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The nitric oxide-specific fluorescent probe Fluorescent Nitric Oxide Cheletropic Trap (FNOCT) 8a was applied to intact tobacco (Nicotiana tabacum cv. Samsun) roots to detect sites of nitric oxide formation and NO distribution. Three week old tobacco seedlings were gently removed from the sand culture pots with intact roots and transferred to small Petri dishes, whose base was replaced by a thin coverslip. Intact roots were subjected to FNOCT 8a to localize NO-dependent fluorescence in these roots; controls with an exogenous NO donor confirmed the presence and distribution of the probe in the roots. To confirm the NO-dependent fluorescence, roots were incubated with the three different NO scavengers cPTIO {2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-L-oxyl-3-oxide}, methylene blue and sodium diethyl dithiocarbamate (DCC) followed by incubation with FNOCT 8a. Methylene blue and DCC were able to completely quench NO-dependent fluorescence, cPTIO quenched partially. The roots were incubated in the presence of NaNO₂ and NaNO₃, which are substrates for nitrite:nitric oxide reductase (NI-NOR) and plasma membrane-bound nitrate reductase (PM-NR), respectively. The NO-dependent fluorescence was more or less same at the root tips upon treatment with NaNO₂, while the overall fluorescence was reduced in the presence of NaNO. Fluorescence from the living roots was visualized by inverted confocal laser scanning microscope (CLSM) using UV laser (excitation 360 nm and emission 408 nm). A specialized apparatus has been devised by the authors for analysis of intact roots as described in the methods section of this paper. Intact roots were chosen for microscopic observation rather than incised roots to avoid production of NO due to stress or physical injury., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Mammalian (Clade 3) catalases utilize NADPH as a protective cofactor to prevent one-electron reduction of the central reactive intermediate Compound I (Cpd I) to the catalytically inactive Compound II (Cpd II) species by re-reduction of Cpd I to the enzyme's resting state (ferricatalase). It has long been known that ascorbate/ascorbic acid is capable of reducing Cpd I of NADPH-binding catalases to Cpd II, but the mode of this one-electron reduction had hitherto not been explored. We here demonstrate that ascorbate-mediated reduction of Cpd I, generated by addition of peroxoacetic acid to NADPH-free bovine liver catalase (BLC), requires specific binding of the ascorbate anion to the NADPH binding pocket. Ascorbate-mediated Cpd II formation was found to be suppressed by added NADPH in a concentration-dependent manner, for the achievement of complete suppression at a stoichiometric 1:1 NADPH:heme concentration ratio. Cpd I → Cpd II reduction by ascorbate was similarly inhibited by addition of NADH, NADP(+), thio-NADP(+), or NAD(+), though with 0.5-, 0.1-, 0.1-, and 0.01-fold reduced efficiencies, respectively, in agreement with the relative binding affinities of these dinucleotides. Unexpected was the observation that although Cpd II formation is not observed in the presence of NADP(+), the decay of Cpd I is slightly accelerated by ascorbate rather than retarded, leading to direct regeneration of ferricatalase. The experimental findings are supported by molecular mechanics docking computations, which show a similar binding of NADPH, NADP(+), and NADH, but not NAD(+), as found in the X-ray structure of NADPH-loaded human erythrocyte catalase. The computations suggest that two ascorbate molecules may occupy the empty NADPH pocket, preferably binding to the adenine binding site. The biological relevance of these findings is discussed.

The synthesis and the structural and spectroscopic characterization of nonfluorescent, pyrene-based cyclic o-quinodimethanes are reported. These compounds react efficiently with nitric oxide (NO) in a formal cheletropic manner, by which the fluorescent aromatic pyrene system is regenerated. The NO trapping capabilities and kinetics of the fluorescent nitric oxide cheletropic traps (FNOCTs) are assessed in THF and buffered aqueous solution by ESR, UV/Vis, and fluorescence spectroscopy, by employing NO solutions and NO released from N-diazeniumdiolates (NONOates). Prototypal biological applications include the quantitation of NO production from cultured rat alveolar macrophages and the endothelium of porcine aorta, which demonstrate a sensitivity for NO detection in the nanomolar range.

In previous studies we reported on the catalase-like activity and antioxidative properties of a non-heme Fe(III)-tetraaza[14]annulene complex, 5,4-didehydro-5,9,14,18-tetraaza-di(2,2-dimethyl-[5,6]benzo[1,3]dioxolo)[a,h]cyclotetradecene--Fe(III) chloride (TAA-1/Fe). We proposed that intracellular application of the parent, iron-free tetraaza[14]annulene ligand, TAA-1, as precursor would allow antioxidative defense along two lines, i.e. by chelation of potentially toxic cellular iron ions and, subsequently, by catalase-mimic activity. We here set out to establish whether the active catalase mimic is indeed formed intracellularly when cells are loaded with the ligand. When isolated rat hepatocytes were preloaded with TAA-1, they were protected against iron-induced cell injury and oxidative stress elicited by exposure to the membrane-permeable iron complex Fe(III)/8-hydroxyquinoline. After lysis of the cells, followed by ultrafiltration to remove endogenous catalase, the lysate exhibited catalase-like activity, while lysates of control cells not treated with TAA-1 showed no catalase-like activity. By comparison with authentic TAA-1/Fe, an intracellular formation of 2.0 +/- 0.3 microm of the active catalase mimic in native hepatocytes exposed to TAA-1 and of 6.5 +/- 1.0 microm in hepatocytes exposed to both TAA-1 and iron ions was estimated. The intracellular formation of the active catalase mimic thus renders TAA-1 an attractive compound for protection against iron- and/or hydrogen peroxide-dependent cell injuries.

