5-LOX Activity Research Articles

Anti-inflammatory activity of myricetin-3-O-beta-D-glucuronide and related compounds.

The anti-inflammatory effect of myricetinglucuronide (MGL) was investigated and structurally-related compounds were compared to examine the structure/activity-relationship in carrageenan-induced rat paw edema. In vitro studies were performed using rat basophilic leukemia (RBL-1) cells, human polymorphonuclear leukocytes (PMNL), COX-1 from ram seminal vesicle, COX-2 from sheep placenta and human venous blood. For the in vivo tests male Wistar rats were used, for the ex vivo test perfused rabbit ears. 1-300 microg/kg MGL or myricetinmethylglucuronate and 0.1-5 mg/kg other related compounds administered p.o. (carrageenan edema). 5, 50 and 150 microg/kg MGL p.o. for 14 days (Freund's adjuvant arthritis), 5 and 50 microg/kg p.o. for 6 days (ulceration). Anti-inflammatory effects were measured in carrageenan edema and in adjuvant arthritis. Incidence of gastric lesions was tested in an ulcerogenicity model in vivo. Influence on COX was determined in the perfused rabbit ear, in PMNL and in a test assay using COX-1 and COX-2. 5-LOX activity was studied using PMNL and RBL-1. The influence on platelet aggregation was evaluated measuring light transmission. MGL exerted a marked and dose-dependent anti-inflammatory effect in acute (carrageenan edema, ED50 15 microg/kg, indomethacin ED50 10 mg/kg) and chronic (adjuvant arthritis, inhibition at 150 microg/kg 18.1 % left paw, 20.6% right paw, indomethacin 3 mg/kg 18.0% and 19.4%)) models of inflammation. In the perfused rabbit ear 1 microg MGL inhibited the release of PGI2, PGD2 and PGE2 to the same extent as 1 microg indomethacin. The inhibition of COX-1 in the intact cell system was IC50 = 0.5 microM, that of indomethacin 0.0038 microM. In the isolated enzyme preparations of COX-1 and COX-2 the IC50 was 10 microM and 8 microM, that of indomethacin 9.2 mM and 2.4 microM. In the RBL-1 and PMNL test assay the inhibition of 5-LOX was 0.1 microM and 2.2 microM. An orally administered dose of 50 microg/kg/day induced no gastric ulcers in rats treated for 6 days. The investigations on carrageenan edema showed a close relationship between the structure of MGL and the anti-inflammatory effect. MGL is a COX-1, COX-2 and 5-LOX inhibitor. In view of the moderate in vitro activity and the very potent in vivo activity an additive mechanism must be involved. Small changes in the molecular structure lead to the loss or reduction of the anti-inflammatory activity.

Distinct signal transduction pathways mediate nuclear factor-kappaB induction by IL-1beta in epithelial and lymphoid cells.

We previously demonstrated that IL-1beta-mediated induction of the nuclear factor-kappaB (NF-kappaB) transcription factor proceeds through the production of reactive oxygen intermediates in lymphoid cells, while it occurs independently of any oxidative stress in epithelial transformed cells. Indeed, inhibition of receptor internalization as well as NH4Cl and chloroquine blocked IL-1beta-mediated induction of NF-kappaB in OVCAR-3 and in other epithelial cell lines but not in lymphoid cells, indicating that distinct pathways are involved. Conversely, while we observed phospholipase A2 activity in both cell types following IL-1beta stimulation, specific inhibitors of this enzyme inhibited NF-kappaB induction only in lymphoid cells. Moreover, expression of the 5-lipoxygenase (5-LOX) enzyme was not detected in epithelial cells, and inhibition of this enzyme blocked NF-kappaB induction by IL-1beta only in lymphoid cells. This study thus indicates that the activation of NF-kappaB following IL-1beta treatment involves the activation of phospholipase A2 and 5-LOX and the production of reactive oxygen intermediates (ROIs) in lymphoid cells, while in epithelial cells, another pathway predominates and could involve the acid sphingomyelinase. Moreover, arachidonic acid could induce NF-kappaB in epithelial and lymphoid cells, but this activation involved the 5-LOX enzyme and the production of ROIs only in lymphoid cells. The inefficiency of the ROI pathway in epithelial cells is probably the consequence of both low ROI production due to undetectable expression of 5-LOX and rapid degradation of hydrogen peroxide due to high catalase activity.

