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Endoplasmic reticulum/mitochondria calcium cross-talk
Abstract and Figures
The interaction of mitochondria with the endoplasmic reticulum (ER) Ca2+ store plays a key role in allowing these organelles to rapidly and effectively respond to cellular Ca2+ signals. In this contribution, we will briefly discuss: (i) old and new concepts of mitochondrial Ca2+ homeostasis; (ii) the relationship between mitochondrial 3D structure and Ca2+ homeostasis; (iii) the modulation by cytoplasmic signalling pathways; and (iv) new data suggesting that mitochondria and ER Ca2+ channels are assembled in a macromolecular complex in which the inositol-1,4,5-trisphosphate receptor directly stimulates the mitochondrial Ca2+ uptake machinery.
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... Initially, the increase in hASM ATP demand is met by increased mitochondrial O 2 consumption and ATP production, but at the expense of reactive oxygen species (ROS) formation and oxidative stress. There is increasing evidence that mitochondria and the ER, although structurally separate organelles, are functionally interdependent units, which must establish links for normal cellular function, including [Ca 2+ ] cyt regulation, energy production and cell proliferation (Hajnoczky et al., 2000;Franzini-Armstrong, 2007;Romagnoli et al., 2007;Liesa et al., 2009;Kornmann and Walter, 2010;Antico Arciuch et al., 2012;Glancy and Balaban, 2012;Dorn and Maack, 2013;Kornmann, 2013;Raturi and Simmen, 2013;van Vliet et al., 2014;Filadi et al., 2017). These links are established through specialized ER-mitochondria encounter structures (ERMES) comprising both ER and mitochondrial transmembrane proteins that provide a tethering force between the two organelles to ensure proximity and communication (Franzini-Armstrong, 2007;Kornmann and Walter, 2010;Dorn and Maack, 2013;Kornmann, 2013;Raturi and Simmen, 2013;van Vliet et al., 2014;Filadi et al., 2017). ...
... Based on biochemical studies, it is well known that mitochondrial production of ATP (oxidative phosphorylation) depends on dehydrogenase enzyme activities of the tricarboxylic acid (TCA) cycle (or citric acid cycle). Some of these dehydrogenase enzymes [i.e., pyruvate dehydrogenase (PDH), NAD-isocitrate dehydrogenase (ICDH), and oxoglutarate dehydrogenase (OGDH)] are Ca 2+ dependent (Rizzuto et al., 2000;Parekh, 2003;Franzini-Armstrong, 2007;Maack and O'Rourke, 2007;Romagnoli et al., 2007). Additionally, an increase in [Ca 2+ ] cyt stimulates mitochondrial shuttle systems such as the glycerol phosphate shuttle and the aspartate/glutamate transporters resulting in an increase in NADH levels in the mitochondria (Palmieri et al., 2001;Denton, 2009). ...
Inflammatory airway diseases such as asthma affect more than 300 million people world-wide. Inflammation triggers pathophysiology via such as tumor necrosis factor α (TNFα) and interleukins (e.g., IL-13). Hypercontraction of airway smooth muscle (ASM) and ASM cell proliferation are major contributors to the exaggerated airway narrowing that occurs during agonist stimulation. An emergent theme in this context is the role of inflammation-induced endoplasmic reticulum (ER) stress and altered mitochondrial function including an increase in the formation of reactive oxygen species (ROS). This may establish a vicious cycle as excess ROS generation leads to further ER stress. Yet, it is unclear whether inflammation-induced ROS is the major mechanism leading to ER stress or the consequence of ER stress. In various diseases, inflammation leads to an increase in mitochondrial fission (fragmentation), associated with reduced levels of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2). Mitochondrial fragmentation may be a homeostatic response since it is generally coupled with mitochondrial biogenesis and mitochondrial volume density thereby reducing demand on individual mitochondrion. ER stress is triggered by the accumulation of unfolded proteins, which induces a homeostatic response to alter protein balance via effects on protein synthesis and degradation. In addition, the ER stress response promotes protein folding via increased expression of molecular chaperone proteins. Reduced Mfn2 and altered mitochondrial dynamics may not only be downstream to ER stress but also upstream such that a reduction in Mfn2 triggers further ER stress. In this review, we summarize the current understanding of the link between inflammation-induced ER stress and mitochondrial function and the role played in the pathophysiology of inflammatory airway diseases.
