Calcium efflux parallel to total phosphate retention in rat liver mitochondria

Phosphate efflux from uncoupled rat liver mitochondria was completely inhibited when mersalyl plus butylmalonate and ATP were added to a sucrose suspending medium. Despite the total retention of phosphate a calcium efflux was observed even in presence of ruthenium red. Under the above conditions no phosphate is transported in association with the ADP/ATP carrier. While mersalyl completely blocked the phosphate release induced by ruthenium red or EGTA from coupled mitochondria it only partially inhibited the CA2+-efflux. The inhibition of Ca2+ efflux was almost completely abolished in the presence of acetate. The existence of a co-transport of Ca2+ associated with phosphate is discussed.     

The divalent cation requirement of the mitochondrial glycerol-3-phosphate dehydrogenase

Coupled respiration by blowfly mitochondria has been utilized to demonstrate an absolute divalent cation requirement for glycerol 3-phosphate respiration. With ADP, phosphate and EGTA, the respiration rate (state 3) decreases as a function of the amount of oxygen reduced, to approximately 15% of its maximum value, even at 40 mM dl-glycerol 3-phosphate; it can be increased to its maximum value by the addition of Ca²⁺, Sr²⁺ or Mn²⁺. The decline in state 3 rate is not due to the removal of membrane-bound calcium into the matrix by the calcium carrier, since it occurs in the presence of ruthenium red. The effect is energy-dependent since the state 3 respiration does not decrease in the presence of uncouplers. The increase in respiration upon the addition of calcium is not due to the energy-dependent calcium transport since it is sensitive to oligomycin and insensitive to ruthenium red.The divalent cation effector site is located on the glycerol-3-phosphate dehydrogense, since state 3 (or state 4) pyruvate-proline respiration (NAD-linked) is not affected by EGTA. Yet the state 3 pyruvate-proline respiration removes calcium so effectively from the glycerol-3-phosphate dehydrogenase in the presence of EGTA, that added calcium stimulates glycerol 3-phosphate (26.4 mM) respiration about 22-fold.Since uncouplers stimulate the inhibited glycerol 3-phosphate respiration only to a very small extent, a calcium stimulation of the rate of phenazine methosulfate reduction by glycerol 3-phosphate (26.4 mM) which bypasses all phosphorylation sites, should be detectable. Only a 3-fold stimulation was observed.The present experiments suggest that upon complete removal of divalent cations from the dehydrogenase, glycerol 3-phosphate does not act as a homotropic effector in the coenzyme Q reductase reaction.     

Calcium-dependent release of acetyl-coenzyme A from liver mitochondria

Calcium ions (10 microM) enhance the permeability of the hepatic inner mitochondrial membrane to acetyl-CoA (and CoA). This effect is suppressed by the absence of phosphate ions or by the presence of EGTA, La3+, or ruthenium red. Exposure of mitochondria to Ca2+ for short intervals of time (e.g., 10 min) results in a reversible permeability change with release of acetyl-CoA and retention of sensitivity to EGTA. It is suggested that conditions resulting in an increase of the calcium ion concentration in the cytoplasm may thereby increase the rate of transfer of acetyl-CoA from the mitochondria to the cytoplasm.     

Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane enabled us to study Ca2+ transport and oxidative phosphorylation in mitochondria in situ. Addition of Ca2+ to these preparations evoked a cycle of respiratory stimulation. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by antimycin A, and stimulated by inorganic phosphate. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone to digitonin-permeabilized T. cruzi epimastigotes under steady-state conditions was followed by Ca2+ release. Antimycin A- and carbonyl cyanide p-trifluoromethoxyphenylhydrazonein-sensitive Ca2+ uptake was also detected in digitonin-permeabilized epimastigotes. Accordingly, ATP stimulated Ca2+ uptake by preparations de-energized by oligomycin and antimycin A. In conclusion, in contrast to previous reports indicating that a Ca2+ transport system occurs only in mitochondria from vertebrate tissues, T. cruzi epimastigotes also possess a similar system. In addition, these protozoan mitochondria have an extremely high resistance to the deleterious effects of massive Ca2+ loads in comparison with most types of mammalian mitochondria.     

