Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2*- production in the intact coronary endothelium

The present study tested the hypothesis that endostatin stimulates superoxide (O2*-) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]i was unchanged. With the conversion rate of L-[3H]arginine to L-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2*-. production was enhanced in endothelial cells. O2*-. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2*-. production through activation of NAD(P)H oxidase. This ceramide-O2*-. signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.     

L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen.

Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water.     

Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

Effect of olive oil minor components on oxidative stress and arachidonic acid mobilization and metabolism by macrophages RAW 264.7

Minor components of virgin olive oil may explain the healthy effects of the Mediterranean diet on the cardiovascular system and cancer development. The uncontrolled production of reactive oxygen species (ROS) and arachidonic acid (AA) metabolites contributes to the pathogenesis of cardiovascular disease and cancer, and inflammatory cells infiltrated in the atheroma plaque or tumor are a major source of ROS and eicosanoids. We aimed to determine the effects of squalene, beta-sitosterol, and tyrosol, which are representative of the hydrocarbons, sterols, and polyphenols of olive oil, respectively, on superoxide anion (O2(-)), hydrogen peroxide (H2O2), and nitric oxide (*NO) levels. We also studied AA release and eicosanoid production by phorbol esters (PMA)-stimulated macrophages RAW 264.7. beta-Sitosterol and tyrosol decreased the O2(-) and H2O2 production induced by PMA, and tyrosol scavenged the O2(-) released by a ROS generating system. These effects were correlated with the impairment of [3H]AA release, cyclooxygenase-2 (COX-2) expression, and prostaglandin E(2)/leukotriene B(4) synthesis in RAW 264.7 cultures stimulated by PMA. beta-Sitosterol exerted its effects after 3-6 h of preincubation. Tyrosol inhibited the [3H]AA release induced by exogenous ROS. beta-Sitosterol and tyrosol also reduced the *NO release induced by PMA, which was correlated with the impairment of inducible nitric oxide synthase (iNOS) levels. This may be correlated with the modulation of NF-kappaB activation. Further studies are required to gain more insight into the potential healthy effects of minor components of extra virgin olive oil.     

Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production

The synthesis of nitric oxide (NO) is limited by the intracellular availability of L-arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular L-arginine which supports an increase in the production of NO. Although L-[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin-layer chromatography analysis showed that free intracellular L-[3H]arginine was six times higher in CHX-treated than in control cultures. Extracellular L-[3H]citrulline levels increased threefold in CHX-treated groups, an effect blocked by NG-nitro-L-arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular L-[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2-amino 5-phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40-50% in the incorporation of 35[S]methionine or L-[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular L-arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin-dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin-dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.     

Superoxide activates constitutive nitric oxide synthase in a brain particulate fraction

Nitric oxide (*NO) can act as an antioxidant by directly scavenging reactive free radicals, inhibiting the oxidative chemistry of iron, and signaling the up-regulation of antioxidant enzymes. However, the cellular utility of *NO as an antioxidant requires that constitutive nitric oxide synthase (NOS) be activated rapidly by a signal(s) for oxidant formation. We report here that superoxide (O2*-), added directly as potassium superoxide (KO2), produced a superoxide dismutase-sensitive and hydrogen peroxide-independent stimulation of NOS activity, measured by the conversion of [3H]arginine to [3H]citrulline and nitrite formation, in a synaptic particulate fraction from rat brain cerebral cortex. O2*- produced maximal activation of NOS in the presence of the antioxidant urate and ATP. Stimulation of NOS activity by O2*- was abolished by N-monomethyl-L-arginine and by the Ca2+ chelator EGTA but not by 7-nitroindazole, which would be expected to inhibit neuronal NOS. We propose that limited activation of NOS by O2*- may be an important contributor to brain oxidant defenses and, more generally, a signal for cellular adaptation and survival, although excessive generation of nitrogen oxides would be expected to produce neurotoxicity.     

Time course of nitric oxide production after endotoxin challenge in mice

Nitric oxide (NO) regulates numerous processes during endotoxemia and inflammation. However, the sequential changes in whole body (Wb) nitric oxide (NO) production during endotoxemia in vivo remain to be clarified. Male Swiss mice were injected intraperitoneally with saline (control group) or lipopolysaccharide (LPS group). After 0, 2, 4, 6, 9, 12, and 24 h, animals received a primed constant infusion of L-[guanidino-(15)N(2)-(2)H(2)]arginine, L-[ureido-(15)N]citrulline, L-[5-(15)N]glutamine, and L-[ring-(2)H(5)]phenylalanine in the jugular vein. Arterial blood was collected for plasma arginine (Arg), citrulline (Cit), glutamine (Gln), and phenylalanine (Phe) concentrations and tracer-to-tracee ratios. NO production was calculated as plasma Arg-to-Cit flux, Wb de novo Arg synthesis as plasma Cit-to-Arg flux, and Wb protein breakdown as plasma Phe flux. LPS reduced plasma Arg and Cit and increased Gln and Phe concentrations. Two peaks of NO production were observed at 4 and 12 h after LPS. Although LPS did not affect total Arg production, de novo Arg production decreased after 12 h. The second peak of NO production coincided with increased Wb Cit, Gln, and Phe production. In conclusion, the curve of NO production in both early and late phases of endotoxemia is not related to plasma Arg kinetics. However, because Wb Cit, Gln, and Phe fluxes increased concomitantly with the second peak of NO production, NO production is probably related to the catabolic phase of endotoxemia.     

