3-Morpholino-sydnonimine-induced suppression of human neutrophil degranulation is not mediated by cyclic GMP, nitric oxide or peroxynitrite: inhibition of the increase in intracellular free calcium concentration by N-morpholino-iminoacetonitrile, a metabolite of 3-morpholino-sydnonimine

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.     

Inhibitory effect of several nitric oxide-generating compounds on purified Na+,K(+)-ATPase activity from porcine cerebral cortex

Nitric oxide (NO)-generating compounds (NO donors) such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, S-nitroso-L-glutathione, 3-morpholino-sydnonimine (SIN-1), (DL)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-5-3-hexenamide, and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene inhibited the Na+,K(+)-ATPase activity purified from porcine cerebral cortex. NO-reducing or -scavenging agents, such as superoxide dismutase or N-(dithiocarbamate)-N-methyl-D-glucamine sodium salt, L-ascorbic acid; and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not alpha-tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, L-ascorbic acid, and alpha-tocopherol. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (.NO2), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN-1). PTIO did not potentiate, but rather attenuated, the SIN-1-induced inhibition. SIN-1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO-). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, L-ascorbic acid, and alpha-tocopherol. These results suggest that NO donors may release NO or NO-derived products, presumably .NO2 and ONOO-, and may inhibit the Na+,K(+)-ATPase activity by interacting with a SH group at the active site of the enzyme.     

Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO-), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-L-arginine methyl ester (L-NAME) blocked IL-8 release by approximately 90% in response to LPS (1 microg/ml), but did not affect the production of IL-1beta or TNF-alpha. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO- (2.5-80 microM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO- formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-gammaB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO-. Our study identifies ONOO- as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO- formation or scavenging ONOO- may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.     

Peroxynitrite-induced toxicity in cultured astrocytes

The exposure of cultured astrocytes to peroxynitrite (ONOO-) for 40 min resulted in a concentration-dependent increase in the release of lactate dehydrogenase from the cells into the bathing medium over the following 24 h. Control experiments showed that the breakdown products of ONOO- contribute, to some extent, to its ability to cause cell death but that the drug vehicle (0.3 M NaOH), which increased the pH of the bathing medium to 9.4, had little effect. The cytotoxic action of ONOO- was mimicked by 3-morpholinosydnonimine (SIN-1) which liberates both nitric oxide (NO) and superoxide but not by S-nitrosoglutathione which liberates only NO. SIN-1-induced cytotoxicity was reversed in a concentration-dependent manner by superoxide dismutase and attenuated by haemoglobin suggesting that the effect of SIN-1 is due, at least in part, to the formation of ONOO-.     

Oxidation and antioxidation of human low-density lipoprotein and plasma exposed to 3-morpholinosydnonimine and reagent peroxynitrite

As peroxynitrite is implicated as an oxidant for low-density lipoprotein (LDL) in atherogenesis, we investigated this process using reagent peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1, which produces peroxynitrite via generation of NO. and O2.-). LDL oxidation was assessed by the consumption of ubiquinol-10 (CoQ10H2) and alpha-tocopherol (alpha-TOH), the accumulation of cholesteryl ester hydro(pero)xides, the loss of lysine (Lys) and tryptophan (Trp) residues, and the change in relative electrophoretic mobility. Exposure to ONOO- or SIN-1 resulted in rapid (<1 min) and time-dependent oxidation, respectively, of LDL's lipids and protein. Manipulating the alpha-TOH content by in vivo or in vitro means showed that when ONOO- or SIN-1 was used at oxidant-to-LDL ratios of <100:1 the extent of LDL lipid peroxidation increased with increasing initial alpha-TOH content. In contrast, in vivo enrichment with the co-antioxidant CoQ10H2 decreased LDL lipid peroxidation induced by SIN-1. At oxidant-to-LDL ratios of >200:1, alpha-TOH enrichment decreased LDL lipid peroxidation for both SIN-1 and ONOO-. In contrast to lipid peroxidation, altering the alpha-TOH content of LDL did not affect Trp or Lys loss, independent of the amounts of either oxidant added. Aqueous antioxidants inhibited ONOO--induced lipid and protein oxidation with the order of efficacy: 3-hydroxyanthranilate (3-HAA) > urate > ascorbate. With SIN-1, these antioxidants inhibited Trp consumption, while only the co-antioxidants ascorbate and 3-HAA prevented alpha-TOH consumption and lipid peroxidation. Exposure of human plasma to SIN-1 resulted in the loss of ascorbate followed by loss of CoQ10H2 and bilirubin. Lipid peroxidation was inhibited during this period, though proceeded as a radical-chain process after depletion of these antioxidants and in the presence of alpha-TOH and urate. Bicarbonate at physiological concentrations decreased ONOO--induced lipid and protein oxidation, whereas it enhanced SIN-1-induced lipid peroxidation, Trp consumption, and alpha-tocopheroxyl radical formation in LDL. These results indicate an important role for tocopherol-mediated peroxidation and co-antioxidation in peroxynitrite-induced lipoprotein lipid peroxidation, especially when peroxynitrite is formed time-dependently by SIN-1. The studies also highlight differences between ONOO-- and SIN-1-induced LDL oxidation with regards to the effects of bicarbonate, ascorbate, and urate.     

