Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB2 and HHT formation at concentrations of 8 microM and below, and at 50 microM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.     

Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets

Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by the cytosol fractions of rat platelets. The more polar products are formed via the lipoxygenase pathways in the same way as are 12-HPETE and 12-HETE, since their formation is not inhibited by indomethacin but by eicosa-5,8,11,14-tetraynoic acid (ETYA). The presence of 0.5-1.5mm-reduced glutathione (GSH) in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. l-Cysteine has the same effect as GSH. However, oxidized glutathione (GSSG) and l-cystine are not able to prevent the formation of the more polar products. The results indicate that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE. Thus the presence of GSH in the reaction mixture offers a convenient and precise cell-free assay system for 12-lipoxygenase in rat platelets. Routine assays of 12-lipoxygenase are carried out in the presence of 1mm-GSH in the reaction mixture. The synthesis of 12-HETE by 12-lipoxygenase is linear during the first 4 min of incubation at 37 degrees C, and has a pH optimum of 7.7. The 12-lipoxygenase reaches half-maximal activity at an arachidonate concentration of 20mum. Fractionation of cell homogenates indicates that the cytosol fraction possesses almost all the 12-lipoxygenase activity, whereas the microsomal fraction exhibits little enzyme activity.     

Modulation of insulin secretion by lipoxygenase products of arachidonic acid. Relation to lipoxygenase activity of pancreatic islets

Glucose (16.7 mM)-induced insulin secretion from isolated pancreatic islets of rats was inhibited by nordihydroguaiaretic acid (NDGA), 1-phenyl-3-pyrazolidinone (phenidone), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 2,6-di-tert-butyl-4-methylphenol (BHT). Indomethacin and aspirin, however, failed to inhibit the glucose-induced insulin secretion but rather tended to enhance it. The glucose-induced insulin secretion was inhibited by 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) (50 microM), 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) (100 microM), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) (100 microM), but not by 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) (100 microM). Exogenous 5-HETE (10 microM) induced significant insulin secretion in a low glucose (3.3 mM) medium. Racemic 5-HETE also showed insulinotropic effect in a concentration-dependent manner with the concentrations 20 microM or above, whereas 12-HETE, 15-HETE, 15-HPETE, 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 alpha failed to induce insulin secretion. Although significant insulin release was observed with arachidonic acid (greater than or equal to 100 microM), reduce cell viability was evident at 200 microM. When the 10,000 X g supernatant of isolated pancreatic islet homogenate was incubated with [3H]arachidonic acid at 37 degrees C in the presence of GSH and Ca2+, and the labeled metabolites then extracted with ethyl acetate and subjected to reverse phase high pressure liquid chromatography, several radioactive peaks, coeluted with authentic 15-, 12-, and 5-HETE, were observed. The radioactive peaks were completely suppressed by the addition of either NDGA, BW755C, or phenidone into the medium. The results support our contention i.e. the involvement of lipoxygenase product(s) in the secretory mechanism of insulin, and further suggest that 5-lipoxygenase system may play a role.     

Preferential utilization of endogenous arachidonate by cyclo-oxygenase in incubations of human platelets

Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.     

Arachidonic acid metabolism in isolated pancreatic islets. III. Effects of exogenous lipoxygenase products and inhibitors on insulin secretion

Isolated pancreatic islets from the rat have been demonstrated by stable isotope dilution-mass spectrometric methods to synthesize the 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) in amounts of 1.7 to 2.8 ng per 10(3) islets. No detectable amounts of 5-HETE and only trace amounts of 15-HETE could be demonstrated by these methods. Nordihydroguaiaretic acid (NDGA) and BW755C have been demonstrated to inhibit islet 12-HETE synthesis and also to inhibit glucose-induced insulin secretion. Inhibition of insulin secretion and of 12-HETE synthesis exhibited similar dependence on the concentration of these compounds. Eicosa-5,8,11,14-tetrynoic acid (ETYA) also inhibited glucose-induced insulin secretion, as previously reported, at concentrations which inhibit islet 12-HETE synthesis. Exogenous 12-HETE partially reversed the suppression of glucose-induced insulin secretion by lipoxygenase inhibitors, but exogenous 12-hydroperoxyeicosatetraenoic acid (12-HPETE), 15-HPETE, 5-HPETE, 15-HETE, or 5-HETE did not reverse this suppression. These observations argue against the recently suggested hypothesis that islet synthesis of 5-HETE modulates insulin secretion. Suppression of glucose-induced insulin secretion by ETYA, BW755C and NDGA may be due to inhibition of the islet 12-lipoxygenase by these compounds. The possibility that other processes involved in glucose-induced insulin secretion are inhibited by ETYA, BW755C and NDGA cannot yet be excluded.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Arachidonic acid metabolism in isolated pancreatic islets. V. The enantiomeric composition of 12-hydroxy-5,8,10,14-eicosatetraenoic acid indicates synthesis by a 12-lipoxygenase rather than a monooxygenase