Zwitterionic diazeniumdiolates of the form RN[N(O)NO(-)](CH(2))(2)NH(2) (+)R, where R=CH(3) (1), (CH(2))(3)CH(3) (2), (CH(2))(5)CH(3) (3), and (CH(2))(7)CH(3) (4) were synthesized by reaction of the corresponding diamines with nitric oxide. Spectrophotometrically determined pK(a)(O) values, attributed to protonation at the terminal oxygen of the diazeniumdiolate group, show shifts to higher values in dependence of the chain lengths of R. The pH dependence of the decomposition of NO donors 1-3 was studied in buffered solution between pH 5 and 8 at 22 degrees C, from which pK(a)(N) values for protonation at the amino nitrogen, leading to release of NO, were estimated. It is shown that the decomposition of these diazeniumdiolates is markedly catalyzed by anionic SDS micelles. First-order rate constants for the decay of 1-4 were determined in phosphate buffer pH 7.4 at 22 degrees C as a function of SDS concentration. Micellar binding constants, K(SM), for the association of diazeniumdiolates 1-3 with the SDS micelles were also determined, again showing a significant increase with increasing length of the alkyl side chains. The decomposition of 1-3 in micellar solution is quantitatively described by using the pseudo-phase ion-exchange (PIE) model, in which the degree of micellar catalysis is taken into account through the ratio of the second-order rate constants (k(2m)/k(2w)) for decay in the micelles and in the bulk aqueous phase. The decay kinetics of 1-3 were further studied in the presence of cosolvents and nonionic surfactants, but no effect on the rate of NO release was observed. The kinetic data are discussed in terms of association to the micelle-aqueous phase interface of the negatively charged micelles. The apparent interfacial pH value of SDS micelles was evaluated from comparison of the pH dependence of the first-order decay rate constants of 2 and 3 in neat buffer and the rate data obtained for the surfactant-mediated decay. For a bulk phase of pH 7.4, an interfacial pH of 5.7-5.8 was determined, consistent with the distribution of H(+) in the vicinity of the negatively charged micelles. The data demonstrate the utility of 2 and 3 as probes for the determination of the apparent pH value in the Stern region of anionic micelles.

The enzymatic system mainly responsible for the reduction of labile iron ions in mammalian cells is still unknown. Using isolated organelles of the rat liver, i.e. mitochondria, microsomes, nuclei and the cytosol, we here demonstrate that Fe(III), added as Fe(III)-ATP complex, is predominantly reduced by an NADH-dependent enzyme system associated with mitochondria (65% of the overall enzymatic Fe(III) reduction capacity within liver cells). Microsomes showed a significantly smaller Fe(III) reduction capacity, whereas the cytosol and nuclei hardly reduced Fe(III). Studying the mitochondrial iron reduction, this NADH-dependent process was not mediated by superoxide, ascorbic acid, or NADH itself, excluding low-molecular-weight reductants. No evidence was found for the involvement of complex I and III of the respiratory chain. Submitochondrial preparations revealed the highest specific activity reducing Fe(III) in the outer membrane fraction. In conclusion, an NADH-dependent mitochondrial enzyme system, most likely the NADH-cytochrome c reductase system, located at the outer membrane, should decisively contribute to the enzymatic reduction of labile iron within liver cells, especially under pathological conditions.

X-ray structures of the 13 different monofunctional heme catalases published to date were scrutinized in order to gain insight in the mechanism by which NADPH in Clade 3 catalases may protect the reactive ferryloxo intermediate Compound I (Cpd I; por (*+)Fe (IV)O) against deactivation to the catalytically inactive intermediate Compound II (Cpd II; porFe (IV)O). Striking similarities in the molecular network of the protein subunits encompassing the heme center and the surface-bound NADPH were found for all of the Clade 3 catalases. Unique features in this region are the presence of a water molecule (W1) adjacent to the 4-vinyl group of heme and a serine residue or a second water molecule hydrogen-bonded to both W1 and the carbonyl group of a threonine-proline linkage, with the proline in van der Waals contact with the dihydronicotinamide group of NADPH. A mechanism is proposed in which a hydroxyl anion released from W1 undergoes reversible nucleophilic addition to the terminal carbon of the 4-vinyl group of Cpd I, thereby producing a neutral porphyrin pi-radical ferryloxo (HO-por (*)Fe (IV)O) species of reduced reactivity. This structure is suggested to be the elusive Cpd II' intermediate proposed in previous studies. An accompanying proton-shifting process along the hydrogen-bonded network is believed to facilitate the NADPH-mediated reduction of Cpd I to ferricatalase and to serve as a funnel for electron transfer from NADPH to the heme center to restore the catalase Fe (III) resting state. The proposed reaction paths were fully supported as chemically reasonable and energetically feasible by means of density functional theory calculations at the (U)B3LYP/6-31G* level. A particularly attractive feature of the present mechanism is that the previously discussed formation of protein-derived radicals is avoided.

