Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D4 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 microM), and by arachidonic acid (AA), melittin and LTD4. FPL55712 (0.1 and 1 microM) inhibited contractions induced by AA (100 microM) and the phospholipase A2 activator melittin (3 micrograms/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 micrograms/ml) more than by A23187 (1 microgram/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 microM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonism of leukotriene (LT) C4-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cyclooxygenase inhibitor indomethacin. NDGA (30 microM) as well as indomethacin (5 microM) inhibited LTC4-induced contractions. But, in the presence of indomethacin NDGA was ineffective to inhibit the LTC4 response, whereas two other lipoxygenase inhibitors, phenidone (3-30 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM), markedly inhibited it. The antagonist action of an LTD4 receptor antagonist FPL55712 against LTC4-induced contractions was significantly reduced by NDGA (10-30 microM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect on the LTC4 antagonism in the presence of indomethacin, but 0.3 microM phenidone and 1 microM ETYA which did not inhibit the LTC4 response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC4, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LTC4 response was not inhibited by indomethacin, 0.3 microM phenidone and 1 microM ETYA. In the presence of a gamma-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC4 antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 microM histamine- and 30 microM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H1-blocker pyrilamine. These results suggest that some putative non-prostanoids are involved in LTC4-induced contractions of guinea-pig trachea and which regulate the effects of LTD4 antagonism and beta-adrenoceptor activation.     

Rat hypothalamic corticotropin-releasing hormone secretion in vitro is stimulated by interleukin-1 in an eicosanoid-dependent manner

We studied the effect of interleukin-1 alpha (IL-1) on corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami in vitro. We also assessed possible mediation of arachidonic acid metabolites on IL-1-stimulated CRH secretion, by preincubating hypothalami with the cyclooxygenase inhibitor indomethacin (INDO, 1 microM), the lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid (ETYA, 10 microM), or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, up to 30 microM). In additional experiments, prostaglandins (PG) E2 and F2 alpha were added to the cultures treated with INDO or ETYA. Finally, we investigated the effect of dexamethasone (DEX) on IL-1-stimulated CRH secretion. IL-1 stimulated immunoreactive CRH (iCRH) secretion by explanted hypothalami in a concentration-dependent fashion. Both INDO and ETYA inhibited IL-1-(10nM)-stimulated iCRH secretion, whereas NDGA did not have any effect. The addition of PGF2 alpha (10 nM) restored the secretion of iCRH inhibited by INDO. DEX treatment significantly inhibited IL-1-stimulated iCRH release. Our results suggest that the stimulatory effect of IL-1 on the hypothalamic CRH neuron is mediated by the cyclooxygenase metabolites of arachidonic acid, and, among others, by PGF2 alpha.     

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.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to inositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA), a tumor promoting agent, could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA (100 mU and 12.5 microM, respectively) augmented hCG induced T secretion, while at higher concentrations (PLC: 500 mU and AA: 200 microM) they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5, 8, 11, 14 Eicosatetraynoic acid (ETYA), a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. We conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclooxygenase products.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D₄ receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 μM), and by arachidonic acid (AA), melittin and LTD₄. FPL55712 (0.1 and 1 μM) inhibited contractions induced by AA (100 μM) and the phospholipase A₂ activator melittin (3 μg/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) was a more effective inhibitor of the melittin response than the response. FPL55712 inhibited contractions induced by OA (100 μg/ml) more than by A23187 (1 μg/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC₄ conversion to LTD₄. NDGA (10 μM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 μM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD₄-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD₄-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Effects of antioxidants on cyclooxygenase and lipoxygenase activities in intact human platelets: Comparison with indomethacin and etya

Five antioxidative agents (BW755C, 1-naphtol, NDGA, propylgallate and quercetin) were compared with indomethacin and ETYA for their effects on (¹⁴C) arachidonic acid metabolism by cyclooxygenase (CO) and lipoxygenase (LPO) enzymes in intact human platelets. All tested compounds inhibited CO activity in a concentration-dependent manner. LPO activity was suppressed by NDGA, propylgallate, quercetin and ETYA but strongly enhanced by BW755C, 1-napthol and indomethacin. Whereas NDGA and ETYA showed almost equipotent inhibitory effects towards both fatty acid oxygenases, propylgallate and quercetin were found to be respectively 6.5 and 4 times better inhibitors of LPO than of CO activities.These data indicate that antioxidants affect arachidonic acid metabolism in intact human platelets in different ways: BW755C and 1-naphtol exerted the same activity as indomethacin, a selective CO blocker, whereas NDGA, propylgallate and quercetin behaved as ETYA, a dual CO-LPO inhibitor. Considering their inhibition selectivity, propylgallate and quercetin may serve as prototypes for more specific blockers of LPO activity.     

