Addition Of Leukotrienes Research Articles

Impact of Arachidonic Acid and the Leukotriene Signaling Pathway on Vasculogenesis of Mouse Embryonic Stem Cells

Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB₄ receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.

Ecdysis of Oesophagostomum: possible involvement of eicosanoids and development of a bioassay

Bioassays were developed and applied to test the role of eicosanoids and pH changes in ecdysis of Oesophagostomum dentatum. Exsheathment (80-100%) was achieved by subjecting third-stage larvae (L3) either to chlorine (hypochlorite assay) for 5 min or by incubating them in HCl followed by addition of NaHCO3 (pH-change assay) with subsequent cultivation at 38.5 degrees C/10% CO2 for 1 week. Addition of the lipoxygenase (LOX) inhibitor diethylcarbamacine (DEC) to the larvae resulted in a reduction of the exsheathment rates which could be restored by the addition of leukotrienes (LT)B4, LTC4, LTD4 and LTE4. Addition of the cyclooxygenase (COX) inhibitor acetylsalicylic acid (ASA) also resulted in decreased exsheathment rates both in the hypochlorite and in the pH-change assays in a dose-dependent manner. However, the primary COX products (prostaglandins) were not able to reverse this effect, in contrast to LTC4. It was concluded that: (1) both tests are suitable for bioassaying the effect of substances on exsheathment, and (2) eicosanoids involved in the control of exsheathment of L3 of O. dentatum are primarily LT.

PROGRESSION OF MOUSE TRANSFORMED LEYDIG-CELLS FROM ESTROGEN-SENSITIVE TO ESTROGEN-INSENSITIVE GROWTH PHENOTYPE CONCOMITANT WITH LOSS OF LEUKOTRIENE D-4 RECEPTOR

We have previously shown that growth enhancement of murine transformed Leydig cells (B-1F) by estrogen is partly mediated through inhibition of leukotriene formation due to suppression of 5-lipoxygenase activity; leukotrienes, which inhibit the proliferation of B-1F cells, play an important role in an autocrine loop for B-1F cells to proliferate in an estrogen-sensitive manner. An estrogen-insensitive cell line, termed Cl 4(-), was established from B-1F cells and maintained in serum-free culture medium without addition of estrogen. The proliferation of Cl 4(-) cells was not affected by the addition of 10(-11)-10(-6) M 17 beta-estradiol, whereas an estrogen receptor in Cl 4(-) cells appeared to be normal, as judged by its binding to estrogens. 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone (AA861), an inhibitor of 5-lipoxygenase, and indomethacin, a cyclooxygenase inhibitor, did not stimulate the proliferation of Cl 4(-) cells, while AA861 markedly stimulated the proliferation of B-1F cells. Analyses of extracts of medium and cells showed that estrogen and AA861 inhibited the production of leukotriene B-4, C-4, D-4 and E(4) in estrogen-insensitive Cl 4(-) cells as well as in estrogen-sensitive B-1F cells. We were able to demonstrate that no inhibition of the proliferation is observed in Cl 4(-) cells by addition of leukotriene D-4, which inhibited the proliferation of B-1F cells, and that the high affinity binding site for leukotriene D-4 is not present in Cl 4(-) whereas it is present in B-1F cells. These results suggest that the insensitivity of Cl 4(-) cell proliferation to estrogen is at least partly due to deficient binding of leukotriene D-4, since, in Cl 4(-) cells, estrogen inhibits the production of leukotriene D-4, which inhibits the proliferation of estrogen-sensitive parental cell line B-1F.

Epidermal growth factor activates calcium channels by phospholipase [formula omitted] leukotriene C 4 production

Epidermal growth factor (EGF) induces a Ca 2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca 2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C 4 can fully mimic EGF in its ability to activate Ca 2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C 4. In addition, we show that EGF-induced, Ca 2+-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA 2 5 - lipoxygenase-mediated leukotriene C 4 production constitutes a novel and specific signal transduction pathway in growth factor action.

