cyclic-gmp and arachidonyltrifluoromethane

Cytosolic phospholipase A2 (cPLA2) preferentially liberates arachidonic acid (AA), which is known to be elevated in Alzheimer's disease (AD). The aim of this study was to investigate the possible relationship between enhanced nitric oxide (NO) generation observed in AD and cPLA2 protein level, phosphorylation, and AA release in rat pheochromocytoma cell lines (PC12) differing in amyloid beta secretion. PC12 control cells, PC12 cells bearing the Swedish double mutation in amyloid beta precursor protein (APPsw), and PC12 cells transfected with human APP (APPwt) were used. The transfected APPwt and APPsw PC12 cells showed an about 2.8- and 4.8-fold increase of amyloid beta (Abeta) secretion comparing to control PC12 cells. An increase of NO synthase activity, cGMP and free radical levels in APPsw and APPwt PC12 cells was observed. cPLA2 protein level was higher in APPsw and APPwt PC12 cells comparing to PC12 cells. Moreover, phosphorylated cPLA2 protein level and [3H]AA release were also higher in APP-transfected PC12 cells than in the control PC12 cells. An NO donor, sodium nitroprusside, stimulated [3H]AA release from prelabeled cells. The highest NO-induced AA release was observed in control PC12 cells, the effect in the other cell lines being statistically insignificant. Inhibition of cPLA2 by AACOCF3 significantly decreased the AA release. Inhibitors of nNOS and gamma-secretase reduced AA release in APPsw and APPwt PC12 cells. The basal cytosolic [Ca2+](i) and mitochondrial Ca2+ concentration was not changed in all investigated cell lines. Stimulation with thapsigargin increased the cytosolic and mitochondrial Ca2+ level, activated NOS and stimulated AA release in APP-transfected PC12 cells. These results indicate that Abeta peptides enhance the protein level and phosphorylation of cPLA2 and AA release by the NO signaling pathway.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Arachidonic Acid; Arachidonic Acids; Blotting, Western; Calcium; Cyclic GMP; Humans; Mutation; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; PC12 Cells; Phospholipases A2, Cytosolic; Phosphorylation; Rats; Reactive Oxygen Species; Signal Transduction; Transfection

Resveratrol (RSVL) is a well-established chemopreventive agent in human breast cancer models. The molecular basis of its action is far less characterized. We investigated the effects of RSVL on activity of adenylate- and guanylate-cyclase (AC, GC) enzymes; two known cytostatic cascades in MCF-7 breast cancer cells. RSVL increased cAMP levels in both time- and concentration-dependent manners (t(1/2), 6.2 min; EC(50) 0.8 micro M). In contrast, it had no effect on cGMP levels. The stimulatory effects for RSVL on AC were not altered either by the protein synthesis inhibitor (actinomycin-D, 5 micro M) or the estrogen-receptor (ER) blockers (tamoxifen and ICI182,780, 1 micro M each). Likewise, cAMP formation by RSVL was insensitive to either the broad-spectrum phosphodiesterase (PDE) inhibitor (IBMX, 0.5 mM) or the cAMP-specific PDE inhibitor (rolipram, 10 micro M). Instead, these PDE inhibitors significantly augmented maximal cAMP formation by RSVL. Parallel experiments showed that either RSVL or rolipram inhibited the proliferation of these cells in a concentration-responsive manner. Further, concurrent treatment with RSVL and rolipram significantly enhanced their individual cytotoxic responses. The antiproliferative effects were appreciably reversed by the kinase-A inhibitors, Rp-cAMPS (100-300 micro M) or KT-5720 (10 micro M). Pretreatment with the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone (10 micro M) markedly antagonized the cytotoxic effects of RSVL, but had no effect on that of rolipram. Altogether, the present study demonstrates, for the first time, that the chemotherapeutic agent RSVL is an agonist for the cAMP/kinase-A system, a documented pro-apoptic and cell-cycle suppressor in breast cancer cells.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Antineoplastic Agents, Phytogenic; Arachidonic Acids; Breast Neoplasms; Cell Division; Cyclic AMP; Cyclic GMP; Dactinomycin; Enzyme Activation; Enzyme Inhibitors; Female; Guanylate Cyclase; Humans; Receptors, Estrogen; Resveratrol; Rolipram; Stilbenes; Tamoxifen; Tumor Cells, Cultured

Pathologic microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially neurotoxic agents, such as pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). Using cultured N9 microglial cells, we have examined the regulation of TNF-alpha following endotoxic insult with lipopolysacharide (LPS), focusing on the role of the pro-inflammatory phospholipase A2/mitogen activated protein kinase/arachidonic acid/cyclo-oxygenase-2 cascade and the nitric oxide/cGMP pathway. Data show that various inhibitors of the PLA2 cascade markedly inhibit LPS-induced TNF-alpha release, supporting a key role of this pathway in the regulation of microglial activation. We also investigated the putative effects of cGMP-elevating agents on blocking microglial activation induced by LPS. Data show that each member of this class of cGMP-elevating compounds that we employed opposed microglial TNF-alpha release, suggesting that strengthening intracellular cGMP signaling mitigates against microglial activation. Taken together, our results suggest novel strategies for reducing microglial activation.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Cells, Cultured; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dipyridamole; Enzyme Inhibitors; Flavonoids; Guanylate Cyclase; Imidazoles; Indazoles; Isoenzymes; Lipopolysaccharides; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; Microglia; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; p38 Mitogen-Activated Protein Kinases; Phospholipases A; Phospholipases A2; Phosphorylcholine; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Signal Transduction; Tumor Necrosis Factor-alpha