valinomycin and beauvericin

Topics: Anti-Bacterial Agents; Cations; Chemical Phenomena; Chemistry; Depsipeptides; Ionophores; Models, Molecular; Peptides; Peptides, Cyclic; Valinomycin

Beauvericin, a cyclic hexadepsipeptide with antibiotic properties, has been shown to reduce contraction force and to affect action potential parameters of guinea pig papillary muscles. Its potential to form cation-selective channels in mammalian membranes has been demonstrated. Patch clamp and fluorescence imaging techniques were used to investigate its effects in enzymatically isolated ventricular myocytes. Application of 10 microM beauvericin caused a large [Ca2+]i increase in Fura 2AM-loaded cardiomyocytes leading to cell shortening. The effect could be partially inhibited by ryanodine pretreatment and was largely dependent on external Ca2+ and blocked by 5 mM Ni2+. Beauvericin initiated a progressive increase in [Mg2+]i, the time course of which developed similarly upon increasing the external chemical gradient of Mg2+ 10-fold, to produce an ionophoric challenge. Monitoring of pH(i) with BCECF showed that beauvericin caused cytosolic acidification. Confocal microscopy revealed mitochondrial depolarization in TMRM-loaded cardiomyocytes, which resembled the effect of classical mitochondrial uncouplers. However, the NADH autofluorescence signal followed a biphasic pattern, in contrast to the NADH response to the uncouplers FCCP and the K+-ionophore valinomycin. These results suggest that beauvericin, possibly via its ionophoric properties, acts as an atypical mitochondrial uncoupler, greatly disturbs the physiological ionic balance and pH, challenges cellular metabolism, and causes ATP depletion.

Topics: Adenosine Triphosphate; Animals; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Cells, Cultured; Cytosol; Depsipeptides; Guinea Pigs; Heart Ventricles; Hydrogen-Ion Concentration; Magnesium; Membrane Potentials; Mitochondria; Myocytes, Cardiac; NAD; Nickel; Rats; Rats, Sprague-Dawley; Ryanodine; Valinomycin

Two ionophores specific for K+, valinomycin and beauvericin, induce a type of cell death very similar to apoptosis due to tumor necrosis factor (TNF alpha). Both ionophores cause cytolysis accompanied by internucleosomal DNA fragmentation of the dying cell into units of 200 base pairs. Morphologically, the cell death appears to consist of a mixture of nuclear apoptotic changes and cytoplasmic necrotic changes. As in the case for TNF alpha-mediated death, metabolic inhibitors have no effect on the course of cell death, but DNA fragmentation and cytolysis are decreased by the endonuclease inhibitor, zinc. Beauvericin and valinomycin trigger an increase in the cytoplasmic calcium concentration, most likely due to release of calcium from intracellular stores, and chelation of cytoplasmic calcium with quin-2 inhibits DNA fragmentation. Thus, these ionophores set off apoptosis through a calcium-activatable endonuclease, suggesting that other nonphysiological toxins might also cause apoptosis through their ability to indirectly elevate the cytoplasmic calcium concentration, without the need to invoke specific surface receptors.

Topics: Animals; Anti-Bacterial Agents; Calcium; Cell Death; Depsipeptides; DNA; Ionomycin; Ionophores; Macrolides; Mice; Microscopy, Electron; Nigericin; Peptides; Peptides, Cyclic; Tumor Cells, Cultured; Valinomycin

The configurations of the 13,14 double bond of the retinylidene chromophore in bacteriorhodopsin (bR) were investigated during the photocycle. The cycle was monitored by measuring the extent of formation of the M intermediate absorbing at 400-420 nm in the light and its rate of decay in the dark. It was inhibited by forming purple membrane films on glass and by using the synergistic ionophores beauvericin and valinomycin, in an equimolar ratio with bR, at 7 degrees C. These systems afforded enrichment in the M intermediate and a rate of decay that could be conveniently studied. The configuration was analyzed by extracting the chromophore with methylene chloride and measuring the ratio of 13-cis-to all-trans-retinals by high-performance liquid chromatography. The relationship between the percent bR cycling and percent 13-cis-retinal extracted was measured for the films by varying the light intensity; the molar ratio of bR cycling to 13-cis-retinal extracted was 1.11. This confirms previous studies which showed photoisomerization of the chromophore during the pumping cycle. The decay kinetics of the blue-shifted intermediate absorbing at 400-420 nm and of the 13-cis-retinal extracted were also measured for the purple membrane films and purple membrane suspensions containing the ionophores. The decay of the 13-cis-retinal extracted followed first-order kinetics with t 1/2 = 0.6 times the decay of the fastest measured component of the 400-420-nm decay. We conclude that the proton pumping photocycle of bR contains a photoisomerization about the 13,14 double bond of the chromophore from the all-trans to the 13-cis configuration and that the thermal rearrangement back to the all-trans form may occur more rapidly than the decay of the M photointermediate.

Topics: Anti-Bacterial Agents; Bacteriorhodopsins; Carotenoids; Depsipeptides; Hydrogen-Ion Concentration; Isomerism; Kinetics; Light; Peptides; Peptides, Cyclic; Valinomycin