12-hydroxy-5-8-10-14-eicosatetraenoic-acid and esculetin

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Arachidonate Lipoxygenases; Arachidonic Acid; Carcinoma, Squamous Cell; Cell Proliferation; Flavanones; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Nitrobenzenes; Salicylamides; Sulfonamides; Tumor Cells, Cultured; Umbelliferones

Bovine aortic endothelial and smooth-muscle cells down-regulate the rate of glucose transport in the face of hyperglycaemia, thus providing protection against deleterious effects of increased intracellular glucose levels. When exposed to high glucose concentrations these cells reduced the mRNA and protein content of their typical glucose transporter, GLUT-1, as well as its plasma-membrane abundance. Inhibition of the lipoxygenase (LO) pathway, and particularly 12-LO, reversed this glucose-induced down-regulatory process and restored the rate of hexose transport to the level seen in vascular cells exposed to normal glucose levels. This reversal was accompanied by increased levels of GLUT-1 mRNA and protein, as well as of its plasma-membrane content. Exposure of the vascular cells to elevated glucose concentrations increased by 2-3-fold the levels of cell-associated and secreted 12-hydroxyeicosatetraenoic acid (12-HETE), the product of 12-LO. Inhibition of 15- and 5-LO, cyclo-oxygenases 1 and 2, and eicosanoid-producing cytochrome P450 did not modify the hexose-transport system in vascular cells. These results suggest a role for HETEs in the autoregulation of hexose transport in vascular cells. 8-Iso prostaglandin F(2alpha), a non-enzymic oxidation product of arachidonic acid, had no effect on the hexose-transport system in vascular cells exposed to hyperglycaemic conditions. Taken together, these findings show that hyperglycaemia increases the production rate of 12-HETE, which in turn mediates the down-regulation of GLUT-1 expression and the glucose-transport system in vascular endothelial and smooth-muscle cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antioxidants; Biological Transport; Cattle; Cell Membrane; Cells, Cultured; Endothelium, Vascular; Gene Expression Regulation; Glucose; Glucose Transporter Type 1; Hyperglycemia; Kinetics; Monosaccharide Transport Proteins; Muscle, Smooth, Vascular; Umbelliferones

We previously found that the inhibition of lipoxygenases resulted in delayed epithelial wound closure in organ-cultured rat corneas. The present study was undertaken to determine the lipoxygenase enzyme and metabolite(s) responsible for regulating reepithelialization and their mechanism of action.. The effects of esculetin--an established lipoxygenase inhibitor--on endogenous hydroxyeicosatetraenoic acids (HETEs) production, epithelial wound closure, filamentous-actin (F-actin) cytoskeleton, and mitotic rate were investigated using a cell-culture assay and an organ-culture assay of rat corneal epithelium.. Lipoxygenase inhibition by esculetin, which resulted in the disruption of F-actin organization and a decrease in the mitotic rate, delayed wound closure in both cell- and organ-culture assays. Normal corneoscleral rims metabolized [3H]arachidonic acid to 12-HETE (major metabolite), 8-HETE, and 9-HETE. HETE synthesis was inhibited by esculetin in a dose-dependent fashion. Chiral-phase analysis revealed that they contained only (S)-enantiomers, which indicated that they were lipoxygenase metabolites. The inhibitory effects of esculetin on F-actin organization and epithelial wound closure in an organ-culture assay were totally reversed by exogenously added 8(S)-HETE, whereas 12- and 9-HETE had no effect. However, none of the HETEs reversed the decreased mitotic rate or achieved complete wound closure in the cell-culture assay.. These results suggest that 8(S)-HETE is the key metabolite of arachidonic acid that regulates corneal epithelial cell migration during wound healing. The metabolite responsible for cell proliferation remains to be determined.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actins; Animals; Arachidonic Acid; Cell Movement; Cells, Cultured; Cornea; Dose-Response Relationship, Drug; Epithelial Cells; Female; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Mitotic Index; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Umbelliferones; Wound Healing