The solution structure of human endothelin-2 a 1H-NMR and CD study

OBJECTIVE: Peroxynitrite (ONOO-) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO- in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO- on human platelets, a system in which we have previously shown that ONOO- has both pro- and anti-aggregatory effects. METHODS: Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO- was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. RESULTS: Peroxynitrite (50-400 microM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO- acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma, ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50-100 microM) of ONOO- inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. CONCLUSIONS: Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO- to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO- in biological systems. We hypothesize that the conversion of ONOO- to NO is the critical factor determining the outcome of ONOO- exposure in vivo.     

Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation of rat isolated mesenteric artery

1. The NO donor 3-morpholino-sydnonimine (SIN-1; 0.01-10 microM) evoked concentration-dependent relaxation of rat isolated mesenteric arteries pre-constricted with phenylephrine (1-3 microM). The relaxation to SIN-1 was not significantly different between endothelium-intact or denuded arterial segments or segments in which basal nitric oxide (NO) synthesis was inhibited (n = 8; P > 0.05). In contrast, the membrane permeable analogue of guanosine 3':5'-cyclic monophosphate (cyclic GMP), 8-Br-cyclic GMP (0.01-1 mM), was much less effective in relaxing intact than denuded arterial segments or intact arterial segments pre-incubated with NO synthase blockers (n = 4; P < 0.01). 2. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min) alone, did not alter SIN-1-evoked relaxation in any tissues (n = 5; P > 0.05). However, in parallel experiments, ODQ almost completely inhibited both basal and SIN-1-stimulated production of cyclic GMP in both the presence and absence of NO synthase blockers (n = 6; P < 0.01) indicating that full relaxation to SIN-1 can be achieved in the absence of an increase in cyclic GMP. 3. Exposure of endothelium-intact arterial segments to the potassium channel blocker charybdotoxin (50 nM; 10 min), significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 90% (n = 5; P < 0.01). In contrast, in arterial segments in which either the endothelial cell layer had been removed or basal NO synthesis inhibited, relaxation to SIN-1 was not reduced in the presence of charybdotoxin (n = 6; P > 0.05). However, in the presence of NO synthase blockers and L-arginine (300 microM) together, charybdotoxin did significantly inhibit SIN-1-evoked relaxation to a similar extent as intact tissues (maximum response induced by around 80%; n = 4; P < 0.01). 4. Pre-incubation with apamin (30 nM; 10 min) or glibenclamide (10 microM; 10 min) did not alter SIN-1-evoked relaxation of phenylephrine-induced tone in any tissues (n = 4 and n = 6, respectively; P > 0.05). However, in the presence of either ODQ and apamin, or ODQ and glibenclamide, SIN-1-evoked relaxation was significantly attenuated in intact arterial segments and segments in which NO synthesis was blocked. 5. Exposure of intact arterial segments to charybdotoxin and apamin, in the presence of NO synthase blockers, also significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 80% (n = 4; P < 0.01). 6. Addition of superoxide dismutase (SOD; 30 u ml-1), potentiated relaxations to SIN-1 in all tissues, but did not alter the effects of charybdotoxin and ODQ and SIN-1-evoked relaxation. 7. These data show that although relaxation to the NO-donor SIN-1 is not significantly different between endothelium-intact and denuded arterial segments, the mechanisms which mediate SIN-1-evoked relaxation in the rat isolated mesenteric artery appear to be modulated by the basal release of endothelium-derived NO. In the presence of an intact endothelial cell layer, the major mechanism for SIN-1-evoked relaxation appears to be the activation of charybdotoxin-sensitive potassium channels. In contrast, when basal NO synthesis is inhibited, SIN-1 appears to cause full relaxation by both the activation of a charybdotoxin-sensitive pathway and the stimulation of soluble guanylyl cyclase.     

