A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells.

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).     

Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by sodium of A23187-mediated calcium translocation

Sodium inhibits in a dose-related fashion the translocation of calcium from an aqueous milieu into an organic phase containing the divalent-cation ionophore A23187. This inhibitory effect is reproduced by other monovalent cations, modulated by the nature of the anion in the sodium halide, and inversely related to the absolute amount of calcium translocated. The inhibitory effect cannot be attributed to a change in osmolarity or ionic strength, to sequestration of the ionophoretic molecule at the interface between the aqueous and organic phases, or to translocation of sodium or chloride. These findings indicate that sodium may directly affect the handling of calcium by ionophoretic systems specifically mediating the transport of divalent cations.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187

Challenge of human peripheral blood leukocytes with ionophore A23187 resulted in leukotriene (LT) synthesis, a decrease in total cellular 5-lipoxygenase activity, and a change in the subcellular localization of the enzyme. In homogenates from control cells, greater than 90% of the 5-lipoxygenase activity and protein was localized in the cytosol (100,000 X g supernatant). Ionophore challenge (2 microM) resulted in a loss of approximately 55% of the enzymatic activity and 35% of the enzyme protein from the cytosol. Concomitantly, there was an accumulation of inactive 5-lipoxygenase in the membrane (100,000 X g pellets) which accounted for at least 45% of the lost cytosolic protein. There was a good correlation between the quantities of LT synthesized and 5-lipoxygenase recovered in the membrane over an ionophore concentration range of 0.1-6 microM. The time course of the membrane association was similar to that of LT synthesis. Furthermore, although the pellet-associated enzyme recovered from ionophore-treated leukocytes was inactive, an irreversible, Ca2+-dependent membrane association of active 5-lipoxygenase could be demonstrated in cell-free systems. To determine whether ionophore treatment induced proteolytic degradation of 5-lipoxygenase, the total activity and protein content of 10,000 X g supernatants from control and ionophore-treated cells were examined. These supernatants, which included both cytosolic and membrane-associated enzyme, showed a 35% loss of 5-lipoxygenase activity but only an 8% loss of enzyme protein as a result of ionophore challenge (2 microM). Therefore, the majority of the loss of 5-lipoxygenase activity was most likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Together these results are consistent with the hypothesis that ionophore treatment results in a Ca2+-dependent translocation of 5-lipoxygenase from the cytosol to a membrane-bound site, that the membrane-associated enzyme is preferentially utilized for LT synthesis, and that it is consequently inactivated. Thus, membrane translocation of 5-lipoxygenase may be an important initial step in the chain of events leading to full activation of this enzyme in the intact leukocyte.     

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Inhibition of 5-lipoxygenase and leukotriene C4 synthase in human blood cells by thymoquinone

Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 microM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 microM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 microM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.     

Feedback activation of ferrous 5-lipoxygenase during leukotriene synthesis by coexisting linoleic acid

Ferrous lipoxygenases seem to be activated through a feedback control mechanism via FA hydroperoxides generated from PUFAs by partially existing ferric lipoxygenases. However, during leukotriene synthesis, feedback activation of ferrous 5-lipoxygenase in the presence of arachidonic acid (AA) was not observed. In the present study, we examined the feedback activation of ferrous 5-lipoxygenase in the 5-lipoxygenase/AA system in the presence of linoleic aicd (LA), which is a predominant component of membrane phospholipids. When potato 5-lipoxygenase was incubated with AA and LA in the presence of nitroxyl radical, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP), one-electron redox cycle reaction between ferric and ferrous 5-lipoxygenase was detected. For each revolution of the cycle, one molecule of PUFA and one molecule of its hydroperoxide were converted into PUFA-allyl radical-CmDeltaP adduct ([PUFA-H].-CmDeltaP) and PUFA-epoxyallyl radical-CmDeltaP adduct ([PUFA-H+O].-CmDeltaP), respectively. The ratios, [AA-H].-CmDeltaP/[LA-H].-CmDeltaP and [AA-H+O].-CmDeltaP/[LA-H+O].-CmDeltaP, were estimated to be 1.7 and 0.13, respectively. These facts indicate that ferrous 5-lipoxygenase is activated through feedback control in the presence of LA, and that resulting ferric 5-lipoxygenase catalyzes the stoichiometric synthesis of leukotrienes from AA. In conclusion, the biosynthesis of leukotrienes is remarkably efficient.     

