Purification and Properties of a New l-Fucose-specific Lectin Produced by Streptomyces No. 100–2

The inhibitory effects of 3-nitro-2,4,6-trihydroxybenzamide derivatives on human 5-lipoxygenase (5-LO), a key enzyme in arachidonic acid cascades, were examined using 5-LO produced by Escherichia coli. Some of the tested compounds inhibited the conversion of arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), and in particular the N-phenylbutyl derivative was about 30 times more active (IC₅₀ = 35 μm) than caffeic acid (IC₅₀ = 1000 μm), a known selective 5-LO inhibitor.     

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BackgroundSubcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.MethodsFLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.Results5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca^2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB₄. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.ConclusionThe cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.General significanceWe present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.     

Distinct parts of leukotriene C4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein

Leukotriene C₄ is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C₄ synthase (LTC₄S) participate in its biosynthesis. We report evidence that LTC₄S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC₄S. FLAP bound to the N-terminal part/first hydrophobic region of LTC₄S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC₄S. Fluorescent FLAP- and LTC₄S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC₄S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC₄S. Direct interaction of 5-LO and LTC₄S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC₄S.     

4-Hydroxynonenal enhances CD36 expression on murine macrophages via p38 MAPK-mediated activation of 5-lipoxygenase

Increased levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) coexist in atherosclerotic lesions but their relationship in atherogenesis is unclear. This study investigated the role of 5-LO in HNE-induced CD36 expression and macrophage foam cell formation, and the link between HNE and 5-LO. In J774A.1 murine macrophages, HNE (10 μM) enhanced CD36 expression in association with an increased uptake of oxLDL, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor, or with 5-LO siRNA. In peritoneal macrophages from 5-LO-deficient mice, HNE-induced CD36 expression was markedly attenuated, confirming a pivotal role of 5-LO in HNE-induced CD36 expression. In an assay for 5-LO activity, stimulation of macrophages with HNE led to increased leukotriene B₄ production in the presence of exogenous arachidonic acid in association with an increased association of 5-LO to the nuclear membrane. Among the mitogen-activated protein kinase (MAPK) pathways involved in 5-LO phosphorylation, HNE predominantly activated p38 MAPK in macrophages, and the p38 MAPK inhibitor SB203580, but not an extracellular signal-regulated kinase inhibitor, suppressed HNE-induced LTB₄ production. Collectively, these data suggest that p38 MAPK-mediated activation of 5-LO by HNE might enhance CD36 expression, consequently leading to the formation of macrophage foam cells.     

5-Lipoxygenase plays an essential role in 4-HNE-enhanced ROS production in murine macrophages via activation of NADPH oxidase

Abstract

4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes; however, its role in the generation of reactive oxygen species (ROS) in macrophages is still unclear. Thus, this study investigated the sources and mechanisms of ROS generation in macrophages stimulated with HNE. Exposure of J774A.1 cells to HNE showed an increased production of ROS, which was attenuated by NADPH oxidase as well as 5-lipoxygenase (5-LO) inhibitors. Linked to these results, HNE increased membrane translocation of p47phox promoting NADPH oxidase activity, which was attenuated in peritoneal macrophages from 5-LO-deficient mice as well as in J774A.1 cells treated with a 5-LO inhibitor, MK886 or 5-LO siRNA. In contrast, HNE-enhanced 5-LO activity was not affected by inhibition of NADPH oxidase. Furthermore, leukotriene B₄, 5-LO metabolite, was found to enhance NADPH oxidase activity in macrophages. Altogether, these results suggest that 5-LO plays a critical role in HNE-induced ROS generation in murine macrophages through activation of NADPH oxidase.     

Partial purification and characterization of 12-lipoxygenase in bullfrog erythrocytes

12-Lipoxygenase (12-LO) in bullfrog (Rana catesbeiana) erythrocytes was purified partially by ion exchange chromatography and affinity chromatography. Bullfrog 12-LO was a single chain protein with a pI of 7.1–7.8 and MW of 7.77 kDa. This enzyme did not show typical Michaelis–Menten type kinetics. At low substrate concentrations, it had a lag phase and at higher substrate concentrations, the activity was inhibited. The product of linoleic acid (LA), 13-hydroperoxy-9, 11-octadecadienoic acid (13-HpODE), was an activator for the enzyme. When arachidonic acid (AA) was used as substrate, 13-HpODE also affected the K_m of bullfrog 12-LO towards AA. The affinity of LA towards bullfrog 12-LO was higher than the affinity of AA. Suicide inactivation was much more rapid than that of any mammalian 12-LO reported. Hemoglobin (Hb) inhibited the activity of 12-LO partially and removing Hb eliminated this inhibition. Both Hb and Met-Hb inhibited the 12-LO activity but did not denatured completely the Hb, suggesting that the inhibition was a direct interaction between 12-LO and Hb protein chain and was not due to competition between 12-LO and Hb for oxygen. This study characterizes bullfrog 12-LO with respect to stability, optimal pH, suicide inactivation and interaction with Hb and provides important evolutionary information about this enzyme.     

