Quantitative Analysis of Mitochondrial Ca2+ Uptake and Release Pathways in Sympathetic Neurons

Rate equations for mitochondrial Ca2+ uptake and release and plasma membrane Ca2+ transport were determined from the measured fluxes in the preceding study and incorporated into a model of Ca2+ dynamics. It was asked if the measured fluxes are sufficient to account for the [Ca2+]i recovery kinetics after depolarization-evoked [Ca2+]i elevations. Ca2+ transport across the plasma membrane was described by a parallel extrusion/leak system, while the rates of mitochondrial Ca2+ uptake and release were represented using equations like those describing Ca2+ transport by isolated mitochondria. Taken together, these rate descriptions account very well for the time course of recovery after [Ca2+]i elevations evoked by weak and strong depolarization and their differential sensitivity to FCCP, CGP 37157, and [Na+]i. The model also leads to three general conclusions about mitochondrial Ca2+ transport in intact cells: (1) mitochondria are expected to accumulate Ca2+ even in response to stimuli that raise [Ca2+]i only slightly above resting levels; (2) there are two qualitatively different stimulus regimes that parallel the buffering and non-buffering modes of Ca2+ transport by isolated mitochondria that have been described previously; (3) the impact of mitochondrial Ca2+ transport on intracellular calcium dynamics is strongly influenced by nonmitochondrial Ca2+ transport; in particular, the magnitude of the prolonged [Ca2+]i elevation that occurs during the plateau phase of recovery is related to the Ca2+ set-point described in studies of isolated mitochondria, but is a property of mitochondrial Ca2+ transport in a cellular context. Finally, the model resolves the paradoxical finding that stimulus-induced [Ca2+]i elevations as small as ∼300 nM increase intramitochondrial total Ca2+ concentration, but the steady [Ca2+]i elevations evoked by such stimuli are not influenced by FCCP.

Download Full-text

Depolarization-induced Calcium Responses in Sympathetic Neurons: Relative Contributions from Ca2+ Entry, Extrusion, ER/Mitochondrial Ca2+ Uptake and Release, and Ca2+ Buffering

The Journal of General Physiology ◽

10.1085/jgp.200609660 ◽

2006 ◽

Vol 129 (1) ◽

pp. 29-56 ◽

Cited By ~ 23

Author(s):

Michael Patterson ◽

James Sneyd ◽

David D. Friel

Keyword(s):

Plasma Membrane ◽

Voltage Clamp ◽

Diffusion Model ◽

Time Course ◽

Sympathetic Neurons ◽

Cell Types ◽

Specific Cell ◽

Modeling Studies ◽

Uniform Model ◽

Uptake And Release

Many models have been developed to account for stimulus-evoked [Ca2+] responses, but few address how responses elicited in specific cell types are defined by the Ca2+ transport and buffering systems that operate in the same cells. In this study, we extend previous modeling studies by linking the time course of stimulus-evoked [Ca2+] responses to the underlying Ca2+ transport and buffering systems. Depolarization-evoked [Ca2+]i responses were studied in sympathetic neurons under voltage clamp, asking how response kinetics are defined by the Ca2+ handling systems expressed in these cells. We investigated five cases of increasing complexity, comparing observed and calculated responses deduced from measured Ca2+ handling properties. In Case 1, [Ca2+]i responses were elicited by small Ca2+ currents while Ca2+ transport by internal stores was inhibited, leaving plasma membrane Ca2+ extrusion intact. In Case 2, responses to the same stimuli were measured while mitochondrial Ca2+ uptake was active. In Case 3, responses were elicited as in Case 2 but with larger Ca2+ currents that produce larger and faster [Ca2+]i elevations. Case 4 included the mitochondrial Na/Ca exchanger. Finally, Case 5 included ER Ca2+ uptake and release pathways. We found that [Ca2+]i responses elicited by weak stimuli (Cases 1 and 2) could be quantitatively reconstructed using a spatially uniform model incorporating the measured properties of Ca2+ entry, removal, and buffering. Responses to strong depolarization (Case 3) could not be described by this model, but were consistent with a diffusion model incorporating the same Ca2+ transport and buffering descriptions, as long as endogenous buffers have low mobility, leading to steep radial [Ca2+]i gradients and spatially nonuniform Ca2+ loading by mitochondria. When extended to include mitochondrial Ca2+ release (Case 4) and ER Ca2+ transport (Case 5), the diffusion model could also account for previous measurements of stimulus-evoked changes in total mitochondrial and ER Ca concentration.

