Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.     

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.     

Sites of action of glucagon and other Ca2+ mobilizing hormones on the malate aspartate cycle

Data from a number of laboratories suggest that the exchange of glutamate for aspartate across the mitochondrial inner membrane is stimulated by glucagon and by Ca2+-mobilizing hormones. The purpose of this study was to determine the site of action of these hormones. Two possibilities were considered and tested. The first hypothesis is that the mitochondrial membrane electrical potential gradient (delta psi m) in the cells is increased by the hormones; and that the putative increase in delta psi m stimulates aspartate efflux. The second possibility is that Ca2+ mediates decreases in cellular levels of alpha-ketoglutarate, secondary to stimulation of alpha-ketoglutarate dehydrogenase, and that the decrease in alpha-ketoglutarate stimulates aspartate production by mitochondria. The effect of glucagon on delta psi m was estimated in intact hepatocytes using the lipophilic cation tetraphenyl phosphonium. No increase in delta psi m was observed due to hormone treatment. On the other hand, alpha-ketoglutarate was found to be an effective competitive inhibitor of aspartate formation via glutamate transamination by isolated liver mitochondria (Ki = 0.55 mM).     

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.     

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.     

Induction of 2H+/Me2+ exchange in rat-liver mitochondria

The time dependency of CA2+ efflux from Ca2+-loaded rat liver mitochondria has been investigated. The rate of ruthenium-red-insensitive Ca2+ efflux is continuously increased during the retention as a result of induction of an electroneutral H+ Ca2+ exchange system. The activation of the Ca2+ efflux pathway takes place under the constant value of the membrane potential and is accompanied by oxidation of mitochondrial pyridine nucleotides. It has also been found that the ruthenium-red-insensitive H+/Sr2+ exchange occurs in mitochondria during Sr2+-induced oscillation of ion fluxes. The rate of H+/Sr2+ exchange is variable and depends on the stage of the oscillatory cycle.     

Quantitative and mechanistic aspects of the hydroperoxide-induced release of Ca2+ from rat liver mitochondria

We have previously demonstrated in rat liver mitochondria a hydroperoxide-induced hydrolysis of pyridine nucleotides and release of Ca2+ [L?tscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1979) Proc. Natl Acad. Sci. USA 76, 4340-4344, and L?tscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1980) J. Biol. Chem. 255, 9325-9330]. Here we investigate pyridine nucleotide hydrolysis and Ca2+ release under conditions of minimized Ca2+ cycling and with smaller Ca2+ loads. The extent of pyridine nucleotide hydrolysis, measured by pyridine-nucleotide-derived nicotinamide release from intact mitochondria, and the Ca2+ release rate show a very similar sigmoidal dependence on the mitochondrial Ca2+ load. The hydrolysis of oxidized pyridine nucleotides is limited under non-cycling conditions. Whereas pyridine nucleotide hydrolysis as measured by nicotinamide release is extensive, net loss of mitochondrial pyridine nucleotides is observed only at relatively high Ca2+ loads. Our results indicate the ability of mitochondria to resynthesize pyridine nucleotides after hydrolysis. Neither a decrease of reduced, nor an increase of oxidized, mitochondrial glutathione favour Ca2+ release. From these and previous findings it is concluded that the hydroperoxide-induced Ca2+ release is triggered by a factor which is distal to the oxidation of mitochondrial pyridine nucleotides. Ca2+ release is stimulated when the movement of protons across the inner mitochondrial membrane is facilitated, giving evidence for the operation of the hydroperoxide-induced release pathway as a Ca2+/H+ antiport.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Menadione- (2-methyl-1,4-naphthoquinone-) dependent enzymatic redox cycling and calcium release by mitochondria

The results presented in this paper reveal the existence of three distinct menadione (2-methyl-1,4-naphthoquinone) reductases in mitochondria: NAD(P)H:(quinone-acceptor) oxidoreductase (D,T-diaphorase), NADPH:(quinone-acceptor) oxidoreductase, and NADH:(quinone-acceptor) oxidoreductase. All three enzymes reduce menadione in a two-electron step directly to the hydroquinone form. NADH-ubiquinone oxidoreductase (NADH dehydrogenase) and NAD(P)H azoreductase do not participate significantly in menadione reduction. In mitochondrial extracts, the menadione-induced NAD(P)H oxidation occurs beyond stoichiometric reduction of the quinone and is accompanied by O2 consumption. Benzoquinone is reduced more rapidly than menadione but does not undergo redox cycling. In intact mitochondria, menadione triggers oxidation of intramitochondrial pyridine nucleotides, cyanide-insensitive O2 consumption, and a transient decrease of delta psi. In the presence of intramitochondrial Ca2+, the menadione-induced oxidation of pyridine nucleotides is accompanied by their hydrolysis, and Ca2+ is released from mitochondria. The menadione-induced Ca2+ release leaves mitochondria intact, provided excessive Ca2+ cycling is prevented. In both selenium-deficient and selenium-adequate mitochondria, menadione is equally effective in inducing oxidation of pyridine nucleotides and Ca2+ release. Thus, menadione-induced Ca2+ release is mediated predominantly by enzymatic two-electron reduction of menadione, and not by H2O2 generated by menadione-dependent redox cycling. Our findings argue against D,T-diaphorase being a control device that prevents quinone-dependent oxygen toxicity in mitochondria.     

