The effect of extracellular Ca and Mg on mitochondrial ultrastructure in isolated intact neurons

The ultrastructural transformations of mitochondria in isolated crayfish neurons were studied after incubation of the cells in saline media containing different Ca2+ and Mg2+ concentrations. Incubation in a 5-fold higher Ca concentration resulted in the swelling of mitochondria that was prevented by the addition of the calcium channel blocker, verapamil. Exposure of the cells to Mg2+-depleted medium induced swelling of all the mitochondria, followed by substantial shrinkage of most of them. The absence of Ca as well as the presence of verapamil in Mg2+-free medium led to the inhibition of mitochondrial swelling and to a strong contraction of the mitochondria after 1 h incubation. The omission of Ca2+ from the saline medium or the addition of Ca2+-ionophore A23187 in the presence of Ca2+ resulted in strong mitochondrial shrinkage. These structural alterations of mitochondria are interpreted as an osmotic response of the inner mitochondrial membranes to changes in their potassium transport, induced by a disturbance in the cellular and mitochondrial Ca2+-Mg2+ homeostasis.     

Dynamics of structural and functional changes in wheat root cells under the action of protonophore

Structural and functional changes in wheat root cells during long-term action of a protonophore--carbonyl cyanide 3-chlorophenylhydrazone (CCCP)--were studied. It was demonstrated that CCCP affected the electrical potential and inward resistance of cells, increased K+ ions release to the incubation medium, inhibits oxygen uptake for 1-4 h, which was followed by oxygen uptake stimulation for 6 h of treatment. These changes of physiological processes were accompanied with a variety of ultrastructural changes in cell organization, namely in the structure of mitochondria, endoplasmic reticulum canals, and the nucleus. The role of protons is discussed, in particular, in the regulation of metabolic state of mitochondria, and in general regulation of structural and functional conditions of cells.     

T channels and steroid biosynthesis: in search of a link with mitochondria

The activity of T-type Ca2+ channels has been associated for a long time with steroid biosynthesis in adrenal cortical cells. Because Ca2+-dependent, rate-limiting steps of steroidogenesis have been shown to occur within the mitochondria, a functional link between these organelles and T-type channels has been thoroughly investigated. Based on several experimental data, a model has been proposed in which plasma-membrane-embedded T channels specifically bring calcium entering the cell in proximity of a pumping site of the endoplasmic reticulum. The quasi direct transfer of Ca2+ from the extracellular medium into the lumen of the e.r. would be a specific feature insured by T channels, not by other voltage-operated calcium channels. The e.r. would then act as a sort of Ca2+ pipeline, carrying the cation to the proximity of mitochondria, where it would be released, upon activation of the inositol 1,4,5-trisphosphate receptor, before being immediately and avidly taken up by the organelle. A strict structural organization must be maintained at each extremity of the pipeline in order to optimize the specificity and the efficacy of this signal transduction. Both functional and structural evidences supporting this model of calcium transport within steroidogenic glomerulosa cells are reviewed in the present article.     

Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress

Functional states of mitochondria are often reflected in characteristic mitochondrial morphology. One of the most fundamental stress conditions, hypoxia-reoxygenation has been known to cause impaired mitochondrial function accompanied by structural abnormalities, but the underlying mechanisms need further investigation. Here, we monitored bioenergetics and mitochondrial fusion-fission in real time to determine how changes in mitochondrial dynamics contribute to structural abnormalities during hypoxia-reoxygenation. Hypoxia-reoxygenation resulted in the appearance of shorter mitochondria and a decrease in fusion activity. This fusion inhibition was a result of impaired ATP synthesis rather than Opa1 cleavage. A striking feature that appeared during hypoxia in glucose-free and during reoxygenation in glucose-containing medium was the formation of donut-shaped (toroidal) mitochondria. Donut formation was triggered by opening of the permeability transition pore or K(+) channels, which in turn caused mitochondrial swelling and partial detachment from the cytoskeleton. This then favored anomalous fusion events (autofusion and fusion at several sites among 2-3 mitochondria) to produce the characteristic donuts. Donuts effectively tolerate matrix volume increases and give rise to offspring that can regain ΔΨ(m). Thus, the metabolic stress during hypoxia-reoxygenation alters mitochondrial morphology by inducing distinct patterns of mitochondrial dynamics, which includes processes that could aid mitochondrial adaptation and functional recovery.     

