Linoleic acid peroxidation by Solanum tuberosum lipoxygenase was activated in the presence of human 5-lipoxygenase-activating protein

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100000xg membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase (S.t.LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t.LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase (S.t.LOX) by using different polyunsaturated fatty acids as substrates.     

5-Lipoxygenase: a target for antiinflammatory drugs revisited

Arachidonate 5-lipoxygenase is the key enzyme in leukotriene biosynthesis and catalyzes the initial steps in the conversion of arachidonic acid to biologically active leukotrienes. Leukotrienes are considered as potent potent mediators of inflammatory and allergic reactions which are locally released by leukocytes and other 5-LO expressing cells and exert their effects via binding to specific membrane receptors and, as suggested recently, the nuclear receptor PPARa. Because of the proinflammatory profile of leukotrienes it was assumed that leukotriene biosynthesis inhibitors and leukotriene receptor antagonists have a therapeutical potential in a variety of inflammatory diseases. Clinical studies confirmed the therapeutic value of the antileukotriene therapy in asthma but the results with leukotriene biosynthesis inhibitors in psoriasis, arthritis and inflammatory bowel disease were more or less disappointing. This review summarizes the biochemistry of the 5-lipoxygenase pathway, the pharmacology of FLAP and 5 lipoxygenase inhibitors and discusses possible criteria for the development of these drugs.     

Tenidap inhibits 5-lipoxygenase product formation in vitro, but this activity is not observed in three animal models

OBJECTIVE AND DESIGN: The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo. MATERIALS AND TREATMENT: In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively. METHODS: 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA. RESULTS: Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models. CONCLUSIONS: Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.     

Thiopyrano[2,3,4-cd]indoles as 5-lipoxygenase inhibitors: synthesis, biological profile, and resolution of 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5 -dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]butanoic acid

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.     

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.     

Regulatory interactions between inducible nitric oxide synthase and eicosanoids in glomerular immune injury

In a rat model of glomerular immune injury induced by administration of anti-glomerular basement membrane antibody and resembling human rapidly progressive glomerulonephritis, we explored whether activation of inducible nitric oxide synthase (iNOS) regulates synthesis of eicosanoids originating from cyclooxygenation or lipoxygenation of arachidonic acid. At early stages (24 hr) of injury, inhibition of iNOS using the selective inhibitor L-N6-(1-iminoethyl) lysine (L-NIL) at doses sufficient to reduce urinary excretion of nitrate/nitrite, reduced glomerular synthesis of the prostaglandins PGE2 and PGI2, but had no effect on that of thromboxane A2 (TxA2). The syntheses of 5-hydroxyeicosatetraenoic acid (HETE), 15-HETE and leukotriene B4 (LTB4) were also reduced. That of 12-HETE remained unchanged. We also explored the effect of arachidonate cyclooxygenation and lipoxygenation eicosanoids on iNOS expression. Administration of the cyclooxygenase (COX) inhibitor, indomethacin, at doses sufficient to inhibit glomerular prostaglandin synthesis, increased iNOS mRNA levels in glomeruli. Administration of the 5-lipoxygenase (5-LO) inhibitor, MK-0591, at doses sufficient to inhibit glomerular LTB4 synthesis also increased iNOS mRNA. The effect of 5-LO inhibition on iNOS expression was more pronounced than that of COX inhibition. In nephritic animals given the iNOS inhibitor, L-NIL, or indomethacin proteinuria worsened. In those given the 5-lipoxygenase inhibitor there was no change in urine protein excretion. These observations point to regulatory interactions between the arachidonic acid and the L-arginine: NO pathways in glomerulonephritis. These interactions are of importance in considering antiinflammatory strategies based on inhibition of iNOS or of specific eicosanoids.     

