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.     

Liquid ventilation for treatment of meconium aspiration syndrome in a piglet model

OBJECTIVES: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. MATERIALS AND METHODS: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. RESULTS: Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. CONCLUSION: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.     

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.     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nomega-monomethyl-L-arginine (L-NMMA), Nomega-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1beta (rhIL-1beta) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1beta with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na2(35)SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1beta challenge (0 to 250 U ml(-1) and 0 to 15 U ml(-1) respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 microM) on NO production by cartilage cells stimulated with IL-1beta (25 U ml(-1) or 5 U ml(-1)), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU > L-NMMA > or = SMT > or = AG > or = L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1beta, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME > or = L-NMMA > > AG > SMT > > AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range. D-isomers of L-arginine analog inhibitors (1000 microM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1beta treated cells. L-arginine (5000 microM) tended to reverse the correcting effect of L-NMMA (1000 microM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100 microM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1beta on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1beta on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.     

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.     

Antisense oligonucleotides, a novel tool for the control of cytokine effects on human cartilage. Focus on interleukins 1 and 6 and proteoglycan synthesis

OBJECTIVE: To prevent the negative effects of interleukin-1 (IL-1) and IL-1-induced IL-6 on cartilage proteoglycan (PG) synthesis, we used an antisense oligonucleotide (ASO) specific for IL-6 messenger RNA (mRNA) to inhibit IL-6 production. METHODS: Explants of human articular cartilage were cultured in the presence or absence of IL-6-ASO, IL-1, and exogenous IL-6. As metabolic parameters, cartilage production of IL-6 was determined in the B9 bioassay and PG as incorporation of 35SO4. RESULTS: The IL-6-ASO prevented IL-1-induced production of IL-6 in the cartilage explants, as well as IL-1-induced inhibition of PG synthesis. This inhibition was restored, despite the presence of IL-6-ASO, when exogenous IL-6 was added. A control ASO (not specific for IL-6 mRNA) was not effective. CONCLUSION: The IL-6-ASO used can penetrate the extracellular matrix of articular cartilage, enter the chondrocytes, and inhibit the IL-1-induced production of IL-6. Furthermore, IL-6-ASO can prevent the IL-1-induced inhibition of cartilage PG synthesis. The effect of exogenous IL-6 shows that IL-1 requires IL-6 for inhibition of PG synthesis.     

Effect of interleukin 1 and leukaemia inhibitory factor on chondrocyte metabolism in articular cartilage from normal and interleukin-6-deficient mice: role of nitric oxide and IL-6 in the suppression of proteoglycan synthesis

We studied the role of IL-6 and nitric oxide (NO) in IL-1 and leukaemia inhibitory factor (LIF) induced suppression of proteoglycan synthesis. Cartilage explants of patellae and femoral heads were incubated with IL-1 or LIF. Conditioned media were analysed for IL-6 activity (B9-assay) and NO content (Griess). Proteoglycan synthesis was assessed using [35S]sulfate incorporation. IL-1 dose dependently induced IL-6 synthesis and neutralizing IL-6 with antibodies did not reduce proteoglycan synthesis suppression, neither in explants nor in isolated chondrocytes. IL-6 independence was confirmed using cartilage from IL-6 deficient mice. IL-1 significantly increased NO release in normal and IL-6 deficient chondrocytes and addition of the NO synthase inhibitor, N(G)-monomethyl-L-arginine markedly alleviated proteoglycan synthesis suppression. LIF also induced proteoglycan synthesis suppression in cartilage from normal and IL-6 deficient mice, but the suppression was neither accompanied by nor dependent on NO release. Furthermore, proteoglycan synthesis suppression during experimental arthritis was similar in both normal and IL-6 deficient mice. We concluded that IL-6 is not a necessary cofactor in IL-1 and LIF induced suppression of proteoglycan synthesis. Furthermore, only the IL-1 induced suppression was mediated by NO, suggesting that inhibition of proteoglycan synthesis may occur through different pathways.     