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.     

Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.     

Effects of homocysteine on intracellular nitric oxide and superoxide levels in the renal arterial endothelium

The present study was designed to test the hypothesis that homocysteine (Hcys) reduces intracellular nitric oxide (NO) concentrations ([NO](i)) and stimulates superoxide (O.) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. With the use of fluorescence microscopic imaging analysis, a calcium ionophore, A-23187 (2 microM), and bradykinin (2 microM) were found to increase endothelial [NO](i) in freshly dissected lumen-opened small renal arteries loaded with 4,5-diaminofluorescein diacetate (DAF-2DA; 10 microM). Preincubation of the arteries with L-Hcys (20-40 microM) significantly attenuated the increase in endothelial [NO](i). However, L-Hcys had no effect on NO synthase activity in the renal arteries, as measured by the conversion rate of [(3)H]arginine to [(3)H]citrulline, but it concentration dependently decreased DAF-2DA-sensitive fluorescence induced by PAPA-NONOate in the solution, suggesting that L-Hcys reduces endothelial [NO](i) by its scavenging action. Because other thiol compounds such as L-cysteine and glutathione were also found to reduce [NO](i), it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hyperhomocysteinemia (hHcys). By analysis of intracellular O. levels using dihydroethidium trapping, we found that only L-Hcys among the thiol compounds studied markedly increased O. levels in the renal endothelium. These results indicate that L-Hcys inhibits the agonist-induced NO increase but stimulates O. production within endothelial cells. These effects of L-Hcys on [NO](i) and [O.] may contribute to endothelial injury associated with hHcys.     

Hydrogen peroxide induces nitric oxide and proteosome activity in endothelial cells: a bell-shaped signaling response

We investigated nitric oxide (*NO)-mediated proteosomal activation in bovine aortic endothelial cells (BAEC) treated with varying fluxes of hydrogen peroxide (H(2)O(2)) generated from glucose/glucose oxidase (Glu/GO). Results revealed a bell-shaped *NO signaling response in BAEC treated with Glu/GO (2-20 mU/ml). GO treatment (2 mU/ml) enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and *NO release in BAEC. With increasing GO concentrations, phospho eNOS and *NO levels decreased. Bell-shaped responses in proteasomal function and *NO induction were observed in BAEC treated with varying levels of GO (2-10 mU/ml). Proteosomal activation induced in GO-treated BAEC was inhibited by N(omega)-nitro-L-arginine-methyl ester pretreatment, suggesting that *NO mediates proteasomal activation. Intracellular *NO induced by H(2)O(2) was detected by isolating the 4,5-diaminoflourescein (DAF-2)/*NO/O(2)-derived "green fluorescent product" using the high-performance liquid chromatography-fluorescence technique, a more rigorous and quantitative methodology for detecting the DAF-2/*NO/O(2) reaction product. Finally, the relationships between H(2)O(2) flux, proteasomal activation/inactivation, endothelial cell survival, and apoptosis are discussed.     

Nitric oxide involvement in Drosophila immunity.

The augmented production of nitric oxide (NO) was observed during the hemocyte-mediated melanotic encapsulation responses of Drosophila melanogaster and D. teissieri. When introduced into the hemocoel of D. melanogaster larvae, NO activated the gene encoding the antimicrobial peptide Diptericin. These observations, together with previous studies documenting the production of superoxide anion (O(*-)(2)) and H(2)O(2) in immune-challenged Drosophila, provide evidence that reactive intermediates of both oxygen (ROI) and nitrogen (RNI) constitute a part of the cytotoxic arsenal employed by Drosophila in defense against both microbial pathogens and eukaryotic parasites. These ROI and RNI appear to represent an evolutionarily conserved innate immune response that is mediated by regulatory proteins that are homologous to those of mammalian species.     

L-Arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs.

