Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

Parallel measurements of Ca²⁺ uptake, oxygen consumption, endogenous Mg²⁺ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca²⁺ in the presence of phosphate than rat liver mitochondria. The extent of Mg²⁺ efflux and swelling induced by Ca²⁺ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg²⁺ as a consequence of Ca²⁺ recycling across the membrane as induced by phosphate. Exogenous Mg²⁺ protects liver mitochondria against the deleterious effects of Ca²⁺ by inhibiting a ruthenium red-insensitive Ca²⁺ efflux induced by phosphate. Phosphate does not induce recycling of Ca²⁺ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca²⁺ recycling. In heart mitochondria the recycling of Ca²⁺ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca²⁺ is subject to metabolic control. In addition it has been observed that Sr²⁺does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca²⁺ than the ruthenium red-sensitive influx transporter.     

The effect of Ca2+ on the oxidation of exogenous NADH by rat liver mitochondria.

The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl₂ (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca²⁺ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca²⁺ is also observed in mitochondria partially swollen in a hypotonic medium.     

Stimulation of Ca efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10⁻¹⁰ M to 10⁻⁷ M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Tri-Calciphor (16,16-dimethyl-15-dehydroprostagalndin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B₁ (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca²⁺ efflux and swelling in mitochondria in the absence of phosphate, Mg²⁺ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca²⁺ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg²⁺ added together, and partially inhibited with phosphate plus either ATP or Mg²⁺. Between 0 and 7 μM added Ca²⁺ and in the presence of phosphate, ATP and Mg²⁺, tri-Calciphor stimulated the uptake of Ca²⁺ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca²⁺. Thus depending on the assay conditions, two different effects involving Ca²⁺ movements and mitochondria are observed with tri-Calciphor.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

The losses of adenine nucleotide accompanying efflux of Ca2+ from heart,liver and kidney mitochondria

Mitochondria prepared from either rat liver, kidney or heart were loaded with Ca²⁺ and then the efflux was observed after adding Ruthenium Red. The efflux of Ca²⁺ was stimulated by adding either NaCl, lysolecithin, or palmitoyl CoA or oleate. Measurements were made of the quantities of previously [¹⁴C]-labelled adenine nucleotide lost from the mitochondria as function of the loss of Ca²⁺. The two losses are correlated with coefficients between 0.85 and 0.92 in absence of bongkrekic acid. With bongkrekic acid present the adenine nucleotide loss tends to be diminished with respect to the Ca²⁺ loss; the most significant effect is seen with heart mitochondria. The bongkrekic acid potentiates the stimulation of Ca²⁺ efflux by Na⁺     

Modeling the calcium sequestration system in isolated guinea pig cardiac mitochondria

Under high Ca²⁺ load conditions, Ca²⁺ concentrations in the extra-mitochondrial and mitochondrial compartments do not display reciprocal dynamics. This is due to a paradoxical increase in the mitochondrial Ca²⁺ buffering power as the Ca²⁺ load increases. Here we develop and characterize a mechanism of the mitochondrial Ca²⁺ sequestration system using an experimental data set from isolated guinea pig cardiac mitochondria. The proposed mechanism elucidates this phenomenon and others in a mathematical framework and is integrated into a previously corroborated model of oxidative phosphorylation including the Na⁺/Ca²⁺ cycle. The integrated model reproduces the Ca²⁺ dynamics observed in both compartments of the isolated mitochondria respiring on pyruvate after a bolus of CaCl₂ followed by ruthenium red and a bolus of NaCl. The model reveals why changes in mitochondrial Ca²⁺ concentration of Ca²⁺ loaded mitochondria appear significantly mitigated relative to the corresponding extra-mitochondrial Ca²⁺ concentration changes after Ca²⁺ efflux is initiated. The integrated model was corroborated by simulating the set-point phenomenon. The computational results support the conclusion that the Ca²⁺ sequestration system is composed of at least two classes of Ca²⁺ buffers. The first class represents prototypical Ca²⁺ buffering, and the second class encompasses the complex binding events associated with the formation of amorphous calcium phosphate. With the Ca²⁺ sequestration system in mitochondria more precisely defined, computer simulations can aid in the development of innovative therapeutics aimed at addressing the myriad of complications that arise due to mitochondrial Ca²⁺ overload.     

