[Frontiers in Bioscience 14, 4427-4435, January 1, 2009]
NO spin trapping in biological systems
Anatoly Vanin, Alexander Poltorakov
Semyonov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia
TABLE OF CONTENTS
1. ABSTRACT
The most recent data on mechanisms of spin trapping of nitric oxide (NO) by iron dithiocarbamate complexes in animal and plant cells and tissues are considered. The rationale is as follows: 1 In the absence of NO in cells and tissues, iron binds primarily to compounds others than dithiocarbamate ligands, e.g., tricarbonic acids. 2. Predominant binding of iron to dithiocarbamate ligands takes place only after its binding to NO, since nitrosylated iron manifests much higher affinity for these ligands that for any non-thiol compounds. 3.Within the composition of mononitrosyl dithiocarbamate complexes, iron exists predominantly in the oxidized (Fe3+) form, i.e., these complexes are originally diamagnetic. Their subsequent single-electron reduction to the paramagnetic, EPR -detectable form is mediated by endogenous or exogenous (e.g., dithionite) reducing agents. 4.Superoxide-mediated transition of paramagnetic mononitrosyl dithiocarbamate iron complexes into EPR-silent state can be accompanied by significant reduction of EPR-detectable complexes. This defect can be overcome through the use of the so-called ABC method. 5. In contrast to hydrophobic complexes fast decomposition of water-soluble mononitrosyl iron complexes in animal organisms testifies to low efficiency of these complexes in determination of NO content in animal cells and tissues.
2.INTRODUCTION
In 1984, Vanin et al. suggested the use of a bivalent iron complex with diethyldithiocarbamate (DETC) as a trap for nitric oxide (NO) in animal cells and tissues (1). Binding of one NO molecule to this complex results in the formation of a paramagnetic mononitrosyl iron complex with DETC (MNIC- DETC) ({NO-Fe2+- (DETC)2}), which at 77 К generates an anisotropic electron paramagnetic resonance (EPR) signal with the following values of the g-factor: g^ = 2.047, gô ÷ = 2.022, gav=2.04. At room temperature and at g = 2.04, this signal represents an isotropic triplet of a hyperfine structure (HFS) and splitting at 12.5 G (Figure 1). High values of rate constants (£ 108 М-1s-1) for the reactions of iron- dithiocarbamate complexes with NO (2,3) are suggestive of a competition between these complexes and the superoxide anions responsible for the oxidation of free NO molecules in animal cells. The rate of NO binding to iron- dithiocarbamate complexes used at millimolar concentrations exceeds the rate of NO oxidation by the superoxide by the orders of magnitude. High efficiency of using Fe-DETC complexes in NO detection in biological systems and its quantitative determination in vivo was confirmed in numerous studies (1, 4-34).
MNIC- DETC are related to the category of water-insoluble hydrophobic complexes. They are localized in hydrophobic compartments of the cell membrane and are not excreted into the hydrophilic cell environment or the blood stream (4-34). The solubility of DETC molecules in H2O is due to the presence in their structure of a negatively charged ionized thiol group. Binding of positively charged Fe2+ ions to the thiol groups in two DETC molecules results in the formation of an electroneutral water-insoluble Fe2+ (DETC)2 complex, while binding of neutral NO molecules to the Fe2+ (DETC)2 complex has no effect on the hydrophobic characteristics of the MNIC-DETC complex formed. In 1993, it was suggested to use the water-soluble iron complex with N-methyl-D-glucamine dithiocarbamate (MGD) for NO trapping in biological systems (35). The presence in MGD of a carbohydrate residue provides for high solubility of MGD molecules and their iron complexes in water in the presence and in the absence of NO. More recently, the use of other dithiocarbamate derivatives able to form water-soluble and water-insoluble complexes with iron and to bind NO was proposed by Japanese investigators (30). Water-soluble iron complexes with dithiocarbamates proved to be valuable tools for detecting NO not only in the animal tissue samples and cell cultures, but also in aqueous solutions of NO donors, such as NO synthases (NOS) (35-54).
In this review, we attempted to generalize an extensive body of evidence concerning the use of iron- dithiocarbamate complexes as NO traps in the analysis of biological systems with special emphasis on the advantages and disadvantages of the EPR method. The data available allow reconsideration of the current views on the molecular mechanisms of formation of paramagnetic MNIC with dithiocarbamate derivatives and NO detection in living systems.
