Effect of Mitochondrial Inhibitors On Metaphase-Telophase Progression and Nuclear Membrane Formation In Chinese Hamster Cells

Chinese hamster Don cells in log-phase were exposed to Colcemid during the G₂ period with and without a combination of divalent cation chelators and mitochondrial inhibitors. Isolated metaphase cells were incubated as follows: (i) without Colcemid but with other agents and the progression was monitored from metaphase (M) to telophase (Tel) and to cell division; (ii) with Colcemid and other agents and the rate of micronuclei formation in the absence of anaphase was studied. Both EDTA and EGTA accelerated the progression from M to Tel, but did not affect the overall rate of cell division. Chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, blocked the effect of the chelators and also retarded the progression. an inhibitor of mitochondrial respiration, Antimycin A (AA), also retarded the progression in the absence of the chelators and prevented the promoting effect of the chelators. A stimulator of ATPase for ATP breakdown. 2,4-dinitrophenol (DNP), accelerated the M to Tel progression. Chloramphenicol (CAP) and AA, as well as DNP, appeared to have little effect on the formation of micronuclei in the presence of Colcemid. EGTA, which affects cell surface Ca²⁺, stimulated the formation of micronuclei. This study indicates that Ca²⁺ ions and mitochondrial function are involved in the regulation of a certain segment of mitosis beyond metaphase, with Ca²⁺ sequestration in the mitochondria and chelation of Ca²⁺ by EGTA as dominant factors.     

Effect of divalent cations and chelators on metaphase to telophase progression and nuclear envelope formation in Chinese hamster cells

Chinese hamster DON cells in log phase were treated with Colcemid in the G₂ period with or without divalent cation chelating agents. The metaphase cells were isolated and incubated in two ways: 1) without Colcemid but with chelating agents or La³⁺ and observed for metaphase to telophase progression, and 2) with Colcemid, with or without chelating agents and the rate of micronuclei formation in the absence of anaphase monitored. The effect of the chelating agents on cellular ⁴⁵Ca²⁺ during metaphase to telophase progression was also studied.The results indicate that Ca²⁺ and possibly Mg²⁺ ions are involved in the regulation of certain segments of mitosis. The reduction of environmental and plasma membrane associated Ca²⁺ with the chelators and La³⁺ promoted the metaphase to telophase progression as well as nuclear envelope and micronuclei formation.     

Effects of divalent-cation chelators and chloramphenicol on the spatial relationship of the nuclear envelope to chromatin in micronuclei of chinese hamster cells

In the presence of the spindle poison Colcemid in the culture medium to prevent anaphase, approximately 20% of Chinese hamster metaphase cells were converted to micronucleated cells during 7 h. In the micronuclei the chromosomes had become enclosed by a nuclear envelope (NE). In the light-microscope the micronuclei were of two kinds: with either visible chromatids or with decondensed chromosomes. In the electron microscope (EM) the spatial relationship of the NE to the chromatin was of two kinds only in the presence of Colcemid. In about 90% of the micronucleated cells the spatial relationship was normal, ie, the NE was immediately adjacent to the chromatin. In the remaining cells, the NE was distended so that the outer NE was separated from the inner one. In the presence of the drivalent cation chelator, (ethylenedinitrilo) tetraacetic acid (EDTA) or the Ca²⁺-chelator [ethylenebis (oxyethylenenitrilo)] tetraacetic acid (EGTA), in addition to Colcemid, the amount of cells with micronuclei increased to 40%. The light-microscope appearance was the same as that found in the absence of the chelating agents. However, after Colcemid plus EGTA, EM revealed that only about 50% of the micronucleated cells had NE that was immediately adjacent to the chromatin and about 10% of them had distended outer NE. In the remaining 40% a third kind of spatial relationship was seen: the NE was intact but most of it was not adjacent to the chromatin. Furthermore, this type of micronucleus often contained mitochondria within the confines of NE. Thus, Ca²⁺ and possibly Mg²⁺ may regulate the rate of formation of the NE and also its ultrastructural relation to the chromatin. Mitochondrial function also appears to be involved in this relationship. In the presence of chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, in addition to Colcemid, only about 50% of the micronucleated cells exhibited the normal relationship. The outer NE was separated from the inner NE in about 46% of the micronucleated cells and the third kind of NE-chromatin relationship was observed only in 2%. In the case of the third kind of relationship produced by CAP, inclusion of mitochondria within the micronuclei was not observed, in contrast to the finding with EGTA.     

