Induced Nitric Oxide Release Research Articles

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann–Pick C1 fibroblasts

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.

The free-radical scavenger, edaravone, augments NO release from vascular cells and platelets after laser-induced, acute endothelial injury in vivo

In vitro and in vivo experimental models have demonstrated that vascular endothelial function is significantly impaired as a result of oxidative stress, mediated by the generation of oxygen-derived free radicals in response to chronic or acute inflammation. In particular, super-oxide () at specific concentrations leads to the impairment of nitric oxide (NO) bioactivity, and it is known that NO plays a fundamental role in the maintenance of vascular homeostasis. The relationship between reactive oxygen species (ROS) and NO release in thrombosis-related endothelial damage in the peripheral microvasculature remains unclear, however. The purpose of the present study was to investigate the effect of the free-radical scavenger, edaravone, on NO synthesis and thrombotic potential in arterioles after exposure to laser irradiation. Highly sensitive electrochemical NO microsensors were positioned in femoral arterioles of mice, and the kinetics of NO release were recorded in response to standardized laser irradiation in vivo. In addition, images of NO release from damaged vascular cells were investigated in a similar rat model using the NO-sensitive dye 4,5-diaminofluorescein diacetate (DAF-2DA). Thrombogenesis was assessed in carotid arterioles by continuous video microscopy using image analysis software. Laser irradiation led to NO release from perturbed endothelial cells and from platelet-rich thrombi. Edaravone had no significant effect on NO release in non-laser treated, intact endothelium compared with placebo. In contrast, edaravone demonstrated a dose-dependent effect on NO release and thrombogenicity. At a concentration of 10.5 mg/kg per h, edaravone promoted a 5-fold increase in NO and a reduction in platelet-rich thrombus volume to 58% of the placebo values. Our data provide direct evidence to confirm that acute endothelial damage in peripheral microvessels initially induces NO release and that the free-radical scavenger, edaravone, augments NO synthesis leading to suppression of platelet thrombus formation.

ROLE OF INTERLEUKIN 18 IN NITRIC OXIDE PRODUCTION AND PANCREATIC DAMAGE DURING ACUTE PANCREATITIS

The release of the immunomodulator, interleukin 18 (IL-18) into sera early in acute pancreatitis (AP) corresponds to disease severity. IL-18 induces nitric oxide (NO), which is involved in the pathophysiology of pancreatitis. The objective of this study was to clarify the role of IL-18 in pathogenesis and NO production during early AP using recombinant mouse (rm) IL-18 protein and IL-18 gene knockout (KO) mice. After pretreatment with phosphate-buffered saline or rmIL-18, wild-type (WT) or KO mice were injected intraperitoneally with phosphate-buffered saline (sham) or cerulein (AP) hourly for 3 h. Blood, pancreas, spleen, and liver were collected until 24 h after the first dose. Main outcome measures were serum IL-18, amylase and lipase levels, histological evaluation of the pancreas with parenchyma vacuolization of acinar cells, mRNA expression of inducible NO synthase (iNOS) in the pancreas, and spleen, liver, and plasma NO metabolite level. Serum IL-18 was significantly increased immediately after induction of AP in WT mice. Serum amylase, lipase, and the numbers of acinar cells with parenchyma vacuolization were significantly higher in the group AP/KO than in the group AP/WT, but these parameters were improved by dose-dependent pretreatment with rmIL-18 administration in both groups. Pancreatic iNOS gene expression and plasma NO metabolites were significantly increased by 6 h after the initiation of AP, but were significantly lower in the group AP/KO than in the AP/WT mice. Pretreatment with rmIL-18 also significantly increased these levels in both groups. Splenic and hepatic iNOS expression was not changed after the initiation of AP in WT mice, whereas pretreatment with rmIL-18 also increased these levels. Administration of aminoguanidine, a selective iNOS inhibitor, before AP induction abolished the protective effect of pretreatment with rmIL-18 on pancreatic injury. IL-18 appears to protect the pancreas during early induced-induced AP in mice, probably through induction of NO release from an iNOS source. IL-18 may be a target for new AP therapeutics.

