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1.
Despite extensive research, the regulation of mitochondrial function is still not understood completely. Ample evidence shows that cytosolic Ca 2+ has a strategic task in co-ordinating the cellular work load and the regeneration of ATP by mitochondria. Currently, the paradigmatic view is that Ca cyt2+ taken up by the Ca 2+ uniporter activates the matrix enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase. However, we have recently found that Ca 2+ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier. Since this activation is not affected by ruthenium red, glutamate transport into mitochondria is controlled exclusively by extramitochondrial Ca 2+. Therefore, this discovery shows that besides intramitochondrial also extramitochondrial Ca 2+ regulates oxidative phosphorylation. This new mechanism acts as a mitochondrial “gas pedal”, supplying the OXPHOS with substrate on demand. These results are in line with recent findings of Satrustegui and Palmieri showing that aralar as part of the malate–aspartate shuttle is involved in the Ca 2+-dependent transport of reducing hydrogen equivalents (from NADH) into mitochondria. This review summarises results and evidence as well as hypothetical interpretations of data supporting the view that at the surface of mitochondria different regulatory Ca 2+-binding sites exist and can contribute to cellular energy homeostasis. Moreover, on the basis of our own data, we propose that these surface Ca 2+-binding sites may act as targets for neurotoxic proteins such as mutated huntingtin and others. The binding of these proteins to Ca 2+-binding sites can impair the regulation by Ca 2+, causing energetic depression and neurodegeneration. 相似文献
2.
Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt(51Q)), modeling the adult form of HD. Ca(free)(2+) up to 2 mum activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca(2+) uniporter, did not affect the Ca(2+)-dependent activation of respiration but reduced Ca(2+)-induced inhibition. Thus, Ca(2+) activation was mediated exclusively by extramitochondrial Ca(2+), whereas inhibition was promoted also by intramitochondrial Ca(2+). In contrast, htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca(2+) activation, and a higher susceptibility to Ca(2+)-dependent inhibition. Furthermore htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca(2+), lower capacities and rates of Ca(2+) accumulation, and a decreased Ca(2+) threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca(2+)-induced inhibition of respiration of htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca(2+). In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca(2+). We suggest that specific regulatory Ca(2+) binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt(51Q) and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD. 相似文献
3.
Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca 2+-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca 2+ effect in BAT mitochondria thermogenesis. We found that Ca 2+ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca 2+ concentration needed for half-maximal activation varied between 0.08 and 0.11 µM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN.Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca 2+ strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca 2+ activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver. 相似文献
4.
Mitochondria act as potent buffers of intracellular Ca 2+ in many cells, but a more active role in modulating the generation of Ca 2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca 2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca 2+ store with thapsigargin (TG) activates Ca 2+ release-activated Ca 2+ (CRAC) channels in T cells, and the ensuing influx of Ca 2+ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca 2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na + depletion, which inhibits Na +-dependent Ca 2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca 2+ levels that are consistent with its localization in the TG-insensitive store. Ca 2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca 2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca 2+ uptake is sensitive to extracellular [Ca 2+], indicating that mitochondria sense Ca 2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na + depletion prevent the ability of T cells to maintain a high rate of capacitative Ca 2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca 2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca 2+ that enters T cells via store-operated Ca 2+ channels, but also play an active role in modulating the rate of capacitative Ca 2+ entry. 相似文献
5.
The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle. 相似文献
6.
Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca 2+ uptake and extrusion modulate free cytosolic [Ca 2+] (Ca c) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca 2+ entry, and to monitor Ca 2+-activated currents that report near-surface [Ca 2+]. We show that rapid recovery from elevations of Ca c requires both the mitochondrial Ca 2+ uniporter and the mitochondrial energization that drives Ca 2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca 2+] (Ca m). The indicated resting Ca m of 100–200 nM is similar to the resting Ca c reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Ca m and Ca c at high temporal resolution shows that, although Ca m increases less than Ca c, mitochondrial sequestration of Ca 2+ is fast and has high capacity. We find that mitochondrial Ca 2+ uptake limits the rise and underlies the rapid decay of Ca c excursions produced by Ca 2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca 2+ from mitochondria, seen as declining Ca m, prolongs complete Ca c recovery and that suppressing export of Ca 2+, by inhibition of the mitochondrial Na +/ Ca 2+ exchanger, reversibly hastens final recovery of Ca c. We conclude that mitochondria are active participants in cellular Ca 2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca 2+. 相似文献
7.
BackgroundWithin the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general. Methodology/Principal FindingsWe applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses.Oxidative phosphorylation (OXPHOS) capacity (pmol O 2•s −1•mg −1 wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses ( N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 ( N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness. Conclusions/SignificanceThe scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology. 相似文献
8.
