Jan Åke Lindgren

Identification and biological activity of novel ω-oxidized metabolites of leukotriene B4 from human leukocytes

The leukotrienes constitute a new group of biologically active compounds derived from polyunsaturated fatty acids [ 11. Thus, arachidonic acid can be oxygenated by a lipoxygenase to SS-hydroperoxyeicosatetraenoic acid [2], which is further converted to an unstable epoxide, 5,6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene Aq, LTA4) [3,4]. This intermediate is transformed enzymatically by addition of glutathione into a ‘slow-reacting substance of anaphylaxis’ (SRS-A), LTC4 [5,6]. The biological activity of most SRS-A preparations is due to LTC4 and the two metabolites LTD4 and LTE4 [7-91. LTA4 can also be hydrolyzed enzymatically to 5S,12R-dihydroxy-6-cis,8,1O-trans,l4-cis-eicosatetraenoic acid (LTB4) [ 1 O-l 2] or non-enzymatically to isomeric 5,12and 5,6-dihydroxy-eicosatetraenoic acids [ 131. We have reported the formation of a novel dihydroxy-acid, 5S,12S-dihydroxy-6-trans,8-cis,lOtrans,l4-cis-eicosatetraenoic acid (SS,12SDHETE), in preparations of human leukocytes [ 14). This dihydroxy-acid was not formed via an epoxide intermediate, but by a double oxygenation of arachidonic acid. In addition, an w-hydroxylated metabolite, 5S,12S,20trihydroxy-6-trans,8-cis,lO-trarzs,lLF-cis-eicosatetraenoic acid (5S,12S,20-THETE), was identified. This report describes the formation of an w-hydroxylated metabolite of LTB4, 5S,12R,20-trihydroxy-6cis

Chapter 4 Coexistence of neuronal messengers — an overview

,lO-trans,l4&s-eicosatetraenoic acid (20-OHLTB4) in human leukocyte preparations and further conversion of this trihydroxy acid to a dicarboxylic acid (20-COOH-LTB4). In addition, the biological activity of LTB4, 20-OH-LTB4 and 20-COOH-LTB4, on guinea pig lung strips is reported.

Stimulation of human leukocyte degranulation by leukotriene B4 and its ω-oxidized metabolites

Publisher Summary This chapter discusses results demonstrating that neurons often contain more than one chemical compound. The different types of coexistence situations are described, including (1) a classical transmitter and one or more peptides, (2) more than one classical transmitter, and (3) a classical transmitter, a peptide, and adenosine triphosphate (ATP). The functional significance of these histochemical findings is at present difficult to evaluate, but in studies on the peripheral nervous system evidence has been obtained that classical transmitter and peptide are coreleased and interact in a cooperative way on effector cells. In addition to enhancement, there is evidence that other types of interactions may occur—for example, the peptide may inhibit the release of the classical transmitter. Also in the central nervous system (CNS), indirect evidence is present for similar mechanisms—that is, to strengthen transmission at synaptic (or non-synaptic) sites and for the peptide inhibition of release of a classical transmitter. Multiple messengers may provide the means for increasing the capacity for information transfer in the nervous system.

Formation of novel hydroxylated eicosatetraenoic acids in preparations of human polymorphonuclear leukocytes

and adhesion to post- capillary venules [3]. In [4-61 LTB., was suggested to be a weak stimulator of polymorphonuclear leuko- cyte (PMNL) degranulation. However, it is now clear that previous purification procedures for biosyntheti- tally prepared LTB4 were inadequate. The newly discovered LTB4 isomer, SQ,12Q-DHETE, co-chro- matographs with LTB4 in the reverse phase high- pressure liquid chromatography (HPLC) systems commonly used [7]. As a consequence, earlier studies probably tested preparations which contained a mix- ture of both isomers with the non-leukotriene product predominating. Therefore, we have investigated the role of LTB4, SQ,12(S)-DHETE and their o-oxida- tion products (fig.1) [8] in leukocyte degranulation in vitro. In addition, the activity of the non-enzymati- tally formed LTB4 isomers was investigated. Cytochalasin B, phenolphthalein glucuronic acid, and dried

Novel leukotrienes: Products formed by initial oxygenation of arachidonic acid at C-15

