On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content.

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.     

Inhibition of mitochondrial-matrix inorganic pyrophosphatase by physiological [Ca2+], and its role in the hormonal regulation of mitochondrial matrix volume.

1. The pyrophosphatase activity in cytosolic and mitochondrial fractions of rat liver was 1.7 and 0.26 units/mg of protein respectively when assayed at 37 degrees C in the presence of physiological [Mg2+] (0.3 mM). 2. Approx. 80% of the mitochondrial pyrophosphatase was inaccessible to extramitochondrial PPi, of which 40% represented soluble matrix enzyme (0.38 unit/mg of matrix protein). 3. Ca2+ inhibited the soluble matrix enzyme; the effective K0.5 for inhibition increased as [Mg2+], an essential cofactor of the enzyme, increased. Measured values were 0.39, 1.15, 3.7, 8.3 and 12.5 microM at 0.04 mM-, 0.1 mM-, 0.3 mM-, 0.6 mM- and 1 mM-Mg2+ respectively. 4. The data were analysed by a kinetic model similar to that for yeast pyrophosphatase, which assumes the substrate to be MgPPi (Km 5 microM) with Mg2+ also activating at an additional site (K0.5 23 microM). Ca2+ inhibits through the formation of CaPPi, a strong competitive inhibitor (Ki 0.067 microM). 5. Heart mitochondria also contain a soluble matrix pyrophosphatase of similar activity to that of liver mitochondria and with the same sensitivity to [Ca2+]. 6. The data provide an explanation for the increase in mitochondrial PPi, mediated by Ca2+, which is responsible for the increase in matrix volume induced by gluconeogenic hormones [Davidson & Halestrap (1988) Biochem. J. 254, 379-384].     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.     

Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.     

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.     

Effects of the general anaesthetic Propofol on the Ca2(+)-induced permeabilization of rat liver mitochondria.

The molecular mechanism of the Ca2(+)-induced permeabilization of rat liver mitochondria was evaluated by studying a new effect of the commonly used general anaesthetic Propofol (2,6-diisopropylphenol). The compound was found to induce an apparent uptake of Ca2+ at steady-state in the Ca2(+)-distribution between the medium and the mitochondria, and to inhibit swelling and release of accumulated Ca2+ induced by inorganic phosphate, t-butyl hydroperoxide, diamide or FCCP plus Ruthenium red. The compound did not stimulate the activity of the Ca2(+)-uniporter and it is concluded that the effects seen are due to the inhibition of the Ca2(+)-dependent, unspecific permeability increase. The results suggest two mechanisms whereby Propofol stabilizes the mitochondrial membrane in the presence of Ca2+: (i) by interaction with the putative pore, thus causing its closure; and (ii) by scavenging of free radicals thus inhibiting its opening during oxidative stress.     

Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria.

The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.     

The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues.

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+-induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+-influx. The possible role of the Na+-dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent efflux of Ca2+.     

Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37 degrees C.

Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed.     

Calcium-independent cell volume regulation in human lymphocytes. Inhibition by charybdotoxin

The properties of the K+ pathway underlying regulatory volume decrease (RVD) in human blood lymphocytes were investigated. Evidence is presented for the existence of three types of K+ conductance in these cells. Ionomycin, a Ca2+ ionophore, induced a K(+)-dependent hyperpolarization, indicating the presence of Ca2(+)-activated K+ channels, which were blocked by charybdotoxin (CTX). CTX also induced a depolarization of the resting membrane potential, even at subphysiological cytosolic [Ca2+]([Ca2+]i), which suggests the existence of a second CTX-sensitive, but Ca2(+)-independent conductance. A CTX-resistant K+ conductance was also detected. RVD in blood lymphocytes was partially (approximately 75%) blocked by CTX. However, volume regulation was not accompanied by detectable changes in [Ca2+]i, nor was it prevented by removal of extracellular Ca2+ and depletion or buffering of intracellular Ca2+. These observations suggest that K+ loss during RVD is mediated by Ca2(+)-independent, CTX-sensitive channels or that Ca2(+)-dependent channels can be activated by cell swelling at normal or subnormal [Ca2+]i. The former interpretation is supported by findings in rat thymic lymphocytes. These cells also displayed a CTX-sensitive Ca2(+)-dependent hyperpolarization. However, CTX did not significantly alter the resting potential, suggesting the absence of functional Ca2(+)-independent, toxin-sensitive channels. Volume regulation in thymic lymphocytes was less efficient than in human blood cells. In contrast to blood lymphocytes, RVD in thymocytes was not affected by CTX. These observations indicate that, though present in lymphocytes, Ca2(+)-activated K+ channels do not play an important role in volume regulation. Instead, RVD seems to be mediated by Ca2(+)-independent K+ channels. We propose that two types of channels, one CTX sensitive and the other CTX insensitive, mediate RVD in human blood lymphocytes, whereas only the latter type is involved in rat thymocytes.     

Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration.

(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+]m) was determined from the fluorescence of internalized indo-1. The value of the Kd of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+]m with the extramitochondrial free Ca2+ ([Ca2+]o) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+]m responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+]m/[Ca2+]o was found to be near unity (0.82 +/- 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 +/- 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDHA) was found to increase with [Ca2+]m, with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+]m/[Ca2+]o concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+]m concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+]m, as inferred in earlier work.     

Possible Mechanism for Formation and Regulation of the Palmitate-Induced Cyclosporin A-Insensitive Mitochondrial Pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the cyclosporin A-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate-induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, P(i)). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 microM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.     

Intramitochondrial K+ as activator of carboxyatractyloside-induced Ca2+ release.

The role of intramitochondrial K+ content on the increase in membrane permeability to Ca2+, as induced by carboxyatractyloside was studied. In mitochondria containing a high K+ concentration (83 nmol/mg), carboxyatractyloside induced a fast and extensive mitochondrial Ca2+ release, membrane de-energization, and swelling. Conversely, in K(+)-depleted mitochondria (11 nmol/mg), carboxyatractyloside was ineffective. The addition of 40 mM K+ to K(+)-depleted mitochondria restored the capability of atractyloside to induce an increase in membrane permeability to Ca2+ release. The determination of matrix free Ca2+ concentration showed that, at an external free-Ca2+ concentration of 0.8 microM, control mitochondria contained 3.9 microM of free Ca2+ whereas K(+)-depleted mitochondria contained 0.9 microM free Ca2+. It is proposed that intramitochondrial K+ affects the matrix free Ca2+ concentration required to induce a state of high membrane permeability.     

Multiple mechanisms of transmitter release evoked by "pathologically" elevated extracellular [K+]: involvement of transporter reversal and mitochondrial calcium

The release of [3H]GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes. Elevating [K+] produced release of [3H]GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A. Accordingly, the sensitivity to clostridial toxins of the depolarization-evoked amino acid release was inversely correlated to the concentration of KCl used. However, at 50 mM K+, one-third of the stimulated release remained which was external Ca2+-independent but insensitive to SKF 100330 A. This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+-dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal [Na+]. In fluorescence experiments with fura-2/AM, 50 mM K+ (in Ca2+ free medium) caused elevation of cytosolic [Ca2+] that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects. When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mM K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low-Na+ media. To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis. These include carrier-mediated release and internal Ca2+-dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.     

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.     

[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

Ruthenium red inhibits mitochondrial Na+ and K+ uniports induced by magnesium removal.

Removal of bound magnesium from the outer surface of the inner mitochondrial membrane opens up a Na+ and Li+ selective electrophoretic uniport pathway whereas simultaneous depletion of intramitochondrial magnesium induces an electrogenic K+ flux as well. In order to clarify the nature of these cation movements we tested the effect of ruthenium red, a potent and specific inhibitor of the mitochondrial Ca2+ uniporter on different Na+ and K+ uniport-associated phenomena. Ruthenium red efficiently inhibited mitochondrial swelling and depolarization induced by either EDTA in a NaCl-based medium (Na+ uniport) or by EDTA plus A23187 in a KCl-based medium (K+ uniport). For both cation uniports half-maximal inhibition was attained at a ruthenium red concentration as low as 40 nM. Complete inhibition was found above 200 nM. Neither the Na+/H+ nor the K+/H+ exchange was affected by ruthenium red. In light of these observations the possibility is raised that the electrogenic Na+ and K+ fluxes provoked by magnesium reduction or depletion may be mediated through the Ca2+ uniporter. It is suggested that intactness of the mitochondrial magnesium pools is necessary for maintaining the Ca2+ selectivity of the Ca2+ uniporter, and alterations of the membrane-associated magnesium content would make this transport route available also for monovalent cations.     

The reversible Ca2+-induced permeabilization of rat liver mitochondria.

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.     

The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter.

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.