Klaus-Dietrich Kröncke

Inducible nitric oxide synthase in human diseases

Since its discovery as a biologically active molecule in the late 1980s, nitric oxide (NO) has been found to play an important role as signal molecule in many parts of the organism as well as cytotoxic or regulatory effector molecule of the innate immune response. The signal molecule NO is synthesized on demand for short periods of time (seconds to minutes) following enzyme activation of constitutively expressed endothelial NO synthase (eNOS) or neuronal NO synthase (nNOS). In contrast, the inducible NO synthase (iNOS) is expressed after cell activation only and then produces NO for comparatively long periods of time (hours to days). Thus, regulated short pulsative synthesis versus constant NO production differentiates between physiological and pathophysiological actions of NO (for review see [1]). As human monocytes in contrast to rodent ones do not produce large amounts of NO when activated in vitro, iNOS expression in human diseases has long been questionable. However, in the last 3 years data have accumulated on iNOS expression in a variety of human diseases or disorders. We here try to review our current understanding of the role of iNOS in human diseases.

Activated macrophages kill pancreatic syngeneic islet cells via arginine-dependent nitric oxide generation

IL-1 and TNF alpha are assumed to be major mediators of islet cell destruction during the pathogenesis of type 1 diabetes. Here we show by neutralization of the two cytokines with excess antibody that IL-1 and TNF alpha do not contribute to the cytotoxic activity of activated macrophages towards isolated islet cells. However, islet cells can be protected from lysis by depleting the culture medium of L-arginine or by adding the antagonist NG-monomethyl-L-arginine, both of which inhibit the generation of nitric oxide by activated macrophages. These results indicate a role of nitric oxide or its equivalent, the endothelium-derived relaxing factor in the development of type 1 diabetes. This is the first report showing that nitric oxide may damage normal cells and thus may be a hitherto unrecognized pathogenetic factor in tissue inflammation and autoimmune disence.

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.

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation.

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitorl-N 5-(1-iminoethyl)-ornithine (0.25 mm). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate at concentrations from 10 to 1000 μm as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.

Nitric oxide mediates intracytoplasmic and intranuclear zinc release

© 1997 Federation of European Biochemical Societies.

Inducible nitric oxide synthase-derived nitric oxide in gene regulation, cell death and cell survival

Studies from many laboratories have demonstrated the complex role of NO in inflammatory processes. Prolonged exposure to NO shifts the cellular redox potential to a more oxidized state and this is critically regulated by intracellular levels of reduced glutathione. NO-mediated stress will alter gene expression patterns, and the number of genes known to be involved is steadily increasing. Indeed, due to its S-nitrosating activity in the presence of oxygen, NO can modify the activity of transcription factors containing zinc finger motifs or cysteines within the DNA-binding domain. In addition, we are faced with not only NO acting as a powerful inducer of apoptosis or of necrosis in some cells, but also representing an equally powerful protection from cell death in many instances. Some of these apparent discrepancies may be explained by different capacities of cells to cope with the stress of NO exposure. Here, we review our findings on the complex impact of NO on transcriptional regulation of genes, cell death and cell survival. These NO-mediated actions will contribute to a better understanding of the impact of inducible nitric oxide synthase (iNOS) enzyme activity during inflammatory reactions.

Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide

Nitric oxide (NO) is known to induce Zn2+ release from the zinc‐storing protein metallothio‐nein and to induce Zn2+ release within the nuclei and cytoplasm of cells. This suggests that zinc finger proteins may be primary targets of NO‐induced stress. In this study, the specific interaction of the heterodimeric complex of two zinc finger transcription factors, 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) receptor (VDR) and retinoid × receptor (RXR) with 1α,25(OH)2D3 response elements (VDREs), was used as a model system. NO was applied to this system via the NO donors SNOC and MAMA/NO and caused a dose‐dependent inhibition of VDR‐RXR‐VDRE complex formation (IC50 values 0.5–0.8 mM). Ligand‐bound or preformed complexes displayed less sensitivity to NO‐induced stress. These in vitro effects of NO were found to be reversible. Functional assays in transiently trans‐fected cells indicated that NO can also act in vivo as a repressor of 1α,25(OH)2D3 signaling (IC50 value of the slow NO donor DETA/NO, 0.5 mM). These findings suggest that NO has a modulatory role on transcription factors depending on their sensitivity to NO‐induced stress, thus providing a mechanism for a gene regulatory function of NO.—Krõncke, K. D., Carlberg, C. Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide. FASEB J. 13, 166–173 (2000)

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.

Toxicity of chemically generated nitric oxide towards pancreatic islet cells can be prevented by nicotinamide

Previous studies have indicated that nitric oxide is involved in the lysis of pancreatic islet cells by inflammatory macrophages. Here we show that the incubation of islet cells with chemical NO-donors leads to cell lysis in a concentration and time dependent way. Islet cell death could be prevented by nicotinamide and 3-aminobenzamide, which are known to inhibit ADP-ribosylation, while several scavengers of oxygen radicals, N-acetylcysteine, dihydrolipoic acid, dimethylthiourea and citiolone, provided no protection.

Cytotoxic action of IL-1β against pancreatic islets is mediated via nitric oxide formation and is inhibited by N G-monomethyl-l-arginine

IL‐1β has been previously shown to act as a cytotoxic agent in islets. Here we show by electron microscopy of alginate encapsulated islets, that islet cell lysis is induced by culturing islets for 24 or 48 h in the presence of IL‐1β. The extent of lysis depends on the IL‐1β concentration and is slightly enhanced by the addition of TNF‐α. Cells can be protected from lysis by N G‐monomethyl‐l‐arginine. Lysis is paralleled by an increase in nitrite concentration in culture supernatants of whole islets but not in supernatants of isolated endocrine cells. The results indicate that IL‐1β toxicity occurs via inducing in non‐endocrine islet cells the synthesis and release of nitric oxide, which has been shown earlier to be highly toxic for islet cells.