Interactions between prostaglandin E 1 and calcium at the level of the mitochondrial membrane

Low concentrations of PGE₁ facilitate the exit of actively accumulated Ca²⁺ from rat liver mitochondria. The effect is evident at pH 6,4 and disappears at neutral pH. Ca²⁺ bound to the mitochondrial membrane in the absence of energy is not discharged by PGE₁.Under conditions that lead to its active accumulation, Ca²⁺ stimulates the binding of PGE₁ to mitochondria. The effect is concentration dependent (maximal at 500 μM Ca²⁺), is evident only at slightly acid pH, and is transitory. The binding of PGE₁ reaches a maximum between 30 sec and 2 min and then declines very rapidly, returning to the baseline 2–5 min after the addition of Ca²⁺. The maximal amount of PGE₁ bound is 1.3 nmoles per mg of mitochondrial protein, i.e., about 1% of the Ca²⁺ taken up by mitochondria. No PGE₁ is bound when permeant anions are tranported into mitochondria together with Ca²⁺. Sr²⁺ and Mn²⁺ also stimulate the binding of PGE₁.Aspirin and indomethacin are powerful inhibitors of the binding of PGE₁ to mitochondria. This effect appears to be secondary to the inhibition of mitochondrial Ca²⁺ transport by the antiinflammatory drugs.     

Ca 2+ transport activity in mitochondria from some plant tissues

Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca²⁺. Additions of Ca²⁺ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca²⁺ was largely accumulated. Accumulation of Ca²⁺ required phosphate and, in most cases, was stimulated by Mg²⁺ and ADP or ATP. Ca²⁺ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca²⁺ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca²⁺ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca²⁺ binding sites sensitive to uncouplers, La³⁺, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca²⁺, probably due to lack of a specific Ca²⁺ carrier.     

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⁺.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Mechanisms of antioxidant activities of quercetin and catechin

Abstract

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 cyclo-sporine 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.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

Role of anion exchange and thiol groups in the regulation of potassium efflux by lead in human erythrocytes

Pb²⁺ is thought to enter erythrocytes through anion exchange (AE) and to remain in the cell by binding to thiol groups. To define the role of AE mechanism and thiol groups in Pb²⁺ toxicity, we studied the effects of drugs and conditions that modify AE and that modify thiol groups on the ability of Pb²⁺ to stimulate potassium efflux as measured with ⁸⁶Rb. The most potent stimulation of ⁸⁶Rb efflux by Pb²⁺ occurred when conditions were optimal for the AE mechanism—that is, when bicarbonate was included in the buffer or a buffer made with Nal or NaCl rather than NaClO₄ or NaNO₃ was used. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid, potent inhibitors of the AE mechanism, completely inhibited stimulation of the ⁸⁶Rb efflux by Pb²⁺. These conditions or inhibitors did not affect stimulation of the ⁸⁶Rb efflux by ionomycin plus Ca²⁺. A role for Ca²⁺ channels was dismissed because the inorganic Ca²⁺ channel blockers, Cd²⁺ or Mn²⁺, did not prevent stimulation of ⁸⁶Rb efflux by Pb²⁺ but did inhibit stimulation by ionomycin plus Ca²⁺. ⁸⁶Rb efflux was more sensitive to Pb²⁺ if erythrocytes were treated for 15 min with thiol-modifying reagents that enter cells, such as iodoacetamide, N-ethylmaleimide, or dithiothreitol, than to reduced glutathione, a thiol-modifying reagent that is not permeable to the cell. Thus, in erythrocytes the AE mechanism and internal thiol groups are critical factors that affect the stimulation of a Ca²⁺-dependent process by Pb²⁺. © 1996 Wiley-Liss, Inc.     

