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.     

Preeclampsia, lipid peroxidation, and calcium adenosine triphosphatase activity of red blood cell ghosts

1. The effects of oxatriazole-type (GEA 3162 and GEA 5624) nitric oxide (NO) donors on mitogenesis and proliferation were studied in vascular smooth muscle cell (VSMC) culture. The effects of the GEA-compounds were compared with well-known NO-donors 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 2. All NO-donors released NO and increased the production of cyclic GMP concentration-dependently. The production of cyclic GMP was inhibited by the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). 3. The NO-donors inhibited basal and serum-induced DNA synthesis concentration-dependently. The GEA-compounds were needed in concentrations 10 times lower than SIN-1 and SNAP. GEA 3162, SIN-1 and SNAP were also able to inhibit serum-induced cell proliferation. GEA 5624 was ineffective. The antimitogenic effect of NO-donors was not reduced by inhibiting the guanylate cyclase. 4. These results suggest that NO inhibits serum-induced DNA synthesis and proliferation of VSMC by a cyclic GMP-independent mechanism. The oxatriazole-type NO-donor GEA 3162 was found to be a more potent inhibitor of mitogenesis and cell proliferation than SIN-1 and SNAP.     

The intravenous administration of tumor necrosis factor alpha, interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.     

Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils

1. The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2. A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 microM) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 microM) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.68 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectively. 3. A role for cyclic GMP as a mediator of chemotaxis was supported by the finding that the guanylyl cyclase inhibitor LY 83583 (100 microM) completely inhibited chemotaxis and suppressed the maximal response; EC50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 microM); EC50 for fMLP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823, respectively. 4. The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 microM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/10(6) cells with and without DPG, respectively. 5. Although the NOS inhibitors L-NMMA and L-canavanine (500 microM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36.15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response from 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O2-/10(6) cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6. A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 microM) and Rp-8-pCPT-cGMPS (100 microM) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS. 7. The phosphatase inhibitor DPG (500 microM) inhibited SAG; EC50 for fMLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respectively. 8. The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 microM) > GEA 5024 (IC50 = 18.44 +/- 0.43 microM) > SIN-1 (IC50 > 1000 microM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most effective (SIN-1 (EC50 = 37.62 +/- 0.9 microM) > GEA 3162 (EC50 = 39.7 +/- 0.53 microM) > GEA 5024 (EC50 = 89.86 +/- 1.62 microM)). 9. In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for these agents in neutrophil activation. However, the increases in cyclic GMP and NO induced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as observed with NO releasing compounds, inhibit chemotaxis.     

Nitric oxide as a regulator of prostacyclin synthesis in cultured rat heart endothelial cells

The effects of nitric oxide (NO) and its second messenger cyclic guanosine monophosphate (cGMT) on prostacyclin (PGI2) synthesis were studied in cultured rat heart endothelial cells using three different non-enzymatic nitric oxide releasing substances as well as inhibitors of nitric oxide synthase and of soluble guanylate cyclase. Production of prostacyclin, measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), was stimulated up to 1.7 fold in endothelial cells treated with the NO donors SIN-1 (3-morpholino sydnonimine), GEA 3162 (3-aryl-substituted oxatriazole imine) and GEA 3175 (3-aryl-substituted oxatriazole sulfonyl), chloride). In each case the synthesis of cGMP increase as much as 40-100 fold. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), decreased the basal production of 6-keto-PGF1 alpha in non-stimulated endothelial cells, an effect that could be reversed by the NO donors SIN-1, GEA 3162 and GEA 3175. cGMP formation in the L-NAME treated endothelial cells was unaltered. The guanylate cyclase inhibitors, methylene blue (100 mumol/l) and LY83583 (100 mumol/l), caused a 1.5-10 fold increase in 6-keto-PGF1 alpha production while NO-donor-stimulated endothelial cGMP production was decreased by 10 to 90%. However, when SIN-1 was used as a stimulant, LY83583 had no significant effect on the production of cGMP. These findings support the hypothesis that NO stimulates prostacyclin production directly by activating cyclooxygenase. The results also suggest that NO could have an indirect effect on prostacyclin production via cGMP.     

