Biomedical subjects

Christoph V Suschek

Publications and source records attributed to Christoph V Suschek.

18 recordsLinked to original sources

PubMed2006

In hepatocytes the regulation of NOS-2 activity at physiological L-arginine levels suggests a close link to the urea cycle.

High-output synthesis of nitric oxide (NO) by the inducible isoform of NO-synthases (NOS-2) plays an important role in hepatic pathophysiological processes and may contribute to both organ protection and organ destruction during inflammatory reactions. As they compete for the same substrate, L-arginine, an interdependence of NOS-2 and arginase-1 has been repeatedly observed in cells where arginase-1 is cytokine-inducible. However, in hepatocytes, arginases are constitutively expressed and thus, their impact on hepatic NOS-2-derived NO synthesis as well as the influence of L-arginine influx via cationic amino acid transporters during inflammatory reactions are still under debate. Freshly isolated rat hepatocytes were cultured for 24h in the presence of various L-arginine concentrations with or without cytokine addition and nitrite and urea accumulation in culture supernatants was measured. We find that both, cytokine-induced NOS-2 and arginase activities strongly depend on extracellular L-arginine concentrations. When we competed for L-arginine influx via the cationic amino acid transporters by addition of L-lysine, we find a 60-70% inhibition of arginase activity without significant loss of NOS-2 activity. Addition of L-valine, as an arginase inhibitor, leads to a 25% increase in NO formation and an 80-90% decrease in arginase activity. Interestingly, product inhibition of arginase and competitive inhibition of CATs through the addition of L-ornithine leads to a highly significant increase in hepatocytic NOS-2 activity with a concomitant and complete abolishment of its dependence on extracellular L-arginine concentrations. In conclusion, hepatocytic NOS-2 activity shows a surprising pattern of dependence on exogenous L-arginine concentrations. Inhibition and competition experiments suggest a relatively tight link of NOS-2 and urea cycle activities. These data stress the hypothesis of a metabolon-like organization of the urea cycle together with NOS-2 in hepatocytes as excess L-ornithine will be metabolized to l-arginine and thereby increases NO production.

Amino Acid Transport Systems, Basic↗

PubMed2006

Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: hypermethylation-independent mechanisms of inactivation.

Death-associated protein kinase (DAPK) is a pro-apoptotic Ca(2+)/calmodulin-dependent serine/threonine kinase that is widely expressed in tissues but kept silent in growing cells. Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, providing a selective growth advantage during tumour progression. As the in vivo expression of DAPK in human renal cell carcinomas (RCCs) has not previously been analysed, 72 RCCs were investigated using semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We found that almost 92% (66/72) of all primary RCCs express DAPK mRNA and results obtained from methylation-specific PCR analyses suggest that aberrant CpG methylation of the DAPK promoter is absent even in DAPK non-expressing tumours. Comparison of early/intermediate with advanced tumour stages of clear cell RCCs showed that no significant changes in the expression levels of DAPK were evident. Chromophilic/papillary RCCs display no significantly different expression patterns of DAPK compared with stage-adjusted clear cell RCCs. Furthermore, on analysing the DAPK enzyme activity in RCC cell lines with DAPK mRNA and protein expression, only 1 out of 11 cell lines showed basal DAPK activity in kinase activity assays, suggesting that DAPK, although expressed in RCC, remains largely inactive. Our study demonstrates the in vivo expression of DAPK in RCCs and reveals that, in contrast to other tumour types, RCCs may not downregulate DAPK mRNA expression during tumour progression. Despite persistent DAPK transcription and translation, however, the markedly reduced DAPK enzyme activity in our RCC cell lines suggested a post-translational inactivation of DAPK in RCCs.

Apoptosis Regulatory Proteins↗

PubMed2006

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.

Animals↗

PubMed2005

Arginase-1 overexpression induces cationic amino acid transporter-1 in psoriasis.

