Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease.

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the nitric oxide (NO)-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Using immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-(3)H-arginine to L-(3)H-citrulline in a Ca(2+)/calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform, the activity of which is compromised in patients with coronary artery disease.

Redox-mediated mechanisms and biological responses of copper-catalyzed reduction of the nitrite ion in vitro.

During ischemia nitrite may be converted into nitric oxide (NO) by reaction with heme-carrying proteins or thiol-containing enzymes. NO acts as a regulator of vasodilation and protector against oxidative stress-induced tissue injuries. As a result of ischemia-induced oxidative stress, hypoxia and/or acidosis bivalent copper ions (Cu(2+)) can dissociate from their physiological carrier proteins. Reduced by the body's own antioxidants, the resultant Cu(1+) might represent an effective reductant of nitrite. Here we have evaluated in vitro copper-dissociation from copper/BSA (bovine serum albumin) complexes under ischemic conditions. Furthermore, using physiological concentrations, we have characterized the capacity of antioxidants and bivalent copper ions to serve as Cu(1+)-agitated catalytic sites for nitrite reduction and also the biological responses of this mechanism in vitro. We found that as a consequence of an acidic milieu and/or oxidative stress the copper-binding capacity of serum albumin strongly declined, leading to significant dissociation of copper ions into the ambient solution. At physiologically relevant pH-values Cu(2+) ions in combination with physiologically available copper reductants (i.e., ascorbate, glutathione, Fe(2+)) significantly enhanced nitrite reduction and subsequent non-enzymatic NO generation under hypoxic but also normoxic conditions. Our data demonstrate for the first time that upon ischemic conditions carrier protein-dissociated copper ions combined with appropriate reductants may serve as Cu(1+)-driven catalytic sites for nitrite reduction, leading to the formation of biologically relevant NO formation. Thus, in addition to the action of heme proteins, copper-catalyzed non-enzymatic NO formation from nitrite might represent a further physiologically relevant vasodilating and NO-dependent protective principle to ischemic stress.

Development and evaluation of a questionnaire to measure the perceived implementation of the mission statement of a competency based curriculum.

BACKGROUND: A mission statement (MS) sets out the long-term goals of an institution and is supposed to be suited for studying learning environments. Yet, hardly any study has tested this issue so far. The aim of the present study was the development and psychometric evaluation of an MS-Questionnaire (MSQ) focusing on explicit competencies. We investigated to what extent the MSQ captures the construct of learning environment and how well a faculty is following--in its perception--a competency orientation in a competency-based curriculum. METHODS: A questionnaire was derived from the MS "teaching" (Medical Faculty, Heinrich-Heine University Düsseldorf) which was based on (inter-) nationally accepted goals and recommendations for a competency based medical education. The MSQ was administered together with the Dundee Ready Education Environment Measure (DREEM) to 1119 students and 258 teachers. Cronbach's alpha was used to analyze the internal consistency of the items. Explorative factor analyses were performed to analyze homogeneity of the items within subscales and factorial validity of the MSQ. Item discrimination was assessed by means of part-whole corrected discrimination indices, and convergent validity was analyzed with respect to DREEM. Demographic variations of the respondents were used to analyze the inter-group variations in their responses. RESULTS: Students and teachers perceived the MS implementation as "moderate" and on average, students differed significantly in their perception of the MS. They thought implementation of the MS was less successful than faculty did. Women had a more positive perception of educational climate than their male colleagues and clinical students perceived the implementation of the MS on all dimensions significantly worse than preclinical students. The psychometric properties of the MSQ were very satisfactory: Item discrimination was high. Similarly to DREEM, the MSQ was highly reliable among students (α = 0.92) and teachers (α = 0.93). In both groups, the MSQ correlated highly positively with DREEM (r = 0.79 and 0.80, p < 0.001 each). Factor analyses did not reproduce the three areas of the MS perfectly. The subscales, however, could be identified as such both among teachers and students. CONCLUSIONS: The perceived implementation of faculty-specific goals can be measured in an institution to some considerable extent by means of a questionnaire developed on the basis of the institution's MS. Our MSQ provides a reliable instrument to measure the learning climate with a strong focus on competencies which are increasingly considered crucial in medical education. The questionnaire thus offers additional information beyond the DREEM. Our site-specific results imply that our own faculty is not yet fully living up to its competency-based MS. In general, the MSQ might prove useful for faculty development to the increasing number of faculties seeking to measure their perceived competency orientation in a competency-based curriculum.