3. ADVANTAGES
The indisputable advantage of the novel method is the possibility to establish unequivocally the nature of compounds used as NO sources in animal organisms, cell cultures and isolated biochemical systems. This is achieved using a hypothetical NO source, in which the nitrogen atoms (14N) are replaced by the nitrogen isotope, 15N. If such a substitution is accompanied by a complete substitution of triplet HFS (which in the EPR spectra of MNIC- dithiocarbamate complexes is a result of hyperfine splitting on 14N nuclei (nuclear spin I = 1)) for doublet HFS (as a result of hyperfine splitting on the 15N nucleus (I = 1/2)), there are all grounds to believe that the 15N-labeled compound can indeed produce NO.
This approach proved its worth in experiments designed to investigate the role of L-arginine as a unique source of NO production in animal tissues and cell cultures via a NOS-catalyzed oxidative pathway. The substitution of 14N for 15N in the guanidine residue of L-arginine was followed by complete replacement of triplet HFS by doublet HFS in the EPR spectra of MNIC-DETC isolated from mouse liver or macrophage cultures during augmented synthesis of inducible NOS oxide (5, 9, 37, 38, 40). The involvement of the nitrite in NO generation in isolated animal hearts by a nonenzymatic reducing mechanism was established using a similar approach (39). The participation of dioxygen molecules in NO formation catalyzed by NOS was proven using 17O (O2) (I = 5/2) in the experiments with activated macrophages. The additive HFS from 17O was detected in triplet EPR signal from 14NO17O-Fe2+-MGD2 (38).
The possibility of unique detection of NO in complex systems (e.g., living organisms) both ex vivo and in vivo is yet another prominent advantage of the iron- dithiocarbamate method. The nature of enzymatic or nonenzymatic systems involved in the generation of NO can be established using specific inhibitors, e.g., NOS inhibitors. Their application, similar to isotopic substitution of L-arginine, made it possible to minimize MNIC- DETC concentrations and provided additional support in favour of the involvement of the L-arginine- NOS-dependent pathway in NO generation in animal cells and tissues (5-7,16,21,37,40-42,48 ).
The problem of quantitative determination of NO in living systems has not yet been adequately explored and needs unambiguous solution. Apparently, the binding of all NO produced by living organisms to iron- dithiocarbamate traps is impossible in principle, since part of free NO molecules bypass the traps instead of binding to them. Nonetheless, their complete capture is theoretically possible and can be achieved through the use of superhigh concentrations of NO traps in cells and tissues. However, this approach entails significant disturbances in cell metabolism. Recent studies have demonstrated that such efforts are a priori unsuccessful, since paramagnetic MNIC containing dithiocarbamate ligands are exposed to the damaging effects of superoxide anions (О2-.), which convert them into an EPR-nondetectable form (50). This problem similar to the other pitfalls of using iron- dithiocarbamate complexes as NO traps, will be discussed in the subsequent sections. Here, it is sufficient to remind that the aim of this paper is to revise some commonly held views on the mechanisms of formation of paramagnetic MNIC- dithiocarbamate complexes in biological systems.
4. MECHANISMS OF FORMATION OF PARAMAGNETIC MNIC- DITHIOCARBAMATE COMPLEXES IN BIOLOGICAL SYSTEMS
The use of iron- dithiocarbamate complexes as NO traps was first proposed in our publication, which appeared in press as early as 1984 (1). In this study we proposed that the reduced state of ferro- DETC complexes appeared, at least partly, by reducing agents present in animal organisms. By binding to ferro- DETC complexes, NO favors the formation of electroneutral paramagnetic MNIC - {NO+-Fe+- (DETC-S-)2} that can be detected by the EPR method.