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.     

Prostaglandin E2 synthesis is differentially affected by reactive nitrogen intermediates in cultured rat microglia and RAW 264.7 cells

We studied the effects of nitric oxide (NO) on prostanoid production, cyclooxygenase (COX-2) expression and [3H]arachidonic acid (AA) release in RAW 264.7 macrophagic cells and rat microglial primary cultures. Inhibition of NO synthesis enhanced microglial prostanoid production without affecting that of RAW 264.7 cells. Both 3-morpholinosydnonimine (SIN-1), (which, by releasing NO and superoxide, leads to the formation of peroxynitrite), and S-nitroso-N-acetylpenicillamine (SNAP), (which releases only NO), inhibited microglial prostanoid production, by preventing COX-2 expression. In contrast, in RAW 264.7 cells, SIN-1 enhanced both basal and LPS-stimulated prostanoid production by upregulating COX-2, while SNAP stimulated basal production and slightly inhibited the LPS-induced production, as a cumulative result of enhanced AA release and depressed COX-2 expression. Thus, reactive nitrogen intermediates can influence prostanoid production at distinct levels and in different way in the two cell types, and results obtained with RAW 264.7 cells can not be extrapolated to microglia.     

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.     

Expression of nerve growth factor and its high-affinity receptor Trk-A in the rat pancreas during embryonic and fetal life

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes. To clarify the mechanism of the IL-1-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-1-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it. Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3. The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release. An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-1-stimulated bFGF release even if increased production of bFGF is related to the release. IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order. NG-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.     

SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes

Nitric oxide (NO) is implicated in a number of inflammatory processes and is an important mediator in animal models of rheumatoid arthritis and in in vitro models of cartilage degradation. The pyridinyl imidazole SB 203580 inhibits p38 mitogen-activated protein (MAP) kinase in vitro, blocks proinflammatory cytokine production in vitro and in vivo, and is effective in animal models of arthritis. The purpose of this study was to determine whether SB 203580 could inhibit p38 MAP kinase activity, NO production, and inducible NO synthase (iNOS) in IL-1 stimulated bovine articular cartilage/chondrocyte cultures. The results indicated that SB 203580 inhibited both IL-1 stimulated p38 MAP kinase activity in isolated chondrocytes and NO production in bovine chondrocytes and cartilage explants with an IC50 value of approximately 1 microM. To inhibit NO production, SB 203580 had to be present in cartilage explant cultures during the first 8 h of IL-1 stimulation, and activity was lost when it was added 24 h following IL-1. SB 203580 did not inhibit iNOS activity, as measured by the conversion of arginine to citrulline, when added directly to cultures where the enzyme had already been induced, but had to be present during the induction period. Using a 372-bp probe for bovine iNOS we demonstrated inhibition of IL-1-induced mRNA by SB 203580 at both 4 and 24 h following IL-1 treatment. The iNOS mRNA levels were consistent with NO levels in 24-h cell culture supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA.     

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-.     

Probenecid inhibits transforming growth factor-beta 1 induced pyrophosphate elaboration by chondrocytes