Diets rich in linoleic acid (LA) stimulate the metastasis of MDA-MB-435 human breast cancer cells from the mammary fat pads of nude mice. This omega-6 fatty acid is metabolized to various cyclo-oxygenase and lipoxygenase products, several of which have been previously associated with tumor cell invasion and metastasis. We now report that MDA-MB-435 cells secreted increased levels of prostaglandin E2 (PGE2), and 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE when cultured in the presence of 2.7 microM (0.75 micrograms/ml) LA; 5-HETE secretion was unchanged. The 12-lipoxygenase inhibitor esculetin (20 microM) completely blocked the LA-stimulated 12-HETE secretion. Linoleic acid also increased MDA-MB-435 cell invasion in an in vitro assay; this stimulation was abolished by 20 microM esculetin, but was unaffected by piroxicam, a selective cyclooxygenase inhibitor. The effect of LA on invasion was replicated by 0.1 microM 12-HETE, but not by 5-HETE or PGE2; 15-HETE was stimulatory only at a concentration of 1.0 microM. Zymographic and Northern blot analyses showed that these events are accompanied by the induction of 92 kDa isoform type IV collagenase (metalloproteinase-9) enzymic activity and mRNA expression by exogenous LA and 12-HETE, and their suppression by the 12-lipoxygenase inhibitor. These results suggest that the effects of dietary LA on breast cancer cell metastasis in the nude mouse model are due, at least in part, to enhanced 12-HETE biosynthesis, with an associated increase in proteolytic enzyme activity and tumor cell invasiveness.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Breast Neoplasms; Collagenases; Eicosanoids; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase Inhibitors; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Piroxicam; RNA, Messenger; Tumor Cells, Cultured; Umbelliferones

Recent evidence suggests that lipoxygenase (LO) metabolites inhibit renin production in vitro. However, the physiological significance of this effect has not been determined. This study examined the role of the LO pathway in the regulation of plasma renin concentration (PRC) in vivo. The acute administration of two structurally unrelated LO inhibitors, phenidone (30 and 60 mg/kg) and esculetin (60 mg/kg), resulted in suppression of platelet 12 hydroxyeicosatetraenoic acid (12HETE) production, reduction in systemic arterial pressure and a 2- to 3-fold increase in PRC. To determine whether the esculetin-induced increase in PRC was secondary to hypotension, esculetin was also administered to rats preinfused with a pressor dose of norepinephrine. In these acutely hypertensive rats, esculetin still induced a 2.5-fold increase in PRC, whereas blood pressure remained over 40 mm Hg above basal levels. Further, esculetin (10(-6)M) increased renin release in renal slices from 150 +/- 10 to 310 +/- 20 ng/ml.h (P < 0.05) and this rise was entirely blocked in the presence of 12HETE (10(-7)M; 130 +/- 40 ng/ml.h). In rats placed on high salt intake, 12HETE concentration in renal slices from the outer cortex was considerably higher than in renal slices from salt-restricted rats (116.5 +/- 15.7 vs. 65 +/- 12 pg/mg protein; P < 0.05). Chronic administration of the LO inhibitor phenidone also resulted in an increase of PRC, which was independent of changes in blood pressure. On either high salt (3.15%0 or low salt (0.05%) diet phenidone-treated rats had higher PRC levels than the respective control groups [high salt 9.7 +/- 3.5 vs. 1.9 +/- 1.4 ng/ml.h; P < 0.05; low salt 33.2 +/- 5.3 vs. 19.4 +/- 3.10 ng/ml.h; P < 0.05]. The finding that LO blockers are potent stimulators of PRC in vivo suggests the existence of a physiological tonic inhibition of renin secretion by LO products that is operative under a wide range of salt intake. High salt intake enhances this inhibitory tone by increasing renal cortical 12 LO activity and, in fact, normal suppression of PRC during high salt diet does not occur in LO-blocked animals. Thus, the LO pathway exerts a tonic inhibitory effect on renin release, which appears particularly important for renin suppression during high salt intake.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Blood Platelets; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Kidney; Lipoxygenase Inhibitors; Male; Norepinephrine; Pyrazoles; Rats; Rats, Sprague-Dawley; Renin; Sodium, Dietary; Umbelliferones

The eicosanoid generating potential of the brain, gills, skin, ovary, muscle, eye, liver, spleen, heart, and alimentary canal in the rainbow trout, Oncorhynchus mykiss, was examined. All the organs/tissues examined synthesized the 12-lipoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), and 12-hydroxyeicosapentaenoic acid (12-HEPE), implying the widespread nature of this enzyme in trout. Both prostaglandin E and LTC were also found in variable amounts in the organs, with the greatest amount of PGE found in the gill. Leukotriene (LT) B4 and LTB5 were found in supernatants from calcium ionophore-challenged brain, skin, ovary, liver, spleen, and heart, but the lipoxins A4 and A5 were only present in brain, ovary, and spleen in relatively small amounts. As lipoxins have previously been shown to be synthesized by macrophages in rainbow trout [Pettitt et al., J. Biol. Chem. 266, 8720-8726 (1991)], and related cells (microglial cells) are found in the brain of mammals, the localization of macrophage-like cells in trout brain was investigated immunocytochemically. Monoclonal antibodies specific for trout leucocytes failed to identify any microglial-like cells in sections of the brain, although microvessels containing immuno-positive reaction products were observed. A number of distinct lipoxygenase products were found in supernatants of ionophore-challenged gill, including 14-hydroxydocosahexaenoic acid, 12-HETE, and 12-HEPE, and a large number of dihydroxy fatty acid derivatives with conjugated triene chromophores. One of these products was tentatively identified as 8(R),15(S)-dihydroxyeicosatetraenoic acid, a dual 12- and 15-lipoxygenase product, but apparently no LTB4 was generated by this tissue.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Brain; Captopril; Eicosanoids; Eicosapentaenoic Acid; Fatty Acids; Female; Gills; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene C4; Masoprocol; Oncorhynchus mykiss; Prostaglandins E; Tissue Distribution; Umbelliferones

