13-cis-Retinoic Acid Affects Oxidation and DNA Damage in Oxidative-Positive SLE Lymphocytes But May Not Be Useful for Therapy

13-cis-Retinoic acid (13-CRA), a water-soluble vitamin A analog and 5′-lipoxygenase inhibitor, was tested in vitro for effects on excess oxidative metabolism and DNA damage in mitogen-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE), because other 5′-lipoxygcnase enzyme inhibitors were shown to lower the excess oxidative metabolism in SLE cells. Excess chemiluminescence (CL) was abolished within minutes after the addition of 1 × 10⁻⁶ M 13-CRA in five of five CL-positive mitogen-stimulated SLE lymphocytes, and was lowered in five of eight samples after 48 to 72 h culture, Similarly, low concentrations of 13-CRA for 48-72 h largely prevented the S1 nuclease-sensitive DNA changes/DNA damage observed in CL-positive lupus lymphocytes in vitro. However, 13-CRA did not affect DNA damage in four of four CL-negative lymphocyte samples. 13-CRA, like other retinoic acid compounds, was known to stimulate B-cell activities in vivo and in vitro but effects on dividing lupus T cells had not been studied. 13-CRA further inhibited the diminished PHA-stimulated lupus T-cell growth in tissue culture at a concentration of 9 × 10⁻⁶ M in three of five lupus lymphocyte samples. 13-CRA has positive and negative effects on multiple aspects of the Immune system and it is not clear whether 13-CRA will have positive or adverse clinical effects on SLE patients. Close attention to vitamin A and vitamin "supplements" in patients with SLE may answer this question.     

Modulation of rat polymorphonuclear leukocyte 5-lipoxygenase activity by 5-HPETE and NADH-dependent flavin inhibition

The effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.     

Dihydrolipoic acid inhibits 15-lipoxygenase-dependent lipid peroxidation

The potential antioxidant effects of the hydrophobic therapeutic agent lipoic acid (LA) and of its reduced form dihydrolipoic acid (DHLA) on the peroxidation of either linoleic acid or human non-HDL fraction catalyzed by soybean 15-lipoxygenase (SLO) and rabbit reticulocyte 15-lipoxygenase (RR15-LOX) were investigated. DHLA, but not LA, did inhibit SLO-dependent lipid peroxidation, showing an IC(50) of 15 microM with linoleic acid and 5 microM with the non-HDL fraction. In specific experiments performed with linoleic acid, inhibition of SLO activity by DHLA was irreversible and of a complete, noncompetitive type. In comparison with DHLA, the well-known lipoxygenase inhibitor nordihydroguaiaretic acid and the nonspecific iron reductant sodium dithionite inhibited SLO-dependent linoleic acid peroxidation with an IC(50) of 4 and 100 microM, respectively, while the hydrophilic thiol N-acetylcysteine, albeit possessing iron-reducing and radical-scavenging properties, was ineffective. Remarkably, DHLA, but not LA, was also able to inhibit the peroxidation of linoleic acid and of the non-HDL fraction catalyzed by RR15-LOX with an IC(50) of, respectively, 10 and 5 microM. Finally, DHLA, but once again not LA, could readily reduce simple ferric ions and scavenge efficiently the stable free radical 1,1-diphenyl-2-pycrylhydrazyl in ethanol; DHLA was considerably less effective against 2,2'-azobis(2-amidinopropane) dihydrochloride-mediated, peroxyl radical-induced non-HDL peroxidation, showing an IC(50) of 850 microM. Thus, DHLA, at therapeutically relevant concentrations, can counteract 15-lipoxygenase-dependent lipid peroxidation; this antioxidant effect may stem primarily from reduction of the active ferric 15-lipoxygenase form to the inactive ferrous state after DHLA-enzyme hydrophobic interaction and, possibly, from scavenging of fatty acid peroxyl radicals formed during lipoperoxidative processes. Inhibition of 15-lipoxygenase oxidative activity by DHLA could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects.     

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.     

Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.

Purified recombinant human 5-lipoxygenase was used to investigate the catalytic properties of the protein in the presence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 x 10(8) cells) and a specific activity of 3-6 mumol of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to the selective 5-lipoxygenase inhibitors BW755C (IC50 = 13 microM) and L-656,224 (IC50 = 0.8 microM). The reaction products of arachidonic acid oxidation were 5-HPETE and 6-trans- and 12-epi-6-trans-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4), indicating that the purified protein expressed both the 5-oxygenase and leukotriene A4 synthase activities (ratio 6:1). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+, and phosphatidylcholine. These factors were found to stabilize 5-lipoxygenase during preincubation of the enzyme at 37 degrees C with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect against enzyme inactivation but are not essential for enzyme activity.     