The development of enzyme mimics of catalase which decompose hydrogen peroxide to water and molecular oxygen according to the 2:1 stoichiometry of native catalase and in aqueous solution at pH 7 and at micromolar concentrations of the enzyme model and hydrogen peroxide is reported. For this purpose, iron(III) complexes of 1,4,8,11-tetraaza[14]annulenes are prepared by various procedures. Efficacious preparations utilize reaction of the [N4] macrocyles with FeII salts in the presence of triphenylamine, followed by gentle oxidation of the FeII complexes by molecular oxygen or by tris(4-bromophenyl)aminium hexachloroantimonate. The complexes are characterized by SQUID magnetometry and by Mössbauer, EPR, and UV/vis spectrometry. In the solid state, the iron(III) center of the catalytically active complexes exists in the intermediate (quartet, S = 3/2) spin state. Several of these complexes decompose hydrogen peroxide in aqueous buffer solution at pH 7.2 at room temperature with turnover numbers between 40 and 80. The apparent second-order rate constant for hydrogen peroxide decomposition is in the range of 1400-2400 M(-1) s(-1), about 3 orders of magnitude lower than the value for native catalase. Besides oxygen production, a non-oxygen releasing pathway of hydrogen peroxide decomposition is unveiled.

A new pyrene-based fluorescent probe for the determination of critical micelle concentrations (CMC) is described. The title compound 1 is obtained in five steps, starting from pyrene. Fluorescence spectroscopic properties of 1 are studied in homogeneous organic solvents and aqueous micellar solutions. In a wide range of organic solvents, probe 1 exhibits a characteristic monomer emission of the pyrene fluorophore, with three distinct peak maxima at 382, 404, and 425 nm. The spectra change dramatically in aqueous solution, where no monomer emission of the pyrene fluorophore is detected. Instead, only strong excimer fluorescence with a broad, red-shifted emission band at lambda(max) = 465 nm is observed. In micellar aqueous solution, a superposition of the monomer and excimer emission is found. The appearance of the monomer emission in micellar solution can be explained on the basis of solubilization of 1 by the surfactant micelles. The ratio of the monomer to excimer fluorescence intensities of 1 is highly sensitive to changes in surfactant concentration. This renders 1 a versatile and sensitive probe molecule for studying the micellization of ionic and nonionic surfactants. For a representative selection of common surfactants, the critical micelle concentrations in aqueous solution are determined, showing excellent agreement with established literature data.

Chelatable cellular iron, and chelatable mitochondrial iron in particular, has yet to be well characterized, so the overall strength with which these "loosely bound" iron ions (presumably mainly Fe(II)) are intracellularly/intramitochondrially bound is unclear. We have previously reported the first selective mitochondrial iron indicator: rhodamine B 4-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). With this compound as a model, we have now developed two additional mitochondrial iron indicators with very different iron-binding affinities and have applied these to the study of the chelatable iron pool in the mitochondria of isolated rat liver cells. With the new indicator rhodamine B 4-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester (RDA), with 2,2'-bipyridine as chelating unit (log beta(3)=17.5), essentially the same iron concentration (16.0+/-1.9 microM) was determined as with RPA (log beta(3)=21.1), despite the four orders of magnitude difference in Fe(II)-binding affinity. This not only demonstrates the reliability of the procedure, but also confirms that iron complexation by these indicators does not induce any significant release of iron from the iron-storage proteins on the timescale of the experiment. In contrast, the indicator rhodamine B 4-[bis(pyridin-2-ylmethyl)aminomethyl]benzyl ester (PIRO), with an N,N-bis(pyridin-2-ylmethyl)amine group as chelating component (log beta(2)=12.2), could not compete against the array of endogenous ligands. The intramitochondrial concentrations of the three indicators were determined to be in the range of 100 microM: that is, about three orders of magnitude lower than the total concentration of endogenous compounds that might chelate iron ions. It is therefore estimated that chelatable mitochondrial iron ions are bound by endogenous ligands with apparent stability constants (log K(app)) of between 9 and 14.

Non-heme iron(III) complexes of 14-membered tetraaza macrocycles have previously been found to catalytically decompose hydrogen peroxide to water and molecular oxygen, like the native enzyme catalase. Here the mechanism of this reaction is theoretically investigated by DFT calculations at the (U)B3LYP/6-31G* level, with focus on the reactivity of the possible spin states of the FeIII complexes. The computations suggest that H2O2 decomposition follows a homolytic route with intermediate formation of an iron(IV) oxo radical cation species (L.+FeIV==O) that resembles Compound I of natural iron porphyrin systems. Along the whole catalytic cycle, no significant energetic differences were found for the reaction proceeding on the doublet (S=1/2) or on the quartet (S=3/2) hypersurface, with the single exception of the rate-determining O--O bond cleavage of the first associated hydrogen peroxide molecule, for which reaction via the doublet state is preferred. The sextet (S=5/2) state of the FeIII complexes appears to be unreactive in catalase-like reactions.

