Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

Biosynthesis of subviral oncogenic particles (virosomes) in mitochondria of rous sarcoma and Rauscher murine leukemia cells

Summary Experimental evidence for the presence and biosynthesis of subviral, leukemogenic particles in the isolated mitochondria of spleen cells of mice infected with Rauscher murine leukemia (RML) virus is presented. These subviral particles sediment at a density of 1.27–1.29 g/ml and induce splenomegaly and RML three weeks after i.v. or i.p. administration to white mice. Virosomes have been labelled with [³²P]phosphate in the isolated mitochondria from RML spleen cells and high molecular weight (70S) [³²P]RNA has been isolated from these subviral, leukemogenic particles. Rauscher virus group specific antigens were detected by immunodiffusion in the inner membrane and matrix fraction of the mitochondria of RML spleen cells. These results together with our earlier findings strongly suggest that mitochondria of the transformed cells participate in the biosynthesis of RNA tumor viruses. Possible mechanism of the penetration of viral genetic information of RNA tumor viruses into mitochondria of tumor cellsin vivo is discussed.     

Effect of the α-agonist noradrenaline on total and 45Ca2+ movements in mitochondria of rat liver cells

Ca²⁺ movements triggered by noradrenaline were determined in isolated cells and mitochondria from rat livers. It has been shown that these depend on experimental conditions. In cells incubated in 1.8mm-Ca²⁺, results suggest that noradrenaline mobilizes Ca²⁺ from reticulum before releasing Ca²⁺ from mitochondria.     

The brain-specific protein, p42IP4 (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42^IP4 (centaurin‐α1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf‐GTPase activating proteins) is mainly expressed in neurons. p42^IP4 operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42^IP4 is localized in mitochondria, isolated from rat brain and from cells transfected with p42^IP4. In rat brain mitochondria we additionally found interaction of p42^IP4 with 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase and α‐tubulin by pull‐down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42^IP4 is predominantly associated with the intermembrane space and the inner membrane. This localization of p42^IP4 indicates that p42^IP4 might have a still unknown mitochondrial function. We studied whether p42^IP4 is involved in Ca²⁺‐induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42^IP4 in mitochondria. In mitochondria isolated from p42^IP4‐transfected mouse neuroblastoma cells, over‐expression of p42^IP4 significantly decreased Ca²⁺ capacity and lag time for Ca²⁺ retention. Thus, we suggest that p42^IP4 is involved in the regulation of Ca²⁺ transport in mitochondria. We propose that p42^IP4 promotes Ca²⁺‐induced permeability transition pore opening and thus destabilizes mitochondria.     

Retention of calcium by mitochondria isolated from Ehrlich ascites tumor cells

Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells accumulate and retain high concentrations of Ca²⁺ in the presence of ATP for periods up to at least 20 min at 25 °C. The presence of inorganic phosphate up to 20 mm does not prevent such Ca²⁺ retention. The tumor mitochondria accumulate Ca²⁺ in the presence of succinate as an energy source but lose the Ca²⁺ after 1–2 min. Addition of ATP (K_m approx 1 mm) to the incubation medium after Ca²⁺ release, induces reaccumulation of the ion. Thus, the ability of the tumor mitochondria to retain Ca²⁺ differs markedly from that of rat liver mitochondria and is seen as being of potential biological significance to the unique metabolic behavior of the ascites tumor cells.     

Biogenesis of mitochondria. The effects of catabolite repression on the in vitro phospholipid synthetic capacities of subcellular fractions of respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae.

A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2-³H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.     

Functional state of cultured chick embryo cell mitochondria investigated in situ

The cell membrane of cultured chick embryo cells is permeable to Ca²⁺ after treatment with mannitol. Ca²⁺ uptake by mannitol-treated cells can be attributed to the mitochondria. $\text{Ca}{}^{\mathrm{2+}}\text{O}$ quotients and acceptor control ratios of such cells and isolated mitochondria are identical.Digitonin exerts two time-dependent effects on mannitol-treated cells: it increases the maximum extent of Ca²⁺ uptake by 5 – 20 percent. It also partially uncouples respiration from Ca²⁺ accumulation.It is suggested that mannitol treatment of cultured cells provides an easy way to study Ca²⁺ uptake by mitochondria $\text{in situ}$.     

Interaction of calcium with mitochondria isolated from Ehrlich ascites tumour cells

Calcium ions are accumulated by intact mitochondria isolated from Ehrlich ascites tumour cells in a buffered system supplemented with ATP or succinate. In the ATP-supplemented system, the tumour mitochondria, in contrast to rat liver mitochondria, retain the accumulated Ca²⁺, do not exhibit a marked “irreversible” ATPase and do not swell. In the succinate-supplemented system, added Ca²⁺ stimulates respiration in either the absence or presence of added inorganic phosphate. Whereas respiration by rat liver mitochondria, measured in the presence of added phosphate, remains continuously activated after the addition of only a small amount of Ca²⁺, that by the tumour mitochondria can be stimulated by several successive additions of 100 μM Ca²⁺ and at all times exhibit appreciable activation ratios.     