Aminoacetone induces iron-mediated oxidative damage to isolated rat liver mitochondria

Aminoacetone (AA) is a threonine metabolite accumulated in threoninemia, cri-du-chat, and diabetes, where it contributes toward the formation of cytotoxic and genotoxic methylglyoxal (MG). Oxyradicals yielded from iron-catalyzed AA aerobic oxidation to MG are shown here to promote Ca2+ -mediated mitochondrial membrane permeabilization in an AA dose-dependent way. The inhibitory effect of added EGTA, cyclosporin A, Mg2+, and DTT observed in this study suggests the formation of transition pores in the inner mitochondrial membrane by AA, associated with thiol protein aggregation. That the mitochondrial iron pool plays a coadjutant role in the transition of mitochondrial permeability is indicated by the dramatic inhibitory effect of added o-phenanthroline. Iron released from ferritin by AA oxidation products--superoxide anion and AA enolyl radicals--is shown to act as an alternative source of ferrous iron, intensifying the mitochondrial damage. These findings may contribute to clarify the role of accumulated AA and iron overload in the mitochondrial oxidative damage reportedly occurring in diabetes mellitus.     

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.     

小鼠卵受精和人工激活过程中胞质游离Ca^2＋变化及其在卵激活中的作用

休止于第二次成熟分裂中期（ＭＩ）的小鼠卵母细胞分别乙醇,钙离子载体Ａ２３１８７、电刺激或精子激活并用Ｃａ＾２＋特异荧光探针－Ｆｕｒａ２／ＡＭ测定细胞内游离Ｃａ＾２＋的变化。结果表明,受精诱导ＭⅡ卵内游离Ｃａ＾２＋浓度多次跃升（ｏｓｃｉｌｌａｔｉｏｎ）乙醇,钙离子载体及１次电刺激仅诱导胞内Ｃａ＾２＋１次升高,人工诱导激活的卵可象正常受精卵一样卵裂并发育至囊胚,用ＥＧＴＡ阻止受精和人工激活过程中卵内游     

A specific role for Ca2+ in the oxidation of exogenous NADH by Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.     

Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 mM EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca2+ ion levels are kept low, but when Ca2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.     

Role of Ca2+ for protein turnover in isolated rat hepatocytes.

Experiments with bivalent-cation chelators (EGTA and EDTA), a Ca2+ ionophore (A23187) and a Ca2+-channel blocker (verapamil) indicate that Ca2+ is required for the lysosomal degradation of endogenous protein in hepatocytes. A distinction is made between lysosomal and non-lysosomal degradation by using the lysosomotropic agent methylamine. As Ca2+ does not appear to be required for the lysosomal degradation of endocytosed asialo-fetuin, the Ca2+-dependence for the degradation of endogenous protein is probably connected with the formation of autophagic vacuoles or the fusion of autophagic vacuoles with lysosomes. EGTA and EDTA had a slight inhibitory effect on the non-lysosomal degradation. This effect could be due to the activity of non-lysosomal Ca2+-dependent thiol proteinases. Together with previous experiments with thiol-proteinase inhibitors, the present experiments indicate that these proteinases have a very limited impact on the bulk protein degradation in the isolated hepatocytes.     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane.

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca2+ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate. 2. Plots relating the initial rate of calcium ion (Ca2+) uptake and the decrease in membrane potential (delta psi) to the Ca2+ concentration show a half-maximal change at less than 10 micron Ca2+ and a saturation above 20 micron Ca2+. 3. Plots relating the initial rate of Ca2+ uptake to delta psi are linear. 4. Addition of Ca2+ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca2+ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca2+ efflux. 5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca2+ ion is transferred across the membrane with one net positive charge in present conditions.     