Regulated uptake of extracellular l-arginine by cationic amino acid transporters (CATs) is required for inducible nitric oxide synthase and arginase activity. Both enzymes were recently recognized as important in the pathophysiology of psoriasis because of their contribution to epidermal hyperproliferation. We here characterize the expression pattern of CATs in psoriatic skin compared to healthy skin. CAT-1 mRNA expression was strongly upregulated in lesional and nonlesional areas of psoriatic skin compared to healthy skin, whereas expression of CAT-2A and the inducible isoform CAT-2B was unaltered in psoriatic skin. Furthermore, we tested the hypothesis that arginase-1 overexpression regulates CAT expression via intracellular l-arginine concentration. In in vitro experiments with arginase-1 overexpressing HaCaT cells, CAT-1 mRNA expression was increased. Likewise, this occurs in l-arginine-starved HaCaT cells. Both CAT-2 isoforms were not affected. Arginase-1 overexpression limits the synthesis of NO at physiological, but not supraphysiological, l-arginine levels. Plasma l-arginine concentration was diminished in psoriasis patients and the arginase product l-ornithine was significantly increased compared to healthy controls. In summary, arginase-1 overexpression leads to upregulated CAT-1 expression in psoriatic skin, which is due to lowered intracellular l-arginine levels and limits NO synthesis at physiological l-arginine concentrations.

Arginase↗

PubMed2005

Enzyme-independent nitric oxide formation during UVA challenge of human skin: characterization, molecular sources, and mechanisms.

Many of the local UV-induced responses including erythema and edema formation, inflammation, premature aging, and immune suppression can be influenced by nitric oxide synthase (NOS)-produced NO which is known to play a pivotal role in cutaneous physiology. Besides NOS-mediated NO production, UV radiation might trigger an enzyme-independent NO formation in human skin by a mechanism comprising the decomposition of photo-reactive nitrogen oxides. Therefore, we have examined the chemical-storage forms of potential NO-generating agents, the mechanisms and kinetics of their decomposition, and their biological relevance. In normal human skin specimens we find nitrite and S-nitrosothiols (RSNO) at concentrations 25- or 360-fold higher than those found in plasma of healthy volunteers. UVA irradiation of human skin leads to high-output formation of bioactive NO due to photo-decomposition of RSNO and nitrite which represents the primary basis for NO formation during UVA exposure. Interestingly, reduced thiols strongly augment photo-decomposition of nitrite and are essential for maximal NO release. The enzyme-independent NO formation found in human skin opens a completely new field in cutaneous physiology and will extend our understanding of mechanisms contributing to skin aging, inflammation, and cancerogenesis.

Animals↗

PubMed2005

What sense lies in antisense inhibition of inducible nitric oxide synthase expression?

The impact of nitric oxide (NO) synthesized after activation by proinflammatory cytokines and/or bacterial products by an inducible NO synthase (iNOS) is still contradictory. Expression of iNOS in inflammatory reactions is often found predominantly in cells of epithelial origin, and in these cases NO may serve as a protective agent limiting pathogen spreading, downregulating local inflammatory reactions by inducing production of Th2-like responses in a classical feedback circle, or limiting tissue damage during stress conditions. However, an abundant amount of data on chronic human disorders with predominant proinflammatory Th1-like reactions points to a destructive role of iNOS activity calling for a specific inhibition. Various methods to inhibit iNOS have been established to elucidate a protective versus a destructive role of NO during various stresses. In this review, we focus on antisense (AS)-mediated gene knock-down as a relatively new method to inhibit NO production and summarize the techniques applied and their successes. At least in theory, it provides a specific, rapid, and potentially high-throughput method for inhibiting gene expression and function. We here discuss the opportunities of iNOS-directed AS-ODN, and extensively deal with limitations and experimental problems.

Animals↗

PubMed2005

Antisense-mediated knockdown of iNOS expression in the presence of cytokines.

The impact of nitric oxide (NO) synthesized after activation by proinflammatory cytokines and/or bacterial products by an inducible NO synthase (iNOS) is still contradictory. Various methods to inhibit iNOS expression or activity have been established. A relatively new approach to inhibit iNOS-derived NO production is the antisense (AS) technique, which theoretically provides a specific and efficient method for inhibiting gene expression and function. This chapter focuses on the application of iNOS-specific AS-oligodeoxynucleotide (ODN) and highlights some of the pitfalls that must be considered to use this technique effectively.

Animals↗

PubMed2005

Nonenzymatic nitric oxide formation during UVA irradiation of human skin: experimental setups and ways to measure.