Direct evidence that (-)-epicatechin increases nitric oxide levels in human endothelial cells.

BACKGROUND: The dietary flavanol (-)-epicatechin has been suggested to mediate its vasodilatory effect by increasing nitric oxide levels in endothelial cells. AIM OF THE STUDY: To directly prove the formation of nitric oxide (NO) in human endothelial cells (HUVEC) in vitro by trapping NO to yield a fluorescent nitrosamine. METHODS: HUVEC were treated with (-)-epicatechin; nitrite and NO formation were determined by reductive chemiluminescence detection and the NO-sensitive fluorophore 5-methoxy-2-(1H-naphthol[2,3-d]imidazol-2-yl)-phenol copper complex (MNIP-Cu), respectively. MNIP was synthesized in a rapid and convenient one-step microwave reaction. Endothelial nitric oxide synthase (eNOS) mRNA levels and mRNA stability were measured. RESULTS: Incubation with (-)-epicatechin (0.3-10 µM) led to elevated NO levels in HUVEC measured via reductive chemiluminescence detection and visualized as the fluorescent NO derivative of MNIP. Expression of eNOS mRNA and mRNA stability were not affected by (-)-epicatechin treatment within the time frame studied. CONCLUSION: (-)-Epicatechin augments the level of NO in endothelial cells, a process suggested to be responsible for the vasodilatory properties of the compound.

Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes.

BACKGROUND AIMS: Obesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3',5'-cyclic monophosphate (cGMP) as well as adenosine 3',5'-cyclic monophosphate (cAMP) on preadipocytes in vitro. METHODS: Preadipocytes were isolated from human subcutaneous adipose tissue, cultured until confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30-150 microm) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2'-5'-dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated. RESULTS: DETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant decrease in adipogenic conversion. CONCLUSIONS: Our results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced inflammatory condition could be regulated to the desired level.

New aspects of adipogenesis: radicals and oxidative stress.

Preadipocytes are multipotent adipogenic precursor cells that can be isolated from mature adipose tissue. They have been receiving increasing attention in the context of obesity, type 2 diabetes, and other nutrition-associated diseases. Understanding the physiological and pathophysiological processes in fat neo-formation, energy homeostasis, and adipose tissue physiology is the basis for research on metabolic diseases and the respective pharmaceutical intervention. While the hormonal influence on intracellular signaling in adipogenesis has been intensively investigated, the effects of free radical formation and oxidative stress have just started to gain scientific attention. This review summarizes the present knowledge on the main molecular pathways in preadipocyte maturation and focuses on recent findings indicating that besides hormonal stimuli reactive oxygen species (ROS) and free radicals may also interact with preadipocyte differentiation.

Computer-based learning versus practical course in pre-clinical education: acceptance and knowledge retention.

BACKGROUND: Computer-based learning is a tool that, when designed appropriately, can be used to effectively meet worthy educational goals. AIMS: This study aimed to compare acceptance and effectiveness of a tutored computer-based practical course (CPC) with a traditional biochemical laboratory experiment in pre-clinical medical education. METHOD: Under quasi-randomised conditions two cohorts of second-year medical students performed either a CPC or a laboratory experiment. Students' perceptions were obtained by assessment questionnaires, knowledge retention was investigated by post test. RESULTS: The students evaluated the CPC highly significantly better than the laboratory experiment. Students performing the CPC demonstrated a statistically significant greater knowledge retention compared to students who performed the laboratory experiment. CONCLUSIONS: These findings show that learning objectives concerning basic biochemical knowledge can be acquired in a CPC and that medical students accept such a CPC as a substitute for a real laboratory experiment.

