Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase

We used a variety of nitric oxide (NO) donors to demonstrate that NO inhibits the activities of tobacco catalase and ascorbate peroxidase (APX). This inhibition appears to be reversible because removal of the NO donor led to a significant recovery of enzymatic activity. In contrast, APX and catalase were irreversibly inhibited by peroxynitrite. The ability of NO and peroxynitrite to inhibit the two major H2O2-scavenging enzymes in plant cells suggests that NO may participate in redox signaling during the activation of defense responses following pathogen attack.     

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes.

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.     

Immortalization of human melanocytes does not alter the de novo properties of nitric oxide to induce cell detachment from extracellular matrix components via cGMP

Nitric oxide (NO) is an important mediator in many (patho)physiological processes including inflammation and skin cancer. A key transducer in NO signaling is the soluble guanylyl cyclase (sGC) that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP). The basic mechanism of NO-cGMP signaling in melanocytic cells is, however, not well elucidated. A setback for such studies is the limited availability of patient-derived melanocytes. Here, we report that immortalized human normal and vitiliginous cell lines generated via cell transfection with human papilloma virus 16 genes E6 and E7 express NO synthase and guanylyl cyclase isoforms and the multidrug resistance-associated proteins 4 and 5 as selective cGMP exporters. Donors of NO (e.g., the NONOate (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NO) and reactive nitrogen oxygen species (RNOS) like 3-morpholino-sydnonimine (SIN-1) as a donor of peroxynitrite as well as YC-1 as a NO-independent sGC stimulator increased intracellular cGMP levels in immortalized melanocytes (up to eightfold over controls), indicating the expression of functional sGC in these cells. PAPA-NO and SIN-1 also reduced the attachment of immortalized melanocytes to extracellular matrix (ECM) components like fibronectin which was dependent on cellular melanin content and cGMP. Such effects on melanoma cells were positively related to metastatic potential and were cGMP independent. Intriguingly, nonpigmented metastatic melanoma cells were more sensitive to exogenous sources of RNOS than of NO. Thus, immortalized melanocytes can be used as a tool for further research on differences in cell signaling between the different melanocytic lineages in particular towards impairment of cell-ECM adhesion by NO or RNOS, which may be important in metastasis and vitiligo pathogenesis.     

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.     

2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO(2)(.) and was used as a tool to investigate NO(2)(.) potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline ((14)C-N-NO-IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 microM NO/min) for 7.5 min, did not convert 10 microM (14)C-IQ to N-NO-IQ. However, the presence of 15 muM CPTIO resulted in 3 microM N-NO-IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 microM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N-NO-IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO(2)(.) or a RNOS like NO(2)(.) potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.     

Muscular nitric oxide synthase (muNOS) and utrophin.

Duchenne muscular dystrophy (DMD), the severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. Three types of treatment are envisaged: pharmacological (glucocorticoid), myoblast transplantation, and gene therapy. An alternative to the pharmacological approach is to compensate for dystrophin loss by the upregulation of another cytoskeletal protein, utrophin. Utrophin and dystrophin are part of a complex of proteins and glycoproteins, which links the basal lamina to the cytoskeleton, thus ensuring the stability of the muscle membrane. One protein of the complex, syntrophin, is associated with a muscular isoform of the neuronal nitric oxide synthase (nNOS). We have demonstrated an overexpression of utrophin, visualised by immunofluorescence and quantified by Western blotting, in normal myotubes and in mdx (the animal model of DMD) myotubes, as in normal (C57) and mdx mice, both treated with nitric oxide (NO) donor or L-arginine, the NOS substrate. There is evidence that utrophin may be capable of performing the same cellular functions as dystrophin and may functionally compensate for its lack. Thus, we propose to use NO donors, as palliative treatment of Duchenne and Becker muscular dystrophies, pending, or in combination with, gene and/or cellular therapy. Discussion has focussed on the various isoforms of NOS that could be implicated in the regeneration process. Dystrophic and healthy muscles respond to treatment, suggesting that although NOS is delocalised in the cytoplasm in the case of DMD, it conserves substantial activity. eNOS present in mitochondria and iNOS present in cytoplasm and the neuromuscular junction could also be activated. Lastly, production of NO by endothelial NOS of the capillaries would also be beneficial through increased supply of metabolites and oxygen to the muscles.     

Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide.