Ca2+ releasing effect of perezone on adrenal cortex mitochondria

Ca2+ energy-coupled transport was analized in adrenal cortex mitochondria using the sesquiterpenic drug perezone. Perezone promotes Ca2+ efflux by inducing collapse of the membrane potential and oxidation of pyridine nucleotides. The effect of perezone on mitochondrial Ca2+ release follows a dose-response relationship and is dependent of the reduction of the drug. These data suggest that perezone may produce a cytotoxic effect through an impairment in Ca2+ homeostasis.     

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.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

Regulation of the mitochondrial Na+/Ca2+ antiport by matrix pH

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.     

Alterations in mitochondrial Ca2+ flux by the antibiotic X-537A (lasalocid-A).

A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results.     

Effects of the membrane potential upon the Ca(2+)- and cumene hydroperoxide-induced permeabilization of the inner mitochondrial membrane.

A protonophore-induced delta psi decrease in a 180-140 mV range causes an increase in the lag-period of Ca(2+)-induced mitochondrial permeabilization but has little effect on the cumene hydroperoxide-induced permeability transition of mitochondria. Suppression of the non-specific permeability induction seems to be mediated by an increase in [ADP] in the mitochondrial matrix. A further decrease in delta psi leads to additional suppression of the non-specific permeability as a result of a partial ruthenium red-sensitive efflux of the previously accumulated Ca2+. On the other hand, complete dissipation of delta psi causes immediate induction of the non-specific permeability. It is concluded that only complete dissipation of delta psi caused by H+ leakages may act as a trigger for non-specific permeability induction.     

The influence of pH on Ca2+ exchange in ferret heart mitochondria

The effect of pH changes on Ca2+ transport by isolated heart mitochondria was measured. Two components of Ca2+ transport were identified, an accumulation dependent on mitochondrial respiration and a Na+-dependent efflux. A decrease of pH over the range 7.7-6.7 reduced the initial rate and the total amount of respiration dependent Ca2+ accumulation. At pH 7.2 the [Na+] required to activate half-maximal efflux, k1/2, was 7.5 +/- 1.1 mM. Decreasing the pH over the range 7.7 to 6.9 increased the k1/2 from 3.6 to 11.6. The effect of acidosis was more profound on the respiration dependent Ca2+ uptake than the Na+-dependent efflux.     

The effect of a ferric iron complex on isolated rat-liver mitochondria. III. Mechanistic aspects of iron-induced calcium efflux

Addition of iron(III)-gluconate complex to isolated rat liver mitochondria induced a net efflux of Ca2+ which was not inhibited by ruthenium red. This process resulted in the enhancement of Ca2+ cycling and a consequent membrane potential drop. Under these experimental conditions the content of mitochondrial glutathione did not appear to be critically modified, whereas an extensive oxidation of mitochondrial pyridine nucleotides was parallelly detected. Iron failed to induce appreciable changes in the oxidation level of pyridine nucleotides in mitochondria isolated from rats fed a selenium deficient diet, a condition in which mitochondrial glutathione peroxidase resulted inhibited by 80%. The iron-induced Ca2+ release in Se-deficient mitochondria appeared largely delayed and the membrane potential of these mitochondrial did not present gross alterations. Iron was also found to induce a transient increase in the mitochondrial cyanide-insensitive oxygen consumption. This effect was largely prevented by the addition of the hydrogen peroxide scavenger catalase. It was concluded that iron induced the activation of a specific Ca2+ efflux pathway via the oxidation of pyridine nucleotides due to the hydrogen peroxide metabolism by glutathione enzyme system.     

Mobilization of mitochondrial Ca2+ by hydroperoxy-eicosatetraenoic acid

Hydroperoxy-eicosatetraenoic acids (HPETEs) and less effectively, also hydroxy-eicosatetraenoic acids (HETEs) stimulated Ca2+ release from rat liver mitochondria. Ca2+ release is accompanied by intramitochondrial pyridine nucleotide oxidation and hydrolysis. Both Ca2+ release and pyridine nucleotide oxidation are impeded when the flow of electrons between pyridine nucleotides and HPETE is impaired. Measurements of the mitochondrial membrane potential indicate that HPETE-stimulated Ca2+ release is not due to uncoupling of mitochondria. It is suggested that HPETEs and HETEs may act as mobilizers of mitochondrial Ca2+ during signal transduction related to proliferation and tumor promotion.     

Studies on the energy-linked Ca2+ accumulation in pig heart mitochondria - role of Mg2'ons

Comparative intracellular distribution of Ca2+, Mg2+ and adenine nucleotides has been studied in pig heart by differential centrifugation or fractional extraction and has shown that Mg2+ and ATP are associated mainly with soluble fractions whereas Ca2+ and ADP are more tightly bound to subcellular structures. Ca2+ accumulation and Ca2+ stimulated respiration were studied in pig heart mitochondria under different energetic conditions in the absence or presence of phosphate. Ca2+ concentrations of about 1200 nmoles/mg protein inhibit Ca2+ accumulation, site I substrate oxidation and induce an efflux of mitochondrial Mg2+. These deleterious effects of Ca2+ on respiration occur even in the absence of phosphate or oxidizable substrate; they are completely prevented by ruthenium red only, and partially prevented by the addition of M2+ to the medium. The kinetics of Ca2+ uptake become of the sigmoidal type when Mg2+ is present. This cation strongly inhibits the rate of Ca2+ uptake in the presence of added phosphate and decreases the affinity of Ca2+ for its transport system. In the absence of phosphate, Mg2+ has no effect on Ca2+ uptake. The possible physiological implications of these findings are discussed