Biomedical subjects

Dieter Steinhilber

Publications and source records attributed to Dieter Steinhilber.

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

The coding sequence mediates induction of 5-lipoxygenase expression by Smads3/4.

5-Lipoxygenase (5-LO) expression is strongly induced by transforming growth factor-beta (TGFbeta) and 1alpha,25-dihydroxyvitamin D(3) in Mono Mac 6 cells. Since Smads have been described as downstream effectors of TGFbeta, we have investigated the role of the TGFbeta/Smad signalling system in the regulation of 5-LO gene expression. The rapid induction of 5-LO mRNA, determined with real-time quantitative RT-PCR, suggests that 5-LO is a primary TGFbeta target gene. In reporter gene assays with plasmids containing the 5-LO promoter plus different parts of the gene, Smads3/4 mediate a prominent upregulation of reporter activity that strongly depends on the coding sequence and to a lesser extent on the 3'-UTR and introns J-M. Deletion studies revealed the most profound decrease of inducibility by Smads3/4 when exons 10-14 are deleted. Sequence analysis and deletion studies indicate the existence of up to four Smad binding elements and at least one TGFbeta responsive element far downstream of the transcriptional start site.

Arachidonate 5-Lipoxygenase↗

PubMed2006

Peroxisome proliferator-activated receptor alpha (PPAR alpha) down-regulation in cystic fibrosis lymphocytes.

BACKGROUND: PPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response. METHODS: PPARalpha, beta and gamma mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARalpha protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARalpha was analyzed by gel shift assay. RESULTS: In lymphocytes, the expression of PPARalpha mRNA, but not of PPARbeta, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARalpha was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARalpha and PPARbeta mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARgamma mRNA levels were below the detection limit. CONCLUSION: Lymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARalpha may therefore contribute to the inflammatory processes that are observed in CF.

Adult↗

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

Intracellular localization of RORalpha is isoform and cell line-dependent.

The retinoid-related orphan receptor alpha (RORalpha) belongs to the nuclear receptor superfamily and comprises four isoforms generated by different promotor usage and alternative splicing. To better understand its function, the subcellular distribution of RORalpha was investigated. We could show that subcellular distribution of RORalpha is cell line and isoform-dependent. Isoform specific differences were mediated by the A/B domains which with the exception of RORalpha1 contain a signal that mediates cytoplasmic localization. The lack of this signal in RORalpha1 results in a complete nuclear localization and prevents cell membrane association observed for RORalpha2, 3, and 4. The region responsible for membrane association was identified as the C-terminal alpha-helix 12. Furthermore, the hinge region/ligand binding domain mediates nuclear localization. Our results show that isoform specific activity of RORalpha is not only regulated by different expression and DNA binding affinities but also by different subcellular distribution. Different access to the nucleus reveals an important mechanism regulating the activity of this constitutively active nuclear receptor.

Base Sequence↗

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

Overexpression, refolding, and purification of polyhistidine-tagged human retinoic acid related orphan receptor RORalpha4.

RORalpha4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORalpha4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6M guanidinium-HCl and renatured by gradual removal of guanidinium-HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20mg of pure and native RORalpha4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORalpha in mice with high sensitivity and specificity.

Animals↗

PubMed2006

Synthesis of thieno[2,3-b]pyridinones acting as cytoprotectants and as inhibitors of [3H]glycine binding to the N-methyl-D-aspartate (NMDA) receptor.

The standard glycine site antagonist of the N-methyl-D-aspartate (NMDA) receptor, 3-phenyl-4-hydroxyquinolin-2(1H)-one (21), was used as a template for bioisostere benzene/thiophene exchange. Phenylacetylation of aminothiophene carboxylic acid methyl esters and subsequent cyclization delivered the three possible thienopyridinone isomers. 4-Hydroxy-5-phenylthieno[2,3-b]pyridin-6(7H)-one (3a), with the shortest distance between the sulfur and the nitrogen atom, was the most potent isomer (K(i) against the binding of [(3)H]glycine to rat membranes 16 microM), comparable in potency to the model quinolinone (21, 12 microM). Replacement of the phenyl substituent of 21 by a 2-thienyl residue resulted in a 2-5-fold loss in potency and was abandoned. In the thieno part of the thienopyridinone nucleus, the most successful substituents were halogen (Cl or Br) close to the sulfur atom and short alkyl chains at the other position, resulting in 7h, 8h, 8i, and 8m, with K(i) values between 5.8 and 10.5 nM. Introduction of a 3'-phenoxy moiety yielded several compounds with still higher potencies (18h, 18i, 18l, and 18m; K(i) between 1.1 and 2.0 nM). Quantitative structure-activity relationship (QSAR) calculations resulted in a consistent interpretation of the potencies of most compounds. Several of these 3'-phenoxy derivatives protected mouse fibroblast cell lines with transfected NMDA receptors from glutamate-induced toxicity. In addition, we report in vivo results for four of these compounds.

Animals↗

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↗

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

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

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

Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

Alkynes↗

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↗