Biomedical subjects

Oliver Werz

Publications and source records attributed to Oliver Werz.

At least 19 recordsLinked to original sources

PubMed2006

Coactosin-like protein supports 5-lipoxygenase enzyme activity and up-regulates leukotriene A4 production.

Regulation of 5-lipoxygenase (5LO) activity is a key determinant for the biosynthesis of proinflammatory leukotrienes. Coactosin-like protein (CLP) is an F-actin-binding protein that can also bind 5LO. Here, we report that CLP can up-regulate and modulate 5LO activity [formation of 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE)], 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HETE), and 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA(4)) in vitro. Three findings are presented. First, CLP up-regulates Ca(2+)-induced 5LO activity, in the absence of phosphatidylcholine (membrane). Apparently, CLP can function as a scaffold for 5LO, similar to membranes. Second, CLP gives a considerable (3-fold) increase in the amount of LTA(4) formed by 5LO, when present together with phosphatidylcholine. Third, CLP increases the ratio of 5-HETE/5-HPETE. These effects require protein interaction by Trp residues in ligand-binding loops of the 5LO beta-sandwich; both binding and stimulatory effects of CLP were abolished for the mutant 5LO-W13/75/102A. In polymorphonuclear leukocytes stimulated with Ca(2+) ionophore, both CLP and 5LO associated with the nucleus, whereas in resting cells, CLP and 5LO were cytosolic. These findings establish CLP as a factor relevant for 5LO product formation. Functioning as a 5LO scaffold, CLP may provide a basis for the formation of 5-HETE in the cytosol of different cell types. Furthermore, in stimulated cells, CLP appears to function in a complex together with 5LO and membranes, increasing the capacity of 5LO for leukotriene biosynthesis.

Arachidonate 5-Lipoxygenase↗

PubMed2006

Design and synthesis of novel 2-amino-5-hydroxyindole derivatives that inhibit human 5-lipoxygenase.

Compounds that inhibit 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of leukotrienes (LTs), possess potential for the treatment of inflammatory and allergic diseases as well as of atherosclerosis and cancer. Here we present the design and the synthesis of a series of novel 2-amino-5-hydroxyindoles that potently inhibit isolated human recombinant 5-LO as well as 5-LO in polymorphonuclear leukocytes, exemplified by ethyl 2-[(3-chlorophenyl)amino]-5-hydroxy-1H-indole-3-carboxylate (3n, IC(50) value congruent with 300 nM). Introduction of an aryl/arylethylamino group or 4-arylpiperazin-1-yl residues into position 2 of the 5-hydroxyindoles was essential for biological activity. Whereas the 4-arylpiperazin-1-yl derivatives were more potent in cell-free assays as compared to intact cell test systems, aryl/arylethylamino derivatives inhibited 5-LO activity in intact cells and cell-free assays almost equally well. On the basis of their 5-LO inhibitory properties, these novel 2-amino-5-hydroxyindoles represent potential candidates for the pharmacological intervention with LT-associated diseases.

Amines↗

PubMed2006

Therapeutic options for 5-lipoxygenase inhibitors.

5-Lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into leukotriene (LT) A(4) and 5-hydroperoxyeicosatetraenoic acid. LTA(4) can then be converted into LTB(4) by LTA(4) hydrolase or into LTC(4) by LTC(4) synthase and the LTC(4) synthase isoenzymes MGST2 and MGST3. LTB(4) is a potent chemoattractant for neutrophils, eosinophils and monocytes leading to adherence of phagocytes to vessel walls, neutrophil degranulation and release of superoxide anions. LTC(4) and its metabolite, LTD(4), are potent bronchoconstrictors that increase vascular permeability and stimulate mucus secretion from airways. Recent data also suggest that LT have an immunomodulatory role. Due to these properties, the increased biosynthesis of LT in asthma, and based upon clinical data obtained with CysLT(1) receptor antagonists in asthma patients, there is a consensus that CysLT play a prominent role in asthma. In this review, we summarize the knowledge on possible functions of the 5-LO pathway in various diseases like asthma, cancer and cardiovascular events and review the corresponding potential therapeutic roles of 5-LO inhibitors.

Animals↗

PubMed2006

Boswellic acids stimulate arachidonic acid release and 12-lipoxygenase activity in human platelets independent of Ca2+ and differentially interact with platelet-type 12-lipoxygenase.