Download Full-text

Oxygen affinity of the respiratory chain of Acanthamoeba castellanii

Biochemical Journal ◽

10.1042/bj2140047 ◽

1983 ◽

Vol 214 (1) ◽

pp. 47-51 ◽

Cited By ~ 15

Author(s):

D Lloyd ◽

H Mellor ◽

J L Williams

Keyword(s):

Plasma Membrane ◽

Cytochrome C ◽

Cytochrome B ◽

Respiratory Chain ◽

Oxygen Affinity ◽

Acanthamoeba Castellanii ◽

Intact Cells ◽

Cytochrome Aa3 ◽

Isolated Mitochondria ◽

Soil Amoeba

Apparent Km values for O2 for the soil amoeba Acanthamoeba castellanii determined polarographically and by bioluminescence gave similar values (0.37 and 0.41 microM respectively). Mitochondria oxidizing succinate or NADH in the presence or absence of ADP gave values in the range 0.21-0.36 microM-O2. Oxidation of respiratory-chain components to 50% of the aerobic steady states in intact cells was observed at the following O2 concentrations: cytochrome aa3, 0.1-0.25 microM; cytochrome c, 0.3-0.6 microM; cytochrome b, 0.35-0.45 microM; flavoprotein, 2 microM. In isolated mitochondria corresponding values for a-, c- and b-type cytochromes were 0.007, 0.035-0.05 and 0.06-0.09 microM-O2. It is concluded that an O2 gradient exists between plasma membrane and mitochondria in A. castellanii.

Download Full-text

Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts

Biochemical Journal ◽

10.1042/bj2500653 ◽

1988 ◽

Vol 250 (3) ◽

pp. 653-658 ◽

Cited By ~ 238

Author(s):

J P Slotte ◽

E L Bierman

Keyword(s):

Plasma Membrane ◽

Cholesteryl Ester ◽

Time Course ◽

Plasma Membranes ◽

Mass Movement ◽

Extracellular Medium ◽

Acyltransferase Activity ◽

Intact Cells ◽

Cultured Fibroblasts ◽

Time Course Study

This study examines the relationship between cellular sphingomyelin content and the distribution of unesterified cholesterol between the plasma-membrane pool and the putative intracellular regulatory pool. The sphingomyelin content of cultured human skin fibroblasts was reduced by treatment of intact cells with extracellularly added neutral sphingomyelinase, and subsequent changes in the activities of cholesterol-metabolizing enzymes were determined. Exposure of fibroblasts to 0.1 unit of sphingomyelinase/ml for 60 min led to the depletion of more than 90% of the cellular sphingomyelin, as determined from total lipid extracts. In a time-course study, it was found that within 10 min of the addition of sphingomyelinase to cells, a dramatic increase in acyl-CoA:cholesterol acyltransferase activity could be observed, whether measured from the appearance of plasma membrane-derived [3H]cholesterol or exogenously added [14C]oleic acid, in cellular cholesteryl esters. In addition, the cholesteryl ester mass was significantly higher in sphingomyelin-depleted fibroblasts at 3 h after exposure to sphingomyelinase compared with that in untreated fibroblasts [7.1 +/- 0.4 nmol of cholesterol/mg equivalents of esterified cholesterol compared with 4.2 +/- 0.1 nmol of cholesterol/mg equivalents of cholesteryl ester in control cells (P less than 0.05)]. The sphingomyelin-depleted cells also showed a reduction in the rate of endogenous synthesis of cholesterol, as measured by incorporation of sodium [14C]acetate into [14C]cholesterol. These results are consistent with a rapid movement of cholesterol from sphingomyelin-depleted plasma membranes to the putative intracellular regulatory pool of cholesterol. This mass movement of cholesterol away from the plasma membranes presumably resulted from a decreased capacity of the plasma membranes to solubilize cholesterol, since sphingomyelin-depleted cells also had a decreased capacity to incorporate nanomolar amounts of [3H]cholesterol from the extracellular medium, as compared with control cells. These findings confirm previous assumptions that the membrane sphingomyelin content is an important determinant of the overall distribution of cholesterol within intact cells.

