Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.     

Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.     

Nitric oxide inactivates glyoxalase I in cooperation with glutathione

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.     

The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.     

Role of S-nitrosothiol transport in the cardioprotective effects of S-nitrosocysteine in rat hearts

The objective of this study was to determine if prior exposure of rat hearts to S-nitrosocysteine (CysNO) was able to provide protection against reperfusion injury. We probed NO release using the extracellular NO scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid transport system L (L-AT), a known transporter of CysNO, using the L-AT competitor, L-leucine (L-Leu). Isolated (9- to 12-week-old Wistar male) rat hearts (six to eight per group) were perfused with CysNO (10 microM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of ischemia. Cardiac function was assessed before, during, and after treatment and during 120 min of reperfusion after ischemia. Functional recovery (rate-pressure product) was significantly improved in the CysNO group compared to hearts in the CysNO+L-Leu group and the control group (p<0.05). Necrosis, measured by triphenyltetrazolium chloride staining, was significantly reduced in CysNO hearts (p<0.05) and this improvement was reversed by L-Leu. The NO scavenger oxyHb (20 microM) was perfused either concomitant with CysNO or just before ischemia. In neither case did oxyHb affect the cardioprotection afforded by CysNO. OxyHb alone, given in either time window, did not alter the course of ischemia-reperfusion injury. When nitrite was used in place of CysNO, no protective effects were observed. Perfusion with CysNO increased tissue S-nitrosothiol (RSNO) levels from an unmeasurable background to a value of about 15.7+/-4.1 pmol RSNO/mg protein, as measured by triiodide-based chemiluminescence in the presence and absence of mercury(II) chloride. In the presence of L-Leu, this value dropped to 0.4+/-0.3 pmol RSNO/mg protein. This study demonstrates that exposure to CysNO before ischemia increases tissue S-nitrosothiol levels, improves postischemic contractile dysfunction, and attenuates necrosis. The mechanism of cardioprotection requires the uptake of CysNO via the L-AT and does not seem to involve NO release either during CysNO exposure or during ischemia. This suggests that the protective effects of CysNO are mediated through the posttranslational modification of cellular proteins through an NO-independent transnitrosation mechanism.     

Pulmonary vasoconstriction by serotonin is inhibited by S-nitrosoglutathione

Nitric oxide (NO) functions as an endothelium-derived relaxing factor by activating guanylate cyclase to increase cGMP levels. However, NO and related species may also regulate vascular tone by cGMP-independent mechanisms. We hypothesized that naturally occurring NO donors could decrease the pulmonary vascular response to serotonin (5-HT) in the intact lung through chemical interactions with 5-HT(2) receptors. In isolated rabbit lung preparations and isolated pulmonary artery (PA) rings, 50-250 microM S-nitrosoglutathione (GSNO) inhibited the response to 0.01-10 microM 5-HT. The vasoconstrictor response to 5-HT was mediated by 5-HT(2) receptors in the lung, since it could be blocked completely by the selective inhibitor ketanserin (10 microM). GSNO inhibited the response to 5-HT by 77% in intact lung and 82% in PA rings. In PA rings, inhibition by GSNO could be reversed by treatment with the thiol reductant dithiothreitol (10 mM). 3-Morpholinosydnonimine (100-500 microM), which releases NO and O simultaneously, also blocked the response to 5-HT. Its chemical effects, however, were distinct from those of GSNO, because 5-HT-mediated vasoconstriction was not restored in isolated rings by dithiothreitol. In the intact lung, neither NO donor altered the vascular response to endothelin, which activates the same second-messenger vasoconstrictor system as 5-HT. These findings, which did not depend on guanylate cyclase, are consistent with chemical modification by NO of the 5-HT(2) G protein-coupled receptor system to inhibit vasoconstriction, possibly by S-nitrosylation of the receptor or a related protein. This study demonstrates that GSNO can regulate vascular tone in the intact lung by a reversible mechanism involving inhibition of the response to 5-HT.     

Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings.

The actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (P(o)) of the calcium release channel at activating calcium concentrations (20-100 microM Ca(2+)) by 60-100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6-2 mM) decreased P(o). Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced P(o) at activating Ca(2+) (20-100 microM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca(2+) (0.6 microM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca(2+). High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca(2+). The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.     

Chloramphenicol inhibition of denitrifying enzyme activity in two agricultural soils.

Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N(2)O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter. We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration. Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays.     

Specific reactions of S-nitrosothiols with cysteine hydrolases: A comparative study between dimethylargininase-1 and CTP synthetase

S-Transnitrosation is an important bioregulatory process whereby NO(+) equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O(-))-S-R' and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-l-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-l-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC(50) value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.     

Inhibitory effects of S-nitrosoglutathione on cell proliferation and DNA synthesis: possible role of glyoxalase I inactivation

We investigated the inhibitory effects of S-nitrosoglutathione (GSNO) on cell proliferation, DNA synthesis and several enzymatic activities using spontaneously immortalized human endothelial cells (ECV304). Proliferation of ECV304 was inhibited by GSNO in a dose-dependent manner (125-1000 microM). DNA synthesis was decreased 2 h after addition of GSNO to cells and was markedly repressed from 20 h after the addition. The activity of ribonucleotide reductase, a rate-limiting enzyme for DNA synthesis, was unchanged in GSNO-treated cells. GSNO inhibited less than 40% of mitochondrial respiration activity, and the membrane potential and cellular levels of ATP were not significantly decreased by GSNO. GSNO had no inhibitory effect on activities of glutathione peroxidase, glutathione S-transferase and glutathione reductase. However, glyoxalase I (Glo I) activity was decreased to 20% of the control level within 60 min, and was consistently repressed during exposure to GSNO for 20 h. A membrane-permeable Glo I inhibitor, S-bromobenzylglutathione diethylester, inhibited proliferation of ECV304 cells, while methylglyoxal (MG), a toxic metabolite generated during glycolysis and a substrate for Glo I, failed to inhibit the cell growth even at 100 microM. Glo I in several mammalian cell lines was inactivated by GSNO with a pI shift. Although we failed to detect accumulation of MG under conditions of Glo I inactivation, these results suggest that the inhibitory effects of GSNO on cell proliferation and DNA synthesis might be at least partly due to inactivation of Glo I.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

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.     

Peroxynitrite inhibits the activity of ornithine decarboxylase

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.     

Cathepsin B is a differentiation-resistant target for nitroxyl (HNO) in THP-1 monocyte/macrophages

We previously showed that the one-electron reduction product of nitric oxide (NO), nitroxyl (HNO), irreversibly inhibits the proteolytic activity of the model cysteine protease papain. This result led us to investigate the differential effects of the nitrogen oxides, such as nitroxyl (HNO), NO, and in situ-generated peroxynitrite on cysteine modification-sensitive cellular proteolytic enzymes. We used Angeli's salt, diethylaminenonoate (DEA/NO), and 3-morpholinosydnoniminehydrochloride (SIN-1), as donors of HNO, NO, and peroxynitrite, respectively. In this study we evaluated their inhibitory activities on the lysosomal mammalian papain homologue cathepsin B and on the cytosolic 26S proteasome in THP-1 monocyte/macrophages after LPS activation or TPA differentiation. HNO-generating Angeli's salt caused a concentration-dependent (62 +/- 4% at 316 muM) inhibition of the 26S proteasome activity, resulting in accumulation of protein-bound polyubiquitinylated proteins in LPS-activated cells, whereas neither DEA/NO nor SIN-1 showed any effect. Angeli's salt, but not DEA/NO or SIN-1, also caused (94 +/- 2% at 316 muM) inhibition of lysosomal cathepsin B activity in LPS-activated cells. Induction of macrophage differentiation did not significantly alter the inhibitory effect of HNO on lysosomal cathepsin B activity, but protected the proteasome from HNO-induced inhibition. The protection awarded by macrophage differentiation was associated with induction of the GSH synthesis rate-limiting enzyme gamma-glutamylcysteine synthetase, as well as with increased intracellular GSH. In conclusion, HNO abrogates both lysosomal and cytosolic proteolysis in THP-1 cells. Macrophage differentiation, associated with upregulation of antioxidant defenses such as increased cellular GSH, does not protect the lysosomal cysteine protease cathepsin B from inhibition.     

