Effects of dauricine on the metabolism of arachidonic acid in rat pleural neutrophils

The effects of dauricine (Dau), an isoquinoline alkaloid and anti-arrhythmic agent used in China recently, on the biosynthesis of metabolites of arachidonic acid in rat pleural neutrophils, comparing with dazoxiben, indomethacin and BW-755 c, were studied. The major products of metabolism by 5-lipoxygenase (5-LPO), measured by HPLC, were LTB4 and 5-HETE, whereas the major cyclooxygenase products measured by HPLC and RIA, were HHT and TXB2. The formation of all products by neutrophils was significantly depressed by Dau in a dose-dependent manner. The concentration of Dau required to obtain 50% inhibition (IC50) of formation of HHT, TXB2, LTB4 and 5-HETE was 25.3, 59.3, 31.8 and 59.5 mumol/L, respectively. These results indicate that the two major metabolic pathways of arachidonic acid in rat pleural neutrophils are inhibited by Dau.     

CGS 22745: a selective orally active inhibitor of 5-lipoxygenase.

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) synthesis in guinea pig leukocytes (IC50 = 0.6 microM). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB4 in blood (IC50's of 4.3, 0.56 and 3.2 microM for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB4 formation ex vivo after 5 min. Its effective biological half-life was greater than 160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibited ex vivo A23187-stimulated LTB4 formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.     

Stimulation of 5-lipoxygenase activity in polymorphonuclear leukocytes of rats by caseinate treatment

In the present study, the lipoxygenase activity in polymorphonuclear (PMN) leukocytes of rats was characterized, and the change in lipoxygenase activity in peritoneal and peripheral PMN leukocytes challenged by caseinate was investigated. Peritoneal PMN leukocytes produced 5-HETE and LTB4 as the major 5-lipoxygenase metabolites from arachidonic acid. The 5-lipoxygenase activity was calcium dependent. Caseinate treatment in rats significantly stimulated the 5-lipoxygenase activity in peripheral and peritoneal PMN leukocytes. Although it also stimulated the cyclooxygenase metabolic pathway, the effect was not as evident as that on the 5-lipoxygenase pathway. Since LTB4 is a potent chemotactic factor for PMN leukocyte migration, the present results might explain the mechanism on the PMN leukocyte infiltration caused by caseinate treatment.     

Auranofin stimulates LTA hydrolase and inhibits 5-lipoxygenase/LTA synthase activity of isolated human neutrophils

The effect of auranofin on the 5-lipoxygenase pathway was studied in human neutrophils stimulated with either fMLP or A23187 (with or without arachidonic acid). The synthesis of leukotriene B4 (LTB4), 5-HETE and the all-trans isomers of LTB4 was measured by HPLC. At low concentrations (0.5-2.0 microM), auranofin stimulated LTB4 synthesis, but inhibited it at higher concentrations (100% inhibition at less than 10 microM). In contrast auranofin caused dose-dependent inhibition of the synthesis of 5-HETE and the all-trans isomers of LTB4. Similar observations were made with each agonist. The stimulation of LTB4 synthesis and inhibition of the trans isomer production suggests that auranofin at low concentrations stimulates LTA hydrolase--the enzyme that converts LTA4 to LTB4, whereas the inhibition of synthesis of all lipoxygenase products at higher auranofin concentrations, suggests inhibition of 5-lipoxygenase/LTA synthase.     

Stimulation of lipoxygenase product synthesis in human leukocytes and platelets by melittin

The effects of melittin on the synthesis of lipoxygenase metabolites of arachidonic acid in human leukocytes and platelets were studied using high performance liquid chromatography. Melittin was found to stimulate strongly the formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs) in a concentration-dependent fashion. The metabolites detected were LTB4, omega-OH-LTB4, omega-COOH-LTB4, LTC4, 5-HETE, 12-HETE, 15-HETE, 5S,12S-DiHETE, and 5S,15S-DiHETE. These results suggest that the action of melittin on the formation of arachidonic acid metabolites might be involved in its ability to release endogenous substrates required for the synthesis of 5-, 15-, and 12-lipoxygenase products in leukocytes and platelets, respectively.     

Effect of age on leukotriene B4 production in guinea pig whole blood.