OBJECTIVE: The elaboration of excess extracellular inorganic pyrophosphate (ePPi) by cartilage contributes to calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. Transforming growth factor-beta 1 (TGF beta 1) is the only defined physiologic stimulant of cartilage ePPi elaboration. The mechanism of ePPi generation by chondrocytes is unknown, but current evidence suggests that TGF beta 1 induced ePPi is made intracellularly. An active transport mechanism such as an anion transporter would then be necessary to export ePPi to the matrix where crystals form. We determined the effect of probenecid (PB), an anion transport inhibitor, on TGF beta 1 induced ePPi elaboration. METHODS: Porcine hyaline articular chondrocytes in high density monolayer cultures were exposed to serum-free media with and without TGF beta 1 and/or PB. ePPi was measured in the media after 48-96 h of exposure. Cell injury was measured by examining the release of 3H-deoxyglucose from chondrocytes. The activity of the ePPi generating ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH) and media lactate concentrations were measured with standard colorimetric assays. As PB may inhibit phosphodiesterase (PDE), its effects on ePPi generation were compared with isobutylmethylxanthine (IBMX), a specific PDE inhibitor. RESULTS: PB inhibited TGF beta 1 induced ePPi elaboration by chondrocytes. PB did not cause membrane injury or decrease NTPPPH activity. Lactate production was decreased by PB but did not correlate with the effects of PB on ePPi elaboration. IBMX did not inhibit TGF beta 1 effect on ePPi elaboration. CONCLUSION: PB blocks TGF beta 1 induced ePPi elaboration. This effect is independent of cell membrane injury, decreased NTPPPH activity, or PDE inhibition. Our data implicate a role for anion transport in TGF beta 1 induced ePPi elaboration, and suggest a potential therapy for CPPD disease.     

Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite

Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.     

A new screening method to detect water-soluble antioxidants: acetaminophen (Tylenol) and other phenols react as antioxidants and destroy peroxynitrite-based luminol-dependent chemiluminescence

This study is based on a simple chemical interaction of peroxynitrite (O = N-O-O-) and luminol, which produces blue light upon oxidation. Since peroxynitrite has a half-life of about 1 s, a drug known as linsidomine (SIN-1) is used as a peroxynitrite generator. Peroxynitrite can oxidize lipids, proteins and nucleic acids. Upon the stimulation of inflammation and/or infection, macrophages and neutrophils can be induced to produce large amounts of peroxynitrite, which can oxidize phenols and sulphhydryl-containing compounds. Therefore, phenols and sulphhydryls eliminate peroxynitrite. This is an example of the Yin-Yang hypothesis e.g. oxidation-reduction. Acetaminophen (Tylenol) can inhibit fever and some types of pain without being a particularly effective anti-inflammatory. Since it is a phenol, it could act as a nitration target for peroxynitrite. Then peroxynitrite, the possible cause of pain and elevated temperature, might be destroyed in the reaction. Acetaminophen is a phenolic compound which produces a clear inhibitory dose-response curve with peroxynitrite in its range of clinical effectiveness. Whether acetaminophen actually works as we suggest is to be proven. Three different types of reaction could decrease the amount of peroxynitrite: (a) interference with base-catalysed opening of the SIN-1 molecule; (b) destruction of one or both substances needed to form it--superoxide and/or nitric oxide; when the SIN-1 degrades to superoxide and nitric oxide, the former may be destroyed by superoxide dismutase (SOD); (c) peroxynitrite may react directly with phenols (mono-, di-, tri- and tetraphenols), possibly by nitration. Nordihydroguaiaretic acid and 2-hydroxyestradiol (catechol estrogen) are potent inhibitors of luminol light emission. Epineprine, isoproterenol, pyrogallol, catechol and ascorbic acid (a classic antioxidant) are all inhibitors of luminol chemiluminescence. Isoproterenol, norepinephrine/and epinephrine first inhibit light but overall stimulate the light production. Initially, SIN-1 degrades to produce peroxynitrite, which reacts with luminol to produce blue light. If any of three catecholamines are present with the reaction that produces light, there is an initial inhibition of light production, and then a marked stimulation. A possible reason for this is that these catechols are oxidized and the metabolized phenol stimulates the production of light from luminol. Also, during oxidation of catecholamines superoxide is sometimes formed, which could stimulate production of peroxynitrite. This simple screening system is introduced to find useful antioxidants against peroxynitrite.     

Genetic and clinical features of sensorineural hearing loss associated with the 1555 mitochondrial mutation

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-beta 1. Type-I and type-II collagen, heat-denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-beta 1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [35S]sulfate and [3H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-beta 1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-beta 1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-beta 1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-beta 1.     

Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.     

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.