A phospholipase A2 isoenzyme provokes lipoxin B formation from endogenous sources of arachidonic acid in porcine leukocytes

doi:10.1016/S0006-291X(87)80484-9

Biochemical and Biophysical Research Communications

Volume 144, Issue 1, 14 April 1987, Pages 123-131

https://doi.org/10.1016/S0006-291X(87)80484-9 Get rights and content

Summary

Porcine leukocytes incubated with an isoenzyme of phospholipase A₂ (PLA₂) (isolated from snake venom) produced several trihydroxytetraene- containing compounds which were derived from endogenous sources of arachidonic acid. The formation of these compounds was dose-dependent with an EC₅₀ of approximately 1.25 × 10⁻⁸M. At this concentration of the isoenzyme and time of explosure the cells remained viable as determined by the exclusion of trypan blue. The compounds were purified by HPLC and their identities were determined by physical criteria which included U.V. spectrometry, GC/MS and by comparison with both synthetic and authentic materials. The biologically derived compounds proved to be lipoxin B (5S, 14R, 15S-trihydroxy-6, 10, 12-trans-8-ciseicosatetraenoic acid) and its two structural isomers (8-trans-LXB and 14S-8-trans-LXB). Of interest, only small amounts of lipoxin A and its isomers were found in these incubations. Results of the present study indicate that porcine leukocytes can generate lipoxin B and its isomers from endogenous sources of arachidonic acid. Moreover, they suggest that certain PLA₂ isoenzymes may initiate the formation of lipoxins and related compounds.

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Cited by (19)