Dual inhibitory activities of tannins from Hamamelis virginiana and related polyphenols on 5-lipoxygenase and lyso-PAF: acetyl-CoA acetyltransferase.

In the present study, the effects of tannins obtained from various plant sources on the activity of 5-LOX and AT were examined. With IC50 values ranging from 1.0 to 18.7 muM, hamamelitannin and the galloylated proanthocyanidins isolated from Hamamelis virginiana L. were found to be most potent inhibitors of 5-LOX. Unlike the 5-LOX study, hamamelitannin proved to be ineffective in the AT assay. Potent candidates are represented by the group of B-type proanthocyanidins. Structure-activity relationships regarding the in vitro inhibitory potency of the polyphenols in the biological assays are discussed.

Correlation between arachidonic acid oxygenation and luminol-induced chemiluminescence in neutrophils: Inhibition by diethyldithiocarbamate

Neutrophils from allergic subjects were hypersensitive to stimulation by low calcium ionophore concentration (0.15 μM), resulting in an increased formation of leukotriene B 4 (LTB 4), 5S-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE), and other arachidonic acid metabolites through the 5-lipoxygenase pathway. In parallel, luminol-dependent chemiluminescence was also higher in neutrophils from allergic patients at the basal state and after stimulation by calcium ionophore, revealing an enhancement of radical oxygen species and peroxide production. The activity of glutathione peroxidase, the main enzyme responsible for hydroperoxide reduction, was lowered in these cells. Diethyl-dithiocarbamate (DTC) induced a concentration-dependent decrease in chemiluminescence and arachidonic acid metabolism after neutrophil stimulation. These data show that the elevation of arachidonic acid metabolism in neutrophils from allergic patients is strongly correlated with oxidative status. This elevation may be the consequence of an increased cellular hydroperoxide known to activate 5-lipoxygenase (5-LOX) activity and/or an increased arachidonic acid availability, due either to phospholipase A 2 (PLA 2) activation or inhibition of arachidonate reesterification into phospholipids. Lowering this oxidative status was associated with a concomitant decrease of this metabolism. Our results suggest that the effect of DTC may be the consequence of an inhibition of peroxyl radical and cellular lipid hydroperoxide production. Thus, DTC may modulate arachidonic acid metabolism in neutrophils by modulating the cellular hydroperoxide level.

Acetyl-11-keto-beta-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity.

1. 5-Lipoxygenase (5-LOX) products from endogenous arachidonic acid in ionophore-stimulated peritoneal polymorphonuclear leukocytes (PMNL) and from exogenous substrate (20 microM) in 105,000 g supernatants were measured. 2. The effects of natural pentacyclic triterpenes and their derivatives on 5-LOX activity were compared with the inhibitory action of acetyl-11-keto-beta-boswellic acid (AKBA), which has been previously shown to inhibit the 5-LOX by a selective, enzyme-directed, non-redox and non-competitive mechanism. 3. The 5-LOX inhibitory potency of AKBA was only slightly diminished by deacetylation of the acetoxy group or reduction of the carboxyl function to alcohol in intact cells (IC50 = 1.5 vs. 3 and 4.5 microM, respectively) and in the cell-free system (8 vs. 20 and 45 microM). 4. beta-Boswellic acid (beta-BA), lacking the 11-keto function, inhibited 5-LOX only partially and incompletely, whereas the corresponding alcohol from beta-BA, as well as amyrin, acetyl-11-keto-amyrin, 11-keto-beta-boswellic acid methyl ester had no 5-LOX inhibitory activity up to 50 microM in either system. 5. beta-BA only partially prevented the AKBA-induced 5-LOX inhibition, whereas the non-inhibitory compounds, amyrin and acetyl-11-keto-amyrin, almost totally antagonized the AKBA effect and shifted the concentration-inhibition curve for the incomplete inhibitor beta-BA to the right. In contrast, the non-inhibitory 11-keto-beta-BA methyl ester exerted no antagonizing effect. 6. The results demonstrate that the pentacyclic triterpene ring system is crucial for binding to the highly selective effector site, whereas functional groups (especially the 11-keto function in addition to a hydrophilic group on C4 of ring A) are essential for 5-LOX inhibitory activity.