... Under pathological conditions, such as endoplasmic reticulum stress, drug action and surgery, the increase of calcium released from endoplasmic reticulum leads to long-term mitochondrial calcium overload. 25 It has been reported that mitochondrial calcium overload can induce oxidative stress by increasing ROS production. 26 MCU overexpression can lead to higher sensitivity of cells to oxidative stress. ...
Purpose
Ru360, a selective inhibitor of mitochondrial calcium uptake, maintains mitochondrial calcium homeostasis. To evaluate whether mitochondrial calcium uniporter (MCU)-mediated mitochondrial function is associated with the pathological process of Postoperative cognitive dysfunction (POCD), elucidate its relationship with neuroinflammation, and observe whether the relevant pathological process can be improved with Ru360.
Methods
Aged mice underwent experimental open abdominal surgery after anesthesia. Open field tests, Novel object recognition tests and Y Maze Tests were used to conduct behavioral experiments. The reactive oxygen species (ROS) content, the levels of inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), intra-mitochondrial calcium, mitochondrial membrane potential (MMP) and the activity of antioxidant superoxide dismutase (SOD) in the hippocampus of mice were detected using kits. The expression of proteins was detected using Western blot.
Results
After treatment with Ru360, MCU-mediated mitochondrial dysfunction was inhibited, neuroinflammation was reduced, and the learning ability of the mice was improved after surgery.
Conclusion
Our study demonstrated that mitochondrial function plays a crucial role in the pathology of POCD, and using Ru360 to improve mitochondrial function may be a new and necessary direction for the treatment of POCD.
... At least some of these processes operate at a defined range of ER-OMM gap sizes: lipid transfer requires at short distance of < 10 nm; Ca 2+ transfer operates at an average of 15 nm gap size; phagosome formation requires ~ 50 nm distance, while the gap size for the contacts of mitochondria with rough ER is between 50 and 80 nm [15]. Detailed analysis of structure and function of these domains is beyond the scope of this contribution and can be found in recent reviews [7,15,16,70,[105][106][107]. Here we shall discuss structure-function relationships of the ER-mitochondria Ca 2+ transfer toolkit and its alteration by the disease-related proteins. ...
Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are morpho-functional units, formed at the loci of close apposition of the ER-forming endomembrane and outer mitochondrial membrane (OMM). These sites contribute to fundamental cellular processes including lipid biosynthesis, autophagy, apoptosis, ER-stress and calcium (Ca²⁺) signalling. At MERCS, Ca²⁺ ions are transferred from the ER directly to mitochondria through a core protein complex composed of inositol-1,4,5 trisphosphate receptor (InsP3R), voltage-gated anion channel 1 (VDAC1), mitochondrial calcium uniporter (MCU) and adaptor protein glucose-regulated protein 75 (Grp75); this complex is regulated by several associated proteins. Deregulation of ER-mitochondria Ca²⁺ transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of Ca²⁺ transfer between ER and mitochondria depends on the protein composition of MERCS, which controls ER-mitochondria interaction regulating, for example, the transversal distance between ER membrane and OMM and the extension of the longitudinal interface between ER and mitochondria. These parameters are altered in neurodegeneration. Here we overview the ER and mitochondrial Ca²⁺ homeostasis, the composition of ER-mitochondrial Ca²⁺ transfer machinery and alterations of the ER-mitochondria Ca²⁺ transfer in three major neurodegenerative diseases: motor neurone diseases, Parkinson disease and Alzheimer's disease.