Recent studies of arachidonic acid metabolism in polymorphonuclear leukocytes have led to the discovery of the biologically active leukotrienes [ 11. The synthesis of these compounds is initiated by oxygenation of arachidonic acid at C-5 forming 5hydroperoxyeicosatetraenoic acid [2], which is further converted to the unstable 5 ,6-epoxy-7,9,11 ,14-eicosatetraenoic acid (leukotriene &, LTA4) [3,4]. The epoxide is hydrolysed either enzymatically to SS,12R-dihydroxy6_cis,8-rruns,lO-trans,l4_cis-eicosatetraenoic acid (LTB,) [S-7] or non-enzymatically to additional isomeric S,12and 5,6-dihydroxy eicosatetraenoic acids [8]. LT& is also converted by addition of glutathione into LTC4 [9,10]. This compound and two metabolites, LTD4 and LTE4, are responsible for the biological activity of most preparations of slowreacting substance of anaphylaxis (SRS-A) [ 11,121. We have reported that a new group of leukotrienes can also be formed by initial oxygenation at C-l 5 [13,14]. Here, we describe the formation of 5S,12Sdihydroxy_6,8,10,14eicosatetraenoic acid and 5S,12S,20-trihydroxy-6,8,10,14-eicosatetraenoic acid in preparations of human leukocytes. These novel metabolites are not formed via the leukotriene pathway. Instead arachidonic acid is transformed by a double oxygenation to the dihydroxy acid and further w oxidized to the trihydroxy acid.

Transcellular conversion of endogenous arachidonic acid to lipoxins in mixed human platelet-granulocyte suspensions

Abstract A preparation of human leukocytes was incubated with arachidonic acid. Two new dihydroxy acids with conjugated triene structures, were isolated and characterized as 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-leukotriene B4) and 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene B4).

Lipoxin formation in human nasal polyps and bronchial tissue

Incubation of mixed human platelet/granulocyte suspensions with ionophore A23187 led to a platelet dependent formation of several lipoxin isomers from endogenous substrate. The major metabolite coeluted with authentic lipoxin A4 (5(S), 6(R), 15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) in several HPLC-systems and showed an identical UV-spectrum. Furthermore, a similar profile of lipoxins was formed in pure platelet suspensions incubated with exogenous leukotriene A4 (5(S) -5, 6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid). The conversion of exogenous leukotriene A4 to lipoxin A4 was markedly increased in the presence of ionophore A23187.

Regional distribution of leukotriene and mono‐hydroxyeicosatetraenoic acid production in the rat brain Highest leukotriene C4 formation in the hypothalamus

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15‐hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15‐hydroxyeicosatetraenoic acid was 3–5‐times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co‐incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15‐lipoxygenase in lipoxin formation in human airways.

Formation and proliferative effects of lipoxins in human bone marrow.

The regional distribution of ionophore A23187‐induced synthesis of leukotrienes and mono‐hydroxyeicosatetraenoic acids in the rat brain in vitro was investigated. Pronounced differences in leukotriene C4 formation were observed, with the highest synthetic capacity in the hypothalamus. The formation of leukotriene C4 was about 12‐times higher in the hypothalamus as compared to the cerebellum. This finding is in agreement with a possible neuroendocrine role for leukotriene C4. In contrast, the activity of leukotriene B4 synthesis was widely distributed without pronounced regional differences in the rat brain. Formation of 5‐, 9‐, 11‐, 12‐ and 15‐monohydroxyeicosatetraenoic acid was detected in all regions. The major lipoxygenase product in the hypothalamus and thalamus was 5‐hydroxyeicosatetraenoic acid, while other monohydroxyeicosatetraenoic acids predominated in the remaining regions tested.

A sensitive and specific radioimmunoassay for leukotriene C4.

Lipoxins A4 and B4 together with the all-trans lipoxin (LX) isomers were produced by normal human bone marrow cell suspensions after incubation with ionophore A23187. Both LXA4 and LXB4 enhanced the growth of myeloid progenitor cells in semisolid agar in the presence of suboptimal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lipoxin A4 at 10(-10) M stimulated the colony formation in 13 out of 15 tested human bone marrows with a mean (+/- SEM) increase of 47 +/- 11% (p = 0.001). A similar stimulatory effect was observed after addition of LXB4 (10(-10) M). The monohydroxyeicosatetraenoic acids 5-, 12- and 15-HETE did not affect colony growth. In addition, LXA4 (10(-8) M) efficiently counteracted the increased colony formation induced by leukotriene C4 (10(-10) M), suggesting an antagonistic relationship between these lipoxygenase products. The results support a role for lipoxins in the regulation of human myelopoiesis.