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

Andrzej Burewicz, Hazem Dawoud, Lu-Lin Jiang, Tadeusz Malinski

Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio, USA.

DOI: 10.4236/ajac.2013.410A1004                

Abstract

The cytoprotective messenger nitric oxide (NO) and cytotoxic peroxynitrite (ONOO^-) are the main components of oxidative 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, functional, 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 oxide 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 redox 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 nitroxidative/ oxidative stress, and may be useful in early medical diagnosis of the cardiovascular system.

Keywords

Nanosensors; Nitric Oxide; Peroxynitrite; Endothelium; Oxidative Stress

Conflicts of Interest

The authors declare no conflicts of interest.

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