Differential effects of anti-inflammatory drugs on lipoxygenase and cyclo-oxygenase activities of neutrophils from a reverse passive Arthus reaction

Rat neutrophils isolated from four-hour reverse passive Arthus reaction pleural exudates actively metabolize arachidonic acid. Production of 11-hydroxy- and 15-hydroxy-icosatetraenoic acid and 12-hydroxy-heptadecatrienoic acid is inhibited by indomethacin, benoxaprofen, BW 755C, piroxicam, ibuprofen, timegadine, and naproxen, suggesting that production of these arachidonic acid metabolites occurs at similar enzymic active sites. In addition, in the presence of the calcium inophore A23187 or the non-ionic detergent, BRIJ 56, rat neutrophils also produce the lipoxygenase products 5-hydroxy-icosatetraenoic acid and leukotriene B. The production of these metabolites is calcium dependent. Moreover, the calcium ionophore A23187 and BRIJ 56 synergistically act to augment the metabolism of exogenously added arachidonic acid via lipoxygenase. The formation of these metabolites is inhibited by BW 755C, benoxaprofen and timegadine but not by other non-steroidal anti-inflammatory drugs tested. In fact, at doses which inhibit cyclo-oxygenase activity, indomethacin, naproxen, and ibuprofen stimulate arachidonic acid metabolism via lipoxygenase.     

Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets.

In the present study the effects of collagen on platelet aggregation and arachidonic acid (AA) mobilization, specifically from phosphatidylcholine (PC), were investigated in the presence and absence of BW755C, a selective inhibitor of cyclo-oxygenase and lipoxygenases. The inhibition of cyclo-oxygenase and lipoxygenase(s) by BW755C (75 microM) resulted in severe impairment in collagen-induced platelet aggregation. In the presence of BW755C, the aggregation response amounted to 14, 26, 37 and 49% of the corresponding controls (without BW755C) at 10, 25, 50 and 100 micrograms of collagen respectively. On the contrary, the amount of AA released from PC, which ranged from 3.5 to 8.6 nmol/10(9) platelets, in response to the action of collagen (10-100 micrograms) remained unaffected by the presence of BW755C. Substantial amounts of non-esterified AA were detected in the free fatty acid fractions obtained from collagen-stimulated platelets in the presence as well as in the absence of BW755C. However, the presence of BW755C caused a greater accumulation of free AA (mass) and this ranged from 4 to 16 nmol, depending upon the amount of collagen. In addition, small increases in free stearic and oleic acids were observed in collagen-stimulated platelets as compared with unstimulated platelets. The amount of AA lost from PC represented 67, 80, 49 and 52% of the free AA obtained at 10, 25, 50 and 100 micrograms of collagen respectively. Our results adhesion of platelets to collagen fibres may be responsible for much of the AA release from PC Furthermore, these results demonstrate that aggregation and/or cyclo-oxygenase/lipoxygenase metabolites are not obligatory for the release of AA from PC in collagen-stimulated human platelets.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A₂₃₁₈₇, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: possible pitfall of using the calcium ionophore A23187 to stimulate 5' lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5' lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5' lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B4 by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC50 1.6 X 10(-4)M) was approximately 100 times less potent than BW755C (IC50 1.7 X 10(-6)M) at inhibiting leukotriene B4 synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 X 10(-6)M). The data obtained using STZ as stimulus are consistent with previous in vivo studies and indicate that benoxaprofen is a relatively selective inhibitor of cyclo-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5' lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.     

Effect of n-3 and n-6 dietary fats on the lipoxygenase products from stimulated rat neutrophils.

Fish oil was fed to rats in combination with an equal amount of olive, sunflower or linseed (flax) oil in semisynthetic diets for 3 weeks. Following stimulation of isolated neutrophils with calcium ionophore the levels of leukotrienes (LT) were determined by HPLC. Graphical presentation of the resultant data show a direct linear relationship between LTB production and substrate concentration with no preferential conversion of n-3 or n-6 substrates. In addition the results highlighted the greater conversion of eicosapentaenoic acid (EPA) and arachidonic acid (AA) to 5-hydroxy metabolites in stimulated neutrophils. There is no suggestion in our results of inhibition of any of the enzymatic conversion steps between EPA or AA and LTB production by any of the dietary fatty acids except by altering the EPA/AA ratio in neutrophil membranes.     

The effect of calcium ionophore A23187 on the metabolism of arachidonic and eicosapentaenoic acids in neutrophils from a marine teleost fish rich in (n-3) polyunsaturated fatty acids

1. [1-14C]AA and [1-14C]EPA were incorporated equally into plaice neutrophil phospholipids. 2. Incubation with A23187 resulted in the loss of label from total phospholipids and increased label released from the neutrophils. 3. Labelled LTB4 and LTB5 production was increased 2.5- and 3-fold, respectively, by A23187 treatment. 4. However, the incorporated AA was generally more metabolically active than the incorporated EPA with approx. 3-4 times as much LTB4 produced than LTB5 by cells in the presence or absence of A23187, respectively. 5. The results obtained in this (n-3) PUFA-rich species were discussed in comparison with current knowledge of AA and EPA metabolism in mammalian neutrophils.     