Anoxia induces a rapid elevation of the cytosolic Ca 2+ concentration ([Ca 2+] cyt) in maize ( Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca 2+ release is important for gene activation and survival in O 2-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca 2+ to the anoxic [Ca 2+] cyt elevation in maize cells. Imaging of intramitochondrial Ca 2+ levels showed that a majority of mitochondria released their Ca 2+ in response to anoxia and took up Ca 2+ upon reoxygenation. We also investigated whether the mitochondrial Ca 2+ release contributed to the increase in [Ca 2+] cyt under anoxia. Analysis of the spatial association between anoxic [Ca 2+] cyt changes and the distribution of mitochondrial and other intracellular Ca 2+ stores revealed that the largest [Ca 2+] cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca 2+] cyt under aerobic conditions but prevented a [Ca 2+] cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca 2+] cyt and participate in the signaling of O 2 deprivation. 相似文献
9.
Dysregulation of intracellular Ca 2+ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer''s Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ 1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca 2+ in neurons and promote mitochondrial Ca 2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca 2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca 2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca 2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca 2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD. 相似文献
10.
Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria–sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5–6 months). Mitochondrial membrane potential and resting O 2 consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O 2 consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca 2+, we investigated the effect of age on mitochondrial Ca 2+ uptake. Although no age-dependent differences were found in Ca 2+ uptake kinetics in isolated mitochondria, mitochondrial Ca 2+ uptake secondary to SR Ca 2+ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca 2+ handling. Age-dependent alterations in SR Ca 2+ transfer to mitochondria and in Ca 2+ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR–mitochondria communication underlies inefficient interorganelle Ca 2+ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.Age is the main independent risk factor for cardiovascular morbidity and mortality. 1 It increases heart vulnerability to cardiac diseases as well as the severity of their clinical manifestations, and reduces the efficacy of cardioprotective interventions. 2 At the cellular level, some of the structural and functional age-dependent changes resemble those of failing cardiac myocytes. 3, 4 Specifically, disturbed Ca 2+ homeostasis and excitation–contraction coupling, 5 as well as deficient mitochondrial energetics 6 and excessive ROS production, 7 have been consistently reported in senescent cardiomyocytes. These subcellular alterations likely contribute to the reduced adaptive capacity to stress (exercise, β-adrenergic stimulation) and increased vulnerability to disease of the aged hearts.In cardiac cells, electrochemical coupling and metabolic adaptations are based upon the coordination between sarcoplasmic reticulum (SR) and mitochondria tightly interconnected forming an interface to support local ionic exchange and signal transduction in a beat-to-beat basis. 8 This privileged interorganelle communication facilitates mitochondrial ATP transport for SR Ca 2+ cycling and ensures energy replenishment by reciprocal Ca 2+ and ADP exchange. Ca 2+ is taken up by mitochondria using a low-affinity uniporter whose activity is driven by the elevated Ca 2+ concentration in the microenvironment present around ryanodine receptors (RyR). 9 Indeed, the kinetics of mitochondrial Ca 2+ uptake is more dependent on the concentration of Ca 2+ at the SR–mitochondria contact points than on bulk cytosolic Ca 2+ concentration. 8 Mitochondrial Ca 2+ uptake allows energy supply–demand matching through the activation of Krebs cycle dehydrogenases and electron transport chain activity, and at the same time it regulates the regeneration of Krebs-coupled antioxidative defenses (NAD(P)H). 10Defective SR–mitochondria cross talk has been causally linked to the abnormal mitochondrial Ca 2+ uptake in failing hearts and may underlie their increased oxidative stress. 11 Also, in diabetic cardiomyopathy, intracellular Ca 2+ overload and depletion of energy stores appear to develop as a consequence of sequential SR–mitochondria dysfunction. 12 Atrial fibrillation has been associated with an increased fusion of mitochondria and a subsequent increased colocalization of giant mitochondria with SR, a subcellular remodeling process that contributes to the perpetuation of the arrhythmia. 13 Because mitochondria are highly dynamic structures, some molecular links have been proposed to provide a stable physical interorganelle bridge 14, 15 while others appear to facilitate direct tunneling of Ca 2+ and other signaling mediators. 16 In the present study, we hypothesized that aging may negatively impact on mitochondria–SR communication by mechanisms involving defective Ca 2+ transmission, and we identified reduced physical interaction between RyR and mitochondrial voltage-dependent anion channel (VDAC) as the main responsible of this effect. 相似文献
11.
The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca 2+. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca 2+ homeostasis during ER stress responses is inositol-requiring enzyme 1 α (IRE1 α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca 2+ dysregulation via the IRE1 α-dependent signaling pathway. In this study, we show that inactivation of IRE1 α by RNA interference increases cytosolic Ca 2+ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca 2+ through the InsP3 receptor (InsP3R). The Ca 2+ efflux in IRE1 α-deficient cells correlates with dissociation of the Ca 2+-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1 α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca 2+ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca 2+ dysregulation-induced mitochondrial abnormalities and cell death in IRE1 α-deficient cells can be blocked by depleting ROS or inhibiting Ca 2+ influx into the mitochondria. These results demonstrate the importance of IRE1 α in Ca 2+ homeostasis and cell survival during ER stress and reveal a previously unknown Ca 2+-mediated cell death signaling between the IRE1 α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion. 相似文献
12.