Download Full-text

Differential Regulation of ER Ca2+ Uptake and Release Rates Accounts for Multiple Modes of Ca2+-induced Ca2+ Release

The Journal of General Physiology ◽

10.1085/jgp.20028484 ◽

2002 ◽

Vol 119 (3) ◽

pp. 211-233 ◽

Cited By ~ 27

Author(s):

Meredith A. Albrecht ◽

Stephen L. Colegrove ◽

David D. Friel

Keyword(s):

Plasma Membrane ◽

Sympathetic Neurons ◽

Surface Membrane ◽

Central Element ◽

Differential Regulation ◽

Transport Systems ◽

Excitable Cells ◽

Release Rates ◽

Multiple Modes ◽

Uptake And Release

The ER is a central element in Ca2+ signaling, both as a modulator of cytoplasmic Ca2+ concentration ([Ca2+]i) and as a locus of Ca2+-regulated events. During surface membrane depolarization in excitable cells, the ER may either accumulate or release net Ca2+, but the conditions of stimulation that determine which form of net Ca2+ transport occurs are not well understood. The direction of net ER Ca2+ transport depends on the relative rates of Ca2+ uptake and release via distinct pathways that are differentially regulated by Ca2+, so we investigated these rates and their sensitivity to Ca2+ using sympathetic neurons as model cells. The rate of Ca2+ uptake by SERCAs (JSERCA), measured as the t-BuBHQ-sensitive component of the total cytoplasmic Ca2+ flux, increased monotonically with [Ca2+]i. Measurement of the rate of Ca2+ release (JRelease) during t-BuBHQ-induced [Ca2+]i transients made it possible to characterize the Ca2+ permeability of the ER (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{P}}_{\mathrm{ER}}\) \end{document}), describing the activity of all Ca2+-permeable channels that contribute to passive ER Ca2+ release, including ryanodine-sensitive Ca2+ release channels (RyRs) that are responsible for CICR. Simulations based on experimentally determined descriptions of JSERCA, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{P}}_{\mathrm{ER}}\) \end{document}, and of Ca2+ extrusion across the plasma membrane (Jpm) accounted for our previous finding that during weak depolarization, the ER accumulates Ca2+, but at a rate that is attenuated by activation of a CICR pathway operating in parallel with SERCAs to regulate net ER Ca2+ transport. Caffeine greatly increased the [Ca2+] sensitivity of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{P}}_{\mathrm{ER}}\) \end{document}, accounting for the effects of caffeine on depolarization-evoked [Ca2+]i elevations and caffeine-induced [Ca2+]i oscillations. Extending the rate descriptions of JSERCA, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{P}}_{\mathrm{ER}}\) \end{document}, and Jpm to higher [Ca2+]i levels shows how the interplay between Ca2+ transport systems with different Ca2+ sensitivities accounts for the different modes of CICR over different ranges of [Ca2+]i during stimulation.

