Lipoxins: Eicosanoids carrying intra-and intercellular messages

The lipoxins are a recent addition to the family of biologically active products derived from arachidonic acid. Compounds of this series contain a conjugated tetraene structure and can be generated by the actions of the major lipoxygenases of human tissues (5-, 12-, and 15-LO's). Biosynthesis of the lipoxins from cellular sources of unesterified arachidonic acid is triggered by the initial actions of either the 15-LO or 5-LO followed by additional reactions. Recent results indicate that lipoxins are also generated by receptor-mediated events during cell-cell interactions with the transcellular metabolism of key intermediates. Lipoxin A₄ and lipoxin B₄ each possess a unique spectrum of biological activities unlike those of other eicosanoids in bothin vivo andin vitro systems. Lipoxin A₄ stimulates changes in the microvasculature and can block some of the proinflammatory effects of leukotrienes (in vivo). Lipoxin A₄ and lipoxin B₄ both inhibit natural killer cells (in vitro), and lipoxin B₄ displays selective actions on hematopoietic cells. The finding that lipoxin A₄ activates isolated protein kinase C suggests that it may also serve an intracellular role in its cell of origin before it is released to the extracellular milieu. Thus, cell-cell interactions, along with multiple oxygenations by lipoxygenases, generate compounds that can regulate cellular responses by serving as both intra- and intercellular messages.     

Antidepressant- and Anxiolytic-Like Effects of New Dual 5-HT1A and 5-HT7 Antagonists in Animal Models

The aim of this study was to further characterize pharmacological properties of two phenylpiperazine derivatives: 1-{2-[2-(2,6-dimethlphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazynine hydrochloride (HBK-14) and 2-[2-(2-chloro-6-methylphenoxy)ethoxy]ethyl-4-(2- methoxyphenyl)piperazynine dihydrochloride (HBK-15) in radioligand binding and functional in vitro assays as well as in vivo models. Antidepressant-like properties were investigated in the forced swim test (FST) in mice and rats. Anxiolytic-like activity was evaluated in the four-plate test in mice and elevated plus maze test (EPM) in rats. Imipramine and escitalopram were used as reference drugs in the FST, and diazepam was used as a standard anxiolytic drug in animal models of anxiety. Our results indicate that HBK-14 and HBK-15 possess high or moderate affinity for serotonergic 5-HT₂, adrenergic α₁, and dopaminergic D₂ receptors as well as being full 5-HT_1A and 5-HT₇ receptor antagonists. We also present their potent antidepressant-like activity (HBK-14—FST mice: 2.5 and 5 mg/kg; FST rats: 5 mg/kg) and (HBK-15—FST mice: 1.25, 2.5 and 5 mg/kg; FST rats: 1.25 and 2.5 mg/kg). We show that HBK-14 (four-plate test: 2.5 and 5 mg/kg; EPM: 2.5 mg/kg) and HBK-15 (four-plate test: 2.5 and 5 mg/kg; EPM: 5 mg/kg) possess anxiolytic-like properties. Among the two, HBK-15 has stronger antidepressant-like properties, and HBK-14 displays greater anxiolytic-like activity. Lastly, we demonstrate the involvement of serotonergic system, particularly 5-HT_1A receptor, in the antidepressant- and anxiolytic-like actions of investigated compounds.     

Eicosanoids and their role in immune modulation in fish—a brief overview

Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E₂, leukotriene B₄ and lipoxin A₄. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B₄ and lipoxin A₄ have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.     

Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro.

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.     

4-Hydroxynonenal enhances CD36 expression on murine macrophages via p38 MAPK-mediated activation of 5-lipoxygenase

Increased levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) coexist in atherosclerotic lesions but their relationship in atherogenesis is unclear. This study investigated the role of 5-LO in HNE-induced CD36 expression and macrophage foam cell formation, and the link between HNE and 5-LO. In J774A.1 murine macrophages, HNE (10 μM) enhanced CD36 expression in association with an increased uptake of oxLDL, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor, or with 5-LO siRNA. In peritoneal macrophages from 5-LO-deficient mice, HNE-induced CD36 expression was markedly attenuated, confirming a pivotal role of 5-LO in HNE-induced CD36 expression. In an assay for 5-LO activity, stimulation of macrophages with HNE led to increased leukotriene B₄ production in the presence of exogenous arachidonic acid in association with an increased association of 5-LO to the nuclear membrane. Among the mitogen-activated protein kinase (MAPK) pathways involved in 5-LO phosphorylation, HNE predominantly activated p38 MAPK in macrophages, and the p38 MAPK inhibitor SB203580, but not an extracellular signal-regulated kinase inhibitor, suppressed HNE-induced LTB₄ production. Collectively, these data suggest that p38 MAPK-mediated activation of 5-LO by HNE might enhance CD36 expression, consequently leading to the formation of macrophage foam cells.     

