2-2-5-5-tetramethyl-1-pyrrolidinyloxy-3-carboxylic-acid and tempol

The relations among hypertensive response, oxidative stress, and mitogen-activated protein kinase (MAPK) in cardiovascular tissues have not been fully established. We investigated the involvement of reactive oxygen species on changes in the hemodynamics and cardiovascular MAPKs activities induced by acutely administered angiotensin II (Ang II) in conscious normotensive rats with or without treatment with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol). Intravenous infusion of a pressor dose of Ang II rapidly increased mean arterial blood pressure (MBP) by 53+/-5 mm Hg. After a 30-minute treatment with Ang II, phosphorylated MAPKs (ERK1/2, JNK, p38) as well as thiobarbital reactive substances (T-BARS) were increased in the aorta and cardiac left ventricle. Tempol had no significant effect on the elevation of MBP elicited by Ang II; however, it dose-dependently suppressed the augmented phosphorylation of cardiovascular MAPKs and increased T-BARS levels in plasma and tissues induced by Ang II. An acutely administered pressor dose of phenylephrine, an alpha-adrenoceptor agonist, also showed tempol-sensitive cardiovascular MAPK activation and tempol-insensitive blood pressure elevation. These in vivo data indicate that acute administration of Ang II or phenylephrine provoked an increase in oxidative stress in the cardiovascular tissues leading to the activation of MAPKs, whether it was mediated by pressure overload or the direct action of these vasoconstrictors, and that oxidative stress might not have a major contribution to the acute hypertensive responses elicited by the vasoconstrictors.

Topics: Acute Disease; Adrenergic alpha-Agonists; Angiotensin II; Animals; Antioxidants; Aorta; Benzimidazoles; Biphenyl Compounds; Cyclic N-Oxides; Enzyme Activation; Heart Ventricles; Hemodynamics; Hypertension; Male; Mitogen-Activated Protein Kinases; Oxidative Stress; Phenylephrine; Prazosin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Spin Labels; Tetrazoles; Thiobarbituric Acid Reactive Substances; Vasoconstrictor Agents

Measurements of 7Li nuclear spin-lattice relaxation times are made at applied magnetic field strengths from 0.25 mT to 7.05 T, in order to determine directly the form of the frequency-dependent spectral densities that modulate relaxation. This magnetic resonance dispersion (MRD) technique provides detailed information regarding molecular dynamics down to the picosecond time scale. 7Li MRD measurements made on aqueous lithium ion in the presence of small concentrations of nitroxide free radicals give direct evidence supporting the formation of a coordination complex. The dipole-dipole electron-nuclear coupling is modulated by both translational and rotational diffusive motions, and both of these contributions are resolved. However, scalar coupling arising from the presence of paramagnetic electron spin density at the nucleus dominates the nuclear relaxation. Changes in the pH and free radical moiety are compared with dynamical variables, geometric constraints, and formation constants obtained from a model of nuclear relaxation. The calculated bimolecular formation constants are on the order of 2 x 10(-3) M-1, and the relative accuracy of this parameter is tested.

Topics: Algorithms; Cyclic N-Oxides; Diffusion; Electron Spin Resonance Spectroscopy; Free Radicals; Hydrogen-Ion Concentration; Isotopes; Lithium; Magnetics; Models, Chemical; Molecular Conformation; Rotation; Spectrum Analysis; Spin Labels; Water

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.

Topics: Animals; Calcimycin; Cattle; Cells, Cultured; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Molecular Mimicry; Molsidomine; Nitric Oxide; Nitrogen Oxides; Spin Labels; Spin Trapping; Superoxide Dismutase; Superoxides

The nitroxide Tempol, a stable free radical, has recently been shown to protect mammalian cells against several forms of oxidative stress including radiation-induced cytotoxicity. To extend this observation, six additional water-soluble nitroxides with different structural features were evaluated for potential radioprotective properties using Chinese hamster V79 cells and clonogenic assays. Nitroxides (10 mM) were added 10 min prior to radiation exposure and full radiation dose-response curves were determined. In addition to Tempol, five of the six nitroxides afforded in vitro radioprotection. The best protectors were found to be the positively charged nitroxides, Tempamine and 3-aminomethyl-PROXYL, with protection factors of 2.3 and 2.4, respectively, compared with Tempol, which had a protection factor of 1.3. 3-Carboxy-PROXYL, a negatively charged nitroxide, provided minimal protection. DNA binding characteristics as studied by nonequilibrium dialysis of DNA with each of the nitroxides demonstrated that Tempamine and 3-amino-methyl-PROXYL bound more strongly to DNA than did Tempol. Since DNA is assumed to be the target of radiation-induced cytotoxicity, differences in protection may be explained by variabilities in affinity of the protector for the target. This study establishes nitroxides as a general class of new nonthiol radioprotectors and suggests other parameters that may be exploited to find even better nitroxide-induced radioprotection.

Topics: Animals; Cell Line; Cell Survival; Cricetinae; Cyclic N-Oxides; Dose-Response Relationship, Radiation; Protein Synthesis Inhibitors; Pyrrolidines; Radiation-Protective Agents; Spin Labels; Triacetoneamine-N-Oxyl

Clearance of the nitroxide radicals, hydroxy-TEMPO and carboxy-PROXYL, in whole-mouse lung was directly measured by in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the lung was confirmed by ESR imaging. The ESR signal of hydroxy-TEMPO was reduced in the lung and the clearance obeyed first-order kinetics, whereas the signal of carboxy-PROXYL remained constant. Comparison of the clearance rates of live and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the blood circulation system.

Topics: Animals; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Female; Free Radicals; Lung; Metabolic Clearance Rate; Mice; Muscles; Nitrogen Oxides; Spin Labels

Contrast agents have been investigated in an effort to increase the differences between the proton relaxation times of normal and pathologic tissue, and thus improve the ability to detect regions of pathology. Data on the oxygen-dependent metabolism of three nitroxides that are potential MRI contrast agents are presented. The amount of active, paramagnetic nitroxide remaining in suspensions of mammalian cells was followed as a function of time using electron-spin resonance spectroscopy. Data on the rates of nitroxide reduction over a wide range of oxygen concentrations are presented and show these rates as a function of the intracellular oxygen concentration. When intracellular oxygen concentrations were measured, only cells that were severely hypoxic reduced these potential contrast agents at increased rates. Because regions of some types of pathology are often hypoxic, the oxygen-dependent metabolism of nitroxides should allow contrast to be achieved on the basis of differences in metabolism between normal and diseased tissues. Further, the data suggest that future studies of nitroxide metabolism need to take into account the O2 concentration in order to understand the observed reduction rates and to predict the rates under other conditions.

Topics: Animals; Cells, Cultured; Contrast Media; Cyclic N-Oxides; Magnetic Resonance Spectroscopy; Mice; Oxygen; Spin Labels