Coupling of phagocytic NADPH oxidase activity and mitochondrial superoxide production

Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are “primed” for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions

Antihypertensive effect of mitochondria-targeted proxyl nitroxides

Superoxide (O2-•) has been implicated in the pathogenesis of many human diseases including hypertension. Mitochondria-targeted superoxide scavenger mitoTEMPO reduces blood pressure; however, the structure–functional relationships in antihypertensive effect of mitochondria-targeted nitroxides remain unclear. The nitroxides are known to undergo bioreduction into hydroxylamine derivatives which reacts with O2-• with much lower rate. The nitroxides of pyrrolidine series (proxyls) are much more resistant to bioreduction compared to TEMPOL derivatives suggesting that mitochondria-targeted proxyls can be effective antioxidants with antihypertensive activity. In this work we have designed and studied two new pyrrolidine mitochondria targeted nitroxides: 3-[2-(triphenyphosphonio)acetamido]- and 3-[2-(triphenyphosphonio) acetamidomethyl]-2,2,5,5-tetramethylpyrrolidine-1-oxyl (mCP2) and (mCP1). These new mitochondria targeted nitroxides have 3- to 7-fold lower rate constants of the reaction with O2-• compared with mitoTEMPO; however, the cellular bioreduction of mCP1 and mCP2 was 3- and 2-fold slower. As a consequence incubation with cells afforded much higher intracellular concentration of mCP1 and mCP2 nitroxides compared to mitoTEMPO nitroxide. This has compensated for the difference in the rate of O2-• scavenging and all nitroxides similarly protected mitochondrial respiration in H2O2 treated endothelial cells. Treatment of hypertensive mice with mCP1 and mCP2 (1.4 mg/kg/day) after onset of angiotensin II-induced hypertension significantly reduced blood pressure to 133±5 mmHg and 129±6 mmHg compared to 163±5 mmHg in mice infused with angiotensin II alone. mCP1 and mCP2 reduced vascular O2-• and prevented decrease of endothelial nitric oxide production. These data indicate that resistance to bioreduction play significant role in antioxidant activity of nitroxides. Studies of nitroxide analogs such as mCP1 and mCP2 may help in optimization of chemical structure of mitochondria-targeted nitroxides for improved efficacy and pharmacokinetics of these drugs in treatment of hypertension and many other conditions including atherosclerosis, diabetes and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role

Feasibility of in vivo three-dimensional T-2(*) mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging

Objectives The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T-2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. Materials and methods Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(H-2(3)) methylpyrrolidin-( 3,4-H-2(2))-(1-N-15)-1-oxyl (H-2, N-15-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(H-2(3)) methylpyrrolidin-(3,4-H-2(2))1- oxyl (H-2-DCP), were used in the study. A clonogenic cell survival assay was performed with the H-2-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected H-2, N-15-DCP and H-2-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T-2* mapping. Results H-2-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of H-2, N-15-DCP in tumor tissues was 24.7 +/- 2.9 min (mean +/- standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the H-2, N-15-DCP radical showed a plateau of 10.2 +/- 1.2 min (mean +/- SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T-2* maps with H-2, N-15-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. Conclusion EPR signals of H-2, N-15-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T-2* mapping with CW-EPR-based SPI

4-Dialkylamino-2,5-dihydroimidazol-1-oxyls with Functional Groups at the Position 2 and at the Exocyclic Nitrogen: The pH-Sensitive Spin Labels

Local acidity and electrostatic interactions are associated both with catalytic properties and the adsorption activity of various materials, and with the vital functions of biomolecules. The observation of acid–base equilibria in stable free radicals using EPR spectroscopy represents a convenient method for monitoring pH changes and the investigation of surface electrostatics, the advantages of which are especially evident in opaque and turbid samples and in porous materials such as xerogels. Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. In this work, several new derivatives of 4-(N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyl, with functional groups suitable for specific binding, were synthesized. The dependence of the parameters of their EPR spectra on pH was studied. Several showed a pKa close to 7.4, following the pH changes in a normal physiological range, and some demonstrated a monotonous change of the hyperfine coupling constant by 0.14 mT upon pH variation by four units

Peek Inside the Water Mixtures of Ionic Liquids at Molecular Level: Microscopic Properties Probed by EPR Spectroscopy

Many ionic liquids (ILs) can be mixed with water, forming either true solutions or emulsions. This favors their applications in many respects, but at the same time might strongly alter their physicochemical properties. A number of methods exist for studying the macroscopic properties of such mixtures, whereas understanding their characteristics at micro/nanoscale is rather challenging. In this work we investigate microscopic properties, such as viscosity and local structuring, in binary water mixtures of IL [Bmim]BF4 in liquid and glassy states. For this sake, we use continuous wave and pulse electron paramagnetic resonance (EPR) spectroscopy with dedicated spin probes, located preferably in IL-rich domains or distributed in IL- and water-rich domains. We demonstrate that the glassy-state nanostructuring of IL-rich domains is very similar to that in neat ILs. At the same time, in liquid state the residual water makes local viscosity in IL-rich domains noticeably different compared to neat ILs, even though the overwhelming amount of water is contained in water-rich domains. These results have to be taken into account in various applications of IL-water mixtures, especially in those cases demanding the combinations of optimum micro- and macroscopic characteristics

Effect of Sterical Shielding on the Redox Properties of Imidazoline and Imidazolidine Nitroxides

The oxidant properties of the series of 2,2,5,5-tetraalkyl imidazoline and imidazolidine nitroxides were investigated. An increase in the number of bulky alkyl substituents leads to a decrease in the rate of reduction with ascorbate, which makes the electrochemical reduction potential more negative and shifts the equilibrium in the mixture of nitroxide and reference hydroxylamine (3-carboxy-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-1-¹⁵N) toward the starting compounds. The effect of structural factors on these reactions was analyzed by means of multiple regression using the Fujita steric constant <i>E</i>_s and the inductive Hammett constant σ_I. Satisfactory statistical outputs were obtained in all of the biparameter correlations, denoting that the oxidant properties of the nitroxides are determined by steric and electronic effects of the substituents. The data imply that bulky substituents can stabilize nitroxide and/or destabilize hydroxylamine