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1. A comparison of the effects of dietary and genetically-induced hypercholesterolaemia on the vasodilator and antiaggregatory capacity of the endothelium was made in rabbit isolated subclavian artery rings. 2. Dietary-induced hypercholesterolaemia in NZW rabbits decreased the maximum relaxation to carbachol (0.01-10 microM) and calcimycin (0.01-0.1 microM) in vessel rings precontracted with 5-hydroxytryptamine (5-HT), 0.1 microM), when compared to responses observed in rings obtained from control normocholesterolaemic NZW rabbits. The relaxant responses to SIN-1 (3-(4-morpholinyl)-sydnonimine hydrochloride) were attenuated but were not significantly different from controls. In Froxfield genetically hypercholesterolaemic (FHH) rabbits, the maximum relaxations to carbachol, calcimycin and SIN-1 were all reduced significantly. 3. Neither genetic nor dietary-induced hypercholesterolaemia modified the contractile responses of vessel rings to either KCl (10-100 mM) or 5-HT (0.01-10 microM). 4. Endothelium-dependent inhibition of collagen-induced platelet aggregation in whole blood was demonstrated by stimulation of a vessel ring, incorporated into the blood sample, with carbachol (10 microM, final blood concentration). This effect was inhibited by NG-nitro-L-arginine (L-NOARG, 100 microM). SIN-1 (10 microM, final blood concentration) also decreased whole blood platelet aggregation, but only in the presence of an unstimulated vessel ring, and this was unaffected by L-NOARG. Superoxide dismutase (150 u ml-1) did not influence the inhibition of aggregation by either a carbachol-stimulated vessel ring or by SIN-1. 5. Carbachol-stimulated artery rings from FHH rabbits inhibited platelet aggregation to a similar extent to that seen with rings from control normocholesterolaemic rabbits. Rings from hypercholesterolaemic NZW rabbits, however, did not significantly inhibit platelet aggregation when stimulated with carbachol. SIN-1 inhibited platelet aggregation in the presence of rings from either group of hypercholesterolaemic rabbits. 6. Hypercholesterolaemia induced by dietary modification induces changes in endothelial function which are characteristically different from those seen in genetically hypercholesterolaemic rabbits. It appears that dietary-induced hypercholesterolaemia primarily decreases NO release from the endothelium, while in genetically-induced hypercholesterolaemic vessel rings NO is released but there is a decreased responsiveness of the vascular smooth muscle cells to NO. This may reflect differences in the age and severity of the atherosclerotic lesions in the two groups of rabbits.     

Effect of Cu2+ on relaxations to the nitrergic neurotransmitter, NO and S-nitrosothiols in the rat gastric fundus

1. The effects of superoxide anion generators before and after treatment with inhibitors of Cu/Zn superoxide dismutase (Cu/Zn SOD) and the effects of thiol-modulating agents were investigated on nitrergic relaxations to electrical stimulation of non-adrenergic non-cholinergic (NANC) nerves of the rat gastric fundus and on relaxations to authentic nitric oxide (NO) and nitroglycerin. 2. The superoxide anion generators, pyrogallol (30 microM) and duroquinone (30-60 microM), significantly inhibited the relaxations to NO (0.03-3 microM) but not nitrergic relaxations to NANC nerve stimulation (0.5-8 Hz) or those to ATP (10 microM). Treatment of the rat gastric fundus with the inhibitors of Cu/Zn SOD, diethyldithiocarbamate (DETC, 1 mM for 2 h) or triethylenetetramine (TETA, 100 microM for 2 h) had no effect on the relaxations to NANC nerve stimulation (1-8 Hz), NO (0.03-3 microM) or on those to ATP (10 microM). 3. After treatment of the rat gastric fundus with DETC (1 mM) but not after treatment with TETA (100 microM), pyrogallol (30 microM) and duroquinone (30-60 microM) significantly inhibited the nitrergic relaxations to electrical stimulation (0.5-8 Hz) and those to NO (0.03-3 microM). This inhibitory effect of pyrogallol and duroquinone was prevented by addition of exogenous SOD (250 units ml-1). Pyrogallol but not duroquinone also inhibited the NO-independent relaxations to ATP (10 microM). 4. The thiol modulators, buthionine sulphoximine (1 mM for 2 h) and ethacrynic acid (30 microM for 2 h), significantly inhibited the relaxations to nitroglycerin (0.03-3 microM) but had no effect on the nitrergic relaxations to electrical stimulation (0.5-8 Hz) or on those to NO (0.03-10 microM) and ATP (10 microM). The thiol modulators, sulphobromophthalein (100 microM for 2 h) and diamide (30-100 microM for 2 h) did not affect the relaxations to nitroglycerin, or those to NANC nerve stimulation and NO. 5. In summary, thiol modulators significantly inhibited the thiol-dependent relaxations to nitroglycerin but not those to NANC nerve stimulation or NO. Relaxations to nitrergic stimulation were decreased by superoxide anion generators only after inhibition of Cu/Zn SOD. These results suggest that the nitrergic NANC neurotransmitter in the rat gastric fundus is not a nitrosothiol but more likely free NO, which is protected from breakdown by tissue SOD.     

