melitten and diphenyleneiodonium

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

beta-Amyloid protein (A beta) is a member of a small group of proteins that accumulate as amyloid deposits in various tissues. It has recently been demonstrated that the toxicity of A beta toward some neural cells is caused by oxidative damage. Since all of the amyloid diseases are characterized by protein deposited in the antiparallel beta-sheet conformation, it was asked whether there is a common toxic mechanism. It is shown here that the protein components of other human amyloidoses, including amylin, calcitonin, and atrial natriuretic peptide, are all toxic to clonal and primary cells. The toxicity is mediated via a free radical pathway indistinguishable from that of A beta. Experiments with synthetic peptides suggest that it is the amphiphilic nature of the peptides generated by their beta structure rather than their beta structure per se that causes toxicity. These results tend to rule out the alternative that amyloid toxicity is exclusively mediated via specific cell surface receptors.

Topics: Amino Acid Sequence; Amyloid; Amyloid beta-Peptides; Animals; Atrial Natriuretic Factor; Brain Neoplasms; Calcitonin; Cell Line; Cell Survival; Flow Cytometry; Humans; Hydrogen Peroxide; Intercellular Signaling Peptides and Proteins; Islet Amyloid Polypeptide; L-Lactate Dehydrogenase; Melitten; Molecular Sequence Data; NADH, NADPH Oxidoreductases; Oligopeptides; Onium Compounds; p-Methoxy-N-methylphenethylamine; Peptide Fragments; Peptides; Protein Structure, Secondary; Rats; Structure-Activity Relationship; Tumor Cells, Cultured; Wasp Venoms