leukotriene-b4 and 1-oleoyl-2-acetylglycerol

We previously showed that, in vitro, hyperforin from St. John's wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B(4) formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.

Topics: Animals; Arachidonate 5-Lipoxygenase; Binding Sites; Bridged Bicyclo Compounds; Carrageenan; Cells, Cultured; Diglycerides; Humans; Hypericum; Leukotriene B4; Lipoxygenase Inhibitors; Male; MAP Kinase Signaling System; Microfilament Proteins; Neutrophils; Oxidation-Reduction; Phloroglucinol; Phospholipids; Pleurisy; Protein Structure, Tertiary; Protein Transport; Rats; Rats, Wistar; Terpenes; Tryptophan

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Arachidonic Acid; Biological Transport; Calcimycin; Diglycerides; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene Antagonists; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Tetradecanoylphorbol Acetate

Dispersed rat gastric mucosal cells (F0) were separated into five fractions (F1-F5) by counterflow elutriation, with F1 representing the smallest and F5 the largest cell diameters. In F0-F5, leukotriene B4 (LTB4) release in response to 10(-5) M calcium ionophore A-23187 was 7.68 +/- 1.26, 51.6 +/- 10.8, 72.4 +/- 10.4, 7.1 +/- 0.7, 5.7 +/- 0.6, and 11.6 +/- 3.4 pg.10(6) cells-1 x 30 min-1. In the identical fractions, sulfidopeptide release in response to A-23187 was 200.6 +/- 20.5, 1,116.0 +/- 166.6, 1,309.4 +/- 163.2, 189.8 +/- 25.8, 108.0 +/- 18.0, and 158.4 +/- 54.0 pg.10(6) cells-1 x 30 min-1. High-pressure liquid chromatography verified the radioimmunologically determined LTB4 and identified LTC4 as the only sulfidopeptide LT released. LT release from F2 cells in response to A-23187 was time and dose dependent, reaching maximal stimulation at 10(-5) M A-23187. This response was blocked by the dual inhibitor of cyclooxygenase and lipoxygenases, BW755C (2 x 10(-5)-2 x 10(-4) M), by the selective 5-lipoxygenase inhibitor L-651,392 (10(-7)-10(-5) M), and by MK-886 (10(-9)-10(-7) M), which blocks translocation of 5-lipoxygenase. The postreceptor stimuli dibutyryl adenosine 3',5'-cyclic monophosphate, forskolin, 12-O-tetradecanoyl-phorbol-13-acetate, and oleyl-acetyl-glycerol failed to induce LT release. However, 10(-4) M arachidonic acid increased basal LT release up to eightfold and increased A-23187-stimulated LT release by an additional 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Arachidonic Acid; Bucladesine; Calcimycin; Cell Separation; Colforsin; Diglycerides; Female; Gastric Mucosa; Histamine; In Vitro Techniques; Interleukin-1; Interleukin-2; Kinetics; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; N-Formylmethionine Leucyl-Phenylalanine; Pentagastrin; Phenothiazines; Platelet Activating Factor; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate

We investigated the regulation of the adhesiveness of the human promonocytic cell line U-937, differentiated along the monocytic pathway either by 1,25-(OH)2-cholecalciferol or a combination of retinoic acid and dibutyryl cAMP. Adhesion to untreated polystyrene plastic was induced by inflammatory agents like PAF, fMLP or LTB4. The response to PAF first appeared after 48hr of differentiation and was inhibited by PAF antagonists and protein kinase C inhibitors indicating involvement of the phosphatidyl-inositol pathway in the stimulating effect. On the other hand, all the c-AMP raising agents tested inhibited PAF-induced cell adhesion, whatever their target membrane receptors, the Gs transducing protein, the catalytic unit of adenylate cyclase or cAMP phosphodiesterase. Direct stimulation of protein kinase A by Br8-cAMP had a similar effect. Moreover, PAF was able to increase cAMP levels. This suggests the existence of a cAMP based negative control mechanism limiting the action of PAF.

Topics: 2-Chloroadenosine; 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Alprostadil; Bucladesine; Calcitriol; Cell Adhesion; Cell Differentiation; Cell Line; Cyclic AMP; Diglycerides; Humans; Kinetics; Leukotriene B4; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Platelet Activating Factor; Tretinoin

Membrane IL-1 (mIL-1) expression was compared with release of soluble IL-1 (sIL-1) by C3H/HeNCrl mouse peritoneal macrophages. Selective antagonists of protein kinase C (PKC) (H-7) and calmodulin (W-7) in combination inhibited LPS-induced mIL-1 and sIL-1 production, suggesting a role for these activation pathways in IL-1 induction. Low levels of A23187, when combined with OAG (a direct activator of PKC), stimulated mIL-1 expression in the absence of sIL-1 release. Induction of mIL-1 by LPS was inhibited by PGE2 and dibutyryl cAMP, but higher concentrations were required to inhibit mIL-1 expression compared with sIL-1 release. LTB4 alone did not induce mIL-1 or sIL-1 production. LTB4 did enhance LPS-induced mIL-1 expression but not sIL-1 release. These results indicate that mIL-1 expression and sIL-1 release are differentially regulated. Membrane IL-1 is induced by lower levels of certain stimuli and is less effectively inhibited than is sIL-1 release. This differential regulation is further evidence to support the existance of membrane IL-1.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antibodies; Calcimycin; Calmodulin; Cell Membrane; Diglycerides; Dinoprostone; In Vitro Techniques; Interleukin-1; Isoquinolines; L-Lactate Dehydrogenase; Leukotriene B4; Macrophages; Mice; Mice, Inbred C3H; Nucleotides, Cyclic; Piperazines; Protein Kinase C; Solubility; Sulfonamides; Thymus Gland

We have developed an improved method to study the directed migration, or chemotaxis, of rat peripheral blood large granular lymphocytes (LGL) in vitro. A modified Boyden chamber technique was used to measure chemotaxis of LGL through polycarbonate filters that had been coated with different basement membrane components. LGL were found to adhere to collagen types I and IV, laminin and fibronectin. However, only collagen type IV was not in itself chemotactic for LGL. Migrated cells could be identified both morphologically and phenotypically as LGL on collagen type IV-coated filters after incubation with a chemotactic stimulus. LGL were found to display chemotaxis to a number of different stimuli, including the classical chemoattractant agents N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and complement fragments present in activated sera. However, the degree of response to these stimuli was much less than that of isolated peripheral blood neutrophils or monocytes. In contrast, all three cell types showed increased chemotaxis to the diacyl glycerol analog 1-oleoyl 2-acetyl glycerol (OAG), which induced a 4-14 fold stimulation of migration. Induction of chemotaxis of LGL by OAG was time and dose-dependent, as confirmed using checkerboard assays. In summary, we have developed a rapid, quantitative method to measure chemotaxis of LGL in vitro. This technique may now be utilized to identify naturally occurring chemoattractants for LGL and to study the intracellular and regulatory events associated with LGL migration.

Topics: Animals; Cell Adhesion; Cell Movement; Chemotaxis, Leukocyte; Collagen; Diglycerides; Fibronectins; Immunoenzyme Techniques; Immunophenotyping; In Vitro Techniques; Killer Cells, Natural; Laminin; Leukotriene B4; Lymphocyte Subsets; Lymphocytes; Male; N-Formylmethionine Leucyl-Phenylalanine; Rats; Rats, Inbred F344

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cytochalasin B; Diglycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2