At the same time, our more recent data suggest that water-soluble and water-insoluble ferro- dithiocarbamate complexes are both rapidly and virtually completely oxidized to the ferri-form in the air, in cultural media or directly in animal tissues (32). It is noteworthy that water-soluble (but not water-insoluble) complexes are reduced to the ferro-form only in the presence of cysteine. The efficiency of such reduction is very low because of extremely low (£ 100 microM) concentration of cysteine in living systems (55). Other endogenous reducing agents, e.g., glutathione and ascorbate, are fairly ineffective, since the reduction of the aforementioned complexes by glutathione proceeds at a very low rate, while ascorbate does not reduce them at all (32). These findings suggest that it is the ferri-form of iron- dithiocarbamate complexes that is responsible for the binding of NO at a rate close to the rate of binding of NO with ferro- dithiocarbamate complexes in animal cells and tissues (20,32,56). This reaction results in the formation of diamagnetic {NO+-Fe2+- (DETC-S-)2} complexes, which themselves do not generate any EPR signals, but can effectively accept electrons from endogenous reducing agents (ascorbate included) to be further converted into an EPR-detectable reduced form (20, 32, 50, 56, 57). Therefore, for biological systems the "clockwise" mechanism of paramagnetic MNIC- dithiocarbamate formation seems to be a more common route (Figure 2 in (32)). It should be noted in this connection that in biological systems the recovery/reduction of nitroso-ferri-dithiocarbamate complexes by endogenous compounds may be incomplete as it can be evidenced from the results of experiments, in which the concentration of paramagnetic MNIC- dithiocarbamate complexes was increased 2- 3-fold after dithionite treatment of animal tissues defrozen in 150 mM HEPES (рН 7.4) (58). This indicates that the use of strong buffers is necessary in order to prevent nitrite reduction to NO by dithionite treatment as it is generally the case with weak buffers, e.g., 15 mM HEPES. Addition of dithionite powder to buffer solutions stimulates the appearance of local low-pH zones, where the nitrite conversion into nitric acid is accompanied by NO release (32,58).
The retention of ferri- dithiocarbamate complexes in animal tissues in the absence of NO also presents a problem. Thus, it was found that some endogenous iron-binding compounds (e.g., citrates, ascorbates, etc.) compete with dithiocarbamates for iron ions up to complete degradation of iron- dithiocarbamate complexes (32,58). This situation changes drastically in the presence of NO, since the affinity of the nitroso-ferri-dithiocarbamate complexes for dithiocarbamates markedly exceeds the affinity of other ligands for iron ions (32,58). As a consequence, dithiocarbamate-containing diamagnetic MNIC retain their activities over sufficiently long periods of time and undergo further reduction to paramagnetic forms under the influence of endogenous compounds.
According to present-day views, iron- dithiocarbamate complexes, especially those involving iron ferri-ions, contain at least three dithiocarbamate molecules (59,60). However, the results of optical analysis of an equilibrium of the Fe3+- MGD mixture strongly suggest that these complexes contain only two MGD molecules (32). The stability constants for these complexes (beta2) are equal to 1.6 х107 М-2 (32) . Our recent studies have demonstrated that at DETC:Fe £ 3 the majority of ferri- DETC complexes contain two DETC molecules (the paper under preparation); their stability constants are by orders of magnitude higher than those for ferri- MGD complexes. At Fe3+:DETC ratio = 1:2, virtually all iron ions appear to be involved in the complex structure. The true reason for the drastic increase in the rate of NO trapping in isolated animal tissues, e.g., in isolated blood vessels, in the presence of water-insoluble colloid iron-DETC complexes formed in aqueous solutions upon mixing of DETC and iron (1:2) thus becomes apparent (61). The concentration of the DETC- iron complexes formed is approximately equal to the concentration of exogenous iron, while all DETC molecules appears to be bound to the complexes. This finding is consistent with the lack of DETC molecules to form paramagnetic DETC complexes with bivalent copper; otherwise, MNIC-DETC signal would be masked by EPR signals (62). Furthermore, the absence of free DETC molecules compensates by the inhibiting effect of the latter towards superoxide dismutase , an endogenous superoxide inhibitor responsible for the oxidation of MNIC- DETC and the resulting increase in the complex yields (61,63).
5. REACTIONS OF PARAMAGNETIC MNIC- DITHIOCARBAMATE COMPLEXES WITH SUPEROXIDE ANIONS AND PEROXYNITRITE
It was found that the paramagnetic form of MNIC- MGD complexes reacts with superoxide anions in phosphate buffers (рН 7.4) (50). This reaction proceeds with the second-order rate constant equal to 3х107 М-1s-1 and results in the conversion of MNIC- MGD into an EPR-silent form. The latter is reconstituted into paramagnetic complexes in the presence of reducing agents (e.g., sodium ascorbate) added to the reaction solution not later than within 5- 7 min after superoxide addition; otherwise the regeneration of the original complex does not take place. It is thought that reaction of the superoxide with NO yields an intermediate peroxynitrite, which remains bound to the complex within 5- 7 min. Subsequent isomerization of peroxynitrite into a nitrate and its decomposition with the release of NO are due to degradation and further conversion of MNIC- MGD complexes into ferri-MGD (50). Similar reactions are characteristic for water-insoluble MNIC (50). The results of treatment of MNIC with peroxynitrite suggest that the latter can itself destroy these complexes (50).