Boswellic acids inhibit the transformation of arachidonic acid to leukotrienes via 5-lipoxygenase but can also enhance the liberation of arachidonic acid in human leukocytes and platelets. Using human platelets, we explored the molecular mechanisms underlying the boswellic acid-induced release of arachidonic acid and the subsequent metabolism by platelet-type 12-li-poxygenase (p12-LO). Both beta-boswellic acid and 3-O-acetyl-11-keto-boswellic acid (AKBA) markedly enhanced the release of arachidonic acid via cytosolic phospholipase A2 (cPLA2), whereas for generation of 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], AKBA was less potent than beta-boswellic acid and was without effect at higher concentrations (> or =30 microM). In contrast to thrombin, beta-boswellic acid-induced release of ara-chidonic acid and formation of 12-H(P)ETE was more rapid and occurred in the absence of Ca2+. The Ca2+-independent release of arachidonic acid and 12-H(P)ETE production elicited by beta-boswellic acid was not affected by pharmacological inhibitors of signaling molecules relevant for agonist-induced arachidonic acid liberation and metabolism. It is noteworthy that in cell-free assays, beta-boswellic acid increased p12-LO catalysis approximately 2-fold in the absence but not in the presence of Ca2+, whereas AKBA inhibited p12-LO activity. No direct modulatory effects of boswellic acids on cPLA2 activity in cell-free assays were evident. Therefore, immobilized KBA (linked to Sepharose beads) selectively precipitated p12-LO from platelet lysates but failed to bind cPLA2. Taken together, we show that boswellic acids induce the release of arachidonic acid and the synthesis of 12-H(P)ETE in human platelets by unique Ca2+-independent routes, and we identified p12-LO as a selective molecular target of boswellic acids.

Arachidonate 12-Lipoxygenase↗

PubMed2006

Analysis of the 5-lipoxygenase promoter and characterization of a vitamin D receptor binding site.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and transforming growth factor beta (TGFbeta) potently induce 5-lipoxygenase (5-LO) in myeloid cells. We analyzed vitamin D receptor (VDR) binding to putative vitamin D response elements within the 5-LO promoter and analyzed its function by reporter gene analysis. Binding of VDR and retinoid X receptor to the promoter region was shown in DNase I footprinting, electrophoretic mobility shift and chromatin immunoprecipitation assays. However, the identified VDR binding region did not mediate induction of reporter gene activity by 1,25(OH)(2)D(3)/TGFbeta, neither in the 5-LO promoter context nor with the thymidine kinase (tk) promoter. Insertion of the rat atrial natriuretic factor VDRE in reporter plasmids containing the 5-LO promoter diminished induction by 1,25(OH)(2)D(3)/TGFbeta as compared with the tk promoter. Similarly, low inductions were obtained when cells were transiently or stably transfected with constructs containing various 5-LO promoter regions. Concerning basal promoter activity, we identified a positive regulatory region (-779 to -229), which includes the VDR binding region, in 5-LO-positive MonoMac6 cells. In summary, the VDR/RXR complex binds to putative VDREs in the 5-LO promoter, but other sequences outside the 5-LO promoter seem to be responsible or additionally required for the prominent induction of 5-LO mRNA expression by 1,25(OH)(2)D(3) and TGFbeta.

Arachidonate 5-Lipoxygenase↗

PubMed2006

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells.

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.

Anions↗

PubMed2006

Boswellic acids: biological actions and molecular targets.

Gum resin extracts of Boswellia species have been traditionally applied in folk medicine for centuries to treat various chronic inflammatory diseases, and experimental data from animal models and studies with human subjects confirmed the potential of B. spec extracts for the treatment of not only inflammation but also of cancer. Analysis of the ingredients of these extracts revealed that the pentacyclic triterpenes boswellic acids (BAs) possess biological activities and appear to be responsible for the respective pharmacological actions. Approaches in order to elucidate the molecular mechanisms underlying the biological effects of BAs identified 5-lipoxygenase, human leukocyte elastase, toposiomerase I and II, as well as IkappaB kinases as molecular targets of BAs. Moreover, it was shown that depending on the cell type and the structure of the BAs, the compounds differentially interfere with signal transduction pathways including Ca(2+/-) and MAPK signaling in various blood cells, related to functional cellular processes important for inflammatory reactions and tumor growth. This review summarizes the biological actions of BAs on the cellular and molecular level and attempts to put the data into perspective of the beneficial effects manifested in animal studies and trials with human subjects related to inflammation and cancer.

Animals↗

PubMed2006

Inhibition of human 5-lipoxygenase and anti-neoplastic effects by 2-amino-1,4-benzoquinones.

