3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.     

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.     

Protective properties of neoechinulin A against SIN-1-induced neuronal cell death

Peroxynitrite (ONOO-) is thought to be involved in the neurodegenerative process. To screen for neuroprotective compounds against ONOO- -induced cell death, we developed 96-well based assay procedures for measuring surviving cell numbers under oxidative stress caused by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1), a generator of ONOO-, and sodium N,N-dietyldithiocarbamate trihydrate (DDC), an inhibitor of Cu/Zn superoxide (O2-) dismutase. Using these procedures, we obtained a microbial metabolite that rescued primary neuronal cells from SIN-1-induced damage, but not from DDC-induced damage. By NMR analysis, the compound was identified as neoechinulin A, an antioxidant compound that suppresses lipid oxidation. We found that the compound rescues neuronal cells such as primary neuronal cells and differentiated PC12 cells from damage induced by extracellular ONOO-. However, non-neuronal cells, undifferentiated PC12 cells and cells of the fibroblast cell line 3Y1 were not rescued. Neoechinulin A has scavenging, neurotrophic factor-like and anti-apoptotic activities. This compound specifically scavenges ONOO-, but not O2- or nitric oxide (NO). Similar to known neuroprotective substances such as nerve growth factor and extracts of Gingko biloba leaves, neoechinulin A inhibits the SIN-1-induced activation of caspase-3-like proteases and increases NADH-dehydrogenase activity. These results suggest that neoechinulin A might be useful for protecting against neuronal cell death in neurodegenerative diseases.     

Chemical properties of water-soluble porphyrins. 5. Reactions of some manganese (III) porphyrins with the superoxide and other reducing radicals

Solution properties of three manganese porphyrins, in monomeric form, were investigated. These were the 'picket-fence-like' porphyrin Mn(III)-alpha,alpha,alpha,beta- tetra-ortho(N-methylisonicotinamidophenyl)porphyrin (Mn(III)PFP) and two 'planar unhindered' porphyrins, the Mn(III)TMPyP (tetrakis (4-N-methylpyridyl)porphyrin) and Mn(III)TAP (tetra(4-N,N,N-trimethylanilinium)porphyrin). The porphyrin properties studied were: the absorption spectra in their manganic and manganous forms; acid/base properties of the aquo complexes; the effect of potential axial ligands (up to a concentration of 0.1 mol dm-3) and their one electron reduction potentials. Knowing these properties, the reaction of the Mn(III) porphyrins with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the Mn(III) porphyrins, which governs the catalytic efficiency of the metalloporphyrins upon the disproportionation of the superoxide radical, was 5.1 X 10(7) to 4.0 X 10(5) dm3 mol-1 s-1, depending on the pH and the nature of the metalloporphyrin. These values are at least one order of magnitude lower than found for Fe(III)TMPyP. One electron reduction of the three Mn(III) porphyrins by eaq-, CO2-, CH2OH and (CH3)2COH had similar second-order rate constants (10(9)-10(10) dm3 mol-1 s-1). That for (CH3)2(CH2)COH was about 10(5) dm3 mol-1 s-1. Reduction in all cases produced the corresponding Mn(II) porphyrin and no intermediate was found. The oxidation reaction of the Mn(II) porphyrins by O2- was approximately two orders of magnitude faster when compared to the reduction of Mn(III) porphyrins with the same radical. Since the reactivities of O2- towards the three manganese (III) compounds follow their reduction potentials, it is suggested that these reactions are governed by an outer-sphere mechanism. This suggestion is corroborated by the finding that water molecules acting as axial ligands, in these aqueous solution systems, are not replaced by another potential ligand when the latter is in the concentration range of 100 mM or less.     

