NaCN inhibits the stereospecific depletion of LTB4 by guinea pig polymorphonuclear leukoxytes

Caseinate elicited suspension of guinea pig peritoneal PMNs synthesized LTB₄, 6t-LTB₄, 12-epi-6t-LTB₄ and 5HETE after incubations with A23187 and arachidonic acid. Concentrations of LTB₄ peaked in 3 minutes and were then rapidly depleted. 6t-LTB₄ and 12-epi-6t-LTB₄ also peaked in concentrations in 3 min but were depleted slower than LTB₄. NaCN inhibited the depletion of LTB₄ in a dose dependent fashion without dramatically affecting biosythesis.     

Metabolism of 6-trans isomers of leukotriene B4 to dihydro products by human polymorphonuclear leukocytes

The major dihydroxy metabolites of arachidonic acid formed by human polymorphonuclear leukocytes (PMNL) are leukotriene B4 (LTB4), 6-trans-LTB4, and 12-epi-6-trans-LTB4. LTB4, and to a lesser extent its 6-trans isomers, are metabolized to 20-hydroxy products by a hydroxylase in PMNL. We have recently reported the existence of a second pathway involving a reductase which, combined with the hydroxylase, results in the conversion of 6-trans-LTB4 to dihydro-6-trans-LTB4. We have now investigated some of the characteristics of this novel triene reductase pathway in human PMNL and have characterized some of the products and their mechanism of formation. At low substrate concentrations, the major pathway for the initial metabolism of both 6-trans-LTB4 and 12-epi-6-trans-LTB4 is reduction of the conjugated triene chromophore to give dihydro products with single absorption maxima at about 230 nm. Dihydro-6-trans-LTB4 is rapidly converted to its 20-hydroxy metabolite by LTB4 20-hydroxylase. However, 20-hydroxy-6-trans-LTB4 is not a substrate for the reductase. Neither 12-epi-6-trans-LTB4 nor its dihydro metabolite, 5,12-dihydroxy-7,9,14-eicosatrienoic acid, which was identified by gas chromatography-mass spectrometry, were very good substrates for the hydroxylase. The dihydro metabolites of 6-trans-LTB4 and 12-epi-6-trans-LTB4 were formed rapidly during the initial phase of the reaction, whereas the corresponding dihydro-20-hydroxy metabolites were formed only after a lag phase. Experiments utilizing deuterium-labeled 12-epi-6-trans-LTB4 indicated that a hydrogen atom is lost from the 5-position of the substrate, suggesting that the initial step in the formation of the dihydro products is the formation of a 5-oxo intermediate. LTB4 is metabolized very rapidly by LTB4 20-hydroxylase in PMNL, and we have not yet identified dihydro products derived from this substance. However, LTB4 strongly inhibits the conversion of 12-epi-6-trans-LTB4 to dihydro products, suggesting that it may also interact with the reductase.     

Conversion of stereoisomers of leukotriene B4 to dihydro and tetrahydro metabolites by porcine leukocytes

We have previously shown that porcine leukocytes convert leukotriene B4 (LTB4) to two major products, 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4. Although we did not detect these products after incubation of LTB4 with human polymorphonuclear leukocytes, these cells converted 12-epi-6-trans-LTB4 to the corresponding 6,11-dihydro metabolite (i.e., there appeared to be a shift in the positions of the remaining double bonds). The objective of the present investigation was to determine whether 6-trans isomers of LTB4 are metabolized by porcine leukocytes by a pathway similar to LTB4, or whether they are metabolized by a pathway analogous to that in human leukocytes. We found that 6-trans-LTB4 and 12-epi-6-trans-LTB4 are metabolized more much extensively than LTB4 by porcine leukocytes. 6-trans-LTB4 appears to be converted by two different reductase pathways to two dihydro products differing in the positions of the two remaining double bonds between carbons 5 and 12. Dihydro-12-oxo and dihydro-5-oxo metabolites are also formed from this substrate. Porcine leukocytes also convert 6-trans-LTB4, presumably by a combination of the above two pathways, to tetrahydro, tetrahydro-12-oxo and tetrahydro-5-oxo metabolites, none of which possesses any conjugated double bonds. 12-epi-6-trans-LTB4 is also converted to tetrahydro metabolites by these cells. Experiments with deuterium-labeled 6-trans-LTB4 indicated that the deuterium in the 5-position was almost completely lost during the formation of tetrahydro-6-trans-LTB4, whereas about 80-85% of the deuterium in the 12-position was lost, suggesting a requirement for a 5-oxo intermediate. As with LTB4, 12-epi-8-cis-6-trans-LTB4, the product of the combined actions of 5-lipoxygenase and 12-lipoxygenase, was converted principally to dihydro and dihydro-12-oxo metabolites. Only a relatively small amount of the tetrahydro metabolite was detected.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Products of the lipoxygenase pathway in human natural killer cell cytotoxicity