Sesamol induces nitric oxide release from human umbilical vein endothelial cells

Sesamol, which is derived from sesame seed lignans, is reportedly an antioxidant. Nitric oxide (NO), the most important vascular relaxing factor, is regulated in the endothelium. In addition, NO is involved in protecting endothelium and has antiatherosclerotic and antithrombotic activities. The endothelium produces NO through the regulation of both endothelial NO synthase (eNOS) expression and activity in endothelial cells. This study sought to investigate the effect of sesamol on NO released from human umbilical vein endothelial cells (HUVEC) and to examine the expression and activity of eNOS. Sesamol induced NO release from endothelial cells in a dose-dependent manner (from 1 to 10 microM), as measured 24 h after treatment; the expression of the eNOS gene at both transcription and translation levels; and NOS activity in endothelial cells. The content of cGMP was also increased by sesamol through NO signaling. The transcription of eNOS induced by sesamol was confirmed through the activation of PI-3 kinase-Akt (protein kinase B) signaling. The results demonstrate that sesamol induces NOS signaling pathways in HUVEC and suggest a role for sesamol in cardiovascular reactivity in vivo.

Essential Role for Membrane Lipid Rafts in Interleukin-1β−Induced Nitric Oxide Release From Insulin-Secreting Cells

We recently reported that the activation of H-Ras represents one of the signaling steps underlying the interleukin-1beta (IL-1beta)-mediated metabolic dysfunction of the islet beta-cell. In the present study, we examined potential contributory roles of membrane-associated, cholesterol-enriched lipid rafts/caveolae and their constituent proteins (e.g., caveolin-1 [Cav-1]) as potential sites for IL-1beta-induced nitric oxide (NO) release in the isolated beta-cell. Disruption of lipid rafts (e.g., with cyclodextrin) markedly reduced IL-1beta-induced gene expression of inducible NO synthase (iNOS) and NO release from beta-cells. Immunologic and confocal microscopic evidence also suggested a transient but significant stimulation of tyrosine phosphorylation of Cav-1 in beta-cells briefly (for 15 min) exposed to IL-1beta that was markedly attenuated by three structurally distinct inhibitors of protein tyrosine phosphorylation. Overexpression of an inactive mutant of Cav-1 lacking the tyrosine phosphorylation site (Y14F) or an siRNA-mediated Cav-1 knock down also resulted in marked attenuation of IL-1beta-induced iNOS gene expression and NO release from these cells, thus further implicating Cav-1 in this signaling cascade. IL-1beta treatment also increased (within 20 min) the translocation of H-Ras into lipid rafts. Here we provide the first evidence to suggest that tyrosine phosphorylation of Cav-1 and subsequent interaction among members of the Ras signaling pathway within the membrane lipid microdomains represent early signaling mechanisms of IL-1beta in beta-cells.

Release of Nitric Oxide and Endothelium-Derived Hyperpolarizing Factor (EDHF) in Porcine Coronary Arteries Exposed to Hyperkalemia: Effect of Nicorandil

Although the detrimental effect of hyperkalemia on coronary endothelium has been reported, there is no direct evidence regarding the effect of hyperkalemic exposure on nitric oxide (NO) release from the coronary endothelium. In addition, it is unclear whether nicorandil, a KATP channel opener, used as hyperpolarizing cardioplegia or added in hyperkalemic cardioplegic solution may protect endothelial function during cardiac surgery. The present study was designed to clarify NO release and the function of endothelium-derived hyperpolarizing factor (EDHF) in coronary circulation with respect to the effect of hyperkalemia and nicorandil. Nitric oxide was measured by using a NO-specific electrode, and EDHF-mediated relaxation was investigated in a myograph. Substance P- and calcium ionophore A23187-induced NO release was compared in porcine left circumflex coronary arteries before and after 1-hour exposure to 20 mM potassium (K+) at 37 degrees C. In coronary microarteries (diameter 200 to 450 microm), precontracted with U46619, in the presence of indomethacin (7 microM), NG-nitro-L-arginine (300 microM), and oxyhemoglobin (20 microM), EDHF-mediated relaxation was induced by bradykinin (-10 to -6.5 log M) after incubation with Krebs (control) or 20 mM K+ with or without 10 microM nicorandil at 37 degrees C for 1 hour. Neither substance P (58.8 +/- 5.0 versus 66.2 +/- 7.2 nmol/L) nor A23187 (86.6 +/- 9.0 versus 82.4 +/- 9.2 nmol/L in control) induced NO release was altered by hyperkalemic exposure (p > 0.05). In contrast, EDHF-mediated relaxation was decreased from 84.2% +/- 3.8% to 42.3% +/- 6.0% (p < 0.001) that was partially restored by nicorandil (50.7% +/- 5.5%, p < 0.05). Exposure to potassium at 20 mM does not affect NO release but impairs EDHF-mediated relaxation in coronary arteries. Supplementation of nicorandil in hyperkalemic cardioplegia may provide a protective effect on EDHF-related endothelial function.

