The effect of butacaine on adenine nucleotide binding and translocation in rat liver mitochondria.

The effect of the local anaesthetic, butacaine, on adenine nucleotide binding and translocation in rat liver mitochondria partially depleted of their adenine nucleotide content was investigated. The range of butacaine concentrations that inhibit adenine nucleotide translocation and the extent of the inhibition are similar to the values obtained for native mitochondria. Butacaine does not alter either the total number of atractyloside-sensitive binding sites of depleted mitochondria, or the affinity of these sites for ADP or ATP under conditions where a partial inhibition of the rate of adenine nucleotide translocation is observed. The data are consistent with an effect of butacaine on the process by which adenine nucleotides are transported across the mitochondrial inner membrane rather than on the binding of adenine nucleotides to sites on the adenine nucleotide carrier. The results are briefly discussed in relation to the use of local anaesthetics in investigations of the mechanism of adenine nucleotide translocation.     

Relationship between the systems of activation of fatty acids and ademine nucleotide translocase in the mitochondria]

Experiments were conducted on freshly isolated rat liver mitochondria and mitochondria subjected to ageing by two different methods. It was shown that the work of the mitochondrial system of fatty acid activation could lead to inhibition of the adenine nucleotide transport through the internal mitochondrial membrane. Inhibition of adenine nucleotide translocase was eliminated by preincubation of mitochondria with carnitine. The presence in the mitochondrial preparations of fatty acids in the concentration adequate for induction of inhibition of addition of CoA and ATP served as a preculiarity of adenine nucleotide translocase inhibition of the ageing mitochondria. The data obtained permitted to make a supposition on the participation of acyl-CoA formed by the mitochondrial acyl-CoA-synthetase in the regulation of adenine nucleotide transport into the mitochondria.     

Interaction of adenine nucleotides with the adenine nucleotide translocase regulates the developmental changes in proton conductance of the inner mitochondrial membrane.

2-h-old neonatal liver mitochondria, when depleted of adenine nucleotides, showed an 'ohmic' current-voltage relationship and a higher passive proton permeability of the membrane, resembling fetal mitochondrial behaviors for the proton conductance. Incubation of fetal mitochondria with ATP, GDP or carboxyatractyloside promoted a significant reduction in the passive proton permeability of the membrane and the appearance of the characteristic biphasic behavior for the proton conductance. It is concluded that the postnatal increase in intramitochondrial adenine nucleotide concentration promotes, by the interaction of the nucleotides with the adenine nucleotide translocase, the reduction in the passive proton permeability of the mitochondrial membrane, allowing efficient energy conservation in the neonatal liver.     

The Effects of Atractyloside and Bongkrekic Acid on Jerusalem Artichoke Mitochondria in Relation to Adenine Nucleotide Translocation

The translocation of ADP and ATP across the inner membrane ofJerusalem artichoke mitochondria is demonstrated. Atractyloside,a competitive inhibitor of the adenine nucleotide translocatorof mammalian mitochondria, is without effect in artichoke mitochondriawhen added at concentrations normally inhibitory in the animalsystem. Bongkrekic acid, a noncompetitive inhibitor, inhibitsthe adenine nucleotide-dependent processes of artichoke andmammalian mitochondria at similar concentrations. The effectsof atractyloside and bongkrekic acid in relation to adeninenucleotide translocation are discussed.     

Distinct characteristics of Ca(2+)-induced depolarization of isolated brain and liver mitochondria

Ca(2+)-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca(2+) concentrations (about 30--100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca(2+); 20 microm Ca(2+) was required to depolarize liver mitochondria. Ca(2+) did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca(2+)-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.     

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.     

Effects of ionic strength and sulfhydryl reagents on the binding of creatine phosphokinase to heart mitochondrial inner membranes

The concept that creatine phosphokinase is bound to the outer surface of the heart mitochondrial inner membrane originated from observations that the enzyme is retained by water-swollen heart mitochondria and by digitonintreated heart mitochondria suspended in isotonic sucrose. The present study establishes that digitonin-treated mitochondria release creatine phosphokinase in isotonic KCl, and other investigators have reported an identical response for the water-swollen organelles. These observations suggest that mitochondrial creatine phosphokinase is not bound to the outer surface of the inner membrane at a site adjacent to the adenine nucleotide translocase under physiologic conditions.     

Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Distinct characteristics of Ca-induced depolarization of isolated brain and liver mitochondria

Ca²⁺-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca²⁺ concentrations (about 30-100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca²⁺; 20 μM Ca²⁺ was required to depolarize liver mitochondria. Ca²⁺ did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca²⁺-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.     

Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through altered membrane fluidity

Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.     

Inhibition of oxidative phosphorylation by a Ca2+-induced diminution of the adenine nucleotide translocator

The mechanism through which internal Ca²⁺ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca²⁺-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca²⁺ accumulated by the mitochondria and on the time that Ca²⁺ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca²⁺, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca²⁺-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca²⁺.     