Respiration and phosphorylation by mitochondria from the hepatopancreas of the blue crab (Callinectes sapidus)

Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show high rates of oxidation of pyruvate + proline and of various intermediates of the tricarboxylic acid cycle in a 280- to 380-mOsm sucrose-mannitol medium supplemented with bovine serum albumin. The respiratory control ratio ranged from 6 to 10. Respiration was accompanied by phosphorylation of ADP, with the expected ADP:O ratio for all substrates tested except α-ketoglutarate, indicating that all three energy-conserving sites were functional. Fatty acids were also oxidized, but no oxidation of β-hydroxybutyrate, glycerol 3-phosphate, or NADH was observed. The crab mitochondria showed a relatively low affinity for phosphate, but a normal affinity for ADP. Respiration and phosphorylation gave the normal responses to respiratory chain inhibitors, uncoupling agents, oligomycin, and ionophores. Crab mitochondria have an exceptionally high content of phosphate, exceeding 1000 nmoles per mg protein, but a normal energy charge of the adenylic system. An unusual feature is the presence of considerable arginine kinase activity, which is usually thought to be restricted to muscle and nerve tissue in anthropods. This enzyme allows arginine to act as secondary phosphate acceptor. The arginine kinase is located on the cytosol side of the atractyloside-sensitive barrier and is thus unable to transfer the terminal phosphate group of matrix ATP directly to arginine.     

On the state of calcium ions in isolated rat liver mitochondria IV. Prevention of phosphate-induced mitochondrial destruction by ruthenium red-insensitive calcium release

Ruthenium red prevented the spontaneous calcium release and the accompanying mitochondrial destruction occurring in calcium-loaded mitochondria in the presence of phosphate. Under these conditions delta pH and membrane potential delta psi were preserved and the ruthenium red-induced calcium efflux was low and at a constant rate. On prolonged incubation with calcium prior to addition of ruthenium red increasingly more mitochondrial calcium developed into a pool rapidly dischargeable by ruthenium red. This development was accompanied by stimulation of respiration which was, however, not abolished by ruthenium red as could have been expected if it had been caused by calcium cycling. Calcium therefore altered mitochondria by a different mechanism than by cycling across the inner membrane.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Control of the induction of ion transport through mitochondrial membranes by the enzymes of the oxidative phosphorylation system

It has been shown that the induction of earlier described system of potassium-dependent transport of hydrogen ions in mitochondria at low pH values of the incubation medium is inhibited by the inhibitors of mitochondria respiratory chain and ATPase. It has been found that antimycin and oligomycin suppress the efflux of potassium ions from mitochondria in the presence of succinic acid. The uncoupler (FCCP) turns the effect of ATPase inhibitors to the efflux of potassium ions and acceleration of mitochondria respiration under experimental conditions. At the same time TMPD removes the effect of antimycin on potassium ion efflux from uncoupled FCCP of mitochondria. The data obtained are explained in terms of the postulate that under experimental conditions along with the system of potassium-dependent ion transport there appears leakage of protons through the ATPase channel. A conclusion is made concerning the control of ion transport induction in mitochondria by the enzymes of oxidative phosphorylation system.     

Role of the Ca2+ cycle in uncoupling of oxidative phosphorylation in liver mitochondria of cold-acclimated rats

Cold acclimation of Wistar rats for 2-4 weeks at about 3 degrees C resulted in an increased respiration rate and a reduced ADP/O ratio in liver mitochondria. With increasing duration of acclimation up to 10-12 weeks, these parameters returned to a normal level. The increase in the respiration rate and the decline of the mitochondrial ADP/O ratio were associated with a significant activation of the electroneutral release of Ca2+. When the animals were acclimated for 10-12 weeks the rate of Ca2+ release reduced to control values. The addition of 1 microM ruthenium red resulted in a decrease in the rates of mitochondrial respiration in control and cold-acclimated rats to approximately equal values and in a partial restoration of the ADP/O ratio in liver mitochondria of rats kept in the cold for 2-4 weeks. The respiratory activity of mitochondria isolated in the presence of 1 mM EGTA unaffected by ruthenium red.     

Calcium ion accumulation and volume changes of isolated liver mitochondria. Reversal of calcium ion-induced swelling

1. The excessive accumulation of Ca²⁺ by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca²⁺ from the mitochondria. When the Ca²⁺ was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.     

