Eicosanoids and the large intestine

During the past three decades, many studies have been conducted to determine the precise role of eicosanoids in colorectal physiology and pathophysiology. This research has increased our understanding of bioactive lipid signaling, and may contribute to the development of more effective therapeutic modalities for digestive diseases in the future. The purpose of this report is to provide a brief overview of the role of eicosanoids in the colon and rectum. This information has been organized according to both functional and disease-related categories. The role of eicosanoids in colonic secretion, motility, inflammatory bowel disease, and colorectal neoplasia will be discussed.     

Control of phospholipase A2 activities for the treatment of inflammatory conditions

Phospholipase-A2 (PLA2) enzymes hydrolyze cell membrane phospholipids to produce arachidonic acid (AA) and lyso-phospholipids (LysoPL), playing a key role in the production of inflammatory lipid mediators, mainly eicosanoids. They are therefore considered pro-inflammatory enzymes and their inhibition has long been recognized as a desirable therapeutic target. However, attempts to develop suitable PLA2 inhibitors for the treatment of inflammatory diseases have yet to succeed. This is due to their functional and structural diversity, and their homeostatic and even anti-inflammatory roles in certain circumstances. In the present review we outline the diversity and functions of PLA2 isoforms, and their interplay in the induction and inhibition of inflammatory processes, with emphasis on discussing approaches for therapeutic manipulation of PLA2 activities.     

Hydroxyeicosanoids bind to and activate the low affinity leukotriene B4 receptor, BLT2

Leukotriene B(4), an arachidonate metabolite, is a potent chemoattractant of leukocytes involved in various inflammatory diseases. Two G-protein-coupled receptors for leukotriene B(4) have been cloned and characterized. BLT1 (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624) is a high affinity receptor exclusively expressed in leukocytes, and BLT2 (Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000) J. Exp. Med. 192, 421-432) is a low affinity receptor expressed more ubiquitously. Here we report the binding profiles of various BLT antagonists and eicosanoids to either BLT1 or BLT2 using the membrane fractions of Chinese hamster ovary cells stably expressing the receptor. BLT antagonists are grouped into three classes: BLT1-specific U-75302, BLT2-specific LY255283, and BLT1/BLT2 dual-specific ZK 158252 and CP 195543. We also show that 12(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroperxyeicosatetraenoic acid, and 15(S)-hydroxyeicosatetraenoic acid competed with [(3)H]LTB(4) binding to BLT2, but not BLT1, dose dependently. These eicosanoids also cause calcium mobilization and chemotaxis through BLT2, again in contrast to BLT1. These findings suggest that BLT2 functions as a low affinity receptor, with broader ligand specificity for various eicosanoids, and mediates distinct biological and pathophysiological roles from BLT1.     

Inhibition of phospholipase A(2) as a therapeutic target

The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.     

Rat basophilic leukemia cells (RBL-2H3) generate prostaglandin D2 (PGD2) after regulated upon activation, normal T-cell expressed and secreted (RANTES) activation

Increasing evidence indicates that local neurogenic inflammation, possibly in response to different stimuli, may be involved in sensory nerve sensitization, migraine generation and some other precipitating events leading to neuronal dysfunction in the brain. In addition, mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. Regulated upon activation, normal T-cell expressed and secreted (RANTES) is a small protein and a prototype member of the CC chemokine-beta subfamily with chemoattractant and inflammatory properties. In this study we used the RBL-2H3 cell line to determine whether or not these cells generate prostaglandin D2 (PGD2) after treatment with RANTES. After 4 hours of incubation, RBL-2H3 cells cultured with RANTES at 20 ng/mL released large amounts of PGD2 in a dose-response manner compared to control. Moreover, RBL-treated RANTES generated a large quantity of histamine. Our study confirms once again the proinflammatory action of RANTES, in this case acting on the stimulation of the arachidonic acid cascade product PGD2.     