The so-called АBC method was devised with a unique purpose to overcome the understated estimation of MNIC- dithiocarbamate concentrations in the presence of the superoxide and peroxynitrite (50). The measurements were performed in two steps. In the first step, an aliquot of MNIC, which additionally fulfilled the function of an exogenous trap for the superoxide and peroxynitrite, was added to animal cells or cell cultures. The binding of endogenous NO was induced by adding bivalent iron and DETC (MGD) to the tested samples. In the second step, aliquots of MNIC and the NOS inhibitor were added to the samples. The intensity of EPR signals (S) was established from the ratio:
S1 = А + В - С (in the absence of the NOS inhibitor, Exp. No.1)
S2 = А - С (in the presence of the NOS inhibitor, Exp. No.2)
where А is the signal generated by exogenous MNIC, В is the signal generated by MNIC formed in the presence of endogenous NO and С is the reduction of the MNIC signal resulting from the degradation of the complexes induced by superoxide or peroxynitrite. By subtracting S2 from S1, we obtain the value of В. Under these conditions, the intensity of А should be greater than the value of С in order to minimize the concentrations of the superoxide and peroxynitrite. At the same time, the intensity of A should not be too high with respect to the intensity of В and for animal cells and cell cultures should be selected in the range 1-50 microМ (50).
In the paradigm of notations based on the use of сonventional methods for NO detection, e.g., the selective use of iron- dithiocarbamate complexes as NO traps, the value of В- С is determined from their EPR spectra. The use of high concentrations of superoxide and peroxynitrite at high values of С results in the degradation of the MNIC formed manifested as a lack of EPR signals.
The АВС method based on the use of iron- dithiocarbamate traps is distinguished for high sensitivity of iron complex assays in the presence of superoxide anions and peroxynitrite. When NO assays were carried out in cultured endothelial cells by conventional methods, e.g., by the addition of Fe- DETC to cell cultures and the 15-min incubation of MNIC- DETC, the reaction yield was 0.5 nmoles/5х106 of cultured cells (data from EPR spectral analysis). For comparison, the yields from the ABC method were 2.5 nmoles (50). In experiments with isolated bean leaves, the difference between experimentally determined values was even more pronounced (64). if the measurements were performed by conventional methods, the rate of MNIC- DETC accumulation in the leaf tissues was increased to 0.9 nmoles/g of wet tissue after 1-hour incubation, whereas in other studies the complexes could not be identified at all. Low yields of MNIC- DETC complexes can be attributed to enhanced production of superoxide anions in bean leaves, which is more characteristic for stress situations e.g., defoliation or incubation in buffer solutions (65). Measurements based on the АВС method revealed that the rate of MNIC- DETC formation in the presence of endogenous NO (the intensity of В) did not change within 1 h and was equal to 20 nmoles/g of wet tissue in all tested samples as can be seen from the difference between the EPR signals corresponding to А + В -С and А - С. Inhibition of endogenous NO synthesis in bean leaves was achieved either through addition of a nitrate as the inhibitor of nitrate reductase responsible for this synthesis in plants or deoxyhemoglobin, which competed with Fe- DETC complexes for endogenously produced NO (64) .
6. ON QUANTITATIVE DETERMINATION OF NO IN ANIMAL ORGANISMS BY THE IRON- dithiocarbamate method
The damaging effects of superoxide anions and peroxynitrite on MNIC- dithiocarbamate complexes invalidate the assumption that the use of iron- dithiocarbamate complexes as NO traps provides quantitative information about the generation, or, more precisely, the rate of generation of NO in animal organisms. Even if MNIC contain the whole amount of endogenously produced NO, they can partly be converted into the nondegradable form, nondetectable by EPR and thus fall out of the scope of consideration. The key issue in quantitative determination of NO in animal cells by the iron-dithiocarbamate method is to elucidate whether these changes affect a negligibly small part or a major part of the paramagnetic MNIC formed.
