Studies of the effects of trimethyltin, tripropyltin, tributyltin and triphenyltin chlorides on the permeability of the inner mitochondrial membrane by the osmotic swelling technique in NaCl and NH,C1 media show that these compounds mediate transport of chloride ions. Studies in KC1 media show that these compounds mediate a strictly coupled chloride-hydroxide antiporter transport. This exchange allows rapid titration of mitochondrial contents by pulses of HCl. Similar results on erythrocytes and smectic mesophases indicate that this is a direct action of the tin compounds and is not due to modification ofa pre-existing anion carrier in the membrane.The highly toxic trialkyltin compounds have been shown to be potent inhibitors of mitochondrial oxidative phosphorylation by Aldridge and Cremer[1], and Sone and Hagihara [2]. Aldridge et al. [3--51 have shown that these compounds also produce several other effects on mitochondria, for example, uncoupling of oxidative phosphorylation, inhibition of the 2,4 dinitrophenol-stimulated ATPase but stimulat'ion of the ATPase in the absence of 2,4 dinitrophenol, a time-dependent inhibition of 2,4 dinitrophenolstimulated respiration and of state 4 respiration. Unlike inhibitors of oxidative phosphorylation such as oligomycin and aurovertin, the trialkyltin compounds are of known structure and chemical investigation of their mode of action may be possible.A preliminary communication by Selwyn, Stockdale and Dawson [6] outlined investigations intended to define the mode of action of trialkyltin compounds on mitochondria and reported that these compounds produced a rapid chloride-hydroxide exchange across the mitochondrial membrane. This paper describes the effect of trialkyltin compounds on the permeability properties of mitochondrial membranes, erythrocytes and smectic mesophase (liposome) artificial membrane systems.Unusual Abbreviations. Carbonylcyanide p-trifluoromethoxy phenylhydrazone, FCCP ; ethylene glycol-bis-(aminoethy1)-tetraacetic acid, EGTA; N-2-hydroxymethylpiperazine-N'-2-ethanesulphonic acid, HEPES.Enzymes. Carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.1). MATERIALS AND METHODS MitochondriaMitochondria prepared from the livers of adult (150 to 250 g) Wistar rats were used throughout this investigation. The rats were killed by decapitation and the mitochondria prepared by the method of Hogeboom et al. [7] with the following modifications. The isolation medium was 0.25 M sucrose, deionised by passage through a column of Permutit "Deminrelit" mixed bed resin, and contained 5 mM HEPES adjusted to pH 7.5 with sodium hydroxide. The final centrifugation was performed a t 14 500 x g for 10 min. A light fluffy layer on top of the mitochondrial pellet was poured off with the supernatant fluid and the tightly packed mitochondrial pellet resuspended in the same medium t o give a final concentration of about 80 mg/ml mitochondrial protein. ErythrocytesErythrocytes were prepared from human blood (containing acid-citr ate dextrose anti-coagulant) obtained from the r...
