coelenterazine and 10-10--dimethyl-9-9--biacridinium

The radical anion of dioxygen superoxide (O2.-) is a physiological free radical formed in various enzymatic processes. On the one hand superoxide is a precursor of reactive oxygen and nitrogen species (hydroxyl radicals, peroxy radicals, hydrogen peroxide, peroxynitrite, etc.), -the initiators of cellular damage; on the other hand it is a signaling molecule regulating numerous physiological processes including apoptosis, aging, and senescence. Therefore, the detection and measurement of superoxide in cells, tissues, and whole organisms is of a vital importance for in vitro and in vivo studies of many physiological and pathophysiological processes. At present different efficient methods were developed, which allow to identificate and measure superoxide in biological systems. In present review the credibility and efficiency of principal mostly applied methods of superoxide detection based on one-electron transfer and nucleophilic reactions are discussed, and spectrophotometrical, chemiluminescent, fluorescent, and ESR spin trapping methods are compared.

Topics: Acridines; Cytochromes c; Imidazoles; Luminescent Measurements; Luminol; Oxygen; Pyrazines; Spectrophotometry; Spin Trapping; Superoxides

The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude. Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.

Topics: Acridines; Animals; Cell Membrane; Firefly Luciferin; Hypoxanthine; Imidazoles; Intracellular Membranes; Luminescent Measurements; Mitochondria, Liver; Molecular Probes; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Paracoccus denitrificans; Pyrazines; Rats; Superoxides; Time Factors; Xanthine Oxidase

Available evidence for oxidative stress after angioplasty is indirect or ambiguous. We sought to characterize the pattern, time course, and possible sources of free radical generation early after arterial balloon injury. Ex vivo injury performed in arterial rings in buffer with lucigenin yielded a massive oxygen-dependent peak of luminescence that decayed exponentially and was proportional to the degree of injury. Signals for injured vs. control arteries were 207. 1 +/- 17.9 (n = 13) vs 4.1 +/- 0.7 (n = 22) cpm x 10(3)/mg/min (p <. 001). Data obtained with 0.25 mmol/l lucigenin were validated with 0. 005-0.05 mmol/l lucigenin or the novel superoxide-sensitive probe coelenterazine (5 micromol/l). Gentle removal of endothelium prior to injury scarcely affected the amount of luminescence. Lucigenin signals were amplified 5- to 20-fold by exogenous NAD(P)H, and were >85% inhibited by diphenyliodonium (DPI, a flavoenzyme inhibitor). Antagonists of several other potential free radical sources, including xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport, were without effect. Overdistension of intact rabbit iliac arteries in vivo (n = 7) induced 72% fall in intracellular reduced glutathione and 68% increase in oxidized glutathione, so that GSH/GSSG ratio changed from 7.93 +/- 2.14 to 0. 81 +/- 0.16 (p <.005). There was also 28.7% loss of the glutathione pool. Further studies were performed with electron paramagnetic resonance spectroscopy. Rabbit aortas submitted to ex vivo overdistension in the presence of the spin trap DEPMPO (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide, 100 mmol/l, n = 5) showed formation of radical adduct spectra, abolished by DPI or superoxide dismutase. Computer simulation indicated a mixture of hydroxyl and carbon-centered radical adducts, likely due to decay of superoxide adduct. Electrical mobility shift assays for NF-kappaB activation were performed in nuclear protein extracts from intact or previously injured rabbit aortas. Balloon injury induced early NF-kappaB activation, which was decreased by DPI. In conclusion, our data show unambiguously that arterial injury induces an immediate profound vascular oxidative stress. Such redox imbalance is likely accounted for by activation of vessel wall NAD(P)H oxidoreductase(s), generating radical species potentially involved in tissue repair.

Topics: Acridines; Animals; Biphenyl Compounds; Catheterization; Cyclic N-Oxides; Cyclooxygenase Inhibitors; Electron Spin Resonance Spectroscopy; Electron Transport; Endothelium, Vascular; Free Radicals; Glutathione; Imidazoles; Lipoxygenase Inhibitors; Luminescent Measurements; Male; Metalloporphyrins; NAD; NADH, NADPH Oxidoreductases; NADP; NADPH Oxidases; NF-kappa B; Nitric Oxide Synthase; Onium Compounds; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption; Pyrazines; Rabbits; Reactive Oxygen Species; Recombinant Fusion Proteins; Spin Labels; Superoxide Dismutase; Superoxides; Transcription, Genetic; Wound Healing; Xanthine Oxidase

Lucigenin-amplified chemiluminescence has frequently been used to assess the formation of superoxide in vascular tissues. However, the ability of lucigenin to undergo redox cycling in purified enzyme-substrate mixtures has raised questions concerning the use of lucigenin as an appropriate probe for the measurement of superoxide production. Addition of lucigenin to reaction mixtures of xanthine oxidase plus NADH resulted in increased oxygen consumption, as well as superoxide dismutase-inhibitable reduction of cytochrome c, indicative of enhanced rates of superoxide formation. Additionally, it was revealed that lucigenin stimulated oxidant formation by both cultured bovine aortic endothelial cells and isolated rings from rat aorta. Lucigenin treatment resulted in enhanced hydrogen peroxide release from endothelial cells, whereas exposure to lucigenin resulted in inhibition of endothelium-dependent relaxation in isolated aortic rings that was superoxide dismutase inhibitable. In contrast, the chemiluminescent probe coelenterazine had no significant effect on xanthine oxidase-dependent oxygen consumption, endothelial cell hydrogen peroxide release, or endothelium-dependent relaxation. Study of enzyme and vascular systems indicated that coelenterazine chemiluminescence is a sensitive marker for detecting both superoxide and peroxynitrite.

Topics: Acetylcholine; Acridines; Animals; Cells, Cultured; Cytochrome c Group; Endothelium, Vascular; Firefly Luciferin; Hydrogen Peroxide; Imidazoles; Luminescent Measurements; Male; NAD; Nitrates; Oxidants; Oxygen Consumption; Pyrazines; Rats; Rats, Sprague-Dawley; Superoxides; Vasodilator Agents; Xanthine Oxidase