Detection of ATP-induced nitric oxide in a biomimetic circulatory vessel containing an immobilized endothelium

Conditions for the adhesion of bovine pulmonary artery endothelial cells (bPAECs) in microbore tubing of 250-mum 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 mug 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 similar to3.5 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 muM ATP is similar to0.80 muM. Based on the injection volume of ATP and the estimated number of cells on the tubing lumen, this value corresponds to similar to1 amol of NO/cell. Moreover, shear stress alone does not provide the agonistic effect required for NO production in the submicromolar range.