Nitrotyrosine and protein carbonyls are equally distributed in HT22 cells after nitrosative stress.

The generation of reactive oxygen and nitrogen species is an inevitable result of cellular metabolism and environmental influence. Such oxidation processes are always combined with the formation of various protein oxidation products. Environmental oxidants might either be activated inside the cell or act by themselves. Therefore, differences in the localization of oxidant formation might change the major compartment of oxidant action. Therefore, we employed NO donors (SNOC, DETA/NO, and Spe/NO) alone or in combination with the redox-cycling bipyridinium compound paraquat, the superoxide- and NO-releasing compound SIN-1, the relatively more lipophilic oxidants tert-butyl and cumene hydroperoxide, and peroxynitrite itself to test the ability of these compounds to generate oxidized and nitrated proteins in various cellular compartments. Combined treatment with oxidants and nitrating compounds led to the formation of protein carbonyls and nitrotyrosine with a severalfold higher concentration in the cytosol, compared to the nucleus. In fluorescence microscopy studies, the resulting protein modifications show a similar distribution of oxidized proteins and nitrotyrosine with highest concentrations in the perinuclear area. Studying the time- and concentration-dependent formation and degradation of protein carbonyls and nitrated proteins large similarities could be measured. Therefore, it can be concluded that formation, localization, and kinetics of protein carbonyl and nitrotyrosine formation parallel each other depending on the stress-inducing agent.

The presence of nitrite during UVA irradiation protects from apoptosis.

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite-mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite-mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.

Mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic nitric oxide generation from photolabile nitric oxide derivates in human skin in vitro and in vivo.

Human skin contains photolabile nitric oxide (NO) derivates such as nitrite and S-nitrosothiols, which upon UVA radiation decompose under high-output NO formation and exert NO-specific biological responses such as increased local blood flow or reduced blood pressure. To avoid the injurious effects of UVA radiation, we here investigated the mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic NO generation from photolabile nitric oxide derivates in human skin in vitro and in vivo. As quantified by chemiluminescence detection (CLD), at physiological pH blue light at 420 or 453 nm induced a significant NO formation from S-nitrosoalbumin and also from aqueous nitrite solutions by a to-date not entirely identified Cu(1+)-dependent mechanism. As detected by electron paramagnetic resonance spectrometry in vitro with human skin specimens, blue light irradiation significantly increased the intradermal levels of free NO. As detected by CLD in vivo in healthy volunteers, irradiation of human skin with blue light induced a significant emanation of NO from the irradiated skin area as well as a significant translocation of NO from the skin surface into the underlying tissue. In parallel, blue light irradiation caused a rapid and significant rise in local cutaneous blood flow as detected noninvasively by using micro-light-guide spectrophotometry. Irradiation of human skin with moderate doses of blue light caused a significant increase in enzyme-independent cutaneous NO formation as well as NO-dependent local biological responses, i.e., increased blood flow. The effects were attributed to blue-light-induced release of NO from cutaneous photolabile NO derivates. Thus, in contrast to UVA, blue-light-induced NO generation might be therapeutically used in the treatment of systemic and local hemodynamic disorders that are based on impaired physiological NO production or bioavailability.

Circulating microparticles carry a functional endothelial nitric oxide synthase that is decreased in patients with endothelial dysfunction.

