Dynamics of Regulating Oxygen Supply by Erythrocytes

可以说，血管系统最重要的功能是向身体的每个细胞输送足够的氧气(O2)。由于氧气可以从血管中的红细胞(RBC)扩散到细胞中消耗能量产生能量的距离为0.1毫米，血管系统必须适当地将氧气供应分配到单个末端小动脉(非常小的动脉)供应支持这些细胞的少量毛细血管的水平。在发育过程中，这种氧分布的准确性不能建立在血管结构中，它必须被动态调节，以响应所服务组织的不同需求(例如，根据体力活动水平在骨骼肌或心肌中)。我们首次提出，作为O2的主要载体的红细胞也是能够如此精细地控制O2分布的传感器。红细胞通过红细胞中血红蛋白携带的氧气量的变化来感觉氧气供应和氧气消耗之间的平衡。当O2与血红蛋白结合时，分子的形状发生变化，这既激活了红细胞内的酶以产生三磷酸腺苷(ATP)，又触发了红细胞膜中的信号通路，使ATP从红细胞中释放出来。ATP激活血管衬里内皮细胞内表面的专门受体(P2y)，导致小动脉直径增加，局部是通过释放一氧化氮或前列腺素，或者是通过通过内皮细胞传导到小动脉的电信号从毛细血管和小静脉远程增加。*关于依赖氧气释放ATP的问题，现有技术无法解决。主要的问题是，在血红蛋白O2饱和度发生变化后，红细胞需要多长时间才能释放出ATP？答案很重要，因为释放时间决定了向血管内皮细胞报告氧饱和度水平的空间准确性。现有技术仅提供氧饱和度变化时的ATP释放量，不能用来确定ATP的快速释放。我的博士生理查德·索维最近取得的进展使我们有可能回答这个问题，并在这样做的同时，对调节ATP释放的机制获得新的见解。SOVé开发并发表了一个计算模型，用于设计一种可用于测量ATP释放动力学的微流控装置。他在他的微流控模型中加入了ATP释放的动态信号通路模型，并证明了这种微流控方法能够揭示信号通路中每个阶段的速度。SOVé的S的方法也可以回答这样一个问题：红细胞释放三磷酸腺苷的速率是由氧饱和度水平还是由氧饱和度变化的速率决定的？另一种模型预测，只有当ATP的释放取决于氧饱和度变化的速度时，氧的分布才能被红细胞调节；因此，这个答案对于我们理解红细胞在调节氧气供应中的作用是至关重要的。*这一建议侧重于发展由Sové提出的微流控系统，以全面描述红细胞对氧依赖的ATP释放的动力学。所有研究都将在老鼠血中进行，以保持与先前研究的一致性。这项技术为其他研究生项目创造了机会，调查红细胞在体内微血管系统中可能遇到的情况的影响，并为未来的合作健康研究项目奠定基础，将这项研究转化为心血管疾病患者的筛查工具。

Arguably the most important function of the vasculature is the delivery of adequate oxygen (O2) to every cell of the body. Since the distance O2 can diffuse from red blood cells (RBCs) in a vessel to where it is consumed in the cell to produce energy is <0.1-mm, the vasculature must properly distribute O2 supply down to the level of a single terminal arteriole (very small artery) supplying a small number of capillaries supporting these cells. This level of accuracy in distributing O2 cannot be built into the vascular structure during development, it must be dynamically regulated to respond to varying needs of the tissue being served (e.g. in skeletal or cardiac muscle depending on the level of physical activity). We were the first to propose that the red blood cell, which is the primary carrier of O2, is also the sensor which enables this fine level of control of the distribution of O2. RBCs sense the balance between the O2 supply and O2 consumption by the change in the amount of O2 carried by hemoglobin in the RBC. As O2 binds to hemoglobin the shape of the molecule changes which both activates enzymes within the RBC to produce adenosine triphosphate (ATP) and triggers a signalling pathway in the RBC membrane for ATP to be released from the RBC. The ATP activates specialized receptors (P2y) on the inner surface of endothelial cells lining the vessel which result in an increase in arteriolar diameter, either locally in arterioles by the release of nitric oxide or prostaglandins or remotely from capillaries and venules via a electrical signal conducted through endothelial cells to the arterioles.*There are unresolved issues about the O2 dependent release of ATP which existing technologies cannot answers. The primary question is how long does it take for ATP to be released from the RBC following a change in hemoglobin O2 saturation? The answer is important since the release time determines the spatial accuracy with which O2 saturation levels can be reported to the vascular endothelium. Existing techniques only provide the how much ATP is released for a change in O2 saturation and cannot be used to determine fast ATP is released. Recent advances by my Ph.D. student, Richard Sové, make it possible for us to answer this question and, in doing so; gain new insights into the mechanisms regulating ATP release. Sové developed and published a computational model for the potential design of a microfluidic device which could be used to measure the dynamics of ATP release. He added a dynamic signaling pathway model for ATP release into his microfluidic model and has demonstrated that this microfluidic approach is able to reveal how the speed of each stage in the signaling pathway. Sové's approach can also answer the question is the release rate of ATP from the RBC governed by the level of O2 saturation or by the rate at which O2 saturation is changing? Another model predicts that O2 distribution can only be regulated by RBCs if ATP release depends on the rate of O2 saturation change; thus the answer is critical to our understanding of the RBC's role in regulating O2 supply.*This proposal focuses on developing the microfluidic system proposed by Sové for fully characterizing the dynamics of O2 dependent ATP release from RBCs. All research will be carried out on rat blood to maintain consistency with previous research. This technology creates the opportunity for other graduate student projects investigating the impact of conditions the RBC would encounter in the microvasculature in vivo and forms the basis for a future collaborative health research projects to translate this research into a screening to tool for patients with cardiovascular disease.