As earlier data suggested the importance of lipoxygenase activation for expression of human NK cell cytotoxicity, four different lipoxygenase inhibitors were tested for suppression of natural killer (NK) cell lysis. All inhibitors were found active at nontoxic concentrations with 50% inhibition at approximately 15 microM for nordihydroguaiaretic acid (NDGA). NK cell lysis could be reconstituted to NDGA-suppressed cells with leukotriene B4 (LTB4), the all-trans isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4, and 20-COOH-LTB4. LTB4 reconstitution was best in the concentration range 1-100 pM and near control levels at both higher and lower concentrations. Herpesvirus Ateles-transformed killer T cells could also be inhibited by NDGA. These data indicate that lipoxygenase activity is required for human NK cell lysis and that several different LTB4-related products can restore NK activity in inhibited cells; they also suggest that the lipoxygenase pathway is present in the killer cell population.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B (LTB), 6-trans-LTB4, 12-epi-6-trans-LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohydroxyeicosatetraenoic acids (i.e., 5-HETE) and omega-oxidation products (i.e., 2O -COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 microM), LTB4 but not 5-HETE formation was impaired. (1-14C)Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate, (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose: the chemical synthesis of isotopically labeled 2-epi-5-epi-valiolone analogs

2-epi-5-epi-valiolone is a cyclization product of the C(7) sugar phosphate, sedoheptulose 7-phosphate, involved in the biosynthesis of the aminocyclitol moieties of acarbose, validamycin, and pyralomicin. As part of our investigation into the pathway from 2-epi-5-epi-valiolone to the valienamine moiety of acarbose, we prepared 1-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-6], 5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-17], 1-epi-2-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-12] and 2-epi-5-epi-(6-(2)H(2))valiolamine [(6-(2)H(2))-11]. Compounds (6-(2)H(2))-6 and (6-(2)H(2))-17 were synthesized from 2,3,4,6-tetra-O-benzyl-D-glucopyranose in 10 and seven steps, respectively, whereas (6-(2)H(2))-12 and (6-(2)H(2))-11 were synthesized from 2,3,4,6-tetra-O-benzyl-D-mannopyranose in eight and 10 steps, respectively.     

Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase

LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.     

Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product

Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.     

Highly potent cell differentiation-inducing analogues of 1alpha,25-dihydroxyvitamin D3: synthesis and biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-chain modifications

Eight 2-methyl substituted analogues of 20-epi-22R-methyl-1alpha,25-dihydroxyvitamin D3 (5) and 20-epi-24,26,27-trihomo-22-oxa-1alpha,25-dihydroxyvitamin D3 (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modified side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a-d), (3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded two sets of four A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D3 (7a-d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-dihydroxyvitamin D3 (8a-d). The biological profiles of the hybrid analogues were assessed in terms of affinity for vitamin D receptor (VDR) and HL-60 cell differentiation-inducing activity in comparison with the natural hormone. The combined modifications of the A-ring at the 2-position and the side chain yielded analogues with high potency.     

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.     

Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.     

Tissue differences in vascular permeability induced by leukotriene B4 and prostaglandin E2 in the rat

The activity of synthetic LTB4 and PGE2, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with 125I-albumin, 51Cr-erythrocytes, and 57Co-EDTA. The rats were then injected through the carotid artery cannula with LTB4, PGE2 or a combination of LTB4 and PGE2. Eight minutes later the rats were killed and the activity of 125I, 51Cr, and 57Co measured in different organs. Changes in vascular permeability were inferred from changes in the ratios of the isotope activities. LTB4 (15 micrograms/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB4, 3 micrograms/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE2 (1 microgram/kg) enhanced vascular permeability only in the caecum. A combination of LTB4 (3 micrograms/kg) and PGE2 (1 microgram/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB4 than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB4 induced increases in permeability.     