We evaluated the effect of age on eicosanoid production in guinea pig blood. Heparinized blood from 7-10-day, 6-week, or 6-month-old guinea pigs was incubated with 150 microM arachidonic acid (AA) for 5 min, followed by stimulation with A23187 (20 micrograms/mL) for an additional 10 min at 37 degrees. The reaction was terminated by centrifugation, and the production of plasma leukotriene (LT) B4 and C4, prostaglandin E2 (PGE2), and thromboxane B2 (TXB2) was determined by enzyme-linked immunoassay (ELISA). LTC4, PGE2, and TXB2 formation were unaffected by age. In marked contrast, production of LTB4 was increased 4- to 5-fold as age increased from 7-10 days (9.51 +/- 2.07 ng/mL) or 6 weeks (8.83 +/- 1.81 ng/mL) to 6 months (40.57 +/- 9.66 ng/mL). To determine the effect of age on the total eicosanoid product profile, blood was stimulated in the presence of [14C]AA, and plasma metabolites were separated by reverse-phase high pressure liquid chromatography (RP-HPLC) and quantitated using on-line radiochemical detection. In addition to increased LTB4 production, a modest increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was also observed in the 6-month-old animals. Previous studies have demonstrated interference of 12-HETE in the immunoassay of LTB4. Therefore, to validate the authenticity of the plasma leukotriene ELISA measurements, samples were precipitated with methanol and fractionated by RP-HPLC. The fractions co-eluting with [3H]LTB4 or [3H]LTC4 were dried under vacuum and reconstituted in ELISA buffer, and leukotrienes were quantitated. As seen previously, following HPLC purification LTB4 production remained significantly elevated in the 6-month-old guinea pigs, whereas LTC4 production was unaffected by age. To further document the selectivity of this effect on LTB4 production, we evaluated the effect of increasing age on cyclooxygenase or phospholipase A2 (PLA2) activity. Neither cyclooxygenase nor PLA2 activity was elevated as animals matured. In conclusion, the capacity of whole blood to produce LTB4, but not LTC4, TXB2, or PGE2, was elevated markedly in older animals.     

Selective inhibition of arachidonate 5-lipoxygenase by novel acetohydroxamic acids: biochemical assessment in vitro and ex vivo

1. The chemically novel acetohydroxamic acids, BW A4C, BW A137C and BW A797C, are potent inhibitors of the synthesis of leukotriene B4 (LTB4) from arachidonic acid by human leucocyte homogenates: the concentrations required for 50% inhibition (IC50) were 0.1 microM, 0.8 microM and 0.5 microM respectively. Inhibition was less at higher concentrations of arachidonic acid. 2. These compounds also inhibited the synthesis of [14C]-5-HETE from [14C]-arachidonic acid and the calcium-dependent synthesis of LTB4 from 5-HPETE. This, therefore, suggests that they inhibit 5-lipoxygenase and LTA4 synthase. 3. Concentrations of acetohydroxamic acids required to inhibit metabolism of arachidonic acid by cyclo-oxygenase, 12-lipoxygenase and 15-lipoxygenase were 10 to 100 times higher than those required to inhibit 5-lipoxygenase. 4. The compounds were potent inhibitors of LTB4 synthesis induced by the ionophore, A23187, in human intact leucocytes. This inhibition was reversed by washing the cells. They were also potent, selective inhibitors of LTB4 synthesis induced by A23187 in whole rat blood: binding to rat plasma proteins did not greatly reduce the effectiveness of the compounds. 5. The effects of the acetohydroxamic acids, administered either intravenously or orally to rats, on the synthesis of LTB4, and thromboxane B2 (TXB2) in A23187-stimulated blood ex vivo was studied. The three compounds caused dose-dependent inhibition of the synthesis of LTB4 but not TXB2. Inhibition of LTB4 synthesis persisted for up to 6 h after a single oral dose of 50 mg kg-1. 6. The plasma concentrations of unchanged compound determined by h.p.l.c. correlated with the inhibition of LTB4 synthesis ex vivo.     

Modulation of arachidonic acid metabolism by orally administered morniflumate in man.

Unlike other classic NSAIDs, some fenamates given at therapeutic concentrations, have been shown to inhibit, both in vitro and in vivo, the 5-lipoxygenase pathway of arachidonic acid cascade as well as the synthesis of cyclooxygenase products. This dual inhibitory property might represent an improvement in anti-inflammatory therapy. The aim of this work was to characterize the effect of morniflumate, administered at therapeutic dosages to normal human volunteers, on leukotriene B4 (LTB4) and thromboxane (TXB2) synthesis, both in purified PMNs and in whole blood. PMNs, isolated two hours after a single oral administration of morniflumate and at steady-state condition, fully retain their capacity to release LTB4 and TXB2. Since intracellular concentrations of the drug were undetectable, in spite of its elevated concentrations in platelet poor plasma, the results obtained using PMNs suggest a drug loss during the cells purification procedure. In whole blood experiments, morniflumate reduced blood LTB4 synthesis induced by Ca-ionophore A23187 Bx approximately 50%, both after single dose and at steady state; the degree of inhibition showed a pattern similar to the plasma levels of the bioactive metabolite of morniflumate (M1). The inhibition of serum TXB2 levels was higher than 85%. Hence, morniflumate is capable of reducing arachidonic acid metabolism acting both on cyclooxygenase and 5-lipoxygenase. This characteristic might provide a better approach in anti-inflammatory therapy.     

