An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Nitroxyl anion regulation of the NMDA receptor

Nitric oxide (NO) is an important regulator of NMDA channel function in the CNS. Recent findings suggest that nitroxyl anion (NO(-)) may also be generated by nitric oxide synthase, which catalyzes production of NO. Using recombinant NMDA receptors (NMDA-r) transfected into human embryonic kidney cells, our data demonstrate that the nitroxyl anion donor, Angeli's salt (AS; Na(2)N(2)O(3)) dramatically blocked glycine-independent desensitization in NMDA-r containing NR1-NR2A subunits. AS did not affect glycine-dependent desensitization, calcium dependent inactivation or glutamate affinity for the NMDA-r. This effect could be mimicked by treatment with DPTA, a metal chelator and was not evident under hypoxic conditions. In contrast, receptors containing the NR1-NR2B subunits demonstrated an approximate 25% reduction in whole cell currents in the presence of AS with no apparent change in desensitization. Our data suggest that the regulation of NMDA-r function by nitroxyl anion is distinctly different from NO and may result in different cellular outcomes compared with NO.     

Effect of Angeli's salt on the glutamate/glutamine cycle activity and on glutamate excitotoxicity in the hamster retina

Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when present in excessive amounts. It is well known that NO is involved in glutamate excitotoxicity, but information regarding the possibility that NO-related species could reciprocally affect glutamate synaptic levels was not previously provided. The dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. The aim of the present work was to comparatively analyze the effect of nitroxyl and NO on the retinal glutamate/glutamine cycle in vitro activity. For this purpose, Angeli's salt (AS) and diethylamine NONOate (DEA/NO) were used as nitroxyl and NO donor, respectively. AS and DEA/NO significantly decreased retinal l-glutamate uptake and glutamine synthetase activity, but only AS decreased l-glutamine influx. Dithiothreitol prevented all the effects of AS and DEA/NO. The intravitreal injection of DEA/NO (but not AS) or a supraphysiological concentration of glutamate induced retinal histological alterations. Although AS could increase glutamate synaptic concentration in vitro, the histological alterations induced by glutamate were abrogated by AS. These results suggest that nitroxyl could regulate the hamster retinal glutamatergic pathway by acting through differential mechanisms at pre- and postsynaptic level.     

Discriminative EPR detection of NO and HNO by encapsulated nitronyl nitroxides

Abstract

Nitric oxide, ^?NO, is one of the most important molecules in the biochemistry of living organisms. By contrast, nitroxyl, NO^?, one-electron reduced analog of ^?NO which exists at physiological conditions in its protonated form, HNO, has been relatively overlooked. Recent data show that HNO might be produced endogenously and display unique biological effects. However, there is a lack of specific and quantitative methods of detection of endogenous HNO production. Here we present a new method for discriminative ^?NO and HNO detection by nitronyl nitroxides (NNs) using electron paramagnetic resonance (EPR). It was found that NNs react with ^?NO and HNO with similar rate constants of about 10⁴ M^{? 1}s^{? 1} but yield different products: imino nitroxides and the hydroxylamine of imino nitroxides, correspondingly. An EPR approach for discriminative ^?NO and HNO detection using liposome-encapsulated NNs was developed. The membrane barrier of liposomes protects NNs against reduction in biological systems while is permeable to both analytes, ^?NO and HNO. The sensitivity of this approach for the detection of the rates of ^?NO/HNO generation is about 1 nM/s. The application of encapsulated NNs for real-time discriminative ^?NO/HNO detection might become a valuable tool in nitric oxide-related studies.     

Consequences of MnSOD interactions with nitric oxide: nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^- ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^-  species initiate the formation of peroxynitrite (ONOO^- ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^-  decomposition and ONOO^- -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^-  is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

In vivo EPR measurement of glutathione in tumor-bearing mice using improved disulfide biradical probe

Disulfide nitroxide biradicals, DNB, have been used for glutathione, GSH, measurements by X-band electron paramagnetic resonance, EPR, in various cells and tissues. In the present paper, the postulated potential use of DNB for EPR detection of GSH in vivo was explored. Isotopic substitution in the structure of the DNB was performed for the enhancement of its EPR spectral properties. (15)N substitution in the NO fragment of the DNB decreased the number of EPR spectral lines and resulted in an approximately two-fold increase in the signal-to-noise ratio, SNR. An additional two-fold increase in the SNR was achieved by substitution of the hydrogen atoms with deuterium resulting in narrowing the EPR lines from 1.35 G to 0.95 G. The spectral changes of DNB upon reaction with GSH and cysteine were studied in vitro in a wide range of pHs at room temperature and "body" temperature, 37 degrees C, and the corresponding bimolecular rate constants were calculated. In in vivo experiments the kinetics of the L-band EPR spectral changes after injection of DNB into ovarian xenograft tumors grown in nude mice were measured by L-band EPR spectroscopy, and analyzed in terms of the two main contributing reactions, splitting of the disulfide bond and reduction of the NO fragment. The initial exponential increase of the "monoradical" peak intensity has been used for the calculation of the GSH concentration using the value of the observed rate constant for the reaction of DNB with GSH, k(obs) (pH 7.1, 37 degrees C)=2.6 M(-1)s(-1). The concentrations of GSH in cisplatin-resistant and cisplatin-sensitive tumors were found to be 3.3 mM and 1.8 mM, respectively, in quantitative agreement with the in vitro data.     

Generation of Nitric Oxide and Possibly Nitroxyl by Nitrosation of Sulfohydroxamic Acids and Hydroxamic Acids

Diazeniumdiolates (NONOates) and sulfohydroxamic acids are chemical entities that spontaneously generate nitric oxide (NO) and nitroxyl (HNO), respectively, at physiological pH and temperature. By combining the functional aspects of the NONOates with the hydroxamic acids and sulfohydroxamic acids, hybrid NONOate-type compounds that could theoretically generate nitroxyl or nitric oxide can be rationalized. Although the instability of these compounds, viz., the N-nitrosohydroxamic acids and the N-nitrososulfohydroxamic acids, precluded their chemical characterization by actual isolation, their transient existence was deduced by identification of the products of their decomposition. Thus, treatment of benzohydroxamic acid (BHA) with limiting or excess nitrous acid (from NaNO(2) and H(3)PO(4)) gave rise to quantitative generation of N(2)O, possibly via HNO, based on the limiting reactant. Nitrosation of N-t-butyloxycarbonyl hydroxylamine gave similar results. The organic acid produced from BHA was identified as benzoic acid. No nitric oxide was detected from these reactions. In contrast, treatment of Piloty's acid (benzenesulfohydroxamic acid) and methanesulfohydroxamic acid (MSHA) with nitrous acid under the same conditions as above gave 36% of the theoretical quantity of NO from Piloty's acid and 47% of NO from MSHA, although finite quantities of HNO (measured as N(2)O) were also formed. The organic acid produced from Piloty's acid was identified by reverse-phase HPLC as the redox product, benzenesulfinic acid.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.