Inhibition of superoxide generation from fMLP-stimulated leukocytes by high concentrations of nitric oxide or peroxynitrite: characterization by electron spin resonance spectroscopy

This present study examined the effects of high concentrations of nitric oxide (NO*) and peroxynitrite (ONOO-) on superoxide (O2*-) production from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leukocytes (PMNs) by using electron spin resonance (ESR) and spin trapping with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). We demonstrated that ONOO- (100 microM) decreased the ESR signal of DEPMPO-OOH from fMLP-activated PMNs, indicating the inhibition of O2*- generation, while it enhanced the signal of DEPMPO-OH. Inhibition of the respiratory burst was also observed when PMNs were pre-exposed to high concentrations of NO* (100 microM), generated by the NO* donor NOR-1, 30 min prior to stimulation with fMLP. NOR-1 inhibited O2*- generation more effectively under conditions in which ONOO-was formed concurrently. The ability of high concentrations of either ONOO- or NO* to inhibit O2*-generation from fMLP-stimulated PMNs is relevant to pathophysiological conditions, such as severe inflammation, in which NO* or ONOO- production can be significantly elevated.     

Reactions of manganese porphyrins with peroxynitrite and carbonate radical anion

We have studied the reaction kinetics of ten manganese porphyrins, differing in their meso substituents, with peroxynitrite (ONOO-) and carbonate radical anion (CO3.) using stopped-flow and pulse radiolysis, respectively. Rate constants for the reactions of Mn(III) porphyrins with ONOO- ranged from 1 x 10(5) to 3.4 x 10(7) m(-1) s(-1) and correlated well with previously reported kinetic and thermodynamic data that reflect the resonance and inductive effects of the substituents on the porphyrin ring. Rate constants for the reactions of Mn(III) porphyrins with CO3. ranged from 2 x 10(8) to 1.2 x 10(9) m(-1)s(-1) at pH 相似文献   

Opposite Effects of Mn(III) and Fe(III) Forms of meso-Tetrakis(4-N-methyl pyridiniumyl) Porphyrins on Isolated Rat Liver Mitochondria

The relevance of porphyrins as therapeutic drugs targeted to mitochondria has been widely recognized. In this work, we studied the action of meso-tetrakis porphyrins (TMPyP) on respiring rat liver mitochondria. Mn(III)TMPyP exerted a protective effect against lipid peroxidation induced by Fe(II) or the azo initiator 4,4-azobis(4-cyanopentanoic acid) (ABCPA), which partition in the hydrophobic phospholipid moiety, and 2,2-azobis(2-amidinepropane)dihydrochloride (ABAP), which partitions in the aqueous phase. In contrast, Fe(III)TMPyP itself induced an intense lipid peroxidation, accompanied by mitochondrial permeability transition. Both mesoporphyrins studied promoted a release of mitochondrial state-4 respiration, in the concentration range of 1.0–20 M. Based on the relative effects of Mn(III)TMPyP against ABAP and ABCPA-induced lipid peroxidation, we believe that meso-tetrakis porphyrins must concentrate preferably at membrane–water interfaces.     

Peroxynitrite enhances astrocytic volume-sensitive excitatory amino acid release via a src tyrosine kinase-dependent mechanism

Volume-regulated anion channels (VRACs) are critically important for cell volume homeostasis, and under pathological conditions contribute to neuronal damage via excitatory amino (EAA) release. The precise mechanisms by which brain VRACs are activated and/or modulated remain elusive. In the present work we explored the possible involvement of nitric oxide (NO) and NO-related reactive species in the regulation of VRAC activity and EAA release, using primary astrocyte cultures. The NO donors sodium nitroprusside and spermine NONOate did not affect volume-activated d-[3H]aspartate release. In contrast, the peroxynitrite (ONOO-) donor 3-morpholinosydnomine hydrochloride (SIN-1) increased volume-dependent EAA release by approx. 80-110% under identical conditions. Inhibition of ONOO- formation with superoxide dismutase completely abolished the effects of SIN-1. Both the volume- and SIN-1-induced EAA release were sensitive to the VRAC blockers NPPB and ATP. Further pharmacological analysis ruled out the involvement of cGMP-dependent reactions and modification of sulfhydryl groups in the SIN-1-inducedmodulation of EAA release. The src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2), but not its inactive analog PP3, abolished the effects of SIN-1. A broader spectrum tyrosine kinase inhibitor tyrphostin A51, also completely eliminated the SIN-1-induced EAA release. Our data suggest that ONOO- up-regulates VRAC activity via a src tyrosine kinase-dependent mechanism. This modulation may contribute to EAA-mediated neuronal damage in ischemia and other pathological conditions favoring cell swelling and ONOO- production.     