In a sucrose medium, free from anions which are effective in the anion-hydroxide exchange catdysed by trialkyltins, the effects of these compounds on mitochondrial respiration are similar to those of oligomycin, i.e. inhibition of respiration coupled to phosphorylation, and of arsenate stimulated respiration. They do not inhibit respiration stimulated by uncouplers nor respiration stimulated by uptake of divalent cations. They do not induce significant light scattering changes in this medium.I n a medium containing potassium chloride similar oligomycin-like effects are observed but in addition some release of respiratory control and marked swelling of the mitochondria are observed. A time-dependent inhibition of uncoupler stimulated respiration appears to be a secondary effect consequent upon extensive swelling. The maximal rate of respiration attainable by the uncoupling effect of the trialkyltin compounds is independent of which compound is used. The concentration of trialkyltin compound required to produce uncoupling is correlated with the concentration required to mediate the chloride hydroxide exchange.In media. containing other anions the maximal rate of respiration produced by trialkyltin compounds is correlated with the rate of electrogenic uniport of the anions through the mitochondrial membrane.Uncoupling by trialkyltin compounds is attributed to the action of the anion-hydroxide exchange followed by leakage of the anion out of the mitochondria. These two processes result in the discharge of both the pH differential and electrical potential difference across the mitochondrial membrane.Apart from a lytic action of triphenyltin chloride and a potent respiratory inhibition in the presence of iodide or thiocyanate ions, it is proposed that all the observed effects of those compounds can be accounted for by the oligomycin-like inhibition and the mediation of an anion hydroxide-exchange across the mitochondrial membrane. shown that they produce a greater inhibition of phosphorylation than of oxidation and also that there is, in the absence of ADP, some degree of release of respiratory control [2]. However this uncoupling activity was not found to stimulate respiration to the same extent as uncouplers such as 2,4-dinitrophenol. Other effecb which distinguish these compounds from uncouplers such as 2,4-dinitrophenol and from inhibitors of the coupling mechanism such as oligomycin are the marked swelling of mitochondria observed under certain conditions Aldridge and Rose [5] published an attractive hypotheais which accounted for these complex effects and provided a molecular basis for the connection between electron transport, phosphorylation of ADP and ion transport. As noted by these authors the precise nature of their hypothesis suggested experimental tests of its validity and this stimulated our investigations of the mode of action of these compounds.All previous observations on the effects of these compounds on mitochondria have been made in media which contained potassium chloride and in view of our repo...
The effects of substituents on the activity of phenols as uncouplers and inhibitors of mitochondrial respiration have been examined. These activities have been correlated with constants describing the effects of the substituents on the electronic and hydrophobic-bonding properties of the phenol. The coefficients in these correlation equations have been compared with each other and with the coefficients in similar correlations for the effects of phenols on the solubilised ATPase from mitochondria, and their activity in mediating proton conduction across artificial lipid membranes and as inhibitors of a variety of enzymes.Differences in the coefficients in the correlation equations for uncoupling and inhibition of respiration indicate that these depend on different properties of the phenol and hence that they are separate effects and that inhibition of substrate accumulation is not a satisfactory explanation for the inhibition of respiration.High concentrations of uncouplers induce swelling of mitochondria. I n the case of 2,4-dibromophenol, or dicoumarol, this occurs a t concentrations similar to those producing inhibition of respiration. With other phenols inhibition of respiration is observed a t concentrations lower than those required to induce swelling.The observations reported here support the hypothesis that phenols uncouple by mediating proton conduction across the inner mitochondrial membrane and that, in general, the inhibition of respiration by high concentrations of phenols is due to direct action on a protein component of the respiratory chain.The classical view that uncouplers of oxidative phosphorylation act by catalysing the breakdown of high energy intermediates [1,2] appears to be supported by the observation that 2,4-dinitrophenol stimulates the activity of the coupling factor ATPase from mitochondria [3,4]. Mitchell's hypothesis that uncouplers act by mediating the passage of protons across the mitochondrial membrane [51 does not account for the effects of uncouplers on the isolated ATPase and it is of interest to determine whether or not these effects are related to uncoupling.A complicating factor is that at high concentrations many uncouplers inhibit mitochondrial respiration and the way in which this inhibition is produced is not certain, alternative proposals being that they inhibit accumulation of substrates [6--81 or that they act directly on components of the respiratory chain [9].One method of investigating the mechanism of action of uncouplers, particularly phenols, has been the study of the relation between their chemical Unusual Abbreviations. HEPES, N-2-hydroxymethylpiperazine-N-2-ethane sulphonic acid; FCCP, p-trifluoromethoxy carbonylcyanide phenylhydrazone. ~-properties and biological activity. It has been found that both the dissociation constant, pKa (the logarithm of the reciprocal of the acid dissociation constant) and lipid solubility have some relation to the uncoupling activity of phenols 110-141.I n chemical correlations it has become customary to use constants charac...
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