Peroxynitrite augments fMLP-stimulated chemiluminescence by neutrophils in human whole blood

The neutrophil respiratory burst was examined by the technique of luminol-dependent chemiluminescence (LDCL) triggered by submaximal concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP) in diluted whole blood. We sought to identify the chemical species responsible for LDCL in whole blood, to examine the role of leukotriene B4 (LTB4) and other arachidonic acid metabolites as mediators of the fMLP signaling pathway, and to investigate the effect of peroxynitrite on this response. Both sodium azide and taurine significantly inhibited LDCL (93% inhibition with 100 microM azide, 52% inhibition with 10 mM taurine). More modest inhibition was seen with superoxide dismutase (SOD), catalase, the nitric oxide synthase inhibitor monomethyl-L-arginine (L-NMMA), and with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (AA-861; no effect), and cytochrome P-450 (SKF 525-A) pathways of arachidonic acid metabolism. The nitric oxide donor SIN-1 (1-100 microM) and peroxynitrite (10-300 microM) also augmented fMLP-induced LDCL. The augmentation seen with peroxynitrite and SIN-1 was attenuated by SOD. Despite the increase in LDCL, peroxynitrite caused a dose-related inhibition of fMLP-stimulated LTB4 release. In summary, our results indicate that (1) LDCL elicited by fMLP in diluted whole blood appears primarily mediated by hypochlorous acid derived from myeloperoxidase; (2) pretreatment with the nitric oxide donor SIN-1 or with peroxynitrite augments LDCL; and (3) LTB4 release does not contribute to fMLP-stimulated LDCL or in the modulation of LDCL by SIN-1 or peroxynitrite.     

Inhibitory effects of nitric oxide donors on nitric oxide synthesis in rat gastric myenteric plexus

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.     

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.     

Effect of some cyclooxygenase inhibitors on the increase in guanosine 3':5'-cyclic monophosphate induced by NO-donors in human whole platelets

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate. 2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC50 being 13.7 microM and 15.8 microM, respectively. 3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC50=32.7 microM, NaNP and 25.0 microM, SNAP), while acetylsalicylic acid (up to 1000 microM) and diclofenac (up to 100 microM) were ineffective. 4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production. 5. Indomethacin (10 microM), indoprofen (30 microM), diclofenac (100 microM) and acetylsalicylic acid (1000 microM) showed a comparable efficacy in inhibiting platelet thromboxane B2 (TXB2) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase. 6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP. 7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.     

Antidepressants and suicide mortality

The aim of this study was to determine the effect of the soluble guanylyl cyclase inhibitors methylene blue and LY83583 (6-anilino-5,8-quinolinedione) on relaxation and increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration ([cGMP]i) induced by sodium nitroprusside, 3-morpholinosydnonimine (SIN-1) and diethylamine-nitric oxide (NO) in porcine tracheal smooth muscle in vitro. We measured (1) the effect of NO donors on isometric force and [cGMP]i and (2) the ability of methylene blue and LY83583 to antagonize these effects. In muscle strips contracted with carbachol (0.1-0.3 microM), both sodium nitroprusside and diethylamine-NO caused relaxation and an increase in [cGMP]i. By contrast, SIN-1 caused a relaxation which was not associated with a concomitant increase in [cGMP]i. Methylene blue (10 microM) and LY83583 (10 microM) completely blocked the increase in [cGMP]i induced by sodium nitroprusside and diethylamine-NO; however substantial relaxation remained. It is concluded that in porcine airway smooth muscle, (1) relaxation induced by some NO donors may occur without a concomitant increase in [cGMP]i; and (2) whereas relaxation induced by some NO donors may be associated with increases in [cGMP]i, the relaxation is not completely dependent upon it.     

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.     