Screening of Indonesian medicinal plants for inhibitor activity on nitric oxide production of RAW264.7 cells and antioxidant activity

Traditional Indonesian medicinal plants were screened for their inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and for the antioxidant activity through the evaluation of free radical scavenging effect and reducing power. The results of screening indicated that 50 methanolic extracts inhibited (>50%) lipopolysaccharides (LPS)-induced NO release from RAW264.7 cells at 50 μg/ml, with 18 having greater than 100% inhibition. At 200 μg/ml, 61 methanol extracts exhibited inhibitory activity (>50%), with 45 showing greater than 100% inhibition. In addition, the free radical scavenging effects of 6 methanolic extracts were found to be more than 50% for extract concentration of 0.5 μg/ml. The results indicate that the extracts contain active compounds that inhibit NO release and scavenge free radical.

Signal transduction pathways of nitric oxide release in primary microglial culture challenged with gram-positive bacterial constituent, lipoteichoic acid

Between one-third and one-half of all cases of sepsis are known to be caused by gram-positive microorganisms through the cell wall component, e.g. lipoteichoic acid (LTA). Gram-positive bacteria are also known to induce encephalomyelitis and meningeal inflammation, and enhance the production of nitric oxide (NO) via expression of inducible nitric oxide synthase (iNOS) in murine tissue macrophages. It remains to be explored if LTA could activate microglia considered to be resident brain macrophages. We report here that LTA derived from gram-positive bacteria ( Staphylococcus aureus) significantly induces NO release and iNOS expression in primary microglia. LTA-induced NO accumulation was detected at 2 h in microglial culture and was significantly attenuated by pretreatment with anti-CD14, complement receptor type 3 (CR3) or scavenger receptor (SR) antibodies. LTA activated mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, p38 MAPK or c-Jun N-terminal kinase in cultured microglia. LTA-elicited microglial NO production was also drastically suppressed by SB203580 (p38 MAPK inhibitor) or pyrrolidine dithiocarbamate (an inhibitor of nuclear factor κB), indicating that p38 MAPK and nuclear factor κB were involved in microglial NO release after LTA challenge. These results suggest that gram-positive bacterial product such as LTA can activate microglia to release NO via the signal transduction pathway involving multiple LTA receptors (e.g. CD14, CR3 or SR), p38 MAPK and nuclear factor κB. The in vivo study further confirmed that administered intracerebrally LTA induced considerable noticeable iNOS, phospho-IκB and phospho-p38 MAPK expression in microglia/macrophages.

Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production

The mechanism of endothelin-1 (ET-1)-induced nitric oxide (NO) production, MMP-1 production and MMP-13 production was investigated in human osteoarthritis chondrocytes. The cells were isolated from human articular cartilage obtained at surgery and were cultured in the absence or presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP). MMP-1, MMP-13 and NO levels were then measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and phosphorylated forms of p38 mitogen-activated protein kinase, p44/42, stress-activated protein kinase/Jun-N-terminal kinase and serine-threonine Akt kinase were determined by western blot. Results show that ET-1 greatly increased MMP-1 and MMP-13 production, iNOS expression and NO release. LY83583 decreased the production of both metalloproteases below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and was completely suppressed by the protein kinase A kinase inhibitor KT5720 and by LY83583, suggesting the involvement of these enzymes in relevant ET-1 signalling pathways. In human osteoarthritis chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. ET-1 and NO should thus become important target molecules for future therapies aimed at stopping cartilage destruction.

