calcimycin and 5-5-dimethyl-1-pyrroline-1-oxide

Endothelium produces oxygen-derived free radicals (nitric oxide, NO&z.rad;; superoxide anion, O(2)(*-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O(2)(*-) production, particularly due to the difficulty in assessing O(2)(*-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O(2)(*-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O(2)(*-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O(2)(*-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O(2)(*-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 microm rather than at 100 microm appears more suitable to detect O(2)(*-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O(2)(*-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O(2)(*-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.

Topics: Acridines; Animals; Aorta; Artifacts; Calcimycin; Cattle; Cells, Cultured; Cyclic N-Oxides; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Indicators and Reagents; Luminescent Measurements; Oxidation-Reduction; Spectrometry, Fluorescence; Spin Labels; Superoxides

Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO* production from endothelium has been extensively characterized, little is known about endothelium-derived O2-*. In the present study, we determined the O2-* production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy.. An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O2-*, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O2-* production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O2-* production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO* did not alter O2-* production. Finally, the amount of O2-* generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.

Topics: Animals; Aorta; Arginine; Calcimycin; Cattle; Cells, Cultured; Culture Media, Conditioned; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Enzyme Inhibitors; Ionophores; NAD; NADP; Nitric Oxide Synthase; Onium Compounds; Rats; Spin Labels; Superoxide Dismutase; Superoxides; Tetradecanoylphorbol Acetate

To understand the direct involvement of free radicals causing reduction in endothelium-dependent relaxation of isolated canine coronary ring preparations, this study was undertaken to examine the effect of free radicals generated from dihydroxy fumarate (DHF) plus Fe(3+)-ADP or from H2O2 plus FeSO4. The vasodilators (acetylcholine, bradykinin, A23187, and nitroglycerin) were given after DHF/Fe(3+)-ADP or H2O2/FeSO4 was removed from the organ chamber. The earlier DHF/Fe(3+)-ADP exposure produced an attenuation of the relaxation of the rings induced by acetylcholine, bradykinin, or A23187 but not of the relaxation induced by nitroglycerin. The observed effect of previous DHF/Fe(3+)-ADP exposure was significantly protected in the vessels isolated from the dogs treated with alpha-tocopherol. In the experiments for assessing the effect of various scavengers, 1O2 scavenger histidine or iron chelator deferoxamine effectively protected the attenuation induced by DHF/Fe(3+)-ADP exposure of the relaxation elicited by acetylcholine; superoxide dismutase (SOD), catalase, or dimethyl sulfoxide (DMSO) had no effect on this system. Furthermore, the relaxation elicited by acetylcholine, but not nitroglycerin, was significantly attenuated by the earlier exposure to .OH generated by Fenton's reagent (H2O2+FeSO4); the attenuation was significantly protected by DMSO. These results are consistent with the view that .OH, 1O2, and/or iron-dependent reactive species selectively damage endothelium-dependent relaxation as opposed to endothelium-independent relaxation in endothelium-intact coronary ring preparations. It is also postulated that lipid peroxidation may be responsible for this effect.

Topics: Acetylcholine; Animals; Bradykinin; Calcimycin; Coronary Vessels; Cyclic N-Oxides; Dogs; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Female; Free Radicals; Fumarates; In Vitro Techniques; Iron; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Oxygen; Spin Labels; Superoxides; Vasodilation; Vasodilator Agents

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Concanavalin A; Cyclic N-Oxides; Cytochrome c Group; Cytoplasmic Granules; Humans; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Opsonin Proteins; Spin Labels; Superoxides; Tetradecanoylphorbol Acetate; Zymosan