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BackgroundSubcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.MethodsFLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.Results5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca^2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB₄. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.ConclusionThe cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.General significanceWe present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.     

Control of experimental pulmonary tuberculosis depends more on immunostimulatory leukotrienes than on the absence of immunosuppressive prostaglandins

Prostaglandins (PGs) and leukotrienes (LTs) are produced in Mycobacterium tuberculosis (Mtb)-infected lungs and have immune suppressive and protective effects, respectively. Considering that both of these mediators are produced during mycobacterial infection, we investigated the specific and relative biological importance of each in regulating host response in experimental tuberculosis. Administration of celecoxib, which was found to reduce lung levels of PGE₂ and increase LTB₄, enhanced the 60-day survival of Mtb-infected mice in 14%. However administration of MK-886, which reduced levels of LTB₄ but did not enhance PGE₂, reduced 60-day survival from 86% to 43% in Mtb-infected mice, and increased lung bacterial burden. MK-886 plus celecoxib reduced survival to a lesser extent than MK-886 alone. MK-886- and MK-886 plus celecoxib-treated animals exhibited reduced levels of the protective interleukin-12 and gamma-interferon. Our findings indicate that in this model, the protective effect of LTs dominates over the suppressive effect of PGs.     

Inhibition of leukotriene biosynthesis abrogates the host control of Mycobacterium tuberculosis

Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B(4) and the anti-inflammatory substance lipoxin A(4) by approximately 50%, increased 60-day mortality from 14% to approximately 57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by approximately 15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) - presumably leukotrienes - positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB(4)) rather than a suppressor (LXA(4)) of antimicrobial defense, suggesting the major effect of leukotrienes.     

5-Lipoxagenase deficiency attenuates L-NAME-induced hypertension and vascular remodeling

BackgroundAbnormalities of the L-arginine-nitric oxide pathway induce hypertension. 5-Lipoxygenase (5-LO) is the key enzyme involved in synthesis of leukotrienes (LTs). However, whether nitricoxide synthase dysfunction induces hypertensive vascular remodeling by regulating 5-LO activity and its downstream inflammatory metabolites remains unknown.Methods and resultsSix-week L-NAME treatment significantly induced hypertension and vascular remodeling in both wild-type (WT) and 5-LO–knockout (5-LO–KO) mice, and blood pressure in caudal and carotid arteries was lower in 5-LO–KO than WT mice with L-NAME exposure. On histology, L-NAME induced less media thickness, media-to-lumen ratio, and collagen deposition and fewer Ki-67–positive vascular smooth muscle cells (VSMCs) but more elastin expression in thoracic and mesenteric aortas of 5-LO–KO than L-NAME–treated WT mice. L-NAME significantly increased LT content, including LTB4 and cysteinyl LT (CysLTs), in plasma and neutrophil culture supernatants from WT mice. On immunohistochemistry, L-NAME promoted the colocalization of 5-LO and 5-LO–activating protein on the nuclear envelope of cultured neutrophils, which was accompanied by elevated LT content in culture supernatants. In addition, LTs significantly promoted BrdU incorporation, migration and phenotypic modulation in VSMCs.ConclusionL-NAME may activate the 5-LO/LT pathway in immune cells, such as neutrophils, and promote the products of 5-LO metabolites, including LTB4 and CysLTs, which aggravate vascular remodeling in hypertension. 5-LO deficiency may protect against hypertension and vascular remodeling by reducing levels of 5-LO downstream inflammatory metabolites.     

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).     

5-Lipoxygenase plays an essential role in 4-HNE-enhanced ROS production in murine macrophages via activation of NADPH oxidase

Abstract

4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes; however, its role in the generation of reactive oxygen species (ROS) in macrophages is still unclear. Thus, this study investigated the sources and mechanisms of ROS generation in macrophages stimulated with HNE. Exposure of J774A.1 cells to HNE showed an increased production of ROS, which was attenuated by NADPH oxidase as well as 5-lipoxygenase (5-LO) inhibitors. Linked to these results, HNE increased membrane translocation of p47phox promoting NADPH oxidase activity, which was attenuated in peritoneal macrophages from 5-LO-deficient mice as well as in J774A.1 cells treated with a 5-LO inhibitor, MK886 or 5-LO siRNA. In contrast, HNE-enhanced 5-LO activity was not affected by inhibition of NADPH oxidase. Furthermore, leukotriene B₄, 5-LO metabolite, was found to enhance NADPH oxidase activity in macrophages. Altogether, these results suggest that 5-LO plays a critical role in HNE-induced ROS generation in murine macrophages through activation of NADPH oxidase.     