Peripheral blood monocytes show morphological pattern of activation and decreased nitric oxide production during acute Chagas’ disease in rats

Peripheral blood monocytes (PBM) recruitment is a rapid and remarkable phenomenon during acute infection with the intracellular protozoan parasite Trypanosoma cruzi, the causative agent of Chagas’ disease. The functional capabilities of these cells during the infection, however, are poorly understood. The purpose of the present study was to determine whether PBM are morphologically activated and produce nitric oxide (NO), a mediator of host cell defense when challenged with the parasite at different time points of acute disease. In parallel, the parasite load was monitored in the blood and heart, a target organ of the disease, as well as the PBM numbers. The infection did not induce NO release by PBM, although these cells exhibited a clear morphological pattern of activation characterized by irregular surface, increase of organelle amount, especially Golgi complex, and cell size. On the contrary, there was significant inhibition of NO production by PBM at the beginning (day 6) and end of acute disease (day 20). At this time, the levels of NO were inversely related to the arginase activity, an enzyme that affects the NO synthesis. The mobilization process of PBM occurred in parallel to parasite load and was associated with the resolution mechanism of parasitemia and heart parasitism. Our results showed that activated PBM are notably involved in the host response to the acute T. cruzi infection in rats. However, the in vivo NO production by these cells seems to be inhibited during the acute Chagas’ disease through a mechanism involving the arginase pathway.

Interaction of NO and VIP in gastrointestinal smooth muscle relaxation.

Gastrointestinal (GI) smooth muscle cell activity is controlled by contractile cholinergic neurons and relaxant non-adrenergic non-cholinergic (NANC) neurons in the myenteric plexus between the circular and longitudinal muscle layer. Decreased or increased NANC relaxation might be involved in the pathophysiology of functional GI motility disorders. Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) are the primary inhibitory NANC neurotransmitters. As classic neurotransmitters, VIP is stored in vesicles in the nerve endings, while NO is synthetized on demand by the neuronal isoform of NO synthase (nNOS). The VIP/nNOS co-localization in myenteric neurons, reported for various regions of the GI tract in different species, suggests that VIP and NO are co-transmitters. At the presynaptic level, VIP and NO can induce each others release. Most clear-cut evidence for this mechanism was obtained in isolated myenteric ganglia where VIP induced NO release, and NO facilitated VIP release. At the postsynaptic level, many studies support that VIP and NO are parallel co-transmitters, acting via the adenylate cyclase/3'5' adenosine cyclic monophosphate (cAMP) and guanylate cyclase/3'5' cyclic guanosine monophosphate pathway respectively. Mainly based on results obtained in isolated GI smooth muscle cells, a serial postsynaptic VIP/NO interaction model was proposed, whereby VIP is the principle neurotransmitter, acting partially via a VPAC receptor and the adenylate cyclase/cAMP pathway but also by induction of muscular NO production. Recent results suggest that the capacity of VIP to release NO from isolated smooth muscle cells is related to the induction of inducible NOS (iNOS) in the cells during the isolation procedure. The relative contribution of NO and VIP to GI NANC relaxation differs upon tissue and nerve firing frequency, so that interference with either of them will lead to varying effects.

Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis.

In view of the presence of a large number of epithelial cells in the alveoli of the lung and their ability to produce various cytokines and chemokines, the possible role of alveolar epithelial cells in the innate immune response to tuberculosis was examined. The human alveolar epithelial cell line A549 was used as a model. The ability of A549 cells to induce nitric oxide (NO) in response to Mycobacterium tuberculosis infection was taken as an in vitro correlate of innate immunity. M. tuberculosis infection induced A549 cells to produce significant levels of NO and to express inducible nitric oxide synthase mRNA at 48 hr of infection. However, the amount of NO released at this point was not mycobactericidal. Cytokine stimulation (interferon-gamma, tumour necrosis factor-alpha, interleukin-1beta, alone or in combination) of the infected A549 cells induced a higher concentration of NO. The study of colony-forming units (CFU) as a measure of the mycobactericidal capacity of A549 cells revealed a reduction in CFU of M. tuberculosis by 39.29% (from 10.62 +/- 0.48 - 6.392 +/- 0.54) following cytokine stimulation of the infected cells. Interestingly gamma-irradiated M. tuberculosis H37Rv could also induce higher than basal level of NO. Therefore we examined mycobacterial antigenic components for their possible role in NO production. We observed that A549 cells produced significantly higher amounts of NO at 48 hr when treated with mycobacterial whole cell lysates, cell wall or cell membrane preparations. The release of NO and the resultant mycobactericidal activity could be further enhanced by simultaneously conditioning the M. tuberculosis infected A549 cells with cytokine and mycobacterial components. These results suggest that alveolar epithelial cells respond to their microenvironment, which is constituted of various cytokines and macrophage-processed antigens and may contribute to the innate immune response to tuberculosis.

