N.E. Lofrumento

Coupling mechanisms in anionic substrate transport across the inner membrane of rat-liver mitochondria

The translocation of Pi, malate, α-oxoglutarate, and citrate across the inner membrane of rat-liver mitochondria has been studied. Investigation on the effect of pH on anionic substrate translocation across the mitochondrial membrane shows that their distribution across the inner membrane can be governed by transmembrane pH difference. However, evidence is presented that the translocation of Pi, but not that of malate, α-oxoglutarate, or citrate can bedirectly coupled to an OH− counterflux (H2PO4−−OH− exchange-diffusion). and malate-tricarboxylate exchange-diffusion reactions is directly demonstrated. The study of the effect of uncouplers on the efflux from mitochondria of substrate anions, in the absence of counteranion, and on the anion exchange-diffusions shows that uncouplers act in at least two ways: they promote the efflux of Pi from mitochondria and inhibitdirectly the exchange-diffusion reactions. The kinetics of this inhibition are described. These results are discussed in the light of previous work on the effect of uncouplers on the distribution of substrate anions across the inner membrane of isolated mitochondria. Coupling mechanisms in substrate anion translocation and aspects of the energetics of anion translocation are discussed.

New aspects of the mechanism of inorganic phosphate and dicarboxylate transport in mitochondria

1. Introduction It is generally accepted that the transport of inor- ganic phosphate in mitochondria is mediated by two distinct translocating systems localized in the inner membrane [l-7]. The first mediates the exchange- diffusion of Pi with OH-(phosphate carrier) and the second that of Pi with dicarboxylates (dicarboxyla- te carrier). The observation that Pi -Pi exchange is inhibited when both N-ethylmaleimide (NEM) and butylmalonate (BM) are present [4,7], together with the finding that dicarboxylate-dicarboxylate or Pi- dicarboxylate exchanges are not inhibited by NEM [8,9], are considered as main evidence that the two carriers are well distinct functional entities. We have however, found that butymalonate interfers with the mechanism of action of NEM since it removes or pre- vents in intact mitochondria the extrabinding of Pi in- duced by NEM [lo]. These findings have prompted us to reinvestigate the effect of NEM and BM on the transport of Pi and dicarboxylic acids in rat-liver mito- chondria. The results reported in this paper indicate that the mitochondrial membrane contains only one transporting system which, depending on the prevailing conditions, mediates either Pi-OH-or Pi-dicarboxy- late exchange. 2. Methods Rat-liver mitochondria were loaded with Pi, malate or succinate by preincubation for 10 min at 4” C in the presence of 250 mM sucrose, 5 I.cg/ml oligomycin, 0.34 m 200 mM sucrose; 20 mM Tris-HCI; oligomycin; rotenone and antimycin at the concentrations indicated above. The pH was ad- justed to 7.2. At the time specified in the legends nige- kin, butylmalonate, N-ethylmaleimide and the coun- ter-anions were added. Incubation was carried out at 4°C directly in small centrifuge tubes in a total vol of 1 ml and mitochondria separated from the suspen- ding medium by rapid centrifugation at 20 000 g. Before incubation Pi was determined chemically [ 1 l] and malate enzymatically [ 121. Their specific activity was calculated by measuring the corresponding total radioactivity of 12% HC104 extracts of loaded mito- chondria. Pi, malate and [14C] succinate content of mitochondrial pellet was corrected for that present in the sucrose space. 3. Results The sensitivity of the Pi carrier to NEM and BM was studied by following the efflux of P, from mito- chondria induced by the addition of nigericin [6]. Fig. 1 shows that NEM at 200 PM fully inhibited the Pi-OH-exchange whilst BM, even at a very high con- centration as 5 mM (not shown), had no effect at all. However when these two compounds were added to- gether different results, according to the sequence of additions and NEM concentration, were obtained. BM, added after NEM, increased the inhibition of Pi efflux

Mechanism of inhibition by uncouplers of succinate oxidation in isolated mitochondria

Abstract 1. The inhibition by uncouplers of succinate oxidation in isolated rabbit-kidney mitochondria has been studied. 2. In the absence of rotenone, the oxidation of 10 mM succinate was inhibited by uncouplers. This inhibition could be prevented by adding glutamate, palmitoylcarnitine or ATP. Activation by ATP occurred in the presence of oligomycin but not of atractyloside. 3. In the presence of rotenone, the uncoupler dicoumarol inhibited the oxidation of succinate when the substrate concentration was 1 mM but not when it was 10 mM. ATP had no effect on the inhibited respiration. Removal of dicoumarol with albumin under conditions in which the mitochondria could subsequently become energized led to a restoration of succinate oxidation. 4. In the absence of rotenone and presence of uncoupler, the activation of respiration by ATP was accompanied by a decline in the level of oxaloacetate and by the formation of phosphoenolpyruvate. 5. In the absence of rotenone and presence of uncoupler, there was an inverse correlation between the rate of respiration and the level of oxaloacetate. The activation of respiration by ATP or palmitoylcarnitine was accompanied by a decline in the level of oxaloacetate. 6. The addition of phosphoenolpyruvate, CO 2 and ADP to mitochondria in the presence of uncoupler and rotenone led to an inhibition of succinate oxidation, presumably due to oxaloacetate formation. 7. It is concluded that the inhibition by uncouplers of succinate oxidation in the absence of rotenone and at relatively high succinate concentrations is due to the formation of oxaloacetate.

