蝙蝠葛碱对血小板聚集及花生四烯酸代谢的影响

蝙蝠葛碱(Dau) 抑制AA及ADP诱导的大鼠血小板聚集,也能抑制AA,ADP及Adr诱导的人血小板聚集。这种抑制作用与Dau剂量呈依赖关系。Dau抑制大鼠洗涤血小板对[1-¹⁴C]AA经环氧酶途径的代谢,TXB₂与HHT的形成均呈剂量依赖性减少。当Dau浓度达到0.1 mmol/L时亦能抑制12-HETE的形成。Dau对AA代谢的上述影响可能是其抑制血小板聚集的机理之一。     

山莨菪碱对大鼠胸水中性白细胞花生四烯酸代谢的影响

本文研究了654-2对大鼠胸水中性白细胞代谢[1-¹⁴C]AA及内源性AA的影响。大鼠白细胞AA经5-LPO代谢途径形成的主要产物为LTB₄及5-HETE,经CO途径的主要产物为HHT及TXB₂。654-2对白细胞代谢[1-¹⁴C]AA无抑制作用,但显著减少白细胞从内源性AA形成的LTB₄,5-HETE,HHT及TXB₂。这种抑制作用与654-2呈剂量依赖关系。本实验结果表明,654-2抑制PG及LT的形成可能是影响了AA从胞膜的释放,而并非直接抑制CO及5-LPO。     

Oxidative transformations of arachidonic acid in human dispersed lung cells: disparity between the utilization of endogenous and exogenous substrate

Eicosanoid release from human dispersed lung cells (HDLC) containing ca 5% mast cells was studied before and after cell activation with ionophore A23187 or anti-IgE. Basal release of eicosanoids synthesized from endogenous arachidonate was measured by radioimmunoassay. In descending order of abundance the products were: 5-hydroxyeicosatetraenoic acid (5-HETE) greater than thromboxane B2 (TXB2) greater than prostaglandin F2 alpha (PGF2 alpha) approximately immunoreactive (i)-PGE2 greater than PGD2 greater than 6-keto-PGF1 alpha approximately i-LTC4. Stimulation of HDLC with ionophore A23187 or, after passive sensitization, with anti-IgE resulted in 2-10 fold increases in the generation of individual eicosanoids. In terms of net generation the most abundant products were PGD2 and TXB2 with either stimulus. Activation with A23187 caused net release of i-LTC4 and 5-HETE, but these products were not measured after immunological activation. A more complete profile of lipoxygenase products released from HDLC dispersed from one lung was obtained after separation by high performance liquid chromatography combined with ultra violet spectroscopy and bioassay. The major products released from the cells from this lung with ionophore stimulation were 13-hydroxylinoleic acid greater than LTB4 greater than 5-HETE greater than 12-HETE greater than LTC4 greater than 15-HETE greater than 11-HETE approximately 9-HETE. When the utilization of exogenous [14C]-arachidonic acid for prostanoid biosynthesis was compared to that of endogenous unlabelled arachidonate the formation of TXB2 was consistently underestimated. These results imply compartmentalization of arachidonic acid utilization in Ca2+-activated HDLC. In unstimulated cells the proportional formation of PGD2 was overestimated when exogenous arachidonic acid was substrate. After activation with A23187 the proportions of PGD2 were similar with both substrate sources. The large proportions of PGD2 and TXB2 generated by HDLC further supports the view that these eicosanoids may be important inflammatory mediators in lung tissue.     

Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood

We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB4, 20-OH-LTB4, and 20-COOH-LTB4 are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB4, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.     

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.     

咪苯嗪酮对TXA2和HHT生成的选择性抑制作用

咪苯嗪酮(CI-914)能抑制大鼠血小板环氧酶和TXA₂合成酶产物HHT的生成,而对脂氧酶产物12-HETE的生成仅高浓度药物才有弱的抑制作用,提示CI-914主要影响花生四烯酸(AA)环氧酶途径,而对脂氧酶途径影响较少。在大鼠血小板和中性白细胞CI-914能抑制TXA₂的生成,同时CI-914还可使白细胞6-keto-PGF_1a和血小板PGE₂的产生量显著增加,提示CI-914在这两种细胞引起了AA的转向合成。上述结果基本证实,CI-914在大鼠中性白细胞和血小板对TXA₂合成酶具有选择性抑制作用。     

Eicosanoid formation by mammalian intestine. Effects of some intestinal secretagogues

Intestinal tissues of man, rat, mouse, guinea-pig and rabbit were preincubated with laxatives, homogenised, and incubated with [14C]arachidonic acid. After extraction into chloroform, the eicosanoids were separated by thin layer chromatography. Metabolism of [14C]arachidonic acid into prostaglandins (PGs), and the lipoxygenase products LTB4 and 5-HETE, was stimulated by ricinoleic acid (100 micrograms/ml) or phenolphthalein (100 micrograms/ml), and to a lesser extent by picosulphate (125 micrograms/ml) and sulfosuccinate (200 micrograms/ml). Mannitol (500 micrograms/ml) had no effect. Indomethacin (1 microgram/ml) inhibited the stimulation of PG formation. The dual pathway inhibitor BW755C (1 microgram/ml) reduced the formation of prostaglandins, LTB4 and 5-HETE. In some experiments on rat colon, prostanoids were separated from lipoxygenase products, characterised by their chromatographic mobility and quantitated (relative amounts PGE2 greater than PGF2 alpha greater than TXB2 greater than PGD2). Their formation was enhanced by ricinoleic acid (100 micrograms/ml) and inhibited by either indomethacin or BW 755C (1 microgram/ml). The present results indicate that mammalian isolated gut tissue can convert [14C]arachidonic acid into both cyclo-oxygenase and lipoxygenase products, and support the suggestion that eicosanoids may participate in the laxative effect of some secretagogues.     