EGTA inhibits reverse uniport-dependent Ca2+ release from uncoupled mitochondria. Possible regulation of the Ca2+ uniporter by a Ca2+ binding site on the cytoplasmic side of the inner membrane

When rat liver mitochondria are allowed to accumulate Ca2+, treated with ruthenium red to inhibit reverse activity of the Ca2+ uniporter, and then treated with an uncoupler, they release Ca2+ and endogenous Mg2+ and undergo large amplitude swelling with ultrastructural expansion of the matrix space. These effects are not produced by Ca2+ plus uncoupler alone. Like other "Ca2+-releasing agents" (i.e. N-ethylmaleimide, t-butylhydroperoxide, oxalacetate, etc.), the development of nonspecific permeability produced by ruthenium red plus uncoupler requires accumulated Ca2+ specifically and is antagonized by inhibitors of phospholipase A2. The permeability responses are also antagonized by ionophore A23187, indicating that a rapid pathway for Ca2+ efflux from deenergized mitochondria is necessary to prevent the development of nonspecific permeability. EGTA can be substituted for ruthenium red to produce the nonspecific permeability change in Ca2+-loaded, uncoupler-treated mitochondria. The permeability responses to EGTA plus uncoupler again require accumulated Ca2+ specifically and are antagonized by inhibitors of phospholipase A2 and by ionophore A23187. The equivalent effects of ruthenium red and EGTA on uncoupled, Ca2+-containing mitochondria indicate that reducing the extramitochondrial Ca2+ concentration to the subnanomolar range produces inhibition of reverse uniport activity. It is proposed that inhibition reflect regulation of the uniporter by a Ca2+ binding site which is available from the cytoplasmic side of the inner membrane. EDTA cannot substitute for EGTA to induce nonspecific permeability in Ca2+-loaded, uncoupled mitochondria. Furthermore, EDTA inhibits the response to EGTA with an I50 value of approximately 10 microM. These data suggest that the uniporter regulatory site also binds Mg2+. The data suggest further that Mg2+ binding to the regulatory site is necessary to inhibit reverse uniport activity, even when the site is not occupied by Ca2+.     

The sequestration of Ca2+ by mitochondria in rat heart cells

Rat heart ventricular cells, purified by Percoll density gradient centrifugation, were incubated in the presence of 1.3 mM CaCl2. After 20 min incubation, samples of the cells were lysed in medium containing 0.3 mM digitonin, ruthenium red and EGTA, and a mitochondrial fraction was isolated at intervals thereafter. Extrapolation of the mitochondrial 45Ca2+ contents to zero time enabled the endogenous 45Ca2+ to be estimated at the time of cell lysis. The lysis conditions yielded essentially complete release of lactate dehydrogenase from the cells, but caused negligible damage to the mitochondria as judged by their retention of glutamate dehydrogenase, and their ability to accumulate and retain Ca2+ in the absence of ruthenium red and EGTA. The data indicate that about 13% of total cell Ca2+ only may be mitochondrial in vivo.     

Calcium buffering in presynaptic nerve terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism

A latent ATP-dependent Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. The data indicate that a fraction of the Ca uptake (measured with 45Ca) is associated with the intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondrial) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. The proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the ATP-dependent Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca2+ concentration (buffered with EGTA) is less than 0.3 micrometer; brain mitochondria take up little Ca when the ionized Ca2+ level is this low. Preliminary subfractionation studies indicate that the nonmitochondrial Ca storage system does not sediment with synaptic vesicles. We propose that this Ca storage system, which has many properties comparable to those of skeletal muscle sarcoplasmic reticulum, may be associated with intraterminal smooth endoplasmic reticulum. This Ca-sequestering organelle may help to buffer intracellular Ca.     

Spatial structures in mitochondrial suspension induced by cation efflux

The formation of spatial structures in a thin unstirred layer of a mitochondrial suspension has been studied. It is shown that the structure formation depends on the state of the ion-transporting systems of mitochondria and that pattern development coincides with the activation of cation efflux from preloaded mitochondria. Spatial structure formation is an energy-dependent process and is suppressed by respiratory chain inhibitors. Patterning is also inhibited by EGTA, EDTA and ruthenium red, reflecting the requirement for divalent cation translocation in mitochondria for the studied phenomenon.     