Etazolate rescues behavioral deficits in chronic unpredictable mild stress model: Modulation of hypothalamic–pituitary–adrenal axis activity and brain-derived neurotrophic factor level

Preliminary study in our laboratory showed that etazolate produced antidepressant- and anxiolytic-like effects in rodent models, however, the ability of etazolate to produce antidepressant- and anxiolytic-like effects and underlying mechanism(s) in chronic unpredictable mild stress (CUMS) model have not been adequately addressed. This study was aimed to investigate the beneficial effects of etazolate on CUMS-induced behavioral deficits (depression- and anxiety-like behaviors). In addition, the possible underlying mechanism(s) of etazolate in CUMS model was also investigated by measuring serum corticosterone (CORT) and brain-derived neurotrophic factor (BDNF) levels. Mice were subjected to a battery of stressors for 28 days. Etazolate (0.5 and 1 mg/kg, p.o.) and fluoxetine (20 mg/kg, p.o.) were administered during the last 21 days (8–28th) of the CUMS paradigm. The results showed that 4-weeks CUMS produces significant depression-like behavior in tail suspension test (TST) and partial anxiety-like behavior in elevated plus maze (EPM) and open field test (OFT). Stressed mice have also shown a significant high serum CORT and low BDNF level. Chronic treatment with etazolate (0.5 and 1 mg/kg., p.o.) and fluoxetine (20 mg/kg., p.o.) produced significant antidepressant-like behavior in TST (decreased duration of immobility), whereas, partial anxiolytic-like behavior in EPM (increased percentage of open arm entries) and OFT (increased % central ambulation score, total ambulation score and time spent in center zone). In addition, etazolate and fluoxetine treatment significantly (p < 0.05) increased the BDNF level and inhibited the hypothalamic–pituitary–adrenocortical (HPA) axis hyperactivity, as evidenced by low serum CORT level in stressed mice. In addition, etazolate and fluoxetine also showed significant antidepressant- and anxiolytic-like effects in normal control mice. In this study no significant changes were observed in locomotor activity in actophotometer test. Moreover, we did not find any effect of etazolate and fluoxetine on CORT and BDNF levels in normal control mice. In conclusion, the results of the present study suggested compelling evidences that etazolate has more marked effect on depression-like behavior in mice, which is atleast in part may be related to their modulating effects on the HPA axis and BDNF level.     

Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice

The present work evaluated the anxiolytic activity of an aqueous extract of Apocynum venetum L. (Apocynaceae) and bioguided its fractionation using the elevated plus maze (EPM) in mice as a model of anxiety. A single treatment of AV extract markedly increased the percentage time spent on the open arms of the EPM in two distinct concentration ranges of 22.5–30 and 100–125 mg/kg p.o., respectively, indicating a putative anxiolytic-like activity. Fractions showing anxiolytic effects in concentrations equal to 30 or 125 mg/kg of whole extract were antagonized using the benzodiazepine antagonist flumazenil (3 mg/kg i.p.) or the 5-HT_1A receptor antagonist WAY-100635 (0.5 mg/kg i.p.). All active fractions in a concentration equal to 125 mg/kg were effectively blocked by the benzodiazepine antagonist flumazenil, while the anxiolytic activities of fractions in the lower dose equivalent to 30 mg/kg of whole extract were inhibited by the 5-HT_1A receptor antagonist WAY-100635. Through further separation of AV fractions it was possible to isolate and characterize the flavonol kaempferol which showed an anxiolytic-like activity in concentrations from 0.02 to 1.0 mg/kg p.o. The anxiolytic activity of kaempferol was partially antagonized by concomitant administration of flumazenil, but not by WAY-100635. In conclusion, our study clearly demonstrates that AV extract possesses anxiolytic-like activity and that at least one of its flavonoids, kaempferol, can elicit the same kind of neuropharmacological activity.     

Dipeptide Tyr-Leu (YL) exhibits anxiolytic-like activity after oral administration via activating serotonin 5-HT1A, dopamine D1 and GABAA receptors in mice

We found that Tyr-Leu (YL) dose-dependently exhibits potent anxiolytic-like activity (0.1-1 mg/kg, i.p.) comparable to diazepam in the elevated plus-maze test in mice. YL was orally active (0.3-3 mg/kg). A retro-sequence peptide or a mixture of Tyr and Leu was inactive. The anxiolytic-like activity of YL was inhibited by antagonists for serotonin 5-HT_1A, dopamine D₁ and GABA_A receptors; however, YL had no affinity for them. We also determined the order of their activation is 5-HT_1A, D₁ and GABA_A receptors using selective agonists and antagonists. Taken together, YL may exhibit anxiolytic-like activity via activation of 5-HT_1A, D₁ and GABA_A receptors.     

