THE ACCUMULATION AND THE RELEASE OF DIVALENT CATIONS ACROSS MITOCHONDRIAL MEMBRANES

Accumulated divalent cations and phosphate (P₁) in isolated bean mitochondria are released by conditions which inhibit respiration, including anaerobiosis and KCN, or by conditions which divert conserved energy from divalent cation uptake. These include ATP synthesis, K^T transport in the presence of valinomycin, and the presence of the uncouplers, 2,4-dinitrophenol and oleic acid. The results indicate that plant mitochondria are not permanent deposit sites for divalent cation and P₁ salts but, rather, function as temporary sequestering sites for these ions. It is suggested that mitochondria may play a role in the control of the movement as well as a regulation of the concentrations of these ions within the cell.     

Role of calcium ions in the control of embryogenesis of Xenopus. Changes in the subcellular distribution of calcium in early cleavage embryos after treatment with the ionophore A23187.

Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

Effects of ionic compositions of the medium on monosodium glutamate binding to taste epithelial cells

Monosodium glutamate and nucleotides are umami taste substances in animals and have a synergistic effect on each other. We studied the ligand-binding properties of the glutamate receptors in taste epithelial cells isolated from bovine tongue. Specific glutamate binding was observed in an enriched suspension of taste receptor cells in Hanks' balanced salt solution, while no specific glutamate binding was apparent in the absence of divalent ions or when the cells had been depolarized by a high content of potassium in Hanks' balanced salt solution. There was no significant difference between the release of glutamate under depolarized or divalent ion-free conditions and under normal conditions. However, glutamate was easily released from the depolarized cells in the absence of divalent ions. These data suggest that the binding of glutamate to receptors depends on divalent ions, which also have an effect on maintaining binding between glutamate and receptors.     

ELECTRON MICROSCOPE STUDIES ON THE ACTIVE ACCUMULATION OF SR++ BY RAT-LIVER MITOCHONDRIA

Electron-opaque granules are deposited in isolated rat-liver mitochondria concomitant with the energy-linked accumulation of Sr⁺⁺ by these organelles. High temperature microincineration (600°C) of thin sections of mitochondria containing different amounts of Sr⁺⁺ shows that a clear qualitative correlation exists between the number of inorganic residues remaining after incineration and the amount of Sr⁺⁺ translocated into the mitochondria. By loading the mitochondria with consecutive pulses of small amounts of Sr⁺⁺ ("multiple-pulse" loading), very early stages of granule formation can be detected; the first detectable deposits are seen closely associated with the cristae. The evidence presented supports the hypothesis that mineral deposition following or during the in vitro accumulation of ions by mitochondria occurs, at least initially, at sites on these membranes and not as nonspecific precipitates in the mitochondrial matrix. The large number of electron-opaque deposits (100 to 200) seen in single thin sections of individual mitochondria having accumulated intermediate levels of Sr⁺⁺ clearly exceeds the number of normal dense granules in rat-liver mitochondria, indicating that the normal matrix granules per se do not constitute sites essential for deposition. At the highest levels of Sr⁺⁺ uptake studied in the multiple-pulse loading experiments, needlelike deposits are seen, a result which suggests that the structural form ("crystallinity") of the mineral deposits may be determined by the rate of accumulation.     

EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

Rat liver mitochondria allowed to accumulate maximal amounts of Ca⁺⁺ and HPO₄⁼ ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca⁺⁺ and HPO₄⁼ from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca⁺⁺-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca⁺⁺-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca⁺⁺-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca⁺⁺ and phosphate from the mitochondria into the medium.     

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.     

Properties of mitochondria from Penicillium cyclopium and their response to calcium and other divalent cations

The respiratory properties of isolated mitochondria from P. cyclopium were studied with particular attention to their response to calcium ions. The results obtained indicate concentration dependent stimulation of NADH oxidation by calcium ions. Similar effects could also be obtained with other divalent cations.     

Effect of divalent metal ions on annexin-mediated aggregation of asolectin liposomes

Annexins belong to a family of Ca2+- and phospholipid-binding proteins that can mediate the aggregation of granules and vesicles in the presence of Ca2+. We have studied the effects of different divalent metal ions on annexin-mediated aggregation of liposomes using annexins isolated from rabbit liver and large unilamellar vesicles prepared from soybean asolectin II-S. In the course of these studies, we have found that annexin-mediated aggregation of liposomes can be driven by various earth and transition metal ions other than Ca2+. The ability of metal ions to induce annexin-mediated aggregation decreases in the order: Cd2+ > Ba2+, Sr2+ > Ca2+ > Mn2+ > Ni2+ > Co2+. Annexin-mediated aggregation of vesicles is more selective to metal ions than the binding of annexins to membranes. We speculate that not every type of divalent metal ion can induce conformational change sufficient to promote the interaction of annexins either with two opposing membranes or with opposing protein molecules. Relative concentration ratios of metal ions in the intimate environment may be crucial for the functioning of annexins within specialized tissues and after treatment with toxic metal ions.     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

