calcimycin and 13-hydroxy-9-11-octadecadienoic-acid

We examined the mechanisms underlying the activation of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) contributing to the supply of fatty acids required for the formation of cholesteryl ester in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages. The possible involvement of oxidized lipids was also examined. In [(3)H]arachidonic acid-labeled mouse peritoneal macrophages, oxLDL stimulated the release of arachidonic acid, which was suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA(2)alpha inhibitor. oxLDL induced an increase in PLA(2)alpha levels in the membrane fraction without affecting those in whole cells or the activity in the lysate. Among 13-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, and 25-hydroxycholesterol, oxidized lipids present in oxLDL particles, only 13-HODE induced the release of arachidonic acid, which was also sensitive to MAFP. Under conditions where addition of Ca(2+) to the cell lysate induced an increase in cPLA(2)alpha protein in the membrane fraction, preincubation with 13-HODE facilitated the Ca(2+)-dependent translocation of cPLA(2)alpha. Furthermore, 13-HODE increased cholesteryl ester formation in the presence of [(3)H]cholesterol. These results suggest that 13-HODE mediates the oxLDL-induced activation of cPLA(2)alpha through an increase in cPLA(2)alpha protein in the membranes, thus contributing, in part, to the supply of fatty acids required for the esterification of cholesterol in macrophages.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cell Membrane; Cells, Cultured; Cholesterol; Cholesterol Esters; Cytosol; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Ionophores; Linoleic Acids; Lipoproteins, LDL; Macrophages, Peritoneal; Mice; Organophosphonates; Oxidation-Reduction; Phospholipases A; Phospholipases A2; Protein Transport

We have investigated lipid peroxidation in the skin of CD1 mice following single or repeated topical applications of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). A substantial accumulation of hydroxyphospholipids, to levels 3-5 times control values, followed exposure to two or more TPA treatments (24-72 h intervals), whereas single applications were ineffective. Sodium borohydride reduction increased the yield of product by approximately 50%, suggesting the additional presence of phospholipid hydroperoxides in the oxidized lipids. Straight phase HPLC analysis of the constituent hydroxy fatty acids, followed by gas chromatography/mass spectrometry, revealed that oxidized derivatives of linoleic acid, including 9- and 13-hydroxyoctadecadienoic acids (9- and 13-HODE), were the primary products. Stereochemical analysis showed ratios of S to R stereoisomers of 1.3 for 13-HODE and 1.27 for 9-HODE, which implied that TPA-induced peroxidation was primarily due to free radical oxidation, although a partial contribution of enzyme (lipoxygenase) activity is possible. The TPA-induced peroxidation was greater in the epidermis than in the dermis. Pre-exposure of mouse skin to the anti-inflammatory agent fluocinolone acetonide, antioxidants and enzyme (phospholipase A2 and lipoxygenase) inhibitors lowered the peroxidation response to subsequent exposure to TPA. Phospholipid peroxidation products may be useful markers of oxygen radical production in TPA-exposed mouse skin with possible relevance to tumor promotion.

Topics: Aminobenzoates; Animals; Antioxidants; Borohydrides; Calcimycin; Chlorobenzoates; Chromatography, High Pressure Liquid; Cinnamates; Epidermis; Female; Fluocinolone Acetonide; Free Radicals; Gas Chromatography-Mass Spectrometry; Hydrolysis; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxidation; Lipoxygenase Inhibitors; Masoprocol; Mice; Mice, Inbred SENCAR; ortho-Aminobenzoates; Phospholipases A; Phospholipases A2; Phospholipids; Pregnatrienes; Stereoisomerism; Tetradecanoylphorbol Acetate; Thiobarbituric Acid Reactive Substances

