Comparison of oxygen consumption rates in minimally transformed BALB/3T3 and virus-transformed 3T3B-SV40 cells.

In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great attention because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3B-SV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as "phosphorylating" respiration coupled to ATP synthesis, "free" respiration not coupled to ATP synthesis, and "reserve" or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3B-SV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. "Phosphorylating" respiration was found to be reduced to 54% and "reserve" respiration, on the contrary, increased up to 160% in virus-transformed 3T3B-SV40 cells. The low rate of "phosphorylating" respiration and high "reserve" respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virus-transformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high "reserve" respiration is suggested to play an important role in preventing apoptosis of 3T3B-SV40 cells.

Generation of reactive oxygen species by polymorphonuclear leukocytes and its modulation by calcium ions during tumor growth.

The generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) and the role of Ca2+ in regulating their activity during Zajdela hepatoma growth in the animal peritoneal cavity were studied. We found a marked increase in the ROS-generating activity of PMNL in circulating blood, the result of increases in both the specific activity of leukocytes and total number of PMNL in circulating blood. The ROS-generating activity of PMNL was substantially activated by Ca2+ ions and a calcium ionophore (ionomycin), but this effect virtually completely disappeared during tumor growth. Perhaps the high ROS-generating activity of PMNL and the lack of the sensitivity to extracellular Ca2+ during tumor growth in the organism are due to an accumulation of intracellular Ca2+ ions.

Regulation of oxidative phosphorylation in the inner membrane of rat liver mitochondria by calcium ions.

The effect of accumulation of Ca2+ at physiological concentrations (10(-8)-10(-6) M) on the rates of ATP synthesis and hydrolysis in rat liver mitochondria was studied. An addition of 5 x 10(-7) M Ca2+ resulted in the maximal rates of synthesis and hydrolysis of ATP. Decrease in the concentration of Ca2+ to 10-8 M or its increase to 5 x 10(-6) M inhibited oxidative phosphorylation and ATP hydrolysis. It was found that the rate of oxidative phosphorylation correlated with the phosphorylation level of a 3.5-kD peptide in the mitochondrial inner membrane on varying the Ca2+ concentration. The possible regulation of oxidative phosphorylation in mitochondria by Ca2+ is discussed.

Sustained oscillations of transmembrane Ca2+ fluxes in mitochondria and their possible biological significance.

Sustained oscillations of transmembrane fluxes of Ca2+ and other ions in isolated mitochondria are described. The data are presented that the major cause of the oscillations is the Ca2+-induced Ca2+ efflux from the mitochondrial matrix and spontaneous opening/closing of the permeability transition pore in the inner mitochondrial membrane. Conditions favourable for the generation of oscillations are considered. The role of phospholipid peroxidation and hydrolysis in the generation of [Ca2+] oscillations is emphasized. Literature data concerning [Ca2+] changes in the mitochondrial matrix in intact cells and the data on the participation of mitochondria in intracellular Ca2+ oscillation and in the Ca2+ wave propagation are reviewed. The hypothesis that mitochondria are able to generate [Ca2+] oscillations in intact cells is put forward. It is assumed that Ca2+ oscillations can protect mitochondria of resting cells from osmotic shock and oxidative stress.

Ca(2+)-modulated phosphorylation of a low-molecular-mass polypeptide in rat liver mitochondria: evidence that it is identical with subunit c of F(0)F(1)-ATPase.

A 3.5-kDa polypeptide associated with the inner membrane of rat liver was found to be phosphorylated by [gamma-(32)P]ATP, presumably via a cAMP-dependent kinase. The phosphorylation was modulated by [Ca(2+)] in the physiological range, with a minimum at 1 microM and rising fourfold toward lower (10 nM) and higher (10 microM) concentrations. Further characterization of the 3.5-kDa component showed that the polypeptide has the same electrophoretic mobility as subunit c of F(0)F(1)-ATPase and that it selectively binds to antibodies against subunit c.

Combined vitamins Bl2b and C induce the glutathione depletion and the death of epidermoid human larynx carcinoma cells HEp-2.