The effect of nitric oxide and peroxynitrite on apoptosis in human polymorphonuclear leukocytes

In acute lung injury, neutrophil apoptosis may be important in regulating the inflammatory process by controlling neutrophil numbers and thus activity. Exogenous inhaled nitric oxide is now a widely used therapy in patients with acute lung injury, and its effects on apoptosis may be important. We investigated the effect of nitric oxide and peroxynitrite on apoptosis in lipopolysaccharide stimulated polymorphonuclear leukocytes as a model of nitric oxide-treated lung injury. Cells were incubated for up to 16 h with and without 1.7 microg/ml lipopolysaccharide and the nitric oxide donor GEA-3162 or the peroxynitrite donor SIN-1. Apoptosis was assessed using flow cytometry following annexin-V staining, after 4, 6, 8, and 16 h. Data were assessed using Kruskal-Wallis analysis of variance or Mann-Whitney U-test as appropriate. Annexin-V staining increased spontaneously over 16 h in untreated cells (p = .0002) and incubation with either 1000 microM SIN-1 or 10 microM GEA-3162 increased annexin staining at early time points in nonactivated cells. Apoptosis was attenuated when cells were exposed to lipopolysaccharide and both nitric oxide and peroxynitrite dose dependently inhibited this suppression at all time points and was most apparent at 16 h (p = .004 and .001, respectively). Exposure of activated neutrophils to exogenous nitric oxide or peroxynitrite has marked influences on apoptosis. This work has implications for the modulation of neutrophil function within the lung in patients with lung injury who receive inhaled nitric oxide therapy.     

Nitric oxide modulates cGMP levels in neurons of the inner and outer retina in opposite ways

In the mammalian retina, neuronal nitric oxide synthase (NOS) is mainly localized in subpopulations of amacrine cells. One function of nitric oxide (NO) is to stimulate soluble guanylate cyclases which in turn synthesize cGMP. We used an antibody specific for cGMP to demonstrate cGMP-like immunoreactivity (cG-IR) in bovine, rat, and rabbit retinae and investigated the effects on cGMP levels of both exogenously applied NO and of endogenously released NO. We found that cGMP levels in inner and outer retina were controlled in opposite ways. In the presence of the NO-donors SNP, SIN-1 or SNAP, cG-IR was prominent in neurons of the inner retina, mainly in cone bipolar cells, some amacrine and ganglion cells. Retinae incubated in IBMX showed weak cG-IR in bipolar cells. Glutamate increased cG-IR in the inner retina, presumably by stimulating endogenous NO release, whereas NOS inhibitors or GABA and glycine decreased cG-IR in bipolar cells by reducing NO release. In somata, inner segments and spherules of rod photoreceptors the situation was reversed. cG-IR was undetectable in the presence of NO-donors or glutamate, was moderate in IBMX-treated retinae, but increased strongly in the presence of NOS inhibitors or GABA/glycine. We conclude that NO is released endogenously in the retina. In the presence of NO, cGMP levels are increased in neurons of the inner retina, but are decreased in rods.     

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.     

Oxygen modulation of guanylate cyclase-mediated retinal pericyte relaxations with 3-morpholino-sydnonimine and atrial natriuretic peptide

PURPOSE: This study explores at which level of the guanylate cyclase pathway oxygen modulates retinal pericyte relaxation induced by nitric oxide (NO). METHODS: Bovine retinal microvascular pericytes were grown on silicone. On silicone, pericyte contractile tone induces wrinkles. Drug-induced relaxation was quantified as a reduced number of wrinkles after exposure to 3-morpholino-sydnonimine (SIN-1) or atrial natriuretic peptide (ANP) in the absence or in the presence of either 0.3 microM methylene blue (MB), a guanylate cyclase inhibitor, or 10 microM hemoglobin, a NO scavenger; and under 100% oxygen (hyperoxia), ambient air (normoxia), or 100% nitrogen (hypoxia). RESULTS: Pericytes were relaxed with SIN-1 and ANP in a concentration-dependent manner (EC50: 0.1 microM and 0.01 microM, respectively). Relaxations induced by SIN-1 or ANP were inhibited (P < 0.001) by MB, whereas hemoglobin inhibited only SIN-1 relaxations (P < 0.001). Relaxations induced by SIN-1, but not by ANP were increased (P < 0.001) under hypoxia and decreased (P = 0.002) under hyperoxia. CONCLUSIONS: SIN-1 and ANP relax pericytes through the activation of guanylate cyclase (inhibited by MB), but only SIN-1 through an extracellular release of NO (inhibited by hemoglobin). That oxygen only modulates pericyte relaxations induced by SIN-1 (NO-mediated) but not those induced by ANP suggests that an interaction between oxygen and NO might participate in the capillary network's blood-flow modulation according to local tissue oxygen tension.     

