oligomycins and mesoxalonitrile

S-15176 is a new inhibitor of the permeability transition pore (PTP) which has been shown to display anti-ischemic properties. We show here that S-15176 prevented PTP, cytochrome c release and maintained mitochondrial membrane potential when low concentrations of S-15176 were used (not exceeding 50 nmol/mg protein). For higher concentrations S-15176 is able to collapse mitochondrial potential. This effect was reversed by the recoupling agent 6-ketocholestanol (6-KCh) suggesting that S-15176 has uncoupling properties. In addition, S-15176 is able to inhibit ATP synthase activity and to stimulate the hydrolytic activity of the enzyme but none of these effects appears to be related to its PTP inhibiting property. These data demonstrate that S-15176 interacts with several targets in mitochondria and these pharmacological properties should be considered in the examination of its health benefits as well as its potential cytotoxicity.

Topics: Adenosine Diphosphate; Animals; ATP Synthetase Complexes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen-Ion Concentration; Ion Channels; Ketocholesterols; Membrane Potentials; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Mitochondrial Proteins; Mitochondrial Proton-Translocating ATPases; Nitriles; Oligomycins; Piperazines; Proton Pumps; Rats; Rats, Wistar; Time Factors; Uncoupling Protein 1

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin. 2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13-20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique. 3. After 2.0 min of incubation ATP decreased from the control level to 43% with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 microM), and 69% with oligomycin (10 microM). 4. Under normal conditions V was -74.4 +/- 1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2 +/- 1.3 and 19.7 +/- 3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve. 5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1 +/- 2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8 +/- 3.5 mV and increased g by 45.5 +/- 9.6%. The action of FCCP was not affected by the presence of TEA. 6. Perfusion of the astrocytes with a Cl- free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2 +/- 1.5 mV in DNP and 3.7 +/- 0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl-]. 7. Block of tentative KATP channels with tolbutamide (1 mM) or Cl- channels with Zn2+ (1 mM) did not inhibit the depolarization caused by DNP or FCCP. 8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl- channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Antimetabolites; Astrocytes; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Dinitrophenols; Ion Transport; Membrane Potentials; Nitriles; Oligomycins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Tetraethylammonium; Tolbutamide; Zinc

The unique compound 2,3-cyclopyrophosphoglycerate occurs at a detectable concentration in the genera Methanobacterium and Methanobrevibacter but not in Methanococcus, Methanospirillum and Methanosarcina, as shown by a 31P-NMR survey of several different methanogens. Metabolic poisons (carbonyl cyanide m-chlorophenylhydrazone and valinomycin) do not decrease the level of the cyclic pyrophosphate in Methanobacterium thermoautotrophicum; therefore, it cannot be a phosphagen, i.e., an energy storage material. 13CO2 is rapidly incorporated into this cyclic compound which represents the major soluble carbon as well as the phosphorus component of this methanobacteria. 13C-NMR analysis demonstrates that the pKa of the 2,3-cyclopyrophosphoglycerate carboxyl group is 2.55. The unusual pseudomurein cell wall structure of methano- and methanobrevibacteria necessitates a high demand on carbohydrate metabolism. For this reason, and the fact that when its concentration is decreased no new phosphorus resonances appear in the high resolution spectra, it is suggested that 2,3-cyclopyrophosphoglycerate has a function in carbohydrate metabolism.

Topics: 2,3-Diphosphoglycerate; Anaerobiosis; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Wall; Euryarchaeota; Magnetic Resonance Spectroscopy; Nitriles; Oligomycins; Organophosphorus Compounds; Species Specificity; Valinomycin