Inhibition of leukotriene-induced contraction of guinea pig trachea by 5-lipoxygenase inhibitors

The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC4 or LTD4 were examined on the isolated guinea pig trachea. Responses to either LTC4 or LTD4 were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to inhibit metabolic conversion of the leukotrienes. NDGA (30 microM) and ETYA (100 microM) produced a selective competitive antagonism of LTD4-induced contractions, while phenidone antagonized both LTC4- and LTD4-induced responses in a non-competitive manner. In contrast, BW-755c (30 microM) did not significantly antagonize LTC4 or LTD4 concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors.     

Inhibition of leukotriene-induced contraction of guinea pig trachea by 5-lipoxygenase inhibitors

The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC₄ or LTD₄ were examined on the isolated guinea pig trachea. Responses to either LTC₄ or LTD₄ or LTD₄ were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to exhibit metabolic conversion of the leukotrienes. NDGA (30 μM) and ETYA (100 μM) produced a selective competitive antagonism of LTD₄ - induced contractions, while phenidone antagonized both LTC₄- and LTD₄ - induced responses in a non-competitive manner. In contrast, BW-755c (30 μM) did not significantly antagonize LTC₄ or LTD₄ concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors.     

Correlation between expression of 5-lipoxygenase-activating protein, 5-lipoxygenase, and cellular leukotriene synthesis

Previous studies involving transfection of cDNAs for 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase into osteosarcoma cells have shown that both these proteins are essential for leukotriene synthesis (Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers, P. J., Evans, J. F., Gillard, J. W., and Miller, D. K. (1990) Nature 343, 282-284). In the present study we show that FLAP is present in a variety of cells known to produce leukotrienes, but is absent from a number of cells which do not synthesize leukotrienes. Furthermore, differentiation of the human promyelocytic HL-60 cell line towards granulocytic cells following exposure to dimethylsulfoxide is associated with the concurrent induction of both FLAP and 5-lipoxygenase and an increased capacity to synthesize leukotrienes. Cellular leukotriene synthesis in this system is functionally dependent on FLAP as shown by its inhibition by the leukotriene biosynthesis inhibitor MK-886, a compound which specifically binds to FLAP.     

Stimulation of leukotriene production and membrane translocation of 5-lipoxygenase by cross-linking of the IgE receptors in RBL-2H3 cells.

Recent studies in rat basophilic leukemia cells (RBL-2H3) have shown that two pharmacological agents, ionomycin and thapsigargin, induce leukotriene C4 production and translocation of 5-lipoxygenase from cytosol to membrane, primarily by causing an influx of extracellular calcium. In the present study, we investigate the induction of these events by receptor activation. Cross-linking of high-affinity IgE receptors (Fc epsilon RI) by antigen in RBL-2H3 cells leads to leukotriene C4 production and membrane translocation of 5-lipoxygenase. As in the ionomycin-stimulated cells, leukotriene C4 production in antigen-stimulated cells is calcium-dependent since the amount of leukotriene C4 produced correlates quantitatively with the increase in intracellular free calcium concentration ([Ca2+]i). However, the increase in [Ca2+]i required for equivalent leukotriene C4 production by antigen is not as high as it is using ionomycin. In addition, no threshold [Ca2+]i level is required for leukotriene production by antigen, which is in contrast to the ionomycin stimulation that a [Ca2+]i level of 300-400 nM is required. Furthermore, antigen causes an additive increase in leukotriene C4 production in cells stimulated by the ionomycin. These results suggest that another as yet unidentified intracellular pathway acts in conjunction with Ca2+ for leukotriene synthesis in antigen-stimulated cells. Antigen stimulation causes 20-30% of the total cell 5-lipoxygenase to associate with membranes (compared with 10% in unstimulated cells) as demonstrated by enzyme activity assay and by Western Blot using antibodies to 5-lipoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and synthesis of achiral 5-lipoxygenase inhibitors employing the cyclobutyl group

The cyclobutyl group is an effective replacement for the linking group required in hydroxyureas for good in vivo activity against 5-Lipoxygenase. The principle is illustrated in two 5-LO inhibitors: 858C, a more potent inhibitor than Zileuton and, 862C, a very poteng 5-LO inhibitor with good oral persistence.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A₄ (actually detected as 6-trans-leukotriene B₄, 12-epi-6-trans-leukotriene B₄, abd 5,6-duhydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-D_R-³H]- or [10-L_S-³H]- labeled arachidonic acid, and 6-trans-LTB₄ and its 12-epimer were analyzed. More than 90% of 10-D_R-hydrogen was lost while about 100% of 10-L_S-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A₄.     

