Trialkyl(vinyl)phosphonium Chlorophenol Derivatives as Potent Mitochondrial Uncouplers and Antibacterial Agents.

Trialkyl phosphonium derivatives of vinyl-substituted p-chlorophenol were synthesized here by a recently developed method of preparing quaternary phosphonium salts from phosphine oxides using Grignard reagents. All the derivatives with a number (n) of carbon atoms in phosphonium alkyl substituents varying from 4 to 7 showed pronounced uncoupling activity in isolated rat liver mitochondria at micromolar concentrations, with a tripentyl derivative being the most effective both in accelerating respiration and causing membrane potential collapse, as well as in provoking mitochondrial swelling in a potassium-acetate medium. Remarkably, the trialkyl phosphonium derivatives with n from 4 to 7 also proved to be rather potent antibacterial agents. Methylation of the chlorophenol hydroxyl group suppressed the effects of P555 and P444 on the respiration and membrane potential of mitochondria but not those of P666, thereby suggesting a mechanistic difference in the mitochondrial uncoupling by these derivatives, which was predominantly protonophoric (carrier-like) in the case of P555 and P444 but detergent-like with P666. The latter was confirmed by the carboxyfluorescein leakage assay on model liposomal membranes.

Zwitterionic Protonophore Derived from 2-(2-Hydroxyaryl)alkenylphosphonium as an Uncoupler of Oxidative Phosphorylation.

2-(2-Hydroxyaryl)alkenylphosphonium salts (here coined as PPR) representing derivatives of quaternary phosphonium with two phenyl (P) and one alkyl (R) substituents linked through alkenyl bridge to substituted phenol were applied here to planar bilayer lipid membranes (BLM), isolated mitochondria, and cell culture. PPR with six carbon atoms in R (PP6) induced proton-selective currents across BLM and caused mitochondrial uncoupling. In particular, PP6 at submicromolar concentrations accelerated respiration, decreased membrane potential, and reduced ATP synthesis in isolated rat liver mitochondria (RLM). Methylation of a hydroxyl group substantially suppressed the protonophoric activity of PP6 on BLM and its uncoupling potency in RLM. Of note, the methylated derivative PP6-OMe was synthesized here via a new synthetic route including cyclization of PP6 with subsequent ring opening. PPR were considered as protonophoric uncouplers of a zwitterionic type, capable of penetrating membranes both as a zwitterion composed of a deprotonated phenol and a cationic quaternary phosphonium, and as a protonated cation. The protonophoric and uncoupling properties of PPR found here were speculated to account for their strong antibacterial activity described previously.

Protonophoric action of triclosan causes calcium efflux from mitochondria, plasma membrane depolarization and bursts of miniature end-plate potentials.

The formerly widely used broad-spectrum biocide triclosan (TCS) has now become a subject of special concern due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that TCS is an uncoupler of oxidative phosphorylation in mitochondria, there have been so far no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). Yet only few works have indicated the relationship between TCS impacts on mitochondria and nerve cell functioning. Here, we for the first time report data on a high protonophoric activity of TCS on planar BLM. TCS proved to be a more effective protonophore on planar BLM, than classical uncouplers. Correlation between a strong depolarizing effect of TCS on bacterial membranes and its bactericidal action on Bacillus subtilis might imply substantial contribution of TCS protonophoric activity to its antimicrobial efficacy. Protonophoric activity of TCS, monitored by proton-dependent mitochondrial swelling, resulted in Ca2+ efflux from mitochondria. A comparison of TCS effects on molluscan neurons with those of conventional mitochondrial uncouplers allowed us to ascribe the TCS-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Also similar to the action of common uncouplers, TCS caused a pronounced increase in frequency of miniature end-plate potentials at neuromuscular junctions. Thus, the TCS-induced mitochondrial uncoupling could alter neuronal function through distortion of Ca2+ homeostasis.

Participation of ATP/ADP antiporter in oleate- and oleate hydroperoxide-induced uncoupling suppressed by GDP and carboxyatractylate.

In experiments on isolated kidney and liver mitochondria, it is shown that oleate hydroperoxide induces a much smaller increase in the controlled respiration rate and DeltaPsi decrease than the same concentrations of oleate. Palmitate appears to be less efficient than oleate but more efficient than oleate hydroperoxide. In all cases, GDP and CAtr cause some recoupling, CAtr being more effective. Addition of 0.2 mM GDP before CAtr does not prevent further DeltaPsi increase by subsequent CAtr addition. On the other hand, GDP added after CAtr is without any effect. GDP partially prevents the DeltaPsi lowering by ADP at the State 4--State 3 transition if small amounts of CAtr are present. The data are consistent with the suggestion of F. Goglia and V.P. Skulachev (FASEB J. 17, 1585-1591, 2003) that fatty acid anions are translocated by mitochondrial anion carriers much better than their hydroperoxides. As to GDP recoupling, it cannot be regarded as a specific probe for uncoupling by UCPs since it can be mediated by the ATP/ADP antiporter.