Control of energy transformation of mitochondria. Analysis by a quantitative model

A mathematical model of control of energy transformation in mitochondria is presented. The considered processes are: the proton translocation by the respiratory chain, the production of ATP by ATPase, the translocation of adenine nucleotides and of phosphate by their translocators, and a passive backflow of protons through the mitochondrial membrane. The mathematical equations expressing the steady-state kinetics of these processes and the relations between them were derived on the basis of current experimental data. The model predicts fairly well the values of the proton electrochemical gradient, of the ATP/ADP ratios within and outside mitochondria and of the distribution of phosphate between both compartments in different metabolic states of mitochondria. From the general agreement of model computations with experimental data, it is suggested that the electron flux through the respiratory chain is immediately controlled by the energy back-pressure of the proton electrochemical gradient, that the ATPase reaction is near equilibrium in phosphorylating mitochondria but that the adenine nucleotide exchange across the mitochondrial membrane requires some loss of energy. The latter is caused by an inhibition of the translocator by ATP from the outer side or by ADP from the inner side depending on the actual ATP/ADP in both compartments. It explains that no fixed relation exists between the rate of respiration and the phosphorylation state of extramitochondrial adenine nucleotides. The relation is modified by the concentration of phosphate and by intramitochondrial energy utilization.     

The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.     

Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.     

Oxidative phosphorylation in yeast IX. Modification of the mitochondrial adenine nucleotide translocation system in the oxidative phosphorylation-deficient mutant op1

The properties of intact cells and isolated mitochondria of the op₁ mutant of Saccharomyces cerevisiae, which had been shown previously to be deficient in oxidative phosphorylation, have been studied further.

1. 1. When isolated mutant mitochondria were preincubated with substrate and phosphorylation was started by the addition of external ADP, low P/O ratios were obtained under standard conditions. The P/O ratios could be raised to normal values approaching 2 with citrate and succinate in the presence of unusually high concentrations of ADP. Under these conditions the Michaelis constant for ADP of respiration and phosphorylation was found to be 2.9 mM. When isolated mitochondria were added to a medium containing substrate and adenine nucleotide, the Michaelis constant for ADP was found to be lower, about 0.5 mM and maximal P/O ratios of only 0.8 were achieved.

2. 2. Adenine nucleotide translocation across the membrane of the mutant mitochondria was found to be different from that in wild-type mitochondria and dependent on the energy level in mitochondria. When the intramitochondrial nucleotide pool consisted mostly of ADP and AMP, the rate of adenine nucleotide translocation was approx. 30 times lower than in wild-type mitochondria and the Michaelis constants for ADP of the translocation process were similar in the two types of mitochondria, being lower than 10 μM. When the nucleotide pool was enriched in ATP, the translocation rate in mutant mitochondria was as high as in wild-type mitochondria but the Michaelis constant for external adenine nucleotide was more than 100 times higher in the former than in the latter.

3. 3. An examination of the effects of the uncoupler, oligomycin, valinomycin and nigericin on the translocation process in the mutant mitochondria provided additional evidence that varying energization of mutant mitochondria was responsible for the variations of the translocation rates and the Michaelis constants under different experimental conditions.

4. 4. The properties of the adenine nucleotide carrier of mutant mitochondria were studied and found to be different from those of wild-type mitochondria.

5. 5. It has been concluded that the modification of adenine nucleotide translocation across the mitochondrial membrane is responsible for oxidative phosphorylation deficiency in the op₁ mutant. The implications of these findings for the understanding of the adenine nucleotide translocation mechanism and the role of the translocation system in the control of cellular syntheses and growth are discussed.

The role of the adenine nucleotide translocator in oxidative phosphorylation. A theoretical investigation on the basis of a comprehensive rate law of the translocator

A minimum model of adenine nucleotide exchange through the inner membrane of mitochondria is presented. The model is based on a sequential mechanism, which presumes ternary complexes formed by binding of metabolites from both sides of the membrane. The model explains the asymmetric kinetics of ADP-ATP exchange as a consequence of its electrogenic character. In energized mitochondria, a part of the membrane potential suppresses the binding of extramitochondrial ATP in competition with ADP. The remaining part of the potential difference inhibits the back exchange of internal ADP for external ATP. The assumption of particular energy-dependent conformational states of the translocator is not necessary. The model is not only compatible with the kinetic properties reported in the literature about the adenine nucleotide exchange, but it also correctly describes the response of mitochondrial respiration to the extramitochondrial ATP/ADP ratio under different conditions. The model computations reveal that the translocation step requires some loss of free energy as driving force. The size of the driving force depends on the flux rate as well as on the extra- and intramitochondrial ATP/ADP quotients. By both quotients the translocator controls the export of ATP formed by oxidative phosphorylation in mitochondria.     

Control of oxidative phosphorylation in rat heart mitochondria. The role of the adenine nucleotide carrier

Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F1-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). Concomitantly with the inhibition of O2 consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : II. Effect of Bongkrekic Acid

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.