Antibodies against 13-hydroxyoctadecadienoic acid (13-HODE) were produced in rabbits by immunizing the animal with 13-HODE-thyroglobulin conjugate. The antibodies appeared to be rather specific for 13-HODE since other hydroxy fatty acids showed minimal crossreaction. The radioimmunoassay was capable of detecting 50 pg per assay tube and was applied to the study of the biosynthesis of 13-HODE in platelets and leukocytes. In contrast to reported findings from endothelial cells, A-23187, thrombin and collagen stimulated synthesis and release of 13-HODE from platelets. However, insignificant synthesis of 13-HODE was found in leukocytes following A-23187 stimulation. Exogenous addition of linoleic acid stimulated the synthesis of 13-HODE from both platelets and leukocytes. The majority of 13-HODE synthesized was found in the medium. These studies suggest that both types of blood cells possess active (omega-6) lipoxygenase. Platelets may use endogenously released linoleic acid to synthesize 13-HODE, whereas leukocytes may utilize linoleic acid released from other cell types for 13-HODE synthesis.

Topics: Animals; Antibodies; Antibody Specificity; Antigens; Antithrombins; Blood Platelets; Calcimycin; Collagen; Leukocytes; Linoleic Acid; Linoleic Acids; Rabbits; Radioimmunoassay; Thrombin

Mammalian tissues contain 5-, 12- and 15-lipoxygenases. Only the 15-lipoxygenase can act on linoleic acid, the predominant essential fatty acid of tissues and plasma, producing 13-hydroxyoctadecadienoic acid (13-HODE). Intracellular production of 13-HODE renders endothelial cells resistant to platelet adhesion, while its hydroperoxy precursor, 13-HPODE, synergises with the platelet anti-aggregatory factor prostacyclin. We have found that a 15-lipoxygenase activity is induced in aortas of cholesterol-fed and Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Aortic tissue from WHHL rabbits incubated with 3H-linoleic acid produced a major metabolite identified as 13-HODE, which was formed with an efficiency comparable to the synthesis 15-HETE from arachidonic acid. These findings indicate that the increased aortic 15-lipoxygenase in vascular tissue is capable of producing 13-HODE in vivo. Since platelet adhesion is increased in atherogenesis, and thrombogenesis is a major complication of advanced atherosclerosis, it is suggested that induction of this enzyme may be a protective response to hypercholesterolemia.

Topics: Animals; Antithrombins; Aorta; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Arteriosclerosis; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Linoleic Acids; Rabbits

Reticulocytes from various species (rat, mouse, rhesus monkey) obtained by phenylhydrazine treatment of the animals metabolized polyenoic fatty acids via a lipoxygenase pathway. Linoleic acid was converted to 13-hydro(pero)xy-9,11(Z,E)octadecadienoic acid [13-H(P)ODE] and 9-hydro(pero)xy-10,12(E,Z)octadecadienoic acid [9-H(P)ODE], whereas arachidonic acid was oxygenated to 15-hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid (15-HETE) as shown by straight-phase high-pressure liquid chromatography (SP-HPLC). Addition of calcium and ionophore A 23,187 strongly enhanced the formation of lipoxygenase products, whereas 5,8,11,14eicosatetraenoic acid (ETYA) completely inhibited their formation. Estimates of the specific radioactivities of the lipoxygenase products indicate differences in the metabolization of externally added and endogenously released polyenoic fatty acids. These results strongly suggest that lipoxygenases generally occur in immature red blood cells.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Macaca; Mice; Phenylhydrazines; Rats; Reticulocytes; Species Specificity

Leukotriene B4 (LTB4) release by calcium ionophore-stimulated human leucocytes was measured by use of selective solvent partition of reaction mixtures and an agarose microdroplet chemokinesis assay, and the inhibitory effects of four monohydroxy fatty acids were determined. 15-Hydroxy-eicosatetraenoic acid (15-HETE) was the most effective inhibitor of LTB4 production with an approximate IC50 value of 6 microM and 99% inhibition at 50 microM, whereas 13-hydroxy-octadecadienoic acid (13-HODD) and 12-HETE were weaker inhibitors with approximate IC50 values of 32 microM and 23 microM, and 59% and 68% inhibition at 50 microM, respectively. We suggest that 13-HODD and 12-HETE, which are present in large amounts in the lesions of the skin disease psoriasis, may act as endogenous modulators of 5-lipoxygenase activity in skin.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Skin; Time Factors