oligomycins and 5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.

Topics: Animals; Benzimidazoles; Calcium Signaling; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cholecystokinin; Cytochalasin D; Cytoskeleton; Depsipeptides; Dose-Response Relationship, Drug; Hydrogen Peroxide; Indoles; Intracellular Fluid; Male; Maleimides; Membrane Potential, Mitochondrial; Mice; Mitochondria; Oligomycins; Organotin Compounds; Pancreas, Exocrine; Protein Kinase C; Ruthenium Compounds; Salicylates; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sincalide; Thapsigargin

Increasing evidence indicates that mitochondrial alterations contribute to the neuronal death in Alzheimer's disease (AD). Presenilin 1 (PS1) and Presenilin 2 (PS2) mutations have been shown to sensitize cells to apoptosis by mechanisms suggested to involve impaired mitochondrial function. We have previously detected active gamma-secretase complexes in mitochondria. We investigated the impact of PS/gamma-secretase on mitochondrial function using mouse embryonal fibroblasts derived from wild-type, PS1-/-, PS2-/- and PS double knock-out (PSKO) embryos. Measurements of mitochondrial membrane potential (DeltaPsim) showed a higher percentage of fully functional mitochondria in PS1-/- and PSwt as compared to PS2-/- and PSKO cells. This result was evident both in whole cell preparations and in isolated mitochondria. Interestingly, pre-treatment of isolated mitochondria with the gamma-secretase inhibitor L-685,458 resulted in a decreased population of mitochondria with high DeltaPsim in PSwt and PS1-/- cells, indicating that PS2/gamma-secretase activity can modify DeltaPsim. PS2-/- cells showed a significantly lower basal respiratory rate as compared to other cell lines. However, all cell lines demonstrated competent bioenergetic function. These data point toward a specific role of PS2/gamma-secretase activity for proper mitochondrial function and indicate interplay between PS1 and PS2 in mitochondrial functionality.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Fibroblasts; Flow Cytometry; Ionophores; Membrane Potentials; Membrane Proteins; Mice; Mice, Knockout; Mitochondrial Membranes; Oligomycins; Oxygen Consumption; Presenilin-1; Presenilin-2

The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate-aspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Aspartic Acid; Benzimidazoles; Carbocyanines; Cells, Cultured; Diclofenac; Dogs; Glutamate Dehydrogenase; Kidney; Malate Dehydrogenase; Malates; Membrane Potentials; Mitochondria; Mitochondrial Membranes; NADH Dehydrogenase; Oligomycins; Rats

The direction of the chemical reaction of ATP synthetase is reversible. The present study was designed to determine whether mitochondria produce or consume ATP during ischemia. For this purpose, changes in mitochondrial membrane potential were measured in vivo at the site of a direct current (DC) electrode using a potentiometric dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and a rat model of focal ischemia. Two microL of dye (control group) or dye with oligomycin, an ATP synthetase inhibitor (oligomycin group), was injected into the parietotemporal cortex through the DC electrode. With the initiation of ischemia, a decrease in mitochondrial potential was observed within 20 seconds in the oligomycin group (earlier than the onset of DC deflection, P = 0.02). In contrast, in the control group, mitochondrial potential was maintained at 91 +/- 5% of the preischemia level for 118 +/- 38 seconds before showing full depolarization simultaneously with DC deflection. During the period of ischemia, the mitochondrial potential was higher in the control group (66 +/- 9%) than in the oligomycin group (46 +/- 8%, P = 0.0002), whereas DC potential was lower in the control group (-18 +/- 3) than in the oligomycin group (-15 +/- 2 mV, P = 0.04). These observations suggest that mitochondria consume ATP during ischemia by reversing ATP synthetase activity, which compromises cellular membrane potential by consuming ATP.

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Brain Ischemia; Carbocyanines; Cerebral Cortex; Enzyme Inhibitors; Fluorescent Dyes; Hemoglobins; Membrane Potentials; Microinjections; Mitochondria; Mitochondrial Proton-Translocating ATPases; Oligomycins; Rats; Rats, Sprague-Dawley

Scabrosin esters (SEs), which have been recently isolated from the lichen Xanthoparmelia scabrosa, belong to the epipolythiodioxopiperazine (ETP) class of secondary metabolites characterized by possession of a reactive disulfide bond. Colony forming assays show that these toxins are active against human tumor cell lines at nanomolar concentrations. Other members of the ETP class of toxins such as gliotoxin have been shown to induce apoptosis in cells, although the cellular target(s) of the ETP toxins is currently unknown. ETP toxins have been shown to inhibit a variety of enzymes via interaction with sensitive cysteine residues. Here we show that the typical scabrosin ester acetate butyrate induces early mitochondrial membrane hyperpolarization assessed by JC-1 staining accompanied by apoptotic cell death. The toxin lowers ATP in intact cells and inhibits the rate of ATP synthesis in permeabilzed cells. Comparison with the effects of the known ATP synthase inhibitor oligomycin B is consistent with ATP synthase as an early target in scabrosin ester-induced cell death.

Topics: Animals; Apoptosis; Benzimidazoles; Carbocyanines; Caspase 3; Caspases; Cells, Cultured; DNA Fragmentation; Esters; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Intracellular Membranes; Lichens; Macrophages; Membrane Potentials; Mitochondria; Mitochondrial Proton-Translocating ATPases; Oligomycins; Plant Extracts

The relative magnitude of the inner mitochondrial membrane potential (DeltaPsim) has been suggested to indicate the competence of mammalian gametes and early embryos. This study examined the response of cultured somatic cells and mouse oocytes to inhibitors and conditions that affect DeltaPsim or metabolism, or both, and measured treatment-specific changes in ATP and cytoplasmic free Ca(2+).. During and after treatment, relative DeltaPsim, free Ca(2+), and ATP levels and cortical granule density were determined.. Comparable responses of somatic cells and metaphase II mouse oocytes to experimental manipulations that affect DeltaPsim and metabolism were observed and reversible loss of DeltaPsim was associated with increased intracellular free Ca(2+), which in certain instances resulted in parthenogenetic activation.. The findings support a mitochondrial basis for pericortical J-aggregate fluorescence but not for a direct association between high DeltaPsim and metabolism. The results extend previous findings indicating that high-polarized (high DeltaPsim, JC-1 J-aggregate-forming) mitochondria occur in pericortical domains in mouse and human oocytes and early preimplantation stage embryos and support the notion that this spatial distribution may be related to localized ionic and metabolic regulation.

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Bongkrekic Acid; Calcimycin; Calcium; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cytoplasm; Exocytosis; Fibroblasts; Fluorescent Dyes; Humans; Intracellular Membranes; Ionophores; Membrane Potentials; Metaphase; Mice; Mitochondria; Oligomycins; Oocytes; Temperature

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.

Topics: Animals; Benzimidazoles; Carbocyanines; Cerebral Cortex; Enzyme Inhibitors; Fluorescent Dyes; Guinea Pigs; Hydrogen Peroxide; Intracellular Membranes; Ketoglutarate Dehydrogenase Complex; Membrane Potentials; Mitochondria; NADP; Nerve Endings; Oligomycins; Oxidative Stress; Proton-Translocating ATPases; Rotenone; Spectrometry, Fluorescence; Synaptosomes; Uncoupling Agents