Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.     

Guanylyl cyclase and protein kinase G mediate nitric oxide suppression of 5-lipoxygenase metabolism in rat alveolar macrophages

We have previously demonstrated that exogenous nitric oxide (NO) directly inhibits alveolar macrophage (AM) cell-free activity of the enzyme 5-lipoxygenase (5-LO), thereby inhibiting metabolism of arachidonic acid to the important proinflammatory lipid mediators, leukotrienes (LT). Here, we explored the possibility that NO indirectly inhibited AM LT synthesis via activation of soluble guanylyl cyclase (sGC) in rat AM. The selective sGC inhibitor, LY83583, abrogated the suppression of cellular LT synthesis elicited by either exogenous or endogenous NO. A non-NO-dependent activator of sGC, YC-1, also inhibited macrophage LT synthesis. We next determined if sGC-mediated suppression of AM LT synthesis was dependent on protein kinase G (cGK). The selective cGK inhibitor, KT5823, reversed the suppression of cellular 5-LO metabolism following treatment with exogenous NO and YC-1. cGK1 activation resulted in phosphorylation of 5-LO. In contrast to peritoneal macrophages, AM exhibited localization of sGC, cGK1 and cGKII to the cell nucleus. In summary, in addition to its direct effects, NO-induced suppression of 5-LO action can be mediated indirectly through activation of the sGC and cGK pathways in AM. The nuclear localization of enzymes sGC, CGK1 and cGKII in the AM, which also demonstrates preferential nuclear 5-LO expression, may confer tighter regulation of LT synthesis.     

Effects of reactive oxygen and nitrogen species on cyclooxygenase-1 and -2 activities

The effects of reactive oxygen species (superoxide anion radical--O(2)*-, hydrogen peroxide--H(2)O(2) and hydroxyl radical--*OH; the reaction products of xanthine plus xanthine oxidase system) and reactive nitrogen species [nitric oxide--NO*; from 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene--NOC7 and peroxynitrite--ONOO(-)] on the activities of purified cyclooxygenase (COX)-1 and -2 were studied. Xanthine plus xanthine oxidase suppressed the COX-1 and -2 activities in a xanthine oxidase concentration-dependent fashion. This effect was reversed by addition of catalase to the reactive oxygen species-generating system but not by superoxide dismutase or mannitol, indicating that H(2)O(2) is the responsible metabolite. NOC7 activated the COX-1 activity but inhibited the COX-2 activity at concentrations ranging from 1 to 50 microM. Experiments utilizing a NO* antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide revealed that the observed effects of NOC7 are caused by NO*.ONOO(-), a product of NO* and O(2)*-, both activated and inhibited the COX-1 and -2 activities, depending on ONOO(-) concentration. At a low concentration of ONOO(-) (5 microM) there was enhancement of the COX-1 and -2 activities, but with higher concentrations there was suppression of these two enzyme activities (COX-1, at 200 microM; COX-2, >50 microM). These results suggest that H(2)O(2), NO* and ONOO(-) can have different modulatory effects on the COX-1 and -2 activities.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.     

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.     

Nitric oxide mediates distinct effects of various LPS chemotypes on phagocytosis and leukotriene synthesis in human neutrophils

We investigated the effect of lipopolysaccharide (LPS) chemotypes differing in their carbohydrate chain length on phagocytosis of serum-opsonized zymosan (OZ) particles and related functions of human polymorphonuclear leukocyte (PMNL, neutrophils). LPS from deep core mutant (Re), complete core (Ra) and smooth (S) phenotypes of Salmonella typhimurium was studied. Priming of neutrophils with various LPSs caused prominent enhancement of OZ phagocytosis, superoxide production and leukotriene (LT) synthesis in neutrophils, with LPS effects increasing as Re < S < Ra. The LPS forms were less potent to activate OZ uptake in the presence of MK-886, 5-lipoxygenase activating protein inhibitor, suggesting the regulatory function of 5-lipoxygenase (5-LO)-derived LTs. Direct measurement of nitrite release from OZ-stimulated neutrophils revealed that the effects of LPS on NO synthesis increased in the range of Ra < S < Re. Nitric oxide synthase (NOS) inhibitor l-NAME increased phagocytosis, LT and superoxide formation by neutrophils, and abolished the difference in the action of the LPSs forms. Further mechanistic studies revealed that NO modulates cellular 5-LO activity in a guanylyl cyclase and protein kinase G dependent manner, as well as interplay between NO and superoxide, and peroxynitrite generation contribute to distinct effects of LPS chemotypes on phagocytosis and LT synthesis in human neutrophils. Our investigation of the three LPS species demonstrates that the LPS polysaccharide core is mostly essential for the PMNL activation and is able to suppress lipid A-induced increase in NOS activity in phagocyting cells by triggering specific signaling cascades.     