During the past years, there has been increasing interest in endogenous nitric oxide storage compounds. Recently, we briefly reported on the ascorbate-dependent release of nitric oxide ((.)NO) from N-nitrosotryptophan derivatives. In the present study, the underlying mechanism of (.)NO release is studied in more detail, primarily utilizing N-acetyl-N-nitrosotryptophan (NANT) as a model compound. The initial rate of the ascorbate-induced release of nitric oxide has been found to correspond to the rate of NANT decay. In this process, N-acetyltryptophan (NAT) is produced almost quantitatively. The final yield of nitrite amounted to around 90 % with respect to the applied amount of NANT. However, the total release of nitric oxide was only 60 %, as determined by using an FNOCT-4(fluorescent nitric oxide cheletropic trap number 4) assay. Besides nitric oxide, a second volatile product, dinitrogen oxide (N(2)O), has been identified by using (15)N NMR spectrometry, strongly indicating the intermediacy of nitroxyl (HNO). The formation of intermediate ascorbyl radical anions during the NANT-ascorbate reaction has been monitored by using ESR spectrometry. Unexpectedly, it was found that the primary oxidized product of vitamin C, dehydroascorbic acid (DHA), efficiently consumes nitric oxide. Since ESR spectrometry further revealed that ascorbyl radical anions are also generated during the spontaneous decay of DHA, the DHA-nitric oxide reaction is related to recombination of (.)NO with the thus formed ascorbyl radical anions. A conclusively established mechanism of the NANT-ascorbate reaction is presented, with O-nitrosoascorbate as a key intermediate, as additionally supported by CBS-QB3 calculations. The present study suggests that vitamin C and its oxidation products can chemically counterbalance endogenous nitric oxide levels.

Heme catalases are considered to degrade two molecules of H(2)O(2) to two molecules of H(2)O and one molecule of O(2) employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H(2)O(2) (relative to catalase concentration), adjusted by H(2)O(2)-generating systems. At a ratio of a H(2)O(2) flux (given in microM/min(- 1)) to catalase concentration (given in microM) of 10 min(- 1) and above, H(2)O(2) degradation occurred via the catalatic cycle. At lower ratios, however, H(2)O(2) degradation proceeded with increasingly diminished production of O(2). At a ratio of 1 min(- 1), O(2) formation could no longer be observed, although the enzyme still degraded H(2)O(2). These results strongly suggest that at low physiological H(2)O(2) fluxes H(2)O(2) is preferentially metabolised reductively to H(2)O, without release of O(2). The pathways involved in the reductive metabolism of H(2)O(2) are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H(2)O(2) fluxes but kinetically outcompete the reaction of compound I with H(2)O(2) at low H(2)O(2) production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H(2)O(2) are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.

Hydrogen peroxide is involved in many types of cell injury and exerts most of its injurious effects in conjunction with chelatable iron. We previously described a synthetic nonporphyrin iron-containing catalase mimic, TAA-1/Fe. Its ligand TAA-1 was designed for application in biological systems in which it is supposed to fulfill a dual task: it should chelate cellular labile iron and thus form the active catalase mimic, thereby decreasing levels of redox-active iron and enhancing the degradation of hydrogen peroxide. Here, we tested these novel compounds in cellular systems, i.e., in cultured hepatocytes and liver endothelial cells. Both the iron complex, i.e., the complete mimic, and the ligand, i.e., the putative precursor of this mimic, provided protection against endothelial cell injury induced by exogenous hydrogen peroxide. Furthermore, the ligand--but not (or less so) the complex--strongly protected both cell types against iron-dependent hypothermic injury and hepatocytes against iron-induced cell injury and against iron-dependent, histidine-induced injury. Together, these results demonstrate that the putative catalase mimic precursor TAA-1 is able to protect cells against iron- and/or hydrogen peroxide-dependent cell injuries and that--in line with our initial concept--it is likely to exert its protection by both iron chelation and hydrogen peroxide degradation.

The effect of pressure on the oxidation of hydroarenes 3-9 with 2,3-dichloro-5,6-dicyano-1,4-quinone (DDQ; 1 a) or o-chloranil (10), leading to the corresponding arenes, has been investigated. The activation volumes were determined from the pressure dependence of the rate constants of these reactions monitored by on-line UV/Vis spectroscopic measurements in an optical high-pressure cell (up to 3500 bar). The finding that they are highly negative and only moderately dependent on the solvent polarity (DeltaV( not equal ) = -13 to -25 in MTBE and -15 to -29 cm(3) mol(-1) in MeCN/AcOEt, 1:1) rules out the formation of ionic species in the rate-determining step and is good evidence for a hydrogen atom transfer mechanism leading to a pair of radicals in the rate-determining step, as was also suggested by kinetic measurements, studies of kinetic isotope effects, and spin-trapping experiments. The strong pressure dependence of the kinetic deuterium isotope effect for the reaction of 9,10-dihydroanthracene 5/5-9,9,10,10-D(4) with DDQ (1 a) can be attributed to a tunneling component in the hydrogen transfer. In the case of formal 1,3-dienes and enes possessing two vicinal C--H bonds, which have to be cleaved during the dehydrogenation, a pericyclic hydrogen transfer has to considered as one mechanistic alternative. The comparison of the kinetic deuterium isotope effects determined for the oxidation of tetralin 9/9-1,1,4,4-D(4)/9-2,2,3,3-D(4)/9-D(12) either with DDQ (1 a) or with thymoquinone 1 c indicates that the reaction with DDQ (1 a) proceeds in a stepwise manner through hydrogen atom transfer, analogously to the oxidations of 1,4-dihydroarenes, whereas the reaction with thymoquinone 1 c is concerted, following the course of a pericyclic hydrogen transfer. The difference in the mechanistic courses of these two reactions may be explained by the effect of the CN and Cl substituents in 1 a, which stabilize a radical intermediate better than the alkyl groups in 1 c. The mechanistic conclusions are substantiated by DFT calculations.

