The transient character of mitochondrial uncoupling by the popular fungicide fluazinam is specific for liver.

The popular fungicide fluazinam is known to exhibit an unusual cyclic pattern of the protonophoric uncoupling activity in isolated rat liver mitochondria (RLM), with membrane deenergization followed by spontaneous recoupling in the minute scale, which is associated with glutathione conjugation of fluazinam catalyzed by glutathione-S-transferase (GST). Here, we compare the fluazinam effect on RLM with that on rat kidney (RKM) and heart (RHM) mitochondria by monitoring three bioenergetic parameters: oxygen consumption rate, mitochondrial membrane potential and reduction of nucleotides. Only in RLM, the uncoupling activity of fluazinam was transient, i.e. disappeared in a few minutes, whereas in RKM and RHM it was stable in this time scale. We attribute this difference to the increased activity of mitochondrial GST in liver. We report data on the detection of glutathione-fluazinam conjugates by mass-spectrometry, thin layer chromatography and capillary electrophoresis after incubation of fluazinam with RLM but not with RKM, which supports the assumption of the tissue specificity of the conjugation.

Ester-stabilized phosphorus ylides as protonophores on bilayer lipid membranes, mitochondria and chloroplasts.

Triphenylphosphonium ylides are commonly used as key intermediates in the Wittig reaction. Based on the known acidities of stabilized ylide precursors, we proposed that a methylene group adjacent to phosphorus in these compounds can ensure proton shuttling across lipid membranes. Here, we synthesized (decyloxycarbonylmethyl)triphenylphosphonium bromide (CMTPP-C10) by reaction of triphenylphosphine with decyl bromoacetate. This phosphonium salt precursor of the ester-stabilized phosphorus ylide along with its octyl (CMTPP-C8) and dodecyl (CMTPP-C12) analogues was found to be a carrier of protons in mitochondrial, chloroplast and artificial lipid membranes, suggesting that it can reversibly release hydrogen ions and diffuse through the membranes in both zwitterionic (ylide) and cationic forms. The CMTPP-C10-mediated electrical current across planar bilayer lipid membranes exhibited pronounced proton selectivity. Similar to conventional protonophores, known to uncouple electron transport and ATP synthesis, CMTPP-Cn (n = 8, 10, 12) stimulated mitochondrial respiration, while decreasing membrane potential, at micromolar concentrations, thereby showing the classical uncoupling activity in mitochondria. CMTPP-C12 also caused dissipation of transmembrane pH gradient on chloroplast membranes. Importantly, CMTPP-C10 exhibited substantially lower toxicity in cell culture, than C12TPP. Thus, we report the finding of a new class of ylide-type protonophores, which is of substantial interest due to promising therapeutic properties of uncouplers.

Alkyl esters of 7-hydroxycoumarin-3-carboxylic acid as potent tissue-specific uncouplers of oxidative phosphorylation: Involvement of ATP/ADP translocase in mitochondrial uncoupling.