The combination of hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) can cause the death of tumor cells at the concentrations of the components at which they are nontoxic when administered separately. This cytotoxic action on epidermoid human larynx carcinoma cells HEp-2 in vitro is shown to be due to the hydrogen peroxide generated by the combination of vitamins B12b and C. The drop in the glutathione level preceding cell death was found to be the result of combined action of the vitamins. It is supposed that the induction of cell death by combined action of vitamins B12b and C is connected to the damage of the cell redox system.

The Ca2+ threshold for the mitochondrial permeability transition and the content of proteins related to Bcl-2 in rat liver and Zajdela hepatoma mitochondria.

Zajdela hepatoma mitochondria were able to accumulate two to five times more Ca2+ than rat liver mitochondria before the permeability transition was induced. Pulses of Ca2+ were given in series to determine the Ca2+ threshold by recording changes in [Ca2+] and membrane potential, the permeability transition causing the release of accumulated Ca2+ and collapse of the membrane potential. Hepatoma mitochondria had lower Ca2+ efflux rates, higher net Ca2+ uptake rates and lower phosphorylation rates than liver mitochondria. Since the differences in regard to induction of the permeability transition might be due to higher expression of the Bcl-2 protein in hepatoma cells than in hepatocytes, the transcription of Bcl-2 and the proteins reacting with a Bcl-2 polyclonal antiserum were estimated by Northern and Western blotting, respectively. Hepatoma cells had two Bcl-2 specific mRNA bands of 7 and 2.4 kb, and substantial amounts of the Bcl-2 protein, whereas in liver cells and mitochondria these were not detected. Both cell lines had a reactive band at 19-20 kDa, and hepatocytes a small band at 31-32 kDa. Bcl-2 antibodies stimulated the permeability transition potently in hepatoma mitochondria.

Effect of glucose and deoxyglucose on the redistribution of calcium in ehrlich ascites tumour and Zajdela hepatoma cells and its consequences for mitochondrial energetics. Further arguments for the role of Ca(2+) in the mechanism of the crabtree effect.

The distribution of Ca(2+) in intact cells was monitored with fluorescent probes: fura-2 for cytosolic [Ca(2+)] and rhod-2 for mitochondrial [Ca(2+)]. It was found that in neoplastic cells, such as Ehrlich ascites tumour and Zajdela hepatoma, but not in non-malignant cells, such as fibroblasts, glucose and deoxyglucose elicited release of Ca(2+) from endoplasmic reticulum stores and an increase in Ca(2+) concentration in the cytosol. Parallel to this, a decrease in the rate of Ca(2+) extrusion from the cell and an enhanced uptake of Ca(2+) by mitochondria were observed. The increase in mitochondrial [Ca(2+)] was accompanied by an increase in the mitochondrial membrane potential and the reduction state of nicotinamide nucleotides. F(1)F(o)-ATPase in submitochondrial particles of Zajdela hepatoma was strongly inhibited in the presence of micromolar Ca(2+) concentrations, whereas this activity in submitochondrial particles from rat liver appeared to be less sensitive to Ca(2+). Indications of glycosylation of Ehrlich ascites tumour cell proteins were also obtained. These data strengthen the proposal [Bogucka, K., Teplova, V.V., Wojtczak, L. and Evtodienko, Y. V. (1995) Biochim. Biophys. Acta 1228, 261-266] that the Crabtree effect is produced by mobilization of cell calcium, which is subsequently taken up by mitochondria and inhibits F(1)F(o)-ATP synthase.

Phosphorylation of a low-molecular-weight polypeptide in rat liver mitochondria and dependence of its phosphorylation on mitochondrial functional state.

We show that incubation of rat liver mitochondria in the presence of [gamma-32P]ATP results in cAMP-dependent phosphorylation of a low-molecular-weight (3.5-kD) polypeptide (LMWP). This component is tightly bound to the mitochondrial membrane. It is not released into solution after freezing and subsequent thawing of the mitochondrial suspension and does not incorporate 32P from [gamma-32P]ATP in the presence of uncouplers of oxidative phosphorylation. Inhibition of adenine nucleotide transport into the mitochondrial matrix by carboxyatractyloside suppresses phosphorylation of the LMWP. Moderate Ca2+ loading of mitochondria increases both phosphorylation and dephosphorylation of the LMWP. Chelation of Ca2+ by incubation in the presence of EGTA suppresses incorporation of 32P into the LMWP.