Download Full-text

Raw Glycerol Based Medium for DHA and Lipids Production, Using the Marine Heterotrophic Microalga Crypthecodinium cohnii

Processes ◽

10.3390/pr9112005 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2005

Author(s):

Patrícia Moniz ◽

Carla Silva ◽

Ana Cristina Oliveira ◽

Alberto Reis ◽

Teresa Lopes da Silva

Keyword(s):

Crude Glycerol ◽

Time Course ◽

Corn Steep Liquor ◽

Lipid Production ◽

Biomass Concentration ◽

Nitrogen Sources ◽

Intact Cells ◽

Raw Glycerol ◽

Dry Cell Weight ◽

The Impact

Crude glycerol, a biodiesel industry byproduct, and corn steep liquor (CSL) derived from a starch industry, were used as carbon and nitrogen sources, respectively, for lipid production, using the heterotrophic microalga C. cohnii grown in a bench bioreactor, in a batch culture. The maximum biomass concentration, lipid content and lipid productivity attained were 5.34 g/L, 24.6% (w/w Dry Cell Weight-DCW) and 0.016 g L−1 h−1, respectively. Flow cytometry analysis was used to evaluate the impact of these substrates on the microalgae cells. A high proportion of intact cells with enzymatic (esterases) activity (>50%) was present throughout the cultivation time course. These results indicate that crude glycerol and CSL can be used in the medium formulation for DHA and lipid production using this microalga, which reduce the process costs in an expected maximum of 84%.

Download Full-text

Dissection of Mitochondrial Ca2+ Uptake and Release Fluxes in Situ after Depolarization-Evoked [Ca2+]i Elevations in Sympathetic Neurons

The Journal of General Physiology ◽

10.1085/jgp.115.3.351 ◽

2000 ◽

Vol 115 (3) ◽

pp. 351-370 ◽

Cited By ~ 83

Author(s):

Stephen L. Colegrove ◽

Meredith A. Albrecht ◽

David D. Friel

Keyword(s):

Plasma Membrane ◽

Initial Phase ◽

Sympathetic Neurons ◽

Low Frequency ◽

Selective Inhibitor ◽

Atp Production ◽

Exchange Inhibitor ◽

Simple Dependence ◽

Uptake And Release

We studied how mitochondrial Ca2+ transport influences [Ca2+]i dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+]i; the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+]i recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+]i, while the net mitochondrial flux (Jmito) is biphasic, indicative of Ca2+ accumulation during the initial phase of recovery when [Ca2+]i is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. Jmito was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na+/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of Jmito increases steeply with [Ca2+]i, as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na+ concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na+/Ca2+ exchanger. Above ∼400 nM [Ca2+]i, net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+]i is ∼200–300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca2+ transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca2+]i to levels below ∼500 nM.

Download Full-text

CD133 Expression in the Nucleus Is Associated with Endometrial Carcinoma Staging and Tumor Angioinvasion

Journal of Clinical Medicine ◽

10.3390/jcm10102144 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2144

Author(s):

Milosz Pietrus ◽

Kazimierz Pitynski ◽

Marcin Waligora ◽

Katarzyna Milian-Ciesielska ◽

Monika Bialon ◽

...

Keyword(s):

Endometrial Cancer ◽

Plasma Membrane ◽

Tumor Progression ◽

Endometrial Carcinoma ◽

Molecular Level ◽

Risk Group ◽

High Risk Group ◽

Cd133 Expression ◽

Transmembrane Glycoprotein ◽

The Impact

Background: (1) Endometrial cancer is one of the most common cancers affecting women, with a growing incidence. To better understand the different behaviors associated with endometrial cancer, it is necessary to understand the changes that occur at a molecular level. CD133 is one of the factors that regulate tumor progression, which is primarily known as the transmembrane glycoprotein associated with tumor progression or cancer stem cells. The aim of our study was to assess the impact of subcellular CD133 expression on the clinical course of endometrial cancer. (2) Methods: CD133 expression in the plasma membrane, nucleus, and cytoplasm was assessed by immunohistochemical staining in a group of 64 patients with endometrial cancer representing FIGO I-IV stages, grades 1–3 and accounting for tumor angioinvasion. (3) Results: Nuclear localization of CD133 expression was increased in FIGO IB-IV stages compared to FIGO IA. Furthermore, CD133 expression in the nucleus and plasma membrane is positively and negatively associated with a higher grade of endometrial cancer and angioinvasion, respectively. (4) Conclusions: Our findings suggest that positive nuclear CD133 expression in the tumor may be related to a less favorable prognosis of endometrial carcinoma patients and has emerged as a useful biomarker of a high-risk group.

