oligomycins and lonidamine

Mitochondria sequester and release calcium (Ca(2+)) and regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) in eukaryotic cells. However, the regulation of different Ca(2+) signalling modalities by mitochondria in smooth muscle cells is poorly understood. Here, we investigated the regulation of Ca(2+) sparks, Ca(2+) waves and global [Ca(2+)](i) by mitochondria in cerebral artery smooth muscle cells. CCCP (a protonophore; 1 microm) and rotenone (an electron transport chain complex I inhibitor; 10 microm) depolarized mitochondria, reduced Ca(2+) spark and wave frequency, and elevated global [Ca(2+)](i) in smooth muscle cells of intact arteries. In voltage-clamped (-40 mV) cells, mitochondrial depolarization elevated global [Ca(2+)](i), reduced Ca(2+) spark amplitude, spatial spread and the effective coupling of sparks to large-conductance Ca(2+)-activated potassium (K(Ca)) channels, and decreased transient K(Ca) current frequency and amplitude. Inhibition of Ca(2+) sparks and transient K(Ca) currents by mitochondrial depolarization could not be explained by a decrease in intracellular ATP or a reduction in sarcoplasmic reticulum Ca(2+) load, and occurred in the presence of diltiazem, a voltage-dependent Ca(2+) channel blocker. Ru360 (10 microm), a mitochondrial Ca(2+) uptake blocker, and lonidamine (100 microm), a permeability transition pore (PTP) opener, inhibited transient K(Ca) currents similarly to mitochondrial depolarization. In contrast, CGP37157 (10 microm), a mitochondrial Na(+)-Ca(2+) exchange blocker, activated these events. The PTP blockers bongkrekic acid and cyclosporin A both reduced inhibition of transient K(Ca) currents by mitochondrial depolarization. These results indicate that mitochondrial depolarization leads to a voltage-independent elevation in global [Ca(2+)](i) and Ca(2+) spark and transient K(Ca) current inhibition. Data also suggest that mitochondrial depolarization inhibits Ca(2+) sparks and transient K(Ca) currents via PTP opening and a decrease in intramitochondrial [Ca(2+)].

Topics: Animals; Bongkrekic Acid; Caffeine; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Calcium-Binding Proteins; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cells, Cultured; Cerebral Arteries; Clonazepam; Cyclosporine; Diltiazem; Female; Hydrogen Peroxide; Indazoles; Ion Channels; Male; Membrane Potentials; Microscopy, Confocal; Mitochondria, Muscle; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Smooth Muscle; Oligomycins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Rhodamines; Rotenone; Ruthenium Compounds; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Thapsigargin; Thiazepines

The effect of the anticancer drug lonidamine (LND) on the pH of intracellular organelles was studied in isolated rat thymocytes by fluorimetric analysis of the (bafilomycin-nigericin sensitive) uptake of the acridine orange dye (AO) into acidic compartments. LND brought about a marked reduction (> 60%) in the above pH gradients with a half maximal decrease at a concentration of 0.25 mM. LND also caused a decrease, although to a lesser extent, in the ATP levels. In isolated rat liver lysosomes, 0.6 mM LND was found to inhibit ATP-driven organelle acidification by about 80%; minor inhibition of ATPase activity was observed in the same conditions. In addition, LND was able to promote proton efflux from isolated lysosomes. On the basis of our results it is suggested that the effect of LND on intracellular proton gradients may be due partially to the decrease in ATP levels, and mostly to a drug-induced increase in the ion (proton) permeability of the membranes.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Enzyme Inhibitors; Hydrogen-Ion Concentration; Indazoles; Lysosomes; Nigericin; Oligomycins; Organelles; Rats; Thymus Gland

The effect of Lonidamine, 1-(2,4 dichlorobenzyl)-1-H-indazol-3-carboxylic acid, on the uptake of Adriamycin by Ehrlich ascites tumor cells has been investigated. The uptake of Adriamycin is greatly stimulated by Lonidamine and the increase depends on the energy sources of the cell. In the presence of glucose the intracellular drug content is remarkably lower than that in its absence. This difference lies in the mechanism by which Lonidamine enhances the uptake of Adriamycin. The Adriamycin efflux is via an active transport process and, in the presence of glucose, both aerobic glycolysis and oxidative phosphorylation contribute to ATP synthesis. Although Lonidamine inhibits both these pathways, there is still sufficient ATP to extrude a certain amount of Adriamycin. The elevated intracellular concentration of Adriamycin depends not only on the Lonidamine-inhibited outward transport but also on higher membrane permeability which allows a low concentration of Adriamycin (18 microM) to interfere also with the oxidative metabolism of Ehrlich ascites tumor cells.

Topics: Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Dinitrophenols; Doxorubicin; In Vitro Techniques; Indazoles; Kinetics; Lactates; Male; Mice; Oligomycins; Oxygen Consumption; Pyrazoles

The action of lonidamine, 1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid, on protein synthesis of neoplastic cells growing both in vivo and in vitro has been investigated. Lonidamine decreases amino acid incorporation in all cells tested, although the inhibition is partially relieved by glucose. The inhibition of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake which, on the contrary, is enhanced by the drug. Tests on cell-free systems showed that lonidamine does not inhibit the tobacco mosaic virus (TMV)-mRNA-directed in vitro protein synthesis, thus indicating that protein synthetic machinery per se is not affected. The inhibition of the rate of protein synthesis achieved by lonidamine must be ascribed to an effect on energy-yielding processes with a mechanism similar to that observed in other metabolic inhibitors. Lonidamine, however, because of its capacity to inhibit both respiration and glycolysis in neoplastic cells, is effective at 10 to 20 times lower concentrations. DNP and oligomycin potentiate the inhibitory effect of lonidamine on the rate of protein synthesis. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.

Topics: Amino Acids; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Line; Cell-Free System; Dinitrobenzenes; Glycolysis; Humans; Indazoles; Kinetics; Lactates; Leukemia, Experimental; Male; Melanoma; Mice; Neoplasm Proteins; Oligomycins; Pyrazoles; Rabbits; Sarcoma, Experimental