Genetic ablation of 12/15-lipoxygenase but not 5-lipoxygenase protects against denervation-induced muscle atrophy

Skeletal muscle atrophy is a debilitating outcome of a number of chronic diseases and conditions associated with loss of muscle innervation by motor neurons, such as aging and neurodegenerative diseases. We previously reported that denervation-induced loss of muscle mass is associated with activation of cytosolic phospholipase A₂ (cPLA₂), the rate-limiting step for the release of arachidonic acid from membrane phospholipids, which then acts as a substrate for metabolic pathways that generate bioactive lipid mediators. In this study, we asked whether 5- and 12/15-lipoxygenase (LO) lipid metabolic pathways downstream of cPLA₂ mediate denervation-induced muscle atrophy in mice. Both 5- and 12/15-LO were activated in response to surgical denervation; however, 12/15-LO activity was increased ~2.5-fold versus an ~1.5-fold increase in activity of 5-LO. Genetic and pharmacological inhibition of 12/15-LO (but not 5-LO) significantly protected against denervation-induced muscle atrophy, suggesting a selective role for the 12/15-LO pathway in neurogenic muscle atrophy. The activation of the 12/15-LO pathway (but not 5-LO) during muscle atrophy increased NADPH oxidase activity, protein ubiquitination, and ubiquitin–proteasome-mediated proteolytic degradation. In conclusion, this study reveals a novel pathway for neurogenic muscle atrophy and suggests that 12/15-LO may be a potential therapeutic target in diseases associated with loss of innervation and muscle atrophy.     

EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B₄ (LTB₄), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB₄ production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB₄ production and activity. We found that treatment with ox-LDL increased the production of LTB₄ and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB₄ receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB₄ receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB₄ production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Age-Dependent Relevance of Endogenous 5-Lipoxygenase Derivatives in Anxiety-Like Behavior in Mice

When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocannabinoid anandamide (AEA) is lost. Therefore, we decided to investigate whether inhibiting this enzyme would influence physiological functions classically described as being under control of the endocannabinoid system. Although 5-LO inhibition by MK-886 reduced lipoxin A₄ levels in the brain, no effect was found in the elevated plus maze (EPM), even at the highest possible doses, via i.p. (10 mg/kg,) or i.c.v. (500 pmol/2 µl) routes. Accordingly, no alterations in anxiety-like behavior in the EPM test were observed in 5-LO KO mice. Interestingly, aged mice, which show reduced circulating lipoxin A₄ levels, were sensitive to MK-886, displaying an anxiogenic-like state in response to treatment. Moreover, exogenous lipoxin A₄ induced an anxiolytic-like profile in the EPM test. Our findings are in line with other reports showing no difference between FLAP KO or 5-LO KO and their control strains in adult mice, but increased anxiety-like behavior in aged mice. We also show for the first time that lipoxin A₄ affects mouse behavior. In conclusion, we propose an age-dependent relevancy of endogenous 5-LO derivatives in the modulation of anxiety-like behavior, in addition to a potential for exogenous lipoxin A₄ in producing an anxiolytic-like state.     

Dual 12/15- and 5-Lipoxygenase Deficiency in Macrophages Alters Arachidonic Acid Metabolism and Attenuates Peritonitis and Atherosclerosis in ApoE Knock-out Mice

