calcimycin and oxophenylarsine

The opening of the cyclosporin-sensitive pore in the inner membrane of mitochondria in rat thymocytes was studied. In thymocytes with digitonin-permeabilized plasma membrane, the mitochondrial pore was induced by Ca2+ overload, by uncoupling, by oxidation or cross-linking of membrane dithiols, and by atractyloside, a specific inhibitor of the adenine nucleotide transporter. Pore opening was prevented by cyclosporin A (CsA) and by its non-immunosuppressive analog MeVal-CsA. The sensitivity of the pore to CsA was decreased by atractyloside and practically disappeared when it was added in combination with uncoupler. The main properties of the pore in mitochondria from thymocytes and from hepatocytes are the same. Release of Ca2+ from thymocyte mitochondria induced by uncoupling is mediated by a specific uniporter and by the pore with similar rates.

Topics: Animals; Arsenicals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane Permeability; Cyclosporine; Digitonin; Enzyme Inhibitors; In Vitro Techniques; Intracellular Membranes; Mitochondria; Oxygen Consumption; Rats; T-Lymphocytes; tert-Butylhydroperoxide; Thymus Gland; Uncoupling Agents

Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.

Topics: Animals; Antimycin A; Arsenicals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Diethyl Pyrocarbonate; Hydrogen-Ion Concentration; Hydroxylamine; Intracellular Membranes; Ionophores; Membrane Proteins; Mitochondria, Liver; Oxidation-Reduction; Permeability; Rats; Rats, Wistar; Serum Albumin, Bovine; Sulfhydryl Compounds

We studied the modulation of the permeability transition pore (MTP), a cyclosporin-A-sensitive channel, in deenergized mitochondria. Rat liver mitochondria were incubated in a potassium gluconate medium and treated with uncoupler, valinomycin, oligomycin and A23187. Under these conditions the membrane and Donnan potentials are collapsed, and no ion gradients are maintained, allowing the study of the dependence of MTP opening on the Ca2+ concentration under a variety of oxidative conditions without the complexities arising from changes of the membrane potential and matrix pH, and from secondary-ion redistribution. Cross-linking of mitochondrial dithiols with arsenite or phenylarsine oxide, or treatment with tert-butylhydroperoxide leading to complete oxidation of glutathione, increased the sensitivity of MTP opening to Ca2+. This effect could be fully prevented by prior treatment of mitochondria with monobromobimane and restored by reduction with dithiothreitol. The effect of tert-butylhydroperoxide was not additive with that of AsO, and it was completely blocked by modification of reduced glutathione with 1-chloro 2,4-dinitrobenzene through glutathione-S-transferase, indicating that oxidized glutathione affects the pore through the AsO-reactive and PhAsO-reactive dithiol. Oxidation of mitochondrial pyridine nucleotides by a variety of treatments also increased the sensitivity of MTP opening to Ca2+ under conditions where glutathione was maintained in the reduced state. This effect could be fully prevented when reduced pyridine nucleotides levels were reestablished with 2-hydroxybutyrate but not by treatment with monobromobimane or dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events, when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events,when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol, or by 2-hydroxybutyrate, respectively. Complete oxidation of pyridine nucleotides did not affect the reactivity of the dithiol towards monobromobimane, indicating that it remained in the reduced state. After transient opening of the MTP, sensitivity to py

Topics: Animals; Arsenicals; Calcimycin; Calcium; Cross-Linking Reagents; Glutathione; Intracellular Membranes; Ion Channel Gating; Mitochondria, Liver; NAD; NADP; Nucleotides; Oligomycins; Oxidants; Oxidation-Reduction; Oxidative Stress; Permeability; Pyridines; Rats; Sulfhydryl Compounds; Valinomycin

Preincubation of rat liver cells (the C-9 cell line) for 25 min with phenylarsine oxide at levels ranging from 0.06 to 0.6 microM amplifies prostaglandin I2 production when subsequently stimulated by platelet activating factor, lysine vasopressin, bradykinin, thapsigargin, and the Ca2+ ionophore, A-23187, but not that stimulated by exogenous arachidonic acid. The amplification is decreased after preincubation for 25 min with 1.8 microM phenylarsine oxide. Preincubation of mouse lymphoma cells (the WEHI-3 cell line) with phenylarsine oxide at levels ranging from 0.06 to 1.8 microM for 60 min does not affect prostaglandin E2 levels but inhibits leukotriene B4 and C4 production stimulated by the Ca(2+)-ionophore, A-23187. Amplification of prostaglandin production by phenylarsine oxide is reversed 100 times more effectively by 2,3-dimercaptopropanol than by 2-mercaptoethanol. Deesterification of lipids appears to be regulated positively in rat liver cells and leukotriene production negatively in mouse lymphoma cells by phosphorylation of tyrosine.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acids; Arsenicals; Bradykinin; Calcimycin; Cell Line; Epoprostenol; Kinetics; Liver; Lymphoma; Mice; Platelet Activating Factor; Rats

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.

Topics: Angiotensin II; Animals; Arsenicals; Brain; Calcimycin; Calcium Radioisotopes; Cells, Cultured; Drug Synergism; Neurons; Protein Kinase C; Rats; Rats, Inbred Strains; Receptors, Angiotensin; Tetradecanoylphorbol Acetate

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Arsenicals; Blood Platelets; Calcimycin; Cyclic AMP; Molecular Weight; Platelet Activating Factor; Rabbits; Sulfhydryl Reagents; Thrombin