BLT1 and BLT2: the leukotriene B(4) receptors

Two receptors for leukotriene B(4) (LTB(4)) have been molecularly identified: BLT1 and BLT2. Both receptors are G protein-coupled seven transmembrane domain receptors, whose genes are located in very close proximity to each other in the human and mouse genomes. The two receptors differ in their affinity and specificity for LTB(4): BLT1 is a high-affinity receptor specific for LTB(4), whereas BLT2 is a low-affinity receptor that also binds other eicosanoids. The two receptors also differ in their pattern of expression with BLT1 being expressed primarily in leukocytes, whereas BLT2 is expressed more ubiquitously. By mediating the activities of LTB(4), these receptors participate both in host immune responses and in the pathogenesis of inflammatory diseases. Reduced disease severity in animal inflammatory models seen with LTB(4) receptor antagonists and in mice with targeted deletion of BLT1 have revealed important roles for LTB(4) and its receptors in regulating pathologic inflammation.     

Atherosclerosis: the eicosanoid connection

This bird's eye view presents connections between the metabolically short-lived local hormones (collectively known as eicosanoids) and atherosclerotic cardiovascular disease. The discussion will be centered around an overview of coronary atherosclerosis with an emphasis on the sequences involved in the formation of atherosclerotic lesions; structure and historical background of oxygenated fatty acids cyclooxygenase and lipoxygenase products — eicosanoids; the generation of free radicals during the formation of endoperoxides by cyclooxygenase; the involvement of eicosanoids in the atherosclerotic inflammatory process, and finally, the effects of non-steroidal and steroidal anti-inflammatory drugs on the synthesis of eicosanoids and experimental atherosclerosis. Little is known about the exact role of eicosanoids in the genesis of atherosclerosis.     

Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release.

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short-chain ceramide analogs for 30 and 60 min leads to a concentration-dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen-activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide-induced arachidonic acid release. By use of photoactivatable ceramide analogs, D- and L-[125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramides (TID-ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D- and L-TID-ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting 'hinge' domain of cPLA2, efficiently bound D- and L-TID-ceramides, whereas the catalytic domain did not interact with TID-ceramides. In vitro binding assays reveal that stearoyl-arachidonyl-phosphatidylcholine (SAPC)-liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC-liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates cPLA2 action in inflammatory diseases.     

P2X7 receptors induce degranulation in human mast cells

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.     

12/15 脂氧酶在脑卒中的作用研究进展

脂氧合酶(LOX)是一类广泛存在于动植物中的非血红素铁蛋白,催化底物生成各种类花生酸物质,与人体的肿瘤、哮喘、炎症、动脉硬化等疾病密切相关。12/15脂氧酶(12/15-LOX)是一种脂质过氧化物酶,可以催化亚油酸,花生四烯酸等多不饱和脂肪酸生成具有生物活性的代谢产物,通过信号转导在许多病理生理过程中发挥着重要的作用,有研究表明,12/15-LOX通路可以刺激炎症因子的产生,参与多种炎性反应,而在脑卒中的发生发展以及病理过程中始终伴随的炎性反应,炎症及细胞因子等对脑卒中有一定的影响,在脑卒中炎症反应继发性脑组织损伤病理发展过程中起着重要的作用。因此,研究12/15-LOX与脑卒中炎症的关系,可以为临床治疗脑卒中提供新的靶点。本文就12/15-LOX在脑卒中后炎症反应中的作用做简要介绍。     

Pharmacotherapy of diseases mediated by 5-lipoxygenase pathway eicosanoids

Inflammatory eicosanoids generated by the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism are now known to have at least 6 receptors: OXE, which recognizes 5-HETE and 5-oxo-ETE; a putative receptor recognizing a potent 5-oxo-ETE metabolite, FOG(7); the LTB(4) receptors, BLT1 and BLT2; the cysteinyl leukotriene receptors, CysLT(1) and CysLT(2), which recognize leukotrienes LTC(4), LTD(4), LTE(4) and LTF(4). The 5-LO pathway is activated in many diseases and invokes inflammatory responses not affected by glucocorticoids, but therapy with selective BLT1 or CysLT(1) antagonists in asthma has met with variable success. Studies show that 5-LO pathway eicosanoids are not primary mediators in all cases of asthma, but may be especially important in severe persistent asthma, aspirin- and exercise-induced asthma, allergic rhinitis, COPD, idiopathic pulmonary fibrosis, atherosclerosis, atopic dermatitis, acne and ischemia-related organ injury. These disorders appear to involve multiple 5-LO pathway eicosanoids and receptor subtypes, suggesting that inhibition of the pathway at the level of 5-LO may be necessary for maximal efficacy.     

