Wheat root cells functioning under inhibition of I and II complexes of mitochondrial respiratory chain

A joint effect of rotenone and malonate on the intensity of respiration, output of K+ and ultrastructure of wheat root cells treated for 6 h was studied. The addition of malonate to rotenone containing solution, in which wheat roots had been incubated for an hour, caused further decrease in respiration intensity and K+ output into external medium. Many mitochondria acquired torus shape in 2h after malonate addition. The increase in respiratory intensity and re-entry of K+ from the incubation medium into the cells were observed during following hours of incubation. We assume that reparation and adaptation processes took place in this case. The observed contacts of endoplasmic reticulum lumens with mitochondria are indicative of possible synthesis of an enzyme able to metabolize malonate to acetyl-CoA and CO2. We propose that torus shape of mitochondria is due to the increase in their outer surfaces, that, in turn, is a result of activation of external NAD(P)H-dehydrogenase. These findings may be evidence of possible adaptation of the root cells to the joint effect of the inhibitors.     

The redox state of pyridine nucleotides controls permeability of uncoupled mitochondria to K+

Heart mitochondria respiring in the presence of P_i release endogenous K⁺ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K⁺, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K⁺-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K⁺. These changes are independent of respiration. The results suggest that a latent pathway for K⁺-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca²⁺ uptake and release is considered.     

Ultrastructural characteristics of mitochondria during cell adaptation to rotenone

A study was made of respiration, heat production, K+ output and ultrastructure of wheat root cells treated for 6 h with rotenone (10 microM), an inhibitor of HADH-ubiquinone oxidoreductase (Complex I). Besides, the involvement of alternative pathways for adaptation to this inhibitor was studied. After 20 min of treatment, a brightened mitochondrial matrix and mitochondria with torus shapes were observed. We propose that the outer area of mitochondria increases due to their torus shapes, and this can point to the activating of extremal NAD(P)H-dehydrogenase, which uses enternal NAD(P)H. Further on the normal ultrastructure of mitochondria was observed, which may result from activation of succinate dehydrogenase and rotenone resistant NAD(P)H-dehydrogenase. After 1 h of treatment, a decrease in respiration, heat production, K+ output and pH increase of incubation medium were observed. Starting from 2 h of incubation and up to the end of the experiment, an increase of respiration and heat production was observed, pointing to the activation of oxidative phosphorilation. Besides, re-entry of K+ and pH decrease in the incubation medium were observed. We conclude that these findings may indicate to a possible adaptation of root cells to this inhibitor. We propose that the torus shape of mitochondria may be associated with function of external NAD(P)H-dehydrogenase.     

Mitochondrial morphology and dynamics in <Emphasis Type="Italic">Triticum aestivum</Emphasis> roots in response to rotenone and antimycin A

Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.     

Monitoring mitochondrial electron fluxes using NAD(P)H-flavoprotein fluorometry reveals complex action of isoflurane on cardiomyocytes

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes.     

Role of Oxidative Metabolism in the Energy Suppply of Active Potassium Transport in Erythrocytes of the Lamprey Lampetra fluviatilis

To study role of glycolysis and oxidative metabolism in providing active transport of monovalent cations, isolated erythrocytes of the lamprey Lampetra fluviatlis were incubated at 20°C in the presence of various metabolic inhibitors. The active (ouabain-sensitive) K⁺ (⁸⁶Rb) influx into erythrocytes did not change after cell incubation for 1–2 h in the absence of glucose or in the presence of 10 mM deoxy-D-glucose or 1 mM monoiodoacetate. Inhibitors of oxidative phosphorylation (antimycin A, rotenone, sodium azide, cyanide) produced a significant decrease (on average, by 74% ) in the active K⁺ transport in the lamprey erythrocytes. All blockers of oxidative phosphorylation produced the same degree of inhibition of the K⁺ transport after the cell pre-incubation with them for 30 and 60 min. In experiments with rotenone, the K⁺ influx was reduced statistically significantly as early as in 5 min of cell incubation and reached a maximal effect after 10–20 min. The intracellular ATP content in erythrocytes decreased by 17, 37, and 45% after 5, 10, and 20 min of cell incubation with rotenone, respectively. The active K⁺ transport in the lamprey erythrocytes is most likely to be closely associated with the intracellular ATP concentration. The data obtained indicate that the energy supply of the Na,K-pump in the lamprey erythrocytes is due exclusively to oxidative phosphorylation processes.     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