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BK(Ca)) in smooth muscle cells isolated from the rat mesenteric artery

1. To assess the action of nitric oxide (NO) and NO-donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2. Inside-out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212+/-5 pS (n = 12) in symmetrical (140 mM) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK(Ca). 3. In five out of six inside-out patches, NO (45 or 67 microM) produced an increase in BK(Ca) activity. In inside-out patches, BK(Ca) activity was also enhanced in some patches with 100 or 200 microM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 microM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4. In inside-out patches, 100 microM SIN-1 failed to increase BK(Ca) activity (in all 4 patches tested), while at a higher (500 microM) concentration SIN-1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside-out patches increased (P < 0.05) BK(Ca) activity in two patches. 5. In the majority of cells (6 out of 7), application of NO (45 or 67 microM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 microM). With SIN-1, the increase in current was blocked with the BK(Ca) channel blocker, iberiotoxin (50 nM). 6. With conventional whole-cell voltage clamp, the increase in the outward K+ current evoked with SIN-1 (50-300 microM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+/-10.2% was recorded at 40 mV. 7. In cell-attached patches, large conductance voltage-dependent K+ channels were stimulated by SIN-1 (100 microM) applied to the cell (n = 5 patches). 8. These data indicate that NO and its donors can directly stimulate BK(Ca) activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK(Ca) channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.     

OH-radical-type reactive oxygen species derived from superoxide and nitric oxide: a sensitive method for their determination and differentiation

Reactive oxygen species are involved in many diseases where the radical species OH, peroxynitrite and the non-radical, hypochlorous acid, play an outstanding role. The formation of OH-type oxidants is essentially confined to a few types of reactions. The most prominent ones are the one-electron reduction of hydrogen peroxide by F2+ or Cu+ -ions (Fenton-type reactions), reaction of hypochlorite with superoxide and finally formation and decay of peroxynitrite (ONOOH), formed from superoxide and NO. In this communication we wish to report on a simple model system allowing to differentiate between these ROS: ethene formation from ACC is only detectable in the presence of hypochlorite (v. Kruedener et al, 1995) and not detectable with Fenton-type oxidants or SIN-1 (3-morpholinosydonimine, a peroxynitrite generator by releasing sequentially superoxide and NO) at 10 microM concentrations. On the other hand, ethene formation from KMB is negligible in the presence of hypochlorite but proceeds rapidly with Fenton-type oxidants (4 microM H2O2; 4 microM Fe2+) as well as with 1 microM SIN-1. Stimulation of Fenton-type oxidants and not of SIN-1 by EDTA and characteristic patterns of inhibition by SOD, catalases, hemoglobin and uric acid allow a differentiation between these two potential precursors of OH-radicals. Synthetic ONOOH shows different reaction kinetics as compared to SIN-1. Inhibition of ONOOH-dependent ethene formation by different compounds occurs more or less "random" indicating an unspecific influence of proteins and also small molecules. Comparison of the individual inhibition types of several selected compounds allows a differential analysis as to the generation pathway of the final oxidants, OH- radical or peroxynitrite.     

Effects of redox-related congeners of NO on apoptosis and caspase-3 activity

Nitric oxide has been shown to inhibit apoptosis of human umbilical venous endothelial cells (HUVEC). Therefore we investigated the effect of different NO donors, PAPA NONOate (NOC-15; NO.) and nitrosodium tetrafluoroborate (NOBF4, NO+), and the reaction product of NO and O2-, peroxynitrite (ONOO- ), on TNF-alpha- or serum depletion-induced apoptosis of HUVEC. TNF-alpha-induced DNA fragmentation, determined by ELISA, was inhibited by NOC-15, NOBF4, and ONOO- in a concentration-dependent manner (maximal effects with 10 microM NO. and ONOO- and 100 microM NO+). The inhibition of apoptosis correlated with a protective effect on cell viability. The caspases, a cysteine protease family, play an important role in apoptotic processes. To determine whether the different NO donors and ONOO- regulate this enzyme, caspase-3-like activity was measured in homogenates of TNF-alpha-treated HUVEC. The TNF-alpha-induced enzyme activity was abrogated by NO., NO+, and ONOO-. Furthermore, caspase-3 activity was determined in vitro by reconstitution of the separately cloned, bacterially expressed, and purified active p17 and p12 subunits. The reconstituted caspase-3 exhibited enzyme activity, which was suppressed by the different NO donors and ONOO- with an IC50 of 50 microM for NOC-15, 1 mM for NOBF4, and 50 microM for ONOO-. The inhibition of caspase-3 activity correlated with a S-nitrosylation of the reactive cysteine residue and was reversed by further addition of dithiothreitol. This study suggests that the cellular regulatory processes of NO to protect cells from apoptosis may be independent of the redox state and that low concentrations of NO and ONOO- inhibit the cellular suicide program in HUVEC via S-nitrosylation of members of the caspase family.     

