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 Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Leucine zipper EF hand-containing transmembrane protein 1 (Letm1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca2+ uptake pathways

Cytosolic Ca(2+) signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca(2+) domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca(2+)/H(+) antiporter that achieved mitochondrial Ca(2+) sequestration at small Ca(2+) increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca(2+) uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca(2+) but strongly diminished the transfer of entering Ca(2+) into mitochondria, subsequently, resulting in a reduction of store-operated Ca(2+) entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca(2+) signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca(2+) uptake at low Ca(2+) conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca(2+) uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca(2+) uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca(2+) uptake pathways in intact endothelial cells.     

Oxidative phosphorylation, Ca(2+) transport, and fatty acid-induced uncoupling in malaria parasites mitochondria

Respiration, oxidative phosphorylation, calcium uptake, and the mitochondrial membrane potential of trophozoites of the malaria parasite Plasmodium berghei were assayed in situ after permeabilization with digitonin. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide. The capacity of trophozoites to sustain oxidative phosphorylation was additionally supported by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. Phosphorylation of ADP could be obtained in permeabilized trophozoites in the presence of succinate, citrate, alpha-ketoglutarate, glutamate, malate, dihydroorotate, alpha-glycerophosphate, and N,N,N',N'-tetramethyl-p-phenylenediamine. Ca(2+) uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca(2+) transport system in these mitochondria. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin, ATP, or GTP and not affected by atractyloside, ADP, glutamate, or malonate. Evidence for the presence of a mitochondrial uncoupling protein in P. berghei was also obtained by using antibodies raised against plant uncoupling mitochondrial protein. Together these results provide the first direct biochemical evidence of mitochondrial function in ATP synthesis and Ca(2+) transport in a malaria parasite and suggest the presence of an H(+) conductance in trophozoites similar to that produced by a mitochondrial uncoupling protein.     

Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue

Calcium-dependent uncoupling of liver mitochondrial oxidative phosphorylation by a non-metabolizable long chain fatty acyl analogue was compared with uncoupling induced by in vivo thyroid hormone treatment. beta,beta'-Methyl-substituted hexadecane alpha, omega-dioic acid (Medica 16) is reported here to induce a saturable 20-30% decrease in liver mitochondrial DeltaPsi, DeltapH and protonmotive force which proceeds in the presence of added Ca(2+) to cyclosporin A-sensitive mitochondrial permeabilization. Ca(2+)-dependent uncoupling by Medica 16 was accompanied by atractylate-enhanced, bongkrekic-inhibited activation of mitochondrial Ca(2+) efflux. The direct mitochondrial effect exerted in vitro by Medica 16 is similar to that induced by in vivo thyroid hormone treatment. Hence, the thyromimetic protonophoric activity of Medica 16 and the uncoupling activity of TH converge onto components of the mitochondrial permeabilization transition pore.     

The mitochondrial Na(+)/Ca(2+) exchanger

Powered by the steep mitochondrial membrane potential Ca(2+) permeates into the mitochondria via the Ca(2+) uniporter and is then extruded by a mitochondrial Na(+)/Ca(2+) exchanger. This mitochondrial Ca(2+) shuttling regulates the rate of ATP production and participates in cellular Ca(2+) signaling. Despite the fact that the exchanger was functionally identified 40 years ago its molecular identity remained a mystery. Early studies on isolated mitochondria and intact cells characterized the functional properties of a mitochondrial Na(+)/Ca(2+) exchanger, and showed that it possess unique functional fingerprints such as Li(+)/Ca(2+) exchange and that it is displaying selective sensitivity to inhibitors. Purification of mitochondria proteins combined with functional reconstitution led to the isolation of a polypeptide candidate of the exchanger but failed to molecularly identify it. A turning point in the search for the exchanger molecule came with the recent cloning of the last member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX (Na(+)/Ca(2+)/Li(+) exchanger). NCLX is localized in the inner mitochondria membrane and its expression is linked to mitochondria Na(+)/Ca(2+) exchange matching the functional fingerprints of the putative mitochondrial Na(+)/Ca(2+) exchanger. Thus NCLX emerges as the long sought mitochondria Na(+)/Ca(2+) exchanger and provide a critical molecular handle to study mitochondrial Ca(2+) signaling and transport. Here we summarize some of the main topics related to the molecular properties of the Na(+)/Ca(2+) exchanger, beginning with the early days of its functional identification, its kinetic properties and regulation, and culminating in its molecular identification.     