Lipoxygenase (LO) enzymes catalyze the conversion of arachidonic acid (AA) into biologically active lipid mediators. Two members, 12/15-LO and 5-LO, regulate inflammatory responses and have been studied for their roles in atherogenesis. Both 12/15-LO and 5-LO inhibitors have been suggested as potential therapy to limit the development of atherosclerotic lesions. Here we used a genetic strategy to disrupt both 12/15-LO and 5-LO on an apolipoprotein E (apoE) atherosclerosis-susceptible background to study the impact of dual LO blockade in atherosclerosis and inflammation. Resident peritoneal macrophages are the major cell type that expresses both LO enzymes, and we verified their absence in dual LO-deficient mice. Examination of AA conversion by phorbol myristate acetate-primed and A23187-challenged macrophages from dual LO-deficient mice revealed extensive accumulation of AA with virtually no diversion into the most common cyclooxygenase (COX) products measured (prostaglandin E₂ and thromboxane B₂). Instead the COX-1 by-products 11-hydroxy-eicosatetraenoic acid (HETE) and 15-HETE were elevated. The interrelationship between the two LO pathways in combination with COX-1 inhibition (SC-560) also revealed striking patterns of unique substrate utilization. 5-LO- and dual LO-deficient mice exhibited an attenuated response to zymosan-induced peritoneal inflammation, emphasizing roles for 5-LO in regulating vascular permeability. We observed gender-specific attenuation of atheroma formation at 6 months of age at both the aortic root and throughout the entire aorta in chow-fed female dual LO-deficient mice. We propose that some of the inconsistent data obtained with single LO-deficient mice could be attributable to macrophage-specific patterns of altered AA metabolism.Lipoxygenase (LO)² enzymes are an important source of lipid mediators throughout the plant and animal kingdoms (1, 2). In mammals, these mediators are predominantly formed from arachidonic acid (AA) and act in various physiological and pathological contexts (1–3). Accordingly 5-LO and 12/15-LO are two members of the LO family involved in cardiovascular and inflammatory diseases expressed to variable degrees in several cell types of the myeloid lineage, and their expression is strictly regulated and incompletely understood (2, 4, 5). Despite considerable structural homology between 5-LO and 12/15-LO, both enzymes generate distinct products. The 5-LO metabolite leukotriene (LT) A₄ is precursor to the proinflammatory LTB₄ and cysteinyl LTs, which regulate leukocyte subset-specific chemotaxis (LTB₄) and vascular permeability (cysteinyl LTs), both crucial events during acute peritonitis (1, 6, 7). 12- and 15-HETE, end products synthesized by 12/15-LO, play potential roles in cellular chemotaxis, cancer growth, and inflammation (2, 8). Transcellular interaction products derived from both 12/15-LO and 5-LO, such as lipoxins and maresins, indicate that these enzymes can possess anti-inflammatory activities in innate immunity and the resolution of inflammation (9, 10).In mice, only one cell type is known to express substantial quantities of both 5-LO and 12/15-LO, the peritoneal macrophage (PMΦ) (2, 11, 12). However, differences in subcellular localization, trafficking, and activation (8, 12–16) of these two LOs indicate that they are independently regulated and not functionally coupled. Tissue-resident MΦ (such as PMΦ) represent the first line of defense against invading pathogens and activate the immunological and inflammatory response (17). These phagocytes are capable of elaborating a wide spectrum of bioactive lipid mediators from the LO and cyclooxygenase (COX) pathways. Little is known about the regulation and putative interdependence of these pathways. Some insight was gained using mice lacking 12/15-LO where substrate shunting from the 12/15-LO into the 5-LO pathway was observed (12).The generation of knock-out mice for 12/15-LO (12) and 5-LO (18) has enabled the study of these lipid mediator pathways in models of health and disease. Because 12/15-LO and 5-LO are primarily expressed in distinct hematopoietic cells, their implication in various inflammatory disorders and models of host defense mechanisms have been investigated (2, 3). Atherosclerosis, an inflammatory disease prevalent in societies with high dietary fat intake, is initiated by low density lipoprotein (LDL) retention in the vascular wall (19) and subsequent oxidative modification. This process greatly enhances the LDL atherogenic potential, and intriguingly 12/15-LO can contribute to lipoprotein oxidation (11, 20). Initial studies using 12/15-LO- and 5-LO-deficient mice indicated proatherogenic roles for these enzymes (20, 21). Additionally mice lacking the LTB₄ receptor BLT-1 exhibit protection in early atherogenesis (22), but subsequent data from our laboratory using 5-LO-deficient mouse models have not supported an involvement of 5-LO in atherogenesis (3, 23, 24). Here we studied the consequences of simultaneous 12/15-LO and 5-LO knock-out on peritoneal inflammation and atherosclerosis in apoE-deficient mice and surmised whether some of the capricious results in atherosclerotic lesion studies could be attributable to variable eicosanoid profiles.     

A novel class of dual mPGES-1/5-LO inhibitors based on the α-naphthyl pirinixic acid scaffold

Dual inhibition of microsomal prostaglandin E₂ synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) represents a promising strategy in the development of novel anti-inflammatory drugs targeting the arachidonic acid cascade. Herein, a class of α-naphthyl pirinixic acids is characterized as dual mPGES-1/5-LO inhibitors. Systematic structural variation was focused on the lipophilic backbone of the scaffold and yielded detailed structure-activity relationships (SAR) with compound 16 (IC₅₀ mPGES-1 = 0.94 μM; IC₅₀ 5-LO = 0.1 μM) showing the most favorable in vitro pharmacological profile.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

Ansaetherone,a New Radical Scavenger from Streptomyces sp.

