AJAC  Vol.4 No.10 A , October 2013
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 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.
Cite this paper: A. Burewicz, H. Dawoud, L. Jiang and T. Malinski, "Nitric Oxide/Peroxynitrite Redox Imbalance in Endothelial Cells Measured with Amperometric Nanosensors," American Journal of Analytical Chemistry, Vol. 4 No. 10, 2013, pp. 30-36. doi: 10.4236/ajac.2013.410A1004.

[1]   T. Malinski and Z. Taha, “Nitric Oxide Release from a Single Cell Measured in Situ by a Porphyrinic-Based Microsensor,” Nature, Vol. 358, No. 6388, 1992, pp. 676-678.

[2]   L. J. Ignarro, “Endothelium-Derived Nitric Oxide: Actions and Properties,” FASEB Journal, Vol. 3, No. 1, 1989, pp. 31-36.

[3]   L. A. Blatter, Z. Taha, S. Mesaros, P. S. Shacklock, W. G. Wier and T. Malinski, “Simultaneous Measurements of Ca2+ and Nitric Oxide in Bradykinin-Stimulated Vascular Endothelial Cells,” Circulation Research, Vol. 76, No. 5, 1995, pp. 922-924.

[4]   J. B. Hibbs, Jr., Z. Vavrin and R. R. Taintor, “L-Arginine Is Required for Expression of the Activated Macrophage Effector Mechanism Causing Selective Metabolic Inhibi-tion in Target Cells,” Journal of Immunology, Vol. 138, No. 2, 1987, pp. 550-565.

[5]   S. H. Snyder and D. S. Bredt, “Nitric Oxide as a Neuronal Messenger,” Trends in Pharmacological Sciences, Vol. 12, No. 4, 1991, pp. 125-128.

[6]   T. Malinski, Z. Taha, S. Grunfeld, S. Patton, M. Kapturczak and P. Tomboulian, “Diffusion of Nitric Oxide in the Aorta Wall Monitored in Situ by Porphyrinic Microsensors,” Biochemical and Biophysical Research Communications, Vol. 193, No. 3, 1993, pp. 1076-1082.

[7]   J. L. Balligand, D. Ungureanu-Longrois, W. W. Simmons, D. Pimental, T. A. Malinski, M. Kapturczak et al., “Cytokine-Inducible Nitric Oxide Synthase (iNOS) Expression in Cardiac Myocytes. Characterization and Regulation of iNOS Expression and Detection of iNOS Activity in Single Cardiac Myocytes in Vitro,” Journal of Biological Chemistry, Vol. 269, No. 44, 1994, pp. 27580-27588.

[8]   W. P. Arnold, C. K. Mittal, S. Katsuki and F. Murad, “Nitric Oxide Activates Guanylate Cyclase and Increases Guanosine 3’:5’-Cyclic Monophosphate Levels in Various Tissue Preparations,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 74, No. 8, 1977, pp. 3203-3207.

[9]   L. J. Ignarro, “Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology,” Bioscience Reports, Vol. 19, No. 2, 1999, pp. 51-71.

[10]   L. J. Ignarro, J. B. Adams, P. M. Horwitz and K. S. Wood, “Activation of Soluble Guanylate Cyclase by NO-Hemoproteins Involves NO-Heme Exchange. Comparison of Heme-Containing and Heme-Deficient Enzyme Forms,” Journal of Biological Chemistry, Vol. 261, No. 11, 1986, pp. 4997-5002.

[11]   S. Grunfeld, C. A. Hamilton, S. Mesaros, S. W. McClain, A. F. Dominiczak, D. F. Bohr et al., “Role of Superoxide in the Depressed Nitric Oxide Production by the Endothelium of Genetically Hypertensive Rats,” Hypertension, Vol. 26, No. 6, 1995, pp. 854-857.

[12]   J. S. Beckman, T. W. Beckman, J. Chen, P. A. Marshall and B. A. Freeman, “Apparent Hydroxyl Radical Production by Peroxynitrite: Implications for Endothelial Injury from Nitric Oxide and Superoxide,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 87, No. 4, 1990, pp. 1620-1624.