Arachidonic acid metabolism and casein secretion in lactating rabbit mammary epithelial cells: effects of inhibitors of prostaglandins and leukotrienes synthesis

The metabolism of arachidonic acid (AA) in fragments of lactating rabbit mammary glands in vitro was studied by considering the distribution of 13-[14C]AA in the cells, and the effects of inhibitors of cyclooxygenase and lipoxygenase pathway on the basal and prolactin (PRL)-stimulated casein secretion. 13-[14C]AA was incorporated in all classes of lipids and PRL increased transiently the percentage of free fatty acid after 1 and 5 min. Ten microM ETYA (5,8,11,14-Eicosatetraynoic acid), a tetrayne analogue of AA inhibited prostaglandins F2 alpha (PGF2 alpha) production but not leukotrienes B4 and C4 (LTB4 and LTC4) production and increased basal casein secretion. 10(-4) M DCHA (Docosahexaenoic acid) a competitive inhibitor of prostaglandin-synthetase inhibited PGF2 alpha production but did not affect basal nor PRL-stimulated casein secretion. Fourteen microM indomethacin inhibited PGF2 alpha and LTC4 production and PRL-stimulated casein secretion. Ten microM NdgA (nordihydroguaiaretic acid) an inhibitor of lipoxygenase pathway, inhibited LTB4 and LTC4 production, increased basal level of casein secretion and inhibited PRL-stimulated casein secretion. Hundred microM caffeic acid, an inhibitor of glutathione-S-transferase (GST), a class of enzymes implied in the transformation of LTA4 into LTC4, had the same effect that NDGA on basal and PRL-stimulated casein secretion. These findings show that inhibitors of AA metabolites alter casein secretion.     

Role of lipoxygenase in the mechanism of acrosome reaction in mammalian spermatozoa

The acrosome reaction (AR) in bull spermatozoa was induced by the Ca2(+)-ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca2+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 microM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20, 40 or 80 micrograms/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 micrograms/ml, respectively. Under identical conditions, 100 microM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 15-HETE, the classical product of 15-lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15-HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa.     

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

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

Possible involvement of arachidonic acid metabolites in the synergistic action of endothelial mitogenesis by basic fibroblast growth factor and phorbol ester.

Our object was to obtain information about the regulatory mechanism which modulates the effect of basic fibroblast growth factor (bFGF) on commitment to growth in human umbilical vein endothelial (HUVE) cells. Firstly, phorbol ester PMA, a known activator of protein kinase C (PKC), was found to be able to act synergistically with bFGF to stimulate 3H thymidine incorporation in HUVE cells. Secondly, bFGF and PMA induced a stimulated phospholipase A2 (PLA2)-catalyzed release of 14C arachidonate. Thirdly, inhibitors of PLA2, PKC and HETE, but not an inhibitor of cyclooxygenase metabolites, inhibited FGF/PMA-stimulated DNA synthesis. Fourth, the stable cyclooxygenase metabolite of prostacyclin was not found to be changed when cells were treated with bFGF plus PMA. The present data suggest that PKC is able of acting synergistically with bFGF in order to stimulate DNA-primary initiation activity in HUVE cells via the PLA2-dependent generation of lipoxygenase metabolites such as HETE.     

Regulation of rat natural killing. II. Inhibition of cytolysis and activation by inhibitors of lipoxygenase: possible role of leukotrienes

Rat splenic natural killer (NK) cell activity against 51Cr-labeled YAC-1 or TMT-081 tumor cells can be augmented by culturing at 37 degrees C for 18 hr. Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism, NDGA, alpha-phenanthroline, quercetin, ETYA, BW755C, esculetin, and timegadine, inhibited this NK activation and also inhibited NK cytotoxicity when added directly to the NK assay. However, there was a partial loss of sensitivity of activated NK cells to suppression by NDGA, BW755C, and esculetin. Indomethacin failed to reverse the inhibition of NK activation caused by NDGA. However, LTB4 and LTC4 (0.01 microgram/ml) were able to reverse the inhibitory effect of NDGA on NK activation. Furthermore, spleen cells cultured for 18 hr synthesized detectable amount of LTC4 in their supernatants. NDGA inhibited the LTC4 synthesis in a dose-dependent manner. These data therefore suggest that leukotrienes are responsible for NK activation, and lipoxygenase activity is essential for NK cytolytic activity.     

Production of Second Messengers Following Chemotactic and Mitogenic Urokinase-Receptor Interaction in Human Fibroblasts and Mouse Fibroblasts Transfected with Human Urokinase Receptor

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transferred with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca²⁺ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG treshold level required to trigger DNA synthesis.     