Direct demonstration of nitric oxide formation in organs of rabbits treated by transdermal glyceryl trinitrate using an in vivo spin trapping technique.

Glyceryl trinitrate (GTN) is commonly delivered by a patch for the treatment of angina pectoris. The idea is now generally accepted that GTN requires a biotransformation process that activates the drug, in particular through nitric oxide (NO) generation. However, the pharmacokinetics of NO delivery from GTN still remains obscure. The objective of this study was to assess GTN-derived NO formation in vascular tissues and organs in rabbit given GTN patches. NO levels were evaluated in rabbits after 3 h of treatment with a 10 mg GTN patch (GTN group; n = 7) or a placebo patch (CTL; n = 7). Nitrosylhaemoglobin (HbNO) was evaluated by electron spin resonance (ESR) spectroscopy in red cell suspension. In vivo spin trapping technique using FeMGD as a spin trap, associated with ESR was used to quantify NO in tissues. The NO-spin trap complex, which is a relatively stable product, has been measured in several tissues. The ESR spectrum corresponding to HbNO was not found in red cell of GTN or CTL rabbits. The spectrum corresponding to the NO-spin trap complex was observed in all analysed tissues of CTL rabbits. The signal was significantly increased in liver, renal medulla, heart left ventricle and spleen of GTN-treated rabbits, and to a lesser extent in right ventricle and lung. No difference was shown between NO-spin trap levels measured in aorta or inferior vena cava from GTN or CTL rabbits. These data suggest that GTN patch treatment induced NO release, and that tissue-specific differences in transdermal GTN-derived NO exist. The GTN-NO pathway appears to be largely involved in organs such as the liver, kidney and heart.

Wortmannin enhances lipopolysaccharide-induced inducible nitric oxide synthase expression in microglia in the presence of astrocytes in rats

Microglia, the primary inflammatory cells in the brain, are activated upon brain injury. Activated microglia produce nitric oxide (NO), a major toxin to neuronal cells. It has been reported that astrocytes inhibit microglial activation. In this study, we found that wortmannin, a natural inhibitor of phosphatidylinositol 3-kinase, significantly increased lipopolysaccharide (LPS)-induced NO release and inducible nitric oxide synthase (iNOS) expression in microglia in the presence but not in the absence of astrocytes. In response to LPS even in the presence of wortmannin, iNOS immunoreactivity was detected in microglia but not in astrocytes. These results suggest that astrocytes could regulate microglia-mediated brain inflammation by inhibiting microglial NO release/iNOS expression via a wortmannin-sensitive mechanism.

Syndecan-4 modulates basic fibroblast growth factor 2 signaling in vivo.