We have recently presented the synthesis of 2-amino-1,4-benzoquinones by nuclear amination of p-hydroquinones with primary aromatic amines using fungal laccases as catalysts. In the present report, a series of selected 2-amino-1,4-benzoquinones was tested for biological activities, such as inhibition of human 5-lipoxygenase and anti-proliferative/anti-neoplastic effects. Compound 9 (2-[4'-(iso-propylphenyl)-amino]-5,6-dimethyl-1,4-benzoquinone) was identified as the most potent aminoquinone derivative, suppressing 5-lipoxygenase in intact human polymorphonuclear leukocytes as well as in crude enzyme preparations in the low micromolar range (IC50 = 6 microM). Structure-activity relationships are discussed. Of interest, the 5-lipoxygenase inhibitory properties of 2-amino-1,4-benzoquinones in intact cells correlated to the anti-neoplastic activities of the compounds in breast and urinary bladder cancer cell lines. Based on these features, bioactive 2-amino-1,4-benzoquinones may possess potential for the pharmacological treatment of diseases associated with elevated 5-lipoxygenase activity, in particular certain types of cancer.

Antineoplastic Agents↗

PubMed2005

GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site.

5-lipoxygenase (5LO) catalyzes formation of leukotrienes, mediators with roles in several inflammatory disorders, including atherosclerosis. The human 5LO gene promoter contain multiple GC-boxes. The relevance of these for expression of 5LO in the human monocytic cell line Mono Mac 6 (MM6) was studied. A downregulating effect of the GC-box binding compound mithramycin indicated that GC-rich sequences in the 5LO gene promoter are important for expression of native 5LO. In DNase I footprinting, mithramycin and Sp1 protected known GC-boxes, but also a novel Sp1 binding site was found, comprising 20 bp upstream of the major transcription initiation site, beside an Initiator-like sequence. Mutation of this site reduced Sp1 binding and expression of reporter genes in MM6 cells, compatible with a function as basal promoter element for the TATA-less 5LO gene. When differentiation was induced by TGFbeta and vitamin D(3), 5LO expression became prominent, but expression levels of Sp1/3 and Egr-1 were the same as for control cells. Also, 5LO reporter gene activity in transiently transfected MM6 cells was insensitive to differentiation. Thus, the GC-rich part of the 5LO gene promoter, including a novel Sp1 site, appear important for basal (rather than upregulated) transcription of 5LO in MM6 cells.

Arachidonate 5-Lipoxygenase↗

PubMed2005

Extraction and visualization of potential pharmacophore points using support vector machines: application to ligand-based virtual screening for COX-2 inhibitors.

Support vector machines (SVM) were trained to predict cyclooxygenase 2 (COX-2) and thrombin inhibitors. The classifiers were obtained using sets of known COX-2 and thrombin inhibitors as "positive examples" and a large collection of screening compounds as "negative examples". Molecules were encoded by topological pharmacophore-point triangles. In retrospective virtual screening, 50-90% of the known active compounds were listed within the first 0.1% of the ranked database. To check the validity of the constructed classifiers, we developed a method for feature extraction and visualization using SVM. As a result, potential pharmacophore points were weighted according to their importance for COX-2 and thrombin inhibition. Known thrombin and COX-2 pharmacophore points were correctly recognized by the machine learning system. In a prospective virtual screening study, several potential COX-2 inhibitors were predicted and tested in a cellular activity assay. A benzimidazole derivative exhibited significant inhibitory activity with an IC(50) of 0.2 microM, which is better than Celecoxib in our assay. It was demonstrated that the SVM machine-learning method can be used in virtual screening and be analyzed in a human-interpretable way that results in a set of rules for designing novel molecules.

Binding Sites↗

PubMed2005

3-O-acetyl-11-keto-boswellic acid decreases basal intracellular Ca2+ levels and inhibits agonist-induced Ca2+ mobilization and mitogen-activated protein kinase activation in human monocytic cells.