Structure and Function of Mitochondria in Crithidia fasciculata*

SYNOPSIS. In correlating mitochondrial structure with composition and function of the electron-transport system in Crithidia fasciculata, failure to find cytochrome oxidase in isolated mitochondria coincided with the presence of longitudinally-oriented lamellar cristae in the mitochondria in intact cells. Cytochromes b and c were detected spectrophotometrically. Respiration of intact cells and mitochondria, measured polarographically, was sensitive to 10⁻⁴ M antimycin A and 5 × 10⁻⁴ M KCN. The difficulty in detecting cytochrome oxidase and the biogenesis of mitochondria are briefly discussed.     

Divalent cation-activated ATP hydrolysis by mitochondrial ATP'ase — Mechanism for energy depletion in ischemic reperfused myocardium

Recovery of high-energy compounds by ischemic myocardium is believed to be important for its return to normal functioning. While it has been previously shown that oxidative phosphorylation is markedly reduced in mitochondria isolated from ischemic myocardium in the presence of all substrates, alterations in ATPase activity have not been confirmed. This study demonstrates that, although the rate of ATP hydrolysis produced by mitochondria isolated from 2-hr ischemic myocardium does not significantly differ from that produced by non-ischemic mitochondria, the rate produced by 2-hr ischemic, 2 hr reperfused mitochondria is significantly higher. Also, Ca⁺⁺ content was observed to be higher in reperfused than in non-reperfused ischemic mitocheondria. The addition of EDTA, EGTA, or oligomycin to the reperfused ischemic mitochondria resulted in the inhibition of ATPase activity. These results indicate that mitochondrial ATPase in ischemic myocardium is activated by Ca⁺⁺ ions and that ischemic reperfused myocardium may contain mitochondria with uncontrolled ATPase activity such that high energy phosphate supplies are excessively depleted when the cells are reperfused.     

On the existence of two populations of mitochondria in a single organ. Respiration, calcium transport and enzyme activities.

Mitochondria isolated from rat heart and kidney cortex by Polytron treatment of the tissues exhibit lower state 3 rates of respiration than mitochondria isolated by Nagarse method. Addition of cytochrome $\text{c}$ to Polytron mitochondria isolated from heart, but not from kidney, increases oxygen uptake to values approaching those of Nagarse-treated preparations. Similar results were observed for Ca²⁺ uptake. Kidney Polytron mitochondria exhibited lower mitochondrial, but higher non-mitochondrial enzyme activities compared to kidney Nagarse mitochondria. Enzyme activities were the same in Polytron and Nagarse mitochondria from heart. The differences between Polytron and Nagarse mitochondria appear to be mainly due to lower cytochrome $\text{c}$ content of Polytron mitochondria from heart and higher contamination of Polytron mitochondria from kidney.     

Microaerobic respiration and oxidative phosphorylation by soybean nodule mitochondria: implications for nitrogen fixation

The infected cells of soybean (Glycine max) root nodules require ATP production for ammonia assimilation and purine synthesis under microaerobic conditions. It is likely that the bulk of this demand is supplied through mitochondrial oxidative phosphorylation. Mitochondria purified from root nodules respired and synthesized ATP in sub-micromolar oxygen concentrations as measured by leghaemoglobin spectroscopy and luciferase luminescence. Both oxygen uptake and the apparent ATP/O ratio declined significantly as the oxygen concentration fell below 100 μmol m^?3. Cytochrome-pathway respiration by root nodule mitochondria had a higher apparent affinity for oxygen (Km 50 μmol m^?3) than did mitochondria isolated from roots (Km 125 μmol m^?3). Electron micrographs showed that mitochondria predominated at the periphery of infected cells adjacent to gas-filled intercellular spaces, where the oxygen concentration is predicted to be highest. Calculations of oxygen concentration and nitrogen fixation rates on an infected cell basis suggest that the measured rates of ATP production by isolated mitochondria are sufficient for the quantifiable in vivo requirements of ammonia assimilation and purine synthesis. The possible roles of mitochondrial respiration in the control of infected cell metabolism are also discussed.     

Influence of specific growth limitation and dilution rate on the phosphorylation efficiency and cytochrome content of mitochondria of Candida utilis NCYC 321

With Candida utilis cells that had been removed directly from a 61 chemostat culture, in steady state, well-coupled mitochondria generally could be isolated. This required a modified snail-gut enzyme procedure that allowed the total processing time to be decreased to 3 h, or less. Examination of these mitochondria in an oxygraph showed the presence of 3 sites of energy conservation when the cells were grown at various dilution rates between 0.1 and 0.45 h^-1 in environments that were, successively, glucose-, ammonia-, magnesium-, phosphate- and sulphate-limited. Potassium-limited cells also apparently possessed 3 sites of oxidative phosphorylation when growing at dilution rates greater than 0.2 h^-1, but only 2 sites when growing at lower dilution rates. Analysis of cytochrome spectra obtained with these intact mitochondria revealed large quantitative (but not qualitative) differences, depending on the environmental conditions under which the yeast had been cultured. In particular, comparison of the ratio of cytochrome b to cytochrome a showed a pattern of change with dilution rate in mitochondria from potassium-limited cells that was distinctly different from those evident in mitochondria from cells that had been limited in their growth by the availability of other nutrients.     