Involvement of arachidonic acid metabolism and EGF receptor in neurotensin-induced prostate cancer PC3 cell growth

Dietary fats, which increase the risk of prostate cancer, stimulate release of intestinal neurotensin (NT), a growth-promoting peptide that enhances the formation of arachidonic acid metabolites in animal blood. This led us to use PC3 cells to examine the involvement of lipoxygenase (LOX) and cyclooxygenase (COX) in the growth effects of NT, including activation of EGF receptor (EGFR) and downstream kinases (ERK, AKT), and stimulation of DNA synthesis. NT and EGF enhanced [3H]-AA release, which was diminished by inhibitors of PLA2 (quinacrine), EGFR (AG1478) and MEK (U0126). NT and EGF phosphorylated EGFR, ERK and AKT, and stimulated DNA synthesis. These effects were diminished by PLA2 inhibitor (quinacrine), general LOX inhibitors (NDGA, ETYA), 5-LOX inhibitors (Rev 5901, AA861), 12-LOX inhibitor (baicalein) and FLAP inhibitor (MK886), while COX inhibitor (indomethacin) was without effect. Cells treated with NT and EGF showed an increase in 5-HETE levels by HPLC. PKC inhibitor (bisindolylmaleimide) blocked the stimulatory effects of NT, EGF and 5-HETE on DNA synthesis. We propose that 5-LOX activity is required for NT to stimulate growth via EGFR and its downstream kinases. The mechanism may involve an effect of 5-HETE on PKC, which is known to facilitate MEK-ERK activation. NT may enhance 5-HETE formation by Ca2+-mediated and ERK-mediated activation of DAG lipase and cPLA2. NT also upregulates cPLA2 and 5-LOX protein expression. Thus, the growth effects of NT and EGF involve a feed-forward system that requires cooperative interactions of the 5-LOX, ERK and AKT pathways.     

Activation of 15-lipoxygenase by low density lipoprotein in vascular endothelial cells. Relationship to the oxidative modification of low density lipoprotein.

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12 + 15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.     

Role of lipoxygenase in the O2-dependent activation of soluble guanylate cyclase from rat lung.

Guanylate cyclase activity in rat lung supernatant fractions is stimulated 3-4 fold by aerobic incubation at 30 degrees C for approx. 30 min ('O2-dependent activation'). This stimulation was blocked by 20 microM-eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of lipoxygenase and cyclo-oxygenase, but not by aspirin or indomethacin, which are cyclo-oxygenase inhibitors. The enzyme activator(s) is presumed to be the fatty acid hydroperoxide(s) formed by lipoxygenase. Removal of lipoxygenase from the supernatant fraction by chromatography on Amberlite XAD-4 also prevented activation, which was restored by the addition of soya-bean lipoxygenase. Bovine serum albumin prevented O2-dependent activation or activation by soya-bean lipoxygenase, through its ability to bind the unsaturated fatty acid substrate of lipoxygenase. The lipoxygenase in the supernatant fraction is inhibited by endogenous glutathione peroxidase plus reduced glutathione (GSH); removal of GSH de-inhibits lipoxygenase and activates guanylate cyclase. This was effected by autoxidation, by cumene hydroperoxide (with GSH peroxidase) and by titration with N-ethylmaleimide (NEM). Activation by NEM was inhibited by serum albumin or ETYA, as was activation by low concentrations (less than 50 microM) of cumene hydroperoxide. Activation by higher concentrations was not so inhibited; therefore, cumene hydroperoxide can also activate by a direct effect on guanylate cyclase. A hypothesis for physiological activation is proposed.     

Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. II. Evidence for a precursor role of arachidonic acid and further purification.

The generation of slow reacting substance (SRS) from ionophore A23187-stimulated rat peritoneal mast cells was enhanced by arachidonic acid (AA). This SRS generation was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analogue of AA and an inhibitor of both fatty acid cyclooxygenase and lipoxygenase. Indomethacin, a fatty acid cyclooxgenase inhibitor, had an enhancing effect upon SRS generation. This suggests SRS generation occurred through an ETYA sensitive step--perhaps a lipoxygenase. Radiolabel from [14C]-AA was incorporated into SRS with comigration of radioactivity and bioreactivity in silicic acid and thin layer chromatographies. Upon silicic acid chromatography, the active principle was eluted in the methanol fraction. Two-dimensional thin layer chromatography revealed chromatographic separation from other known spasmogenic substances and phospholipids. Mast cell SRS was found to display physiochemical properties similar to those of rat basophilic leukemia cell SRS, namely: that mast cell SRS generation was 1) enhanced by arachidonic acid; 2) inhibited by ETYA but not by indomethacin; 3) incorporation of [14C]-AA into the active principle; and 4) similar behavior during purification in silicic acid and thin layer chromatographies.