Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

Substrate-dependent effect of phenolic antioxidants on Ca2+ accumulation by rat liver mitochondria

The effect of different phenolic antioxidants on mitochondrial Ca2+ capacity (maximum amount of Ca2+ mitochondria can accumulate) was studied. Butylated hydroxytoluene substantially enhanced the Ca2+ capacity in mitochondria oxidizing succinate, butylated hydroxyanisole had a moderate effect while 2,5-di-(t-butyl)- 1,4 benzohydroquinone did not affect Ca2+ capacity at all. The analysis of Ca2+ accumulation in mitochondria oxidizing succinate in the presence of 2,5-di-(t-butyl)-1,4 benzohydroquinone revealed inhibition of the rate of Ca2+ accumulation. This effect was absent when ATP hydrolysis or NAD+-dependent substrate oxidation supported Ca2+ transport. Direct measurements of oxygen consumption revealed the concentration-dependent inhibition of succinate oxidation by increasing concentrations of 2,5-di-(t-butyl)- 1,4 benzohydroquinone. When succinate was substituted by NAD+-dependent respiratory substrates, the Ca2+ capacity of mitochondria with 2,5-di-(t-butyl)-1,4 benzohydroquinone was even higher than in the presence of butylated hydroxytoluene.     

Effects of Pentobarbitone on 45Ca2+ Transport by Rat Brain Mitochondria

The effects of pentobarbitone on the transport of 45Ca2+ by rat brain mitochondria were studied, using the Ruthenium Red-EGTA quench technique. In the presence of succinate and inorganic phosphate, mitochondria rapidly accumulate 45Ca2+. Pentobarbitone (0.1-1.0 mM) stimulates the initial rate of Ca2+ transport. In contrast, pentobarbitone (1 mM) did not affect the NaCl (50 mM)-induced efflux of 45Ca2+ from mitochondria. Dibucaine (60 micro M), a clinically used local anaesthetic, inhibits both 45Ca2+ uptake an efflux. The results suggest that barbiturate stimulation of mitochondrial Ca2+ uptake may, in combination with effects on other Ca2+ sequestering processes, contribute to the inhibitor of transmitter release observed at a number of synapses.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

Regulation of oxidative phosphorylation in mitochondria by external free Ca2+ concentrations

The rate of oxidative phosphorylation was studied in rat liver mitochondria incubated with free Ca2+ concentrations that range from 10(-9) to 5 X 10(-6) M. The highest rate was observed between 0.5-1.0 microM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radioactive inorganic phosphate. The concentration of Ca2+ at which maximal rates of ATP synthesis take place is modified by Mg2+ and phosphate. The dependence of oxidative phosphorylation on Ca2+ was observed with alpha-ketoglutarate, glutamate + malate, and succinate, but not with beta-hydroxybutyrate. At 10(-9) M Ca2+ there is a continuous exit of endogenous Ca2+, while with 10(-6) M Ca2+, intramitochondrial Ca2+ levels remained constant throughout time. Apparently the control of the level of internal Ca2+ by external Ca2+ modulates the rate of oxidative phosphorylation. Uncoupler-stimulated respiration also depends on Ca2+ concentration, even though at 10(-9) to 10(-6) M Ca2+ the rate of oxidative phosphorylation is lower than the rate of uncoupled respiration. The contribution of the ADP/ATP carrier and the ATP synthase to the kinetic regulation of ATP synthesis at 10(-9) and 10(-6) M Ca2+ was evaluated by titrations with carboxyatractyloside and oligomycin, respectively. The contribution of the carrier and the synthase to the regulation of the final rate of ATP synthesis was different at the two concentrations of Ca2+; therefore, the concentration of extramitochondrial Ca2+ influences the overall kinetics of oxidative phosphorylation.     