BACKGROUND: Microparticles (MPs) are circulating membrane particles of less than a micrometer in diameter shed from endothelial and blood cells. Recent literature suggests that MPs are not just functionally inert cell debris but may possess biological functions and mediate the communication between vascular cells. As a significant proportion of MPs originate from platelets and endothelial cells, we hypothesized that MPs may harbor functional enzymes including an endothelial NO synthase (eNOS). METHODS AND RESULTS: Using immunoprecipitation and Western blot analysis, we found that human circulating MPs carry an eNOS. Ca(2+) and l-arginine-dependent NOS activity of crude enzyme extract from MPs was determined by measuring the conversion of [(3)H]-L-arginine to [(3)H]-citrulline and NOS-dependent nitrite production. NOS-dependent NO production in intact MPs was assessed by the NO-specific fluorescent probe MNIP-Cu. In patients with cardiovascular disease, endothelial dysfunction was associated with an increase in the total number of circulating MPs as well as a significant decrease in the expression and activity of eNOS in MPs. No difference in reactive oxygen species was noted in MPs isolated from either group. CONCLUSIONS: Our data further support the concept that circulating MPs may not only retain phenotypic markers but also preserve the functionality of enzymes of the cells they originate from, including eNOS.

3,3'-Dihydroxyisorenieratene prevents UV-induced formation of reactive oxygen species and the release of protein-bound zinc ions in human skin fibroblasts.

3,3'-Dihydroxyisorenieratene (DHIR) is a structurally unusual carotenoid exhibiting bifunctional antioxidant properties. It is synthesized by Brevibacterium linens, used in dairy industry for the production of red smear cheeses. The compound protects cellular structures against photo-oxidative damage and inhibits the UV-dependent formation of thymidine dimers. Here we show that DHIR prevents a UV-induced intracellular release of zinc ions from proteins in human dermal fibroblasts. The effect is correlated with a decreased formation of intracellular reactive oxygen species. In contrast, zinc release from cellular proteins induced by hyperthermia is not affected by pretreatment of cells with the antioxidant DHIR. It is suggested that the intracellular zinc release upon UV irradiation is due to oxidative modifications of the zinc ligands in proteins (e.g. cysteine) and that protection by DHIR is due to intracellular scavenging of reactive oxygen species generated in photo-oxidation.

Nitric oxide-mediated protection of endothelial cells from hydrogen peroxide is mediated by intracellular zinc and glutathione.

Oxidative stress may cause endothelial dysfunction and vascular disease. It has been shown that NO protects endothelial cells (EC) against H(2)O(2)-induced toxicity. In addition, it is known that NO within cells induces a zinc release from proteins containing zinc-sulfur complexes. The aim of this study was to investigate whether zinc released intracellularly by NO plays a signaling role in the NO-mediated protection against H(2)O(2) in rat aortic EC. Our results show that the NO-mediated protection toward H(2)O(2) depends on the activities of glutathione peroxidase and glutamate cysteine ligase (GCL), the rate-limiting enzyme of glutathione (GSH) de novo biosynthesis. Moreover, NO increases the synthesis of the antioxidant GSH by inducing the expression of the catalytic subunit of GCL (GCLC). Chelating intracellular "free" zinc abrogates the NO-mediated increase of GCLC and of cellular GSH levels. As a consequence, the NO-mediated protection against H(2)O(2)-induced toxicity is impaired. We also show that under proinflammatory conditions, both cellular NO synthesis and intracellular "free" zinc are required to maintain the cellular GSH levels. Using RNA interference and laser scanning microscopy, we found that the NO-induced expression of GCLC depends on the activation of the transcription factor Nrf2 but not on the activity of the "zinc-sensing" transcription factor MTF-1. These findings show that intracellular "free" zinc plays a signaling role in the protective activity of NO and could explain why maintenance of an adequate zinc status in the endothelium is important to protect from oxidative stress and the development of vascular disease.

Nitrite, a naturally occurring precursor of nitric oxide that acts like a 'prodrug'.