High-performance liquid chromatography separation and determination of lipoxins
1990, Methods in Enzymology
The chapter presents a study on high-performance liquid chromatography (HPLC) separation and determination of lipoxins. This chapter describes the isolation, reversed-phase high-performance liquid chromatography (RP-HPLC) separation, and determination of lipoxins of the four series. The lipoxins are a series of biologically active, acyclic eicosanoids, which contain a conjugated tetraene structure as a characteristic feature. The following two main compounds are positional isomers: one is designated lipoxin A₄ (LXA₄) and the other lipoxin B₄ (LXB₄). Multiple pathways exist for lipoxin biosynthesis that are substrate-, cell type-, and species-specific. The chapter outlines the route that involves the transformation of 15- hydroxyeicosatetraenoic acid (HETE) and formation of a 5(6)-epoxytetraene by human leukocytes. Other routes can involve the transcellular metabolism of various substrates including leukotriene A₄, arachidonic acid, 5,15-DHETE, and 5-HETE. LXA₄ and LXB₄ each display biological activities, which can be distinguished from those of other eiscosanoids. Several isomers of LXA₄ and LXB₄ have been identified, including a novel 7-cis- 11-trans-LXA₄. Because LXA₄, LXB₄, and their isomers differ in biological actions and potencies, their identification within biologically derived materials is of interest. As is the case when identifying other eicosanoids, lipoxins should be identified from biological sources by fulfilling several criteria that may include the following: (1) coelution of the product with authentic materials in more than one HPLC system (retention time); (2) coelution of the derivatized material with that of authentic standard (determination of C value); (3) UV spectral analyses of the isolated material; (4) the presence of diagnostic ions in the mass spectra of several derivatives (that is, n-butyl boronate- trimethylsilyl derivatives); and (5) bioassay of the isolated products and comparison with authentic materials.
Lipoxins are major lipoxygenase products of rainbow trout macrophages
1989, FEBS Letters
Rainbow trout macrophages synthesize lipoxins as major lipoxygenase products entirely from endogenous fatty acids. High-performance liquid Chromatographic analysis of the supernatants from macrophages challenged with calcium ionophore A23187 revealed a range of lipoxygenase products including mono-hydroxy fatty acids, leukotrienes B₄ and B₅ and four major peaks with retention times and UV spectra characteristic of lipoxins (λ_max 302 nm). Cochromatography with authentic standards, UV spectroscopy and radiolabelling with (¹⁴C]arachidonate and eicosapentaenoate allowed tentative identification of the two largest peaks as lipoxin A₄ and A₅.
Agonist-dependent generation of lipoxins from rat basophilic leukemia cell (RBL-1)
1989, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Incubation of RBL-1 cells in the presence of 15-HPETE and various agonists generated lipoxins and several isomers. Addition of either A23187, fMLP or PMA modulated the number of isomers and amount of lipoxins produced. Administration of A23187 yielded the largest amount of product (5.3 ± 1.6 μg per 10⁸ cells and generated a total of six and three isomers of LXA₄ and B₄, respectively. This was 2-fold greater than fMLP, which produced a total of two isomers of LXA₄ and LXB₄. Addition of PMA generated only LXA₄ (0.68 ± 0.26 μg). This is similar to the control receiving only 15-HPETE. Biologically derived LXA₄ (3 nM) isolated from RBL-1 incubations contracted a rat tail artery preparation to 12% of the maximum induced by phenylephrine (0.125 μM), whereas LXA₄ standard (3 nM) elicited 17.6% of the maximum contraction. These results indicate that RBL-1 cells can utilize exogenous 15-HPETE to generate biologically active lipoxins. Further, the yield and isomers of lipoxins can be modified by different agonists.
On the relationship between leukotriene and lipoxin production by human neutrophils: evidence for differential metabolism of 15-HETE and 5-HETE
1989, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Lipoxygenase (LO) products generated by human PMN were examined utilizing a gradient-HPLC and rapid spectral detector which permitted continuous UV-spectral monitoring of leukotrienes, lipoxins and related oxygenated products of arachidonic acid. When exposed to the ionophore A23187, PMN generated LTB₄ and its ω-oxidation products as well as LXA₄, LXB₄, and 7-cis-11-trans-LXA₄ from endogenous sources. Addition of 15-HETE changed the profile of products generated by activated PMN and led to a time- and dose-dependent increase in lipoxins and related compounds while the production of LTB₄ and its ω-oxidation products was inhibited. Results of time-course and radiolabel studies revealed that 15-HETE is rapidly transformed within 15 s to 5,15-DHETE and conjugated tetraene-containing products, and that the inhibition of leukotriene formation followed a similar time-course. In contrast, PMN did not generate either lipoxins or related products from 5-[³H]HETE, nor did 5-HETE block leukotriene formation. Stimulated PMN generated 5,15-DHETE from exogenous 5-HETE, while in the absence of ionophore, 5-HETE was transformed to 5,20-HETE. These results indicate that PMN can generate lipoxins and related products from endogenous sources and that 15-HETE and 5-HETE are transformed by different routes.
Identification of a novel 7-cis-11-trans-lipoxin A<inf>4</inf> generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A<inf>4</inf> and B<inf>4</inf>
1989, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 μM) to human neutrophils led to the formation of both lipoxin A₄ and lipoxin B₄ as well as a novel 3,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A₄ and lipoxin B₄ were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA₄ (7-cis-11-trans-LXA₄). LXA₄, 11-trans-LXA₄, 7-cis-LXA₄ and 7-cis-11-trans-LXA₄ all evoked dose-dependent (0.1–10 μM) contractions of the guinea pig lung strip, whereas 6-cis-LXB₄ and 6-cis-8-trans-LXB₄ relaxed this preparation. LXA₄ and 7-cis-LXA₄ were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA₄ caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA₄ was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.
Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid
1988, FEBS Letters
Incubation of bovine polymorphonuclear leukocytes (PMNs) with arachidonic acid leads to the formation of four lipoxins. The same lipoxins are also formed upon incubation of bovine PMNs with 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5(S)-hydroperoxy, 15(S)-hydroxy-6,13-trans-8, 11-cis-eicosatetraenoic acid or 5(S), 15(S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid. A 5,6-epoxide as intermediate in lipoxin formation in the bovine PMN is highly improbable because the 5-hydroxy compounds are as good substrates as the 5-hydroperoxy compounds. Moreover, the two main lipoxins were found to coelute with the two lipoxins produced via a triple dioxygenation of arachidonic acid by soybean lipoxygenase-1. Hence the bovine PMN is the first cell for which evidence is presented that the formation of lipoxins proceeds mainly via triple dioxygenation and not via 15-hydroxy-leukotriene A₄ as is proposed for human and porcine PMNS.

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⁺: Present address: Hematology Division, Brigham and Womens Hospital, Harvard Medical School, 75 Francis St., Boston, MA.

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A phospholipase A2 isoenzyme provokes lipoxin B formation from endogenous sources of arachidonic acid in porcine leukocytes

Summary

Biochim. Biophys. Res. Commun.

Biochim. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Vit. and Hormones

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS LETTERS

Life Sciences

Toxicon.

A phospholipase A₂ isoenzyme provokes lipoxin B formation from endogenous sources of arachidonic acid in porcine leukocytes