Syndecan-4 is one of the principal heparan sulfate-carrying proteins on the cell surface. Unlike other members of syndecan family, syndecan-4 mediates phosphatidylinositol 4,5-bisphosphate 2 (PIP(2))-dependent PKC-alpha activation, and overexpression of syndecan-4 in vitro results in enhanced FGF2 signaling. The present study was designed to test the functional effect of increased syndecan-4 expression in endothelial cells in transgenic mice. Several transgenic mice lines expressing syndecan-4 cDNA under control of human endothelial nitric oxide (NO) synthase (eNOS) promoter were generated. Exogenous syndecan-4 was mainly expressed in the heart, brain, and lungs. In particular, the heart demonstrated the greatest increase in the ratio of transgenic-to-native syndecan-4 gene expression. Vessels from the eNOS-syndecan-4 mice demonstrated more pronounced vasodilation to FGF2 but not to VEGF-A(165), sodium nitroprusside, and A 23187 compared with wild-type mice. To elucidate the mechanism of this effect, we measured NO release from primary cardiac endothelial cells isolated from transgenic or wild-type adult mice. Cells from the eNOS-syndecan-4 transgenic mice had a significant increase in FGF2- and VEGF-A(165)-induced NO release compared with endothelial cells from the wild-type mice. However, the absolute magnitude of this increase was higher for FGF2 than VEGF-A(165). In conclusion, enhanced syndecan-4 expression in mouse cardiac endothelial cells results in preferential augmentation of FGF2 but not VEGF-A(165)-induced NO release.

The Endocannabinoid System Protects Rat Glioma Cells Against HIV-1 Tat Protein-induced Cytotoxicity: MECHANISM AND REGULATION

Cannabinoids modulate nitric oxide (NO) levels in cells of the central nervous system. Here we studied the effect of cannabinoid CB(1) and CB(2) receptor agonists on the release of NO and cell toxicity induced by the human immuno-deficiency virus-1 Tat protein (HIV-1 Tat) in rat glioma C6 cells. The CB(1) and CB(2) agonist WIN 55,212-2 inhibited the expression of inducible NO synthase (iNOS) and NO release caused by treatment of C6 cells with HIV-1 Tat and interferon-gamma (IFN-gamma). The effect of WIN 55,212-2 was uniquely due to CB(1) receptors, as shown by experiments carried out with selective CB(1) and CB(2) receptor agonists and antagonists. CB(1) receptor stimulation also inhibited HIV-1 Tat + IFN-gamma-induced and NO-mediated cell toxicity. Moreover, cell treatment with HIV-1 Tat + IFN-gamma induced a significant inhibition of CB(1), but not CB(2), receptor expression. This effect was mimicked by the NO donor GSNO, suggesting that the inhibition of CB(1) expression was due to HIV-1 Tat + IFN-gamma-induced NO overexpression. HIV-1 Tat + IFN-gamma treatment also induced a significant inhibition of the uptake of the endocannabinoid anandamide by C6 cells with no effect on anandamide hydrolysis. These findings show that the endocannabinoid system, through the modulation of the l-arginine/NO pathway, reduces HIV-1 Tat-induced cytotoxicity, and is itself regulated by HIV-1 Tat.

Inhibition of LPS-induced p42/44 MAP kinase activation and iNOS/NO synthesis by parthenolide in rat primary microglial cells

Nitric oxide (NO) has been implicated in the etiopathology of central nervous system (CNS) diseases such as multiple sclerosis (MS). Inhibition of NO synthesis has been proposed to be a possible mechanism of action of relevance in the treatment of multiple sclerosis and migraine. Here, we investigated the effect of parthenolide on inducible NO synthase (iNOS) synthesis and NO release using primary rat microglia. We found parthenolide to be an inhibitor of iNOS/NO synthesis. Investigating the molecular mechanisms by which parthenolide prevents iNOS/NO synthesis, we found that parthenolide inhibits the activation of p42/44 mitogen-activated protein kinase (MAPK), but not IkBalpha (IκBα) degradation or nuclear factor-kappaB (NF-κB) p65 activation. The data suggest that parthenolide might have a potential in the treatment of CNS diseases where NO is part of the pathophysiology.