Participation of calcium in flagellar shortening and regeneration in Chlamydomonas reinhardii

Cation chelators cause flagellar shortening in Chlamydomonas reinhardii. Most effective are EDTA and EGTA (1 mM) but pyrophosphate (10 mM) also is effective. Addition of 5 mM Ca2+ after shortening caused by 4 mM EGTA results in flagellar regeneration. Other divalent cations can replace Ca2+ with the following relative activities: Ca2+ greater than Sr2+ = Mn2+ much greater than Ba2+ = Mg2+. Although the specific ion requirement to reverse shortening is not clear, it is possible that all of the ions act by displacing one bound cation, presumably Ca2+. A specific requirement for Ca2+ in flagellar regeneration could be demonstrated, however, because as little as 50 microM EGTA in the presence of 500 microM Mg2+ delayed regeneration and prevented full regeneration. Ca2+ at 100 microM overcame this inhibition.     

Effect of calcium chelators on the Ca2+-dependent luminescence of aequorin.

The luminescence of aequorin, a useful tool for studying intracellular Ca2+, was recently found to be inhibited by the free EDTA and EGTA that are present in calcium buffers. In the present study we have examined the effect of the free forms of various chelators in the calibration of [Ca2+] with aequorin. Free EDTA and EGTA in low-ionic-strength solutions strongly inhibited the Ca2+-triggered luminescence of aequorin, causing large errors in the calibration of [Ca2+] (approx. 2 pCa units), whereas in solutions containing 150mM-KCl, errors were relatively small (0.2-0.3 pCa units). Citric acid in low-ionic-strength solutions and [(carbamoylmethyl)imino]diacetic acid in high-ionic-strength solutions showed no inhibition and did not cause detectable error in the calibration of [Ca2+], indicating that they are better chelators than EDTA and EGTA for use with aequorin.     

Introduction of calcium chelators into isolated rat pancreatic acini inhibits amylase release in response to carbamylcholine

The Ca2+ chelators, EGTA and BAPTA, have been introduced into intact, isolated rat pancreatic acini using a hypotonic swelling method. This resulted in complete inhibition of amylase release, stimulated by carbamylcholine at a submaximal concentration and 82 - 85% inhibition at maximal concentrations. Acini swollen in the absence of Ca2+ chelators showed similar secretory responses to those of unswollen acini. Treatment of unswollen acini with chelators inhibited the maximum response to carbamylcholine by only 23%. The inhibitory effect of intracellular chelators was not due to ATP depletion or a lowering of the total cell Ca2+ content. Thus, these results provide the first direct demonstration that an increase in intracellular Ca2+ concentration is necessary for the stimulation of enzyme release from pancreatic acinar cells.     

Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin

The binding of EGTA and EDTA to alpha-lactalbumin, first demonstrated by Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709) and afterwards regarded as a significant source of error in estimating the binding constant of Ca2+ to the protein, has been investigated by comparison of the thermal unfolding curves of the protein in the absence and presence of the chelators and also by measuring the NMR spectra of the protein, the chelators, and the protein-chelator mixtures. The unfolding curve in the presence of a large excess of each chelator has been found to be identical to that in the absence of chelator, indicating that there is essentially no interaction between the chelators and alpha-lactalbumin. The NMR results have also supported this conclusion, and the innocuous character of these chelators as metal-ion buffers in studying the Ca2+-binding properties of alpha-lactalbumin is demonstrated. In order to re-examine the binding constant for Ca2+ of alpha-lactalbumin without the aid of chelating metal-ion buffers, the thermal unfolding curve of the protein in the presence of 0.1 mM excess Ca2+ but without chelators has been compared with the unfolding curve in the absence of Ca2+ at a constant concentration of Na+ (0.010 or 0.10 M) at pH 7.0. The binding constant of alpha-lactalbumin can be calculated from the increment of melting temperature caused by the presence of Ca2+ and from the enthalpy and heat capacity changes in the unfolding. Because Ca2+ binding to the unfolded protein can be neglected under the conditions employed, the binding constant evaluated corresponds to the binding constant to protein that has native structure. The constant obtained is 3-5 X 10(9) M-1 after corrections for binding of Na+ to the protein and for ionic strength, and this shows excellent agreement with the corresponding value previously estimated (2.9 +/- 1.0 X 10(9) M-1), although the latter value was obtained in the presence of EDTA. The apparent Ca2+-binding constant that has been discussed in most previous studies, without taking account of the folding-unfolding equilibrium associated with the binding process, also depends on the concentration of monovalent cations such as Na+, and the present results lead to values of 1.5 X 10(8) and 8.7 X 10(6) M-1 at 0.01 and 0.1 M Na+, respectively.     

