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.     

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.     

Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB4 concentrations were determined when canine PMNs were incubated with 10 microM A23187. Increasing LTB4 concentrations were determined through 10 min incubation. The maximal LTB4 concentrations (310 +/- 30 pg LTB4/2.5 x 10(5) cells) determined at 10 min did not change through a 55 min incubation period. Greater LTB4 concentrations were synthesized by canine PMNs at 37 degrees C (268 +/- 12 pg LTB4/2.5 x 10(5) cells) than at 25 degrees C (206 +/- 11 pg LTB4/2.5 x 10(5) cells) or 5 degrees C (59 +/- 3 pg LTB4/2.5 x 10(5) cells). The synthesis of LTB4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4 synthesis with an approximate IC50 = 0.1 microM, whereas BW755C inhibited LTB4 synthesis with an approximate IC50 = 10 microM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB4 synthesis in canine PMNs.     

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, abd 5,6-duhydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-D_R-³H]- or [10-L_S-³H]- labeled arachidonic acid, and 6-trans-LTB₄ and its 12-epimer were analyzed. More than 90% of 10-D_R-hydrogen was lost while about 100% of 10-L_S-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A₄.     

Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNL) convert arachidonic acid (20:4) to a number of dihydroxy metabolites, including leukotriene B4 (LTB4) 5S,12R-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid (isomer-1), 5S,12S-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid, 5S,12S-dihydroxy-6,8,10,14-EZEZ-icosatetraenoic acid (5S,12S-dh-20:4), 5,6-dihydroxy-7,9,11,14-icosatetraenoic acid, and 5,15-dihydroxy-6,8,11,13-icosatetraenoic acid. LTB4 was synthesized rapidly after stimulation of PMNL with the divalent cation ionophore, A23187, but its concentration rapidly declined after about 4 min, in contrast to the other dihydroxy metabolites of 20:4 whose concentrations remained stable for at least 20 min. The amounts of polar metabolites (identified primarily as 20-hydroxy-LTB4) increased steadily with time up to 20 min. These results suggest that LTB4 may be specifically converted to its 20-hydroxy metabolite by PMNL. We prepared 3H- and 14C-labeled analogs of the dihydroxyicosatetraenoic acid metabolites described above by incubation of labeled 20:4 with PMNL. Although all of these substances were metabolized to some extent by human PMNL, LTB4 (apparent Km, 1.0 microM) was metabolized the most rapidly, followed by 5S,12S-dh-20:4 (apparent Km, 2.4 microM) and isomer-1 (apparent Km, 4.8 microM). All three substrates were shown by mass spectrometry to be converted to their 20-hydroxy metabolites. LTB4 was also metabolized to its omega-carboxy derivative. Human mononuclear leukocytes and rabbit PMNL metabolized LTB4 very slowly, whereas rat PMNL metabolized this substrate at about one-sixth the rate of human PMNL. These results demonstrate that human PMNL contain an omega-hydroxylase that specifically converts LTB4 to its 20-hydroxy metabolite. This enzyme may be important for the regulation of LTB4 levels in vivo.     

Lipoxin synthesis by arachidonate 5-lipoxygenase purified from porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes produced several more polar compounds from 5,15-dihydroperoxy-eicosatetraenoic acid added as such or generated from 15-hydroperoxy acid. These polar products with absorption maxima at 301-302 nm and shoulders at 289 nm and 316-317 nm were identified as 5S,6R,15S-11-cis-lipoxin A and its 6-epimer, all-trans-lipoxin A isomers, and all-trans-lipoxin B isomers. Most of these lipoxins were presumably degradation products of a 5,6-epoxy intermediate formed by the catalysis of leukotriene A synthase, an integral part of 5-lipoxygenase. The rate of the enzymatic lipoxin synthesis from 15-hydroperoxy acid was about 6% of arachidonate 5-oxygenation.     