Arachidonate 5-lipoxygenase of porcine leukocytes studied by enzyme immunoassay using monoclonal antibodies

The cytosol fraction of porcine leukocytes contained 5-lipoxygenase, the activity of which was masked by a predominant activity of 12-lipoxygenase. The 5-lipoxygenase was partially purified to a specific activity of about 10 nmol of arachidonic acid oxygenated/min/mg of protein and given to mice as an antigen to prepare monoclonal antibodies against the enzyme. Two species of antibodies recognized separate sites of the 5-lipoxygenase protein and did not cross-react with 12-lipoxygenase. They were utilized to develop a peroxidase-linked immunoassay of sandwich-type, which allowed a quantitative determination of the 5-lipoxygenase protein. The assay was applied to a screening of the 5-lipoxygenase content in various porcine tissues. By far the highest content of 5-lipoxygenase was found in leukocytes. About one-tenth the amount of the enzyme was found in lung, pancreas, ileum, and thymus, which could not be attributed to the contaminating leukocytes in these tissues.     

1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

Effects of adenosine and deoxyadenosine on PHA-stimulation of lymphocytes of man, horse and pig

1. Adenosine inhibits thymidine and uridine incorporation of PHA-stimulated lymphocytes of man and horse at concentrations higher than 50 and 10 microM, respectively. Deoxyadenosine is inhibitory at concentrations higher than 100 microM. Thymidine and uridine incorporation of porcine lymphocytes are elevated 5-7-fold by 25-100 microM adenosine, deoxyadenosine, inosine and hypoxanthine. Leucine incorporation of PHA-stimulated lymphocytes was affected by adenosine and deoxyadenosine in the same way, but to a lower extent. 2. Effects of adenosine and deoxyadenosine were more pronounced at shorter cultivation times. 3. EHNA potentiated the effects of adenosine and deoxyadenosine on human and equine lymphocytes. With human lymphocytes inhibition by deoxyadenosine and EHNA was higher than by adenosine and EHNA. With porcine lymphocytes only the combination of deoxyadenosine and EHNA was inhibitory. 4. Homocysteine potentiated the inhibition of thymidine incorporation by the combination of adenosine and deoxyadenosine with equine lymphocytes, but not the inhibition of adenosine or deoxyadenosine alone. 5. Adenosine suppressed the PHA-stimulated elevation of PRPP concentrations. With porcine lymphocytes PRPP remained at the level of 0 hr, while with equine lymphocytes PRPP concentration decreased to below that level. 6. The various effects of adenosine and deoxyadenosine on lymphocytes of man, horse and pig can partially be related to differences in adenosine and deoxyadenosine metabolism.     

5-oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.     

Mechanism of inhibition of porcine leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid

The mechanism of inhibition of porcine leukocyte 12-lipoxygenase by 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was investigated. This compound is selective for the leukocyte form of the 12-lipoxygenase and inhibits the purified recombinant enzyme with an IC(50) value of approximately 2 microM. OPP induced a concentration-dependent lag phase in the oxygenation of arachidonic acid and decreased the maximal rate of reaction. Addition of the fatty acid hydroperoxide 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE) to the reaction greatly reduced the OPP-induced lag. Lineweaver-Burk analysis of the effect of OPP on 12-lipoxygenase kinetics with arachidonic acid indicated that it was a mixed-type inhibitor. OPP was not metabolized by 12-lipoxygenase as evidenced by its quantitative recovery from incubations with stoichiometric amounts of enzyme and 13-HPODE or arachidonic acid. OPP inhibited the pseudoperoxidase activity of the enzyme with 13-HPODE and the reducing agent, BWA137C. Lineweaver-Burk analysis of the effect of OPP on pseudoperoxidase kinetics suggested that OPP was competitive with 13-HPODE. Single-turnover experiments indicated that OPP inhibited the reduction of 13-HPODE by a stoichiometric amount of ferrous 12-lipoxygenase. Addition of 13-HPODE shortened the OPP-induced lag phase but did not affect the maximal rate of enzyme activity. In addition, OPP had no effect on total product formation in either the presence or the absence of 5 microM 13-HPODE when the reaction was allowed to go to completion. All of these observations are consistent with a model for inhibition of 12-lipoxygenase activity in which OPP slows the oxidation of the inactive ferrous enzyme to the active ferric enzyme and competes with arachidonic acid for the ferric enzyme.     