Background/aims: We previously described that the cold-induced apoptosis of cultured hepatocytes and liver endothelial cells is mediated by an increase in the cellular chelatable iron pool-in the absence of any increase in O(2)(.-)/H(2)O(2) formation. As this is an unusual mechanism, we here set out to assess whether an increase in cellular chelatable iron per se is sufficient to trigger cell injury/apoptosis., Methods: Cultured rat hepatocytes were acutely loaded with iron using the membrane-permeable complex Fe(III)/8-hydroxyquinoline and incubated under otherwise 'physiological' conditions., Results: Incubation with Fe(III)/8-hydroxyquinoline (15 microM/30 microM) increased the cellular chelatable iron and induced strong hepatocellular injury with morphological features of apoptosis, but also of necrosis. The iron-induced cell injury was oxygen-dependent, and although it was not inhibitable by extracellular catalase, it was strongly inhibited by the novel membrane-permeable catalase mimic TAA-1/Fe. The experimentally induced increase in cellular chelatable iron triggered a mitochondrial permeability transition (MPT) as assessed using double-staining with calcein and tetramethylrhodamine methyl ester. The MPT inhibitor cyclosporine A partially and the well-known inhibitor combination trifluoperazine+fructose completely inhibited the iron-induced cell injury/apoptosis., Conclusions: These results show that iron per se can induce cell injury/apoptosis and that this injury is mediated via an MPT.

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the 'protein transduction' by beta-oligoarginines are discussed.

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4 degrees C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2'-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.

The cycloaddition of thiobenzophenone S-methylide to thiobenzophenone, an experimentally well-known reaction, was studied, using (U)HF/3-21G* for finding stationary points and (U)B3LYP/6-31G*//(U)HF/3-21G* single-point calculations for energies. Some optimizations were performed by (U)B3LYP/ 6-31G* to check the reliability of the calculations. The comparison of the concerted pathways and stepwise reactions via C,C-biradicals and C,S-zwitterions showed that the formation of a tetraphenyl-substituted C,C-biradical and its ring closure to 4,4,5,5-tetraphenyl-1,3-dithiolane constitutes the energetically most probable pathway of product formation, despite the fact that the regioisomeric 2,2,4,4-tetraphenyl-substituted product is more favorable by 17 kcal mol(-1). Model calculations on bond dissociation energies showed that (U)B3LYP with various basis sets overestimates radical stabilization, whereas CBS-QB3 closely reproduced experimental values. Results with the BLYP functional are similar to those with B3LYP. The consequences of the overestimation of radical stability for the cycloaddition mechanism involving biradicals are discussed. Thiobenzophenone S-methylide, if not captured by a dipolarophile, dimerizes to 2,2,3,3-tetraphenyl-1,4-dithiane. Calculation disclosed likewise a tetraphenyl-substituted C,C-biradical as intermediate.

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).

It has been known for quite some time that tetrahydrobiopterin (H4B) is prone to autoxidation in the presence of molecular oxygen. Evidence has been presented that in this process superoxide radicals may be released, although their intermediacy never has been directly proven. In the present study, the autoxidation of H4B was reinvestigated with the aim to find direct evidence for superoxide formation. By means of two specific assays, namely elicitation of luminescence from lucigenin and ESR-spectrometric detection of the DEPMPO-OOH radical adduct, the release of free superoxide radicals was unequivocally demonstrated. The production of superoxide radicals was further corroborated by interaction with nitric oxide. The kinetics of the autoxidation process was established. Our data fully confirm earlier conclusions that the direct reaction between H4B and oxygen serves as an initiation reaction for the further, rapid reaction of the thus formed superoxide with H4B, thereby very likely establishing a chain reaction process involving reduction of molecular oxygen by the intermediary tetrahydrobiopterin radical. Conclusively, because H4B can per se induce oxidative stress, an in vivo overproduction of this pterin, as is evident in various diseases, may be responsible for the observed acceleration of pathophysiological pathways.

The regiochemistry of 1,3-dithiolanes obtained from thiocarbonyl ylides 9 and thiones 10 shows a striking dependence on substituents. Previously and newly performed experiments indicate that sterically hindered cycloalkanethione S-methylides and dialkylthioketone S-methylides react with alicyclic and aliphatic thiones to give the 2,2,4,4-tetrasubstituted 1,3-dithiolanes 11 exclusively. Aryl groups in one or both reactants lead to a preference for, or even complete formation of, 4,4,5,5-tetrasubstituted 1,3-dithiolanes 12. Several mechanisms appear to be involved, but the paucity of experimental criteria is troubling. Quantum-chemical calculations (see preceding paper) on the cycloaddition between thioacetone S-methylide and thioacetone furnish lower activation energies for the concerted process than for the two-step pathways via C,S- or C,C-biradicals; the favoring of the 2,4-substituted 1,3-dithiolanes over the 4,5-substituted type would be expected to increase with growing bulk of substituents. Aryl groups stabilize intermediate biradicals. Experimental criteria for the differentiation of regioisomeric dithiolanes are discussed. Thiocarbonyl ylides 9 are prepared by 1,3-cycloadditions between diazomethane and thioketones and subsequent N(2) elimination from the usually isolable 2,5-dihydro-1,3,4-thiadiazoles 17; different ratios of the two rate constants lead to divergent product formation scenarios.