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

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

Membrane association of active 5-lipoxygenase in resting cells. Evidence for novel regulation of the enzyme in the rat alveolar macrophage.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes, substances which play pivotal roles both in normal host defense and in pathologic states of inflammation. Recent studies in granulocytic cells have shown that activation of 5-LO involves its Ca(2+)-dependent translocation from cytosol to membrane compartments. However, little information exists about the molecular regulation of 5-LO in macrophages, even though these cells comprise the resident effector cell population of most organs. We therefore examined the levels of 5-LO activity and immunoreactive protein in cytosol and membrane fractions of resident rat alveolar (AM) and peritoneal macrophages (PM) and compared them with the well studied human neutrophil (polymorphonuclear leukocyte). In the resting state, PM resembled polymorphonuclear leukocyte in that most of their cell-free 5-LO activity, as well as protein content, were localized to the cytosol fraction. By contrast, resting AM contained most of their activity and almost half of their immunoreactive protein in the crude membrane fraction. The inability of the drug MK-886 to reverse this membrane association suggested that the 5-LO-activating protein was not the site of binding in the resting cell; however, this drug completely inhibited leukotriene B4 synthesis in ionophore A23187-stimulated AM, indicating that an interaction between 5-LO and 5-LO-activating protein was nonetheless required for product synthesis upon stimulation. Translocation of cytosolic 5-LO protein could not be convincingly demonstrated in A23187-stimulated AM, suggesting that the pool of 5-LO enzyme responsible for product formation originated in the membrane rather than the cytosol fraction of the resting cell. The AM therefore represents the first mammalian cell in which 5-LO has been recovered from the membrane fraction (a) of a resting cell and (b) in active form. These novel findings extend our understanding of the molecular regulation of 5-LO and may be of importance in designing strategies to limit inflammation in the lung and other sites.     

Regulation of xanthine oxidase by nitric oxide and peroxynitrite

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.     

Activation of p38 MAPK induced peroxynitrite generation in LPS plus IFN-gamma-stimulated rat primary astrocytes via activation of iNOS and NADPH oxidase

We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.     

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.     

Alterations in the pattern of arachidonate metabolism accompany rat macrophage differentiation in the lung

The present study was undertaken to investigate the changes in arachidonic acid (AA) metabolism which accompany rat macrophage (m phi) differentiation in the lung in order to determine whether these changes occur in the alveolar space or in the pulmonary interstitium, as well as the mechanisms responsible for such changes. Metabolism of endogenous and exogenous AA by cultured m phi obtained from the peritoneum (PM), the pulmonary interstitium (IM), and the alveolar spaces (AM) was examined by using HPLC and RIA. Although PM and AM released similar amounts of endogenous AA in response to both ionophore A23187 and the particulate zymosan, PM metabolized AA predominantly to cyclooxygenase (CO) products, whereas AM produced predominantly 5-lypoxygenase (5-LO) metabolites. IM synthesized a profile of eicosanoids which more closely resembled that of PM. Studies of the metabolism of exogenously supplied AA demonstrated that AM indeed had less CO activity than did PM. PM, but not AM, CO activity decreased during prolonged culture in air, suggesting the possibility that oxidative inactivation of CO plays a role in the decline in CO capacity which accompanies m phi differentiation in the lung. In contrast, the greater expression of 5-LO metabolism in AM than PM did not reflect mere differences in enzyme capacity, since upon activation of protein kinase C with PMA or oleoylacetylglycerol, ionophore-stimulated PM produced amounts of 5-LO products which were comparable to the amounts produced by AM stimulated with A23187 alone. These results indicate that increases in 5-LO metabolism and decreases in CO metabolism accompany rat m phi differentiation in the lung, that these changes occur largely in the alveolar space, and that the increased 5-LO capacity and decreased CO capacity are independently regulated by different mechanisms.     