The currently available data suggest that in animal cells the overwhelming majority of water-soluble MNIC- MGD complexes undergo irreversible degradation within half an hour, presumably under the action of superoxide anions and peroxynitrite. Therefore, the EPR signals of these complexes detected in animal urine or internal organs provide only qualitative information about NO generation within the first 30 min following administration of MGD- iron complexes as NO traps. This conclusion was based on the results of experiments in which mice and rabbits were injected with 2- 3 micromoles of �preformed� MNIC- MGD (66). In the liver and other internal organs, these complexes stimulated the appearance of small (£ 40 nmoles) concentrations of paramagnetic thiol-containing dinitrosyl iron complexes (DNIC); the content of paramagnetic MNIC- MGD complexes in urine samples did not exceed 1 nmole. Subsequent treatment of animal urine with dithionite increased their content 3- 4-fold, apparently at the expense of nitrite formed upon decomposition of MNIC- MGD to NO. The yield of MNIC-MGD increased by more than two orders of magnitude after treatment of urine samples with gaseous NO, which testifies unambiguously to a virtually complete degradation of exogenous MNIC- MGD complexes. The MGD molecules and iron ions released from the complexes were reassembled into MNIC- MGD complexes in the presence of exogenous NO that was detected by the EPR method.
In urine samples of mice treated with bacterial lipopolysaccharide (LPS), the MNIC- MGD content was decreased 5- 10-fold concomitantly with inflammation and augmented synthesis of inducible NOS responsible for enhanced production of NO and the superoxide (5,9). A significant contribution to the formation of superoxide was made by oxidase systems triggered upon inflammation. In all probability, enhanced production of superoxide anions is one of the reasons for more efficient degradation of MNIC- MGD complexes in comparison with LPS-untreated animals. The decay of MNIC- MGD complexes in animals affected by inflammation can also be provoked by peroxynitrite; its production is enhanced during augmented synthesis of the superoxide and NO.
In the foregoing sections, we considered the possibility to overcome the damaging effects of the superoxide and peroxynitrite using the АВС method. An alternative approach consists in increasing the content of exogenous water-soluble iron- MGD complexes as nitric oxide traps, which made it possible to achieve dramatic increases in the yields of EPR-detectable MNIC- MGD complexes (49). However, the use of higher concentrations of these complexes is undesirable, since they can provoke toxic effects. Thus, MGD hydrolysis is associated with the formation of a highly toxic compound, viz., carbon disulfide.
The use of water-insoluble MNIC-forming traps, e.g., iron- DETC complexes, localized in hydrophobic compartments of cell membranes is a more promising approach to quantitative determination of NO in animal cells and tissues. An obvious advantage of MNIC- MGD complexes is that they are not excreted from the organism with urine, but are accumulated in the organism. For example, in the mouse liver their concentration does not change for at least one hour (5,67). The content of MNIC- DETC in the livers of LPS-treated mice can reach 1 micromole/g of wet tissue, while their total content in all body organs is commensurate with those of nitrite and nitrate, the oxidation products of endogenously produced NO in the urine of LPS-treated mice to whom iron-dithiocarbamate complexes were not given (67, 68). This prompts a conclusion that specific localization of MNIC- DETC complexes in hydrophobic zones underlies their effective protection against superoxide-induced damage, since these zones are inaccessible for superoxide anions. These data altogether suggest that the decay of DETC molecules is the only reason for MNIC- DETC degradation.
In conclusion it may be said that MNIC- dithiocarbamate complexes are formed both upon binding of NO to iron- dithiocarbamate complexes and by the reaction of the latter with endogenous S-nitrosothiols and DNIC resulting in the formation of NO depots (1, 27, 52, 69). However, taking into account the extremely small size of NO depots (70,71), the contribution of S-nitrosothiols and DNIC to MNIC production is negligibly small in comparison with NO produced by NО synthases. As regards the interaction of iron- dithiocarbamate complexes with nitrites, which results in the recovery/reduction of the protonated form of the nitrite by these complexes with subsequent formation of MNIC, this process occurs at a very low rate and does not play any conspicuous role in MNIC formation in animal cells and tissues (47).
7. ACKNOWLEDGMENT
The work was supported by the Russian Foundation of Basic Researches (Grant 07-04-13506 ofi_c).
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Key Words: NO, iron dithiocarbamate complexes, EPR, Review
Send correspondence to: Anatoly Vanin, Semyonov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia, Tel: 7-495-939-7535, Fax: 7-495-651-21-91;, E-mail: vanin@polymer.chph.ras.ru