There are enzymatic and non-enzymatic mechanisms that generate NO* from nitrite in blood, stomach, saliva, urine and skin. In blood vessels, nitrite-derived NO* can provide protection via compensatory vasodilation during hypoxia, and in various body fluids it may have antibacterial activity. In the skin, nitrite-derived NO* may contribute to skin tanning, as well as to protection against UV-induced cell damage. Current knowledge on nitrite acting like an NO* 'prodrug' is presented, emphasizing the role of nitrite in skin.

Nitrosative stress and transcription.

Low NO concentrations synthesized by constitutively expressed NO synthases act on several signaling pathways activating transcription factors (TF), such as NF-kappaB or AP-1, and thereby influence gene expression. In contrast, during inflammatory reactions the inducible NO synthase produces NO for prolonged periods of time. The resulting nitrosative stress directly affects redox-sensitive TF like NF-kappaB, AP-1, Oct-1, c-Myb, or zinc finger-containing TF, but also additional mechanisms have been identified. Nitrosative stress in some cases induces expression of TF (AP-1, p53), indirectly modulates activity or stability of TF (HIF-1, p53) or their inhibitors (NF-kappaB), or modulates accessibility of promoters via increased DNA methylation or histone deacetylation. Depending on the promoter the result is induced, increased, decreased or even totally inhibited expression of various target genes. In unstimulated cells nitrosative stress increases NF-kappaB- or AP-1-dependent transcription, while in activated cells nitrosative stress rather abolishes NF-kappaB- or AP-1-dependent transcription. Sometimes the oxygen concentration also is of prime importance, since under normoxic conditions nitrosative stress activates HIF-1-dependent transcription, while under hypoxic conditions nitrosative stress leads to inhibition of HIF-1-dependent transcription. This review summarizes what is known about effects of physiological NO levels as well as of nitrosative stress on transcription.

Orphan nuclear receptor binding site in the human inducible nitric oxide synthase promoter mediates responsiveness to steroid and xenobiotic ligands.

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are members of the nuclear receptor superfamily that regulate target gene transcription in a ligand-dependent manner. CAR and PXR have a rather broad, overlapping set of ligands that range from natural steroids to xenobiotics and also recognize similar DNA binding sites, referred to as response elements (REs), primarily in promoter regions of cytochrome P450 (CYP) genes. In this study, a CAR and PXR RE, composed of a direct repeat of two GGTTCA motifs in a distance of 4 nucleotides (DR4), was identified in the promoter of the human inducible nitric oxide (NO) synthase (iNOS) gene, which is the first nuclear receptor binding site reported for this promoter. In a heterologous promoter context, the DR4-type sequence also acts as a functional RE for the nuclear receptors for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25OH2D3) and 3,5,3'-triiodothyronine (T3), VDR and T3R. However, in a direct competition of CAR, PXR, VDR, and T3R, the PXR-retinoid X receptor (RXR) complex appears to be the dominant regulator on the iNOS DR4-type RE. In the natural iNOS promoter context, the DR4-type RE specifically mediates downregulation of promoter activity by the testosterone metabolite androstanol through CAR-RXR heterodimers and upregulation by the xenobiotic drug clotrimazole through PXR-RXR heterodimers. These results were confirmed on the level of mRNA expression. Since an iNOS-induced production of NO is known to influence inflammation and apoptosis, a CAR- and PXR-regulated iNOS activity may explain a modulatory effect of steroids and xenobiotics on these cellular processes.

Necrosis is the predominant type of islet cell death during development of insulin-dependent diabetes mellitus in BB rats.

