Astrid Schrammel

S-nitrosation of glutathione by nitric oxide, peroxynitrite, and •NO/O2•−

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.

Cardiac dysfunction in adipose triglyceride lipase deficiency: treatment with a PPARα agonist.

Adipose triglyceride lipase (ATGL) has been identified as a rate‐limiting enzyme of mammalian triglyceride catabolism. Deletion of the ATGL gene in mice results in severe lipid accumulation in a variety of tissues including the heart. In the present study we investigated cardiac function in ATGL‐deficient mice and the potential therapeutic effects of the PPARα and γ agonists Wy14,643 and rosiglitazone, respectively.

Efficient nitrosation of glutathione by nitric oxide

Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO2 or N2O3) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg2+. These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.

Inhibition of purified soluble guanylyl cyclase by l-ascorbic acid

OBJECTIVE L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide (NO)/cGMP signalling. The aim of the present study was to investigate the effect of vitamin C on the most prominent physiological target of endogenous and exogenous NO, i.e. soluble guanylyl cyclase (sGC). METHODS To address this issue we used a highly purified enzyme preparation from bovine lung (from the slaughterhouse). Enzymic activity was measured by a standard assay based on the conversion of [alpha-32P]GTP to [32P]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified using a commercially available Clark-type electrode. RESULTS Stimulation of sGC by the NO donor 2, 2-diethyl-1-nitroso-oxyhydrazine was inhibited by ascorbate with an IC(50) of approximately 2 microM. Maximal enzyme inhibition ( approximately 70%) was observed at 0.1-1 mM vitamin C. Stimulation of sGC by the NO-independent activator protoporphyrin-IX was also inhibited with similar potency. The effect of ascorbate on sGC was largely antagonised by reduced glutathione (1 mM) and the specific iron chelator diethylenetriaminepentaacetic acid (0.1 mM). Electrochemical experiments revealed that NO is potently scavenged by vitamin C. Consumption of NO by ascorbate was prevented by reduced glutathione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide dismutase (500 units/ml) whereas up to 5000 units/ml superoxide dismutase failed to restore sGC activity. CONCLUSIONS Our results suggest that physiological concentrations of L-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC.

ISOFORM-SPECIFIC EFFECTS OF SALTS ON NITRIC OXIDE SYNTHASE ACTIVITY

We investigated the effects of salts on the properties of the neuronal, endothelial, and inducible isoforms of nitric oxide synthase (nNOS, eNOS, and iNOS), and found pronounced isoform-specific effects on NOS-catalyzed L-citrulline formation. Salts inhibited iNOS monotonously, whereas nNOS and eNOS were stimulated up to 3-fold at low, and inhibited at high (>/=0.1-0.2 M) salt concentrations. The effectivities of different ions mostly followed the Hofmeister series, indicating that the effects can for a large part be ascribed to changes in protein solvation. Km(Arg) increased in the presence of NaCl, demonstrating the importance of charge interactions for substrate binding. The coupling of NADPH oxidation to NO production was not affected by KCl. Salts (</=1 M) had no major impact on the tertiary and quaternary structure, or on the state of the heme. Extrapolation of these results to commonly applied experimental conditions for in vitro activity assays suggests that true specific activities of nNOS and eNOS may, in some cases, be underestimated as much as 3-fold.

Cardiac oxidative stress in a mouse model of neutral lipid storage disease.

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.

Nitric oxide-induced autoinhibition of neuronal nitric oxide synthase in the presence of the autoxidation-resistant pteridine 5-methyltetrahydrobiopterin.

Nitric oxide synthase (NOS) catalysis results in formation of NO or superoxide (O(2)(-.)) depending on the presence or absence of the cofactor tetrahydrobiopterin (BH4). In the absence of O(2)(-.) scavengers, net NO formation cannot be detected even at saturating BH4 concentrations, which is thought to be due to O(2)(-.) production by BH4 autoxidation. Because the N-5-methylated analogue of BH4 (5-Me-BH4) sustains NOS catalysis and is autoxidation-resistant, net NO formation by the neuronal isoform of NOS (nNOS) can be observed at saturating 5-Me-BH4 concentrations. Here we compare the effects of 5-Me-BH4 on L-citrulline formation, NADPH oxidation, H(2)O(2) production and soluble guanylate cyclase (sGC) stimulation. All activities were stimulated biphasically (EC(50) approx. 0.2 microM and more than 1 mM), with an intermediate inhibitory phase at the same pterin concentration as that required for net NO generation and sGC stimulation (4 microM). Concomitantly with inhibition, the NADP(+)/L-citrulline stoichiometry decreased from 2.0 to 1.6. Inhibition occurred only at high enzyme concentrations (IC(50) approx. 10 nM nNOS) and was antagonized by oxyhaemoglobin and by BH4. We ascribe the first stimulatory phase to high-affinity binding of 5-Me-BH4. The inhibitory phase is due to low-affinity binding, resulting in fully coupled catalysis, complete inhibition of O(2)(-.) production and net NO formation. At high enzyme concentrations and thus high NO levels, this causes autoinhibition. NO scavenging by 5-Me-BH4 at concentrations above 1 mM, resulting in the antagonization of inhibition of NOS, explains the second stimulatory phase. In agreement with these assignments 5-Me-BH4 was found to stimulate formation of a haem-NO complex during NOS catalysis. The observation of inhibition with 5-Me-BH4 but not with BH4 implies that, unless O(2)(-.) scavengers are present, a physiological role for NO-induced autoinhibition is unlikely.

Endothelial dysfunction in adipose triglyceride lipase deficiency

Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~ 50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.

Intact mitochondrial Ca(2+) uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS).

Abstract Mitochondrial Ca2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO•) production. However, it is not entirely clear if the organelles support or counteract NO• biosynthesis by taking up Ca2+. The objective of this study was to verify whether or not mitochondrial Ca2+ uptake influences Ca2+‐triggered NO• generation by endothelial NO• synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS‐RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO• probes, the geNOps, and Ca2+ sensors to monitor single cell NO• and Ca2+ dynamics upon cell treatment with ATP, an inositol 1,4,5‐trisphosphate (IP3)‐generating agonist. Mitochondrial Ca2+ uptake was specifically manipulated by siRNA‐mediated knock‐down of recently identified key components of the mitochondrial Ca2+ uniporter machinery. In endothelial cells and the eNOS‐RFP expressing HEK293 cells we show that reduced mitochondrial Ca2+ uptake upon the knock‐down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca2+‐triggered NO• increase independently of global cytosolic Ca2+ signals. The knock‐down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca2+ sequestration and Ca2+‐induced NO• signals. The positive correlation between mitochondrial Ca2+ elevation and NO• production was independent of eNOS phosphorylation at serine1177. Our findings emphasize that manipulating mitochondrial Ca2+ uptake may represent a novel strategy to control eNOS‐mediated NO• production. Graphical abstract Figure. No Caption available. HighlightsgeNOps allow real‐time imaging of eNOS‐mediated NO• formation in single cells.Impairment of mitochondrial Ca2+ uptake reduces NO• synthesis by eNOS.Increased mitochondrial Ca2+ uptake facilitates Ca2+‐triggered NO• formation.Mitochondrial Ca2+ uptake does not affect eNOS phosphorylation.The link between mitochondria and eNOS activity remains unidentified.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.