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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL073942-02
Application #
6765882
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Denholm, Elizabeth M
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
2
Fiscal Year
2004
Total Cost
$113,250
Indirect Cost
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Meyer, J A; Froelich, J M; Reid, G E et al. (2008) Metal-activated C-peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter. Diabetologia 51:175-82
Subasinghe, Wasanthi; Spence, Dana M (2008) Simultaneous determination of cell aging and ATP release from erythrocytes and its implications in type 2 diabetes. Anal Chim Acta 618:227-33
Carroll, Jamie S; Ku, Chia-Jui; Karunarathne, Welivitiya et al. (2007) Red blood cell stimulation of platelet nitric oxide production indicated by quantitative monitoring of the communication between cells in the bloodstream. Anal Chem 79:5133-8
D'Amico Oblak, Teresa; Root, Paul; Spence, Dana M (2006) Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device. Anal Chem 78:3193-7
Kovarik, Michelle L; Torrence, Nicholas J; Spence, Dana M et al. (2004) Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses. Analyst 129:400-5
Spence, Dana M; Torrence, Nicholas J; Kovarik, Michelle L et al. (2004) Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel. Analyst 129:995-1000
Price, Alexander K; Fischer, David J; Martin, R Scott et al. (2004) Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels. Anal Chem 76:4849-55
Fischer, David J; Torrence, Nicholas J; Sprung, Robert J et al. (2003) Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo. Analyst 128:1163-8