Ca 2+ ionophore A23187-stimulated secretion of azurophil granules in human polymorphonuclear leukocytes is largely mediated by endogenously formed leukotriene B 4

The mode of action of the new leukotriene synthesis inhibitor BAY X1005 (( R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) and structurally-related quinoline derivatives is reflected by the binding to a high-affinity binding site presumably identical to FLAP (five lipoxygenase activating protein). In addition to FLAP, we have identified a second BAY X1005 (low-affinity) binding site localized in the granule fraction of human PMNL (polymorphonuclear leukocytes). Based on the hypothesis that the corresponding target protein might be involved in the regulation of granule release, the influence of the leukotriene synthesis inhibitors BAY X1005 and MK-886 and the direct 5-LOX (5-lipoxygenase, EC 1.13.11.34) inhibitor A-64077 on the A23187- and fMLP ( N-formyl-methionyl-leucyl-phenylalanin)-stimulated release of β-glucuronidase (as a marker for azurophil granules) and vitamin B 12-binding protein (as a marker for specific granules) was investigated. In contrast to MK-886, neither BAY X1005 nor A-64077 significantly affected fMLP-stimulated granule release. This was also true for the A23187-stimulated release of specific granules; however, under the same conditions the A23187-stimulated release of azurophil granules was almost totally inhibited by all three compounds. No obvious relationship between the corresponding ic 50 values and the ability of these compounds to compete for BAY X1005 binding at the low-affinity binding site existed. Instead, by extending these studies to additional inhibitors, a correlation between the ic 50 values for inhibition of A23187-stimulated (i) β-glucuronidase release and (ii) LTB 4 (leukotriene B 4) synthesis was found ( r = 0.969, N = 7). This relationship was independent of the mode of action of the compounds, namely direct 5-LOX inhibition or indirect 5-LOX inhibition mediated via binding to FLAP. These results suggest that 5-LOX metabolites may be involved in A23187-stimulated azurophil granule release. Of the two main biologically active 5-LOX metabolites synthesized under these conditions (LTB 4 and 5-hydroxyeicosatetraenoic acid), only LTB 4 stimulated β-glucuronidase release to nearly the same extent as A23187. In addition, this metabolite significantly enhanced A23187-stimulated β-glucuronidase release, but only at A23187 concentrations (⩾ 0.25 μmol L ) which by themselves were not sufficient to trigger LTB 4 formation. Moreover, the inhibition of A23187-stimulated β-glucuronidase release by BAY X1005 or A-64077 was totally reversed by the addition of LTB 4. Taken together, these results indicate that: (i) the BAY X1005 low-affinity binding site does not play a crucial role in the granule release process; (ii) compounds interfering with 5-LOX activity inhibit the release of azurophil granules upon A23187 stimulation; and (iii) this effect is largely due to the inhibition of endogenously synthesized LTB 4.

Inversely-correlated inhibition of human 5-lipoxygenase activity by bay X1005 and other quinoline derivatives in intact cells and a cell-free system—implications for the function of 5-lipoxygenase activating protein

A series of quinoline derivatives were analysed for the influence on leukotriene synthesis as a parameter for 5-LOX (EC 1.13.11.34) activity in a cell-free system of the 10,000 g supernatant of human PMNL (polymorphonuclear leukocytes). The ratios of the ic 50 values for leukotriene synthesis inhibition in this cell-free system and in A23187-stimulated intact PMNL ranged from 1–1100. Consequently, plotting of the two values resulted in a random distribution ( r = −0.281, N = 18) suggesting that no relationship between the inhibition of leukotriene synthesis in the cell-free system and in intact cells exists. At first sight this finding was not surprising since we have shown earlier that in intact cells this class of quinoline derivatives shares the same mode of action as MK-886, i.e. an indirect inhibition of 5-LOX activity by binding to FLAP. However, we found that the potency of these compounds in intact cells is strongly influenced by the K value (partition coefficient) which is a parameter for the ability of a substance to accumulate in a lipid (membrane) phase compared to the water phase. Therefore, the ic 50 values for leukotriene synthesis inhibition in intact PMNL were corrected for the corresponding K value of the compounds and the resulting values again plotted against the ic 50 values for inhibition of leukotriene synthesis in the cell-free system. As a result, a significant correlation ( r = −0.878, N = 18) was obtained. In order to simplify this relationship the influence of the partition coefficient was eliminated by comparing compounds with about the same K value ( K = 7243 ± 1646, N = 7). As a result, the ic 50 values for inhibition of leukotriene synthesis in the 10,000 g supernatant fraction (indicative for the affinity of the compounds to 5-LOX) and in intact cells (indicative for the affinity of the compounds to FLAP) were highly, but inversely correlated ( r = −0.992). That means that a compound with a high affinity to 5-LOX will have a low affinity to FLAP and vice versa. We hypothesized that this pharmacologically obtained relationship could be indicative of a physiologically occurring equivalent. We therefore propose a model in which FLAP binds arachidonic acid as its physiological substrate with low affinity and allows 5-LOX to get access to its substrate (assuming a higher affinity of 5-LOX to arachidonic acid) after 5-LOX translocation from the cytosol to the membrane. In support of this model we provide evidence that arachidonic acid and other cis-unsaturated fatty acids, but neither a trans-unsaturated nor a saturated fatty acid, inhibit BAY X1005 binding to FLAP in intact human PMNL.