Plasma membranes from candida tropicalis grown on glucose or hexadecane. I. Isolation,identification and purification

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes.After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation.Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences.The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg²⁺-dependant ATPase was determined.By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)     

Effects of calcium ionophores on the transport and distribution of calcium in isolated cells and in liver and kidney slices

Summary The effects of calcium ionophores on cellular calcium metabolism were studied in cultured kidney cells, in cells freshly isolated from rat kidney, and in liver and kidney slices. In isolated cells, these ionophores decreased the total cellular Ca content and the mitochondrial Ca.⁴⁵Ca efflux from prelabelled cells was also stimulated even in the absence of extracellular Ca. In slices, the ionophore A23187 increased the total slice Ca and the uptake of⁴⁵Ca. However, the mitochondria isolated from these slices treated with the ionophore had a lower total Ca and a depressed relative radioactivity. These results suggest that the increased cytosolic Ca produced by Ca ionophores may be due to mobilization of intracellular Ca stores rather than to a net shift of Ca from the extracellular fluids to the cell.     

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca²⁺ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²⁺-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²⁺. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.     

Changes in mitochondrial protein composition during testicular differentiation in mouse and bull

The morphology of testicular mitochondria changes markedly during spermatogenesis from a form normally seen in somatic cells to a “germ cell” form in which the matrix is diffuse and vacuolated and finally to a form with a condensed matrix seen in spermatozoa. Colloidal silica gel gradients and high-resolution, two-dimensional gel electrophoresis were used to define the changes in density and polypeptide composition that occur in testicular mitochondria during spermatogenesis. Similar densities were observed for mitochondria isolated from the same bovine or murine tissue, but mitochondria from different tissues usually had different densities. Mitochondria from testis of calf, bull, or sexually mature mouse had densities of 1.06 gm/cm³ while liver mitochondria were more dense, having a density of 1.09 gm/cm³. “Somatic-type” testicular mitochondria from calf and “germ cell-type” mitochondria from sexually mature mouse or bull had similar densities, 1.06 gm/cm³, while the density of mitochondria from ejaculated spermatozoa differed, ρ = 1.08 gm/cm³. Analysis of polypeptide composition of somatic and germ cell mitochondria from testes of prepuberal and sexually mature animals and from highly enriched populations of pachytene primary spermatocytes and round spermatids revealed a staining pattern of mitochondrial proteins that was markedly constant throughout development with most polypeptides being conserved and a few specific spots changing in abundance. Marked differences were detected, however, when mitochondria from ejaculated spermatozoa were compared with those from testis with many minor and major polypeptides missing and several new polypeptides present at high concentration.     

COMPARATIVE STUDIES ON MITOCHONDRIA ISOLATED FROM NEURON-ENRICHED AND GLIA-ENRICHED FRACTIONS OF RABBIT AND BEEF BRAIN

Fractions enriched in neuronal and glial cells were obtained from dispersions of whole beef brain and rabbit cerebral cortex by large-scale density gradient centrifugation procedures. The fractions were characterized by appropriate microscopic observation. Mitochondria were then isolated from these fractions by differential centrifugation of their homogenates. The two different types of mitochondria were characterized with respect to certain enzyme activities, respiratory rate, rate of protein synthesis, and their buoyant density in sucrose gradients. The mitochondria from the neuron-enriched fraction were distinguished by a higher rate of incorporation of amino acids into protein, higher cytochrome oxidase activity, and a higher buoyant density in sucrose density gradients. Mitochondria from the glia-enriched fraction showed relatively high monoamine oxidase and Na⁺- and K⁺-stimulated ATPase activities. The rates of oxidation of various substrates and the acceptor control ratios did not differ appreciably between the two types of mitochondria. The difference in the buoyant density of mitochondria isolated from the neuron-enriched and glia-enriched cell fractions was utilized in attempts to separate neuronal and glial mitochondria from the mixed mitochondria obtained from whole brain homogenates in shallow sucrose gradients. The appearance of two peaks of cytochrome oxidase, monoamine oxidase, and protein concentration in such gradients shows the potential feasibility of such an approach.     

Redox-optimized ROS balance: A unifying hypothesis

While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O₂ to superoxide (O₂^?) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD⁺) or ROS scavenging (NADPH/NADP⁺, GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, ?Ψ_m), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low ?Ψ_m) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum.