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.