Effect of KCl Medium on Malate Oxidation in Mitochondria of Sugar Beet Taproot

Isolated mitochondria were obtained from growing and stored sugar beet (Beta vulgaris L.) taproots. These preparations were used to monitor the mitochondrial matrix volume and malate oxidation after the replacement of sucrose with KCl in the reaction medium. The transfer of mitochondria from sucrose-containing isolation medium to the isoosmotic KCl solution initiated spontaneous or energy-dependent (in the presence of respiratory substrate) swelling whose kinetic parameters (the initial rate and amplitude) were virtually independent of the plant age. At the same time, effects of KCl-induced swelling on oxidative and phosphorylating activities of mitochondria were age-dependent. In mitochondria from growing taproots, K⁺ ions stimulated nonphosphorylating malate oxidation, thereby decreasing the respiratory control ratio and the ADP/O coefficient. The incubation of mitochondria from stored taproots in KCl solution induced a short-term activation and subsequent progressive inhibition of malate oxidation but did not inhibit the oxidation of exogenous NADH. The inhibition of malate oxidation was not released by adding ADP or uncouplers and was enhanced in the presence of valinomycin. The swelling of mitochondria in KCl solutions did not impair the integrity of mitochondrial membranes and did not preclude stimulation of malate oxidation by exogenous NAD. It is supposed that the KCl-induced inhibition of respiration is related to a large increase in the matrix volume and a drastic decrease in the concentration of a coenzyme NAD. Previous studies with isolated mitochondria from stored taproots showed that the mitochondrial NAD level was a rate-limiting factor of malate oxidation assayed in the sucrose-containing media. A possible role of K⁺-transporting mechanisms in regulation of mitochondrial matrix volume and metabolic activity of plant mitochondria is discussed.     

Regulation of palmitoylcarnitine oxidation in isolated rat liver mitochondria. Role of the redox state of NAD(H)

The redox-mediated regulation of palmitoylcarnitine oxidation was studied in isolated rat liver mitochondria in which the mitochondrial free NADH/NAD⁺ ratio was controlled by graded concentrations of acetoacetate and ketomalonate in a rotenone and malonate-inhibited system in the presence of ADP. The NADH/ NAD⁺ ratio was buffered kinetically by adjusting the concentrations of the hydrogen acceptor substances and determined by calibrated NAD(P)H fluorometry of the mitochondrial suspension. A two-fold variation in the β-oxidation rate and a five-fold variation in the free NADH/NAD⁺ ratio was obtained in the presence of rotenone. A non-linear negative correlation was found between the acetyl-CoA concentration and the β-oxidation rate and a negative correlation between the long-chain acyl-CoA concentration and the β-oxidation rate. The data indicate that the redox state is a partial controller of the β-oxidation rate in liver mitochondria. The contribution of acetyl-CoA, a putative regulator of β-oxidation at the acyl-CoA thiolase step is small under the conditions used.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.     

Evaluation of electron-transfer flavoprotein and alpha-lipoamide dehydrogenase redox states by two-channel fluorimetry and its application to the investigation of beta-oxidation

A new method permitting the simultaneous evaluation of the redox states of alpha-lipoamide dehydrogenase and electron-transfer flavoprotein in intact rat liver mitochondria by two-channel fluorimetry is described. It is shown that correction for the partial overlap of emission spectra can readily be introduced after a calibration procedure is performed. This method was applied to the investigation into regulation of palmitoylcarnitine oxidation. It was found that in the presence of rotenone, malonate and a redox buffer for the mitochondrial NAD-system, the beta-oxidation flux was sensitive to variations in redox state of respiratory chain electron carriers at low states of NAD reduction. Therefore, the concept of beta-oxidation control caused solely by the NAD redox state can no longer be sustained.     

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

The objective of this study was to detect ATP-sensitive K⁺ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K⁺-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of K_ATP-channel (ATP-sensitive K⁺-channel) – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K⁺ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of K_ATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of K_ATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.     

A malate-oxaloacetate shuttle linking cytosolic fatty acid α-oxidation to the mitochondrial electron transport chain in uncoupled fresh potato slices

The possible existence of a malonate-sensitive dicarboxylate-mediated electron shuttle between microsomal NAD-linked fatty acid α-oxidation and the mitochondrial electron transport chain in uncoupled fresh potato slices was investigated. Uncoupled slice respiration is inhibited by benzylmalonate and butylmalonate, inhibitors of dicarboxylate transport into mitochondria. Uncoupled slice respiration is also inhibited by rotenone, an indication of intramitochondrial NADH oxidation. Since fatty acid α-oxidation per se is rotenone insensitive, rotenone and benzylmalonate inhibition of the oxidation of carboxyl-labeled myristate in slices points to a dicarboxylic acid shuttle linking microsomal fatty acid a-oxidation with intramitochondrial NADH dehydrogenase.
Malonute inhibits both respiration and ¹⁴CO², release from carboxyl-labeled myristate in fresh uncoupled slices, as do inhibitors of dicarboxylate transport. Mitochondrial studies show that malonate inhibits malate oxidation but not malate dehydrogenase per se. Furthermore, malonate inhibits malate transport more severely than malate oxidation. Accordingly, mulonate inhibition of uncoupled slice respiration in the absence of tricarboxylic acid cycle activity is attributed to its interference with mitochondrial malate transport, and its consequent curtailment of a putative malate-OAA shuttle linked to cytosolic NAD-mediated fatty acid α-oxidation.     

