15-hydroxy-5-8-11-13-eicosatetraenoic-acid and 4-bromophenacyl-bromide

Serum-cultured rat W256 carcinosarcoma cells of the monocytoid origin undergo rapid apoptosis in response to the lipoxygenase inhibitor NDGA (nordihydroguaiaretic acid). Exogenous arachidonic acid (AA), in a time- and dose-dependent fashion, suppressed NDGA-induced W256 cell apoptosis as well as DNA fragmentation, with the maximal effect observed at approximately 25 microM. Mobilization of endogenous AA by calcium ionophore A23187 provided an even stronger and longer-lasting protection against NDGA-caused cell death. The A23187 effect on AA release as well as W256 cell death can be blocked by bromophenacyl bromide, thus suggesting involvement of phospholipase A2 activation. Serum withdrawal similarly caused W256 cells to undergo typical apoptosis, which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation-induced W256 cell apoptosis and significantly extended cell survival in a dose-dependent manner. Lipoxygenase products, 12(S)- and 15(S)-, but not 5(S)-hydroxyeicosatetraenoic acid (HETE), in a dose-dependent fashion, also prevented both NDGA- and serum-starvation-induced W256 cell apoptosis. AA appears to suppress W256 cell apoptosis via distinct signaling pathway(s) since it does not prevent cell death triggered by several other inducers. Examination of a panel of polyunsaturated fatty acids revealed that alpha-linolenic and linoleic acid can also suppress NDGA-induced W256 cell apoptosis. Our data suggest that AA and other polyunsaturated fatty acids and/or their metabolites may enhance tumor growth not only by promoting cell proliferation but also by suppressing apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Apoptosis; Arachidonic Acid; Calcimycin; Carcinosarcoma; Cell Line; Cell Survival; Cytokines; DNA Fragmentation; Fatty Acids, Nonesterified; Growth Substances; Hydroxyeicosatetraenoic Acids; Kinetics; Masoprocol; Rats; Tumor Cells, Cultured

Both IFN-alpha/beta and IFN-gamma have recently been demonstrated to induce a rapid but transient activation of phospholipase A2 (PLA2) in BALB/c 3T3 fibroblasts and a human neuroblastoma cell line. We report that IFN-gamma induces the synthesis and prolonged activation of cytosolic phospholipase A2 (cPLA2) in a human bronchial epithelial cell line (BEAS 2B). Treatment of the cells with IFN-gamma (300 U/ml) increased the release of [3H]arachidonic acid (AA) from prelabeled cells with a maximal effect at 12 h after stimulation. The increased [3H]AA release was inhibited by the PLA2 inhibitor p-bromophenacyl bromide (10(-5) M). Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the IFN-gamma-treated cells. Subcellular enzyme activity assay revealed that IFN-gamma increased PLA2 activity in both the cytosol and membrane fractions with a translocation of the cPLA2 to cell membranes in a Ca(2+)-free cell lysing buffer. Treatment with IFN-gamma also induced the release of 15-HETE, an arachidonic acid metabolite. Immunoblot showed that IFN-gamma induced the synthesis of cPLA2 protein. Nuclear run-on assay demonstrated that IFN-gamma initiated cPLA2 gene transcription within 15 min, and this effect was sustained at 4 h and returned to near control level at 12 h. The cPLA2 mRNA level was assayed by reverse transcription and PCR. IFN-gamma was found to increase the cPLA2 mRNA after 2-24 h treatment. Furthermore, the IFN-gamma induced cPLA2 mRNA increase was blocked by inhibitors of protein kinase C and calcium/calmodulin-dependent protein kinases, suggesting the involvement of these protein kinases in IFN-gamma-induced gene expression of cPLA2. This study shows that IFN-gamma induces the synthesis and prolonged activation of cPLA2.

Topics: Acetophenones; Arachidonic Acid; Base Sequence; Bronchi; Calcimycin; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epithelium; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Kinetics; Molecular Sequence Data; Phospholipases A; Phospholipases A2; Polymerase Chain Reaction; Protein Processing, Post-Translational; RNA, Messenger; Transcription, Genetic

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A2-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A2 inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A2 in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclo-oxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A2-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Chronobiology Phenomena; Dose-Response Relationship, Drug; Enzyme Activation; Heart; Hydroxyeicosatetraenoic Acids; Isoproterenol; Lactates; Leukotrienes; Melitten; Phospholipases A; Phospholipases A2; Prostaglandins D; Quinacrine; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 15S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration [( Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 microM whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cyclooxygenase Inhibitors; Hydroxyeicosatetraenoic Acids; Indomethacin; Ion Channel Gating; Ionomycin; Masoprocol; Melitten; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; T-Lymphocytes

The effect of 5-lipoxygenase products of arachidonic acid on 14-C-alkyl-acetyl-glycero-phosphocholine (14C-alkyl-acetyl GPC) production in rat peritoneal macrophages was investigated, using macrophages prelabeled with N-methyl-14C-alkyl-lyso-glycero-phosphocholine (14C-alkyl-lyso GPC) (prelabeled macrophages). Bromophenacyl bromide (BPB: phospholipase A2 inhibitor), and AA861 (5-lipoxygenase inhibitor) suppressed the production of 14C-alkyl-acetyl GPC in the A23187-stimulated prelabeled macrophages in a dose-dependent manner. A23187-induced hydrolysis of 14C-alkyl-acyl-glycero-phosphocholine (14C-alkyl-acyl GPC) and formation of 14C-alkyl-lyso GPC were also reduced by BPB and AA861. However, indomethacin (IND: cyclo-oxygenase inhibitor) had no significant effect on 14C-alkyl-acetyl GPC production in the A23187-stimulated prelabeled macrophages. Exogenously supplied 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) reversed the inhibitory effect of AA861 on 14C-alkyl-acetyl GPC production in A23187-stimulated prelabeled macrophages. Reduced hydrolysis of 14C-alkyl-acyl GPC and formation of 14C-alkyl-lyso GPC in A23187-stimulated prelabeled macrophages, which were pretreated with AA861, were also reversed by the addition of 5-HPETE and 5-HETE. However, LTB4 had no such effects. 5-HPETE and 5-HETE augmented the stimulatory effect of A23187 on 14C-alkyl-acetyl GPC production in prelabeled macrophages, while they could not stimulate alkyl-acetyl GPC production in the absence of A23187. These results suggest that 5-lipoxygenase products, especially 5-HPETE and 5-HETE, may play an important role in alkyl-acetyl GPC production in rat peritoneal macrophages.

Topics: Acetophenones; Animals; Arachidonic Acids; Benzoquinones; Calcimycin; Carbon Radioisotopes; Dose-Response Relationship, Drug; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Macrophages; Male; Platelet Activating Factor; Quinones; Rats; Rats, Inbred Strains