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

1. Introduction

Nitric oxide is a free radical with a half-life of less than five seconds, which has been found as a signaling molecule in both, the cardiovascular system and neurological system [1,2]. NO is involved in the relaxation of the arterial wall [3], acts as a mediator of immune system [4], and is a crucial neurotransmitter in the peripheral and central nervous systems [5]. NO can be produced by five electrons oxidation process of L-arginine by nitric oxide synthase (NOS). There are two constitutive nitric oxide synthase (cNOS): endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS). These two enzymes are dimers. NO production from eNOS is stimulated by Ca²⁺-calmodulin dependent pathway. The time of NO production is about 5 - 20 s and 1 - 5 s by eNOS and nNOS, respectively [6]. Inducible nitric oxide synthase (iNOS) is a Ca²⁺-calmodulin independent enzyme and can produce NO for an extended period of time (up to hours) [7]. Intra-cellular Ca²⁺ flux into an endothelial cell triggers NO production by eNOS. NO is a small lipophilic molecule that can diffuse readily through cellular membranes and plasma to activate the soluble guanylate cyclase (sGC)/guanosine 3,5-cyclic monophosphate (cGMP) pathway in smooth muscle cells, platelets, and leukocytes [8-10]. Therefore, NO plays a vital role in maintaining vascular smooth muscle relaxation, inhibits the adhesion of platelets-leukocytes to endothelial cells and prevents platelet aggregation, also regulates blood flow and blood pressure [3,11]. NO is also a main scavenger of superoxide ion in biological milieu. Under pathological conditions, at high oxidative stress, most of the NO produced by endothelium or neurons is consumed in the reaction with to form peroxynitrite (ONOO⁻) [12,13]. ONOO⁻ is much more powerful oxidant than NO or. High levels of ONOO⁻ can cause DNA strands break, lipid peroxidation, trigger cell apoptosis via the activation of caspase cascade, and deactivate several enzymes [14]. The production of ONOO⁻ not only increases the level of redox toxicity but also diminishes the concentration of bioavailable NO, resulting in endothelial and/or neuronal dysfunction [15,16]. A dysfunctional endothelium has been found in patients with serious cardiovascular conditions, such as: hypertension, diabetes, atherosclerosis, coronary artery diseases, and chronic heart failure [17-19]. In addition, the dysfunction of the endothelium and neurons has also been implicated in several neurodegenerative diseases, such as, Alzheimer’s disease [20], Lou Gehrig’s disease (amyotrophic lateral sclerosis, ALS) [21], Parkinson’s disease and epilepsy [2,22]. We hypothesized that the real-time measurements of balance/imbalance between the vital signaling molecule NO and the cytotoxic ONOO⁻ may be useful as diagnostic tool to detect early dysfunction of cardiovascular or neurological system. Both NO and ONOO⁻ are short living species with a half-life of 3 s and 1 s respectively [13]. Both NO and ONOO⁻ molecules can react or form adducts with many other biological molecules. Therefore, the average distance of diffusion of these molecules is less than 100 µm [23,24]. Nanosensors were applied for in situ measurement of NO and ONOO⁻ concentration produced by a single endothelial cell. These electrochemical nanosensors meet all fundamental requirements for this kind measurement: small enough in size (about 200 - 300 µm) to be placed near the membrane of an endothelial cell. The response time of the sensors is less than 50 µs and the detection limit is about 10⁻⁹ mol∙L⁻¹.

2. Materials and Methods

2.1. Materials

Superoxide dismutase-polyethylene glycol (PEG-SOD); 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-6k) pyrimidine (VAS2870); calcium ionophore (CaI, A23187), N^G-Methyl-L-arginine acetate salt (L-NMMA), Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (Mn(III) porphyrin) were purchased from Sigma-Aldrich (St. Louis, MO).

2.2. Subjects and Cell Cultures

Human umbilical vein endothelial cells (HUVECs) were purchased as proliferating cells from Lonza (Walkersville, MD). Cells were cultured in the recommended MCDB-131 (Vec Technologies) complete medium and maintained at 37˚C in a 5% CO₂ humidified incubator. Cells were supplied with a fresh medium every other day and propagated using an enzymatic dissociation (trypsin) procedure (maximum of 16 population doublings).