Cytochrome c-induced cytosolic nicotinamide adenine dinucleotide oxidation, mitochondrial permeability transition, and apoptosis

A catalytic amount of cytochrome c (cyto-c) added to the incubation medium of isolated mitochondria promotes the transfer of reducing equivalents from extramitochondrial nicotinamide adenine dinucleotide in its reduced state (NADH) to molecular oxygen inside the mitochondria, a process coupled to the generation of a membrane potential. This mimics in many aspects the early stages of those apoptotic pathways characterized by the persistence of mitochondrial membrane potential but with cyto-c already exported into the cytosol. In cyclosporin-sensitive and calcium-induced mitochondrial permeability transition (MPT) a release of cyto-c can also be observed. However, in MPT uncoupled respiration associated with mitochondrial swelling and preceded by the complete dissipation of the membrane potential which cannot be restored with ATP addition or any other source of energy is immediately activated. The results obtained and discussed with regard to intactness of mitochondrial preparations indicate that MPT could be an apoptotic event downstream but not upstream of cyto-c release linked to the energy-requiring processes. In the early stages of apoptosis cytosolic cyto-c participates in the activation of caspases and at the same time can promote the oxidation of cytosolic NADH, making more energy available for the correct execution of the cell death program. This hypothesis is not in contrast with available data in the literature showing that cyto-c is present in the cytosol of both control and apoptosis-induced cultured cell lines.

Phosphate transport in mitochondria action of mersalyl on the binding and transport of inorganic phosphate

Inorganic phosphate moves across the inner mitochondrial membrane either by exchangediffusion with hydroxide ions [ 1,2] this is equivalent to a phosphoric acid uniport [3] or by exchange-diffusion with dicarboxylates [l ,2] . Both reactions are inhibited by mersalyl [4-71. It is unknown whether the two reactions are mediated by two separate systems or if different functional groups of the same system are involved cf. ref. [7]. It is shown in this paper that mersalyl, in the same concentration range at which it inhibits Pi transport, stimulates Pi uptake by rat-liver mitochondria when added after net Pi transport is completed. This finding suggests that mersalyl inhibits the transport of Pi in mitochondria by fixing the anion on binding site(s) in the membrane.

On the inhibition of the transport of inorganic phosphate by N-ethylmaleimide.

The transport of inorganic phosphate in mitochondria is inhibited by sulphydryl-blocking reagents [l-6]. It is reported in this paper that, in intact ratliver mitochondria preincubated with ionophores to minimize the accumulation of Pi in the matrix space, (NEM)*,enhances the uptake of added Pi by mitochondria [7,:.8]. This effect of NEM appears to involve binding of Pi to the mitochondrial membrane. Evidence is presented that the stimulation of Pi uptake by mitochondria induced by NEM is directly related to the inhibition of Pi transport across the mitochondrial membrane. Analogous results have been obtained with mersalyl [7-g].

Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system.

Cytochrome c (cyto‐c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto‐c electron transport pathway. The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto‐c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria. At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto‐c. We suggest that, in physiological low‐amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto‐c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto‐c electron transport pathway.

Conformative response of the mitochondrial Pi-dicar☐ylate transport system to inhibitors and substrates

The distribution of inorganic phosphate between intraand extra-mitochondrial space has been established to be governed by the ApH [l-3] and the A concentration of anionic substrates [l-5] imposed across the inner membrane by respiration and metabolic activity. The hypothesis that the transport of Pi is mediated by two well distinct translocating systems, is mainly based on the differential effect of two classes of inhibitors: SH reagents and dicarboxylate analogues [6-81. Recently we have proposed and presented evidences [9-l l] that the inner mitochondrial membrane contains only one carrier which, irrespective of the driving force, mediates either the Pi-OHor the Pi-dicarboxylate exchange-diffusion reactions. Kinetic studies carried out in presence and absence of N-ethylmaleimide (NEM), butylmalonate (BM) and bathophenanthroline (BP) further substantiate this hypothesis. The results reported in this paper indicate that the SH groups of the membrane are not directly involved in the transport process and that the functional state of the Pi-dicarboxylate translocator is promoted by the binding of specific couples of complementary substrates. Conformational change of the transport system appears to be a suitable mechanism to account for the stimulatory and inhibitory effect of NEM and for the finding that the effectiveness of non-competitive inhibition of BM is related to the nature of intraand extramitochondrial substrates.

Ceramide-induced activation of cytosolic NADH/cytochrome c electron transport pathway: An additional source of energy for apoptosis.

We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long-chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either raft-like platforms or channels, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program.

Calcium release induced by N-ethylmaleimide in rat liver mitochondria

Recently we have found that in rat liver mitochondria the energy-dependent respiration induced by the addition of Ca*’ ions or K’ plus valinomycin is inhibited by NEM [l]. The inhibition could not be ascribed to the extensively studied effect of NEM on the translocase system which mediate Pi transport and citric cycle intermediates [2,3], in that the respiration was supported by endogenous substrates. The finding that the inhibition was also observed when TMPD t ascorbate was used as source of reducing equivalents, excluded the possibility of a direct effect of NEM on both NADand FAD-dependent dehydrogenases. Preliminary experiments on calcium transport have revealed that NEM promotes a back-flow rather than inhibit the efflux of H’ induced by Ca*+ uptake [4,5]. In this paper the effect of NEM on calcium transport follawing the distribution of 45Ca2+ across the inner mitochondrial membrane as well as the pH changes in the external medium, have been studied. The data obtained are inconsistent with an unspecific effect of NEM on the permeability of the membrane. As a working hypothesis it is proposed that NEM may influence the energetic state of inner membrane responsible of calcium gradient betwee/ cytosol and matrix compartments.