Detection of ATP-Induced Nitric Oxide in a Biomimetic Circulatory Vessel Containing an Immobilized Endothelium

Abstract

Conditions for the adhesion of bovine pulmonary artery endothelial cells (bPAECs) in microbore tubing of 250-microm i.d. are described. When immobilized to the lumen of microbore tubing, these cells represent a mimic of a circulatory vessel's endothelium. The microbore tubing is coated with 100 microg mL(-1) fibronectin in order to promote bPAEC adhesion to the lumen of the tubing. A series of micrographs of the cells inside of the tubing indicates that approximately 3.5 h is necessary for cell adhesion. In this study, adenosine triphosphate (ATP) is used to induce the release of nitric oxide from the endothelium mimic. The endothelium-derived NO is detected amperometrically at a parallel flow cell containing a glassy carbon working electrode modified with Nafion. Results indicate that detectable amounts of NO are only produced by the endothelium mimic when ATP is present in the buffer. The typical concentration of NO produced by the endothelium mimic upon the introduction of 100 microM ATP is approximately 0.80 microM. Based on the injection volume of ATP and the estimated number of cells on the tubing lumen, this value corresponds to approximately 1 amol of NO/cell. Moreover, shear stress alone does not provide the agonistic effect required for NO production in the submicromolar range.

Patterned Electrode-Based Amperometric Gas Sensor for Direct Nitric Oxide Detection within Microfluidic Devices

“…Detection of such high levels of NO with such few cells is made possible by direct integration of the NO sensor with microchannels and by use of low gas permeable materials to construct the channels. At the relatively slow flow rate of media used in this study (3 μL/min) it is estimated that the longest travel time for NO produced by cells to reach the planar sensor is about 80 s. This is within the reported lifetime of NO (~ 200 s) in deoxygenated buffers 4, 9. The sensing capability is reversible; the detector's current readout returns to original background levels when the cell-conditioned media is replaced with fresh media.…”

Detection of nitric oxide production in cell cultures by luciferin–luciferase chemiluminescence

“…Evidently, the substantial part of consumed oxygen (about 40%) in these cells is spent on NO production. For endothelial cells, studies report the values from 0.6 to 3 amol/(cell·min) and bradykinin-stimulated increase of 3–6 times over the basal rate [23,29,30]. Our results agree with those data produced by different methods.…”

Purinergic Signaling in the Airways

“…ATP release from endothelial cells induced by NA was greater in the distal (smaller) arteries compared with proximal pulmonary arteries of the rabbit (Takeuchi et al, 1995). In addition to ATP released from endothelial cells, ATP release from erythrocytes contributes to release of NO and subsequent vasodilation (Sprague et al, 1996(Sprague et al, , 2003Kotsis and Spence, 2003). Protein kinase C mediates inhibition of endothelial P2Y 1 and P2Y 2 receptor-mediated phosphoinositide turnover in bovine pulmonary artery (Chen and Lin, 1999).…”