Several reports propose that apoptosis of pancreatic beta cells may play a central role in the pathogenesis of both spontaneous and induced insulin-dependent diabetes mellitus (IDDM) in animal models. Whether apoptosis is a major cell death pathway during diabetes development, however, is highly controversial. The aim of this study was to examine the mode of islet cell death in prediabetic diabetes-prone (dp) BB rats, which spontaneously develop diabetes and serve as an animal model for human IDDM. In addition we investigated the cell death pathway of islet cells treated with the widely used diabetogenic compound streptozotocin or with nitric oxide (NO), which during IDDM development has been found to be present in inflamed islets in high concentrations because of the expression of inducible NO synthase. Islets of prediabetic BBdp rats were analyzed for DNA strand breaks and screened by electron microscopy. The mode of islet cell death in vitro after treatment with cytotoxic concentrations of streptozotocin or of NO was investigated using different methods including morphologic analysis by electron microscopy, detection of DNA strand breaks, poly(ADP-ribose) polymerase cleavage, and annexin V staining. Although cells with DNA stand breaks-often accepted as a proof for apoptosis-could be identified, we did not find apoptosis-specific features during islet cell death. Instead we observed massive necrosis as evidenced by disrupted plasma membranes and spilled-out cellular constituents in vitro as well as during disease manifestation in BBdp rats. These results may have serious consequences with regard to the treatment of humans to prevent the development of IDDM.

Singlet oxygen-induced signaling effects in mammalian cells.

Singlet oxygen, an electronically excited form of molecular oxygen, may be generated photochemically or in dark reactions in vivo. Singlet oxygen is not only toxic to cells and impairs signaling events but is also capable of eliciting a cellular stress response. The signaling processes initiated in this response include the activation of mitogen-activated protein kinases. Two possible activation mechanisms of signaling pathways by singlet oxygen are the generation of positive regulators as well as the inactivation of negative regulators.

Comparing nitrosative versus oxidative stress toward zinc finger-dependent transcription. Unique role for NO.

During inflammatory reactions, cells are under nitrosative and/or oxidative stress. The zinc finger transcription factors vitamin D receptor (VDR) and retinoid X receptor (RXR) were used as a model system to characterize effects of NO. and/or reactive oxygen species on zinc finger-dependent gene expression. Nitric oxide (NO.) as well as H(2)O(2), singlet oxygen ((1)O(2)), peroxyl radicals (ROO.) and peroxynitrite (ONOO-), respectively, were shown to inhibit VDR/RXR-DNA complex formation in vitro in a dose-dependent manner. While NO-induced inhibition of VDR/RXR-DNA complex formation could be restored nearly completely by subsequent treatment with dithiothreitol, inhibition by H(2)O(2) proved to be only partially reversible, and inhibition by (1)O(2), ROO. or ONOO- was found to be irreversible. In cells transiently transfected with VDR and RXR, subtoxic concentrations of NO. or hydroperoxides and intracellular generation of superoxide anion radicals inhibited VDR/RXR-dependent reporter gene activity in a dose-dependent manner. Interestingly, cells can repair the zinc fingers of VDR and RXR after nitrosative stress but not after oxidative stress. The results indicate that, among the reactive species investigated, only NO. may act sufficiently gentle to be considered as a regulator and not only as an inhibitor of gene expression via zinc finger transcription factors.

Role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress.

The trace elements copper, zinc and selenium are linked together in cytosolic defense against reactive oxygen and nitrogen species. Copper, zinc-superoxide dismutase catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide. The latter and other hydroperoxides are subsequently reduced by the selenoenzyme glutathione peroxidase (GPx). Cytosolic GPx can also act as a peroxynitrite reductase. The antioxidative functions of these trace elements are not confined to being constituents of enzymes: 1) copper and zinc ions may stimulate protective cellular stress-signaling pathways such as the antiapoptotic phosphoinositide-3-kinase/Akt cascade and may stabilize proteins, thereby rendering them less prone to oxidation; and 2) selenium does not only exist in the cell as selenocysteine (as in GPx) but also as selenomethionine, which is regularly present in low amounts in proteins in place of methionine. Selenomethionine catalyzes the reduction of peroxynitrite at the expense of glutathione. Also, low-molecular-weight organoselenium and organotellurium compounds of pharmacologic interest catalyze the reduction of hydroperoxides or peroxynitrite with various cellular reducing equivalents.