Previously, we showed that 11-keto-boswellic acid and 3-O-acetyl-11-keto-BA (AKBA) stimulate Ca(2+) mobilization and activate mitogen-activated protein kinases (MAPKs) in human polymorphonuclear leukocytes (PMNLs). Here, we addressed the effects of boswellic acids on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and on the activation of p38(MAPK) and extracellular signal-regulated kinase (ERK) in the human monocytic cell line Mono Mac (MM) 6. In contrast to PMNLs, AKBA concentration dependently (1-30 microM) decreased the basal [Ca(2+)](i) in resting MM6 cells but also in cells where [Ca(2+)](i) had been elevated by stimulation with platelet-activating factor (PAF). AKBA also strongly suppressed the subsequent elevation of [Ca(2+)](i) induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), PAF, or by the direct phospholipase C activator 2,4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide, but AKBA failed to prevent Ca(2+) signals induced by thapsigargin or ionomycin. Suppression of Ca(2+) homeostasis by AKBA was also observed in primary monocytes, isolated from human blood. Moreover, AKBA inhibited the activation of p38(MAPK) and ERKs in fMLP-stimulated MM6 cells. Although the effects of AKBA could be mimicked by the putative phospholipase C (PLC) inhibitor U-73122 (1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), AKBA appears to operate independent of PLC activity since the release of intracellular inositol-1,4,5-trisphosphate evoked by 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide was hardly diminished by AKBA. Inhibitor studies indicate that AKBA may decrease [Ca(2+)](i) by blocking store-operated Ca(2+) and/or nonselective cation channels. Together, AKBA interferes with pivotal signaling events in monocytic cells that are usually required for monocyte activation by proinflammatory stimuli. Interruption of these events may represent a possible mechanism underlying the reported anti-inflammatory properties of AKBA.

Anti-Inflammatory Agents↗

PubMed2005

Inhibitors of actin polymerisation stimulate arachidonic acid release and 5-lipoxygenase activation by upregulation of Ca2+ mobilisation in polymorphonuclear leukocytes involving Src family kinases.

Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 approximately 200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation.

Actins↗

PubMed2005

Caspase-mediated degradation of human 5-lipoxygenase in B lymphocytic cells.

5-Lipoxygenase (5-LO) is a tightly regulated enzyme in the synthesis of bioactive lipids from arachidonic acid. Here, we demonstrate that 5-LO is regulated by caspases, which are signaling molecules that control critical biological processes by means of specific limited proteolysis. Cell splitting of the Epstein-Barr virus-transformed B lymphocytic cell line BL41-E95-A caused a pronounced, but transient, reduction of functional 5-LO protein, accompanied by the appearance of a 62-kDa 5-LO cleavage product. In parallel, splitting of BL41-E95-A cells induced activation of caspase-6 (casp-6) and casp-8. Caspase activation and 5-LO degradation were blocked by the protein-synthesis inhibitor cycloheximide, and cell-permeable peptide inhibitors of casp-6 and casp-8 prevented 5-LO cleavage. Activation of casp-6 and casp-8 was connected to subsequent enhancement of cell proliferation, whereas selective caspase inhibition blocked cell growth. Last, isolated human 5-LO was cleaved by recombinant casp-6 in vitro to a 58-kDa fragment. Based on site-directed mutagenesis studies, 5-LO is cleaved by casp-6 after Asp-170, which in a homology-based 3D model of 5-LO is located on the enzyme periphery. We suggest that splitting of BL41-E95-A cells induces de novo synthesis of a protein involved in the activation of casp-6, which cleaves 5-LO.

Arachidonate 5-Lipoxygenase↗

PubMed2005

Development of 5-lipoxygenase inhibitors--lessons from cellular enzyme regulation.

5-Lipoxygenase (5-LO) catalyzes the first steps in the conversion of arachidonic acid (AA) into leukotrienes (LTs) that are mediators of inflammatory and allergic reactions. Recently, the 5-LO pathway has also been associated with atherosclerosis and osteoporosis. Thus, in addition to the classical applications including asthma and allergic disorders, LT synthesis inhibitors might be of interest for the treatment of cardiovascular diseases and osteoporosis. Recently, it has been shown that cellular 5-LO activity is regulated in a complex manner that can involve different signalling pathways. 5-LO can be activated by an increase in intracellular Ca2+ concentration, diacylglycerols, phosphorylation by MAPKAP kinase-2 and ERK. Previous work could demonstrate that cellular 5-LO activity is repressed in a protein kinase A-dependent manner and by glutathione peroxidases. This comment focuses on the impact of these stimulatory and inhibitory pathways on the efficacy of 5-LO inhibitors and suggests additional criteria for the development of this class of compounds.

Animals↗

PubMed2005

Identification of molecular targets of the oligomeric nonprenylated acylphloroglucinols from Myrtus communis and their implication as anti-inflammatory compounds.