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The effects of peroxynitrite (ONOO-) on cultured cardiac myocytes were examined by simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contractile function. On exposure to 0.2 mM ONOO-, [Ca2+]i increased to beyond the systolic level within 5 min with a concomitant decrease in spontaneous contraction of myocytes followed by complete arrest. Addition of a L-type Ca2+ channel blocker or removal of extracellular Ca2+ prevented the ONOO(-)-induced increase in [Ca2+]i, indicating that the increase in [Ca2+]i was caused by the enhanced influx of Ca2+ through the plasma membrane and not by the enhanced release from sarcoplasmic reticulum (SR). Plasma membrane fluidity and concentration of the thiobarbiturate acid-reactive substance (TBARS) in the cells remained unchanged by the ONOO- treatment. The complete cessation of contraction of myocytes persisted even under the massive increase in [Ca2+]i, which was induced by an additional saponin (5 microM) treatment. In conclusion, ONOO- increases [Ca2+]i in myocytes through disturbance of Ca2+ transport systems in the plasma membrane and impairs contractile protein.     

Nitroxides increase the detectable amount of nitric oxide released from endothelial cells

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.     

Formation of S-nitrosothiols via direct nucleophilic nitrosation of thiols by peroxynitrite with elimination of hydrogen peroxide

Peroxynitrite (ONOO-), a potent oxidant formed by reaction of nitric oxide (NO.) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO. Reaction of ONOO- with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO. Kinetic analysis of NO. production after reaction of ONOO- with GSH established that NO. originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO- does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2. or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO- with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO-. Our results indicate that ONOO- may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO- may represent an important and often overlooked component of NO. biochemistry.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Influence of SIN-1 and sodium nitroprusside (NANP) on ox-LDL metabolism in macrophages

Effects of NO-donors (3-morpholinosydnonimine-SIN-1 and sodium nitroprusside NaNP) on the accumulation and degradation of oxidized LDL (ox-LDL) by macrophages were studied. Ox-LDL, but not native-LDL (n-LDL) suppressed the LPS-stimulated biosynthesis of NO by macrophages. SIN-1 at low concentrations < 100 microM was without any effect while SIN-1 at high concentration (300 microM) and NaNP (30-300 microM) stimulated the accumulation and degradation of ox-LDL by macrophages. The pretreatment of macrophages with NG-monomethyl-L-arginine (L-NMMA, 3 microM) for 24 hours had the same stimulatory effect. The inhibition of endogenous formation of NO, by L-NMMA profoundly changed the pattern of action of NO-donors on ox-LDL catabolism by macrophages; the stimulatory action of SIN-1 was transformed to the inhibitory action on the accumulation and degradation of ox-LDL whereas NaNP lost its stimulatory action entirely. Our interpretation of this unexpected interactions between SIN-1, NaNP and L-NMMA is as follows. Endogenous NO in macrophages inhibits the accumulation of ox-LDL and therefore, the stimulatory effect of L-NMMA has been overcome by exogenous NO from SIN-1. However, NO at high concentrations promotes lipid accumulation in macrophages and thereby, in the absence of L-NMMA, SIN-1 at high concentrations and NaNP produced a paradoxical stimulatory effect in macrophages. NaNP is not a proper NO-donor and its mode of action differed from that of SIN-1. In conclusion, NO at low physiological concentrations keeps scavenger receptors of macrophages downregulated and hence endogenous NO may show anti-atherogenic properties.     

Diffusion-weighted MR imaging in hypertensive encephalopathy: clues to pathogenesis

We investigated the effects of organic buffers on the NO-like biological activities of ONOO-. In HEPES buffer (50 mM), ONOO- (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO. ONOO- produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM). Electrochemical detection of NO showed that the biological effects of ONOO- in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions. CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway. Formation of NO from ONOO- was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO- on cGMP accumulation in this buffer. These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO-.     

The nitric oxide donor SIN-1 protects endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity: possible role for cyclic GMP and heme oxygenase

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.