FNOCT as a fluorescent probe for in vivo localization of nitric oxide distribution in tobacco roots.

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.

Hydrogen peroxide decomposition by a non-heme iron(III) catalase mimic: a DFT study.

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.

Assessment of chelatable mitochondrial iron by using mitochondrion-selective fluorescent iron indicators with different iron-binding affinities.

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.

Protection against iron- and hydrogen peroxide-dependent cell injuries by a novel synthetic iron catalase mimic and its precursor, the iron-free ligand.

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.

Iron-induced mitochondrial permeability transition in cultured hepatocytes.

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.

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron.

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.

Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool.

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 chelatable iron pool in living cells: a methodically defined quantity.

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.

Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor.

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.