The mechanisms of cycloaddition of thioformaldehyde S-methylide and thioacetone S-methylide, as models for an alkyl-substituted ylide, to thioformaldehyde and thioacetone, as well as to ethene as a model for a C=C double bond have been studied by ab initio calculations. Restricted and unrestricted B3LYP/6-31G* calculations were performed for the geometries of ground states, transition structures, and intermediates. Although basis sets with more polarization functions were tested, the 6-31G* basis set was applied throughout. Single-point CASPT2 calculations are reported for analysis of the unsubstituted system. The stabilities of structures with high biradical character seem to be overestimated by DFT methods in comparison to CASPT2. The general trends of the results are independent of the level of theory. Thioformaldehyde adds to thioformaldehyde S-methylide without activation energy, and the activation energies for two-step biradical pathways to 1,3-dithiolane are low. C,S biradicals are more stable than C,C biradicals. The two-step cycloaddition is not competitive with the concerted cycloaddition. Methyl substitution in the 1,3-dipole and the dipolarophile does not change the mechanistic relationships. TSs for the concerted formation of the regioisomeric cycloadducts of thioacetone Smethylide and thioacetone were located. Concerted addition remains the preferred reaction. The reactivity of the C=S double bond is high relative to that of the C=C double bond.

A very small, predominantly cytosolic pool of iron ions plays the central role in the cellular iron metabolism. It links the cellular iron uptake with the insertion of the metal in iron storage proteins and other essential iron-containing molecules. Furthermore, this transit ('labile') pool is essentially involved in the pathogenesis of a number of diseases. Due to its high physiological and pathophysiological significance, numerous methods for its characterization have been developed during the last five decades. Most of these methods, however, influence the size and nature of the transit iron pool artificially, as they are not applicable to viable biological material. Recently, fluorescence spectroscopic methods for measurements within viable cells have become available. Although these methods avoid the artifacts of previous methods, studies using fluorescent iron indicators revealed that the 'intracellular transit iron pool', which is methodically assessed as 'chelatable iron', is substantially defined by the method and/or the iron-chelating indicator applied for its detection, since the iron ions are bound to a large number of different ligands in different metabolic compartments. A more comprehensive characterization of the nature and the role of the thus not uniform cellular transit iron pool therefore requires parallel employment of different indicator molecules, which clearly differ in their intracellular distribution and their physico-chemical characteristics.

Nitrogen dioxide (*NO2) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed *NO2. Peroxynitrite (ONOO-/ONOOH) is believed to be an important endogenous source of *NO2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of *NO2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and beta-carotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O2*-)-dependent formation of peroxynitrate (O2NOO-) and the central role of vitamin C are of special importance.

Mitochondrial chelatable ("redox-active") iron is considered to contribute to several human diseases, but has not yet been characterized in viable cells. In order to determine this iron pool, we synthesized a new fluorescent indicator, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). In a cell-free system, RPA fluorescence was strongly and stoichiometrically quenched by Fe(2+). RPA selectively accumulated in the mitochondria of cultured rat hepatocytes. The intramitochondrial RPA fluorescence was quenched when iron was added to the cells in a membrane-permeant form. It increased when the mitochondrial chelatable iron available to the probe was experimentally decreased by the membrane-permeant transition metal chelators pyridoxal isonicotinoyl hydrazone and 1,10-phenanthroline. The concentration of mitochondrial chelatable iron in cultured rat hepatocytes, quantified from the increase in RPA fluorescence after addition of pyridoxal isonicotinoyl hydrazone, was found to be 12.2 +/- 4.9 microM. Inhibition of haem synthesis with succinylacetone did not alter the signal obtained in hepatocytes, but a rapid increase in the concentration of mitochondrial chelatable iron was observed in human erythroleukaemia K562 cells. In conclusion, RPA enables the selective determination of the highly physiologically and pathophysiologically interesting mitochondrial pool of chelatable iron in intact cells and to record the time course of alterations of this pool.

Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2*) and trioxocarbonate(1-) (CO3*-) radicals. We have studied the mechanism by which glutathione (GSH) inhibits the NO2*/CO3*--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent tyrosine consumption (IC50 = 660 microM) as well as 3-nitrotyrosine formation (IC50) = 265 microM). From the determination of the level of oxygen produced or consumed under various initial conditions, it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS*) through reduction of dioxygen to superoxide (O2*-) and reaction with NO2* to form peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effective in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2* radicals is highly unlikely at physiological levels of GSH.