Increased activity of 5-lipoxygenase in polymorphonuclear leukocytes from asthmatic patients

The formation of 5-lipoxygenase products of arachidonic acid, 5-HETE and 5,12-diHETE, was determined in 100,000 X g supernatant of polymorphonuclear leukocytes from 17 healthy subjects, 17 patients with extrinsic asthma and 15 patients with intrinsic asthma. After the supernatant was incubated with 14C-arachidonic acid in the presence of calcium and indomethacin, the lipoxygenase products of arachidonic acid were separated by thin layer chromatography. The results were expressed as the percentage conversion of 14C-arachidonic acid into the product per 10(7) cells. The formation of 5,12-diHETE, but not of 5-HETE, was significantly increased in the cells from the group of patients with extrinsic asthma (4.38 +/- 0.78%, mean +/- S.E.; p less than 0.01) and intrinsic asthma (6.09 +/- 1.11%; p less than 0.01), when compared to normal subjects (1.74 +/- 0.30%). Both extrinsic and intrinsic asthmatics had significantly enhanced 5-lipoxygenase activity, which was expressed as the sum of percentage conversion of 14C-arachidonic acid into 5-HETE and 5,12-diHETE. The percentage conversion in normal subjects was 4.19 +/- 0.39%, 6.24 +/- 0.84% for 17 patients with extrinsic asthma (p less than 0.05), and 8.59 +/- 1.29% for 15 patients with intrinsic asthma (p less than 0.01). There was no significant difference between these asthmatic groups. These results indicate that 5-lipoxygenase activity is increased in patients with bronchial asthma.     

Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.     

The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.     

A previously unidentified leukotriene produced by human polymorphonuclear leukocytes

A previously unidentified leukotriene was isolated from incubations of human polymorphonuclear leukocytes with ionophore A23187. The compound eluted between LTB4 and 20-carboxy LTB4 on SP-HPLC and between the two 6-trans isomers of LTB4 on RP-HPLC. Ultraviolet spectroscopy revealed three absorption bands at 258 nm, 268 nm and 278 nm. 1802 was incorporated by the OH at both C5 and C12. Oxidative ozonolysis indicated the presence of 5S, 12S configuration. The structure of the newly identified leukotriene is 5S, 12S-dihydroxy-icosatetraenoic acid. Stereochemistry of the double bonds and biologic activity were not investigated.     

Conversion of leukotriene B4 to dihydro and 19-hydroxy metabolites by rat polymorphonuclear leukocytes

Rat polymorphonuclear leukocytes metabolize leukotriene B4 (LTB4) by at least two major pathways. LTB4 is converted by a reductase in these cells to a dihydro metabolite in which one of the three conjugated double bonds has been reduced to give a conjugated diene with a UV absorption maximum at 230 nm. DihydroLTB4 appears to be a key intermediate in the metabolism of LTB4 by rat polymorphonuclear leukocytes, since a number of other metabolites, exhibiting UV absorbance at 235 nm, but not at 280 nm, have been detected by high pressure liquid chromatography. In addition, these cells contain a 19-hydroxylase, which converts LTB4 to 19-hydroxyLTB4, which has a typical leukotriene UV spectrum, exhibiting absorption maxima at 261, 270, and 282 nm.     

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4

Leukotriene B4 stimulated a transient production of superoxide anions (O2-) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B4 and calcium for the half-maximal production were about 1 microM and 200 microM, respectively. 8-(N,N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O2- production stimulated by leukotriene B4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B4 was added to the cells treated with cytochalasin B, the production of O2- was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca2+ concentrations but it could be induced even without the addition of Ca2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca2+-free medium showed a negligible production of superoxide on addition of leukotriene B4, but the production appeared upon addition of CaCl2. These findings suggest that the superoxide production stimulated by leukotriene B4 is associated with the influx of Ca2+.     

Some newly synthesized leukotriene B4 analogs inhibit LTB4-induced lysozyme release from rat polymorphonuclear leukocytes

Antagonistic activities of newly synthesized LTB4 analogs, which possess the same olefinic geometry and the same hydroxyl group stereochemistry as natural LTB4, were investigated by studying lysozyme release from rat polymorphonuclear leukocytes (PMNLs). 14,15-Dihydro LTB4(LTB3) induced lysozyme release from PMNLs as well as LTB4, while 14,15-dehydro LTB4 did not cause lysozyme release but instead clearly inhibit LTB4-induced lysozyme release. Compounds containing aminocarbonyl groups partially retained lysozyme releasing activity. A displacement of the hydrocarbon chain at C13-20 by cyclohexenyl and beta-cyclohexylethyl groups had little effect on lysozyme release, but did strongly inhibit release induced by LTB4. These results may be useful in developing LTB4 antagonists.     