Identification of 5-keto-(7E,9E,11Z,14Z)-eicosatetraenoic acid as a novel nonenzymatic rearrangement product of leukotriene A4

Leukotriene A4 (LTA4), the reaction product of 5-lipoxygenase in human polymorphonuclear (PMN) leukocytes, is transformed both to LTB4 and a mixture of 5,6- and 5,12-dihydroxy-eicosatetraenoic acids (diHETE) via nonenzymatic hydrolysis. Evidence has been obtained that LTA4 is also converted to 5-keto-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE). The compound was isolated from the products of the 5-lipoxygenase reaction and its structure elucidated by UV spectroscopy, LC-MS, two-dimensional [1H]NMR spectroscopy and chemical reduction to the corresponding alcohol. The 5-oxo-ETE represented about 14% of the LTA4 hydrolysis products as compared to 72 and 14% for the 5,12-diHETE and 5,6-diHETE, respectively. A similar profile of hydrolysis products was obtained after incubation of synthetic LTA4 in aqueous buffer. Human PMN leukocytes produced 5-oxo-ETE in an arachidonic acid-dependent and MK-886-inhibitable manner. The 5-oxo-ETE caused 50% inhibition of 5-lipoxygenase activity at 1 microM. These results demonstrate that the nonenzymatic conversion of LTA4, in addition to the previously described hydrolysis products, yields 5-oxo-ETE during both the 5-lipoxygenase reaction and arachidonic acid oxidation by human PMN leukocytes. They indicate that allylic epoxides can rearrange in aqueous media at physiological pH to spontaneously form beta,gamma-unsaturated ketones.     

Mice deficient for 5-lipoxygenase, but not leukocyte-type 12-lipoxygenase, display altered immune responses during infection with Schistosoma mansoni

Periovular granuloma formation during Schistosoma mansoni infection is a complex, multifaceted immunologic response. Products of arachidonic acid metabolism have been shown to contribute to this response through studies in which general inhibitors of lipoxygenase function reduce granulomatous inflammation. To determine which lipoxygenases are important for granuloma development in schistosomiasis, wild type mice or mice deficient for 5-lipoxygenase (5-LO) or "leukocyte-type" 12-lipoxygenase (12-LO) were infected with S. mansoni and studied for responses to schistosome eggs and egg antigens. At the acute stage of infection, when granuloma formation is usually maximal, 5-LO deficient mice developed smaller granulomas around liver-deposited schistosome eggs compared with wild type or 12-LO deficient mice. 5-LO mice also displayed less antibody-mediated (5 h) and cell-mediated, delayed-type (24 h) hypersensitivity to schistosome egg antigens than did the other two infection groups. In an attempt to determine possible mechanisms for the reduced inflammatory responses, we also measured hepatic mRNA levels of cytokines that have been shown to influence granuloma size (IL-4, IL-10, and IFN-gamma). The mRNA levels for IL-10 were significantly lower in 5-LO-deficient mice, but SEA-stimulated spleen cells did not demonstrate a significant difference in IL-10 production between wild type and 5-LO mice. These data suggest that 5-LO plays a role in host responses to schistosomiasis via a mechanism that cannot be explained solely by changes in expression of these cytokines.     

Metabolism of 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human neutrophils

We have previously shown that human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to 6,11-dihydro metabolites (Powell and Gravelle (1988) J. Biol. Chem. 263, 2170-2177). In the present study, we have shown that the first step in the formation of these dihydro metabolites is oxidation of the 5-hydroxyl group to a 5-oxo group, which is catalyzed by an NADP(+)-dependent microsomal dehydrogenase enzyme. All the dihydroxyeicosanoids we investigated which contained a 5(S)-hydroxyl group followed by a 6-trans double bond were good substrates for this reaction. However, LTB4, which contains a 6-cis double bond, was not metabolized to any detectable 5-oxo products. The preferred substrate for the dehydrogenase reaction is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE), which has a Km of about 0.2 microM, compared to approx. 0.9 microM for 12-epi-6-trans-LTB4. In contrast to 5(S)-HETE, 5(R)-HETE as well as a variety of positional isomers of 5(S)-HETE are not metabolized to significant extents by the PMNL dehydrogenase. 5-Oxo-ETE and 5-oxo-15-hydroxy-ETE, which are formed from 5(S)-HETE and 5,15-diHETE, respectively, by this pathway, are potent chemotactic agents for human neutrophils, and raise intracellular calcium levels in these cells.     