Effect of the 5-hydroperoxide of eicosatetraenoic acid and inhibitors of the lipoxygenase pathway on the formation of slow reacting substance by rat basophilic leukemia cells; direct evidence that slow reacting substance is a product of the lipoxygenase pathway

Previous studies in a line of rat basophilic leukemia (RBL 1) cells have indicated that the slow reacting substance (SRS) made during stimulation with the divalent cation ionophore, A23187, is derived from arachidonic acid (AA). In the present report, various inhibitors of AA metabolism were compared with regard to their effects on SRS formation and incorporation of radioactivity from [1-14C]-AA into known metabolites of the lipoxygenase and cyclooxygenase pathways. An apparently close parallel between lipoxygenase product formation and SRS synthesis is demonstrated. In addition, exogenous 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) has been shown to markedly enhance SRS synthesis, even when A23187 is absent. The data provide very strong evidence that SRS is produced through the lipoxygenase pathway.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils

Arachidonic acid metabolism in human neutrophils stimulated in vitro with the calcium ionophore A23187 was studied using combined HPLC and radioimmunoassays. Indomethacin (0.1 and 1.0 microM) caused a 300% increase in LTB4 formation in neutrophils stimulated with A23187. 5-, 12- and 15-HETE levels were also increased. In the presence of exogenous arachidonic acid 1.0 microM Indomethacin caused a 37% increase in LTB4 formation. Acetyl Salicylic Acid and Ibuprofen had no effect on the formation of lipoxygenase metabolites. The effect of indomethacin on LTB4 formation does not appear to be due to a simple redirection of substrate arachidonic acid from the cyclooxygenase to the lipoxygenase pathways.     

Inhibition by Salicylhydroxamic Acid, BW755C, Eicosatetraynoic Acid, and Disulfiram of Hypersensitive Resistance Elicited by Arachidonic Acid or Poly-l-Lysine in Potato Tuber

The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) was more persistent. These results are consistent with previous reports that SHAM and BW755C are reversible inhibitors of lipoxygenase and easily oxidized by potato while ETYA acts as an irreversible inhibitor. Disulfiram (tetraethylthiuram disulfide) also inhibited AA elicitor activity. SHAM was most effective if applied at the time of AA treatment, having no effect if applied 6 hours afterward. SHAM was effective in the presence of MES or MOPS buffers but not in acetate-buffered or unbuffered solutions; neither BW755C nor ETYA exhibited this restriction. However, SHAM, BW755C, and ETYA also were inhibitors of browning and sesquiterpene accumulation elicited in potato by poly-l-lysine, which, unlike AA, is not a lipoxygenase substrate. SHAM effectiveness also was restricted to 6 hours after treatment with poly-l-lysine. While the results with AA support a role for lipoxygenase, those with poly-l-lysine may be evidence that these compounds are having other effects in potato tissue.     

Glucose-stimulated protein phosphorylation in the pancreatic islet

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: Possible pitfall of using the calcium ionophore A23187 to stimulate 5′ lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5′ lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5′ lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B₄ by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC₅₀ 1.6 × 10⁻⁴M) was approximately 100 times less potent than BW755C (IC₅₀ 1.7 × 10⁻⁶M) at inhibiting leukotriene B₄ synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 × 10⁻⁶M). The data obtained using STZ as stimulus are consistent with previous studies and indicate that benoxaprofen is a relatively selective inhibitor of cylco-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5′ lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A2 during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosapentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPA are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of 14C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a 'Max EPA' fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F1 alpha but no difference was seen in leukotriene B4 formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA group than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as 14C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Lipoxygenase and cyclooxygenase blockade by BW755C prevents endotoxin-induced hypotension but not changes in glucose metabolism

Arachidonic acid metabolites are mediators of various pathophysiologic events following endotoxin administration. However, their role in the endotoxin-induced increase in glucose metabolism has not been examined. Rats were administered either saline or BW755C (an inhibitor of both the cyclooxygenase and lipoxygenase pathways) 30 min prior to injection of E. coli endotoxin and whole body glucose kinetics assessed using a constant iv infusion of [6-3H] glucose. Treatment with BW755C prevented the endotoxin-induced hypotension and tachycardia. Endotoxin produced characteristic increases in the plasma glucose (23-70%) and lactate (2- to 9-fold) concentrations, as well as elevations in the rate of glucose appearance (34-63%) and metabolic clearance (40-92%). In contrast to the amelioration in hemodynamics, pretreatment with BW755C did not prevent these alterations in glucose metabolism normally seen after endotoxin. BW755C markedly reduced the endotoxin-induced increase in plasma catecholamine concentrations, but levels were still elevated 2- to 4-fold compared to control values. The results suggest that arachidonic acid metabolites mediate the early hypotensive response following endotoxin, but are not by themselves responsible for the elevated rates of glucose production and utilization.     

The salvage of ischaemic myocardium by BW755C in anaesthetised dogs

BW755C, a dual inhibitor of the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism reduces the size of an infarct produced by 60 min of coronary occlusion followed by 5 hours reperfusion in anaesthetised beagles. This effect of BW755C is observed when the drug is given after the period of occlusion, and is independent of any haemodynamic effect. In contrast, indomethacin, which inhibits only the cyclo-oxygenase pathway, did not influence infarct size. It is suggested that the salvage of acutely ischaemic myocardium by BW755C is due to inhibition of lipoxygenase product formation by migrating cells which invade the damaged myocardium to produce an inflammatory response.     

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.