manoalide and arachidonyltrifluoromethane

4-Bromophenacyl bromide (BPB) is generally used as a phospholipase A(2) (PLA2) inhibitor. In the present study, we demonstrate that BPB induces Ca2+ influx in human gingival fibroblasts. In fura-2-loaded human gingival fibroblasts, BPB evoked a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The BPB-induced Ca2+ mobilization was also shown in a single fluo-3-loaded-fibroblast. The BPB-induced increase in [Ca2+]i was completely abolished by the elimination of the external Ca2+. Ca2+ influx induced by the Ca2+-mobilizing agonist histamine was markedly enhanced in the presence of BPB. These suggest that the BPB-induced Ca2+ mobilization is due to the influx of extracellular Ca2+. However, it is unlikely that the effect of BPB is dependent on the inhibition of PLA2 activity, because other PLA2 inhibitors, such as AACOCF3, quinacrine dihydrochloride and manoalide, failed to induce Ca2+ mobilization. Chemical compounds similar to BPB, but which have no -CH2-Br at position 1 in the benzene ring failed to evoke Ca2+ mobilization, indicating that the position of -CH2--Br in BPB is important for causing the Ca2+ influx.

Topics: Acetophenones; Aniline Compounds; Arachidonic Acids; Bradykinin; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Fluorescent Dyes; Gingiva; Histamine; Humans; Kinetics; Phospholipases A; Phospholipases A2; Quinacrine; Spectrometry, Fluorescence; Structure-Activity Relationship; Terpenes; Xanthenes

Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. M. Francik, P. A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The intracellular pathways involved in this process are, however, unknown. The purpose of the present study was to test the hypothesis that contraction-related formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A(2) (PLA(2)). Studies were performed by using an in vitro hemidiaphragm preparation submerged in an organ bath, and formation of ROS in muscles was assessed by using a recently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations and then determined whether contraction-related ROS generation could be altered by administration of various PLA(2) inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA(2); arachidonyltrifluoromethyl ketone (AACOCF(3)), an inhibitor of 85-kDa PLA(2); and haloenol lactone suicide substrate (HELSS), an inhibitor of calcium-independent PLA(2). We found 1) little ROS formation [2.0 +/- 0.8 (SE) ng/mg] in noncontracting control diaphragms, 2) a high level of ROS (20.0 +/- 2.0 ng/mg) in electrically stimulated contracting diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s(-1)), 3) near-complete suppression of ROS generation in manoalide (3.0 +/- 0.5 ng/mg, P < 0. 001)- and aristolochic acid-treated contracting diaphragms (4.0 +/- 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF(3) or HELSS on ROS formation in contracting diaphragm. During in vitro studies examining fluorescent measurement of ROS formation in response to a hypoxanthine/xanthine oxidase superoxide-generating solution, manoalide, aristolochic acid, AACOCF(3), and HELSS had no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of inhibitors of the 14-kDa isoform of PLA(2) and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction.

Topics: Animals; Arachidonic Acids; Aristolochic Acids; Diaphragm; Electric Stimulation; Enzyme Activation; Enzyme Inhibitors; Ethidium; Fluorescence; Free Radicals; Kinetics; Male; Muscle Contraction; Naphthalenes; Phenanthrenes; Phospholipases A; Pyrones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxides; Terpenes

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Arteries; Cyclohexanones; Enzyme Activation; Enzyme Inhibitors; Estrenes; In Vitro Techniques; Isoenzymes; Lipoprotein Lipase; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Phospholipases A; Pyrones; Pyrrolidinones; Rats; Terpenes; Type C Phospholipases

This investigation was undertaken to clarify the mechanisms of superoxide anion (O2-) generation in rat peritoneal mast cells. Compound 48/80, a typical histamine liberator mediated by calcium influx, elicited O2- generation from the mast cells in a dose-dependent fashion. It was demonstrated by immunohistochemical study and Western blot analysis that the mast cells contained the 47-kDa phagocyte oxidase (p47phox) protein, which was one cytosolic component of the NADPH oxidase system. Arachidonic acid stimulated O2- generation in the mast cells, but other unsaturated fatty acids had no effect. On the other hand, 48/80-induced O2- generation was inhibited by phospholipase A2 inhibitors, such as arachidonyl trifluoromethyl ketone and manoalide. Forskolin, isoprenaline, and dibutyryl cyclic AMP inhibited the O2- generation, and KT-5720, a cyclic AMP-dependent protein kinase (A-kinase) inhibitor, markedly enhanced the O2- generation. These findings suggest that O2- is generated by a NADPH oxidase-like enzyme system in mast cells and that this enzyme system is activated by arachidonic acid released by cytosolic phospholipase A2. Thus, it is regulated by the cyclic AMP-A kinase system.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blotting, Western; Bucladesine; Carbazoles; Colforsin; Cyclic AMP-Dependent Protein Kinases; Granulomatous Disease, Chronic; Immunohistochemistry; Indoles; Isoproterenol; Male; Mast Cells; NADPH Oxidases; p-Methoxy-N-methylphenethylamine; Phosphoproteins; Protein Kinase C; Pyrroles; Rats; Rats, Wistar; Recombinant Proteins; Superoxides; Terpenes