An impressive body of evidence has been accumulated now on sound beneficial effects of mitochondrial uncouplers in struggling with the most dangerous pathologies such as cancer, infective diseases, neurodegeneration and obesity. To increase their efficacy while gaining further insight in the mechanism of the uncoupling action has been remaining a challenge. Encouraged by our previous promising results on lipophilic derivatives of 7-hydroxycoumarin-4-acetic acid (UB-4 esters), here, we use a 7-hydroxycoumarin-3-carboxylic acid scaffold to synthesize a new series of 7-hydroxycoumarin (umbelliferone, UB)-derived uncouplers of oxidative phosphorylation - alkyl esters of umbelliferone-3-carboxylic acid (UB-3 esters) with varying carbon chain length. Compared to the UB-4 derivatives, UB-3 esters proved to be stronger uncouplers: the most effective of them caused a pronounced increase in the respiration rate of isolated rat heart mitochondria (RHM) at submicromolar concentrations. Both of these series of UB derivatives exhibited a striking difference between their uncoupling patterns in mitochondria isolated from liver and heart or kidney, namely: a pronounced but transient decrease in membrane potential, followed by its recovery, was observed after the addition of these compounds to isolated rat liver mitochondria (RLM), while the depolarization of RHM and rat kidney mitochondria (RKM) was rather stable under the same conditions. Interestingly, partial reversal of this depolarization in RHM and RKM was caused by carboxyatractyloside, an inhibitor of ATP/ADP translocase, thereby pointing to the involvement of this mitochondrial membrane protein in the uncoupling activity of both UB-3 and UB-4 esters. The fast membrane potential recovery in RLM uncoupled by the addition of the UB esters was apparently associated with hydrolysis of these compounds, catalyzed by mitochondrial aldehyde dehydrogenase (ALDH2), being in high abundance in liver compared to other tissues. Protonophoric properties of the UB derivatives in isolated mitochondria were confirmed by measurements of RHM swelling in the presence of potassium acetate. In model bilayer lipid membranes (liposomes), proton-carrying activity of UB-3 esters was demonstrated by measuring fluorescence response of the pH-dependent dye pyranine. Electrophysiological experiments on identified neurons from Lymnaea stagnalis demonstrated low neurotoxicity of UB-3 esters. Resazurin-based cell viability assay showed low toxicity of UB-3 esters to HEK293 cells and primary human fibroblasts. Thus, the present results enable us to consider UB-3 esters as effective tissue-specific protonophoric mitochondrial uncouplers.

Protonophoric action of BAM15 on planar bilayers, liposomes, mitochondria, bacteria and neurons.

Small molecules capable of uncoupling respiration and ATP synthesis in mitochondria are protective towards various cell malfunctions. Recently (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (BAM15), a new compound of this type, has become popular as a potent mitochondria-selective depolarizing agent producing minimal adverse effects. To validate protonophoric mechanism of BAM15 action, we examined its behavior in bilayer lipid membranes (BLM). BAM15 proved to be a typical anionic protonophore with the activity on planar membranes being suppressed upon decreasing membrane dipole potential. In both planar BLM and liposomes, BAM15 induced proton conductance with the potency close to that of the classical protonophoric uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). In isolated rat liver mitochondria (RLM), BAM15 caused membrane potential collapse, increased respiration rate and induced Ca2+ efflux at concentrations slightly higher than those for CCCP. Surprisingly, the uncoupling action of BAM15 on isolated RLM, in contrast to that of CCCP, was partially reversed by carboxyatractyloside (CATR), an inhibitor of adenine nucleotide translocase, thereby indicating involvement of this protein in the BAM15-induced uncoupling. BAM15 inhibited growth of Bacillus subtilis at micromolar concentrations. In electrophysiological experiments on molluscan neurons, BAM15 caused plasma membrane depolarization and suppression of electrical activity, but the effect developed more slowly than that of CCCP.

Usnic acid as calcium ionophore and mast cells stimulator.

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.

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.

Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K(0.5)(Trp)=1.3 micromol l(-1) vs K(0.5)(Phe)=430 micromol l(-1)) and [2 or 1 Na(+) or K(+)]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

The invertebrate B(0) system transporter, D. melanogaster NAT1, has unique d-amino acid affinity and mediates gut and brain functions.