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.     

山莨菪碱对洗涤大鼠血小板代谢花生四烯酸的影响

作者建立了一个用大鼠洗涤血小板研究药物对外源性及内源性AA代谢影响的方法。采用HPLC测定血小板AA代谢物HHT及12-HETE,观察了底物(AA)浓度、孵育时间、A23187加量等对血小板代谢AA的影响。并用此法研究了654-2对洗涤血小板AA代谢的影响。654-2显著减少血小板从内源性AA形成HHT及12-HETE,且作用随剂量增加而增强,但不影响血小板对外源性AA的代谢。上述结果表明,654-2是通过抑制AA释放而减少AA代谢物的形成。     

Increased metabolism of arachidonic acid in an immune model of colitis in guinea-pigs.

Inflammation of the guinea-pig colon was produced by skin sensitization and subsequent intracolonic challenge with the chemical hapten, dinitrochlorobenzene. Metabolism of [14C]-arachidonic acid by homogenates of control colon was very low, although metabolites co-migrating on thin layer chromatography (t.l.c.) with prostaglandin E2 (PGE2), PGF2 alpha, PGD2, 6-keto-PGF1 alpha, thromboxane B2 (TXB2), HHT and 11-, 12-, 15-HETE were formed. There was an overall 3 fold increase in metabolism of [14C]-arachidonic acid by homogenates of inflamed mucosa. The greatest increase in metabolite formation was of PGE2, with smaller increases in HHT, 11-, 12-, 15-HETE, PGD2, TXB2, PGF2 alpha and 6-keto-PGF1 alpha. The formation of these metabolites was inhibited both by indomethacin and the dual pathway inhibitor, BW755C. The formation of immunoreactive PGE2, TXB2 and 6-keto-PGF1 alpha was also increased in homogenates of inflamed guinea-pig colon. The small level of immunoreactive LTB4 detected in control colon was not changed in inflamed colonic tissue. The dinitrochlorobenzene model of colitis offers a means of studying arachidonic acid metabolism in an immune-mediated inflammatory response in intestinal tissue.     

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.     

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)     

Comparison of eicosanoids production between rat polymorphonuclear leukocytes and macrophages: detection by high-performance liquid chromatography with precolumn fluorescence labeling.

We developed a procedure for serial measurement of fluorescent derivatives of eicosanoids in biological samples by HPLC. The 9-anthryldiazomethane (ADAM)-derivatized sample was first fractionated through SEP-PAK silica into fraction 1 (eluate of chloroform:toluene) and fraction 2 (eluate of acetonitrile:methanol). Both fractions were loaded separately onto an ODS column, and eluted with a step-gradient of 85% and 95% acetonitrile for Fr-1 (HETE's and arachidonic acid) and with 70% acetonitrile for Fr-2 (PG's and LTB4). The method was applied to the arachidonate products of rat peritoneal leukocytes which were stimulated with A23187. The polymorphonuclear leukocytes (PMNL), which were collected after stimulation with casein, released mainly LTB4, 5-HETE, 6-K-PGF1 alpha, but little arachidonic acid. In contrast to PMNL, rat macrophages, which were collected after peritoneal injection of soluble starch and bacto peptone, released 5-HETE, arachidonic acid, and 6-K-PGF1 alpha, but no LTB4. These differences might be partly caused by the differential rates of uptake or turnover of arachidonic acid into their membrane phospholipids.     

A novel biologically active seleno-organic compound--V. Inhibition by ebselen (PZ 51) of rat peritoneal neutrophil lipoxygenase

Suspensions of rat peritoneal PMNLs elicited with glycogen were stimulated by calcium and an ionophore to produce leukotrienes from endogenous arachidonic acid. We investigated the effect of the non-toxic, anti-inflammatory seleno-organic compound, ebselen (PZ 51). When ethanolic extracts of the medium of stimulated cells were analysed by HPLC, a dose-dependent inhibition by ebselen of LTB4 formation with a concomitant decrease of 5-HETE production was found. Half-maximum inhibition was observed at 20 mumoles/l ebselen. Similar findings were obtained after analysis of chloroform extracts of both cells and medium using a different HPLC system. Under these conditions, enhanced 5-HETE formation was associated with reduced production of LTB4 and other di-HETE isomers, when purified glutathione peroxidase + GSH were present. We conclude that the reported GSH peroxidase-like activity of ebselen, catalysing the reduction of 5-HPETE to 5-HETE, can not account for our findings. Therefore, the lipoxygenase reaction itself apparently represents the site of inhibition of LTB4 formation by ebselen.     

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 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.     

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.     

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.