Previously, our laboratory found that pulmonary hypertension developed and lung nitric oxide (NO) production was reduced when piglets were exposed to chronic hypoxia (Fike CD, Kaplowitz MR, Thomas CJ, and Nelin LD. Am J Physiol Lung Cell Mol Physiol 274: L517-L526, 1998). The purposes of this study were to determine whether L-arginine addition augments NO production and to evaluate whether L-arginine uptake is impaired in isolated lungs of chronically hypoxic newborn piglets. Studies were performed by using 1- to 3-day-old piglets raised in room air (control) or 10% O(2) (chronic hypoxia) for 10-12 days. Lung NO production was assessed in isolated lungs from both groups by measuring the perfusate accumulation of nitrites and nitrates (collectively termed NO(-)(x)) before and after addition of L-arginine (10(-2) M) to the perfusate. The rate of perfusate NO(-)(x) accumulation increased by 220% (from 0.8 +/- 0.4 to 2.5 +/- 0.5 nmol/min, P < 0.05) after L-arginine addition to chronic hypoxic lungs but remained unchanged (3.2 +/- 0. 8 before vs. 3.3 +/- 0.4 nmol/min after L-arginine) in control lungs. In the second series of studies, L-arginine uptake was evaluated by measuring the perfusate concentration of L-[(3)H]arginine at fixed time intervals. The perfusate concentration of L-[(3)H]arginine at each time point was less (P < 0.05) in control than in chronic hypoxic lungs. Thus L-arginine uptake was impaired and may underlie in part the reduction in lung NO production that occurs when piglets are exposed to 10-12 days of chronic hypoxia. Moreover, these findings in isolated lungs lead to the possibility that L-arginine supplementation might increase in vivo lung NO production in piglets with chronic hypoxia-induced pulmonary hypertension.     

Reactions of *NO2 with chromium(III) complexes with histamine and pyridoxamine ligands studied by the stopped-flow technique

This study demonstrated the direct formation of the nitrogen dioxide (*NO2) radical during the decomposition of 3-morpholinosydnonimine (SIN-1) in biological buffer 4-morpholinoethanosulfone acid solution. Consequently, at approximately pH 4, SIN-1 can be used successfully as a source of *NO2. This conclusion is drawn from a comparison of the reactions of cis-[Cr(C2O4)(L- L)(OH2)2]+, where L-L denotes pyridoxamine (Hpm) or histamine (hm), with the gaseous *NO2 radical obtained by two methods: from SIN-1 and from a simple redox reaction. These reactions were investigated using the stopped-flow technique. The measurements were carried out at temperatures ranging from 5 to 25 degrees C over a pH range from 6.52 to 9.11 for cis-[Cr(C2O4)(Hpm) (OH2)2]+ and from 6.03 to 8.15 for cis-[Cr(C2O4)(hm)(OH2)2] +. We also determined the thermodynamic activation parameter (E(a)) and the uptake mechanism for each of the coordination compounds studied.     

The photorelease of nitrogen monoxide (NO) from pentacyanonitrosyl coordination compounds of group 8 metals.

The photodetachment of NO from [M(II)(CN)5NO]2- with M = Fe, Ru, and Os, upon laser excitation at various wavelengths (355, 420, and 480 nm) was followed by various techniques. The three complexes showed a wavelength-dependent quantum yield of NO production Phi(NO), as measured with an NO-sensitive electrode, the highest values corresponding to the larger photon energies. For the same excitation wavelength the decrease of Phi(NO) at 20 degrees C in the order Fe > Ru > Os, is explained by the increasing M-N bond strength and inertness of the heavier metals. Transient absorption data at 420 nm indicate the formation of the [M(III)(CN)5H2O]2- species in less than ca. 1 micros for M = Fe and Ru. The enthalpy content of [Fe(III)(CN)5H2O]2- with respect to the parent [Fe(II)(CN)5NO]2- state is (190 +/- 20) kJ mol(-1), as measured by laser-induced optoacoustic spectroscopy (LIOAS) upon excitation at 480 nm. The production of [Fe(III)(CN)5H2O]2- is concomitant with an expansion of (8 +/- 3) ml mol(-1) consistent with an expansion of the water bound through hydrogen bonds to the CN ligands plus the difference between NO release into the bulk and water entrance into the first coordination sphere. The activated process, as indicated by the relatively strong temperature dependence of the Phi(NO) values and by the temperature dependence of the appearance of the [Fe(III)(CN)5H2O]2- species, as determined by LIOAS, is attributed to NO detachment in less than ca. 100 ns from the isonitrosyl (ON) ligand (MS1 state).     

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.     

Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species

We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (*OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite ((-)OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and superoxide (O2*-) by using HPF or APF alone, but (-)OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluorescein, and for the first time we could visualize (-)OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and (-)OCl in many biological and chemical applications.     

Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism

Introduction

The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way.

Methods

Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[³H]glucose (2-[³H]DG). Statistical analysis was performed using the Mann-Whitney U test.

Results

NOC-12 liberates approximately ten times more NO₂^- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[³H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[³H]DG than healthy cells.

Conclusions

These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.     

NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.     

A comparative study of neuronal and inducible nitric oxide synthases: generation of nitric oxide, superoxide, and hydrogen peroxide

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (.NO), superoxide (O2.-), and hydrogen peroxide (H2O2). Since .NO has been implicated in many physiological processes, the importance of O2.- and H2O2 in regulating cell signaling by .NO cannot be overlooked. Before addressing these questions, we investigated the production of .NO, O2.-, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of .NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of .NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2.- and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production of .NO, O2.-, and H2O2 may define the specific physiologic function of each isozyme.