Umberto De Marchi

Bax Does Not Directly Participate in the Ca2+-induced Permeability Transition of Isolated Mitochondria

The mitochondrial permeability transition pore and Bax have both been proposed to be involved in the release of pro-apoptotic factors from mitochondria in the “intrinsic” pathway of apoptosis. The permeability transition pore is widely thought to be a supramolecular complex including or interacting with Bax. Given the relevance of the permeability transition in vivo, we have verified whether Bax influences the formation and/or the properties of the Ca2+/Pi-induced permeability transition by using mitochondriaisolated from isogenic human colon cancer bax+/– and bax–/– HCT116 cell lines. We used mitochondria isolated from both types of cells and from Bax+ cells exposed to apoptotic stimuli, as well as Bax-less mitochondria into which exogenous Bax had been incorporated. All exhibited the same behavior and pharmacological profile in swelling and Ca2+-retention experiments. Mitochondria from a bax–/bak– cell line also underwent an analogous Ca2+/Pi-inducible swelling. This similarity indicates that Bax hasno major role in regulating the Ca2+-induced mitochondrial permeability transition.

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production.

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca2+ entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca2+ uniporter. To clarify the role of UCPs in mitochondrial Ca2+ handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca2+ and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca2+ uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca2+ elevations evoked by histamine. The reduced Ca2+ elevations were due to inhibition of store-operated Ca2+ entry and reduced depletion of endoplasmic reticulum (ER) Ca2+ stores. UCP3 depletion accelerated the ER Ca2+ refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca2+ (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca2+ responses. Our results indicate that UCP3 is not a mitochondrial Ca2+ uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca2+ thus reflect metabolic alterations that impact on cellular Ca2+ homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca2+ uptake and release sites.

A mitochondriotropic derivative of quercetin: a strategy to increase the effectiveness of polyphenols.

Mitochondria‐targeted compounds are needed to act on a variety of processes that take place in these subcellular organelles and that have great pathophysiological relevance. In particular, redox‐active molecules that are capable of homing in on mitochondria provide a tool to intervene on a major cellular source of reactive oxygen species and on the processes they induce, notably the mitochondrial permeability transition and cell death. We have linked the 3‐OH of quercetin (3,3′,4′,5,7‐pentahydroxy flavone), a model polyphenol, and the triphenylphosphonium moiety, a membrane‐permeant cationic group, to produce proof‐of‐principle mitochondriotropic quercetin derivatives. The remaining hydroxyls were sometimes acetylated to hinder metabolism and improve solubility. The new compounds accumulate in mitochondria in a transmembrane potential‐driven process and are only slowly metabolised by cultured human colon cells. They inhibit mitochondrial ATPase activity much as quercetin does, and are toxic for fast‐growing cells.

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch-clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca(2+)-activated K(+)-selective channel K(Ca)3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca(2+)] will cause mtK(Ca)3.1 opening, thus linking inner membrane K(+) permeability and transmembrane potential to Ca(2+) signalling.

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

Background: Nutrients stimulate calcium dependent activation of energy metabolism, in pancreatic beta cells. Results: Glucose-induced ATP synthase-dependent respiration is strictly calcium-dependent, with little or no effect of calcium on the NAD(P)H response. Conclusion: Calcium coordinates oxidative metabolism and respiration in pancreatic beta cells. Significance: Calcium has novel mitochondrial targets downstream of mitochondrial dehydrogenases. Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio.

Mitochondrial calcium handling during ischemia-induced cell death in neurons

Mitochondria sense and shape cytosolic Ca(2+) signals by taking up and subsequently releasing Ca(2+) ions during physiological and pathological Ca(2+) elevations. Sustained elevations in the mitochondrial matrix Ca(2+) concentration are increasingly recognized as a defining feature of the intracellular cascade of lethal events that occur in neurons during cerebral ischemia. Here, we review the recently identified transport proteins that mediate the fluxes of Ca(2+) across mitochondria and discuss the implication of the permeability transition pore in decoding the abnormally sustained mitochondrial Ca(2+) elevations that occur during cerebral ischemia.

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca2 + -dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca2 +  concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD− mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the “standard” PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca2 + , CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.

Tyramine and monoamine oxidase inhibitors as modulators of the mitochondrial membrane permeability transition.

Incubation of rat liver mitochondria with 100–500 mM tyramine, a substrate for monoamine oxidases A and B (MAOs), in the presence of 30 mM Ca2+ induces matrix swelling, accompanied by collapse of membrane potential, efflux of endogenous Mg2+ and accumulated Ca2+ and oxidation of endogenous pyridine nucleotides. These effects are completely abolished in the presence of cyclosporin A, ADP, dithioerythritol and N-ethylmaleimide, thus confirming the induction of the mitochondrial membrane permeability transition (MPT). The observed partial protective effect exerted by catalase indicates the involvement of both MAO-derived hydrogen peroxide and aldehyde. Higher concentrations of tyramine (1–2 mM) are less effective or even completely ineffective. At these high concentrations tyramine has an inhibitory effect when the MPT is induced by 100 mM Ca2+. The MAO inhibitors clorgyline (50 mM) and pargyline (500 mM) completely protect against MPT induction by 100 mM tyramine but also inhibit the phenomenon, although with different efficacy, when it is induced by 100 mM Ca2+ in the absence of tyramine. Taken together, our data suggest that tyramine, clorgyline and pargyline act as modulators of the MPT either through a direct inducing/protective effect or by controlling hydrogen peroxide and aldehyde generation.

SLP-2 negatively modulates mitochondrial sodium-calcium exchange

Mitochondria play a major role in cellular calcium homeostasis. Despite decades of studies, the molecules that mediate and regulate the transport of calcium ions in and out of the mitochondrial matrix remain unknown. Here, we investigate whether SLP-2, an inner membrane mitochondrial protein of unknown function, modulates the activity of mitochondrial Ca(2+) transporters. In HeLa cells depleted of SLP-2, the amplitude and duration of mitochondrial Ca(2+) elevations evoked by agonists were decreased compared to control cells. SLP-2 depletion increased the rates of calcium extrusion from mitochondria. This effect disappeared upon Na(+) removal or addition of CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, and persisted in permeabilized cells exposed to a fixed cytosolic Na(+) and Ca(2+) concentration. The rates of mitochondrial Ca(2+) extrusion were prolonged in SLP-2 over-expressing cells, independently of the amplitude of mitochondrial Ca(2+) elevations. The amplitude of cytosolic Ca(2+) elevations was increased by SLP-2 depletion and decreased by SLP-2 over-expression. These data show that SLP-2 modulates mitochondrial calcium extrusion, thereby altering the ability of mitochondria to buffer Ca(2+) and to shape cytosolic Ca(2+) signals.

The properties of the mitochondrial megachannel in mitoplasts from human colon carcinoma cells are not influenced by Bax

This paper explores the relationship between Bax and the mitochondrial permeability transition pore (PTP). Isolated human colon tumor (HCT116) Bax− mitochondria exposed to recombinant Bax exhibited a slow, cyclosporin A‐sensitive swelling, but only at [Bax] > 200 nM. The amount of Bax incorporated was much higher than that found in organelles isolated from HCT116 Bax+ staurosporine‐ or etoposide‐treated apoptotic cells, casting doubts on the significance of the putative PT induction for apoptosis. Bax did not influence the electrophysiological properties of an approximately 1 nS channel ascribed to the Ca2+‐dependent mitochondrial permeability transition pore. These observations indicate that the PTP is independent of Bax.