tempo and 10-10--dimethyl-9-9--biacridinium

New copper(I) complexes have been synthesised from the reaction of CuCl with 4-(diphenylphosphane)benzoic acid and lithium tris(1H-pyrazol-1-yl)methanesulfonate, Li(SO(3))C(pz)(3), sodium hydrotris(3-trifluoromethyl-1H-pyrazol-1-yl)borate, NaHB[3-(CF(3))pz](3), potassium dihydrobis(1H-1,2,4-triazol-1-yl)borate, KH(2)B(tz)(2), hydrotris(1H-1,2,4-triazol-1-yl)borate, KHB(tz)(3), sodium hydrotris(1H-pyrazol-1-yl)borate, NaHB(pz)(3), potassium hydrotris(3,5-dimethyl-1H-pyrazol-1-yl)borate KHB(3,5-Me(2)Pz)(3) or potassium hydrotris(4-bromo-1H-pyrazol-1-yl)borate KHB(4-Brpz)(3). The complexes obtained have been characterized by elemental analyses and FT-IR in the solid state, and by NMR (1H and 31P[(1)H]) spectroscopy and conductivity measurements in solution. The solution data are consistent with partial dissociation of the sterically hindered complexes by way of breaking of Cu-P and Cu-N bonds. Electrospray mass spectrometry has been used to investigate the relative properties of the 4-(diphenylphosphane)benzoic acid and of the "scorpionate" ligands towards copper(I) ions. Chemiluminescence technique was used to evaluate the superoxide scavenging activity of these new copper complexes.

Topics: Acridines; Benzoates; Copper; Cyclic N-Oxides; Free Radical Scavengers; Inhibitory Concentration 50; Ligands; Luminescent Measurements; Magnetic Resonance Spectroscopy; Organometallic Compounds; Phosphines; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; Superoxide Dismutase; Superoxides; Xanthine Oxidase

Peroxynitrite (ONOO(-)) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH-oxidase inhibitor apocynin (4-hydroxy-3-methoxy-acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro The murine macrophage cell-line J774A.1, stimulated with IFNgamma/LPS, was used as a model. Conversion of 123-dihydrorhodamine (123-DHR) to its oxidation product 123-rhodamine was used to measure peroxynitrite production. Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH-oxidase dependent superoxide formation. The NADPH-oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

Topics: Acetophenones; Acridines; Animals; Antioxidants; Cell Line; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidines; Interferon-gamma; Lipopolysaccharides; Luminescent Measurements; Macrophages; Mice; Molsidomine; Nitrates; Nitric Oxide Synthase; Oxidation-Reduction; Rhodamines

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide O2(-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2(-.) and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6, 6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2(-.) production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria.

Topics: Acridines; Catalase; Cyclic N-Oxides; Electron Transport; Humans; Luminescent Measurements; Luminol; Malates; Mitochondria; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Pyruvic Acid; Reactive Oxygen Species; Superoxide Dismutase

Direct detection of intramitochondrial superoxide anion radical (O(-*)(2)) production is of critical importance for investigating the pathophysiological consequences resulting from altered cellular reactive oxygen homeostasis. The purpose of this study with isolated mitochondria was to characterize the biochemical basis for lucigenin as a chemiluminescent probe to detect intramitochondrial O(-*)(2) production. Incubation of isolated mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived chemiluminescence (LDCL), which was increased markedly by mitochondrial substrates, pyruvate/malate or succinate. The LDCL was reduced greatly by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. With an ion-pair HPLC method, a concentration-dependent accumulation of lucigenin was detected within mitochondria. The accumulation of lucigenin by mitochondria was reduced markedly in the presence of carbonyl cyanide p-(trifluoromethoxy)phenyhyldrazone, an uncoupler known to dissipate the mitochondrial membrane potential. With submitochondrial particles, we observed that both complexes I and III of the mitochondrial electron transport chain appear to be able to catalyze the one electron reduction of lucigenin, a critical step involved in LDCL. After incubation of mitochondria with lucigenin at non-redox cycling concentrations, formation of N-methylacridone, the proposed end product of the reaction pathway leading to LDCL, within the mitochondrial fraction was also detected. In addition, a significant linear correlation was observed between the LDCL and either the lucigenin accumulation or the N-methylacridone formation within the mitochondria. Taken together, our results conclusively demonstrate that when properly used LDCL can reliably detect intramitochondrial O(-*)(2) production.

Topics: Acridines; Acridones; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations; Cell Line; Chromatography, High Pressure Liquid; Cyclic N-Oxides; Electron Transport; Humans; Intracellular Membranes; Luminescent Measurements; Malates; Membrane Potentials; Metalloporphyrins; Mitochondria; Molecular Mimicry; Oxidation-Reduction; Permeability; Pyruvic Acid; Succinic Acid; Superoxide Dismutase; Superoxides