... Increasing evidence shows that although mitochondria and the ER are two structurally independent organelles, their functions in regulation of Ca 2+ , generation of energy, and cell proliferation are interrelated and interdependent (Filadi et al. 2017;Romagnoli et al. 2007). Mitofusin-2(Mfn2) is an ERmitochondria encounter structure (ERMES) component, which can bind mitochondria to the ER, fuse mitochondria, regulate the mitochondria and the contact between ER-mitochondria, and regulate the UPR and mitochondrial functions through PERK (Basso et al. 2018;. ...
The endoplasmic reticulum (ER) is a multifunctional organelle, which is crucial for correct folding and assembly of secretory and transmembrane proteins. Perturbations of ER function can cause ER stress. ER stress can activate the unfolded protein response (UPR) to cope with the accumulation of misfolded proteins and protein toxicity. UPR is a coordination system that regulates transcription and translation, leading to the recovery of ER homeostasis or cell death. However, cells have an integrated signaling system to cope with ER stress, which helps cells to restore and balance their ER function. The main components of this system are ER-associated degradation (ERAD), autophagy, hypoxia signaling, and mitochondrial biogenesis. If the balance cannot be restored, the imbalance will lead to cell death or apoptosis, or even to a series of diseases. In this review, a series of activities to restore the homeostasis of cells during ER stress are discussed.
Key points
• Endoplasmic reticulum (ER) plays a key role in the biological process of cells.
• Perturbations of ER function can cause ER stress, including the ER overload response (EOR), sterol-regulated cascade reaction, and the UPR.
• Cells have an integrated signaling system (ERAD, autophagy, hypoxia signaling, and mitochondrial biogenesis) to cope with the adverse impact caused by ER stress.
Graphical abstract
... The activation of transmembrane stress-sensing proteins in the ER, including inositol-requiring protein 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF-6), either gives rise to signals for cellular self-recovery or, alternatively, promotes cell death (Garg et al., 2012;Harding et al., 2002;Tabas and Ron, 2011). Calcium homeostasis also plays crucial role in ER/mitochondria cross-talk and hence induces various modes of cell death, autophagy, apoptosis, necrosis (Pinton et al., 2008;Romagnoli et al., 2007;Kaufman and Malhotra, 2014;Malhotra and Kaufman, 2011). ...
The aim of the present study was to evaluate the underlying mechanism of multi-walled carbon nanotubes (MWCNT) induced cellular response and their potential cross-talk, specifically, between endoplasmic reticulum (ER) stress, MAPK activation and apoptosis and how these nano-bio interactions depend on the physico-chemical properties of MWCNT. For this purpose, human bronchial epithelial (Beas2B) and human hepatoma (HepG2) cell lines, were exposed to five kinds of MWCNTs which differ in functionalization and aspect ratios. Tissue-specific sensitivity was evident for calcium homeostasis, ER-stress response, MAPK activation and apoptosis, which further depended on surface functionalization as well as aspect ratios of MWCNT. By applying specific pharmaceutical inhibitors, relevant biomarkers gene and proteins expressions, we found that possibly MWCNT induce activation of IRE1α-XPB1 pathway-mediated ER-stress response, which in turn trigger apoptosis through JNK activation in both type of cells but with variable intensity. The information presented here would have relevance in better understanding of MWCNT toxicity and their safer applications.
... Especially, given the observed interactions between Seps1 suppression and palmitate treatment and that palmitate is known to induce phospho-Jnk (Gorgani-Firuzjaee et al. 2014). Cross-talk between the ER and mitochondria is also important in regulating cellular homeostasis (Romagnoli et al. 2007;Marchi et al. 2014). In response to cellular stress, as part of an adaptive response, mitochondria can buffer the excess calcium released from the ER. ...