Sesquiterpene constituents from the liverwort Bazzania japonica

The hydrodistillation products of the liverwort Bazzania japonica were separated by preparative gas chromatography (GC) and investigated by spectroscopic methods. Seven unknown compounds were isolated and identified by GC-MS and NMR. Four of them, the norsesquiterpene hydrocarbons 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2), 4-epi-11-nor-aristola-9,11-diene (3), and one oxygenated sesquiterpene, (-)-aristol-1(10)-en-12-ol (5) are new natural compounds, and one, (+)-himachala-2,4-diene (7), has for the first time been isolated from liverworts. The absolute configurations of 5 and 7 were derived by chemical correlation reactions and/or enantioselective GC using cyclodextrin phases. 1, 2 and 3 have identical absolute configuration.     

Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.     

Functional properties of guinea pig eosinophil leukotriene B4 receptor.

It is currently thought that pulmonary eosinophils play a proinflammatory role in bronchial asthma. Leukotriene B4 (LTB4) is being considered as an important mediator in regulating eosinophil function because of its potent activities in inducing leukocyte chemotaxis, chemokinesis, degranulation, and aggregation. Because the LTB4 receptor has not been characterized in eosinophils, we report in this study the presence of a functional high affinity receptor for LTB4 on guinea pig (GP) eosinophils. Scatchard analysis of saturation binding studies yielded a Kd of 1.4 +/- 0.2 nM (mean +/- SEM, n = 3) and a Bmax of 1.6 +/- 0.4 pmol/mg of protein for LTB4 in GP eosinophil membranes. A linear Scatchard plot was obtained, suggesting that GP eosinophil membranes expressed only a single high affinity LTB4 receptor population. Saturation binding studies in whole cells also yielded a linear Scatchard plot, with a Kd of 2.8 +/- 0.96 nM (mean +/- SEM, n = 4) and a Bmax of 4 x 10(4) +/- 6 x 10(3) receptors/cell. Competitive binding studies using several compounds with structures similar to that of LTB4 showed that these agents bound to the receptor in the following descending order of affinity (Ki, nM): LTB4 (0.96) less than TB3 (1.0) greater than 20-hydroxy-LTB4 (3.5) greater than 12(R)-hydroxy-5,8,14-cis,10-trans-eicosatetraenoic acid (20) greater than 12(S)-hydroxy-5,8,14-cis,10-trans-eicosatetraenoic acid (231) greater than 20-carboxy-LTB4 (350) greater than 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid (541). This rank order of potency in binding affinity correlates closely with the ability of these compounds to induce both chemotaxis and superoxide anion generation. Analysis of the structure-activity relationship suggests that the 12R-hydroxyl group and a cis double bond at the C-6 position are important for optimal agonist binding to the LTB4 receptor present in GP eosinophil membranes. The results suggest that LTB4 may be an important chemoattractant for eosinophils in GP and may induce the release of reactive oxygen species from this cell.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Effects of leukotriene B4 on permeability, prostacyclin and thromboxane release by normal and oxygen-preexposed isolated, perfused rat lungs

This study assessed the hemodynamic and permeability effects of exogenous, synthetic leukotriene B4 (LTB4) on normal rat lungs and lungs from rats preexposed to oxygen for 48 h, which were isolated and perfused at constant flow in vitro. Adult, Sprague-Dawley rats were exposed to air or greater than 97% O2 for 48 h. After exposure, their lungs were removed from the thorax, ventilated with normoxic gas, and perfused at 12 ml/min with Krebs-Ringer bicarbonate buffer which contained 5 mM glucose and 3 mg/ml albumin. A total of 5.55 micrograms of synthetic LTB4 was infused in three separate boluses over 15 minutes. Perfusion and airway pressures were monitored, and the lungs release of 6-ketoprostaglandin F1 alpha and thromboxane B2 (TXB2) into the effluent from the pulmonary vasculature was measured by radioimmunoassay. The LTB4 had no measureable effects on pulmonary vascular pressures. LTB4 infusion caused a pronounced increase in permeability, indicated by increased albumin concentrations in alveolar lavage fluid from O2-preexposed lungs. Release of TXB2 from both air- and O2-preexposed lungs was increased after LTB4 infusion, while the change in 6-ketoprostaglandin F1 alpha release was not statistically significant. Both the increase in permeability enhanced TXB2 released after LTB4 infusion were inhibited by 10 microM indomethacin in the perfusate. These data indicate that exogenous LTB4 increases microvascular permeability in O2-exposed lungs in association with increased release of TXB2 into the pulmonary vascular effluent.