[13]   J. S. Beckman and W. H. Koppenol, “Nitric Oxide, Superoxide, and Peroxynitrite: The Good, the Bad, and Ugly,” American Journal of Physiology, Vol. 271, No. 5, 1996, pp. C1424-C1437.

[14]   A. J. Kozak, F. Liu, P. Funovics, A. Jacoby, R. Kubant and T. Malinski, “Role of Peroxynitrite in the Process of Vascular Tone Regulation by Nitric Oxide and Prostanoids—A Nanotechnological Approach,” Prostaglandins Leukot Essent Fatty Acids, Vol. 72, No. 2, 2005, pp. 105-113.

[15]   T. Malinski, “Normal and Pathological Distribution of Nitric Oxide in the Cardiovascular System,” Polish Journal of Pharmacology, Vol. 50, No. 6, 1998, pp. 387-391.

[16]   T. Malinski, “Nitric Oxide Signaling in the Cardiovascular System—Physiology and Pathology,” Post?py Higieny i Medycyny Do?wiadczalnej, Vol. 53, No. 2, 1999, pp. 205-207.

[17]   L. Kalinowski and T. Malinski, “Endothelial NADH/ NADPH-Dependent Enzymatic Sources of Superoxide Production: Relationship to Endothelial Dysfunction,” Acta Biochimica Polonica, Vol. 51, No. 2, 2004, pp. 459-469.

[18]   R. P. Mason, L. Kalinowski, R. F. Jacob, A. M. Jacoby and T. Malinski, “Nebivolol Reduces Nitroxidative Stress and Restores Nitric Oxide Bioavailability in Endothelium of Black Americans,” Circulation, Vol. 112, No. 24, 2005, pp. 3795-3801.

[19]   R. P. Mason, R. Kubant, R. F. Jacob, M. F. Walter, B. Boychuk and T. Malinski, “Effect of Nebivolol on Endothelial Nitric Oxide and Peroxynitrite Release in Hypertensive Animals: Role of Antioxidant Activity,” Journal of Cardiovascular Pharmacology, Vol. 48, No. 1, 2006, pp. 862-869.

[20]   T. Malinski, “Nitric Oxide and Nitroxidative Stress in Alzheimer’s Disease,” Journal of Alzheimer’s Disease, Vol. 11, No. 2, 2007, pp. 207-218.

[21]   D. R. Rosen, T. Siddique, D. Patterson, D. A. Figlewicz, P. Sapp, A. Hentati, et al., “Mutations in Cu/Zn Superoxide Dismutase Gene Are Associated with Familial Amyotrophic Lateral Sclerosis,” Nature, Vol. 362, No. 6415, 1993, pp. 59-62.

[22]   R. F. Furchgott, “Endothelium-Derived Relaxing Factor: Discovery, Early Studies, and Identification as Nitric Oxide,” Bioscience Reports, Vol. 19, No. 4, 1999, pp. 235-251.

[23]   D. I. Levy, N. J. Sucher and S. A. Lipton, “Redox Modulation of NMDA Receptor-Mediated Toxicity in Mammalian Central Neurons,” Neuroscience Letters, Vol. 110, No. 3, 1990, pp. 291-296.

[24]   J. Wood and J. Garthwaite, “Models of the Diffusional Spread of Nitric Oxide: Implications for Neural Nitric Oxide Signalling and Its Pharmacological Properties,” Neuropharmacology, Vol. 33, No. 11, 1994, pp. 1235-1244.

[25]   V. Brovkovych, S. Patton, S. Brovkovych, F. Kiechle, I. Huk and T. Malinski, “In Situ Measurement of Nitric Oxide, Superoxide and Peroxynitrite during Endotoxemia,” Journal of Physiology and Pharmacology, Vol. 48, No. 4, 1997, pp. 633-644.

[26]   J. Xue, X. Ying, J. Chen, Y. Xian and L. Jin, “Amperometric Ultramicrosensors for Peroxynitrite Detection and Its Application toward Single Myocardial Cells,” Analytical Chemistry, Vol. 72, No. 21, 2000, pp. 5313-5321.