The antioxidant Selenoprotein S (Seps1, Selenos) is an endoplasmic reticulum (ER)-resident protein associated with metabolic and inflammatory disease. While Seps1 is highly expressed in skeletal muscle, its mechanistic role as an antioxidant in skeletal muscle cells is not well characterized. In C2C12 myotubes treated with palmitate for 24 h, endogenous Seps1 protein expression was upregulated twofold. Two different siRNA constructs were used to investigate whether decreased levels of Seps1 exacerbated lipid-induced oxidative and ER stress in C2C12 myotubes and myoblasts, which differ with regards to cell cycle state and metabolic phenotype. In myoblasts, Seps1 protein knockdown of ~50% or ~75% exacerbated cellular stress responses in the presence of palmitate; as indicated by decreased cell viability and proliferation, higher H2 O2 levels, a lower reduced to oxidized glutathione (GSH:GSSG) ratio, and enhanced gene expression of ER and oxidative stress markers. Even in the absence of palmitate, Seps1 knockdown increased oxidative stress in myoblasts. Whereas, in myotubes in the presence of palmitate, a ~50% knockdown of Seps1 was associated with a trend toward a marginal (3-5%) decrease in viability (P = 0.05), decreased cellular ROS levels, and a reduced mRNA transcript abundance of the cellular stress marker thioredoxin inhibitory binding protein (Txnip). Furthermore, no enhancement of gene markers of ER stress was observed in palmitate-treated myotubes in response to Seps1 knockdown. In conclusion, reduced Seps1 levels exacerbate nutrient-induced cellular stress responses to a greater extent in glycolytic, proliferating myoblasts than in oxidative, differentiated myotubes, thus demonstrating the importance of cell phenotype to Seps1 function.
... The mitochondria can store Ca 2+ , but Ca 2+ will overload under conditions of oxidative stress [46][47][48]. Increased intracellular Ca 2+ concentration may be associated with apoptosis [49]. The intracellular Ca 2+ concentration was measured by Molecular Devices using Fluo-3AM as fluorescent probe. ...
Five Ru(II) polypyridyl complexes [Ru(N–N)2(nfip)](ClO4)2 (N–N = dmb, 1; bpy, 2; ttbpy, 3, phen, 4, dmp, 5) were synthesized and characterized. The cytotoxic activities of the complexes against cancer SGC-7901, Eca-109, HepG2, A549, HeLa and normal LO2 cells were investigated using the MTT method. Complexes 1–4 show high cytotoxicity against SGC-7901 cells with low IC50 values of 12.4 ± 1.4, 6.7 ± 1.6, 1.3 ± 0.5 and 1.1 ± 0.4 µM, respectively. Complex 5 is active against HeLa cells with an IC50 value of 2.2 ± 0.6 µM. Among these complexes, complexes 3 and 4 exhibit higher cytotoxic activity than cisplatin toward SGC-7901 cells under identical conditions. Apoptosis was assayed using the AO/EB staining method and flow cytometry. The location of the complexes at lysosomes and mitochondria, the permeability of lysosomes, the levels of reactive oxygen species, and the changes of mitochondrial membrane potential were studied by fluorescence microscopy. The cell cycle distribution of SGC-7901 cells induced by the complexes was studied by flow cytometry. The expression of caspase-3, PARP and Bcl-2 family proteins was investigated by Western blot. The complexes were shown to accumulate in the lysosomes and then enter into the mitochondria. The mechanism demonstrates that the complexes induce apoptosis in SGC-7901 cells through three pathways: (1) ROS-mediated lysosomal–mitochondrial dysfunction; (2) inhibition of PI3K/AKT/mTOR pathway; and (3) DNA damage and inhibition of the cell growth at G0/G1 or S phase.
... Under normal physiological conditions, the bulk of the Ca 2+ resides within the ER lumen and, during cellular events requiring a Ca 2+ signal, a small proportion crossing the outer mitochondrial membrane. In pathological conditions such as ER stress, increased release of Ca 2+ from the ER result in massive and/or a prolonged mitochondrial Ca 2+ overload [25] In this regard, various pathological conditions including ER stress, oxidative stress, palmitate, and chronic high glucose decreased ER calcium levels in pancreatic β cells [26]. Importantly, the rate of ER calcium-depleted β cells was increased by chronic high glucose [26]. ...