12-hydroxyeicosatetraenoic acid is a long-lived substance in the rabbit circulation

12-Hydroxyeicosatetraenoic acid (12-HETE) is one of the major metabolites formed from arachidonic acid in platelets. We have recently shown that the in vitro metabolism of 12-HETE by human leukocytes, with and without stimulation, is effectively inhibited by the addition of physiological concentrations of albumin, probably by sequestration of the compound. In the present paper, we have studied the in vivo metabolism of 12-HETE in the rabbit, using either [1-14C]- or [14C(U)]12-HETE. Distribution of radioactivity was followed in urine, plasma, and bile, as well as in a number of tissues. In most of the tissues examined, the hydrophilic radioactivity constituted more than 50% of the total radioactivity after 20 min. When the lipophilic fraction was analyzed, around 15% of the radioactivity was shown to be unesterified 12-HETE, and only a very minor part could be detected as metabolites. The dominating lipophilic compound in the circulation after i.v. administration of radiolabeled 12-HETE was at all time points (1-60 min.) the parent compound, as analyzed by HPTLC and HPLC. A comparison of the plasma metabolite profiles obtained when [1-14C]- and [14C(U)]12-HETE were used displayed almost identical patterns, thus indicating that beta-oxidized metabolites either were not formed or were rapidly removed from the circulation. The appearance of large amounts of water-soluble radioactivity with time supported the latter conclusion. Several minor metabolites were seen that chromatographed in the dihydroxy acid region as judged by HPLC and TLC. The major one of these compounds represented about 10% of the lipophilic plasma radioactivity after 60 min., while unmetabolized 12-HETE at this stage still represented about 30%. The metabolite had a polarity similar to 12,20-dihydroxyeicosatetraenoic acid; however, when chromatographed together, these two compounds separated, indicating a different structure of the metabolite. Our findings are in agreement with in vitro data concerning the protective effect of albumin on the metabolism of 12-HETE and is the first extensive metabolic study of 12-HETE in vivo covering all metabolic possibilities involving the carbon skeleton.     

Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene

Leukotrienes constitute a class of potent biological mediators of inflammation and anaphylaxis (for reviews see refs 1 and 2). Their biosynthesis derives from 5-lipoxygenase-catalysed oxygenation of arachidonic acid in granulocytes, macrophages and mast cells. To examine the physiological importance of leukotrienes, we have disrupted the 5-lipoxygenase gene by homologous recombination in embryonic stem cells. 5-Lipoxygenase-deficient (5LX-/-) mice develop normally and are healthy. They show a selective opposition to certain inflammatory insults. Although there is no difference in their reaction to endotoxin shock, the 5LX-/- animals resist the lethal effects of shock induced by platelet-activating factor. Reaction to ear inflammation induced by phorbol ester is normal, whereas inflammation induced by arachidonic acid is markedly reduced. Contrasts were also found in two models of leukocyte chemotaxis in vivo. The phenotype of 5LX-/- mice under injurious insult identifies the role for leukotrienes in the pathophysiology of select inflammatory states.     

Adrenal scintigraphy with 131I-6beta-iodomethyl-19-norcholesterol: good practice guideline

The monohydroxyeicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which is derived from oxygenation of arachidonic acid by 12-lipoxygenase, is one of the major metabolites in platelets. In a recent study, we have showed that this eicosanoid stimulated basal sickle-red-cell-endothelial-cell adhesion. To understand the pathophysiologic significance of 12-HETE, we measured the levels of this eicosanoid in plasma and urine from children with sickle cell disease. We found that as compared with controls, plasma 12-HETE levels are increased in patients with sickle-cell disease in the steady state, and are increased further during vaso-occlusive crises. Urinary 12-HETE levels were also increased during the steady state. We also assessed plasma levels of soluble P-selectin (another potential marker for platelet activation), and found changes in the levels of this marker similar to those seen with plasma 12-HETE. In additional studies, we found that 12-HETE enhanced hypoxia-induced sickle-red-cell-endothelial adherence, and that this effect was mediated by potentiation of agonist-induced upregulation of the expression of the mRNA for vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Because 12-HETE appears to enhance both basal and agonist-induced sickle-red-cell adhesion, this metabolite could potentially play a role in the pathogenesis of the vaso-occlusive crisis (VOC) in sickle-cell disease.     

5-HPETE is a potent inhibitor of neuronal Na+, K(+)-ATPase activity

The effects of 1 microM concentrations of arachidonic acid hydroperoxide (HPETES) products of 5-, 12- and 15-lipoxygenase on Na+, K(+)-ATPase activity were investigated in synaptosomal membrane preparations from rat cerebral cortex. 5-HPETE inhibited Na+, K(+)-ATPase activity by up to 67 %. In contrast, 12-HPETE and 15-HPETE did not inhibit Na+, K(+)-ATPase activity. In addition, neither 5-HETE or LTA4 inhibited Na+, K(+)-ATPase activity. Dose-response studies indicated that 5-HPETE was a potent (IC25 = 10(-8) M) inhibitor of Na+, K(+)-ATPase activity. These findings indicate that 5-HPETE inhibits Na+, K(+)-ATPase activity by a mechanism that is dependent on the hydroperoxide position and independent of further metabolism by 5-lipoxygenase. It is proposed that 5-HPETE production by 5-lipoxygenase and subsequent inhibition of neuronal Na+, K(+)-ATPase activity may be a mechansim for modulating synaptic transmission.     

Probing the substrate alignment at the active site of 15-lipoxygenases by targeted substrate modification and site-directed mutagenesis. Evidence for an inverse substrate orientation

For oxygenation of polyenoic fatty acids by 12- and 15-lipoxygenases the methyl terminus of the substrate constitutes the signal for the initial hydrogen abstraction. In contrast, for 5-lipoxygenases an inverse head to tail substrate orientation has been proposed. However, recent structure-based sequence alignments suggested a conserved uniform substrate orientation for 5S- and 15S-lipoxygenation. Oxygenation of 15S-HETE derivatives by various wild-type and mutant lipoxygenases was investigated, and the evidence proved an inverse substrate orientation: (i) Substrate affinity and Vmax of 15S-HETE oxygenation by arachidonic acid 15-lipoxygenases are >1 order of magnitude lower than the corresponding data for polyenoic fatty acids. 5S,15S- and 14R, 15S-DiH(P)ETE were identified as major reaction products. (ii) Methylation of the carboxylate group of 15S-HETE augmented the reaction rate and shifted the reaction specificity strongly toward 5S-lipoxygenation. In contrast, methyl arachidonate was less effectively oxygenated than the free acid. Methylation of 15S-HETrE(8,11,14), which lacks the C5-C6 double bond, was without major impact on the oxygenation rate and on the product specificity. (iii) Introduction of a bulky glycerol moiety at the carboxylic group of 15S-HETE reversed the kinetic effects of methylation and led to a 14R-oxygenation of the substrate. (iv) When the product pattern of 15S-HETE oxygenation by the recombinant wild-type rabbit 15-lipoxygenase was compared with that formed by the Arg403Leu mutant, 5S- and 8S-lipoxygenations were augmented and 14R, 15S-DiH(P)ETE formation was impaired. (v) Phe353Leu or Ile418Ala mutation of the same enzyme, which favored 12S-HETE formation from arachidonic acid, strongly augmented 8S-lipoxygenation of 15S-HETE methyl ester. These kinetic data and the alterations in the product specificity are consistent with the concept of an inverse head to tail substrate orientation during the oxygenation of 15S-HETE methyl ester and/or of free 15S-HETE by 15-LOXs. For 5S- and 8S-lipoxygenation, 15-HETE may slide into the substrate binding pocket with its carboxy terminus approaching the doubly allylic methylenes C-7 or C-10 to the non-heme iron.     