Temporary Sequestration of Potassium by Mitochondria in Astrocytes

Increases in extracellular potassium concentration ([K⁺]_o), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K⁺_o, the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K⁺ in astrocytes. Astrocytes were loaded with the fluorescent K⁺ indicator PBFI and release of K⁺ from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K⁺]_o led to increases in K⁺ within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K⁺] ([K⁺]_i) transients evoked by brief exposures to CCCP. When mitochondrial K⁺ sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K⁺]_i upon exposure to elevated [K⁺]_o. Blockade of plasmalemmal K⁺ uptake routes by ouabain, Ba²⁺, or a mixture of voltage-activated K⁺ channel inhibitors reduced K⁺ uptake into mitochondria. Also, reductions in mitochondrial K⁺ uptake occurred in the presence of mito-K_ATP channel inhibitors. Rises in [K⁺]_i evoked by brief applications of CCCP following exposure to high [K⁺]_o were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K⁺ uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K⁺_o handling by astrocytes.     

In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity

Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer’s disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.     

Salicylic acid induces the proton conductance in the inner mitochondrial membrane of lupine cotyledons

The influence of salicylic acid (SA) on generation of membrane potential (Δψ) at the inner membrane of isolated mitochondria from cotyledons of lupine seedlings (Lupinus angustifolius L.) was investigated. The mitochondrial preparations conformed to all criteria of the intactness: the organelles were characterized by the integrity of their membranes and by tight coupling of oxidation and phosphorylation. High functional activity of mitochondria was also evident from their ability to generate Δψ during succinate oxidation and from the long-term maintenance of steady-state transmembrane potential by virtue of electrontransport chain (ETC) operation or ATP hydrolysis after the inhibition of respiratory ETC. The addition of SA to the incubation medium (0.5–1.0 mM) induced a fast and complete dissipation of Δψ after a distinct lag period. The Δψ was not restored by subsequent ATP hydrolysis, indicating that the phytohormone SA induced the proton conductance of the inner membrane. The SA-induced collapse of Δψ was observed under suppression of ETC by anaerobiosis, cyanide, or inhibitory concentrations of the phytohormone. The SAinduced dissipation of Δψ was not reversed by cyclosporine A but was prevented in the presence of dithiothreitol (DTT). Conversely, the incubation of mitochondria in the presence of phenylarsine oxide (PAO) known to oxidize the protein thiol groups also elevated the proton conductance and eliminated Δψ at the inner membrane of lupine mitochondria. The PAO-induced Δψ collapse was not reversed in the presence of ATP, but Δψ was restored after the addition of DTT. These results and the literature data suggest that, under suppressed ETC activity, salicylic acid permeabilizes the inner membrane of mitochondria from cotyledons of lupine seedlings due to opening of a specialized mitochondrial uncoupling channel (MUC) that is permeable to protons and, possibly, to other small cations (K⁺, Ca²⁺). An important role in the induction of MUC belongs apparently to oxidative stress resulting in oxidation of thiol groups in protein molecules that constitute this channel or regulate the channel activity.     

Effect of malonate on the structural and functional changes of wheat Triticum aestivum L. root cells

A study was made of respiration, output of K+ and ultrastructure of wheat root cells treated for 6 h with malonic acid (MA) (15 mM), an inhibitor of succinate dehydrogenase. After a 1 h treatment, on the background of a decrease in respiration, and output of K+ an increased number of lumens of smooth endoplasmic reticulum was observed. These changes may be the result of lipid biosynthesis. Within first hours of treatment with MA, the mitochondrial matrix was becoming more brightened, and after 3 h all organelles became transparent. Moreover, mitochondria increased in size and almost lacked cristae. After 4 h mitochondria assumed their normal sizes due, presumably, to a competitive action of malonate. After 5 h the matrix was brightened again, mitochondria augmented in size, several organelles acquired torus shapes, and their outer area was eventually increased. We found contacts of endoplasmic reticulum lumens with mitochondria, which may suggest the synthesis of an enzyme, able to transform to malonate. After a 6 h exposure of MA, we observed the increase of respiration, re-entry of K+ and normal ultrastructure of mitochondria. Based on our experiments, we conclude that adaptation of root cells may be a result of external NADPH-dehydrogenase activity and MA detoxification.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

Ionophoric actions of avenaciolide on mitochondrial cations

Avenaciolide produces an initial stimulation of mitochondrial respiration followed at high doses of the drug by a decline in respiration to less than the unstimulated rate; under these conditions the mitochondria are insensitive to ADP and to uncoupler. At lower avenaciolide concentrations followed by ADP there is a sustained acceleration of respiration which is sensitive to EDTA and oligomycin, pointing to the existence of a Mg-requiring ATPase.Spectrophotometric tests with bromthymol blue and fluorimetry show a similarity between the responses to avenaciolide and divalent cations.Mitochondrial contents of substrate anions and cations are altered by avenaciolide; the extent of the changes depend on the level of the drug used and also on the composition of the medium. If K⁺ is present with an energy source, the uptake of K⁺ at the start of an incubation is enhanced by avenaciolide when supplied at less than 25–30 nmole/mg protein, and the K⁺ gain is accompanied by an uptake of substrate anion; at higher concentrations of avenaciolide the direction of flow is reversed with loss of K⁺, divalent cations, and substrate anions. In potassium-free media, or in the absence of energy only losses of ions are found.Addition of avenaciolide to mitochondria onto which [¹⁴C]octyl malonate had previously been adsorbed results in a discharge of the labeled compound.