Mitochondrial Ca²⁺ is a key regulator of organelle physiology and the excessive increase in mitochondrial calcium is associated with the oxidative stress. In the present study, we investigated the molecular mechanisms linking mitochondrial calcium to inflammatory and coagulative responses in hepatocytes exposed to high glucose (HG) (33mM glucose). Treatment of HepG2 cells with HG for 24 h induced insulin resistance, as demonstrated by an impairment of insulin-stimulated Akt phosphorylation. HepG2 treatment with HG led to an increase in mitochondrial Ca²⁺ uptake, while cytosolic calcium remained unchanged. Inhibition of MCU by lentiviral-mediated shRNA prevented mitochondrial calcium uptake and downregulated the inflammatory (TNF-α, IL-6) and coagulative (PAI-1 and FGA) mRNA expression in HepG2 cells exposed to HG. The protection from HG-induced inflammation by MCU inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). Importantly, MCU inhibition in HepG2 cells abrogated the phosphorylation of p38, JNK and IKKα/IKKβ in HG treated cells. Taken together, these data suggest that MCU inhibition may represent a promising therapy for prevention of deleterious effects of obesity and metabolic diseases.
As an important metal in industry, national defense, and production, nickel widely exists in nature and is also a necessary trace element for human beings and animals. Nickel deficiency will affect the growth and development of animals, the contents of related active substances, enzymes and other essential elements in vivo. However, excessive nickel or longer nickel exposure can induce excessive free radicals (reactive oxygen species and reactive nitrogen) in the body, which can lead to a variety of cell damage, apoptosis and canceration, and ultimately pose negative effects on the health of the body. Among them, the intestinal tract, as the largest interface between the body and the external environment, greatly increases the contact probability between nickel or nickel compounds and the intestinal mucosal barrier, thus, the intestinal structure and function are also more vulnerable to nickel damage, leading to a series of related diseases such as enteritis. Therefore, this paper briefly analyzed the damage mechanism of nickel or its compounds to the intestinal tract from the perspective of four intestinal mucosal barriers: mechanical barrier, immune barrier, microbial barrier and chemical barrier, we hope to make a certain theoretical contribution to the further research and the prevention and treatment of nickel related diseases.
In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared to normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in Drp1 and a decrease in Mfn2 expression, mitochondrial fission and fusion proteins, respectively. Pro-inflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of pro-inflammatory cytokines in mitochondrial remodeling is not clearly established. In non-asthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in 3-D using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor, aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased PGC1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O2 consumption rate. However, when normalized for mitochondrial volume density, O2 consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O2 consumption rate per mitochondrion.
We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.
In a previous report (Macedo, D.V., Ferraz, V. L., Pereira-da-Silva, L., and Vercesi, A. E. (1988) in Integration of Mitochondrial Functions (Lemasters, J. J., et al., eds) pp. 535-542, Plenum Publishing Corp., New York), we proposed that the alterations in the inner mitochondrial membrane permeability caused by Ca2+ plus prooxidants could be the consequence of membrane protein sulfhydryl-disulfide transitions. In this study, we show that Ca2+ plus diamide, a thiol oxidant, significantly decrease the ability of beef heart submitochondrial particles to build up and sustain a membrane potential generated by succinate oxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicates that these effects on the membrane potential are associated with the production of protein aggregates due to thiol cross-linking. Evidence is also presented that these protein aggregates can be produced in mitoplasts previously loaded with Ca2+ and that this is potentiated by the presence of either diamide or t-butylhydroperoxide. Furthermore, dithiothreitol, a disulfide reductant, was found to be much more effective than NAD(P)+ reductants in reversing Ca2+ efflux induced by prooxidants. It is concluded that the perturbation of the inner mitochondrial membrane caused by Ca2+ plus prooxidants is associated with protein polymerization due to thiol cross-linking, resulting in the production of high molecular mass protein aggregates.
