A system for exposing molecules and cells to biologically relevant and accurately controlled steady-state concentrations of nitric oxide and oxygen
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Nitric Oxide
(Impact Factor: 3.52).
06/2012; 27(3):161-8. DOI: 10.1016/j.niox.2012.06.004
Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O(2) over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O(2) into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O(2) in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.
Available from: Luiz Godoy
[Show abstract] [Hide abstract]
ABSTRACT: Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-l-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl l-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.
Chemical Research in Toxicology 09/2012; 25(10):2194-202. DOI:10.1021/tx300274a · 3.53 Impact Factor
Available from: Mahesh S Joshi
[Show abstract] [Hide abstract]
ABSTRACT: Nitric oxide (NO) research in biomedicine has been hampered by the absence of a method that will allow quantitative measurement of NO in biological tissues with high sensitivity and selectivity, and with adequate spatial and temporal resolution. 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) is a NO sensitive fluorescence probe that has been used widely for qualitative assessment of cellular NO production. However, calibration of the fluorescent signal and quantification of NO concentration in cells and tissues using fluorescent probes, have provided significant challenge. In this study we utilize a combination of mathematical modeling and experimentation to elucidate the kinetics of NO/DAF-FM reaction in solution. Modeling and experiments suggest that the slope of fluorescent intensity (FI) can be related to NO concentration according to the equation: ddtF1=2αk(1)NO(2)O(2)DAF-FMkNO+DAF-FM where α is a proportionality coefficient that relates FI to unit concentration of activated DAF-FM, k(1) is the NO oxidation rate constant, and k was estimated to be 4.3±0.6. The FI slope exhibits saturation kinetics with DAF-FM concentration. Interestingly, the effective half-maximum constant (EC(50)) increases proportionally to NO concentration. This result is not in agreement with the proposition that N(2)O(3) is the NO oxidation byproduct that activates DAF-FM. Kinetic analysis suggests that the reactive intermediate should exhibit NO-dependent consumption and thus NO(2)() is a more likely candidate. The derived rate law can be used for the calibration of DAF-FM fluorescence and the quantification of NO concentration in biological tissues.
Nitric Oxide 10/2012; 28. DOI:10.1016/j.niox.2012.10.001 · 3.52 Impact Factor
Available from: Brian Skinn
[Show abstract] [Hide abstract]
ABSTRACT: Nitrogen dioxide is formed endogenously via the oxidation of NO by O(2) or O(2)(-), and from NO(2)(-) via peroxidases, among other pathways. This radical has many potential biological targets and its concentration, like that of NO and other reactive nitrogen species, is thought to be elevated at sites of inflammation. To investigate the specific cytotoxic or mutagenic effects of NO(2), it is desirable to be able to maintain its concentration at constant, predictable, and physiological levels in cell cultures, in the absence of NO. To do this, a delivery system was constructed in which NO(2)-containing gas mixtures contact a liquid within a small (110ml) stirred reactor. In such gas mixtures NO(2) is present in equilibrium with its dimer, N(2)O(4). The uptake of NO(2) and N(2)O(4) was characterized by measuring the accumulation rates of NO(2)(-) and NO(3)(-), the stable products of N(2)O(4) hydrolysis, in buffered aqueous solutions. In some experiments NO(2)-reactive 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonate) (ABTS) was included and formation of the stable ABTS radical was measured. A reaction-diffusion model was developed that predicts the accumulation rates of all three products to within 15% for gas-phase concentrations of NO(2) spanning three orders of magnitude. The model also provides estimates for the NO(2) concentration in the liquid. This system should be useful for exposing cells to NO(2) concentrations similar to those in vivo.
Free Radical Biology and Medicine 10/2012; 56. DOI:10.1016/j.freeradbiomed.2012.10.534 · 5.74 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.