Progestin receptor-mediated reduction of anxiety-like behavior in male rats

Background

It is well known progesterone can have anxiolytic-like effects in animals in a number of different behavioral testing paradigms. Although progesterone is known to influence physiology and behavior by binding to classical intracellular progestin receptors, progesterone''s anxiety reducing effects have solely been attributed to its rapid non-genomic effects at the GABA_A receptor. This modulation occurs following the bioconversion of progesterone to allopregnanolone. Seemingly paradoxical results from some studies suggested that the function of progesterone to reduce anxiety-like behavior may not be entirely clear; therefore, we hypothesized that progesterone might also act upon progestin receptors to regulate anxiety.

Methodology/Principal Findings

To test this, we examined the anxiolytic-like effects of progesterone in male rats using the elevated plus maze, a validated test of anxiety, and the light/dark chamber in the presence or absence of a progestin receptor antagonist, RU 486. Here we present evidence suggesting that the anxiolytic-like effects of progesterone in male rats can be mediated, in part, by progestin receptors, as these effects are blocked by prior treatment with a progestin receptor antagonist.

Conclusion/Significance

This indicates that progesterone can act upon progestin receptors to regulate anxiety-like behavior in the male rat brain.     

Central prostaglandin D2 exhibits anxiolytic-like activity via the DP1 receptor in mice

We found that prostaglandin (PG) D₂, the most abundant PG produced in the central nervous system (CNS), exhibited anxiolytic-like activity at a dose of 10–100 pmol/mouse after intracerebroventricular (i.c.v.) administration in the elevated plus-maze test in mice. A DP₁ receptor-selective agonist, BW245C, mimicked the anxiolytic-like activity of PGD₂, while a DP₂ receptor agonist 13,14-dihydro-15-keto-PGD₂ was inactive. The anxiolytic-like activity of PGD₂ was blocked by a DP₁ antagonist, BWA868C, suggesting that PGD₂-induced anxiolytic-like activity was mediated by the DP₁ receptor. Adenosine A_2A or GABA_A receptor antagonists, SCH58261 or bicuculline, respectively, also blocked its anxiolytic-like activity. Taken together, centrally administered PGD₂ may induce anxiolytic-like activity via the A_2A and GABA_A receptors, downstream of the DP₁ receptor.     

Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A₄ (LTA₄) either by a specific LTC₄ synthase to leukotriene C₄ or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC₄, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA₄ to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC₄, LTD₄ and LTE₄). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA₄ and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100 000 × g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.     

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.     

Lack of adenosine A3 receptors causes defects in mouse peripheral blood parameters

The role of the adenosine A₃ receptor in hematopoiesis was studied using adenosine A₃ receptor knockout (A₃AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A₃AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A₃AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A₃AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A₃ receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A₃AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A₃AR KO mice and their WT counterparts.     

The Effects of N-Methyl-d-Aspartate Receptor Blockade During The Early Neurodevelopmental Period on Emotional Behaviors and Cognitive Functions of Adolescent Wistar Rats

The N-Methyl-d-Aspartate (NMDA) receptor is expressed abundantly in the brain and plays an important role in neuronal development, learning and memory, neurodegenerative diseases, and neurogenesis. In this study, we evaluated the effects of NMDA receptor blockade during the early neurodevelopmental period on exploratory locomotion, anxiety-like behaviors and cognitive functions of adolescent Wistar rats. NMDA receptor hypofunction was induced 7–10 days after birth using MK-801 in rats (0.25 mg/kg twice a day for 4 days via intraperitoneal injection). The open-field (OF), elevated plus maze (EPM) and passive avoidance (PA) tests were used to evaluate exploratory locomotion, anxiety-like behaviors and cognitive functions. In the OF test, MK-801 caused an increase in locomotion behavior (p < 0.01) and in the frequency of rearing (p < 0.05). In the EPM test, MK-801 treatment increased the time spent in the open arms, the number of open arm entries and the amount of head dipping (p < 0.01). MK-801 treatment caused no statistical difference compared to the control group in the PA test (p > 0.05). Chronic NMDA receptor blockade during the critical period of maturation for the glutamatergic brain system (postnatal days 7–10) produces locomotor hyperactivity and decreased anxiety levels, but has no significant main effect on cognitive function during adolescence.