We identified a new radical scavenger, ansaetherone (C₂₆H₃₃NO₇), from a culture of the Streptomyces sp. USF-4727 strain. In our previous study, it was shown that this strain produced four lipoxygenase inhibitors, tetrapetalones A, B, C and D. The chemical structure of ansaetherone was elucidated by the spectroscopic method, indicating that this compound was constructed with an aglycon and a sugar moiety. This chemical structure suggested that ansaetherone was related to the tetrapetalones. This finding provided information regarding tetrapetalone biosynthesis. Ansaetherone showed radical scavenging activity with an ED₅₀ value of 300 μM in our assay.     

New Indophenol-reducing 1,3-Dicarbonyl Compound from Streptomyces

In the course of our search for new indophenol-reducing substances, a strain of Streptomyces was found to produce USF-142A, a novel 1,3-dicarbonyl compound which reduces 2,6-dichlorophenol in-dophenol and exists in keto-enol tautomers in solution. This compound showed antioxidative activity by the Rhodan iron method.     

Bronchipret® syrup containing thyme and ivy extracts suppresses bronchoalveolar inflammation and goblet cell hyperplasia in experimental bronchoalveolitis

Background/purposeAcute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret^® syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action.MethodsBronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24–72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO).ResultsBRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB₄ (IC₅₀ = 36 µg⋅ml⁻¹) and cysLT (IC₅₀ = 10 µg⋅ml⁻¹) and inhibited the activity of isolated 5-LO (IC₅₀ = 19 µg⋅ml⁻¹).ConclusionBRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB.     

Analogs of arachidonic acid methylated at C-7 and C-10 inhibit leukotriene biosynthesis and phospholipase A2 activity

The ability of (all Z)-7,7-dimethyl-5,8,11,14-eico-satetraenoic acid, (all Z)-7,7-dimethyl-5,8,11-eicosatrienoic acid, (Z,Z)-7,7-dimethyl-5,8-eicosadienoic acid, (all Z)-10,10-dimethyl-5,8,11,14-eicosatetraenoic acid, (all Z)-10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac-(Z,Z)-15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid to inhibit ionophore-induced slow-reacting substance of anaphylaxis (SRS-A) biosynthesis in rat peritoneal cells was studied. It was thought that compounds such as these might inhibit proton abstractions at the 7 or 10 carbon positions on arachidonic acid which are thought to be important in the mechanism of catalysis of Δ⁵-lipoxygenase(Δ⁵-LO). All compounds were found to be potent inhibitors of SRS-A biosynthesis in the in vitro rat peritoneal cell system (IC₅₀ < 10 μM). In fact they were more potent inhibitors in the test system than standard Δ⁵-LO inhibitors such as NDGA and quercetin. To determine if the mechanism of inhibition of the dimethyl arachidonic acid analogs did involve gD⁵-LO inhibition these compounds were evaluated in an assay system utilizing the Δ⁵-LO from rat basophilic leukemia (RBL^?1_cells. It was found, however, that these compounds were much less potent inhibitors of this enzyme (IC₅₀ ～ 100 μM) than standard compounds such as NDGA (IC₅₀ 0.14 μM) and quercetin (IC₅₀, 0.2 μM). The arachidonic acid analogs were subsequently found to be potent inhibitors of phospholipase A₂ (PLA₂) enzymes with IC₅₀'s between 10–20 μM as inhibitors of a snake venom enzyme. In fact these compounds are among the most potent inhibitors of PLA₂ yet studied, having potencies better than standards such as p-bromophenacyl bromide (IC₅₀, 87 μM) and U-10029A (IC₅₀, 36 μM). These results suggest that the methylated arachidonic acid analogs may inhibit SRS-A biosynthesis through inhibiting PLA₂.     

Lipoxins: Eicosanoids carrying intra-and intercellular messages

The lipoxins are a recent addition to the family of biologically active products derived from arachidonic acid. Compounds of this series contain a conjugated tetraene structure and can be generated by the actions of the major lipoxygenases of human tissues (5-, 12-, and 15-LO's). Biosynthesis of the lipoxins from cellular sources of unesterified arachidonic acid is triggered by the initial actions of either the 15-LO or 5-LO followed by additional reactions. Recent results indicate that lipoxins are also generated by receptor-mediated events during cell-cell interactions with the transcellular metabolism of key intermediates. Lipoxin A₄ and lipoxin B₄ each possess a unique spectrum of biological activities unlike those of other eicosanoids in bothin vivo andin vitro systems. Lipoxin A₄ stimulates changes in the microvasculature and can block some of the proinflammatory effects of leukotrienes (in vivo). Lipoxin A₄ and lipoxin B₄ both inhibit natural killer cells (in vitro), and lipoxin B₄ displays selective actions on hematopoietic cells. The finding that lipoxin A₄ activates isolated protein kinase C suggests that it may also serve an intracellular role in its cell of origin before it is released to the extracellular milieu. Thus, cell-cell interactions, along with multiple oxygenations by lipoxygenases, generate compounds that can regulate cellular responses by serving as both intra- and intercellular messages.