Role of mitochondrial Ca2+ in the regulation of cellular energetics

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.     

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.     

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.     

A proposed sequence of events for cadmium-induced mitochondrial impairment

Cadmium is a very important environmental toxicant, the cytotoxicity mechanism of which is likely to involve mitochondria as a target. In the present study we addressed the cause/effect relationship between the multiple cadmium-induced responses involving the mitochondrial energetic and oxidative status. Assays were performed with succinate-energized rat liver mitochondria incubated with 5 microM CdCl(2) for 0-25 min, in the absence or presence, respectively, of N-ethylmaleimide (NEM), butylhydroxytoluene (BHT), ruthenium red (RR), and cyclosporine A+ADP. A sequence of events accounting for cadmium-induced mitochondrial impairment is proposed, beginning with an apparent interaction of Cd(2+) with specific protein thiols in the mitochondrial membrane, which stimulates the cation's uptake via the Ca(2+) uniporter, and is followed by the onset of mitochondrial permeability transition (MPT); both effects dissipate the transmembrane electrical potential (Deltapsi), causing uncoupling, followed by an early depression of mitochondrial ATP levels. The respiratory chain subsequently undergoes inhibition, generating reactive oxygen species which together with iron mobilized by the cation, cause late, gradual mitochondrial membrane lipid peroxidation.     

Effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract on heart and liver mitochondrial functions: mechanism(s) of action

Though extracts of Ginkgo biloba leaves (GBE) have a wide pharmacological application, little is known about GBE effects on mitochondria. In this work, effects of ethanolic GBE on the respiration of isolated rat heart and liver mitochondria were investigated. We found that GBE stimulates the pyruvate + malate-dependent State 2 respiration of heart mitochondria and decreases mitochondrial membrane potential. Uncoupling effect of GBE was found to be due to its protonophoric action and is likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. The effect of GBE was less in liver than in heart mitochondria. State 3 respiration of heart mitochondria was slightly stimulated at low and depressed at higher GBE concentrations. Inhibition of State 3 respiration of heart mitochondria was not relieved by uncoupler indicating that GBE may inhibit the respiratory chain complexes or the substrate transport. However, Complex IV of the respiratory chain was not inhibited by GBE. H₂O₂ generation was attenuated by low concentration of GBE probably due to mild uncoupling. The data suggest that mild but not severe uncoupling activity of GBE may be important in providing pharmacological protection of cellular functions in pathological situations.     

G37R SOD1 mutant alters mitochondrial complex I activity, Ca(2+) uptake and ATP production

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the molecular mechanisms whereby these mutations induce motor neuron death remain controversial. Here, we show that stable overexpression of mutant human SOD1 (G37R) - but not wild-type SOD1 (wt-SOD1) - in mouse neuroblastoma cells (N2a) results in morphological abnormalities of mitochondria accompanied by several dysfunctions. Activity of the oxidative phosphorylation complex I was significantly reduced in G37R cells and correlated with lower mitochondrial membrane potential and reduced levels of cytosolic ATP. Using targeted chimeric aequorin we further analyzed the consequences of mitochondrial dysfunction on cellular Ca(2+) handling. Mitochondrial Ca(2+) uptake, elicited by IP(3)-induced Ca(2+) release from endoplasmic reticulum (ER) was significantly reduced in G37R cells, while uptake induced by a brief Ca(2+) pulse was not affected in permeabilized cells. The decreased mitochondrial Ca(2+) uptake resulted in increased cytosolic Ca(2+) transients, whereas ER Ca(2+) load and resting cytosolic Ca(2+) levels were not affected. Together, these findings suggest that the mechanism linking mutant G37R SOD1 and ALS involves mitochondrial respiratory chain deficiency resulting in ATP loss and impairment of mitochondrial and cytosolic Ca(2+) homeostasis.     

Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.     

The KATP channel is critical for calcium sequestration into non-ER compartments in mouse pancreatic beta cells.