2.3. Preparation of Nanosensors for NO and ONOO⁻ Detection

NO and ONOO⁻ were measured with nanosensors (diameter 200 - 300 nm). The design of the nanosensors was based on previously developed chemically modified carbon fiber technology [25-27]. Briefly, the carbon fibers were sealed in glass capillaries with a non-conductive epoxy and electrically connected to copper wires with conductive sliver epoxy. A fiber (original diameter 7 nm) was covered with a film of bee wax and rosin. The diameter of carbon fiber tip was reduced to about 300 nm by gradual burning of the fiber using a propane microburner. The exposed suface of the conical shape of the tip was covered with conductive polymeric porphyrin: polymeric nickel (II) tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin (Frontier Scientific) for the NO sensor, and Mn (III)-[2, 2] paracyclophenyl-porphyrin (Frontier Scientific) for the ONOO⁻ sensor, respectively. The polymeric porphyrinic film was covered with thin layer of Nafion for the NO sensor and with poly (4-vinylpyridine) for the ONOO⁻ sensor.

2.4. Amperometric Measurement of NO and ONOO⁻

A three-electrode system has been utilized for amperometric measurement of NO and ONOO⁻. In this system, NO and ONOO⁻ nanosensors served as working electrode; a silver/silver chloride (Ag/AgCl) as reference electrode, and a platinum wire (diameter 0.1 mm) as a counter electrode. Amperometric measurement was performed by using a computer-controlled Gamry VFP600 multichannel potentiostat, which has been used to measure NO and ONOO⁻ concentrations change from their basal levels with time. The detection limit is 1 nmol L^-1 and the resolution time < 50 ms. Linear calibration curves were constructed for each sensor in the range of 20 nmol∙L⁻¹ to 1 µmol∙L⁻¹ of NO and ONOO⁻ standard solution. A standard addition method was also used to calibrate the sensors after the measurement of NO and ONOO⁻ in endothelial cells. Endothelial cells were placed in 2 cm wells to achieve confluence. A single sensor or tandem of NO and ONOO⁻ sensors were placed in the well with a help of computerized remote controlled micromanipulator (Sensapex, Findland) with x, y, and z space resolution of 1 µm. Sensors were lowered to a membrane level of the endothelial cell, sending a small piezoelectric signal of 6 to 8 pA, lasting 1 to 3 ms, until the surface of the cell membranes was reached. This procedure helped to establish a zero distance from cell membrane on z axis. From this point, the nanosensors were lifted about 5 µm from the cell membrane and shifted horizontally along x and y axises about 30 - 50 µm. The sensors were positioned above a single endothelial cell. The measurements were done in a 0.1 mol∙L⁻¹phosphate buffer (at pH 7.4) in the absence or presence of superoxide dismutase (PEG-SOD), 400 U∙ml⁻¹; Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (Mn(III) porphyrin) 10 µmol∙L⁻¹, a scavenger for ONOO⁻; VAS2870 1 µmol∙L⁻¹, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase selective inhibitor; or N^G-Methyl-Larginine acetate salt (L-NMMA) 0.4 mmol∙L⁻¹, an inhibitor of constitutive NOS. NO/ONOO⁻ release was stimulated by calcium ionophore (CaI, A23187), at a concentration of 1 µmol∙L⁻¹, injected via a nanoinjector positioned with a computer-controlled micromanipulator.

2.5. Calculations and Statistical Analysis

All data are presented as mean ± standard deviation (SD) of the mean of n = 3 - 5. Statistical analysis of the mean difference between multiple groups was performed using one-way analysis of variance (ANOVA) with StudentNewman-Keuls multiple comparisons post hoc analysis; and between two groups, using two-tailed Student’s t-test. The alpha level for all the tests was 0.05. A P value < 0.05 was considered to be statistically significant. All statistical analyses were performed using Origin (v 6.1 for Windows; OriginLab, Northampton, MA).

3. Results and Discussion

Nanosensors were placed near the surface of HUVEC cells. An accurate positioning of the nanosensors in the relation to the cell membrane surface is crucially important in order to obtain reproducible results. The nanosensors cannot be placed directly on the surface of endothelial cells because they will stimulate NO and ONOO⁻ release through activation of mechanical channels and calcium flux to cytoplasm.

Figure 1 depicts a change of NO concentration as a function of the distance from endothelial cell membrane. The NO concentration decreases exponentially with a distance from a surface of the membrane. The process of NO diffusion from a membrane of the cell depends directly on the gradient of concentration between the membrane surface and buffer solution or cytoplasm. In the phosphate buffer, NO concentration is decreased by 50% at the distance of about 30 µm from the membrane

Conflicts of Interest

The authors declare no conflicts of interest.

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