Dagmar Siliprandi

Uptake of spermine by rat liver mitochondria and its influence on the transport of phosphate

Spermine, a polyamine present in the mammalian cells at rather high concentration, has, among other actions, a remarkable stabilizing effect on mitochondria, functions which have generally been attributed to the capability of this and other polyamines to bind to membrane anionic sites. In the present paper evidence is provided that at physiological concentrations spermine may also be transported into rat liver mitochondrial matrix space, provided that mitochondria are energized and inorganic phosphate is simultaneously transported. The close dependence of spermine transport is also demonstrated by the concurrent efflux of spermine and inorganic phosphate when mitochondria preloaded with the two ionic species are deenergized either with uncouplers or respiratory chain inhibitors. Furthermore, Mersalyl, the known inhibitor of phosphate transport, prevents both spermine uptake and release. Mg2+ inhibits the transport of spermine conceivably by competing for the some binding sites on the mitochondrial membrane. The physiological significance of these results is discussed.

Involvement of endogenous phospholipase A2 in Ca2+ and Mg2+ movements induced by inorganic phosphate and diamide in rat liver mitochondria

Abstract Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol oxidizing agent, induced a respiration dependent efflux of Mg2+ which was prevented by both antimycin A and tetracaine. Tetracaine also inhibited the release of respiration induced by phosphate or diamide. Endogenous Ca2+ were retained by mitochondria until 50–60 per cent of endogenous Mg2+ has been lost. Tetracaine retarded Ca2+ release. The involvement of mitochondrial phospholipase A2 is demonstrated both by its inhibition by tetracaine and its activation by diamide or phosphate. The failure of these compounds to activate phospholipase A2 in the presence of added Ca2+ makes reasonable the assumption that the activation of phospholipase A2 is secondary to respiration dependent Ca2+ movements, which would favour the interaction Ca2+-phospholipase A2.

On the mechanism by which Mg2+ and adenine nucleotides restore membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate.

The presence of ATP or ADP in the incubation medium prevents the collapse of membrane potential induced by external Ca2+ and phosphate. The same adenine nucleotides are unable to restore collapsed membrane potential unless Mg2+ are also added. Bongkrekate is also able to prevent the effects of external Ca2+ and phosphate and when added after membrane potential has collapsed strongly potentiates the restorative action of ATP or ADP. Atractyloside has an opposite effect.

Synergic action of calcium ions and diamide on mitochondrial swelling.

Abstract The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg ++ and ruthenium red. Diamide potentiates the swelling action of Ca ++ , while DTE potentiates that of Mg ++ . The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg ++ . The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca ++ and Mg ++ is closely correlated with the redox state of membrane bound thiol groups.

Restoration of some energy linked processes lost during the ageing of rat liver mitochondria.

Abstract Some energy linked processes partially lost during the ageing of rat liver mitochondria, such as respiratory control index, ADP:O ratio and the Ca++ and K+ uptake were restored to approximately the original level prior to ageing by addition of dithioerythritol to aged mitochondria. It is suggested that the maintenance of mitochondrial energy linked processes may depend upon the integrity of specific pairs of vicinal thiol groups of the membrane.

The alterations in the energy linked properties induced in rat liver mitochondria by acetylsalycilate are prevented by cyclosporin A or Mg2

The alterations in rat liver mitochondria induced by acetylsalicylate in the presence of low concentrations of Ca2+ (large amplitude swelling, permeability to 14C]sucrose, collapse of transmembrane potential and effluxes of endogenous Mg2+ and accumulated Ca2+) were fully prevented by either cyclosporin A or Mg2+. Cyclosporin A and Mg2+ were also capable of restoring transmembrane potential upon its decrease induced by acetylsalicylate. The loss of endogenous Mg2+ was the primary effect promoted by acetylsalicylate; the other noxious effects followed. These results indicate that Mg2+ are fundamental components of the mitochondrial permeability barrier and that their loss might be responsible for the membrane transition induced by acetylsalicylate.

A possible mechanism for respiration-dependent efflux of Mg ions from liver mitochondria

Addition of Pi or diamide to a suspension of rat liver mitochondria induced a net efflux of Mg++ which is dependent on coupled respiration. This Mg++ efflux is prevented by EGTA and by Ruthenium red, both of which also prevent the increased rate of state 4 respiration induced by Pi or by diamide. It is assumed that an accelerated recycling of endogenous Ca++ induced by Pi or by diamide through an altered permeability of inner membrane to Ca++ is responsible for Mg++ efflux, and accounts for its apparent dependence on coupled respiration.

Effects of palmitoyl CoA and palmitoyl carnitine on the membrane potential and Mg2+ content of rat heart mitochondria

Palmitoyl CoA and palmitoyl carnitine added to rat heart mitochondria in amounts above 20 and 50nmoles/mg protein, respectively, induced a fall in transmembrane potential and loss of endogenous Mg2+. The dissipation of membrane potential by low concentrations of palmitoyl CoA in the presence of Ca2+, but not that of high concentrations of palmitoyl CoA alone, was prevented by either ruthenium red, Cyclosporin A or Mg2+, but reversed only by Mg2+. The fall of membrane potential induced by palmitoyl carnitine was not prevented by any of these factors. It is suggested that the action of both palmitoyl CoA and palmitoyl carnitine at high concentrations is due to a non specific disruption of membrane architecture, while that of low concentrations of palmitoyl CoA in the presence of Ca2+ is associated specifically with energy dissipation due to Ca2+ cycling. (Mol Cell Biochem 116: 117-123, 1992)

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 Pi 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 Pi 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.

The action of tellurite, a reagent for thiol groups, on mitochondria oxidative processes

Abstract Tellurite, a reagent for thiol groups, added to rat kidney, or liver mitochondria at the concentration of 1 mM, selectively inhibits the oxidation of NAD dependent substrates (pyruvate, α-ketoglutarate, glutamate, etc.) without affecting the oxidation of succinate, α-glycerophosphate and ascorbate. Moreover NADH oxidation by rat heart or by aged liver mitochondria is not affected by tellurite. The inhibitory effect of tellurite, which unlike that of rotenone is not reversed by menadione, is completely reversed by dithioerythritol.