Increased oxidative metabolism in phytohemagglutinin-stimulated lymphocytes from patients with systemic lupus erythematosus is associated with serum SSA antibody.

We have examined oxidative metabolism in phytohemagglutinin (PHA)-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE) because increased oxygen free radicals would explain the DNA abnormality previously observed in these cells. Almost no oxidative activity was found in freshly isolated control or lupus lymphocytes or control lymphocytes stimulated with PHA. However, increased oxidative metabolism, measured by nitroblue tetrazolium (NBT) conversion to formazan, was found in PHA-stimulated lymphocytes from 14 of 21 lupus patients. A time course study showed that NBT activity appeared in positive lupus lymphocytes at 1-2 days of PHA stimulation, increased to a maximum at 2-4 days, and diminished thereafter. NBT activity was not related to specific disease symptoms, drug therapy, or serum dsDNA, Sm, RNP, or SSB (La) antibodies. The selected population of lupus patients studied precluded conclusions about NBT activity and disease severity. However, the intensity of NBT response in stimulated lupus lymphocytes was positively correlated with the presence of serum SSA (Ro) antibody. We suggest that increased oxidative activity of SLE lymphocytes generates a chemical change in endogenous DNA in vivo and may be a primary event in the pathogenesis of autoimmunity. Absence of detectable oxidative activity in stimulated lymphocytes in a subgroup of lupus patients suggests that at least two different mechanisms are associated with the altered DNA profiles observed in this disorder.     

Assessment of a role for phospholipase A2 and arachidonic acid metabolism in human lymphocyte natural cytotoxicity

The reagents quinacrine, hydrocortisone, and dexamethasone have been assumed to affect phospholipase A2 (PA2) when they reduce natural killer (NK) activity. However, these reagents did not reduce lymphocyte incorporation of [14C]arachidonate, which implies that they are not acting as PA2 inhibitors in this lymphocyte system. However, p-bromophenacyl bromide (BPB), which is an active site inhibitor of PA2, irreversibly abrogated NK activity of pretreated lymphocytes, disrupted target cell binding, and reduced [14C]arachidonic acid incorporation by 70-80% as compared to controls. Other observations contrary to expectations for PA2 inhibitors were: (1) quinacrine inhibited NK lysis when lymphocytes were pretreated and (2) the glucocorticoids only inhibited NK activity when continuously present in the assay. Furthermore, NK inhibition by hydrocortisone did not require protein synthesis. The lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosotetraynoic acid (ETYA), and hydroxyphenylretinamide, and not cycloxygenase inhibitors, reduced NK activity. These data suggest that arachidonate must be metabolized through the 5-lipoxygenase pathway in order to function in NK.     

Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation.

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.     

Examination of the effects of piriprost (U-60,257B) on alkaline phosphatase activity of rat endometrial stromal cells in vitro.

Previously, piriprost (U-60,257B; an inhibitor of leukotriene (LT) synthesis) was shown to increase alkaline phosphatase (ALP) activity in cultured endometrial stromal cells (1). The present study investigated the mechanism of action of piriprost in this system. Sensitized rat endometrial stromal cells were isolated and cultured for up to 72 hr with various treatments. Piriprost (100 microM) was found to decrease 5-hydroxyeicosatetraenoic acid (a 5-lipoxygenase product) by 53% after 72 hr which provided evidence that 5-lipoxygenase was being inhibited by piriprost. Lactate dehydrogenase (LDH) activity confirmed that piriprost was not toxic to the cells. The possibility of piriprost acting in an analogous manner with that of PGs was examined. Three microM PGE2 or 20 microM carba-prostacyclin (CP), an analogue of PGI2, maximally increased (p less than 0.01) ALP activity at 72 hr and the further addition of 100 microM piriprost to PGE2 or CP caused an additional, additive increase in ALP activity. This indicated that the mechanism of action of piriprost was probably different from that of PGE2 or PGI2. The possibility that piriprost was shunting arachidonic acid into PG production was examined. Ten microM indomethacin (an inhibitor of PG synthesis) caused a decrease (p less than 0.01) in ALP activity and a 99% reduction in PGE2 at 72 hr. The effects of the combination of 100 microM piriprost and 10 microM IM were statistically additive, suggesting that the effects of piriprost were not due to an increase in PG production. These studies suggest that the effects piriprost on possible in vitro decidualization may be due to inhibition of 5-lipoxygenase.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Effects of leukotrienes and f-Met-Leu-Phe on oxidative metabolism of neutrophils and eosinophils

