The leading role of mitochondrial depolarization in the mechanism of glutamate-induced disorder in Ca(2+)-homeostasis]

Digital fluorescence imaging techniques were employed to monitor changes in the cytoplasmic Ca2+ concentration and mitochondrial potential in fura-2 AM or rhodamine-123 loaded individual cerebellar granule cells during and following the Glu exposure. The data obtained suggests that the MD-induced blockade of the mitochondrial Ca2+ uptake and a reversal of the mitochondrial ATP-synthase play a critical role in the mechanism of the glutamate-induced disorder of neuronal Ca2+ homeostasis.     

The role of cytoplasmic [Ca] in glucose-induced inhibition of respiration and oxidative phosphorylation in Ehrlich ascites tumour cells: a novel mechanism of the Crabtree effect

The effect of Ca²⁺ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (Δψ) and alkalinization of the medium. This typical behaviour is drastically changed if Ca²⁺ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of Δψ, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 μM Ca²⁺ and a preincubation for 2 min with 0.4–1.0 μM Ca²⁺. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca²⁺ concentrations as low as 0.3 μM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca²⁺] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyż A. Evtodienko YuV. Duszyński J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148–1154; Czyż A. Teplova VV. Sabała P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539–544], the present results suggest that cytoplasmic Ca²⁺ plays a crucial role in the Crabtree effect.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca²⁺ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

The role of mitochondrial calcium transport during inflammation and the effect of anti-inflammatory drugs

The effects of inflammation induced by the inoculation of rats with Freund's adjuvant on calcium transport by isolated rat liver mitochondria and on mitochondrial in vivo protein synthesis were investigated. Mitochondria isolated from the liver of inflamed rats exhibited (i) a reduction in 45Ca2+ uptake and, (ii) a reduction in protein synthesis. Addition of ATP to the calcium uptake medium stimulate the uptake in inflamed rat liver mitochondria. After inflammation was controlled by treatment with a mixture of Clerodendron inerme flavonoidal glycosides and indomethacin, rat liver mitochondria showed (i) an increase in 45Ca2+ uptake and, (ii) an increase in mitochondrial in vivo protein synthesis. The mechanism of mitochondrial calcium transport and the mitochondrial protein metabolism during inflammation and after treatment with anti-inflammatory drugs were discussed.     

The binding of cationized ferritin at the surfaces of ehrlich ascites tumor cells: the effect of pH and glutaraldehyde fixation.

The densities of cationized ferritin (CF) particles binding to the surfaces of cultured Ehrlich ascites tumor cells were determined at pH 7.4, where the ferritin stain was applied either prior to or following glutaraldehyde fixation. The densities were also determined with CF adjusted to pH 1.9 and applied after fixation. For all fixed samples there was a higher density of particles bound to microvilli than to the spaces between them. Treatment with neuraminidase removed more particles from microvilli than from the inter-microvillus spaces, but did not reduce the levels of binding to the same value. When cationized ferritin is applied prior to fixation, an aggregation of the CF particles at the cell surface was observed, with the internalization of some clusters. This effect was independent of neuraminidase treatment.     

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate.

Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution ("starved cells") resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.     

Crystal structures of S100A6 in the Ca(2+)-free and Ca(2+)-bound states: the calcium sensor mechanism of S100 proteins revealed at atomic resolution

S100A6 is a member of the S100 family of Ca(2+) binding proteins, which have come to play an important role in the diagnosis of cancer due to their overexpression in various tumor cells. We have determined the crystal structures of human S100A6 in the Ca(2+)-free and Ca(2+)-bound states to resolutions of 1.15 A and 1.44 A, respectively. Ca(2+) binding is responsible for a dramatic change in the global shape and charge distribution of the S100A6 dimer, leading to the exposure of two symmetrically positioned target binding sites. The results are consistent with S100A6, and most likely other S100 proteins, functioning as Ca(2+) sensors in a way analogous to the prototypical sensors calmodulin and troponin C. The structures have important implications for our understanding of target binding and cooperativity of Ca(2+) binding in the S100 family.     