Contribution of ATP synthase to stimulation of respiration by Ca2+ in heart mitochondria

A variety of experimental conditions were applied with the aim to estimate the correlation between the contribution of ATP synthase to the respiratory flux control and the calcium-induced activation of succinate oxidation in heart mitochondria isolated from rat, rabbit and guinea pig. The sensitivity of respiration in heart mitochondria to the decrease in temperature from 37 degrees C to 28 degrees C decreases in the order rabbit > guinea pig > rat. Ca2+ effect on succinate oxidation rate in state 3 respiration was species- and temperature-dependent and ranged from 0 (rat, 37 degrees C) to +44% (rabbit, 28 degrees C). For mitochondria from all experimental animals, the increase of Ca2+ in physiological range of concentration did not change state 2 respiration rate, and the stimulatory effect of Ca2+ on state 3 respiration was more pronounced at 28 degrees C than at 37 degrees C. The respiratory subsystem was sensitive to Ca2+ ions only in rabbit heart mitochondria. A high positive correlation between Ca2+ ability to stimulate succinate oxidation in state 3 and the control exerted by ATP synthase over the respiratory flux provides argument confirming stimulation of ATP synthase by Ca2+ ions.     

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.     

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Properties of a new calcium ion antagonist on cellular uptake and mitochondrial efflux of calcium ions.

Compound YS 035 [NN-bis-(3,4-dimethoxyphenethyl)-N-methylamine] is a new synthetic compound capable of inhibiting Ca2+ uptake by different cells. The inhibition of Ca2+ uptake by muscle cells isolated from chicken embryo is dose-dependent in the compound YS 035 concentration range 10-30 microM. The new compound also inhibits Ca2+ entry into rat brain synaptosomes and less effectively into baby-hamster kidney cells. Compound YS 035 partially inhibits the slow Ca2+ release induced by Ruthenium Red and the rapid Na+-dependent efflux from heart mitochondria. The inhibition of the Na+/Ca2+ exchange appears to be of a non-competitive type with an apparent Ki of 28 microM. The new Ca2+ antagonist totally inhibits the Ca2+ efflux from liver mitochondria induced by Ruthenium Red, but it does not affect the release induced by uncoupler, respiratory inhibitor or chelator, nor the mitochondrial ATP synthesis and membrane potential. The properties shown by the new compound indicate it to be a Ca2+ antagonist and a useful tool for studies on the mitochondrial Ca2+ transport.     

The effect of alkanols on Ca2+ transport in brain mitochondria.

Ethanol stimulates the Na(+)-dependent Ca2+ efflux in brain mitochondria and inhibits the Na(+)-independent Ca(2+)-efflux. Here, we studied the effects of n-alkanols on the various Ca2+ transport processes in brain mitochondria. Only short-chain alcohols (i.e. methanol, ethanol and propanol) stimulated Na+/Ca2+ exchange. The inhibition of H+/Ca2+ exchange was significant only with ethanol. Short-chain alcohols inhibit while long-chain alcohols activate the cyclosporin-sensitive Ca(2+)-efflux. These data suggest that the mechanism of the alkanols' effects on Na+/Ca2+ exchange, H+/Ca2+ exchange and the cyclosporin sensitive pore are entirely different. Alkanols have no effect on the electrogenic Ca2+ uniporter. Ethanol did not affect the apparent K0.5 for Na+ (7.5 mM) of the Na+/Ca2+ exchange. Similarly, the magnitude of the effect of ethanol did not depend on matrix Ca2+ concentration, suggesting that short-chain alkanols do not stimulate the rate of Na+/Ca2+ exchange by increasing the affinity of the carrier to Ca2+in or Na+out. High concentrations of K+, Mg2+ and Ca2+ enhanced the ethanol effect. It is possible that high surface potential attenuates the effect of ethanol. It is suggested that ethanol stimulation of Na+/Ca2+ exchange depends on the modulation of the surface dielectric constant.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Kinetics of Mg2+ flux into rat liver mitochondria.

Unidirectional fluxes of Mg2+ across the limiting membranes of rat liver mitochondria have been measured in the presence of the respiratory substrate succinate by means of the radioisotope 28Mg. Rates of both influx and efflux of Mg2+ are decreased when respiration is inhibited. A linear dependence of the reciprocal of the Mg2+ influx rate on the reciprocal of the Mg2+ concentration is observed. The apparent Km for Mg2+ averages about 0.7 mM. N-Ethyl-maleimide, an inhibitor of transmembrane phosphate-hydroxyl exchanges, enhances the observed pH dependence of Mg2+, influx. In the presence of MalNEt, the apparent Vmax of Mg2+ influx is greater at pH 8 than at pH 7, and there is a linear dependence of the Mg2+ influx rate on the external OH- concentration. The K+ analogue Tl+ inhibits Mg2+ influx, while La3+, an inhibitor of mitochondrial Ca2+ transport, has no effect on Mg2+ influx. Mg2+ competitively inhibits the flux of K+ into rat liver mitochondria. The mechanism(s) mediating mitochondrial Mg2+ and K+ fluxes appear to be similar in their energy dependence, pH dependence, sensitivity to Tl+, and insensitivity to La3+.     