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

Effect of ruthenium red on the induction by Ca2+ ions of the beta- and gamma states of comuton regulation of mitochondrial respiration and oxidative phosphorylation

Preincubation of liver mitochondria (Mch) with Ca2+ ions at inorganic phosphate concentration less than I mM in the presence of liver cell soluble phase (CSP) induced rotenone-independent tissue-specific uncoupling of oxidative phosphorylation (beta state of comuton regulation) and rotenone-stimulated tissue-specific uncoupling (gamma state of comuton regulation). The reduction in K+ ion concentration in the incubation medium entirely inhibited the induction of beta state. Tissue-specific stimulation of the rat liver Mch respiration in substrate-containing medium was increased after rotenone addition. Ruthenium red was added to the medium before and after the end of Mch preincubation with Ca2+ in the presence of CSP. The results suggest that limited Ca2+ transport in Mch is necessary for the induction of beta and gamma states of comuton regulation. Ca2+ ejected from Mch also participates in the induction of beta state of comuton regulation. Comuton receptor on the mitochondrial membrane surface is devoid of glyco- and mucoprotein components bound by ruthenium red.     

Regionally Selective Inhibition of Cerebral Protein Synthesis in the Rat During Hypoglycemia and Recovery

The effect of EGTA on the release of labeled gamma-aminobutyric acid (GABA), glutamate, acetylcholine, and dopamine was studied in superfused synaptosomes from mouse brain. In the absence of both Ca2+ and Mg2+, EGTA and also EDTA at 50 microM or higher concentrations induced a 2.5-5-fold stimulation of [3H]GABA release, similar to that produced by potassium depolarization, whereas only a slight effect, or no effect at all, was observed on the release of the other transmitters studied. The GABA-releasing action of EGTA was practically abolished in the presence of Mg2+. In contrast, the effect of EDTA was also observed when the medium contained Mg2+. Studies on the ionic dependence showed that the stimulation of GABA release by EGTA was abolished in a Na+-free medium. Li+ did not substitute Na+ for the EGTA effect, which was also independent of chloride. This Na+ dependence does not seem to involve voltage-sensitive channels, since tetrodotoxin did not affect the GABA-releasing action of EGTA, whereas in parallel superfusion chambers it blocked over 80% the stimulation of GABA release by veratridine. In contrast, two calcium channel blockers in synaptosomes, La3+ and the cationic dye ruthenium red, greatly inhibited the GABA-releasing effect of EGTA. L-2,4-Diaminobutyric acid, an inhibitor of the Na+-dependent GABA carrier, did not affect the releasing action of EGTA, whereas in a parallel experiment this drug inhibited by more than 90% the exchange of labeled GABA with unlabeled GABA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation-activated ATP hydrolysis by mitochondrial ATP'ase — Mechanism for energy depletion in ischemic reperfused myocardium

Recovery of high-energy compounds by ischemic myocardium is believed to be important for its return to normal functioning. While it has been previously shown that oxidative phosphorylation is markedly reduced in mitochondria isolated from ischemic myocardium in the presence of all substrates, alterations in ATPase activity have not been confirmed. This study demonstrates that, although the rate of ATP hydrolysis produced by mitochondria isolated from 2-hr ischemic myocardium does not significantly differ from that produced by non-ischemic mitochondria, the rate produced by 2-hr ischemic, 2 hr reperfused mitochondria is significantly higher. Also, Ca⁺⁺ content was observed to be higher in reperfused than in non-reperfused ischemic mitocheondria. The addition of EDTA, EGTA, or oligomycin to the reperfused ischemic mitochondria resulted in the inhibition of ATPase activity. These results indicate that mitochondrial ATPase in ischemic myocardium is activated by Ca⁺⁺ ions and that ischemic reperfused myocardium may contain mitochondria with uncontrolled ATPase activity such that high energy phosphate supplies are excessively depleted when the cells are reperfused.     

Evidence for the involvement of dithiol groups in mitochondrial calcium transport: studies with cadmium

The effect of cadmium on some functions of mitochondria isolated from kidneys of rat was studied. Addition of cadmium chloride to mitochondria induced stimulation of both State 4 respiratory rate and ATPase activity, which are prevented by the addition of ruthenium red. We also show that cadmium inhibits competitively calcium translocation; this inhibitory effect of cadmium is reverted by the addition of dithiothreitol. From these results, it is proposed that, similarly to Ca2+, cadmium penetrates mitochondria and binds to a membrane dithiol group, which is essential for the translocation of the cation.