On experimental artifacts in the use of metal ion chelators for the determination of the cation binding constants of alpha-lactalbumin. A reply

The binding constant of Ca2+ to the strong cation site of bovine alpha-lactalbumin has been measured directly by monitoring the free calcium concentration by Quin 2 fluorescence. A dissociation constant of 1-4 nM was calculated, which confirms the strong calcium binding properties of this protein. In order to examine whether the metal ion chelators EDTA or EGTA affect the cation binding equilbria by binding to bovine alpha-lactalbumin, calcium binding equilibria were carefully measured under highly stabilized pH and temperature conditions. Within the concentration ranges required for competitive binding by these ligands (EDTA or EGTA) (less than 1-3 mM) these chelators produced no artifacts, in contradiction to the data of Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709).     

Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition

2,4-dinitrophenol (DNP) compromises ATP production within the cell by disrupting the mitochondrial electron transport chain. The resulting loss of ATP leads to an increase in glucose uptake for anaerobic generation of ATP. In L6 skeletal muscle cells, DNP increases the rate of glucose uptake by twofold. We previously showed that DNP increases cell surface levels of glucose transporter 4 (GLUT4) and hexose uptake via a Ca2+-sensitive and conventional protein kinase C (cPKC)-dependent mechanism. Recently, 5' AMP-activated protein kinase (AMPK) has been proposed to mediate the stimulation of glucose uptake by energy stressors such as exercise and hypoxia. Changes in Ca2+ and cPKC have also been invoked in the stimulation of glucose uptake by exercise and hypoxia. Here we examine whether changes in cytosolic Ca2+ or cPKC lead to activation of AMPK. We show that treatment of L6 cells with DNP (0.5 mM) or hyperosmolar stress (mannitol, 0.6 M) increased AMPK activity by 3.5-fold. AMPK activation peaked by 10-15 min prior to maximal stimulation of glucose uptake. Intracellular Ca2+ chelation and cPKC inhibition prior to treatment with DNP and hyperosmolarity significantly reduced cell surface GLUT4 levels and hexose uptake but had no effect on AMPK activation. These results illustrate a break in the relationship between AMPK activation and glucose uptake in skeletal muscle cells. Activation of AMPK does not suffice to stimulate glucose uptake in response to DNP and hyperosmolarity.     

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.     

Features of structural organization of mouse centrometric heterochromatin, detected during differential decondensation of chromosomes

The centromeric heterochromatin (CH) of mouse metaphase and interphase chromosomes has been shown to be practically devoid of the chromonemal and chromomeric levels of DNP organization. CH decondensation into DNP-fibrils caused by decreasing Ca2+-ions concentration is accomplished without formation of chromonemata and chromomeres. The peripheral regions of CH, immediately contacting the inner surface of kinetochores, display the highest stability towards the factors inducing the artificial decondensation.     

Effects of divalent cations on in vitro maturation of bovine oocytes

Lowering the external concentrations of both Mg+2 and Ca+2 caused failure of meiotic resumption in vitro of bovine, oocyte-cumulus complexes. Lowering of external Ca+2 levels singly had no effect on either meiotic resumption or completion of the first meiotic division. Lowering of external Mg+2 concentrations alone, although having no effect on meiotic resumption in vitro when Ca+2 was present, did interfere with the completion of the first meiotic division. The result was arrest of oocyte maturation between germinal vesicle breakdown and formation of the first metaphase plate.     

Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells

Cyclic AMP (cAMP)is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP3), Ca2+ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of extracellular Ca2+. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8-9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca2+ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycol-bis(b-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P3 -receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca2+ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.