H+-dependent efflux of Ca2+ from heart mitochondria

A rapid loss of accumulated Ca²⁺ is produced by addition of H⁺ to isolated heart mitochondria. The H⁺-dependent Ca⁺ efflux requires that either (a) the NAD(P)H pool of the mitochondrion be oxidized, or (b) the endogenous adenine nucleotides be depleted. The loss of Ca²⁺ is accompanied by swelling and loss of endogenous Mg^2–. The rate of H⁺-dependent Ca²⁺ efflux depends on the amount of Ca²⁺ and P_i taken up and the extent of the pH drop imposed. In the absence of ruthenium red the H⁺-induced Ca²⁺-efflux is partially offset by a spontaneous re-accumulation of released Ca²⁺. The H⁺-induced Ca²⁺ efflux is inhibited when the P_i transporter is blocked withN-ethylmaleimide, is strongly opposed by oligomycin and exogenous adenine nucleotides (particularly ADP), and inhibited by nupercaine. The H⁺-dependent Ca²⁺ efflux is decreased markedly when Na⁺ replaces the K⁺ of the suspending medium or when the exogenous K⁺/H⁺ exchanger nigericin is present. These results suggest that the H⁺-dependent loss of accumulated Ca²⁺ results from relatively nonspecific changes in membrane permeability and is not a reflection of a Ca²⁺/H⁺ exchange reaction.     

Mitochondrial Generation of Oxygen Radicals During Reoxygenation of Ischemic Tissues

Ischemia and reperfusion causes severe mitochondrial damage, including swelling and deposits of hyd-roxyapatite crystals in the mitochondrial matrix. These crystals are indicative of a massive influx of Ca²⁺ into the mitochondrial matrix occurring during reoxygenation. We have observed that mitochondria isolated from rat hearts after 90 minutes of anoxia followed by reoxygenation, show a specific inhibition in the electron transport chain between NADH dehydrogenase and ubiquinone in addition to becoming uncoupled (unable to generate ATP). This inhibition is associated with an increased H₂O₂ formation at the NADH dehydrogenase level in the presence of NADH dependent substrates. Control rat mitochondria exposed for 15 minutes to high Ca²⁺ (200 nmol/mg protein) also become uncoupled and electron transport inhibited between NADH dehydrogenase and ubiquinone. a lesion similar to that observed in post-ischem-ic mitochondria. This Ca²⁺ -dependent effect is time dependent and may be partially prevented by albumin, suggesting that it may be due to phospholipase A₂ activation. releasing fatty acids, leading to both inhibition of electron transport and uncoupling. Addition of arachidonic or linoleic acids to control rat heart mitochondria, inhibits electron transport between Complex I and III. These results are consistent with the following hypothesis: during ischemia, the intracellular energy content drops severely, affecting the cytoplasic concentration of ions such as Na⁺ and Ca²⁺. Upon reoxygenation, the mitochondrion is the only organelle capable of eliminating the excess cytoplasmic Ca²⁺ through an electrogenic process requiring oxygen (the low ATP concentration makes other ATP-dependent Ca^?' lransport systems non-operational). Ca²⁺-overload of mitochondria activates phospholipase A₂ releasing free fatty acids, leading to uncoupling and inhibition of the interactions between Complex I and III of the respiratory chain. As a consequence, the NADH-dehydrogenase becomes highly reduced, and transfers electrons directly to oxygen generating O₂.     

Effects of polyamines on mitochondrial Ca transport

Mammalian mitochondria are able to enhance Ca²⁺ accumulation in the presence of polyamines by activating the saturable systems of Ca²⁺ inward transport and buffering extramitochondrial Ca²⁺ concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca²⁺ concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca²⁺ transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S₁ binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca²⁺ transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S₁ site. Important physiological functions are related to activation of Ca²⁺ transport systems by polyamines and their interactions with the S₁ site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)