Induction of 5-lipoxygenase activation in polymorphonuclear leukocytes by 1-oleoyl-2-acetylglycerol

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold).Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.     

Activation of a 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen

Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.     

Regulation of 5-lipoxygenase activity by the glutathione status in human polymorphonuclear leukocytes

The influence of the glutathione status of human polymorphonuclear leukocytes (PMN) on 5-lipoxygenase activity was studied by treating cells with increasing concentrations of 1-chloro-2,4-dinitrobenzene (Dnp-Cl) or azodicarboxylic acid bis(dimethylamide) (Diamide). Subsequent incubation with arachidonate resulted in an up to tenfold-stimulated formation of 5-hydroxyeicosatetraenoic acid, leukotriene B4, leukotriene B4 isomers and omega-hydroxyleukotriene B4. Higher concentrations of the GSH reagents were inhibitory. At maximal stimulation by Dnp-Cl, 5-hydroperoxyeicosatetraenoic acid started to be built up at the expense of 5-HETE at glutathione levels which were diminished by about 50% compared to resting cells. No increase in cytosolic Ca2+ could be measured under these conditions by the fura-2 method. In PMN homogenates Dnp-Cl and Diamide were without effect and even caused inhibition when 5-lipoxygenase was stimulated by Ca2+ and ATP. 15-Lipoxygenase was either unchanged in the case of Diamide, or even increased after pretreatment with Dnp-Cl. The results allow us to conclude that 5-lipoxygenase activity in intact PMN is regulated not only by Ca2+ but in a complex manner also by the glutathione redox status. Conditions of oxidative stress increase the activity which may reflect the in vivo situation under phagocytosis and oxidative burst.     

Inhibition of leukotriene B4 synthesis in human polymorphonuclear leukocytes after exposure to meningococcal lipopolysaccharide

Polymorphonuclear leukocytes (PMNL) were preincubated in the presence and absence of lipopolysaccharide (LPS) prior to stimulation of arachidonic acid (20:4) metabolism by addition of the divalent cation ionophore, A23187. Analysis of the products by high pressure liquid chromatography showed that LPS inhibited the formation of leukotriene B₄, 5-hydroxy-6,8,11,14- icosatetraenoic acid and 12-hydroxy-5,8,10,14-icosatetraenoic acid by about 70%. In the absence of ionophore, LPS had little effect on the basal synthesis of 20:4 metabolites. Preincubation with LPS also inhibited the formation of the above 3 products in the presence of an excess of exogenous 20:4, suggesting that its action was mediated by the inhibition of lipoxygenases rather than phospholipase.     

12-Lipoxygenase from bovine polymorphonuclear leukocytes, an enzyme with leukotriene A4-synthase activity

Bovine polymorphonuclear leukocytes exhibit a 12-lipoxygenase activity upon sonication. In contrast to bovine platelet 12-lipoxygenase and other 12-lipoxygenases, this enzyme is unable to convert 5(S)-HETE (5(S)-hydroxy,6-trans-8,11,14-cis-eicosatetraenoic acid) or 5(S)-HPETE (5(S)-hydroperoxy,6-trans-8,11,14-cis-eicosatetraenoic acid) into 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid. Surprisingly, the formation of leukotriene A4-derived products namely leukotriene B4 and the leukotriene B4-isomers 12-epi,6-trans- leukotriene B4 and 6-trans-leukotriene B4, was observed upon incubation of this enzyme with 5(S)-HPETE. Hence, the 12-lipoxygenase from bovine polymorphonuclear leukocytes possesses leukotriene A4-synthase activity.     

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.     

Investigations of the biological activity of leukotriene A4 in human polymorphonuclear leukocytes

An investigation was undertaken to compare the responses of human neutrophils to the epoxide leukotriene A4 with those elicited by its stable product leukotriene B4 under identical IN VITRO conditions. LTA4 evokes neutrophil responses similar in nature to those induced by LTB4 but at much higher concentrations. Evidence suggests that LTA4 is important primarily for its role as an intermediate rather than for inherent activity.