Reductive nitrosylation of water-soluble iron porphyrins by S-nitroso-N-acetylpenicillamine: rate constants and EPR characterization

Reductive nitrosylation of the water-soluble iron derivatives of the cationic Fe(III)(TMPyP) and anionic Fe(III)(TPPS) porphyrins [where TMPyP=tetra-meso-(4-N-methylpyridiniumyl)porphinate and TPPS=tetra-meso-(4-sulfonatophenyl)porphinate] by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was studied using optical absorption spectroscopy and electron paramagnetic resonance. Nitrosylation rates were obtained, the reaction was found to be first order in the SNAP concentration and the stoichiometry of the reaction was one to one. The similarity between the obtained second-order rate constants for both porphyrins, k(TMPyP)=0.84 x 10(3)M(-1)s(-1) and k(TPPS)=0.97 x 10(3)M(-1)s(-1), suggested that the reaction mechanism is approximately independent of the nature of the porphyrin meso-substituents. A mechanism was proposed involving the hydrolysis of SNAP by an out of plane liganded H(2)O yielding the sulfenic acid of N-acetylpenicillamine and the transfer of NO(-) to Fe(III). The EPR (electron paramagnetic resonance) spectra of the SNAP- and gaseous NO-treated porphyrins were obtained and compared. The difference between the spectra of the cationic and anionic porphyrins indicates different local symmetry and Fe-N-O bond angle. SNAP-treatment produced much more resolved hyperfine structures than gaseous NO-treatment.     

Mechanisms of peroxynitrite decomposition catalyzed by FeTMPS, a bioactive sulfonated iron porphyrin

Peroxynitrite is a known cytotoxic agent that plays a role in many pathological conditions. Various peroxynitrite decomposition catalysts and pathways are being explored to develop efficient therapeutic agents that can safely remove peroxynitrite from cells and tissues. Water-soluble porphyrins, such as iron(III) meso-tetra(2,4,6-trimethyl-3,5-disulfonato)porphine chloride (FeTMPS) and iron(III) meso-tetra(N-methyl4-pyridyl)porphine chloride (FeTMPyP), have been shown to react catalytically with peroxynitrite (ONOO-). However, their mechanisms are yet to be fully understood. In this study, we have explored the reactivity of FeTMPS in the catalytic decomposition of peroxynitrite. The mechanism of this complex process has been determined. According to this mechanism, Fe(III)TMPS is oxidized by peroxynitrite to produce oxoFe(lV)TMPS and NO2 (k1 = 1.3 x 10(5) M(-1)(s(-1). The porphyrin is then reduced back to Fe(III)TMPS by nitrite, but this rate (k2 = 1.4 x 10(4) M(-1)s(-1)) is not sufficient to maintain the catalytic process at the observed rate. The overall rate of peroxynitrite decomposition catalysis, kcat, was determined to be 6 x 10(4) M(-1)s(-1), under typical conditions. We have postulated that an additional reduction pathway must exist. Kinetic simulations showed that a reaction of oxoFe(IV)TMPS with NO2 (k3 = 1.7 x 10(7) M((-1)s(-1)) could explain the behavior of this system and account for the fast reduction of oxoFe(IV)TMPS to Fe(III). Using the kinetic simulation analysis, we have also shown that two other rearrangement reactions, involving FeTMPS and peroxynitrite, are plausible pathways for peroxynitrite decay. A "cage-return" reaction between the generated oxoFe(IV)TMPS and NO2 (k8 = 5.4 x 10(4) M(-1)s(-1)), affording Fe(III)TMPS and nitrate, and a reaction between oxoFe(IV)TMPS and peroxynitrite (k7 = 2.4 x 10(4) M(-1)s(-1)) that affords oxoFe(IV)TMPS and nitrate are presented. The mechanism of FeTMPS-catalyzed peroxynitrite decay differs markedly from that of FeTMPyP, providing some insight into the reactivity of metal centers with peroxynitrite and biologically important radicals such as NO2.     

Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

SOD-like activity of Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself

Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto beta-pyrrolic positions dramatically improves thermodynamic facilitation for the O2*- dismutation. We have previously characterized the para isomer, Mn(II)Br(8)TM-4-PyP(4+) [Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn(II)Br(8)TM-3-PyP(4+) with respect to UV/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O2*- dismutation, metal-ligand stability, and the ability to protect SOD-deficient Escherichia coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn(III)/Mn(II) reduction potential, E((1/2))=+468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn(III)TM-3-PyP(5+) and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, an increase in E((1/2)) results in a higher catalytic rate constant for the O2*- dismutation, log k(cat)> or =8.85; 1.5-fold increase with respect to the para isomer. The IC(50) was calculated to be < or =3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k(cat) ever reported for a metalloporphyrin, being essentially identical to the k(cat) of superoxide dismutases (log k(cat)=8.84-9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn beta-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5-fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn(II)Br(8)TM-3-PyP(4+) was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow >60% of the growth of wild type; at concentrations > or =5 microM it exhibits toxicity. Our work shows that exceptionally high k(cat) for the O2*- disproportionation can be achieved not only with an N(5)-type coordination motif, as rationalized previously for aza crown ether (cyclic polyamines) complexes, but also with a N(4)-type motif as in the Mn porphyrin case; both motifs sharing "up-down-up-down" steric arrangement.     

Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.     

Irradiated cationic mesoporphyrin induces larger damage to isolated rat liver mitochondria than the anionic form

The action of irradiated cationic Fe(III)TMPyP and anionic Fe(III)TPPS4 forms of mesoporphyrins on mitochondrial functions was investigated using experimental conditions that caused minimal effects on mitochondria in the dark. Treatment of mitochondria with 1 microM Fe(III)TMPyP for 2 min decreased the respiratory control by 3% in the dark and 28% after irradiation. Fe(III)TPPS4 (1 microM) had no significant effect on respiratory control under any of the above conditions. Both porphyrins increased the mitochondrial production of reactive oxygen species in the presence of Ca2+; however, the effect of Fe(III)TMPyP was significantly stronger. In both cases, this overproduction was associated with membrane lipid peroxidation. It was also observed that the association constant of Fe(III)TMPyP with mitochondria was 11 times higher than that of Fe(III)TPPS4. In conclusion, the damage to isolated mitochondria induced by Fe(III)TMPyP under illumination was larger than by Fe(III)TPPS4, probably because its cationic charge favors association with the mitochondrial membrane. This is supported by the decrease in the association constant of Fe(III)TMPyP with mitochondria in higher salt medium.     

Metalloporphyrin probes for antimalarial drug action

Metal-substituted protoporphyrin IXs (Co(III)PPIX (1), Cr(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6) and Sn(IV)PPIX (7)), phthalocyanine tetrasulfonates (PcS (8) and Ni(II)PcS (9)), and anionic and cationic porphyrins (meso-tetra(4-sulfonatophenyl)porphine (TPPS4, 10), meso-tetra(4-carboxyphenyl)porphine (TPPC4, 11), tetrakis(4-N-trimethylaminophenyl)porphine (TMAP, 12) and meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4, 13)) have been used as probes to compare two different assays for the inhibition of beta-hematin formation. The results demonstrate that the efficacy of these probes in either the beta-hematin inhibition assay (9, 7, 6, 5>4>11, 3>10, 8>2, 1; 12 and 13 did not inhibit.) or the bionucleating template assay (8>1>11>9, 2>4>3>7>10>5>6; 12 and 13 did not inhibit.) differ significantly. These differences are examined in light of possible interactions between the inhibitor probes, heme, beta-hematin and the bionucleating template. This detailed analysis highlights the fact that while dominant modes of interactions may be occasionally identified, the precise mechanism of inhibition undoubtedly consists of the interplay between multiple interactions.     