Rapid Stimulation of 5-Lipoxygenase Activity in Potato by the Fungal Elicitor Arachidonic Acid

The activity of lipoxygenase (LOX) in aged potato tuber discs increased by almost 2-fold following treatment of the discs with the fungal elicitor arachidonic acid (AA). Enzyme activity increased above that in untreated discs within 30 min after AA treatment, peaked at 1 to 3 h, and returned to near control levels by 6 h. The majority of the activity was detected in a soluble fraction (105,000g supernatant), but a minor portion was also associated with a particulate fraction enriched in microsomal membranes (105,000g pellet); both activities were similarly induced. 5-Hydroperoxyeicosatetraenoic acid was the principal product following incubation of these extracts with AA. Antibodies to soybean LOX strongly reacted with a protein with a molecular mass of approximately 95-kD present in both soluble and particulate fractions whose abundance generally corresponded with LOX activity in extracts. LOX activity was not enhanced by treatment of the discs with nonelicitor fatty acids or by branched beta-glucans from the mycelium of Phytophthora infestans. Prior treatment of the discs with abscisic acid, salicylhydroxamic acid, or n-propyl gallate, all of which have been shown to suppress AA induction of the hypersensitive response, inhibited the AA-induced increment in LOX activity. Cycloheximide pretreatment, which abolishes AA elicitor activity for other responses such as phytoalexin induction, did not inhibit LOX activity in water- or elicitor-treated discs but enhanced activity similar to that observed by AA treatment. The elicitor-induced increase in 5-LOX activity preceded or temporally paralleled the induction of other studied responses to AA, including the accumulation of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase and phenylalanine ammonia lyase reported here. The results are discussed in relation to the proposed role of the 5-LOX in signal-response coupling of arachidonate elicitation of the hypersensitive response.

The inhibition of 5-lipoxygenase by RG 6866.

The generation of leukotrienes C4, D4 and E4 from arachidonic acid is dependent upon the activity of 5-lipoxygenase (5-LOX). The effects of RG 6866 (N-methyl-4-benzyloxyphenylacetohydroxamic acid) on the activity of guinea pig 5-LOX in vitro and in vivo were determined in the present study. The generation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from arachidonic acid by isolated guinea pig peritoneal polymorphonuclear (PMN) cells was inhibited by incubation with RG 6866 (IC50 = 0.20 microM). A similar effect (IC50 = 0.23 microM) was observed when 5-HETE production was measured in a supernatant fraction from PMNs. Additionally, the compound did not inhibit 3H-LTD4 binding to guinea pig membranes. In actively sensitized guinea pigs pretreated with indomethacin, propranolol and pyrilamine, RG 6866 inhibited antigen-induced systemic anaphylaxis and LTD4-dependent bronchoconstriction in a dose-dependent manner following oral administration. In the pulmonary anaphylaxis model, significant (p less than 0.05) inhibition of the mortality was observed within 30 min and maintained through four hours after treatment with RG 6866 (50 mg/kg i.g.). Finally, orally administered RG 6866 inhibited the formation of LTC4 in these animals with an ED50 = 24.0 mg/kg. These findings indicate that RG 6866 is an inhibitor of 5-LOX both in vitro and in vivo.