Positive inotropic effects of low concentrations of leukotrienes C4 and D4 in rat heart

We examined the effects of leukotrienes (LT) B4, C4, D4, and E4 (0.010-2.5 ng/ml) on contractile and coronary function in isolated rat hearts. Concentration-dependent effects were examined either by the cumulative addition of LTs or by addition of specific concentrations to individual preparations. Neither LTB4 nor LTE4 produced myocardial or coronary effects at any concentration, irrespective of addition protocol. At 0.010 ng/ml, both LTC4 and LTD4 produced an increase in force that was associated with a 30% elevation in coronary pressure. Further cumulative addition of either leukotriene resulted in a negative inotropic effect and a further increase in coronary pressure. In contrast, following single additions of LTC4 or LTD4 (0.01-0.50 ng/ml) a positive inotropic effect and an increased coronary pressure were observed. LTC4 or LTD4 at 0.5 ng/ml produced a negative inotropic effect in hearts pretreated with 0.01 ng/ml of LTD4 or LTC4, respectively. Reversal of this addition protocol resulted in a negative inotropic effect of either 0.01 ng/ml LTD4 or LTC4. Verapamil and nifedipine significantly attenuated the positive inotropic and coronary constricting effect of 0.5 ng/ml LTC4 and LTD4. The addition of either LT following BAY K 8644 resulted in a negative inotropic effect, in contrast to the positive inotropic influence seen with leukotriene alone. Our results demonstrate a positive inotropic effect of low concentrations of LTC4 and LTD4 concomitant with coronary artery constriction, a phenomenon determined by leukotriene addition protocols and suggestive of LTC4/LTD4 receptor interaction. The effects of calcium channel antagonists and BAY K 8644 on the inotropic response suggest a leukotriene-mediated activation of the calcium channel resulting in increased intracellular calcium concentrations.

Regulation of murine hematopoiesis by arachidonic acid metabolites

Arachidonic acid metabolites have been shown to exert a variety of regulatory effects on cellular activation and proliferation. Recently, a role for these products as regulators of hematopoiesis was suggested and evidence provided that products of the lipoxygenase pathway, specifically leukotrienes, are essential for human myeloid colony formation in vitro. In this report the broader role of these metabolites in hematopoiesis was examined using murine bone marrow stem cell assays for both myeloid and lymphoid cell lines. The effects of lipoxygenase and/or cyclooxygenase pathway inhibitors on stem cell colony formation were evaluated and compared to qualitative and quantitative changes in arachidonic acid metabolism that occurred in similarly treated bone marrow cell cultures. Interruption of the lipoxygenase pathway by esculetin or nordihydroguaiaretic acid resulted in decreased colony formation in both lymphoid and myeloid stem cells. This inhibition of colony growth was partly reversed by the addition of leukotrienes and was particularly evident in B-cell progenitor cultures to which was added LTB4. Inhibition of the cyclooxygenase pathway by indomethacin or ibuprofen had a slight stimulatory effect on myeloid colony formation, while slightly inhibiting the formation of lymphoid colonies. These results support a direct role for lipoxygenase products in myeloid colony formation and lymphoid stem cell proliferation. A more complex role for cyclooxygenase metabolites in the hematopoietic process appears probable.

Modulation of interleukin-1 activity on murine thymocytes by various inhibitors of arachidonic acid oxygenation.

A variety of cyclooxygenase (FCO) and 5-lipoxygenase (5-LPO) inhibitors were tested for their ability to modulate murine thymocyte proliferation induced by IL-1 and suboptimal levels of the mitogen phytohemagglutinin (PHA). The contribution of drug toxicity to inhibition of 3H-thymidine incorporation was estimated by measuring MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) formazan production in the thymocyte cultures at the end of the assay. Cyclosporin A and dexamethasone, two positive control compounds, potently inhibited thymocyte proliferation at extremely low concentrations (0.01 and 0.001 micrograms/ml respectively), although activity roughly paralleled toxicity. In contrast, 5-LPO inhibitors (AA-861, BW-755c, and ETYA), but not selective FCO inhibitors (ibuprofen and indomethacin), suppressed lymphoproliferation at nontoxic concentrations, suggesting that products of the 5-LPO pathway may mediate the thymocyte proliferative response induced by IL-1/PHA. Attempts to counteract the suppressive activity of 5-LPO inhibitors by addition of leukotriene (LT) B4, LTC4, LTE4, 5-HETE, and 15-HETE were unsuccessful.