The effect of osmotic pressure on oligomycin-sensitive ATPase activity and the liberation of Mg2+ from liver mitochondria of rats of various ages]

The influence of osmotic pressure of the incubating medium (25-500 mM sucrose) on oligomycin--sensitive, 2,4-dinitrophenyl-stimulated ATP-ase-activity, Mg2+ release and swelling of the liver mitochondria in 1-, 3-, 12-, 24-months Wistar rats is, investigated to determine age changes of structurally functional state of mitochondria. An increase in the sucrose concentration in the medium from 150 to 500 mM causes almost equal and practically absolute inhibition of ATP-ase-activity in different-age groups of rats, regardless of the presence or absence of Mg2+ ions in the medium A fall of the sucrose concentration to 150-25 mM induces a decrease in mitochondria ATP-ase-activity in Mg2+ free medium in 12- and 24-months rats (to 30 and 22%, respectively). No changes are observed in 1- and 3-months animals. Differences in rates of exogenous NADH oxidation by mitochondria of 1- and 12-months rats as a reflection of inner membrane damage degree are not observed under these conditions. Relative changes in ATP-ase-activity in a Mg2+ free medium with sucrose concentration of 25 mM (compared with 150 mM) correlate (r = 0.82) with those of optical density of mitochondria, measured at light wave length of 520 nm. It is obvious that the liver mitochondria of young and old rats sufficiently differ in spontaneous swelling rate in the media with different osmotic pressure: mitochondria of 1-month rats swell much faster than those of old rats. Considerable age differences of osmotic dependence of Mg2+ output from mitochondria are observed. They depend also on peculiarities of spontaneous organelle swelling dynamics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characteristics and oxidative phosphorylation of the isolated mitochondria from the rabbit small intestine epithelium under normal conditions and under the effect of choleragen

Mitochondrial fraction was isolated from the rabbit small intestine epithelium by differential centrifugation in isotonic sucrose medium. The fraction was insignificantly contaminated with fragments of other organellae and microvilli. Respiratory control coefficient of such mitochondria constituted 3-5, when malate-glutamate mixture was used as a substrate. Changes in the functional state of mitochondria were accompanied by stereotypical reconstruction of the "orthodox-condensed" configuration. Introduction of crude choleragen into the mitochondria incubation medium failed to induce any changes in the rate of oxygen consumption in states III and IV (after Chance).     

On the mechanism and functional significance of the ADP/ATP carrier (AAC) dimerization

According to previous studies, ADP/ATP carrier (AAC) can possibly exist as a monomer or in a dimer state in the inner mitochondrial membrane; however, the question on its functional oligomeric state is still open. The aim of the present work is to establish the external factors that could control the functional oligomeric state of AAC (i.e., monomer or dimer). The study is based on the results of our previous work, which revealed that the volume regulation system of mitochondria (MVRS) affects the oxidative phosphorylation (OXPHOS) system: MVRS could transfer OXPHOS system functioning in a state of supercomplex. Consequently, one may expect that the volume regulation system could also control the functional state of AAC during phosphorylation. Here, on rat liver mitochondria we show that, depending on the incubation medium tonicity, AAC functions in two different ways: either as a monomer (in hypotonic and isotonic media) or as a dimer (in a hypertonic medium). Thus, the transition between the monomeric and dimeric forms of AAC is regulated by MVRS, as well as by functioning of OXPHOS. We conclude that the structural reorganization of AAC is associated with the entire OXPHOS reorganization into a supercomplex. It was also found that dimerization of AAC can occur not only due to the action of MVRS (in hypotonic media) but also under hypoxic conditions.     

Correlation of functional and ultrastructural abnormalities of mitochondria in mouse heart carrying a pathogenic mutant mtDNA with a 4696-bp deletion.

We examined the correlation of functional and structural abnormalities of cardiac mitochondria created by pathogenic mutant mtDNAs using mito-mice with hearts carrying 88% mutant DeltamtDNA4696 with a 4696 deletion. COX histochemistry, quantitative PCR analysis, and electronmicrographs showed that accumulation of 91.6% DeltamtDNA4696 in single cardiac muscle fibers induced progressive reduction of COX activity to form COX-negative fibers. Moreover, hearts carrying 88% DeltamtDNA4696 consisted of three types of cardiac muscle fibers with different functional properties, COX-positive, -negative, and -intermediate fibers, which corresponded respectively to three types of fibers with different structural properties; type A fibers containing mitochondria with only lamellar cristae, type B containing mitochondria with only tubular cristae, and type C possessing mitochondria with both lamellar and tubular cristae. These observations suggest that lamellar cristae with COX activity transform into tubular cristae without COX activity along with the accumulation of DeltamtDNA4696, which would be responsible for insufficient supply of mtDNA products required to keep the normal structure and function of mitochondrial cristae. The correlation of these structural and functional abnormalities of cristae should provide important insight into diagnosis of cardiomyopathies caused by accumulation of pathogenic mutant mtDNAs.     

Inhibition of mitochondrial-specific protein symthesis in human lymphocytes and platelets is dependent upon the stage of cellular differentiation

Small lymphocytes differentiate into functionally active blast cells in vitro upon stimulation with such mitogens as phytohemagglutinin and sodium periodate. If stimulated lymphocytes are subsequently treated with the nucleic acid intercalating dye ethidium bromide, electron-dense complexes containing nucleic acid are formed in mitochondria, protein synthesis in mitochondria is inhibited, and lymphoblast division ceases. Formation of complexes and the development of morphologically abnormal mitochondria provide ultrastructural evidence of mitochondrial protein inhibition and serve as markers for mitogen-responsive lymphocytes. The formation of these abnormalities in all mitochondria of treated megakaryocytes and 22% of mitochondria in platelets indicates that platelets contain functional nucleic acid and that the induced structural changes may be occurring in a less-differentiated (i.e., younger) subpopulation of circulating platelets.     

