Nitric Oxide/Peroxynitrite Redox Imbalance in Endothelial Cells Measured with Amperometric Nanosensors

Abstract

The cytoprotective messenger nitric oxide (NO) and cytotoxic peroxynitrite (ONOO −) are the main components of oxi-dative stress and can be generated by endothelial cells. A tandem of electrochemical nanosensors (diameter 200 -300 nm) were used to measure, in situ, the balance between NO and ONOO − produced by human umbilical vein endothelial cells (HUVEC's). The amperometric nanosensors were placed 5 ± 2 µm from the surface of the endothelial cells and the concentration of NO and ONOO − was measured at 630 mV and −300 mV (vs Ag/AgCl) respectively. Normal, func-tional, endothelial cells produced maximal 450 ± 25 nmol·L −1 of NO and 180 ± 15 nmol·L −1 of ONOO − in about 3 s, after stimulation with calcium ionophore. The in situ measurements of NO and ONOO − were validated using nitric ox-ide synthase inhibitor L-NMMA, ONOO − scavenger Mn(III) porphyrin, and superoxide dismutase (PEG-SOD). The ratio of NO concentration to ONOO − concentration ([NO]/[ONOO − ]) was introduced for quantification of both, the re-dox balance and the level of the nitroxidative stress in the endothelium. [NO]/[ONOO − ] was 2.7 ± 0.1 in a functional endothelium. The model of the dysfunctional endothelium was made by the treatment of HUVEC's with angiotensin II for 20 min. Dysfunctional HUVEC's produced only 115 ± 15 nmol·L −1 of NO, but generated a significantly higher concentration of ONOO − of 490 ± 30 nmol·L −1 . The [NO]/[ONOO − ] ratio decreased to 0.23 ± 0.14 in the dysfunctional endothelium. Electrochemical nanosensors can be effectively used for in situ monitoring of changing levels of nitroxi-dative/ oxidative stress, and may be useful in early medical diagnosis of the cardiovascular system.