Download Full-text

Decline of contractility during ischemia-reperfusion injury: actin glutathionylation and its effect on allosteric interaction with tropomyosin

AJP Cell Physiology ◽

10.1152/ajpcell.00419.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. C719-C727 ◽

Cited By ~ 70

Author(s):

Frank C. Chen ◽

Ozgur Ogut

Keyword(s):

Reperfusion Injury ◽

Posttranslational Modifications ◽

Actin Polymerization ◽

Time Course ◽

Troponin I ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

High Concentration ◽

Cross Sectional ◽

The Impact

The severity and duration of ischemia-reperfusion injury is hypothesized to play an important role in the ability of the heart subsequently to recover contractility. Permeabilized trabeculae were prepared from a rat model of ischemia-reperfusion injury to examine the impact on force generation. Compared with the control perfused condition, the maximum force (Fmax) per cross-sectional area and the rate of tension redevelopment of Ca2+-activated trabeculae fell by 71% and 44%, respectively, during ischemia despite the availability of a high concentration of ATP. The reduction in Fmax with ischemia was accompanied by a decline in fiber stiffness, implying a drop in the absolute number of attached cross bridges. However, the declines during ischemia were largely recovered after reperfusion, leading to the hypothesis that intrinsic, reversible posttranslational modifications to proteins of the contractile filaments occur during ischemia-reperfusion injury. Examination of thin-filament proteins from ischemic or ischemia-reperfused hearts did not reveal proteolysis of troponin I or T. However, actin was found to be glutathionylated with ischemia. Light-scattering experiments demonstrated that glutathionylated G-actin did not polymerize as efficiently as native G-actin. Although tropomyosin accelerated the time course of native and glutathionylated G-actin polymerization, the polymerization of glutathionylated G-actin still lagged native G-actin at all concentrations of tropomyosin tested. Furthermore, cosedimentation experiments demonstrated that tropomyosin bound glutathionylated F-actin with significantly reduced cooperativity. Therefore, glutathionylated actin may be a novel contributor to the diverse set of posttranslational modifications that define the function of the contractile filaments during ischemia-reperfusion injury.

Download Full-text

The mitochondrial consequences of uncoupling intact cells depend on the nature of the exogenous substrate

Biochemical Journal ◽

10.1042/bj3550231 ◽

2001 ◽

Vol 355 (1) ◽

pp. 231-235 ◽

Cited By ~ 6

Author(s):

Brigitte SIBILLE ◽

Céline FILIPPI ◽

Marie-Astrid PIQUET ◽

Pascale LECLERCQ ◽

Eric FONTAINE ◽

...

Keyword(s):

Oxidation Rate ◽

Marked Decrease ◽

Electrical Potential ◽

Atp Synthesis ◽

Intact Cells ◽

Isolated Mitochondria ◽

Exogenous Substrate ◽

High Oxidation ◽

Transmembrane Electrical Potential ◽

Sustained Increase

In isolated mitochondria the consequences of oxidative phosphorylation uncoupling are well defined, whereas in intact cells various effects have been described. Uncoupling liver cells with 2,4-dinitrophenol (DNP) in the presence of dihydroxyacetone (DHA) and ethanol results in a marked decrease in mitochondrial transmembrane electrical potential (∆ψ), ATP/ADP ratios and gluconeogenesis (as an ATP-utilizing process), whereas the increased oxidation rate is limited and transient. Conversely, when DHA is associated with octanoate or proline, DNP addition results in a very large and sustained increase in oxidation rate, whereas the decreases in ∆ψ, ATP/ADP ratios and gluconeogenesis are significantly less when compared with DHA and ethanol. Hence significant energy wastage (high oxidation rate) by uncoupling is achieved only with substrates that are directly oxidized in the mitochondrial matrix. Conversely in the presence of substrates that are first oxidized in the cytosol, uncoupling results in a profound decrease in mitochondrial ∆ψ and ATP synthesis, whereas energy wastage is very limited.

Download Full-text