Intracellular oxygen pressure within intact isolated cardiac myocytes is studied as a function of steady state extracellular oxygen pressure. The fractional saturation of myoglobin with oxygen is used to report sarcoplasmic oxygen pressure. The fractional oxidation of cytochrome oxidase, the fractional oxidation of cytochrome c, the rate of respiratory oxygen uptake, and lactate accumulation are used to reflect the availability of oxygen at the inner mitochondrial membrane. These probes of mitochondrial function show no large change with decreasing extracellular oxygen pressure until that pressure is less than 2 torr and intracellular myoglobin is largely deoxygenated. Sarcoplasmic oxygen pressure in resting cells is nearly the same as extracellular oxygen pressure and is about 2 torr less in cells whose respiration has been increased 3.5-fold by mitochondrial uncoupling. Oxygen pressure at the mitochondrial inner membrane differs from sarcoplasmic oxygen pressure by no more than 0.2 torr and from extracellular oxygen pressure by no more than 2 torr. We conclude that differences of oxygen pressure within the cardiac myocyte are very small. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. We conclude also that mitochondria of the cardiac myocyte become oxygen limited only when sarcoplasmic myoglobin is almost entirely deoxygenated.
In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD). Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(1,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.
Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μm with an apparent Km for oxygen of less than 1 μm. In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μm downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic and intramitochondrial because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and .
The human mitochondrial genome is very small and economically packed; the expression of the whole genome is essential for the maintenance of mitochondrial bioenergetic function. Mutation occurs at a much higher rate in the mitochondrial DNA (mtDNA) than in chromosomal DNA. Transient heteroplasmy of mtDNA occurs after a mutational event; the random pattern of cytoplasmic segregation that occurs during subsequent growth gives rise to a mosaic of cells. The variable proportion of mutant mitochondrial genomes per cell results in cells with a range of bioenergetic capacities. It is proposed that the accumulation of mitochondrial mutations and the subsequent cytoplasmic segregation of these mutations during life is an important contributor both to the ageing process and to several human degenerative diseases. Replacement therapy and pharmacological support may be possible for the amelioration of such disorders by means of appropriate redox compounds. Moreover, new compounds with desired redox potentials can be rationally designed for clinical use.
State III (activated) mitochondrial respiration rates with pyruvate/malate, glutamate/malate, and succinate as substrates were assayed in isolated intact skeletal muscle mitochondria in 29 subjects aged 16-92 years. There was a significant negative correlation between respiration rate and age with all substrates tested. A similar trend was seen for respiratory enzyme activities assayed in muscle homogenate. These findings suggest a substantial fall in mitochondrial oxidative capacity in ageing muscle, which may contribute to reduced exercise capacity in elderly people. Mitochondrial respiratory failure may contribute to the ageing process in other organs.
Isolated microtubule-associated protein 2 (MAP2), tau factor, and tubulin were phosphorylated by a purified Ca2+, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and tau factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, tau factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, tau factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca2+, calmodulin system via phosphorylation of MAP2, tau factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.
The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.
Hepatocytes respond to inositol 1,4,5-trisphosphate (InsP3)-linked agonists with frequency-modulated oscillations in the intracellular free calcium concentration ([Ca2+]i), that occur as waves propagating from a specific origin within each cell. The subcellular distribution and functional organization of InsP3-sensitive Ca2+ pools has been investigated, in both intact and permeabilized cells, by fluorescence imaging of dyes which can be used to monitor luminal Ca2+ content and InsP3-activated ion permeability in a spatially resolved manner. The Ca2+ stores behave as a luminally continuous system distributed throughout the cytoplasm. The structure of the stores, an important determinant of their function, is controlled by the cytoskeleton and can be modulated in a guanine nucleotide-dependent manner. The nuclear matrix is devoid of Ca2+ stores, but Ca2+ waves in the intact cell propagate through this compartment. The organization of [Ca2+]i signals has also been investigated in the perfused liver. Frequency-modulated [Ca2+]i oscillations are still observed at the single cell level, with similar properties to those in the isolated hepatocyte. The [Ca2+]i oscillations propagate between cells in the intact liver, leading to the synchronization of [Ca2+]i signals across part or all of each hepatic lobule.