Studies on Scutellariae Radix; XIII1. Effects of Various Flavonoids on Arachidonate Metabolism in Leukocytes.

The effects of various flavonoids [i.e. baicalein, baicalin, wogonin, skullcapflavone II, (2 S),2',5,6',7-tetrahydroxyflavanone, (2 R,3 R),2',3,5,6',7-pentahydroxyflavanone, and 2',5,5',7-tetrahydroxy-6',8-dimethoxyflavone] isolated from Scutellariae Radix on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase products were studied. Baicalein (5,6,7-trihydroxyflavone) was found to inhibit the formation of 5-HETE (lipoxygenase product) more strongly than the formation of HHT (cyclooxygenase product); its concentrations for 50% inhibition (IC (50)) were 7.13 +/- 0.767 microM for the formation of 5-HETE and 55.3 +/- 16.9 microM for the formation of HHT (cyclooxygenase product). Baicalin (baicalein-7- O- D-glucuronide) also inhibited the formation of 5-HETE, though less strongly, while its compound had no effect on the formation of HHT (cyclooxygenase product) in polymorphonuclear leukocytes. In contrast, (2 S),2',5,6',7-tetrahydroxyflavanone inhibited the formation of HHT more strongly than the formation of 5-HETE via 5-lipoxygenase pathway; its concentrations for IC (50) were 5.63 +/- 1.27 microM for the formation of HHT and 670.0 + 85.0 microM for the formation of 5-HETE. Wogonin and (2 R,3 R),2',3,5,6',7-pentahydroxyflavanone also inhibited the formation of HHT; their IC (50) values were respectively 14.6 +/- 3.51 microM and 50.0 +/- 4.04 microM.     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB4 formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipid-dependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B4(LTB4) in FMLP-stimulated neutrophils, the ID50 was approximately 4.5 micrograms/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB4 nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB4 production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

Effects of Nonanal,trans-2-Nonenal and 4-Hydroxy-2,3-trans-nonenal on Cyclooxygenase and 12-Lipoxygenase Metabolism of Arachidonic Acid in Rabbit Platelets

The effects of nonanal, trans-2-nonenal and 4-hydroxy-2,3-trans-nonenal on the formation of thromboxane B₂ (TXB₂), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets were examined. Nonanal and trans-2-nonenal at concentrations ranging from 0.25 to 2 μm inhibited TXB₂, HHT and 12-HETE formation, reducing the amounts of these three arachidonic acid metabolites by 50% at nonanal and trans-2-nonenal concentrations of approximately 0.25 μm. The inhibition of TXB₂, HHT and 12-HETE formation induced by 4-hydroxy-2,3-trans-nonenal (50% inhibition by 4-hydroxy-2,3-trans-nonenal at a concentration of approximately 100 μm) was 400 times weaker than that induced by nonanal and trans-2-nonenal. These results suggest that nonanal and trans-2-nonenal can be modulators of platelet arachidonic acid metabolism by affecting the activity of cyclooxygenase and 12-lipoxygenase.     

Inhibition of leukotriene B4 formation in rat peritoneal neutrophils by an ethanolic extract of the gum resin exudate of Boswellia serrata.

Suspensions of rat peritoneal polymorphonuclear leukocytes (PMNL) elicited with glycogen were stimulated by calcium and ionophore to produce leukotrienes and 5-HETE from endogenous arachidonic acid (AA). We investigated the effect of ethanolic extracts of the gum resin exudate of Boswellia serrata. A concentration-dependent inhibition of LTB4 and 5-HETE production by different charges of exudate extracts were found. All products of the 5-lipoxygenase (5-LOx) from endogenous arachidonic acid (AA) in PMNL were reduced to the same extent by the extracts tested. The ethanolic extract of the gum resin also decreased 5-LOx mediated metabolisation of exogenously added AA to LTB4 and 5-HETE. Since steroidal-type anti-inflammatory drugs do not exert an immediate effect in the test system used, we conclude that the activity of the 5-LOx itself represents the side of inhibition by the gum resin extract. Therefore, an inhibition of 5-LOx catalysed mediator synthesis might be involved in the previously reported anti-inflammatory activity in vivo.     

