12-hydroxy-5-8-10-14-eicosatetraenoic-acid and 1-oleoyl-2-acetylglycerol

The present study was conducted to determine whether protein kinase C was involved in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid (12-HETE) from bovine adrenal glomerulosa cells. Activators of protein kinase C, 12-O-tetradecanoylphorbol 4-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG), significantly increased release of 12-HETE. The effect of OAG was potentiated by BAYK8644, a stimulator of calcium entry. Sphingosine, H-7 and staurosporine, which inhibited the activity of protein kinase C in vitro, almost completely blocked 12-HETE release induced by TPA. These agents also significantly reduced angiotensin II-mediated 12-HETE release. When time course of the liberation of 12-HETE was measured, angiotensin II elicited sustained release of 12-HETE, which was inhibited by staurosporine. These results indicate that angiotensin II induces sustained release of 12-HETE, a feed forward regulator of aldosterone secretion, and that protein kinase C may be involved in this process.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Alkaloids; Angiotensin II; Animals; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Diglycerides; Hydroxyeicosatetraenoic Acids; Isoquinolines; Piperazines; Protein Kinase C; Radioimmunoassay; Sphingosine; Staurosporine; Tetradecanoylphorbol Acetate; Time Factors; Zona Glomerulosa

Modulation of renin synthesis by lipoxygenase products has been studied in cultured human mesangial cells under basal conditions and in the presence of prostaglandin (PG) E2. Total renin and cyclic AMP productions were stimulated in a dose-dependent manner (0.1-10 microM) by PGE2. The stimulatory effect of PGE2 on renin production was inhibited by 12-hydroxyeicosatetraenoic acid (12-HETE) between 0.1 and 100 nM. Extracellular and intracellular renin were affected similarly. Neither basal and PGE2-dependent cyclic AMP nor basal cyclic GMP productions were modified. 15-Hydroxyeicosatetraenoic acid (15-HPETE), 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) had the same effects as 12-HETE. Intracellular calcium concentration was not modified in the presence of 12-HETE. Since oleyl-2-acetylglycerol (OAG), an analog of diacylglycerol, also inhibited PGE2-stimulated renin production, it is hypothesized that the effect of the lipoxygenase products is mediated via protein kinase C stimulation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Angiotensin II; Calcium; Cyclic AMP; Cyclic GMP; Diglycerides; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Cortex; Leukotrienes; Lipid Peroxides; Lipoxygenase; Renin

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cattle; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Diacylglycerol Kinase; Diglycerides; Dose-Response Relationship, Drug; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Indomethacin; Mitosis; Phosphotransferases; Thymidine

Washed human platelets prelabeled with [14C]arachidonic acid and then exposed to the Ca2+ ionophore A23187 mobilized [14C]arachidonic acid from phospholipids and formed 14C-labeled thromboxane B2, 12-hydroxy-5-8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Addition of phorbol myristate acetate (PMA) by itself at concentrations from 10 to 1000 ng/ml did not release arachidonic acid or cause the formation of any of its metabolites, nor did it affect the metabolism of exogenously added arachidonic acid. When 1 microM A23187 was added to platelets pretreated with 100 ng of PMA/ml for 10 min, the release of arachidonic acid, and the amount of all arachidonic acid metabolites formed, were greatly increased (average 4.1 +/- 0.5-fold in eight experiments). This effect of PMA was mimicked by other stimulators of protein kinase C, such as phorbol dibutyrate and oleoyl acetoyl glycerol, but not by 4-alpha-phorbol 12,13-didecanoate, which does not stimulate protein kinase C. However, phosphorylation of the cytosolic 47-kDa protein, the major substrate for protein kinase C in platelets, was produced at lower concentrations of PMA and at a much higher rate than enhancement of arachidonic acid release by PMA, suggesting that 47-kDa protein phosphorylation is not directly involved in mobilization of the fatty acid. PMA also potentiated arachidonic acid release when stimulation of phospholipase C by the ionophore (which is due to thromboxane A2 and/or secreted ADP) was blocked by aspirin plus ADP scavengers, i.e. apyrase or creatine phosphate/creatine phosphokinase. Increased release of arachidonic acid was attributable to loss of [14C]arachidonic acid primarily from phosphatidylcholine (79%) with lesser amounts derived from phosphatidylinositol (12%) and phosphatidylethanolamine (8%). Phosphatidic acid, whose production is a sensitive indicator of phospholipase C activation, was not formed. Thus, the potentiation of arachidonic acid release by PMA appeared to be due to phospholipase A2 activity. These results suggest that diacylglycerol formed in response to stimulation of platelet receptors by agonists may cooperatively promote release of arachidonic acid via a Ca2+/phospholipase A2-dependent pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Diglycerides; Enzyme Activation; Fatty Acids, Unsaturated; Glycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Phorbols; Phospholipases A; Phospholipases A2; Phospholipids; Phosphoproteins; Protein Kinase C; Tetradecanoylphorbol Acetate; Thromboxane B2