Myrtucommulone (MC) and semimyrtucommulone (S-MC) are unique oligomeric, nonprenylated acylphloroglucinols contained in the leaves of myrtle (Myrtus communis). Although extracts of myrtle have been traditionally used in folk medicine for the treatment of various disorders, studies addressing select cellular or molecular pharmacological properties of these extracts or specific ingredients thereof are rare. Here, we show for the first time that MC and S-MC potently suppress the biosynthesis of eicosanoids by direct inhibiting cyclooxygenase-1 and 5-lipoxygenase in vitro and in vivo at IC50 values in the range of 1.8 to 29 microM. Moreover, we show that MC and S-MC prevent the mobilization of Ca2+ in polymorphonuclear leukocytes, mediated by G protein signaling pathways at IC50 values of 0.55 and 4.5 microM, respectively, and suppress the formation of reactive oxygen species and the release of elastase at comparable concentrations. The isobutyrophenone core of MC as well as S-MC was much less potent or even not active at all. In addition, MC or S-MC only partially inhibited peroxide formation or failed to block Ca2+ mobilization and elastase release when polymorphonuclear leukocytes were challenged with ionomycin that circumvents G protein signaling for cell activation. We conclude that, in view of their ability to suppress typical proinflammatory cellular responses, the unique acylphloroglucinols MC and S-MC from myrtle may possess an anti-inflammatory potential, suggesting their therapeutic use for the treatment of diseases related to inflammation and allergy.

Anti-Inflammatory Agents↗

PubMed2005

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.

Arachidonate 5-Lipoxygenase↗

PubMed2005

The aminosteroid phospholipase C antagonist U-73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) potently inhibits human 5-lipoxygenase in vivo and in vitro.

U-73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione) is a widely used antagonist of phosphoinositide-specific phospholipase C (PLC) and is frequently used to define a role of PLC in receptor-mediated elevation of intracellular calcium concentration ([Ca2+]i). In human polymorphonuclear leukocytes (PMNLs), U-73122 inhibited increases in [Ca2+]i induced by G protein-coupled receptor (GPCR) agonists (N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor; IC50 of approximately 2 to 4 microM), but it failed to suppress responses induced by ionomycin or thapsigargin. 5-Lipoxygenase (5-LO) is a Ca(2+)-regulated enzyme that can be activated in leukocytes by stimuli that elevate [Ca2+]i. Attempts to investigate the involvement of PLC in cellular 5-LO activation revealed that U-73122 suppresses 5-LO product synthesis regardless of the stimulus and independently of Ca2+. Thus, U-73122 blocked 5-LO product synthesis induced by cell stress, involving 5-LO phosphorylation pathways in the absence of Ca2+ with an IC50 of approximately 2 microM. Direct inhibition of 5-LO by U-73122 was evident in PMNL homogenates (IC50 of approximately 2.4 microM), and isolated human recombinant 5-LO enzyme was potently inhibited by U-73122 (IC50 of approximately 30 nM). Thiols (glutathione) strongly blunted the effect of U-73122 on isolated 5-LO. On the other hand, depletion of cellular thiols by N-ethylmaleimide strongly increased the efficacy of U-73122 to inhibit 5-LO in intact cells or corresponding homogenates, suggesting that U-73122 may interfere with sulfhydryl groups on 5-LO. Since 5-LO products induce increases in [Ca2+]i via GPCRs, caution should be used when interpreting data where U-73122 is used as tool to determine a direct role of PLC in receptor-mediated Ca2+ mobilization.

Arachidonate 5-Lipoxygenase↗

PubMed2005

Induction of central signalling pathways and select functional effects in human platelets by beta-boswellic acid.

We have recently shown that in polymorphonuclear leukocytes, 11-keto boswellic acids (KBAs) induce Ca2+ mobilisation and activation of mitogen-activated protein kinases (MAPK). Here we addressed the effects of BAs on central signalling pathways in human platelets and on various platelet functions. We found that beta-BA (10 microM), the 11-methylene analogue of KBA, caused a pronounced mobilisation of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal-regulated kinase (ERK)2, and Akt. These effects of beta-BA were concentration dependent, and the magnitude of the responses was comparable to those obtained after platelet stimulation with thrombin or collagen. Based on inhibitor studies, beta-BA triggers Ca2+ mobilisation via the phospholipase (PL)C/inositol-1,4,5-trisphosphate pathway, and involves Src family kinase signalling. Investigation of platelet functions revealed that beta-BA (> or =10 microM) strongly stimulates the platelet-induced generation of thrombin in an ex-vivo in-vitro model, the liberation of arachidonic acid (AA), and induces platelet aggregation in a Ca2+-dependent manner. In contrast to beta-BA, the 11-keto-BAs (KBA or AKBA) evoke only moderate Ca2+ mobilisation and activate p38 MAPK, but fail to induce phosphorylation of ERK2 or Akt, and do not cause aggregation or significant generation of thrombin. In summary, beta-BA potently induces Ca2+ mobilisation as well as the activation of pivotal protein kinases, and elicits functional platelet responses such as thrombin generation, liberation of AA, and aggregation.

Analysis of Variance↗