A mechanism for both capacitative Ca(2+) entry and excitation-contraction coupled Ca(2+) release by the sarcoplasmic reticulum of skeletal muscle cells

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca(2+) uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca(2+) from cytosol to SR, and measurements of cytosolic Ca(2+). When Ca(2+) is added to Ca(2+)-free cells in the presence of Tg, the measured cytosolic Ca(2+) rises. This has been universally interpreted to mean that as SR Ca(2+) is depleted, exogenous Ca(2+) crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca(2+) in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca(2+) in the presence of Tg showed an increase in luminal Ca(2+) concentration in response to added exogenous Ca(2+). While we were able to reproduce the conventional demonstration of CCE-an increase of Ca(2+) in the cytosol in the presence of thapsigargin-we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca(2+) release channel, the ryanodine receptor (RyR). This was also unexpected if Ca(2+) enters the cytosol first. When Ca(2+) was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca(2+) and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca(2+) transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca(2+) inhibited its own uptake, a known feature of the RyR. We confirmed that Ca(2+) enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn(2+) in place of direct Ca(2+) measurements; we showed that Mn(2+) was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca(2+) flow in muscle cells. Our findings are entirely explained by a new model whereby Ca(2+) enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca(2+) subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca(2+) is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.     

Effects of calcium channel blockers on insulin secretion and 45Ca(2+)-uptake of rat islets stimulated by glucose or K(+)-depolarization.

Two calcium channel antagonists, verapamil and nifedipine, have been used to explore the dependence of secretion on voltage-gated influx of calcium. Both antagonists were able to suppress the secretory response to K(+)-depolarization as well as the stimulation of 45Ca(2+)-uptake. However, they inhibited only partially the stimulation of both secretion and 45Ca(2+)-uptake. However, they inhibited only partially the stimulation of both secretion and 45Ca(2+)-uptake induced by glucose, alone or with palmitate. The stimulation of 45Ca(2+)-uptake by K(+)-depolarization, unlike that induced by glucose, was not sensitive to norepinephrine, starvation or fatty acid oxidation inhibitors. Therefore, it is suggested that glucose either modifies the properties of the voltage-dependent calcium channel and/or accelerates the exchange of a particular intracellular pool of calcium.     

Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium.

The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.     

Effects of the membrane potential upon the Ca(2+)- and cumene hydroperoxide-induced permeabilization of the inner mitochondrial membrane.

A protonophore-induced delta psi decrease in a 180-140 mV range causes an increase in the lag-period of Ca(2+)-induced mitochondrial permeabilization but has little effect on the cumene hydroperoxide-induced permeability transition of mitochondria. Suppression of the non-specific permeability induction seems to be mediated by an increase in [ADP] in the mitochondrial matrix. A further decrease in delta psi leads to additional suppression of the non-specific permeability as a result of a partial ruthenium red-sensitive efflux of the previously accumulated Ca2+. On the other hand, complete dissipation of delta psi causes immediate induction of the non-specific permeability. It is concluded that only complete dissipation of delta psi caused by H+ leakages may act as a trigger for non-specific permeability induction.     

Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake.

Cellular uptake of Cd2+ has been monitored using intracellularly trapped dyes, Fura 2 and Quin 2, which bind Cd2+ with extremely high affinity, and digital fluorescence imaging has been used to visualize intracellular free Cd2+. The excitation spectrum of the Cd2+ complex of Fura 2 is similar to that of the Ca2+ complex, whereas Cd2+ displaces Ca2+ from Quin 2 and reduces fluorescence. Fluorescence of Fura 2-loaded cells increased when 50 microM extracellular Cd2+ was added and fluorescence of Quin 2-loaded cells decreased. Cd2+ uptake by GH3 pituitary cells, which occurs in part via voltage-sensitive L-type calcium channels, was increased by BAY K8644 and depolarization and decreased by nimodipine. When Fura 2 and Quin 2 were used to measure Cd2+ uptake by glial C6 cells, which have no L-channel activity, high K+ and BAY K8644 did not change the apparent rate of Cd2+ uptake. GH3 and C6 cells were incubated with Cd2+ for 24 h and loaded with Fura 2, and fluorescence was measured before and after addition of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a membrane permeant chelator with extremely high affinity for metals. TPEN had little effect on fluorescence of Fura 2-loaded GH3 and C6 cells not exposed to Cd2+ but decreased fluorescence of cells that had been incubated with 1-10 microM Cd2+. Fluorescence ratio imaging of Fura 2-loaded cells was used to image intracellular free Cd2+ for both GH3 and C6 cells. Cd2+ uptake over 30-180 min could be followed by the increase in 340/380 fluorescence ratio and the increase in fluorescence ratio was reversed within 5 min by TPEN. The results provide further evidence for the importance of voltage-gated calcium channels to Cd2+ uptake of certain cells.