The survival of skeletal muscle myoblasts in vitro is sensitive to a donor of nitric oxide and superoxide, SIN-1, but not to nitric oxide or peroxynitrite alone.

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2'-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O(-)(2), 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO(-)) or to DETA NONOate in the presence of an O(-)(2)-generating system did not reduce their survival. The results show that NO or ONOO(-) alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H(2)O(2) in this myoblast culture system.     

SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death

3-Morpholinosyndnomine (SIN-1) has been reported to be a peroxynitrite (OONO(-)) donor because it produces both nitric oxide (NO) and superoxide (O(2)(-).) upon decomposition in aqueous solution. However, SIN-1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN-1 caused a concentration-dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3-nitrotyrosine into the culture medium. The increase in 3-nitrotyrosine immunoreactivity, a footprint of OONO(-) production, was specific for SIN-1 as exposure to neurotoxic concentrations of an NO donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN-1-induced injury and 3-nitrotyrosine staining were prevented by the addition of either 5,10,15,20-Tetrakis (4-sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO(-) decomposition catalyst, or uric acid, an OONO(-) scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO(-) from SIN-1 as well as its cytotoxicity. Removal of O(2)(-). and the subsequently formed H(2)O(2) by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein-bound tyrosine but actually enhanced the cytotoxicity of SIN-1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN-1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non-NMDA receptors contributed to the SIN-1-mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN-1 initiates neural cell death in an OONO(-)-dependent or -independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO-related species generated.     

Reactions of *NO2 with chromium(III) complexes with histamine and pyridoxamine ligands studied by the stopped-flow technique

This study demonstrated the direct formation of the nitrogen dioxide (*NO2) radical during the decomposition of 3-morpholinosydnonimine (SIN-1) in biological buffer 4-morpholinoethanosulfone acid solution. Consequently, at approximately pH 4, SIN-1 can be used successfully as a source of *NO2. This conclusion is drawn from a comparison of the reactions of cis-[Cr(C2O4)(L- L)(OH2)2]+, where L-L denotes pyridoxamine (Hpm) or histamine (hm), with the gaseous *NO2 radical obtained by two methods: from SIN-1 and from a simple redox reaction. These reactions were investigated using the stopped-flow technique. The measurements were carried out at temperatures ranging from 5 to 25 degrees C over a pH range from 6.52 to 9.11 for cis-[Cr(C2O4)(Hpm) (OH2)2]+ and from 6.03 to 8.15 for cis-[Cr(C2O4)(hm)(OH2)2] +. We also determined the thermodynamic activation parameter (E(a)) and the uptake mechanism for each of the coordination compounds studied.     

Pindolol is a potent scavenger of reactive nitrogen species

Pindolol is an indolic drug that has been shown to enhance and/or accelerate selective serotonin specific reuptake inhibitors (SSRI)-induced antidepressant (AD) effect, even though the respective mechanism is still unclear. It has been demonstrated that inhibition of nitric oxide (*NO) synthesis in CNS produces anxiolytic and AD-like behavioural effects in a variety of animal paradigms. On the other hand, sustained high levels of *NO may be deleterious to CNS, predominantly due to the formation of peroxynitrite anion (ONOO-), which is generated via reaction of *NO with superoxide radical (O2*-). Therefore, the purpose of the present study was to characterize the putative pindolol scavenging effect on *NO, ONOO-, and O2*-, using in vitro non-cellular systems. The obtained results clearly show that pindolol is a potent scavenger of *NO (IC50 of 449+/-33 microM) and ONOO- (IC50 of 131+/-24 microM). Additionally, the scavenging effect of pindolol increased almost 8 times in the presence of 25 mM NaHCO3 (IC50 of 17+/-3 microM), which indicates that pindolol efficiently scavenges reactive species that are produced from the ONOO-/CO2 reaction such as the nitrogen dioxide radical (*NO2) and the carbonate radical anion (CO3*-). These effects may contribute for the reduction of SSRI antidepressant latency that has been attributed to pindolol and may also constitute an additional value for this drug when depression is associated with pro-oxidant neurodegenerative diseases.