BACKGROUND: Excitotoxicity and excess generation of nitric oxide (NO) are believed to be fundamental mechanisms in many acute and chronic neurodegenerative disorders. Disturbance of Ca2+ homeostasis and protein nitration/nitrosylation are key features in such conditions. Recently, a family of proteases collectively known as caspases has been implicated as common executor of a variety of death signals. In addition, overactivation of poly-(ADP-ribose) polymerase (PARP) has been observed in neuronal excitotoxicity. We therefore designed this study to investigate whether triggering of caspase activity and/or activation of PARP played a role in cerebellar granule cell (CGC) apoptosis elicited by peroxynitrite (ONOO-) or NO donors. MATERIALS AND METHODS: CGC from wild-type or PARP -/- mice were exposed to various nitric oxide donors. Caspase activation and its implications for membrane alterations, Ca2+ homeostasis, intracellular proteolysis, chromatin degradation, and cell death were investigated. RESULTS: CGC exposed to NO donors undergo apoptosis, which is mediated by excess synaptic release of excitotoxic mediators. This excitotoxic mechanism differs from direct NO toxicity in some other neuronal populations and does not involve PARP activation. Inhibition of caspases with different peptide substrates prevented cell death and the related features, including intracellular proteolysis, chromatin breakdown, and translocation of phosphatidylserine to the outer surface of the cell membrane. Increased Ca2+ influx following N-methyl-D-aspartate (NMDA) receptor (NMDA-R) activation was not inhibited by caspase inhibitors. CONCLUSIONS: In CGC, NO donors elicit apoptosis by a mechanism involving excitotoxic mediators, Ca2+ overload, and subsequent activation of caspases.     

Nitric oxide inhibits mammalian methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (.CH(2)-Ado), both of which are generated from the co-factor of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (> or =600 microm) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and .CH(2)-Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and .CH(2)-Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.     

Cell type-dependent release of nitric oxide and/or reactive nitrogenoxide species from intracellular SIN-1: effects on cellular NAD(P)H

SIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (*NO) as monitored by the fluorescent *NO scavenger FNOCT-1 and by means of a *NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1 -derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for *NO formation was found although the intracellular amount of SIN-1 was four-fold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing *NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.     

Intra-mitochondrial poly(ADP-ribosyl)ation: Potential role for alpha-ketoglutarate dehydrogenase

Poly(ADP-ribose) polymerase (PARP) is an intracellular enzyme involved in DNA repair and in building poly-ADP-ribose polymers on nuclear proteins using NAD⁺. While the majority of PARP resides in the nucleus, several studies indicated that PARP may also be located in the cytosol or in the mitochondrial matrix. In this study we found several poly-ADP-ribosylated proteins in isolated rat liver mitochondria following hydrogen peroxide (H₂O₂) or nitric oxide donor treatment. Protein poly-ADP-ribosylation was more intense in isolated mitochondria than in whole tissue homogenates and it was not associated with increased nuclear PARP activity. We identified five poly-ADP-ribose (PAR) positive mitochondrial bands by protein mass fingerprinting. All of the identified enzymes exhibited decreased activity or decreased levels following oxidative or nitrosative stress. One of the identified proteins is dihydrolipoamide dehydrogenase (DLDH), a component of the alpha-ketoglutarate dehydrogenase (KGDH) complex, which uses NAD⁺ as a substrate. This raised the possibility that KGDH may have a PARP-like enzymatic activity. The intrinsic PARP activity of KGDH and DLDH was confirmed using a colorimetric PARP assay kit and by the incubation of the recombinant enzymes with H₂O₂. The KGDH enzyme may, therefore, have a novel function as a PARP-like enzyme, which may play a role in regulating intramitochondrial NAD⁺ and poly(ADP-ribose) homeostasis, with possible roles in physiology and pathophysiology.     

Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.     

Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.     

Nitric oxide evolution and perception

Various experimental data indicate signalling roles for nitric oxide (NO) in processes such as xylogenesis, programmed cell death, pathogen defence, flowering, stomatal closure, and gravitropism. However, it still remains unclear how NO is synthesized. Nitric oxide synthase-like activity has been measured in various plant extracts, NO can be generated from nitrite via nitrate reductase and other mechanisms of NO generation are also likely to exist. NO removal mechanisms, for example, by reaction with haemoglobins, have also been identified. NO is a gas emitted by plants, with the rate of evolution increasing under conditions such as pathogen challenge or hypoxia. However, exactly how NO evolution relates to its bioactivity in planta remains to be established. NO has both aqueous and lipid solubility, but is relatively reactive and easily oxidized to other nitrogen oxides. It reacts with superoxide to form peroxynitrite, with other cellular components such as transition metals and haem-containing proteins and with thiol groups to form S-nitrosothiols. Thus, diffusion of NO within the plant may be relatively restricted and there might exist 'NO hot-spots' depending on the sites of NO generation and the local biochemical micro-environment. Alternatively, it is possible that NO is transported as chemical precursors such as nitrite or as nitrosothiols that might function as NO reservoirs. Cellular perception of NO may occur through its reaction with biologically active molecules that could function as 'NO-sensors'. These might include either haem-containing proteins such as guanylyl cyclase which generates the second messenger cGMP or other proteins containing exposed reactive thiol groups. Protein S-nitrosylation alters protein conformation, is reversible and thus, is likely to be of biological significance.     