Monitoring eicosanoid biosynthesis via lipoxygenase and cyclooxygenase pathways in human whole blood by single HPLC run

Eicosanoids play an important role as lipid mediators for physiological and pathological processes. Inhibitors of their biosynthesis have been developed as drugs for various diseases with major health political relevance. The search for more efficient inhibitors of eicosanoid formation requires simultaneous monitoring of various metabolic pathways. We developed an HPLC-based assay system, which quantifies lipoxygenase metabolites leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and cyclooxygenase metabolite 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) in whole human blood. Eicosanoid formation in blood is initiated with calcium ionophore A23187, arachidonic acid and calcium and magnesium ions. After solid phase extraction the different eicosanoids were separated by isocratic RP-HPLC using prostaglandin B1 as authentic standard. To verify the assay we determined the IC50 of known inhibitors of eicosanoid biosynthesis (zileuton, indomethacin, nordihydroguaiaretic acid). The test system is simple. It does not require extensive methodological experience and can be carried out in any biochemical laboratory. The analytical procedure can be robotized and thus, the assay appears suitable for medium-throughput testing of drugs.     

SK&F 86002: a structurally novel anti-inflammatory agent that inhibits lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H2 (PGH2) synthase activity (IC50 120 microM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells (IC50 70 microM) and its sonicate (IC50 100 microM) and human monocytes (IC50 1 microM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells (IC50 10 microM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B4 (LTB4) generation from human neutrophils (IC50 20 microM), leukotriene C4 (LTC4) generation by human monocytes (IC50 20 microM), and 5-HETE production by RBL-1 cells (IC50 40 microM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.     

Inhibition of leukotriene biosynthesis and polymorphonuclear leukocyte functions by orally active quinolylmethoxyphenylamines.

The N-substituted quinolylmethoxyphenylamines, ETH603, ETH615 and ETH647, inhibited the formation of LTB4 in rat peritoneal leukocytes, human peripheral polymorphonuclear leukocytes and canine whole blood. In rat and human cells, the compounds also inhibited the formation of 5-HETE and stimulated the synthesis of 15-HETE. In rat leukocytes, the compounds were 15-30 times more potent inhibitors of LTB4 synthesis than nordihydroguaiaretic acid, but in canine whole blood they were significantly less potent, possibly due to protein binding. However, after oral administration of the compounds to dogs a long-lasting inhibition of LTB4 production in peripheral blood was observed at serum concentrations much lower than those required in vitro. Furthermore, the compounds inhibited the LTB4-directed chemotaxis and the phagocytosis of C. albicans blastospores by canine polymorphonuclear leukocytes both in vitro and following oral administration. The calcium ionophore A23187-induced release of LTB4 in the peritoneal cavity of rats was also inhibited by systemic administration of the compounds. We therefore conclude that these novel quinolines are orally active 5-lipoxygenase inhibitors which may accumulate in inflammatory cells in vivo, leading to potent inhibition of leukotriene biosynthesis and cell function.     

A novel anti-pancreatic cancer agent, LY293111

Arachidonic acid is metabolized by two major pathways, cyclooxygenases and lipoxygenases. The metabolites catalyzed by these enzymes are important mediators of acute and chronic inflammation. Both enzymes and their metabolites are well recognized to be involved in cancer development and progress. It is well documented that inhibition of cyclooxygenase 2 (COX-2) activity decreases cancer incidence and inhibits tumor growth. It has also been reported that 5-lipoxygenase is involved in cancer cell survival and proliferation. 5-lipoxygenase metabolites including both 5-HETE and leukotriene (LT) B4 directly mediate cancer cell growth. Although 5-HETE receptors are still elusive, two LTB4 receptor subtypes (BLT1 and BLT2) have been characterized. Both 5-lipoxygenase and LTB4 receptors are upregulated in both pancreatic cancer and early pancreatic cancer lesions; hence, these proteins are potential targets for cancer treatment and prevention. Recent studies have shown that an orally stable leukotriene (LT) B4 receptor antagonist, LY293111, has a potent anti-pancreatic cancer effect. LY293111 inhibits pancreatic cancer growth, induces tumor cell apoptosis both in vitro and in vivo, and enhances the anti-pancreatic cancer effect of gemcitabine. LY293111 exhibits its anti-cancer effects through LTB4 receptors and peroxisome-proliferator activated receptor-gamma. A phase I clinical trial indicated that LY293111 is well tolerated by patients with no significant side-effects. LY293111 may be a valuable drug for treatment of pancreatic cancer, especially in combination with gemcitabine. A double-blinded, placebo-controlled phase II clinical trial with LY293111 is currently underway. This review summarizes the current research status of LY293111 as an anti-cancer agent with a focus on pancreatic cancer.     