Structural modifications of the permeability transition pore complex in resealed mitochondria induced by matrix-entrapped disaccharides

Mitochondrial resealing after the opening of the permeability transition (PT) pore was studied in saline- and sugar-based media by following the fluorescence anisotropy changes of mitochondria-bound hematoporphyrin (HP), a probe sensitive to conformational variations of the pore complex [Biochemistry 38 (1999) 9300]. The HP anisotropy changes correlated well with complete mitochondrial resealing in saline media and suggested that the pore complex regained the native structure after closure. Rebuilding of the pore complex structure was also achieved in monosaccharide-based media, thus ruling out a major influence of the swollen state of mitochondria on the reconstitution properties of the pore components. On the contrary, when sucrose or other disaccharides were used as osmotic support, restoration of the native mitochondrial structure, as monitored by HP anisotropy, was not achieved, though the proton barrier of the inner membrane and respiration functions were reestablished. Infrared spectroscopy experiments indicated the occurrence of strong perturbations of the mitochondrial membrane structure after disaccharide entrapment in the matrix space. These data suggest that mitochondria are able to reseal and regain functional activity after opening of the PT pore irrespective of the incubation medium but in sucrose (and other disaccharides) the pore complex adopts a conformation different from that existing before permeabilization. In general, our data indicate that the pore complex can exist in different conformations which are modulated by the nature of the interactions with the medium cosolvents.     

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.     

Homologous and Heterologous Interactions between Hexokinase and Mitochondrial Porin: Evolutionary Implications

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase–mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.     

Acyl-CoA dehydrogenases and acyl-CoA oxidases. Structural basis for mechanistic similarities and differences.

Acyl-CoA dehydrogenases and acyl-CoA oxidases are two closely related FAD-containing enzyme families that are present in mitochondria and peroxisomes, respectively. They catalyze the dehydrogenation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. This review examines the structure of medium chain acyl-CoA dehydrogenase, as a representative of the dehydrogenase family, with respect to the catalytic mechanism and its broad chain length specificity. Comparing the structures of four other acyl-CoA dehydrogenases provides further insights into the structural basis for the substrate specificity of each of these enzymes. In addition, the structure of peroxisomal acyl-CoA oxidase II from rat liver is compared to that of medium chain acyl-CoA dehydrogenase, and the structural basis for their different oxidative half reactions is discussed.     

Ceramide Induces Release of Pro-Apoptotic Proteins from Mitochondria by Either a Ca2+-Dependent or a Ca2+-Independent Mechanism

Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C2- or C16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+ -independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C2-ceramide causes cytochrome c release through a Ca2+ -dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C2-ceramide.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Changes in mitochondrial function in the cells and cytoplasts of a pig embryo kidney culture as affected by the action of actinomycin D

Vital staining of PE kidney cells by fluorescent cationic dye, ethyl rhodamine, (accumulated inside mitochondria by the membrane potential and hence reflecting their functional state) shows rather close level of the fluorescence intensity both in cells and in their cytoplasts for 10 hours of culture survival in the standard medium. In cells and cytoplasts cultivated in the medium with 0.2 mg/ml of actinomycin D inhibiting RNA synthesis, fluorescence intensity of mitochondria sharply decreases after 10 hours as against the control patterns. It is concluded that mitochondria possess a significant degree of autonomy of the nucleus and it is supposed that a considerable part of mitochondrial RNA is under the nucleus control.     

Subcellular localization of VDAC in mitochondria and ER in the cerebellum

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.     

Attenuation of intestinal ischemia/reperfusion injury with sodium nitroprusside: studies on mitochondrial function and lipid changes

Reactive oxygen species have been implicated in cellular injury during ischemia/reperfusion (I/R). Mitochondria are one of the main targets of oxygen free radicals and damage to this organelle leads to cell death. Reports suggest that nitric oxide (NO) may offer protection from damage during I/R. This study has looked at the functional changes and lipid alteration to mitochondria during intestinal I/R and the protection offered by NO. It was observed that I/R of the intestine is associated with functional alterations in the mitochondria as suggested by MTT reduction, respiratory control ratio and mitochondrial swelling. Mitochondrial lipid changes suggestive of activation of phospholipase A(2) and phospholipase D were also seen after (I/R) mediated injury. These changes were prevented by the simultaneous presence of a NO donor in the lumen of the intestine. These studies have suggested that structural and functional alterations of mitochondria are prominent features of I/R injury to the intestine which can be ameliorated by NO.     

Structural and functional link between the mitochondrial network and the endoplasmic reticulum

Mitochondrial and endoplasmic reticulum (ER) networks are fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Recent structural and functional studies revealed the interaction of these networks. These zones of close contact between ER and mitochondria called MAM (mitochondria associated membranes) support communication between the two organelles including bioenergetics and cell survival. The existence of macromolecular complexes in these contact sites has also been revealed. In this contribution, we will review: (i) the ER and mitochondria structure and their dynamics, (ii) the basic principles of ER mitochondrial Ca²⁺ transport, (iii) the physiological/pathological role of this cross-talk.