Gastrointestinal inflammation: a central component of mucosal defense and repair

The mucosal layer of the gastrointestinal (GI) tract is able to resist digestion by the endogenous substances that we secrete to digest foodstuffs. So-called "mucosal defense" is multi-factorial and can be modulated by a wide range of substances, many of which are classically regarded as inflammatory mediators. Damage to the GI mucosa, and its subsequent repair, are also modulated by various inflammatory mediators. In this article, we provide a review of some of the key inflammatory mediators that modulate GI mucosal defense, injury, and repair. Among the mediators discussed are nitric oxide, polyamines, the eicosanoids (prostaglandins and lipoxins), protease-activated receptors, and cytokines. Many of these endogenous factors, or the enzymes involved in their synthesis, are considered potential therapeutic targets for the treatment of diseases of the digestive tract that are characterized by inflammation and ulceration.     

Evidence for LTB4/12-HETE binding sites in a human epidermal cell line

We identified leukotriene B4 (LTB4)/12-hydroxyeicosatetraenoic acid (12-HETE) binding sites in a squamous cell cancer-derived human epidermal cell line. Analysis of the binding data revealed a single class of binding sites with a dissociation constant of 0.16 microM and a Bmax of 3.8 x 10(6) sites per cell. Competitive binding assays with various eicosanoids at 37 degrees C showed nearly equal binding of 12(S)-HETE, 12(R)-HETE and LTB4. 5(S)-HETE and LTB4-analogs bound with lesser affinity. Specific LTB4 binding at 37 degrees C could also be demonstrated in freshly isolated normal human keratinocytes. Since lipoxygenase-derived eicosanoids are thought to play an important role in hyperproliferative and inflammatory skin diseases, the identification of LTB4/12-HETE binding sites in keratinocytes could have implications for the development of new drugs controlling these disease processes.     

The role of mast cell-derived secreted phospholipases A2 in respiratory allergy

Secreted phospholipases A₂ (sPLA₂s) are molecules released in plasma and biological fluids of patients with systemic inflammatory, autoimmune and allergic diseases. These molecules exert proinflammatory effects by either enzymatic-mechanisms or through binding to surface molecules expressed on inflammatory cells. sPLA₂s are released at low levels in the normal airways and tend to increase during respiratory allergies (e.g., rhinitis and bronchial asthma) as the result of local secretion. Several sPLA₂ isoforms are expressed in the human lung and some of them (e.g., group IIA and group X) are released in the airways of patients with rhinitis or asthma. Mast cells play a major role in the pathogenesis of respiratory allergies and other chronic inflammatory lung diseases. Recent evidence indicates that mast cells purified from human lung express most of the sPLA₂ isoforms so far described. IgE-mediated activation of these cells induce the release of sPLA₂s suggesting that mast cells are a main source of extracellular sPLA₂s during allergic reactions. Once released, sPLA₂s may contribute to the generation of eicosanoids (e.g., PGD₂ and LTC₄) and to the release of preformed mediators (e.g., histamine) by an autocrine loop involving the interaction of sPLA₂s with surface molecules such as heparan sulphate proteoglycans or the M-type receptor. Thus, mast cell-derived sPLA₂s may play an important role in the initiation and amplification of the inflammatory reactions in patients with allergic rhinitis and bronchial asthma.     

Gammalinolenic acid-enriched diet alters cutaneous eicosanoids

There are reports that vegetable oils containing gammalinolenic acid (GLA) may exert beneficial effects on inflammatory skin disorders. To determine whether or not dietary GLA exerts any modulatory role on cutaneous eicosanoids, guinea pigs were fed either a control diet containing safflower oil (less than 0.5% GLA) or borage oil, a GLA-rich diet containing 25% GLA. After an 8-week feeding period, epidermal samples from both animal groups were analyzed for fatty acid composition and tissue eicosanoids. Analysis of epidermal neutral lipids and phospholipids in borage oil-fed animals showed a marked increase in GLA and its elongase product, dihomogammalinolenic acid (DGLA). Similarly, analysis of epidermal eicosanoids in the borage oil-fed animals revealed significant increases in the amounts of the 15-hydroxy fatty acid (15-OH-20:3n-6) and prostaglandin PGE1, both metabolites of DGLA. Since these metabolites have anti-inflammatory potential, our results suggest that increased dietary GLA could result in the generation of local anti-inflammatory metabolites thus providing a non-toxic approach to suppression of cutaneous inflammatory skin disorders.     