Effect of adenine nucleotides on cyclooxygenase and lipoxygenase enzyme products of arachidonic acid in human platelets

Nucleotides are known to enhance cyclooxygenase product formation in several tissues and, in addition, are believed to function as cofactors for mammalian 5-lipoxygenases. Since nucleotides are released by stimulated platelets and by damaged tissue, we examined the hypothesis that nucleotides can affect the metabolism of arachidonic acid (AA) in washed human platelets. The various nucleotides were given 15 sec prior to the addition of 3 microM arachidonic acid and 1 muCi [3H]AA. We found that the phosphorylated adenine derivatives (ATP, ADP, and AMP) increased the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) by 2-fold without altering the formation of cyclooxygenase products. Adenosine was without effect on 12-HETE formation. ATP also stimulated 12-HETE formation in lysed platelets. This suggests that the 12-lipoxygenase enzyme of platelets can be regulated by adenine nucleotides. We next determined the portion of the nucleotide molecule responsible for the enhanced 12-lipoxygenase activity of platelets. Alteration of the nucleotide base led to a decrease in stimulation, with GTP less active than ATP, and UTP even less active than GTP. Studies with adenine nucleotides showed that the length of the phosphate chain was not important. We also found that the stable methylene isosters of ATP (alpha, beta-methylene ATP and beta, gamma-methylene ATP) increased 12-HETE formation, suggesting that the conformation and hydrolysis of the phosphate chain are not responsible for the stimulatory activity. Cyclic 3',5'AMP and 3'AMP were inactive, implying the necessity for a free phosphate at the 5' position for nucleotide stimulation of 12-HETE synthesis. In conclusion, platelet 12-lipoxygenase was stimulated by ATP, as is true for several mammalian 5-lipoxygenases. However, cyclooxygenase product formation by platelets was not altered by nucleotide addition. These studies suggest that following in vivo injury or platelet aggregation, when local concentrations of nucleotides are high, platelet lipoxygenase activity may be stimulated.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophils in vitro. Evidence of formation of antiinflammatory compounds.

Enzymatic transformation of the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of other n-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if these n-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 microM) and different concentrations (0-100 microM) of the n-6 fatty acids linoleic acid (LA) and dihomo-gamma-linolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50 = 45 microM) and DGLA (IC50 = 40 microM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50 = 7.5 microM and IC50 = 0.2 microM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50 = 0.75 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of ionophore-stimulated leukotriene B4 production in human leucocytes by monohydroxy fatty acids.

Leukotriene B4 (LTB4) release by calcium ionophore-stimulated human leucocytes was measured by use of selective solvent partition of reaction mixtures and an agarose microdroplet chemokinesis assay, and the inhibitory effects of four monohydroxy fatty acids were determined. 15-Hydroxy-eicosatetraenoic acid (15-HETE) was the most effective inhibitor of LTB4 production with an approximate IC50 value of 6 microM and 99% inhibition at 50 microM, whereas 13-hydroxy-octadecadienoic acid (13-HODD) and 12-HETE were weaker inhibitors with approximate IC50 values of 32 microM and 23 microM, and 59% and 68% inhibition at 50 microM, respectively. We suggest that 13-HODD and 12-HETE, which are present in large amounts in the lesions of the skin disease psoriasis, may act as endogenous modulators of 5-lipoxygenase activity in skin.     

Endotoxin protection against pulmonary oxygen toxicity and its reversal by acetyl salicylic acid: role of eicosanoid production by broncho-alveolar lavage cells

Small doses of endotoxin markedly increase the survival rate of adult rats exposed to 98% oxygen for periods that are normally lethal. The lysine salt of acetyl salicylic acid (L-ASA) partially reverses this protective effect of endotoxin. In this pilot study we investigated the level of eicosanoid production by broncho-alveolar lavage (BAL) cells and found that BAL cells of endotoxin protected rats, present in abundance, have an equal or increased capacity of HHT, 15-HETE, 12-HETE, LTB4 and 5-HETE production. These data suggest that production of the lipoxygenase products by BAL cells does not seem to play an important role in the pathogenesis of pulmonary oxygen toxicity. We did not find any indication for the occurrence of shunting of arachidonic acid metabolism to the lipoxygenase pathway as an explanation for the reversal of endotoxin's protective action by L-ASA.