Many of the local ultraviolet (UV)-induced responses, including erythema and edema formation, inflammation, premature aging, and immune suppression, can be influenced by nitric oxide synthase (NOS)-produced NO, which plays a pivotal role in cutaneous physiology. Besides enzyme-mediated NO production, UV radiation triggers an enzyme-independent NO formation in human skin. This occurs due to decomposition of photoreactive nitrogen oxides like nitrite and S-nitrosothiols, which are present in human skin at relatively high concentrations and lead to high-output formation of bioactive NO. This enzyme-independent NO formation opens a new field in cutaneous physiology and will extend our understanding of mechanisms contributing to skin aging, inflammation, and cancerogenesis but also functional protection. Therefore, it is of high interest to examine the chemical storage forms of these potential NO-generating agents in skin, the mechanisms and kinetics of their decomposition, and their biological relevance.

Humans↗

PubMed2004

The role of iNOS in chronic inflammatory processes in vivo: is it damage-promoting, protective, or active at all?

The expression of the inducible nitric oxide synthase (iNOS) is one of the direct consequences of an inflammatory process. Early studies have focused on the potential toxicity of the ensuing high-output NO-synthesis serving as a means to eliminate pathogens or tumor cells but also contributing to local tissue destruction during chronic inflammation. More recently, however, data are accumulating on a protective effect of high-output NO synthesis and - equally important - on a gene-regulatory function that helps to mount a protective stress response and simultaneously aids in down-regulating the proinflammatory response. These findings appear to contrast to the often observed sustained iNOS-expression during chronic inflammatory diseases, as for instance in Psoriasis vulgaris and other conditions with a chronic Th1-like reactivity. We here pose the question as to whether the iNOS is really active in these diseases. We review the data accumulated on iNOS expression in chronic diseases. We also report on the various factors that potentially interfere with proper NO formation by the expressed enzyme. We also highlight the recent findings of how, why and where evidences emerge that impeded NO formation contributes to chronic disease processes and finally present details on our current understanding of such abnormally low NO synthesis and its contribution to the pathophysiological processes of the human proinflammatory skin disease Psoriasis vulgaris.

Animals↗

PubMed2003

Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release.

Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.

Active Transport, Cell Nucleus↗

PubMed2003

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.

Apoptosis↗

PubMed2003

iNOS activity is essential for endothelial stress gene expression protecting against oxidative damage.

In endothelial cells, the expression of the inducible nitric oxide synthase (iNOS) and the resulting high-output nitric oxide synthesis have often been assumed as detrimental to endothelial function, but recent publications have demonstrated a protective role resulting from iNOS espression and activity. To address this question, we used antisense-mediated iNOS knockdown during proinflammatory cytokine challenge in primary endothelial cell cultures and studied endothelial function by monitoring the expression of stress defense genes. Using antisense oligonucleotides, we achieved a block of iNOS protein formation, accompanied by a strong decrease in the expression of the protective stress response genes bcl-2, vascular endothelial growth factor, and heme oxygenase-1 (HO-1). Additionally, cells were also maintained in the presence of limited exogenous substrate concentrations during cytokine challenge, thereby mimicking a situation of low serum arginine level during inflammation. Under these conditions, cytokine addition results in full iNOS protein expression with minimal nitric oxide formation, concomitant with a significant reduction in stress response gene expression and susceptibility to cell death induced by reactive oxygen species. Taken together, our data suggest that cytokine-induced endogenous iNOS expression and activity have key functions in increasing endothelial survival and maintaining function. Thus suppression of iNOS expression or limited substrate supply, as has been reported to occur in atherosclerosis patients, appears to significantly contribute to endothelial dysfunction and death during oxidative stress.

Animals↗

PubMed2003

Critical role of L-arginine in endothelial cell survival during oxidative stress.

BACKGROUND: Oxidative damage of vascular endothelium represents an important initiation step in the development of atherosclerosis. Recently, we reported about protection of inducible nitric oxide synthase (iNOS)-derived high-output NO in endothelial cells. Because iNOS activity critically depends on the availability of its substrate l-arginine, the present study aims at elucidating iNOS-mediated effects on H2O2-induced apoptosis of cytokine-activated rat aortic endothelial cells (AECs) subject to medium l-arginine concentrations. METHODS AND RESULTS: In cytokine-activated AECs, iNOS activity was found to be half-maximal at 60 micromol/L arginine, which represents the medium serum level in rats but also in humans. Maximal activity is seen at and above 200 micromol/L arginine. Activated cells grown in the absence of arginine with minimal iNOS activity are highly sensitive toward H2O2-induced apoptosis, and increases in medium arginine concentrations result in increased cell survival. Moreover, competition experiments show that iNOS activity is completely dependent on cationic amino acid transporter-mediated arginine uptake. We also find that the arginine-dependent protection includes inhibition of endothelial lipid peroxidation and increases in the expression of vasoprotective stress response genes. CONCLUSIONS: Our data demonstrate that arginine concentrations corresponding to physiological serum levels do not allow for optimal endothelial iNOS activity. Thus, decreases in systemic arginine concentrations, or locally within atherosclerotic plaques, will impair the endothelial iNOS-mediated stress response and will significantly increase the risk of endothelial dysfunction.

