melitten and manoalide

Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca(2+)-independent phospholipase A(2) (iPLA(2)) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA(2)-IVA, cPLA(2)-IVB, cPLA(2)-IVC, iPLA(2)-VIA, iPLA(2)-VIB, and secretory sPLA(2)-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA(2)-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA(2)-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA(2) substrate availability, potentiated arachidonic acid release and osmolyte efflux in a volume-sensitive, 5-lipoxygenase-dependent, cyclooxygenase-independent manner. Melittin-induced arachidonic acid release was inhibited by manoalide and slightly but significantly by BEL. A BEL-sensitive, melittin-induced PLA(2) activity was also detected in lysates devoid of sPLA(2), indicating that both sPLA(2) and iPLA(2) contribute to arachidonic acid release in vivo. Swelling-induced taurine efflux was inhibited potently by BEL and partially by manoalide, whereas the reverse was true for melittin-induced taurine efflux. It is suggested that in NIH3T3 cells, swelling-induced taurine efflux is dependent at least in part on arachidonic acid release by iPLA(2) and possibly also by sPLA(2), whereas melittin-induced taurine efflux is dependent on arachidonic acid release by sPLA(2) and, to a lesser extent, iPLA(2).

Topics: Animals; Arachidonic Acid; Cell Nucleus; Cell Size; Isoenzymes; Melitten; Mice; Naphthalenes; NIH 3T3 Cells; Osmolar Concentration; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Pyrones; Taurine; Terpenes

Contraction-induced respiratory muscle fatigue and sepsis-related reductions in respiratory muscle force-generating capacity are mediated, at least in part, by reactive oxygen species (ROS). The subcellular sources and mechanisms of generation of ROS in these conditions are incompletely understood. We postulated that the physiological changes associated with muscle contraction (i.e., increases in calcium and ADP concentration) stimulate mitochondrial generation of ROS by a phospholipase A(2) (PLA(2))-modulated process and that sepsis enhances muscle generation of ROS by upregulating PLA(2) activity. To test these hypotheses, we examined H(2)O(2) generation by diaphragm mitochondria isolated from saline-treated control and endotoxin-treated septic animals in the presence and absence of calcium and ADP; we also assessed the effect of PLA(2) inhibitors on H(2)O(2) formation. We found that 1) calcium and ADP stimulated H(2)O(2) formation by diaphragm mitochondria from both control and septic animals; 2) mitochondria from septic animals demonstrated substantially higher H(2)O(2) formation than mitochondria from control animals under basal, calcium-stimulated, and ADP-stimulated conditions; and 3) inhibitors of 14-kDa PLA(2) blocked the enhanced H(2)O(2) generation in all conditions. We also found that administration of arachidonic acid (the principal metabolic product of PLA(2) activation) increased mitochondrial H(2)O(2) formation by interacting with complex I of the electron transport chain. These data suggest that diaphragm mitochondrial ROS formation during contraction and sepsis may be critically dependent on PLA(2) activation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Arachidonic Acid; Calcium; Cyanides; Diaphragm; Electron Transport Complex I; Electron Transport Complex IV; Enzyme Inhibitors; Hydrogen Peroxide; Malates; Male; Melitten; Mitochondria; NADH, NADPH Oxidoreductases; NADPH Oxidases; Onium Compounds; Phosphodiesterase Inhibitors; Phospholipases A; Pyruvic Acid; Rats; Rats, Inbred Strains; Reactive Oxygen Species; Rotenone; Sepsis; Terpenes; Uncoupling Agents

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times > or =12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.

Topics: Animals; Arachidonic Acid; Cell Division; Cell Size; Cell Survival; Cyclosporine; Dexamethasone; Digitonin; Dose-Response Relationship, Drug; Enzyme Activation; Immunosuppressive Agents; Interleukin-1; Melitten; Mice; Phospholipases A; Phospholipases A2; Pituitary Neoplasms; Tacrolimus; Terpenes; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitamin E

The phospholipase A2 (PLA2) inhibitors quinacrine, manoalide and scalaradial inhibit the carbachol-stimulated secretion of the amyloid precursor protein (APP) from cells transfected with the human m1 muscarinic receptor. Conversely, activation of PLA2 by melittin increases secretion of an apparently immature species of APP from these cells. These results implicate PLA2 in regulating APP processing and secretion, which may have important implications for understanding the pathogenesis of Alzheimer's Disease.

Topics: Amyloid beta-Protein Precursor; Animals; Carbachol; CHO Cells; Cricetinae; Enzyme Activation; Homosteroids; Melitten; Phospholipases A; Phospholipases A2; Protein Processing, Post-Translational; Quinacrine; Receptors, Muscarinic; Sesterterpenes; Terpenes

The molecular mechanisms of depolarization-induced calcium-dependent acetylcholine (ACh) release and its inhibition by botulinum neurotoxin type A (BoTx) are not clear. We studied these mechanisms in an in vitro cholinergic neuronal pheochromocytoma PC12 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release. Stimulation of these cells with high K+ (80 mM) in the perfusion medium caused a marked increase (three to four times) in [3H]ACh release in a Ca(2+)-dependent manner. K(+)-stimulated [3H]ACh release was totally inhibited by pretreatment of cells with BoTx (2 nM) for 2 h. High K+ also stimulated the release of arachidonic acid ([3H]AA) from the cell membrane, which was inhibited by BoTx (2 nM). Addition of phospholipase A2 (PLA2) inhibitors (quinacrine, 4-bromophenacyl bromide, manoalide) to the perfusion medium inhibited K(+)-stimulated [3H]ACh and [3H]AA release in a dose-dependent manner. Inclusion of exogenous AA, the PLA2 activator melittin, or PLA2 itself prevented the effect of BoTx. These results demonstrate that in NGF-differentiated PC12 cells, AA release is associated with ACh release, BoTx inhibits both processes, and increased AA can protect against BoTx.

Topics: Acetophenones; Acetylcholine; Animals; Arachidonic Acid; Botulinum Toxins; Cell Differentiation; Dose-Response Relationship, Drug; Ethacrynic Acid; Indomethacin; Kinetics; Masoprocol; Melitten; Models, Biological; Nerve Growth Factors; PC12 Cells; Phospholipases A; Phospholipases A2; Potassium; Protein Kinase C; Quinacrine; Terpenes

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostagl

Topics: Acetophenones; Calcimycin; Cell Line; Dinoprostone; Fatty Acids, Unsaturated; Kinetics; Macrophages; Melitten; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Substrate Specificity; Terpenes