The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumour cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Thorne RF
PROVIDER: S-EPMC1168533 | biostudies-other | 1974 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. The modification of pyruvate kinase activity in vitro was examined by altering the environmental [Mg(2+)]/[Ca(2+)] ratio with EDTA on the one hand and isolated rat liver mitochondria on the other. 2. Controlled additions of Ca(2+) and EDTA caused pyruvate kinase activity to be alternately and rapidly switched on and off. 3. By being able to accumulate Ca(2+) in preference to Mg(2+) rat liver mitochondria were able to alter the [Mg(2+)]/[Ca(2+)] ratio in the vicinity of pyruvate kinase and thereby modify the activity of this enzyme. 4. The possible role of mitochondria in modifying pyruvate kinase and other ion-sensitive cytoplasmic enzyme activities is discussed.
| S-EPMC1173777 | biostudies-other
Project description:The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.
| S-EPMC1135547 | biostudies-other
Project description:IntroductionIncidents of myocardial infarction and sudden cardiac arrest vary with time of the day, but the mechanism for this effect is not clear. We hypothesized that diurnal changes in the ability of cardiac mitochondria to control calcium homeostasis dictate vulnerability to cardiovascular events.ObjectivesHere we investigate mitochondrial calcium dynamics, respiratory function, and reactive oxygen species (ROS) production in mouse heart during different phases of wake versus sleep periods.MethodsWe assessed time-of-the-day dependence of calcium retention capacity of isolated heart mitochondria from young male C57BL6 mice. Rhythmicity of mitochondrial-dependent oxygen consumption, ROS production and transmembrane potential in homogenates were explored using the Oroboros O2k Station equipped with a fluorescence detection module. Changes in expression of essential clock and calcium dynamics genes/proteins were also determined at sleep versus wake time points.ResultsOur results demonstrate that cardiac mitochondria exhibit higher calcium retention capacity and higher rates of calcium uptake during sleep period. This was associated with higher expression of clock gene Bmal1, lower expression of per2, greater expression of MICU1 gene (mitochondrial calcium uptake 1), and lower expression of the mitochondrial transition pore regulator gene cyclophilin D. Protein levels of mitochondrial calcium uniporter (MCU), MICU2, and sodium/calcium exchanger (NCLX) were also higher at sleep onset relative to wake period. While complex I and II-dependent oxygen utilization and transmembrane potential of cardiac mitochondria were lower during sleep, ROS production was increased presumably due to mitochondrial calcium sequestration.ConclusionsTaken together, our results indicate that retaining mitochondrial calcium in the heart during sleep dissipates membrane potential, slows respiratory activities, and increases ROS levels, which may contribute to increased vulnerability to cardiac stress during sleep-wake transition. This pronounced daily oscillations in mitochondrial functions pertaining to stress vulnerability may at least in part explain diurnal prevalence of cardiac pathologies.
| S-EPMC8240107 | biostudies-literature
Project description:Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.
| S-EPMC7851384 | biostudies-literature
Project description:The data presented in this article are related to the research article entitled "Rotenone decreases ischemia-induced injury by inhibiting mitochondrial permeability transition in mature brains" (Rekuviene et al., 2017) [1]. Data in this article present the direct effects of rotenone on calcium retention capacity (CRC) in isolated normal cortex and cerebellum mitochondria, effects of rotenone intravenous infusion on leak and phosphorylating respiration rates of isolated cortex and cerebellum mitochondria, on activities of respiratory chain complexes I and II in freezed-thawed/sonicated cortex and cerebellum mitochondria after brain ischemia. In addition, detailed experimental procedures of isolation of brain mitochondria, measurements of CRC, respiration, activities of respiratory chain complexes and H2O2 generation in cortex and cerebellum mitochondria are described.
| S-EPMC5512184 | biostudies-literature
Project description:Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (?p) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology.
| S-EPMC5544316 | biostudies-literature
Project description: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 (??m), the role of the mitochondria in buffering cytosolic Ca(2+) signals was investigated. We show here that rapid loss of ??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.
| S-EPMC4167535 | biostudies-literature
Project description:The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti-tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti-tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME-such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell-based microenvironment therapies-to provide novel ideas for producing breakthroughs in tumour therapy strategies.
| S-EPMC7445401 | biostudies-literature
Project description:Tumours progress despite being infiltrated by effector T cells. Tumour necrosis is associated with poor survival in a variety of cancers. Here, we report that that necrosis causes release of an intracellular ion, potassium, into the extracellular fluid of human and mouse tumours. Surprisingly, elevated extracellular potassium ([K+]e) was sufficient to profoundly suppress mouse and human T cell anti-tumour function. Elevations in [K+]e acted to acutely impair T cell receptor (TCR) dependent Akt-mTOR phosphorylation and effector function. Potassium mediated suppression of Akt-mTOR signalling and T cell effector function required intact activity of PP2A, a serine/threonine phosphatase. The suppressive effect mediated by elevated [K+]e required a T cell-intrinsic increase in intracellular potassium ([K+]i) and was independent of changes in plasma membrane potential (Vm). Finally, ionic reprogramming of tumour-specific T cells via over-expression of the voltage-gated potassium channel Kv1.3 lowered [K+]i and improved effector functions in vitro and in vivo, with this gain of function being dependent on intact channel function. Consequently, Kv1.3 T cell expression enhanced tumour clearance and the survival of melanoma-bearing mice. These results uncover a previously undescribed ionic checkpoint against T cell function within tumours and identify new strategies for cancer immunotherapy. RNA expression was measured by RNA-Seq on day 5 of cultures, maintained in individual biologial triplicates which were stimulated with immobilized anti-CD3/28 antibodies or kept in complete media (no stim) - with equivalent conditions treated with isotonic media containing elevated potassium.
2016-08-13 | E-GEOD-84996 | biostudies-arrayexpress
Project description:Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.
| S-EPMC7660078 | biostudies-literature