Oxaloacetate- and acetoacetate-induced calcium efflux from mitochondria occurs by reversal of the uptake pathway.

1. Addition of oxaloacetate or acetoacetate to isolated rat liver mitochondria results in an efflux of Ca2+. Concomitant with this efflux is an immediate oxidation of endogenous nicotinamide nucleotides, a fall in the mitochondrial membrane potential and an increase in the rate of respiration. The primary effect in this sequence may be either (a) physiologically important stimulation of a Ca2+-efflux carrier, followed by Ca2+ re-uptake, a fall in membrane potential and increased respiration, or (b) physiologically unimportant damage to mitochondrial integrity, followed by a fall in membrane potential, increased respiration and Ca2+ efflux. 2. Ruthenium Red and EGTA will restore the increased respiratory rate to one approximating to the control rate of respiration. However, addition of lanthanide, at a concentration which inhibits the uptake but not the normal efflux of Ca2+, inhibits the rate of Ca2+ efflux induced by oxaloacetate or acetoacetate. Therefore the observed efflux is occurring by a reversal of the uptake pathway (uniporter) and thus follows the fall in membrane potential. 3. From these results we conclude that the decrease in membrane potential and increase in the rate of respiration seen during oxaloacetate- or acetoacetate-induced Ca2+ efflux cannot be accounted for by rapid Ca2+ cycling, but are due to damage to mitochondrial integrity.     

Inhibition of Glutamine Transport in Rat Brain Mitochondria by Some Amino Acids and Tricarboxylic Acid Cycle Intermediates

Glutamine transport into rat brain synaptic and non-synaptic mitochondria has been monitored by the uptake of [³H]glutamine and by mitochondrial swelling. The concentration of glutamate in brain mitochondria is calculated to be high, 5–10 mM, indicating that phosphate activated glutaminase localized inside the mitochondria is likely to be dormant and the glutamine taken up not hydrolyzed. The uptake of [³H]glutamine is largely stereospecific. It is inhibited by glutamate, asparagine, aspartate, 2-oxoglutarate and succinate. Glutamate inhibits this uptake into synaptic and non-synaptic mitochondria by 95 and 85%, respectively. The inhibition by glutamate, asparagine, aspartate and succinate can be explained by binding to an inhibitory site whereas the inhibition by 2-oxoglutarate is counteracted by aminooxyacetic acid, which indicates that it is dependent on transamination. The glutamine-induced swelling, a measure of a very low affinity uptake, is inhibited by glutamate at a glutamine concentration of 100 mM, but this inhibition is abolished when the glutamine concentration is raised to 200 mM. This suggests that the very low affinity glutamine uptake is competitively inhibited by glutamate. Furthermore, glutamine-induced swelling is inhibited by 2-oxoglutarate, succinate and malate, similarly to that of the [³H]glutamine uptake. The properties of the mitochondrial glutamine transport are not identical with those of a recently purified renal glutamine carrier.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states. I. Comparison of liver mitochondria from starved and fed rats.

It has been shown that KM values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The Ki values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 muM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adeninenucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

A kinetic study of mitochondrial calcium transport.

This report describes a kinetic analysis of energy-linked Ca2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy-bis(ethylamine)-tetraacetic acid] quenching technique has been used to measure rates of 45Ca2+ transport. Accurately known concentrations of free 45Ca2+ were generated with Ca2+/nitrilotriacetic acids buffers for the determination of substrate/velocity relationships. The results show that the initial velocity of transport is a sigmoidal function of Ca2+ concentration (Hill coefficient = 1.7), the Km being 4 muM Ca4 at 0 degrees C and pH 7.4. These values for the Hill coefficient and the Km remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration. The Ca2+-binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0 degrees C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN- on Ca2+ transport. Studies on the time-course of Ca2+ uptake and of EFTA-induced Ca2+ efflux from pre-loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids. These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca2+ bound to pairs of interacting acidic sites.     

Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. A model for post-ischemic and toxic mitochondrial damage

With a variety of forms of ischemic and toxic tissue injury, cellular accumulation of Ca2+ and generation of oxygen free radicals may have adverse effects upon cellular and, in particular, mitochondrial membranes. Damage to mitochondria, resulting in impaired ATP synthesis and diminished activity of cellular energy-dependent processes, could contribute to cell death. In order to model, in vitro, conditions present post-ischemia or during toxin exposure, the interactions between Ca2+ and oxygen free radicals on isolated renal mitochondria were characterized. The oxygen free radicals were generated by hypoxanthine and xanthine oxidase to simulate in vitro one of the sources of oxygen free radicals in the early post-ischemic period in vivo. With site I substrates, pyruvate and malate, Ca2+ pretreatment, followed by exposure to oxygen free radicals, resulted in an inhibition of electron transport chain function and complete uncoupling of oxidative phosphorylation. These effects were partially mitigated by dibucaine, a phospholipase A2 inhibitor. With the site II substrate, succinate, the electron transport chain defect was not manifest and respiration remained partially coupled. The electron transport chain defect produced by Ca2+ and oxygen free radicals was localized to NADH CoQ reductase. Calcium and oxygen free radicals reduced mitochondrial ATPase activity by 55% and adenine nucleotide translocase activity by 65%. By contrast oxygen free radicals alone reduced ATPase activity by 32% and had no deleterious effects on translocase activity. Dibucaine partially prevented the Ca2+-dependent reduction in ATPase activity and totally prevented the Ca2+-dependent translocase damage observed in the presence of oxygen free radicals. These findings indicate that calcium potentiates oxygen free radical injury to mitochondria. The Ca2+-induced potentiation of oxygen free radical injury likely is due in part to activation of phospholipase A2. This detrimental interaction associated with Ca2+ uptake by mitochondria and exposure of the mitochondria to oxygen free radicals may explain the enhanced cellular injury observed during post-ischemic reperfusion.     

The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals.

1. Studies on the cytochrome spectra of liver mitochondria from control and glucagon-treated rats in State 4, State 3 and in the presence of uncoupler are reported. 2. The stimulation of electron flow between cytochromes c1 and c observed previously [Halestrap (1978) Biochem. J. 172, 399-405] was shown to be an artefact of Ca2+-induced swelling of mitochondria. 3. When precautions were taken to prevent such swelling, glucagon treatment was shown to enhance the reduction of cytochromes c, c1 and b558 in both State 3 and uncoupled conditions with either succinate or glutamate + malate as substrate. An increase in the reduction of cytochromes b562 and b566 was also seen in some, but not all, experiments. 4. In State 4 with succinate but not glutamate + malate as substrate, cytochromes c, c1, b558, b562 and b566 showed increased reduction. 5. Glucagon stimulated oxidation of duroquinol and palmitoylcarnitine by intact mitochondria and of NADH by disrupted mitochondria. 6. No effect of glucagon on succinate dehydrogenase activity or the temperature-dependence of succinate oxidation could be detected. 7. Glucagon enhanced the inhibition of the respiratory chain by colletotrichin, but not antimycin or 8-heptyl-4-hydroxyquinoline N-oxide. 8. These results are interpreted in terms of a primary stimulation by glucagon of the 'Q cycle' [Mitchell (1976) J. Theor. Biol. 62, 827-367] within Complex III (ubiquinol:cytochrome c oxidoreductase) and a secondary site of action involving stimulation of electron flow into Complex III from the ubiquinone pool. 9. Ageing of mitochondria, hyperosmotic treatment or addition of 20 mM-benzyl alcohol opposed the effects of glucagon treatment on cytochrome spectra and colletotrichin inhibition of respiration. 10. These results support the hypothesis that glucagon exerts its effects on the mitochondria by perturbing the membrane structure.     

T-2 toxin inhibits mitochondrial function in yeast

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.