We assessed the effects of several leukotrienes and of f-Met-Leu-Phe on oxygen consumption in neutrophils and on the initial burst of chemiluminescence (CL) in both neutrophils and eosinophils. It was found that f-Met-Leu-Phe initiated 2.6 times higher oxygen consumption in neutrophils than did leukotriene B4 (LTB4). f-Met-Leu-Phe also stimulated five to 10 times more CL from both types of granulocytes than LTB4, which was at least five times more potent than its omega-hydroxylated metabolite, 20-OH-LTB4, whereas the corresponding 20-COOH derivative was effective only in eosinophils. The double dioxygenation product 5(S), 12(S)- DHETE caused no CL. Neutrophils from patients with chronic granulomatous disease did not respond with CL to any of the agents. The peak of CL occurred 50 to 60 sec after the addition of fMLP, whereas the LTB4-associated peak occurred after 5 to 6 sec and then rapidly subsided. The treatment of cells with sodium azide to inhibit the myeloperoxidase system did not change the kinetics or the rapid decline of the LTB4-induced CL. The CL response to LTB4 could be inhibited to 85% by 0.5 microgram/ml of superoxide dismutase, to 72% by 200 mg/ml of catalase, and to 50% by 80 microM of mannitol. The corresponding figures for f-Met-Leu-Phe-induced CL were 80, 58, and 16%, suggesting that, although a substantial part of the CL appears to be due to superoxide ion production, other oxygen radicals are involved in luminol-enhanced CL production. Thus, in contrast to some previous reports that leukotrienes do not stimulate an oxidative metabolic response in granulocytes despite their potent activity as chemotactic factors, our studies show that leukotrienes are definite inducers of granulocyte oxidative metabolism.     

Activated lymphocytes increase expression of 5-lipoxygenase and its activating protein in THP-1 cells

The aim of this study was to investigate the regulation of the5-lipoxygenase pathway of arachidonic acid metabolism by lymphocytes using the monocyte-like cell line, THP-1. When THP-1 cells were incubated over 4-7 days in 10% supernatant from lectin-activated human lymphocytes, their capacity to synthesize 5-lipoxygenase productswas significantly increased. In contrast, the supernatant fromnonactivated lymphocytes had no effect. The increase in capacity tosynthesize 5-lipoxygenase products was mimicked by the addition ofeither granulocyte macrophage colony-stimulating factor (GM-CSF) orinterleukin-3. These increases in synthetic capacity reflected increased enzymatic activity. Increased immunoreactive protein and mRNAfor the enzymes 5-lipoxygenase and 5-lipoxygenase-activating protein were also found in cells conditioned with activated lymphocyte supernatants. Furthermore, the increase in mRNA for both enzymes wasnot blocked by cycloheximide, suggesting that the effect on steady-state mRNA levels does not require the synthesis of new protein.The increase in mRNA could be reproduced by GM-CSF. We conclude thatlymphocytes can regulate the expression of 5-lipoxygenase in THP-1cells over a period of days via the release of soluble factors.

     

Inhibitory effect of linoleyl-hydroxamic acid on the oxidation of linoleic acid by 12-lipoxygenase from porcine leukocytes]

Linoleic acid oxidation by 12-lipoxygenase from porcine leukocytes has been studied as affected by linoleyl-hydroxamic acid. Linoleyl-hydroxamic acid has been found to be an effective inhibitor of porcine leucocyte 12-lipoxygenase. Aerobic preincubation of 12-lipoxygenase with 0.1-6 microM of linoleyl-hydroxamic acid led to a time- and dose-dependent inhibition of the enzyme. The inhibitor's concentration able to induce a 50% loss of the enzyme activity with and without 15-min preincubation were 3.5 and 0.65 microM, respectively. Experimental results obeyed a kinetic scheme, which supposed 2 extra substrate molecules bounding with the enzyme-substrate complex in the presence of linoleyl-hydroxamic acid.     

Dimerumic acid protected oxidative stress-induced cytotoxicity in isolated rat hepatocytes

Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.     

Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers (Calluna vulgaris).

A compound was isolated and purified from heather flowers (Calluna vulgaris) based on its ability to inhibit lipoxygenase activity. This molecule was characterized as ursolic acid by GC-MS. Ursolic acid was found to be an inhibitor of both potato tuber 5-lipoxygenase and soybean 15-lipoxygenase with IC50 values of 0.3 mM. Ursolic acid also inhibits lipoxygenase activity in mouse peritoneal macrophages at 1 microM and HL60 leukemic cells growth (IC50 = 0.85 microM) as well as their DNA synthesis (IC50 = 1 microM). The possible role of lipoxygenase inhibition in the proliferation of leukemic cells is discussed.