Hydrogen carbonate (bicarbonate, HCO(3)(-)) has been proposed to accelerate the decomposition of N(2)O(3) because N-nitrosation of morpholine via a nitric oxide/oxygen mixture ((*)NO/O(2)) was inhibited by the addition of HCO(3)(-) at pH 8.9 [Caulfield, J. L., Singh, S. P., Wishnok, J. S., Deen, W. M., and Tannenbaum, S. R. (1996) J. Biol. Chem. 271, 25859-25863]. In the study presented here, it is shown that carbon dioxide (CO(2)) is responsible for this kind of protective effect because of formation of amine carbamates, whereas an inhibitory function of HCO(3)(-) is excluded. N-Nitrosation of morpholine (1-10 mM) at pH 7.4-7.5 by the (*)NO-donor compounds PAPA NONOate and MAMA NONOate (0.5 mM each) was not affected by the presence of large amounts of HCO(3)(-) (up to 100 mM) in aerated aqueous solution. Similar results were obtained by replacing the (*)NO-donor compounds with authentic (*)NO (900 microM). In agreement with data from the study cited above, (*)NO/O(2)-mediated formation of N-nitrosomorpholine (NO-Mor) was indeed inhibited by about 45% in the presence of 50 mM HCO(3)(-) at pH 8.9. However, 500 MHz (13)C NMR analysis with (13)C-enriched bicarbonate revealed that significant amounts of morpholine carbamate are formed from reaction of equilibrated CO(2) with morpholine (1-100 mM) at pH 8.9, but only to a minor extent at pH 7. 5. The protective effect of morpholine carbamate formation is explained by a significantly reduced charge density at nitrogen. This view is supported by the results of density functional theory/natural population analysis, i.e., quantumchemical calculations for morpholine and morpholine carbamate. In agreement with its lower pK(a), another secondary amine, piperazine, had already produced significant amounts of piperazine carbamate at pH 7. 4 as shown by (13)C NMR spectrometry. Consequently, and in contrast to morpholine, N-nitrosation of piperazine (2 mM) by both (*)NO/O(2) (PAPA NONOate, 0.5 mM) and the (*)NO/O(2)(-)(*)-releasing compound SIN-1 (1 mM) was inhibited by about 66% in the presence of 200 mM HCO(3)(-).

Organic amine-based buffer compounds such as HEPES (Good's buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (.NO) and superoxide (O-2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mM SIN-1 was low ( approximately 15 microM), incubation of SIN-1 with Good's buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mM SIN-1 with 20 mM HEPES about 190 microM H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mM SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration of p-hydroxyphenylacetic acid were strongly inhibited by 20 mM HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H2O2. Likewise, authentic peroxynitrite (1 mM) also induced the formation of both O-2 and H2O2 upon addition to HEPES (400 mM)-containing solutions in a pH (4.5-7.5)-dependent manner. In accordance with previous reports it was found that at pH >/=5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mM)-induced H2O2 formation ( approximately 80 microM at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mM/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good's buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate alpha-amino alkyl radicals with molecular oxygen leads to the formation of O-2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H2O2 formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.

Cultured rat liver endothelial cells were incubated with 1 and 2 mM spermineNONOate at different O2 concentrations in the incubation atmosphere. (Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropylammonio) butyl]-amino}diazen-1-ium-1,2- diolate (spermineNONOate), at 2 mM, was highly cytotoxic at 21% and 95% O2 (as measured by lactate dehydrogenase release); more than 80% of the cells were damaged after 6 h of incubation. Cytotoxicity induced by 2 mM spermineNONOate was significantly decreased at 10%, 5% and 0% O2; cell death was 54%, 36% and 25% respectively after 6 h of incubation. In contrast, 1 mM spermineNONOate was almost non-toxic towards the cells. Only at 95% O2 was a slight damaging effect, of 25%, observed. The nitric oxide (.NO) concentrations released from 1 and 2 mM spermineNONOate were determined as varying between 5 and 12 microM and between 12 and 22 microM respectively as measured by the oxyhaemoglobin and the NO cheletropic spin-trapping methods. The decomposition rate of spermineNONOate and the resulting .NO concentrations were independent of O2 at all applied concentrations. Likewise, the steady-state concentrations of H2O2 remained at approx. 1 nM at all O2 concentrations, as measured with the fluorescent dye scopoletin. L-Tyrosine and L-ascorbate, both of which are known to scavenge reactive nitrogen species, markedly diminished spermineNONOate-induced cytotoxicity at 95% O2. The formation of 3-nitrotyrosine, indicating the reaction of L-tyrosine with nitrogen dioxide (.NO2) and/or peroxynitrite anions, was enhanced in incubations with spermineNONOate at 21% and 95% O2. The results demonstrate that at O2 partial pressures typically found under physiological conditions and at .NO concentrations that can occur in vivo, .NO alone is almost non-toxic towards cultured rat liver endothelial cells. .NO at these concentrations in vivo, however, exerts toxic effects at supraphysiological O2 partial pressures, owing to its oxidation to reactive nitrogen species such as .NO2.

We have previously demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. In the present study, we report experimental evidence for the involvement of reactive oxygen species in this injury: LDH release during 48 h of cold incubation in UW solution was decreased from 40-55% under aerobic conditions to less than 20% under hypoxic conditions or by the presence of KCN (1 mM). Similar protection was achieved by the addition of the spin trap 5,5-dimethyl-1-pyrroline N-oxide, the hydroxyl radical scavenger dimethyl sulfoxide, or the flavonoid silibinin to UW solution under aerobic conditions. Preincubating the cells with the iron chelator deferoxamine even decreased the injury to less than 5%. The residual injury (as observed after longer incubation times) under hypoxic conditions or in cells preincubated with deferoxamine was no longer energy dependent. The amount of thiobarbituric acid-reactive substances markedly increased during cold incubation of the cells in UW solution. This increase was not observed in UW solution to which KCN had been added, i.e., under the conditions of energy depletion. These results suggest that an iron-dependent generation of reactive oxygen species with subsequent lipid peroxidation is involved in the pathogenesis of the injury to cultured liver endothelial cells in cold UW solution.