5-Lipoxygenase and leukotriene synthesis: effects of calcium ions and of inhibitors

5-Lipoxygenase and leukotriene (LT) A4 synthase, the first two enzymes in the pathway converting arachidonic acid to leukotrienes, can be co-purified. The Ca2+-activated conversion of arachidonic acid and of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to LTB4 have been compared, using cytosol from human leucocytes. The two activities showed identical responses to a number of inhibitors, suggesting that the two catalytic activities may involve the same active centre. The effects of Ca2+ ions were further investigated. With 5-HPETE as substrate, substantial synthesis of LTB4 was given in the absence of Ca2+, and the inhibitor sensitivity of this component was quite different from that of the Ca2+-activated component. This Ca2+-independent synthase activity was, however, very low in saponin-permeabilised washed leucocytes and it may therefore be not significant physiologically. With arachidonic acid as substrate at pH 7, the activity was highly Ca2+-dependent at a low substrate concentration (6.6 microM), but at a high concentration (132 microM) substantial activity was observed without Ca2+. This was also found when 5-lipoxygenase was assayed in cytosol from RBL cells. At pH 8-8.5, however, Ca2+ was required at both high and low concentrations of arachidonic acid. This suggests that Ca2+ is required for 5-lipoxygenase activity on arachidonate ions in solution but possibly not on protonated arachidonic acid or micelles.     

Inhibition of human sPLA2 and 5-lipoxygenase activities by two neo-clerodane diterpenoids.

The inhibitory effect of two neo-clerodane diterpenoids, E-isolinaridial (EI) and its methylketone derivative (EIM), isolated from Linaria saxatilis var. glutinosa, on PLA2 and other enzyme activities involved in the inflammatory process was studied. Both compounds inhibited human synovial sPLA2 in a concentration-dependent manner with IC50 values of 0.20 and 0.49 microM, respectively, similar to scalaradial. Besides, these compounds decreased the cell-free 5-lipoxygenase activity and A23187-induced neutrophil LTB4 biosynthesis. Another function of human neutrophils, such as receptor-mediated degranulation, was also significantly reduced. In contrast, none of the compounds affected superoxide generation in leukocytes, or cyclooxygenase-1, cyclooxygenase-2 and inducible nitric oxide synthase activities in cell-free assays.     

Modulation of apoptosis of proliferating and differentiating HL-60 cells by protein kinase inhibitors: suppression of PKC or PKA differently affects cell differentiation and apoptosis.

The relationship between RA- or dbcaMP-mediated differentiation and subsequent apoptosis in HL-60 cells was assessed by modulating the levels of differentiation suppressing the activity of PKC and PKA with calphostin C or GF 109203X and H89, respectively. Results demonstrated that (1) RA and dbcAMP caused a dose-dependent increase in apoptosis concomitant with progressive differentiation; (2) the suppression of PKC activity resulted in an increase of apoptosis unrelated to the modulated levels of differentiation; (3) the inhibition of PKA decreased granulocytic differentiation, but did not significantly affect apoptosis; (4) the pretreatment of cells with dbcAMP strongly potentiated RA-mediated differentiation without apparent changes in apoptosis; (5) cell differentiation and apoptosis were associated with cell cycle arrest in G1 phase and G2/M phases, respectively. Our findings indicate that the functional maturity of differentiating cells is not directly related to the apoptotic programme, and suggest that induction of cell differentiation and apoptosis are regulated by separate mechanisms in which PKC and PKA are involved.     

Inhibition of tyrosinase activity and protein synthesis in melanoma cells by calcium ionophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. Ionophore at a concentration of 10(-6) g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. Ionophore A23187 also inhibits the PGE1 mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10(-4)M). Ionophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGE1, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. Ionophore causes a rapid and marked (greater than 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.