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.     

Modulation of human T cell responses by nitric oxide and its derivative, S-nitrosoglutathione

OBJECTIVE: To examine the effects of nitric oxide (NO) and its more stable derivative, S-nitrosoglutathione (SNO-GSH), on the response of activated T lymphocytes. METHODS: The effects of NO and SNO-GSH on DNA synthesis, interleukin-2 (IL-2) production, IL-2 receptor expression, and cGMP accumulation were determined in phytohemagglutinin-activated peripheral blood mononuclear cells (PBMC) and spleen T cells. RESULTS: Nitric oxide (half-life [T1/2] < 15 seconds) did not inhibit T cell proliferation. However, the derivative SNO-GSH (25 microM) (T1/2 > 2 hours) inhibited DNA synthesis by a mean +/- SD of 65 +/- 19.6% (P < 0.001) in PBMC and 75 +/- 15% (P < 0.001) in spleen cells. Macrophage depletion of PBMC did not abrogate the inhibition. SNO-GSH had no effect on IL-2 production or IL-2 receptor expression. NO (25 microM) increased the cGMP content of PBMC (0.65 +/- 0.15 pmoles/10(6) cells; P < 0.04), as did SNO-GSH (25 microM) in both PBMC (3.8 +/- 1; P < 0.001) and spleen T cells (5.2 +/- 1.2; P < 0.001). Methylene blue and hemoglobin, which are NO inhibitors, inhibited SNO-GSH-induced cGMP accumulation (P < 0.001). CONCLUSION: SNO-GSH inhibits T cell DNA synthesis independently of IL-2 production and in association with cGMP accumulation via a NO-dependent mechanism. We suggest that NO and its S-nitrosothiol derivatives may act as endogenous inhibitors of T cell-mediated inflammation.     

Binding of hemoglobin by Porphyromonas gingivalis

Nitric oxide which was released in aqueous solutions (> or = 10 microM) of direct NO-donors such as 3-morpholinesydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine (SNAP) consumed avidly sulfhydryl groups of N-acetylcysteine > cysteine > glutathione. In case of SIN-1 generation of nitrites run in parallel to disappearance of sulfhydryl groups of N-acetylcysteine and glutathione, however, for a pair of SIN-1 and cysteine the rate of formation of nitrites was much slower than the rate of consumption of sulfhydryl groups. We infer that kinetics of formation and breakdown of S-nitrosothiols varies depending on the type of a thiol which reacts with a NO-donor. Indirect NO-donors such as glyceryl trinitrate (GTN), molsidomine (MSD) or sodium nitroprusside (NaNP) at concentrations < 100 microM did not consume sulfhydryl groups of cysteine unless pretreated with the xanthine/xanthine oxidase system. We suppose that in this last case superoxide anions react with nitric oxide to form peroxynitrites with a higher potency than nitric oxide itself to destroy sulfhydryl groups. We conclude that out of three studied thiols N-acetylcysteine is the best substrate for the formation of S-nitrosothiols, while S-nitrosocysteine is the slowest releaser of nitric oxide. Moreover, unlike SIN-1 and SNAP, NaNP is not a direct NO-donor but behaves rather like GTN. Minute amounts of nitric oxide released either from NaNP or GTN gain from superoxide anions an amplification as SH-scavengers.