The high calcium ion uptake capacity of Ehrlich ascites tumour cell mitochondria is due to inhibition of the permeability transition and phospholipase A2 activity by magnesium.

Tumour cells frequently have a high Ca2+ threshold for the mitochondrial permeability transition which occurs when a large pore in the inner membrane is opened. We studied whether this was due to the known high content of Mg2+ in Ehrlich ascites tumour cell mitochondria or to the increased expression of the protooncogene bcl-2. The latter was found not to be the case. Mg2+ potently inhibited the permeability transition and the binding of Ca2+ to the inner membrane. Also, phospholipase A2 activity was reduced by Mg2+. It is concluded that the high Ca2+ threshold is due to the high Mg2+ content in these tumour mitochondria.

A model of mitochondrial Ca(2+)-induced Ca2+ release simulating the Ca2+ oscillations and spikes generated by mitochondria.

Recent evidence underlines a key role of mitochondrial Ca2+ fluxes in cell Ca2+ signalling. We present here a kinetic model simulating the Ca2+ fluxes generated by mitochondria during mitochondrial Ca(2+)-induced Ca2+ release (mCICR) resulting from the operation of the permeability transition pore (PTP). Our model connects the Ca2+ fluxes through the ruthenium redsensitive Ca2+ uniporter, the respiration-dependent and passive H+ fluxes, the rate of oxygen consumption, the movements of weak acids across the mitochondrial membrane, the electrical transmembrane potential (delta psi), and operation of the PTP. We find that two factors are crucial to account for the various mCICR profiles that can be observed experimentally: (i) the dependence of PTP opening and closure on matrix pH (pHi), and (ii) the relative inhibition of the respiratory rate consecutive to PTP opening. The resulting model can simulate irreversible Ca2+ efflux from mitochondria, as well as the genesis of damped or sustained Ca2+ oscillations, and of single Ca2+ spikes. The model also simulates the main features of mCICR, i.e. the threshold-dependence of mCICR triggering, and the all-or-nothing nature of mCICR operation. Our model should appear useful to further mathematically address the consequences of mCICR on the spatiotemporal organisation of Ca2+ signals, as a 'plug-in' module for the existing models of cell Ca2+ signalling.

Effect of prooxidants on mitochondrial permeability transition and cell death in Ehrlich ascites tumour cells.

Ca2+ retention in mitochondria, opening of the Cysclosporin A- sensitive permeability transition pore and cell death were studied in Ehrlich ascites tumour cells in the presence of different prooxidants. Low concentrations (1-20 microM) of the prooxidants (menadione, cumenehydroperoxide, t-butylhydroperoxide) induced pore-opening in permeabilized cells at threshold Ca2+ load. Incubation of cells with low concentrations of prooxidants was able to induce cell cycle disturbance and cell death. Under the prooxidant effect, mitochondrial membrane potential drop and Ca2+ retention decrease in mitochondria were found to precede death of Ehrlich ascites tumour cells.

Calcium binding to polypeptides of rat liver and Zajdela hepatoma mitochondrial inner membranes.

Composition and amount of 45Ca2+-binding proteins in the inner membrane fraction of rat liver and Zajdela hepatoma mitochondria were determined. In the inner membrane of liver mitochondria, three major 45Ca2+-binding polypeptides: a protein of approximately 130 kDa (carbamoyl-phosphate synthetase), a glycoprotein of 43-44 kDa (previously considered as the calcium uniporter), and 29-30 kDa protein were found. These components were absent (130 kDa component) or relatively reduced (43-44 kDa and 29-30 kDa components) in the inner membrane of hepatoma mitochondria. Previously unknown low molecular mass polypeptides, having very high Ca2+-binding ability, were found in the inner membrane of hepatoma mitochondria. One of them might be the natural Ca2+-binding inhibitor of H+-ATPase.

Microtubule-active drugs suppress the closure of the permeability transition pore in tumour mitochondria.

