6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2h-pyran-2-one and manoalide

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

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

The polychlorinated biphenyl (PCB) mixture Aroclor 1242 (A1242) increases frequency of contractions of pregnant rat uteri, suggesting a possible mechanism for decreased gestational age and increased spontaneous abortion in women and animals exposed to PCBs. In the present study, we hypothesized that A1242-induced stimulation of uterine contraction is mediated by arachidonic acid released by phospholipase A2 (PLA2) enzymes. Isometric uterine contraction was measured in longitudinal uterine strips isolated from gestation day 10 rat. Pretreatment of uterine strips with the PLA2 inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) or manoalide, or an inhibitor of the G protein of PLA2, isotetrandrine, completely prevented the increase of contractile frequency induced by 50 microM A1242. However, the phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) and neomycin were unable to block stimulation of uterine contraction by A1242. In accordance, A1242 (100 microM) did not release inositol phosphates from myo-[3H]inositol-labeled myometrial cells, whereas myometrial cells prelabeled with [3H]arachidonic acid released arachidonic acid in a concentration- and time-dependent manner after exposure to A1242 (10-100 microM). A1242 significantly stimulated arachidonic acid release in the absence of extracellular calcium, although the release was attenuated. Analysis of the eicosanoids released by A1242 indicated that only 0.83% of released [3H]arachidonic acid was metabolized to eicosanoids and 99.07% remained as free arachidonate. Uterine contraction increased in strips exposed to exogenous arachidonic acid (1-100 microM). This study suggests that A1242 stimulates contraction in pregnant rat uterus by a mechanism involving PLA2-mediated arachidonic acid release, and that arachidonic acid, rather than eicosanoids, may mediate A1242 uterotonic action in the uterus.

Topics: Alkaloids; Animals; Antioxidants; Arachidonic Acid; Aroclors; Benzylisoquinolines; Eicosanoids; Environmental Pollutants; Enzyme Activation; Enzyme Inhibitors; Female; In Vitro Techniques; Inositol Phosphates; Isoenzymes; Isometric Contraction; Myometrium; Naphthalenes; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Pregnancy; Pyrones; Rats; Rats, Sprague-Dawley; Terpenes; Type C Phospholipases; Uterine Contraction; Uterus

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