The tumor suppressor activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10) is thought to be largely attributable to its lipid phosphatase activity. PTEN dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate to directly antagonize the phosphoinositide 3-kinase-Akt pathway and prevent the activating phosphorylation of Akt. PTEN has also other proposed mechanisms of action, including a poorly characterized protein phosphatase activity, protein–protein interactions, as well as emerging functions in different compartment of the cells such as nucleus and mitochondria. We show here that a fraction of PTEN protein localizes to the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), signaling domains involved in calcium ( 2+) transfer from the ER to mitochondria and apoptosis induction. We demonstrate that PTEN silencing impairs ER Ca 2+ release, lowers cytosolic and mitochondrial Ca 2+ transients and decreases cellular sensitivity to Ca 2+-mediated apoptotic stimulation. Specific targeting of PTEN to the ER is sufficient to enhance ER-to-mitochondria Ca 2+ transfer and sensitivity to apoptosis. PTEN localization at the ER is further increased during Ca 2+-dependent apoptosis induction. Importantly, PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and this correlates with the reduction in their phosphorylation and increased Ca 2+ release. We propose that ER-localized PTEN regulates Ca 2+ release from the ER in a protein phosphatase-dependent manner that counteracts Akt-mediated reduction in Ca 2+ release via IP3Rs. These findings provide new insights into the mechanisms and the extent of PTEN tumor-suppressive functions, highlighting new potential strategies for therapeutic intervention. 相似文献
13.
The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca 2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca 2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca 2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca 2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. 相似文献
14.
Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca 2+ concentration was examined. An initial 5–10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200–400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5–10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1β–converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca 2+ cycling and the influx of extracellular Ca 2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca 2+ mobilization. 相似文献
15.
It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca 2+-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/P i carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca 2+ signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca 2+-dependent way with an S 0.5 for Ca 2+ activation of 3.3 ± 0.9 μ m. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca 2+-mobilizing agents, possibly by allowing a Ca 2+-dependent accumulation of mitochondrial AdNs and matrix Ca 2+, events permissive for other glucagon actions. 相似文献
16.
Inhibition of the mitochondrial Na +/Ca 2+ exchanger (NCLX) by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca 2+ homeostasis. However, the Ca 2+ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca 2+ levels are modulated by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 on neuronal Ca 2+ homeostasis using cameleon-based mitochondrial Ca 2+ and cytosolic Ca 2+ ([Ca 2+] i) live imaging. We observed that NCLX-driven mitochondrial Ca 2+ exchange occurs in cortical neurons under basal conditions as {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 induced a decrease in [Ca 2] i concomitant with a Ca 2+ accumulation inside the mitochondria. In turn, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 also inhibited mitochondrial Ca 2+ efflux after the stimulation of acetylcholine receptors. In contrast, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 strongly prevented depolarization-induced [Ca 2+] i increase by blocking voltage-gated Ca 2+ channels (VGCCs), whereas it did not induce depletion of ER Ca 2+ stores. Moreover, mitochondrial Ca 2+ overload was reduced as a consequence of diminished Ca 2+ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca 2+ overload by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 modulates cytosolic and mitochondrial Ca 2+ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs. 相似文献
17.
1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1- 14C]- and [5- 14C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD +, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD +, resulting in preferential oxidation of α-oxoglutarate. 相似文献
18.
The possible contribution of mitochondrial Ca 2+ accumulation and release to contractile phenomena has been investigated. Two intracellular fractions of Ca 2+ sequestration can be identified in cardiac myocytes, one ascribed to mitochondria. Two modes of Ca 2+ transport exist within the mitochondrial fraction, one dependent upon mitochondrial respiration and the other upon extramitochondrial [Na +]. Experiments with trabeculae show that under appropriate conditions, the rate of relaxation and the amount of tension developed is dependent on these two modes of Ca 2+ transport. A model is presented quantifying the contribution of the mitochondria to relaxation. 相似文献
19.
The aim of this study was to investigate the interrelationship between the mitochondrial phospholipid cardiolipin (CL), mitochondrial respiration and morphology in dependence on hypoxia/reoxygenation and Ca 2+. Therefore, we subjected rat liver mitochondria to hypoxia/reoxygenation at different extramitochondrial Ca 2+ concentrations and analysed mitochondrial respiration, morphology, CL content, the composition of molecular CL species, oxidation of CL and two mono-lyso-CL species. Hypoxia/reoxygenation in the presence of elevated extramitochondrial Ca 2+ concentration caused dramatic impairment of mitochondrial respiration and morphology. Concomitantly, increased amounts of oxidised CL were detected in the incubation medium after the treatment. Hypoxia/reoxygenation alone caused degradation of CL. The treatments had no effect on the composition of molecular CL species. Our data support the hypothesis that CL oxidation and CL degradation are involved in mitochondrial injury caused by hypoxia/reoxygenation and Ca 2+. Our results further suggest that prevention of CL oxidation by modification of CL composition may support the beneficial action of antioxidants during hypoxia/reoxygenation in the presence of elevated Ca 2+ concentrations. 相似文献
20.
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
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