K(ATP) channel activity influences beta cell Ca(2+) homeostasis by regulating Ca(2+) influx through L-type Ca(2+) channels. The present paper demonstrates that loss of K(ATP) channel activity due to pharmacologic or genetic ablation affects Ca(2+) storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca(2+) release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca(2+) ATPases by cyclopiazonic acid abolished the FCCP-induced Ca(2+) transient. In beta cells treated with K(ATP) channel inhibitors FCCP elicited a significantly larger Ca(2+) transient. Cyclopiazonic acid did not abolish this Ca(2+) transient suggesting that non-ER compartments are recruited as additional Ca(2+) stores in beta cells lacking K(ATP) channel activity. Genetic ablation of K(ATP) channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca(2+)-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca(2+) evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K(ATP) channel activity conveys Ca(2+) release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca(2+) it is suggested that mitochondria are the recruited store. The change in Ca(2+) sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.     

The interaction of flavonoids with mitochondria: effects on energetic processes

The study addressed aspects of energetics of isolated rat liver mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, taking into account influences of the 2,3 double bond/3-OH group and 4-oxo function on the C-ring, and o-di-OH on the B-ring of their structures, as well as mitochondrial mechanisms potentially involved in cell necrosis and apoptosis. The major findings/hypothesis, were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria, induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca(2+) correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The flavonoids causing substantial respiratory chain inhibition or mitochondrial uncoupling, quercetin and galangin, respectively, also decreased the mitochondrial ATP levels, thus suggesting an apparent higher potential for necrosis induction in relation to the flavonoids inducing MPT, taxifolin and cathechin, which did not decrease significantly the ATP levels, rather suggesting an apparent higher potential for apoptosis induction.     

Arachidonic acid causes cytochrome c release from heart mitochondria

Arachidonic acid interaction with heart mitochondria is known to cause uncoupling as well as inhibition of pyruvate + malate and succinate-supported respiration. Here we present experiments showing that arachidonic acid causes cytochrome c release from Ca(2+)-loaded heart mitochondria. We have also measured mitochondrial matrix swelling and found a fairly good correlation between the two processes, as revealed by the same arachidonic acid concentration dependence and by the same susceptibility toward different free fatty acid species. The effects produced by arachidonic acid are not related to its protonophoric activity since, under the experimental conditions used, saturating concentrations of FCCP did not cause any effect.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (～7.6) and ΔpH(m) (～0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (～5 mm, pH 7.8) compared with the cytosol (～20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.     

Cyclosporin a inhibits the protonophoric uncoupling activity of laurate in liver mitochondria

The effects of cyclosporin A, carboxyatractylate, and glutamate on the protonophoric uncoupling activity of laurate in liver mitochondria have been studied. It was found that 5 μM cyclosporin A partly inhibits laurate-stimulated mitochondrial respiration, which is suggestive of its recoupling effect, i.e., the ability to suppress the protonophoric activity of this fatty acid. Under these conditions, cyclosporin A has no effect on the ability of carboxyatractylate and glutamate to inhibit the uncoupling effect of laurate. In their turn, these compounds do not influence the recoupling activity of cyclosporin A. The recoupling effects of cyclosporin A, carboxyatractylate, and glutamate are additive: acting simultaneously, they fully suppress the uncoupling activity of laurate. It is concluded that the protonophoric uncoupling activity of fatty acids in liver mitochondria is mediated not only by ADP/ATP and aspartate/glutamate antiporters, but also by a system that is sensitive to cyclosporin A, but is not related with cyclophilin D.     

Effect of transforming growth factor-beta on calcium homeostasis in prostate carcinoma cells

Ca(2+) plays a fundamental role in the control of a variety of cellular functions, in particular, in energy metabolism and apoptosis. In this study, we show that TGF-beta at concentrations of 0.1-1.0 ng/ml transiently increases the level of intracellular Ca(2+) ([Ca(2+)](in)) in human prostate carcinoma, PC-3U, cells. Experiments with mitochondrial inhibitors (oligomycin and antimycin A) and an inhibitor of endoplasmic reticulum Ca(2+) uptake (BHQ) implied that the effect of TGF-beta1 was due to an effect on the mitochondria. TGF-beta1 treatment resulted in a decrease in ATP synthesis and to a depolarisation, leading to a release of Ca(2+) from mitochondria and decreased activity of the Ca(2+) pumps. Analysis of the mitochondria within the PC-3U cells by polarography and membrane potential-sensitive dye (Rhodamine 123) confirmed that under these experimental conditions, TGF-beta1 inhibited ATP synthesis and depolarised the mitochondria. The results implicate that TGF-beta1 affects the function of the mitochondria and may be of significance for the understanding of the proapoptotic effect of TGF-beta1 in these cells.