Recent evidence indicates that the arachidonate metabolite 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) or its precursor may act as a second messenger in stimulus-response coupling in a variety of cells including Aplysia neurons, adrenal glomerulosa cells, and pancreatic islets. The compound 12(S)-HETE is generated from the precursor 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12(S)-HPETE), which is a product of the 12-lipoxygenase enzyme. Some cells have recently been found to produce the enantiomer 12(R)-HETE, apparently via a cytochrome P-450 monooxygenase, and the biologic actions of 12(R)-HETE and 12(S)-HETE differ. We have examined the stereochemistry of 12-HETE from isolated pancreatic islets both radiochemically and by a new mass spectrometric method capable of quantitating subnanogram amounts of 12-HETE stereoisomers. Endogenous 12-HETE from islets was found to be exclusively the S-isomer. D-Glucose stimulated both insulin secretion and islet accumulation of 12(S)-HETE but not of 12(R)-HETE. Pharmacologic inhibition of islet 12-HETE biosynthesis also suppressed glucose-induced insulin secretion. These findings suggest that islet 12-HETE is a product of a 12-lipoxygenase rather than of a cytochrome P-450 monooxygenase and further implicate 12-lipoxygenase products in stimulus-secretion coupling.     

Conversion of arachidonic acid into 12-oxo derivatives in human platelets. A pathway possibly involving the heme-catalysed transformation of 12-hydroperoxy-eicosatetraenoic acid

Analysis of arachidonic acid metabolites in human platelets by reverse-phase HPLC with radioactivity and UV detection revealed, besides Thromboxane B2 (TXB2), 12-hydroxy-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (12-HETE) previously described, two peaks of unidentified material absorbing at 280 nm. This material was purified by straight-phase HPLC and characterized by UV spectroscopy and gas chromatography-mass spectrometry. Three carbonyl compounds were identified: 12-keto-5,8,10,14-eicosatetraenoic acid and two geometric isomers of 12-oxo-5,8,10-dodecatrienoic acid. In a 5 min incubation at 37 degrees C in the presence of 9 microM arachidonic acid, the yield was of 0.5 to 1% of added arachidonic acid for the ketonic compound and of 4 to 7% for the sum of the two isomeric fatty acid aldehydes in comparison to 10 to 13% and 25 to 28% for TXB2 and 12-HETE, respectively. Because the three compounds carry a carbonyl group at position 12, their relationship with the 12-lipoxygenase pathway was investigated. It was found that the three compounds were formed when 12-hydroperoxy-eicosatetraenoic acid (12-HPETE) was incubated with intact or heat denaturated platelets or hemoproteins, strongly suggesting that these carbonyl compounds are products of a heme-catalysed transformation of 12-HPETE.     

Altered lipoxygenase metabolism and decreased glutathione peroxidase activity in platelets from selenium-deficient rats

Washed platelets from selenium-deficient and control rats were incubated with [1-¹⁴C]-arachidonic acid and the lipoxygenase and cyclooxygenase products were identified by gas chromatography/mass spectrometry. Platelets from selenium-deficient rats showed a three to four-fold increased synthesis of the lipoxygenase-derived isomeric trihydroxy fatty acids, 8,9,12-trihydroxy-5,10,14-eicosatrienoic acid and 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid. A major reduction in glutathione peroxidase activity was also observed in platelets from deficient rats. These results support the interpretation that these trihydroxy fatty acids arise from breakdown of the primary platelet lipoxygenase product $\text{L}$-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) under conditions in which its reduction to the $\text{L}$-12-hydroxy product (12-HETE) by a selenium-dependent glutathione peroxidase is limited. Further-more, these results indicate a specific function for selenium in platelet metabolism of essential fatty acids.     

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.     

Time-dependent inhibition of the cyclooxygenase pathway by 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid

We examined effects of small dose (1 microM or less) of exogenous 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) on the formation of cyclooxygenase products from exogenous arachidonic acid (AA) in washed human platelets. With a simultaneous addition of AA, 12-HPETE did not affect the formation of thromboxane (TX)B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). However, by being preincubated with platelets before an addition of AA, 0.1 microM or greater of 12-HPETE inhibited the formation of TXB2 and HHT dose-dependently. In addition, the inhibitory effect of 12-HPETE increased as the preincubation time was prolonged. These results suggest that 12-HPETE is a strong inhibitor for the cyclooxygenase pathway.     

Lipoxygenase Metabolism of Arachidonic Acid in Brain

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.     