In order to elucidate the potential of substituted o-quinodimethanes as reagents for the trapping of nitric oxide (NO) in biological systems, the reaction of alkoxyl- and alkyl-substituted 7,8-diphenyl- and 7,7,8-triphenyl-o-quinodimethanes with nitric oxide in solution was investigated by ESR spectroscopic and UV/vis stopped-flow techniques. Photolytic decarbonylation of 1,3-diphenyl- and 1,1,3-triphenylindan-2-ones gave the corresponding phenyl-substituted benzocyclobutenes as the major products and low photostationary concentrations of o-quinodimethanes. During 266-nm laser flash photolysis (LFP) of 1,3-dimethoxy-1,3-diphenylindan-2-one and 1-methoxy-1,3,3-triphenylindan-2-one in acetonitrile, species absorbing in the 400-600 nm range were produced, which were attributed to configurational isomers of the corresponding 7,7,8,8-substituted o-quinodimethanes. The isomeric o-quinodimethanes decayed at significantly different rates, indicating a strong influence of the relative orientation of the terminal substituents on their stability. Reaction of the raw photolysates of the 2-indanones with NO produced strong ESR spectra of the corresponding cyclic nitroxide radicals, isoindolin-2-oxyls. The nitroxide radicals were generated in a two-phase process, the first, rapid phase being attributed to the reaction of NO with the photolytically formed o-quinodimethanes and the second, slow phase reflecting the reaction with small amounts of o-quinodimethanes, generated by thermal ring opening of the phenyl-substituted benzocyclobutenes and probably a direct reaction of NO with the benzocyclobutenes. The kinetics of both steps, as evaluated by stopped-flow UV/vis and ESR spectroscopy, revealed a strong dependence of the rate constants of the o-quinodimethane + NO reaction on the substitution pattern of the o-quinodimethanes, with rate constants spanning a range of 10-4000 M(-)(1) s(-)(1). The rate constants ((0.4-7.5) x 10(-)(4) s(-)(1)) for the reaction of NO with the 7,7,8,8-tetrasubstituted benzocyclobutenes are much less influenced by the substitution pattern. The utility of phenyl-substituted benzocyclobutenes as "reservoirs" for o-quinodimethane-type nitric oxide traps is discussed.

S-Nitroso-N-acetyl-DL-penicillamine (SNAP) and sodium nitroprusside (SNP), both of which are known to release nitric oxide (.NO), exhibited cytotoxicity against cultivated endothelial cells. Under hypoxic conditions 5 mM SNAP and 20 mM SNP induced a loss in cell viability of about 90% and 80% respectively, after an 8 h incubation. Under normoxic conditions, cell death was only 45% and 42% respectively within the same time period. Concentrations of .NO liberated from SNAP and SNP were measured by the oxyhaemoglobin method and by two of the recently developed nitric oxide cheletropic traps (NOCTs). The .NO concentrations from SNAP and SNP increased from 74 microM and 28 microM to 136 microM and 66 microM respectively within 15 min of hypoxic incubation, and then decreased to 36 microM and 28 microM. In the respective normoxic incubations the .NO levels from SNAP and SNP remained in the region of about 30 microM and 20 microM respectively. In contrast, spermine/NO adduct (spermineNONOate) was shown to be more toxic under normoxic than under hypoxic conditions. Under either of these conditions, the concentration of .NO liberated from 2 mM spermineNONOate was about 20 microM. The results demonstrate that the cytotoxicity of SNAP and SNP, but not of spermineNONOate, is significantly enhanced under hypoxic compared with normoxic incubations. Studies on the .NO-releasing behaviour of these compounds indicate that the increased toxicity of SNAP and SNP under hypoxic conditions is related to the influence of O2 on the chemical processes by which .NO is produced from the precursors, rather than to an increased sensitivity of the hypoxic cells towards .NO.

The mechanism of oxidation of N omega-hydroxyl-L-arginine (NHA) by the iron-dioxygen complex in nitric oxide synthase (NOS) is still uncertain. The uncertainty has not been helped by a lack of precision in the notation used to describe the oxidation states and electrical charges on the iron and oxygen in some of the suggested mechanisms. These problems of notation are addressed, and, in addition, a cyclic voltammetric measurement of the oxidation potential of NHA, namely +0.10 +/- 0.04 V versus normal hydrogen electrode, is used to argue that the sometimes postulated oxidation of NHA by the iron-dioxygen complex to form an intermediate radical cation, NHA.+, is very unlikely for thermodynamic reasons. Instead, it is suggested that this oxidation occurs by a thermodynamically favored abstraction of the hydrogen atom from the > C = NOH moiety of NHA to form an intermediate iminoxyl radical, > C = NO(.). A subsequent nucleophilic attack by the iron-hydroperoxide species formed by this H-atom abstraction on the carbon atom of the iminoxyl radical moiety leads to the production of nitric oxide (NO) and citrulline.