O(2)-dependent stimulation of the pentose phosphate pathway by S-nitrosocysteine in human erythrocytes

In the present study we analysed the effects of S-nitrosocysteine (CysNO) on adult human red blood cell metabolism and observed that metabolic response depended on the degree of cell oxygenation. In particular, glucose metabolised through the pentose phosphate pathway (PPP) was higher in treated erythrocytes than in untreated cells only at high O(2) pressure. Since, following the treatment of intact cells with CysNO, glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase (PFK) activities did not evidence any significant alteration, the possibility that the stimulation of PPP was triggered by a CysNO mediated modification of these enzymes was excluded. Intracellular S-nitrosoglutathione (GSNO), detected only in treated red blood cells, may be linked solely to the exposition to the NO donor. A possible rationalisation of the different metabolic behaviour shown by erythrocytes as a function of their oxygenation state is proposed. It takes into account the different route of catabolic degradation observed in vitro for GSNO under aerobic and anaerobic condition.     

Metabolic regulation of aldose reductase activity by nitric oxide donors

Regulation of aldose reductase (AR), a member of the aldo-keto reductase superfamily, by nitric oxide (NO) donors was examined. Incubation of human recombinant AR with S-nitrosoglutathione (GSNO) led to inactivation of the enzyme and the formation of an AR-glutathione adduct. In contrast, incubation with S-nitroso-N-acetyl penicillamine (SNAP) or N-(beta-D-glucopyranosyl)-SNAP (GlycoSNAP) led to an increase in enzyme activity which was accompanied by the direct nitrosation of the enzyme and the formation of a mixed disulfide with the NO-donor. To examine in vivo modification, red blood cells (RBC) and rat aortic vascular smooth muscle cells (VSMC) were incubated with 1 mM GSNO or SNAP. Exposure of VSMC to SNAP and GSNO for 2 h at 37 degrees C led to approximately 71% decrease in the enzyme activity with DL-glyceraldehyde as the substrate. Similarly, exposure of RBC in 5 mM glucose to NO-donors for 30 min at room temperature, followed by increasing the glucose concentration to 40 mM, resulted in >75% decrease in the formation of sorbitol. These investigations indicate that NO and/or its bioactive metabolites can regulate cellular AR, leading to either activation (by nitrosation) or inactivation (by S-thiolation).     

Persistent susceptibility of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the presence of endogenous nitric oxide

Nitrosation of enzyme regulatory cysteines is one of the key posttranslational modification mechanisms of enzyme function. Frequently such modifications are readily reversible; however, cysteine proteases, such as cathepsin B, have been shown to be covalently and permanently inactivated by nitroxyl (HNO), the one-electron reduction product of NO. Owing to the high reactivity of HNO with NO, endogenous NO production could provide direct protection for the less reactive protein cysteines by scavenging HNO. Additionally, endogenous cellular production of NO could rescue enzyme function by protective nitrosation of cysteines prior to exposure to HNO. Thus, we studied the effect of endogenous NO production, induced by LPS or IFN-gamma, on inhibition of cysteine protease cathepsin B in RAW macrophages. Both LPS and IFN-gamma induce iNOS with generation of nitrate up to 9 muM in the media after a 24-h stimulation, while native RAW 264.7 macrophages neither express iNOS nor generate nitrate. After the 24-h stimulation, the HNO-releasing Angeli's salt (0-316 microM) caused dose-dependent and DTT-irreversible loss of cathepsin B activity, and induction of iNOS activity did not protect the enzyme. The lack of protection was also verified in an in vitro setup, where papain, a close structural analogue of cathepsin B, was inhibited by Angeli's salt (2.7 microM) in the presence of the NO donor DEA/NO (0-316 microM). This clearly showed that a high molar excess of DEA/NO (EC(50) 406 microM) is needed to protect papain from the DTT-irreversible covalent modification by HNO. Our results provide first evidence on a cellular level for the remarkably high sensitivity of active-site cysteines in cysteine proteases for modification by HNO.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

Nitric oxide activates the expression of IRAK-M via the release of TNF-α in human monocytes

The activation of interleukin receptor associated kinases (IRAK) is an important event in several inflammatory processes. However, exposing monocytes to a nitric oxide (NO) donor inhibits the activity of IRAK-1 and its molecular interaction with TNF receptor associated factor-6 (TRAF6). Despite the fact that NO is known to regulate many events in the immune and vascular system, the mechanism that underlies this inhibition remains unknown. We have recently demonstrated that IRAK-M inhibits the TLR/IRAK pathway during endotoxin tolerance and thus, we hypothesized that IRAK-M may be involved in the inhibition of IRAK-1 activity in the presence of NO. Hence, we have analyzed the expression of IRAK-M in human monocytes following exposure to a NO donor (GSNO) and we have observed that GSNO was capable of inducing IRAK-M mRNA and protein expression 8 and 20 h after stimulation, respectively. It is known that NO induces the expression of TNF-alpha in monocytes and we found that exposure to TNF-alpha induced IRAK-M mRNA expression in human monocytes within 2 h of stimulation. Furthermore, the expression of IRAK-M induced by GSNO was inhibited by the presence of a blocking antibody raised against TNF-alpha. Thus, our data indicate that stimulation of human monocytes with a NO donor results in a clear induction of IRAK-M and this is dependent on the release of TNF-alpha by this kind of cells.     

Role of mitogen-activated protein kinases in S-nitrosoglutathione-induced macrophage apoptosis

The inflammatory mediator nitric oxide (NO*) promotes apoptotic cell death based on morphological evidence, accumulation of the tumor suppressor p53, caspase-3 activation, and DNA fragmentation in RAW 264.7 macrophages. Since nitrosothiols may actually be the predominant form of biologically active NO* in vivo, we used S-nitrosoglutathione (GSNO) to study activation of extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and p38 kinases. Moreover, we determined the role of mitogen-activated protein kinase signaling in the apoptotic transducing ability of GSNO. ERK1/2 became activated in response to GSNO after 4 h and remained active for the next 20 h. Blocking the ERK1/2 pathway by the mitogen-activated protein kinase kinase inhibitor PD 98059 enhanced GSNO-elicited apoptosis. p38 was activated as well, but inhibition of p38 with SB 203580 left apoptosis unaltered. Activation of JNK1/2 by GSNO showed maximal kinase activities between 2 and 8 h. Attenuating JNK1/2 by antisense-depletion eliminated the pro-apoptotic action of low GSNO concentrations (250 microM), whereas apoptosis proceeded independently of JNK1/2 at higher doses of the NO donor (500 microM). Decreased apoptosis by JNK1/2 depletion prevented p53 accumulation after the addition of GSNO, which positions JNK1/2 upstream of the p53 response at low agonist concentrations. In line, JNK1/2 activation proceeded unaltered in p53-antisense transfected macrophages. However, with higher GSNO concentrations apoptotic transducing pathways, including p53 accumulation, were JNK1/2 unrelated. The regulation of mitogen-activated protein kinases by GSNO may help to define cell protective and destructive actions of reactive nitrogen species.