Nitric oxide selectively inhibits adenylyl cyclase isoforms 5 and 6

We have previously shown that N18TG2 neuroblastoma cells express the type 6 adenylyl cyclase and that preincubation with nitric oxide (NO) attenuates Gs- and forskolin-stimulated activity. Here we show that this inhibition reflects a direct action of NO on the adenylyl cyclase. Preincubation of N18TG2 cell membranes and insect cell membranes expressing recombinant type 5 and type 6 isoforms with NO donors leads to an inhibition of forskolin-stimulated adenylyl cyclase activity. NO donors do not alter the type 1 (representative of the type 1,3,8 family) or type 2 (representative of the type 2,4, 7 family) isoforms expressed in insect cells, even under conditions of compromised assay conditions or a range of temperatures. Thus, the ability of NO to inhibit adenylyl cyclase stimulation is dependent upon the nature of the isoform present, and appears to represent a unique regulation of the type 5,6 isoform family.     

Role of nitric oxide in liver injury

The complex role of nitric oxide (NO) in the liver can be explained by its patterns of regulation and unique biochemical properties. With a broad range of direct and indirect molecular targets, NO acts as an inhibitor or agonist of cell signaling events. In the liver, constitutively generated NO maintains the hepatic microcirculation and endothelial integrity, while inducible NO synthase (iNOS)-governed NO production can be either beneficial or detrimental. For instance, NO potentiates the hepatic oxidative injury in warm ischemia/reperfusion, while iNOS expression protects against hepatic apoptotic cell death seen in models of sepsis and hepatitis. Anti-apoptotic actions are either cyclic nucleotide dependent or independent, including the expression of heat shock proteins, prevention of mitochondrial dysfunction, and inhibition of caspase activity by S-nitrosation. Whether NO protects or injures is probably determined by the type of insult, the abundance of reactive oxygen species (ROS), the source and amount of NO production and the cellular redox status of liver. Through the use of pharmacological NO donors or NOS gene transfer in conjunction with genetically altered knockout animals, the physiological and pathophysiological roles of NO in liver function can be explored in more detail. The purpose of this paper is to review the current understanding of the role of NO in liver injury.     

Nitric oxide is involved in abscisic acid-induced antioxidant activities in Stylosanthes guianensis

Previous studies suggest that abscisic acid (ABA) stimulates the activities of antioxidant enzymes under normal and chilling temperature and enhanced chilling resistance in Stylosanthes guianensis. The objective of this study was to test whether nitric oxide (NO) is involved in the ABA-induced activities of the antioxidant enzymes in Stylosanthes guianensis due to its nature as a second messenger in stress responses. Plants were treated with NO donors, ABA, ABA in combination with NO scavengers or the nitric oxide synthase (NOS) inhibitor and their effects on the activity of antioxidant enzymes and NO production were compared. The results showed that ABA increased the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The effect of ABA on antioxidant enzyme activities was suppressed by the NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA), and the NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (PTIO). NO content increased after 5 h of ABA treatment. The NO-scavenger, PTIO, and the NOS-inhibitor, L-NNA, inhibited the accumulation of NO in ABA-treated Stylosanthes guianensis. NO donor treatment enhanced the activities of SOD, CAT, and APX. The results suggested that NO was involved in the ABA-induced activities of SOD, CAT, and APX in Stylosanthes guianensis. ABA triggered NO production that may lead to the stimulation of antioxidant enzyme activities.     

Neuronal nitric oxide synthase (nNOS) modulates the JNK1 activity through redox mechanism: a cGMP independent pathway

Nitric oxide (NO) is a small, uncharged molecule, which is primarily generated by the nitric oxide synthase (NOS) family of proteins, including neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO has been implicated in diverse roles in biological systems, such as the regulation of cell death and survival signaling pathways of a variety of cell types, including neuronal cells. In this study, we determined that the NO generated from l-arginine by ectopically overexpressed nNOS in HEK293 cells exerted an inhibitory effect against the activity of c-Jun N-terminal kinase (JNK), an important modulator of neuronal cell death and survival signaling pathways. NO repressed the activation of JNK, but exerted no significant effects on the activities of SEK1/MKK4 and MEKK1, which are the upstream MAPKK and MAPKKK of JNK1, respectively. This NO-mediated inhibition of JNK1 was not affected by the addition of ODQ, a guanylyl cyclase inhibitor, indicating that the effect is independent of the level of cyclic GMP. In an in vitro kinase assay, SNAP, a NO donor, was shown to directly suppress JNK1 activity, thereby indicating that NO is a direct modulator of JNK1. Moreover, the NO-mediated suppression of JNK1 was demonstrated to be redox-sensitive and dependent on the cysteine-116 in JNK1. Finally, according to the results of an immunohistochemical study using rat striatal neurons, we were able to determine that nNOS-expressing neurons evidenced significantly reduced JNK1 activation. Collectively, these data suggest that JNK1 is regulated by nNOS-mediated NO production in neurons, via a thiol-redox-sensitive mechanism.     

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.