The role of endogenously produced arachidonic acid metabolites on glomerulonephritis was investigated using cultured rat mesangial cells. The cultured mesangial cells could produce prostaglandin (PG) E2 and F2 alpha and 12-hydroxyeicosatetraenoic acid (12-HETE). The treatment of the mesangial cells with indomethacin enhanced the cell growth stimulated by 10% fetal calf serum (FCS). This stimulatory effect was significantly attenuated by concomitant treatment with PGE2, but not with PGF2 alpha. To test whether the mechanism by which PGE2 inhibited the mesangial cell growth is related to in the increment of cyclic AMP (cAMP), we examined the effect of dibutyryl cAMP on mesangial cell growth. As expected, treatment with dibutyryl cAMP decreased the cell proliferation. Moreover treatment with KT-5720, a protein kinase A (PKA) inhibitor, stimulated the cell growth as well as indomethacin. These data strongly suggest that the inhibition of mesangial cell growth by PGE2 involves an activation of PKA. In contrast, treatment with baicalein, a specific inhibitor of 12-lipoxygenase, inhibited the mesangial cell growth. The up and down regulations by arachidonic acid metabolites were also observed in the growth induced by platelet-derived growth factor (PDGF). These results suggest that endogenously produced arachidonic acid metabolites are involved in the regulation of mesangial cell growth.

Topics: 1-Methyl-3-isobutylxanthine; 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Benzeneacetamides; Bucladesine; Carbazoles; Cell Division; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dinoprost; Dinoprostone; DNA Replication; Flavanones; Flavonoids; Glomerular Mesangium; Hydroxamic Acids; Hydroxyeicosatetraenoic Acids; Indoles; Indomethacin; Lipoxygenase Inhibitors; Male; Platelet-Derived Growth Factor; Pyrroles; Rats; Rats, Sprague-Dawley; Umbelliferones

Microsomes of bovine corneal epithelium metabolized [14C]arachidonic acid extensively to [14C]12-hydroxyeicosatetraenoic acid. The biosynthesis was not stimulated by exogenous NADPH and it could be inhibited by over 90% with two lipoxygenase inhibitors, esculetin and eicosatetraynoic acid. Chiral phase high performance liquid chromatography showed that 12-HETE consisted of the 12S enantiomer. The results indicate biosynthesis by 12S-lipoxygenase of bovine corneal epithelium rather than by cytochrome P450.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Cattle; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Endothelium, Corneal; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxygenase; Lipoxygenase Inhibitors; Microsomes; Molecular Conformation; NADP; Umbelliferones

Arachidonic acid metabolites have been shown to exert a variety of regulatory effects on cellular activation and proliferation. Recently, a role for these products as regulators of hematopoiesis was suggested and evidence provided that products of the lipoxygenase pathway, specifically leukotrienes, are essential for human myeloid colony formation in vitro. In this report the broader role of these metabolites in hematopoiesis was examined using murine bone marrow stem cell assays for both myeloid and lymphoid cell lines. The effects of lipoxygenase and/or cyclooxygenase pathway inhibitors on stem cell colony formation were evaluated and compared to qualitative and quantitative changes in arachidonic acid metabolism that occurred in similarly treated bone marrow cell cultures. Interruption of the lipoxygenase pathway by esculetin or nordihydroguaiaretic acid resulted in decreased colony formation in both lymphoid and myeloid stem cells. This inhibition of colony growth was partly reversed by the addition of leukotrienes and was particularly evident in B-cell progenitor cultures to which was added LTB4. Inhibition of the cyclooxygenase pathway by indomethacin or ibuprofen had a slight stimulatory effect on myeloid colony formation, while slightly inhibiting the formation of lymphoid colonies. These results support a direct role for lipoxygenase products in myeloid colony formation and lymphoid stem cell proliferation. A more complex role for cyclooxygenase metabolites in the hematopoietic process appears probable.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; B-Lymphocytes; Cyclooxygenase Inhibitors; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Hydroxyeicosatetraenoic Acids; Ibuprofen; In Vitro Techniques; Indomethacin; Leukotrienes; Lipoxygenase Inhibitors; Macrophages; Masoprocol; Mice; Prostaglandins; Thromboxane B2; Umbelliferones