Advantages of immunostaining over DNA analysis using PCR amplification to detect p53 abnormality in long-term formalin-fixed tissues of human colorectal carcinomas

The purpose of our studies was to examine differentiation-dependent expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase (PGHS) isoforms in cultured normal human tracheobronchial epithelial cells. In the presence of retinoic acid (RA) the cultures differentiated into a mucociliary epithelium. When cultured in RA-depleted media, the cultures differentiated into a squamous epithelium. In the absence of RA the cultures did not express 15-LO or either of the PGHS isoforms. The PGHS-1 isoform was not expressed in RA-sufficient cultures, but both PGHS-2 messenger RNA (mRNA) and protein were strongly expressed, and prostaglandin E2 (PGE2) was produced during the predifferentiation phase. No PGHS-2 expression or PGE2 could be detected in fully differentiated mucociliary cultures. 15-LO showed the opposite expression pattern: neither mRNA nor protein were detected during the predifferentiation stage, but both were strongly expressed once mucous differentiation had occurred. Cytosolic phospholipase A2 protein was expressed throughout all stages of growth and differentiation. The cultures generated no 15-LO metabolites when incubated with 10 microM to 50 microM arachidonic acid (AA) and stimulated with ionophore. However, lysates prepared from such cultures generated 15-hydroxyeicosatetraenoic acid (15-HETE) and 12-HETE from AA, indicating that the cells contained active enzyme. When cultures expressing 15-LO protein were incubated with 10 microM linoleic acid (LA) instead of AA, and were stimulated with ionophore, they generated 13-hydroxy-9,11-octadecadienoic acid. LA rather than AA appeared to be the preferred substrate for the 15-LO enzyme. Our studies indicated that the expression of 15-LO and PGHS-2 is differentiation dependent in airway epithelial cells.     

Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis-type 12-lipoxygenase (e12-lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4-8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)-HETE from arachidonic acid. By fluorescence in situ hybridization the e12-lipoxygenase gene was localized to chromosome band 11 B1-B3.     

In vivo stimulation of 12(S)-lipoxygenase in the rat skin by bradykinin and platelet activating factor: formation of 12(S)-HETE and hepoxilins, and actions on vascular permeability

In this study we set out to investigate whether the inflammatory agents, bradykinin (BK) and platelet activating factor (PAF), affect the lipoxygenase pathway in rat skin in vivo and whether the main products so formed may be involved in the inflammatory actions of these agents. In vitro preparations of epidermis were also investigated to determine whether lipoxygenases are stimulated by these agents. We also investigated the actions of arachidonic acid and 12(S)-HPETE as substrates for the lipoxygenases. Our results indicated that 12-lipoxygenase is actively and selectively stimulated in a dose-dependent way in both preparations by the administration of BK and PAF; the main product, 12-HETE, was shown by chiral analysis to be exclusively of the S-configuration, indicating that 12(S)-lipoxygenase was present in the rat skin and was stimulated by these inflammatory agents. Hepoxilins were also formed but to a lesser extent in both in vivo and in vitro preparations. In separate experiments, 12(S)-HETE administered intradermally on its own (40 ng/site), increased vascular permeability as also seen with bradykinin (100 ng/site) and PAF (10 ng/site). However, unlike previously observed with hepoxilin A3 administration, 12(S)-HETE did not stimulate the action of BK on vascular permeability, suggesting that the two compounds may have different mechanisms of action to enhance inflammation. These observations suggest that the vascular permeability and plasma extravasation observed with both inflammatory agents (BK and PAF) may be mediated at least in part through the activation of 12(S)-lipoxygenase, resulting in enhanced formation of 12(S)-HETE which causes acute inflammation.     

Biological inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 micromol/L, but both blocked 5-oxo-ETE-induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 micromol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.