A novel metabolic pathway for leukotriene B 4 in different cell types: primary reduction of a double bond

Addition of leukotriene B 4 together with trace amounts of tritiated leukotriene B 4 to different cell types, such as bone marrow-derived macrophages, T-lymphocytes, mesangial cells or fibroblast tumor cells, led to the formation of several hitherto unknown degradation products within hours. None of them could be identified as 20-hydroxy- or 20-carboxyleukotriene B 4, known to be produced by polymorphonuclear leukocytes. The primarily formed transient leukotriene B 4 metabolite was less polar than leukotriene B 4 and was detectable by measuring its ultraviolet absorbance at 232 nm or its radioactivity. Mass spectral analysis showed very similar fragmentation spectra of leukotriene B 4 and its primary metabolite. The most abundant ion and the main fragments of the new metabolite were increased by two mass units compared to leukotriene B 4. These observations suggest that, in a variety of cells, leukotriene B 4 is first reduced to a 5,12-dihydroxyeicosatrienoic acid, which is further converted to secondary hydrophilic degradation products. This raises the question of the major route of leukotriene B 4 metabolism in vivo.

Defective IFN-gamma production in the human neonate. II. Role of increased sensitivity to the suppressive effects of prostaglandin E.

Analysis of endogenous production and effects of exogenous addition of interleukin 2 (IL 2), leukotrienes (LT), and prostaglandin E (PGE) has been used to investigate the dysregulation responsible for impaired PHA-induced IFN-gamma secretion by cord blood leukocytes (CBL). The addition of LT or IL 2 could not reverse the IFN defect of CBL. The production of these two mediators was found to be normal in CBL cultures. CBL and control leukocytes from adult donors produced comparable amounts of PGE2. In contrast, sensitivity to the suppressive effects of PGE2 on IFN-gamma secretion was much higher with CBL than with control leukocytes. Treatment with indomethacin reversed the IFN-gamma defect with most CBL tested, and the addition of physiologic amounts of PGE2 to indomethacin-treated cultures resulted in a profound impairment of IFN-gamma production similar to that of untreated CBL cultures. Preincubation of CBL for 24 hr before PHA stimulation resulted in restoration of a normal sensitivity to exogenous PGE2, in parallel with correction of the IFN-gamma defect. Our observations suggest that the impairment of IFN-gamma secretion in neonates is not due to deficient amplification circuits, but is the consequence of an exaggerated cellular sensitivity to the suppressive effects of PGE produced endogenously in normal amounts.

Leukotriene-induced contraction and thromboxane production in guinea-pig lung parenchymal strips.

Addition of leukotrienes (LTs)C4 and D4 to guinea-pig isolated lung parenchymal strips stimulated the production of thromboxane A2(TxA2) and prostacyclin (PGI2) as determined by radioimmunoassay of their respective degradation products, thromboxane B2(TxB2) and 6-keto-prostaglandin F1 alpha (6-keto PGF1 alpha) in the bathing medium. However, contraction of the lung strips in response to LTD4 preceded the increases in the levels of these products in the organ bath. Pretreatment of the lung strips with aspirin, indomethacin or BW 755C abolished the formation of TxA2 and PGI2 but had no significant effect (10-25% inhibition) on LT-induced contraction. By contrast, a similar concentration of indomethacin significantly inhibited LTD4-induced contractions when the agonist was administered as a bolus to superfused lung strips. It is concluded that the production of metabolites of arachidonic acid in response to the leukotrienes is not a major mechanism mediating their contractile action in peripheral lung tissues at equilibrium, but its contribution to the contractile response may vary with experimental technique.

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B 4

Leukotriene B 4 stimulated a transient production of superoxide anions (O − 2) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca 2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B 4 and calcium for the half-maximal production were about 1 μM and 200 μM, respectively. 8-( N, N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O − 2 production stimulated by leukotriene B 4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B 4 was added to the cells treated with cytochalasin B, the production of O − 2 was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca 2+ concentrations but it could be induced even without the addition of Ca 2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca 2+-free medium showed a negligible production of superoxide on addition of leukotriene B 4, but the production appeared upon addition of CaCl 2. These findings suggest that the superoxide production stimulated by leukotriene B 4 is associated with the influx of Ca 2+.