力血管在完整的循环。具体来说，我们将检查的影响，改变内径和长度的微孔通道，以及流速对ATP释放从红细胞的兔子。此外，我们还将监测在存在和不存在RBC衍生ATP的情况下产生的NO的量。因此，在这个建议中，我们解决的假设是：ATP，释放红细胞在响应机械变形，是一种刺激内源性NO的合成，因此，是一个重要的决定因素，在肺循环血管阻力。在这里，我们打算1）证明通道直径的减小和通道长度和流速的增加刺激从制造的微芯片中的这些细胞释放ATP，2）证明RBC固有的某些性质，即细胞变形性和细胞年龄，可以影响RBC的ATP释放; 3）证明了固定在微芯片通道内腔的内皮细胞可用于模拟体内真实的阻力血管的内皮，并且可以在芯片上用电流法测量由RBC衍生的ATP刺激的固定化内皮细胞的NO产生和释放。这些研究的成功完成将导致对负责控制肺循环中血管阻力的机制的更全面的理解。这一信息将允许发展新的假设红细胞的贡献，以控制血管口径在健康和疾病。

DESCRIPTION (provided by applicant): This research proposal describes a series of experiments that are designed help to determine the exact mechanism by which resistance vessels in the circulatory system, namely arterioles and capillaries, control vascular tone. Specifically, this proposal will help define the role of the red blood cell (RBC) in the control of pulmonary vascular resistance. When traversing microvascular beds, such as in the lung, RBCs are subjected to mechanical deformation. Previous findings indicate the RBCs are required for nitric oxide (NO) synthesis in the lung, and that passage of RBCs through micrometer-sized pores or tubing results in the release of ATP, a known stimulus for endothelial cell NO synthesis. In this proposal, we describe a series of studies designed to quantify both the rate and duration of ATP release from RBCs as they traverse microbore channels fabricated in polydimethylsiloxane (PDMS) chips with internal diameters comparable to those of resistance vessels in the intact circulation. Specifically, we will examine the effect of alterations in the internal diameter and the length of the microbore channel, as well as the velocity of flow on ATP release from RBCs of rabbits. In addition, we will also monitor the amount of NO produced in the presence and absence of RBC derived ATP. Thus, in this proposal we address the hypothesis that: ATP, released from RBCs in response to mechanical deformation, is a stimulus for endogenous NO synthesis and, thereby, is an important determinant of vascular resistance in the pulmonary circulation. Here, we intend to 1) demonstrate that decreases in channel diameter and increases in channel length and flow velocity stimulate ATP release from these cells in a fabricated microchip, 2) demonstrate that certain properties intrinsic to the RBC, namely cell deformability and cell age, can affect ATP release from RBCs and 3) demonstrate that endothelial cells immobilized to the lumen of a microchip channel can be employed to mimic the endothelium of a real resistance vessel in vivo, and that the NO production and release from immobilized endothelial cells, stimulated by RBC-derived ATP, can be measured amperometrically on-chip. The successful completion of these studies will lead to a more comprehensive understanding of those mechanisms that are responsible for he control of vascular resistance in the pulmonary circulation. This information will permit the development of new hypotheses regarding the contribution of RBCs to the control of vascular caliber in health and disease.