Inhibition of sPLA2 enzyme activity by cell-permeable antioxidant EUK-8 and downregulation of p38, Akt,and p65 signals induced by sPLA2 in inflammatory mouse paw edema model

The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A₂ (PLA₂). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA₂s. Among these, we found EUK-8 is a potent inhibitor with its IC₅₀ value ranges 0.7–2.0 µM for sPLA₂s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA₂. In vivo effect of EUK-8 was studied in HSF-sPLA₂-induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA₂ activity, it also plays an important role in the regulation of sPLA₂-induced cell signaling cascades. Apart from the sPLA₂ inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.     

Cyclopentenone-containing oxidized phospholipids and their isoprostanes as pro-resolving mediators of inflammation

Inflammation represents a powerful innate immune response that defends tissue homeostasis. However, the appropriate termination of inflammatory processes is essential to prevent the development of chronic inflammatory disorders. The resolution of inflammation is actively induced by specialized pro-resolving lipid mediators, which include eicosanoids, resolvins, protectins and maresins. The responsible pro-resolution pathways have emerged as promising targets for anti-inflammatory therapies since they mitigate excessive inflammation without compromising the anti-microbial defenses of the host. We have recently shown that the lipid peroxidation of membrane phospholipids, which is associated with inflammatory conditions, generates oxidized phospholipid (OxPL) species with potent pro-resolving activities. These pro-resolving OxPLs contain a cyclopentenone as their common determinant, and are structurally and functionally related to endogenous pro-resolving prostaglandins. Here, we review the regulation of inflammatory responses by OxPLs with particular focus on the bioactivities and structural characteristics of cyclopentenone-OxPLs, and discuss the impact of the responsible signaling pathways on inflammatory diseases. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.     

Selective eicosanoid-generating capacity of cytoplasmic phospholipase A2 in Pseudomonas aeruginosa-infected epithelial cells

Airway neutrophil infiltration is a pathological hallmark observed in multiple lung diseases including pneumonia and cystic fibrosis. Bacterial pathogens such as Pseudomonas aeruginosa instigate neutrophil recruitment to the air space. Excessive accumulation of neutrophils in the lung often contributes to tissue destruction. Previous studies have unveiled hepoxilin A(3) as the key molecular signal driving neutrophils across epithelial barriers. The eicosanoid hepoxilin A(3) is a potent neutrophil chemoattractant produced by epithelial cells in response to infection with P. aeruginosa. The enzyme phospholipase A(2) liberates arachidonic acid from membrane phospholipids, the rate-limiting step in the synthesis of all eicosanoids, including hepoxilin A(3). Once generated, aracidonic acid is acted upon by multiple cyclooxygenases and lipoxygenases producing an array of functionally diverse eicosanoids. Although there are numerous phospholipase A(2) isoforms capable of generating arachidonic acid, the isoform most often associated with eicosanoid generation is cytoplasmic phospholipase A(2)α. In the current study, we observed that the cytoplasmic phospholipase A(2)α isoform is required for mediating P. aeruginosa-induced production of certain eicosanoids such as prostaglandin E(2). However, we found that neutrophil transepithelial migration induced by P. aeruginosa does not require cytoplasmic phospholipase A(2)α. Furthermore, P. aeruginosa-induced hepoxilin A(3) production persists despite cytoplasmic phospholipase A(2)α suppression and generation of the 12-lipoxygenase metabolite 12-HETE is actually enhanced in this context. These results suggest that alterative phospholipase A(2) isoforms are utilized to synthesize 12-lipoxygenase metabolites. The therapeutic implications of these findings are significant when considering anti-inflammatory therapies based on targeting eicosanoid synthesis pathways.