Platelet lipoxygenase-dependent oxygen burst. Evidence for differential activation of lipoxygenase in intact and disrupted human platelets

The metabolism of arachidonic acid in platelets by both cyclooxygenase and lipoxygenase involves the rapid consumption of molecular oxygen. However, selective inhibition of cyclooxygenase completely abolishes the arachidonate-induced oxygen burst in intact platelets. This is in contrast to platelet lysates, in which approximately 50% of the arachidonate-induced oxygen burst remains detectable following inhibition of cyclooxygenase with acetylsalicylic acid. This lipoxygenase oxygen burst is blocked by preincubation of the platelets with ETYA, which inhibits both cyclooxygenase and lipoxygenase. In cell-free 100000 x g supernatants of platelet lysates, which contain only lipoxygenase activity, arachidonate induces an oxygen burst which is not blunted by preincubation with aspirin but is completely abolished by preincubation with ETYA. The finding of a lipoxygenase-dependent oxygen burst in platelet lysates but not in intact platelet suspensions suggests differential activation or differential availability of platelet lipoxygenase in intact and disrupted platelets. This was confirmed by a 5 min lag in the generation of [14C]HETE (the major lipoxygenase product) from [14C]arachidonic acid in intact platelets, but an almost immediate initiation of [14C]HETE production in platelet lysates. In contrast, the synthesis of [14C]thromboxane B2 (the major cyclooxygenase product) from [14C]arachidonic acid began immediately in both intact and disrupted platelet preparations and peaked within 5 min. These observations provide new insight into factors controlling platelet hydroxy acid production and help to explain the nature of the platelet oxygen burst.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Arachidonic acid metabolism in rat pancreatic acinar cells: calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromatography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8-10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Role of glutathione and glutathione peroxidase in human platelet arachidonic acid metabolism

Selective removal of intracellular glutathione (GSH) and inhibition of the GSH-dependent peroxidase (GSH-Px) by 1-chloro-2,4-dinitrobenzene (CDNB) was used to evaluate the role of GSH and GSH-Px in arachidonic acid (AA) metabolism in human platelets. Although total conversion of AA through the lipoxygenase pathway is lowered by GSH depletion, significant 12-HETE formation was observed suggesting that GSH and GSH-Px are not required for the generation of 12-HETE in human platelets. Prolonged treatment of platelets with CDNB (2 h) completely destroyed GSH-Px activity creating a model in which the effects of GSH alone could be determined. Platelet homogenates replenished with GSH, but lacking GSH-Px activity converted significantly higher amounts of AA to 12-HPETE and 12-HETE than control. Platelet cytosolic metabolism of 15-HPETE to 15-HETE decreased after CDNB, while the membrane metabolism remained similar to control due to high GSH-independent peroxidase activity associated with the membranes. These results indicate that GSH and GSH-Px function to enhance lipoxygenase activity, rather than catalyse the reduction of 12-HPETE to 12-HETE.     

Formation and biological activity of 12-ketoeicosatetraenoic acid in the nervous system of Aplysia

12-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), a lipoxygenase product, simulates the synaptic responses produced by the modulatory transmitter, histamine, and the neuroactive peptide, Phe-Met-Arg-Phe-amide (FMRFamide), in identified neurons of the marine mollusk, Aplysia californica (Piomelli, D., Shapiro, E., Feinmark, S. J., and Schwartz, J. H. (1987) J. Neurosci. 7, 3675-3886; Shapiro, E., Piomelli, D., Feinmark, S., Vogel, S., Chin, G., and Schwartz, J. H. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, in press). The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. We therefore began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. Here we report the identification of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE), a metabolite of 12-HPETE formed by Aplysia nervous tissue. This product was identified in incubations of the tissue with arachidonic acid using high performance liquid chromatography, UV spectrometry, and gas chromatography/mass spectrometry. [3H]12-KETE was formed from endogenous lipid stores in nervous tissue, labeled by incubation with [3H]arachidonic acid, when stimulated by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. 12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid, another metabolite of 12-HPETE, is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.     

Role of glutathione and glutathione peroxidase in human platelet arachidonic acid metabolism

Selective removal of intracellular glutathione (GSH) and inhibition of the GSH-dependent peroxidase (GSH-Px) by 1-chloro-2, 4-dinitrobenzene (CDNB) was used to evaluate the role of GSH and GSH-Px in arachidonic acid (AA) metabolism in human platelets. Although total conversion of AA through the lipoxygenase pathway is lowered by GSH depletion, significant 12-HETE formation was observed suggesting that GSH and GSH-Px are not required for the generation of 12-HETE in human platelets. Prolonged treatment of platelets with CDNB (2 h) completely destroyed GSH-Px activity creating a model in which the effects of GSH alone could be determined. Platelet homogenates replenished with GSH, but lacking GSH-Px activity converted significantly higher amounts of AA to 12-HPETE and 12-HETE than control. Platelet cytosolic metabolism of 15-HPETE to 15-HETE decreased after CDNB, while the membrane metabolism remained similar to control due to high GSH-independent peroxidase activity associated with the membranes. These results indicate that GSH and GSH-Px function to enhance lipoxygenase activity, rather than catalyse the reduction of 12-HPETE to 12-HETE.