Amino Acid Transport Systems↗

PubMed2003

Specific iNOS-targeted antisense knockdown in endothelial cells.

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.

Animals↗

PubMed2003

The importance of cationic amino acid transporter expression in human skin.

Inducible nitric oxide synthase and arginase activities are acknowledged as important players in human skin epidermal function. For proper enzyme function the substrate availability of L-arginine for both enzymes and thus its transport across the cell membrane via the y+-system (also named cationic amino acid transporters) is critical. Here, we examine the expression of cationic amino acid transporters and their functional role in modulating inducible nitric oxide synthase and arginase activities in human skin and primary keratinocytes, fibroblasts and endothelial cells as well as their impact on keratinocyte proliferation. Skin biopsies were found to express constitutively both cationic amino acid transporter-1 and cationic amino acid transporter-2 mRNA, an expression pattern known to occur in hepatocytes and muscle cells only. To determine the cellular components expressing cationic amino acid transporter, we analyzed the expression patterns in the different human skin cell types in vitro, i.e., in fibroblasts, dermal endothelial cells, and keratinocytes as well as in the HaCaT cell line. An ubiquitous cationic amino acid transporter-1 mRNA expression was found in all cells, whereas constitutive cationic amino acid transporter-2 mRNA expression occurs in resident keratinocytes and dermal endothelial cells only. De novo induction of cationic amino acid transporter-2 and inducible nitric oxide synthase by proinflammatory cytokines was seen in fibroblasts and HaCaT. Competitive inhibition of the cationic amino acid transporter-mediated L-arginine transport by culturing primary human keratinocytes in the presence of increased L-lysine concentration led to decreased inducible nitric oxide synthase and arginase activities with a concomitant significant decrease in keratinocyte proliferation. In summary, our results demonstrate that human keratinocytes constitutively express cationic amino acid transporters 1 and 2 and that cationic amino acid transporter mediated L-arginine influx, is essential for both inducible nitric oxide synthase and arginase enzyme activities, which in turn modulate proliferation and differentiation of human epidermal skin cells.

Arginase↗

PubMed2003

Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide.

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H2O2). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm2) results in the generation of significant amounts of H2O2, with concentrations of about 100 microM. H2O2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H2O2. Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H2O2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.

Animals↗

PubMed2002

Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas.

Survivin, a novel member of the inhibitor of apoptosis protein (IAP) family, reduces the susceptibility of tumor cells to proapoptotic stimuli, thereby promoting tumor cell survival during tumor progression and treatment with anticancer drugs. Recently, we identified 2 novel alternative splice variants of survivin, survivin-2B and survivin-Delta Ex3, which differ in their antiapoptotic properties. Survivin-2B has lost its antiapoptotic potential and may act as a naturally occurring antagonist of antiapoptotic survivin and survivin-Delta Ex3. Because the in vivo expression of these splice variants in human cancer has not been analyzed so far, 57 renal cell carcinomas (RCCs) were explored using quantitative reverse transcriptase polymerase chain reaction. We found that all RCCs express survivin-Delta Ex3, survivin-2B and survivin, the latter being the dominant transcript. When we compared early and intermediate stages with late stages of clear cell RCCs, no significant changes in the expression levels of survivin and survivin-Delta Ex3 became evident. However, a significant decrease was observed for the mRNA ratio between survivin-2B and survivin in late tumor stages (p = 0.036). Chromophilic/papillary RCCs, which are known to be less aggressive than clear cell RCCs, did not show significantly lower expression levels of antiapoptotic survivin and survivin-Delta Ex3, compared with stage-adjusted clear cell RCCs. Our study demonstrates for the first time in vivo expression of functionally different survivin variants and suggests a role of these survivin splice variants in the progression and clinical behavior of human RCCs.

Alternative Splicing↗

PubMed2002

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.

Base Sequence↗