We report the effects of anticancer drugs, inhibitors of microtubule organisation, on the mitochondrial permeability transition pore (PTP) in Ehrlich ascites tumour cells. Taxol (5-20 microM) and colchicine (100-500 microM) prevented closing of the cyclosporin A-sensitive PTP. No taxol or colchicine effects on oxidative phosphorylation were observed in the range of concentrations used. We suggest that either membrane-bound tubulin per se can be part of PTP and/or the attachment of mitochondria to the microtubular network is essential for PTP regulation. The taxol inhibition of PTP closure, mediated through interaction with the cytoskeleton, sheds new light on the cytotoxic properties of this anticancer drug.

Strontium excitability of the inner mitochondrial membrane: regenerative strontium-induced strontium release.

Regenerative Sr(++)-induced Sr++ release from isolated rat liver mitochondria was studied using ion-selective electrode techniques. Mitochondria, when exposed to a pulse of Sr++, demonstrated a reversible and transient increase in inner membrane permeability to K+ and H+ ions. The increase in permeability was an all-or-none process with a threshold dependent on the amplitude of the Sr++ pulse. The threshold concentration of Sr++ was lowered from 120-150 microM to 20-30 microM when mitochondria were preloaded with 100 nmoles Sr++/mg protein. Release of matrix-stored divalent cations provided a mechanism for amplification of the extramitochondrial Sr++ concentration (regenerative Sr(++)-induced Sr++ release). The mitochondrial inner membrane became refractory, since subsequent cycles of excitation could not be immediately induced. These experiments demonstrate that the inner mitochondrial membrane is an excitable membrane, with threshold-dependent, regenerative and subsequently refractile Sr(++)-induced Sr++ release characteristics.

Effect of cyclosporin A on Ca2+ fluxes and the rate of respiration in Ehrlich ascites tumour cells.

Cyclosporin A at micromolar concentration decreases the respiration of Ehrlich ascites tumour cells (with pyruvate as substrate) but prevents the inhibition of oxygen uptake produced by glucose or deoxyglucose (the Crabtree effect). Cyclosporin A also diminishes the increase of cytoplasmic free Ca2+ concentration elicited by deoxyglucose and almost completely abolishes this increase induced by extracellular ATP and thapsigargin but does not decrease the size of endoplasmic reticulum Ca2+ stores as revealed after addition of ionomycin. It is concluded that cyclosporin A inhibits the inositol trisphosphate-sensitive Ca2+ channel and the passive permeability of the endoplasmic reticulum to Ca2+ in Ehrlich ascites tumour cells.

Inhibition by Ca2+ of the hydrolysis and the synthesis of ATP in Ehrlich ascites tumour mitochondria: relation to the Crabtree effect.

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszynski, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszynski, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

Effect of glucose and deoxyglucose on cytoplasmic [Ca2+] in Ehrlich ascites tumor cells.

Concentration of free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumor cells, measured using fura-2, amounted to 170-300 nM and was increased by 50-160 nM after addition of 10 mM D-glucose or D-2-deoxyglucose but not 3-O-methylglucose at pH 7.4. In the range of external pH between 6.8 and 7.8 the increase was higher at higher pH. This increase occurred within 30-60 s after addition of hexose and lasted for at least 10 min. This [Ca2+]i rise was observed both in presence and virtual absence of Ca2+ in the external medium. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose. The mechanism of [Ca2+] in the external medium. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose. The mechanism of [Ca2+]i rise evoked by glucose and deoxyglucose and its importance in switching cell metabolism from oxidative to glycolytic are discussed.

Effect of glucose and deoxyglucose on cytoplasmic concentration of free Ca2+ in Ehrlich ascites tumour: studies on single cells.

Concentration of free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumour cells loaded with fura-2 was measured in single cells applying a video imaging system. In resting cells [Ca2+]i amounted to 60-340 nM and was increased after addition of 10 mM D-glucose or D-2-deoxyglucose by 80-200 nM. This increase occurred within 30-60 s following addition of the sugars and lasted for several minutes. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose and, vice versa, thapsigargin added after the sugars mobilized less Ca2+ than when added before. A possible relation of the [Ca2+]i rise evoked by glucose and deoxyglucose to the Crabtree effect is discussed.

Properties of different Ca2+ pools in permeabilized rat thymocytes.

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.