The CG3252 gene product, DmNAT1, represents the first Nutrient Amino acid Transporter cloned from Drosophila. It absorbs a broader set of neutral amino acids versus earlier characterized insect NATs and mammalian NATs-B(0) system transporters from the Sodium Neurotransmitter symporter Family (SNF, a.k.a. solute carrier family 6, SLC6). In addition to B(0)-specific l-substrates, DmNAT1 equally or more effectively transports d-amino acids with sub-millimolar affinities and 1:1 sodium:amino acid transport stoichiometry. DmNAT1 is strongly transcribed in the absorptive and secretory regions of the larval alimentary canal and larval brain, revealing its roles in the primary absorption and redistribution of large neutral l-amino acids as well as corresponding d-isomers. The absorption of d-amino acids via DmNAT1 may benefit the acquisition of fermented and symbiotic products, and may support the unique capacity of fruit fly larvae to utilize a diet with substitution of essential amino acids by d-isomers. It also suggests a remarkable adaptive plasticity of NAT-SLC6 mechanisms via alterations of a few identifiable sites in the substrate-binding pocket. The strong transcription in the brain suggests roles for DmNAT1 in neuronal nutrition and clearance of l-neutral amino acids from the fly brain. In addition, neuronal DmNAT1 may absorb synaptic d-serine and modulate NMDA receptor-coupled signal transduction. The characterization of the first invertebrate B(0)-like transporter extends the biological roles of the SLC6 family, revealing adaptations for the absorption of d-isomers of the essential amino acids. These findings suggest that some members of the NAT-SLC6 subfamily are evolving specific properties which contribute to nutrient symbiotic relationships and neuronal functions.

Synergy and specificity of two Na+-aromatic amino acid symporters in the model alimentary canal of mosquito larvae.

The nutrient amino acid transporter (NAT) subfamily is the largest subdivision of the sodium neurotransmitter symporter family (SNF; also known as SLC6; HUGO). There are seven members of the NAT population in the African malaria mosquito Anopheles gambiae, two of which, AgNAT6 and AgNAT8, preferably transport indole- and phenyl-branched substrates, respectively. The relative expression and distribution of these aromatic NATs were examined with transporter-specific antibodies in Xenopus oocytes and mosquito larval alimentary canal, representing heterologous and tissue expression systems, respectively. NAT-specific aromatic-substrate-induced currents strongly corresponded with specific accumulation of both transporters in the plasma membrane of oocytes. Immunolabeling revealed elevated expressions of both transporters in specific regions of the larval alimentary canal, including salivary glands, cardia, gastric caeca, posterior midgut and Malpighian tubules. Differences in relative expression densities and spatial distribution of the transporters were prominent in virtually all of these regions, suggesting unique profiles of the aromatic amino acid absorption. For the first time reversal of the location of a transporter between apical and basal membranes was identified in posterior and anterior epithelial domains corresponding with secretory and absorptive epithelial functions, respectively. Both aromatic NATs formed putative homodimers in the larval gut whereas functional monomers were over-expressed heterologously in Xenopus oocytes. The results unequivocally suggest functional synergy between substrate-specific AgNAT6 and AgNAT8 in intracellular absorption of aromatic amino acids. More broadly, they suggest that the specific selectivity, regional expression and polarized membrane docking of NATs represent key adaptive traits shaping functional patterns of essential amino acid absorption in the metazoan alimentary canal and other tissues.

Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family.

Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B(0) type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

Vestibular compensation in lampreys: restoration of symmetry in reticulospinal commands.

Removal of a vestibular organ (unilateral labyrinthectomy, UL) in the lamprey results in a loss of equilibrium, so that the animal rolls (rotates around its longitudinal axis) when swimming. Owing to vestibular compensation, UL animals gradually restore postural equilibrium and, in a few weeks, swim without rolling. Important elements of the postural network in the lamprey are the reticulospinal (RS) neurons, which are driven by vestibular input and transmit commands for postural corrections to the spinal cord. As shown previously, a loss of equilibrium after UL is associated with disappearance of vestibular responses in the contralateral group of RS neurons. Are these responses restored in animals after compensation? To answer this question, we recorded vestibular responses in RS neurons (elicited by rotation of the compensated animal in the roll plane) by means of chronically implanted electrodes. We found that the responses re-appeared in the compensated animals. This result supports the hypothesis that the loss of equilibrium after UL was caused by asymmetry in supraspinal motor commands, and the recovery of postural control in compensated animals was due to a restoration of symmetry.