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1.
Parvalbumin (PV) is a cytosolic Ca 2+-binding protein acting as a slow-onset Ca 2+ buffer modulating the shape of Ca 2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca 2+ shuttle facilitating transcellular Ca 2+ resorption. In excitable cells, upregulation of mitochondria in “PV-ergic” cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca 2+ transport and membrane potential, uncoupling protein 2 ( Ucp2), mitocalcin ( Efhd1), mitochondrial calcium uptake 1 ( Micu1), mitochondrial calcium uniporter ( Mcu), mitochondrial calcium uniporter regulator 1 ( Mcur1), cytochrome c oxidase subunit 1 ( COX1), and ATP synthase subunit β ( Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca 2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca 2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca 2+-buffering/sequestration capacity. 相似文献
2.
We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca 2+ in physiological concentration ranges (S 0.5 = 360 nM Ca 2+). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca 2+ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca 2+-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca 2+. At micromolar concentrations, Ca 2+ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca 2+ regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca 2+. 相似文献
3.
Recent studies have suggested that mitochondria may play important roles in the Ca 2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca 2+ flux can regulate the generation of Ca 2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca 2+ (Ca i
2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca 2+ release and CaWs were induced in the presence of high (4 mM) external Ca 2+ (Ca o
2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Ca i
2+ levels even after depletion of SR Ca 2+ in the absence of Ca o
2+ , suggesting Ca 2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential ( Δψ
m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ
m) or Ru360 (a mitochondrial Ca 2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca 2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca 2+ release and uptake exquisitely control the local Ca 2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis. 相似文献
4.
The paper examines membranotropic Ca 2+-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca 2+, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca 2+ to HPA-containing liposomes induced their aggregation and/or fusion. Ca 2+ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca 2+-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca 2+ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca 2+ was replaced with Sr 2+ (but not with Ba 2+ or Mg 2+). A supposition is made that HPA can induce a Ca 2+-dependent aggregation of mitochondria, as well as Ca 2+dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles. 相似文献
5.
Mitochondrial Ca 2+ uptake exerts dual effects on mitochondria. Ca 2+ accumulation in the mitochondrial matrix dissipates membrane potential ( ΔΨ m), but Ca 2+ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca 2+ ([Ca 2+] m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca 2+] m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca 2+ indicators with a 20-fold difference in Ca 2+ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca 2+ indicator (D4cpv) calibrated against known Ca 2+ levels. Matrix pH (pH m) and ΔΨ m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca 2+] m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca 2+] m coincided with a rapid elevation in pH m and was followed by an after-stimulus ΔΨ m hyperpolarization. However, pH m decreased and no ΔΨ m hyperpolarization was observed in response to lower levels of [Ca 2+] m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca 2+] m are required to stimulate presynaptic mitochondrial energy metabolism. 相似文献
6.
α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca 2+ oscillations that burden mitochondria, we examined mitochondrial Ca 2+ stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca 2+, promoted Ca 2+-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca 2+. Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca 2+ homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca 2+-handling requirements. 相似文献
7.
Ca 2+ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca 2+ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca 2+-ATPase (PMCA) overexpression on Ca 2+-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca 2+] using a combination of aequorins with different Ca 2+ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca 2+] in the cytosol and subcellular heterogeneities in [Ca 2+] among mitochondria. Overexpression of PMCA decreased [Ca 2+] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca 2+] in the cytosol and subcellular heterogeneities in [Ca 2+] among mitochondria. PMCA overexpression depletes intracellular [Ca 2+] stores and, despite a decrease in mitochondrial [Ca 2+], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca 2+ homeostasis that could decrease β-cell apoptosis in diabetes. 相似文献
8.
The work examines the mechanism of central nerve cell death upon stimulation of brain NMDA receptors with the stimulatory
mediator glutamate. A prolonged stimulation of neurons with glutamate is known to result in the disorder of Ca 2+ homeostasis and severe mitochondrial depolarization followed by cell death. It has been shown that the overload of mitochondria
with Sr 2+ leads to the release of the cation, medium alkalization, decrease of membrane potential and mitochondrial swelling, indicating
a nonspecific permeabilization of the mitochondrial membrane. The permeabilization, in our opinion, is caused by the activation
of Ca 2+/Sr 2+-dependent phospholipase A 2 (PLA 2), resulting in the formation of free palmitic and stearic acids in the mitochondrial membrane. These fatty acids bind Ca 2+ with high affinity and the process of binding is accompanied by the formation of a transient lipid pore—a phenomenon demonstrated
earlier on both artificial and mitochondrial membranes. The inhibitors of PLA 2 have been shown to suppress permeabilization of mitochondrial membranes. In the culture of granular cerebellum neurons, the
PLA 2 inhibitors prolonged the lag of the delayed Sr 2+ deregulation and membrane depolarization. On the basis of data obtained on isolated mitochondria and neurons we suppose that
the initial stages of glutamate-induced Ca 2+ deregulation of neurons are underlain by the opening of lipid pores in brain mitochondria. 相似文献
9.
Oxidative damage of mammalian mitochondria induced by Ca 2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca 2+ uniport, undergo inner membrane permeabilization when treated with Ca 2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg 2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca 2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species. 相似文献
10.
Alloxan at millimolar concentrations slightly inhibited the velocity of Ca 2+ uptake by isolated rat liver mitochondria irrespective of the free Ca 2+ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca 2+ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca 2+ uptake but only at free Ca 2+ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca 2+ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca 2+ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca 2+ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca 2+ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca 2+ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca 2+ uptake. The direct effects of ninhydrin on mitochondrial Ca 2+ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca 2+ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca 2+ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992) 相似文献
11.
Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive
pore induced by low concentrations of palmitic acid (Pal) plus Ca 2+ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca 2+, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the
mitochondrial membrane, the release of Ca 2+ and the swelling of mitochondria. Inhibitors of the Ca 2+ uniporter, ruthenium red and La 3+, as well as EGTA added in 10 min after the Pal/Ca 2+-activated pore opening, prevent the release of Ca 2+ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria
accumulating high [Sr 2+], which leads to the activation of phospholipase A 2 and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca 2+ cycle, in which Ca 2+ uptake is mediated by the Ca 2+ uniporter and Ca 2+ efflux occurs via a short-living Pal/Ca 2+-activated pore. 相似文献
12.
The amination of α-ketoglutarate (α-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca 2+. The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca 2+ in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca 2+ had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca 2+. About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca2+ had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca2+. In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar α-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca2+, 10 and 50 millimolar NH4+, and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation. 相似文献
13.
Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca 2+ and is fueled either by Ca 2+ from the extracellular space when triggered by neuronal activity or by Ca 2+ released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca 2+ pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca 2+ homeostasis in models of ischemia. The proteins include those important for the Ca 2+-dependent motility of mitochondria and for Ca 2+ transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca 2+ release from the ER, voltage-dependent anion channels that allow Ca 2+ entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca 2+ uniporter together with its regulatory proteins that permit Ca 2+ entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca 2+ from the mitochondria such as the mitochondrial Na +/Ca 2+ exchanger or, if the mitochondrial Ca 2+ concentration exceeds a certain threshold, the mitochondrial permeability transition pore. 相似文献
14.
Ca 2+ transport through mitochondrial Ca 2+ uniporter is the primary Ca 2+ uptake mechanism in respiring mitochondria. Thus, the uniporter plays a key role in regulating mitochondrial Ca 2+. Despite the importance of mitochondrial Ca 2+ to metabolic regulation and mitochondrial function, and to cell physiology and pathophysiology, the structure and composition of the uniporter functional unit and kinetic mechanisms associated with Ca 2+ transport into mitochondria are still not well understood. In this study, based on available experimental data on the kinetics of Ca 2+ transport via the uniporter, a mechanistic kinetic model of the uniporter is introduced. The model is thermodynamically balanced and satisfactorily describes a large number of independent data sets in the literature on initial or pseudo-steady-state influx rates of Ca 2+ via the uniporter measured under a wide range of experimental conditions. The model is derived assuming a multi-state catalytic binding and Eyring's free-energy barrier theory-based transformation mechanisms associated with the carrier-mediated facilitated transport and electrodiffusion. The model is a great improvement over the previous theoretical models of mitochondrial Ca 2+ uniporter in the literature in that it is thermodynamically balanced and matches a large number of independently published data sets on mitochondrial Ca 2+ uptake. This theoretical model will be critical in developing mechanistic, integrated models of mitochondrial Ca 2+ handling and bioenergetics which can be helpful in understanding the mechanisms by which Ca 2+ plays a role in mediating signaling pathways and modulating mitochondrial energy metabolism. 相似文献
15.
BackgroundDisorders of mitochondrial Ca 2+ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca 2+ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain. MethodsThe functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique. ResultsAt the doses of 30 and 45 μM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 μM DS caused hyperpolarization of mitochondria of resting neurons, and 45 μM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30–60 μM DS stimulated cellular respiration. DS was shown to suppress Ca 2+ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca 2+, high DS concentrations caused a collapse of the mitochondrial membrane potential. ConclusionsThe data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria. General significanceThe using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis. 相似文献
16.
Polyclonal rabbit antibodies against a Ca 2+-binding mitochondrial glycoprotein were found to inhibit the uniporter-mediated transport of Ca 2+ in mitoplasts prepared from rat liver mitochondria. Spermine, a modulator of the uniporter, decreased the inhibition. This glycoprotein of M
r
40,000, isolated from beef heart mitochondria and earlier shown to form Ca 2+-conducting channels in black-lipid membranes, thus is a good candidate for being a component of the uniporter. Antibody-IgG was found to specifically bind to mitochondria in human fibroblasts. 相似文献
17.
Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca 2+] i) in heart. These buffers can remove up to one-third of the Ca 2+ that enters the cytosol during the [Ca 2+] i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca 2+ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca 2+ signals (i.e., Ca 2+ sparks and [Ca 2+] i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ m), the role of the mitochondria in buffering cytosolic Ca 2+ signals was investigated. We show here that rapid loss of ΔΨ m leads to no significant changes in cytosolic Ca 2+ signals. Second, we make direct measurements of mitochondrial [Ca 2+] ([Ca 2+] m) using a mitochondrially targeted Ca 2+ probe (MityCam) and these data suggest that [Ca 2+] m is near the [Ca 2+] i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca 2+ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca 2+ cycling suggests that mitochondrial Ca 2+ uptake would need to be at least ∼100-fold greater than the current estimates of Ca 2+ influx for mitochondria to influence measurably cytosolic [Ca 2+] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca 2+ uptake does not significantly alter cytosolic Ca 2+ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca 2+] i under physiological conditions in heart. 相似文献
18.
In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca 2+ influx, which triggers insulin exocytosis. The mitochondrial Ca 2+ uniporter (MCU) mediates Ca 2+ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca 2+ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca 2+. Suppression of the putative Ca 2+/H + antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca 2+-induced matrix acidification. These results demonstrate that MCU-mediated Ca 2+ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells. 相似文献
20.
BAX cooperates with truncated BID (tBID) and Ca 2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca 2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/ oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca 2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca 2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca 2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca 2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state. 相似文献
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