Muscle actin filaments bind pituitary secretory granules in vitro

Hog anterior pituitary secretory granules sediment at 3,000 g. When rat or rabbit skeletal muscle actin filaments are present with the granules, the sedimentation decreases markedly. Depolymerized actin or viscous solutions of Ficoll and collagen have no effect on granule sedimentation. With this assay, actin filaments bind secretory granules (consisting of the proteinaceous core plus limiting membrane), secretory granule membranes, mitochondria, artificial lecithin liposomes, and styrene-butadiene microspheres, but have little or no interaction with membrane-free secretory granule cores and albumin microspheres. A secretory granule-actin complex sedimentable between 3,000 g and 25,000 g can be isolated. Metal ions, nucleotides, salts, dithiothreitol, or pretreatment of the granules with trypsin do not destroy the binding, which appears to be a lipophilic interaction.     

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [14C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.     

Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn2+ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn2+, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Significance and mechanism of divalent-ion binding to transfer RNA.

Phosphorus NMR shows that divalent ions (manganese) bind to tRNA phosphates as to those of DNA or isolated phosphodiesters. The time for dissociation of a phosphate-divalent ion complex is in the microsecond range. For no single phosphate is the affinity to divalent ions greater than 10 times that of the average phosphate. It is often stated that a small number of strong binding sites exist and are structurally and functionally important. This concept originates from binding curves whose properties should, instead, be traced to the polyelectrolyte nature of nucleic acids. The 31P NMR data preclude the existence of strong sites to which divalent ions would bind very selectively. The Spectroscopic and crystallographic observations of sites for divalent ions do not in fact demonstrate selective binding to these sites.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron''s damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Biochemical and Cytological Changes Accompanying Growth and Differentiation in the Roots of Zea mays

The apical meristem of the root affords an excellent material with which to study changes in cellular components accompanying growth and differentiation. The ontogeny of cytoplasmic particles can be followed, since the younger cells are constantly dividing and reforming new cytoplasm. Electron microscope pictures of these newly formed cells reveal a dense background of microsomal granules and small, thin walled vesicles of the endoplasmic reticulum. Two types of mitochondria are noted and, as the cells enlarge, mitochondria regarded as immature can no longer be seen, but only mitochondria with well developed cristae. The development of these cristae was found to be associated with an increase in respiration of the tissue as well as with increased rates of oxidation and phosphorylation of isolated mitochondria. As the cells grow and mature, the mitochondria make up an increasing percentage of the total cytoplasmic protein, and this increase probably accounts to a great extent for the increase in tissue respiration. Concomitantly, there is a decrease in microsomal granules. All these changes have been verified by electron microscope pictures of cells in situ, chemical analysis of isolated particulates, and metabolic studies of tissue and isolated fractions.     

Different isotonic density gradients in separation of renin granules from rat kidney cortex

Crude renin granule preparations isolated from the rat renal cortex were further purified in isotonic conditions (300 mOsm/kg) using various density gradient materials. It was not possible to separate renin granules from other subcellular organelles using dextran, 40,000-sucrose or metrizamide-sucrose gradients at about 300 mOsm/kg. When osmolality of dextran-sucrose gradients was increased, some separation was found but both renin granules and mitochondria gained density. During a short centrifugation (4640 X g, 30 min) renin granules remained intact and appeared in two populations in Percoll-sucrose gradients. The apparently heavier (larger) particles (at 1.12-1.13 kg/l) were greatly purified from mitochondria (80 X purification vs. the whole homogenate), protein (120 X) and lysosomes (24 X). Electron micrographs demonstrated many dense core granules. The fraction containing apparently lighter (small) granules (at 1.08-1.09 kg/l) was heavily contaminated with mitochondria and lysosomes. During longer centrifugation (4640 X g, 60 min), only one major peak showing renin activity was observed at 1.12-1.13 kg/l, and other cell organelles were lighter. Hence the two renin populations evidently do not differ in density but rather in size. In the animals kept on a low-sodium diet, both types of renin granules were increased.     

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg²⁺ free medium. Exogenous citrate suppressed H₂O₂ emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate’s effect. Exogenous MgCl₂ strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg²⁺ or other divalent cations.