Different patterns of arachidonate metabolism in autologous human blood monocytes and alveolar macrophages

As peripheral blood monocytes (PBM) differentiate into tissue macrophages, they undergo a variety of functional changes. One such difference which has been described is an enhanced metabolism of arachidonic acid (AA) via the 5-lipoxygenase (5-LO) pathway in alveolar macrophages (AM) as compared to PBM. In order to elucidate a possible mechanism for this difference, we compared the metabolism of endogenously released AA mobilized by agonists and of exogenously supplied fatty acid in adherent autologous PBM and AM obtained from six normal subjects. Exogenous AA was metabolized to larger amounts of both cyclooxygenase (CO) and 5-LO products by PBM as compared with AM. Although the two cell types released similar amounts of endogenous AA in response to ionophore A23187, marked differences in the pattern of its metabolism were observed. In PBM, a large proportion of released AA remained unmetabolized, and that which was metabolized was converted predominantly to CO products. In contrast, arachidonate released by AM was efficiently metabolized, predominantly via the 5-LO pathway. Similar results were obtained when cells were stimulated with the particulate zymosan, with PBM synthesizing mainly CO and AM, mainly 5-LO eicosanoids. In addition, culture of PBM for up to 5 days in an aerobic environment did not alter their response to A23187 stimulation. These results suggest that the lesser 5-LO metabolism by PBM than AM is not explained by lesser phospholipase or 5-LO activities, but rather a compartmentalization of the endogenous AA deacylated by phospholipase and the 5-LO enzyme in the PBM. The acquisition of the capacity to metabolize endogenous AA to large quantities of 5-LO products as mononuclear phagocytes differentiate in the lung may equip them with the ability to mount an inflammatory response in the alveolar space.     

Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria.     

Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.     

Spin trapping agent, phenyl N-tert-butylnitrone, reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125-400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

Sulfatides inhibit leukotriene synthesis in human polymorphonuclear granulocytes by a mechanism involving lipid rearrangement in intracellular membranes

Sulfatides - sulfated derivatives of galactocerebroside - are endogenous ligands for P- and L-selectins and are able to induce intracellular signaling in neutrophils through a L-selectin dependent pathway. Sulfatides are implicated in a variety of physiological functions and have been found to suppress the synthesis of 5-lipoxygenase (5-LO) metabolites and impede 5-LO translocation to the nuclear envelope in adherent human polymorphonuclear leukocytes (PMNs) [Sud'ina, G. F., Brock, T. G., Pushkareva, M. A., Galkina, S. I., Turutin, D. V., Peters-Golden, M., et al. (2001). Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase. The Biochemical Journal, 359, 621-629]. In this study we investigated the mechanism of the leukotriene (LT) synthesis inhibition by sulfatides. Sulfatides neither attenuated the ionophore-induced rise in [Ca(2+)](i) nor promoted PKA activation. We demonstrated that sulfatides directly inhibited 5-LO enzyme activity in a cell-free assay. BODIPY-labeled sulfatides were able to rapidly penetrate into the cells. Sulfatides induced rearrangement and redistribution of cytoskeletal components in adherent PMNs. The lipid incorporation as well as sulfatide-induced inhibition of LT synthesis were abolished by cytochalasin D, an inhibitor of actin polymerization and endocytosis. Importantly, sulfatides caused a prominent intracellular cholesterol redistribution, increasing its abundance at the uropod region. On the basis of these data, we suggest that increased cholesterol accumulation in cell compartments represents a novel mechanism by which sulfatides abrogate 5-LO translocation and activation.