manoalide and diphenyleneiodonium

Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell types, impaired in the presence of BEL and DPI and following restoration of the cell volume. Thus, potentiation of the volume-sensitive taurine efflux pathway following inhibition of tyrosine phosphatase activity reflects increased arachidonic acid mobilization and ROS production for downstream signaling. Vanadate delays the inactivation of volume-sensitive taurine efflux in NIH3T3 cells, and this delay is impaired in the presence of DPI. Vanadate has no effect on the inactivation of swelling-induced taurine efflux in Ehrlich Lettre cells. It is suggested that increased tyrosine phosphorylation of regulatory components of NADPH oxidase leads to increased ROS production and a subsequent delay in inactivation of the volume-sensitive taurine efflux pathway and that NADPH oxidase or antioxidative capacity differ between NIH3T3 and Ehrlich Lettre cells.

Topics: Animals; Antioxidants; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Size; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Hydrogen Peroxide; Hypotonic Solutions; Lipoxygenase Inhibitors; Mice; NADPH Oxidases; Naphthalenes; NIH 3T3 Cells; Onium Compounds; Phospholipases A; Protein Tyrosine Phosphatases; Pyrones; Reactive Oxygen Species; Signal Transduction; Sodium Chloride; Taurine; Terpenes; Time Factors; Vanadates

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