Control of Nitric Oxide Dynamics by Guanylate Cyclase in Its Activated State

Soluble guanylate cyclase (sGC) is the target of nitric oxide (NO) released by nitric-oxide synthase in endothelial cells, inducing an increase of cGMP synthesis in response. This heterodimeric protein possesses a regulatory subunit carrying a heme where NO binding occurs, while the second subunit harbors the catalytic site. The binding of NO and the subsequent breaking of the bond between the proximal histidine and the heme-Fe(2+) are assumed to induce conformational changes, which are the origin of the catalytic activation. At the molecular level, the activation and deactivation mechanisms are unknown, as is the dynamics of NO once in the heme pocket. Using ultrafast time-resolved absorption spectroscopy, we measured the kinetics of NO rebinding to sGC after photodissociation. The main spectral transient in the Soret band does not match the equilibrium difference spectrum of NO-liganded minus unliganded sGC, and the geminate rebinding was found to be monoexponential and ultrafast (tau = 7.5 ps), with a relative amplitude close to unity (0.97). These characteristics, so far not observed in other hemoproteins, indicate that NO encounters a high energy barrier for escaping from the heme pocket once the His-Fe(2+) bond has been cleaved; this bond does not reform before NO recombination. The deactivation of isolated sGC cannot occur by only simple diffusion of NO from the heme; therefore, several allosteric states may be inferred, including a desensitized one, to induce NO release. Thus, besides the structural change leading to activation, a consequence of the decoupling of the proximal histidine may also be to induce a change of the heme pocket distal geometry, which raises the energy barrier for NO escape, optimizing the efficiency of NO trapping. The non-single exponential character of the NO picosecond rebinding coexists only with the presence of the protein structure surrounding the heme, and the single exponential rate observed in sGC is very likely to be due to a closed conformation of the heme pocket. Our results emphasize the physiological importance of NO geminate recombination in hemoproteins like nitric-oxide synthase and sGC and show that the protein structure controls NO dynamics in a manner adapted to their function. This control of ligand dynamics provides a regulation at molecular level in the function of these enzymes.

Articular Chondrocytes from Animals with a Dermatan Sulfate Storage Disease Undergo a High Rate of Apoptosis and Release Nitric Oxide and Inflammatory Cytokines: A Possible Mechanism Underlying Degenerative Joint Disease in the Mucopolysaccharidoses

Mucopolysaccharidosis (MPS) Type VI (Maroteaux-Lamy Disease) is the lysosomal storage disease characterized by deficient arylsulfatase B activity and the resultant accumulation of dermatan sulfate-containing glycosaminoglycans (GAGs). A major feature of this and other MPS disorders is abnormal cartilage and bone development leading to short stature, dysostosis multiplex, and degenerative joint disease. To investigate the underlying cause(s) of degenerative joint disease in the MPS disorders, articular cartilage and cultured articular chondrocytes were examined from rats and cats with MPS VI. An age-progressive increase in the number of apoptotic chondrocytes was identified in the MPS animals by terminal transferase nick-end translation (TUNEL) staining and by immunohistochemical staining with anti-poly (ADP-ribose) polymerase (PARP) antibodies. Articular chondrocytes grown from these animals also released more nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) into the culture media than did control chondrocytes. Notably, dermatan sulfate, the GAG that accumulates in MPS VI cells, induced NO release from normal chondrocytes, suggesting that GAG accumulation was responsible, in part, for the enhanced cell death in the MPS cells. Coculture of normal chondrocytes with MPS VI cells reduced the amount of NO release, presumably because of the release of arylsulfatase B by the normal cells and reuptake by the mutant cells. As a result of the enhanced chondrocyte death, marked proteoglycan and collagen depletion was observed in the MPS articular cartilage matrix. These results demonstrate that MPS VI articular chondrocytes undergo cell death at a higher rate than normal cells, because of either increased levels of dermatan sulfate and/or the presence of inflammatory cytokines in the MPS joints. In turn, this leads to abnormal cartilage matrix homeostasis in the MPS individuals, which further exacerbates the joint deformities characteristic of these disorders.

Sphingomyelinase but not ceramide induces nitric oxide synthase expression in rat brain microglia

Microglia, brain inflammatory cells, are activated in injured brain and function similar to macrophages. The activated microglia produce nitric oxide (NO), a major toxic substance from these cells, by inducing expression of inducible NO synthase (iNOS). In this study, we found that sphingomyelinase (SMase) alone induced NO release/iNOS mRNA expression in cultured rat brain microglia. On the contrary to SMase, however, membrane-permeable c2-ceramide had little effect on NO release/iNOS mRNA expression. Fumonisin B1, an inhibitor of de novo synthesis of ceramide, did not reduce lipopolysaccharide (LPS)-induced NO release. However, neither SMase nor c2-ceramide enhanced LPS- or Aβ (25–35)-induced NO release/iNOS mRNA expression.