Ricerche Sul Rigonfiamento di Piccola Ampiezza in Mitocondri Isolati di Pisello

Abstract

Low amplitude swelling in isolated pea mitochondria. — The authors have studied the volume changes of isolated pea internode mitochondria occurring in a medium which allows the oxidative activity to proceed. With succinate as substrate respiratory control ratios as high as 9 have been obtained. They can be taken as an index of a tight coupling of mitochondria. The adding of succinate induces on the other hand a slight but continuous swelling which is strongly enhanced by ADP or ATP. Inhibitors of the respiratory chain like antimycin A and of the phosphorylation reactions like atractylate or oligomycin block completely the ADP or ATP-induced swelling. 2,4-dinitrophenol at 5×10^?6 M concentration exerts a strong inhibitory action on succinate oxidation and on swelling. Both actions of 2,4-DNP can be reversed by ATP. It can be concluded from these findings that the substances which slow down or abolish the oxidative and phosphorylative reactions inhibit also mitochondrial swelling. This type of volume changes appears therefore to be strictly energy-dependent.     

Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons

Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.     

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.     

Induction of Apoptosis in Rat Myocardium under Anoxic Conditions

The effect of anoxic incubation of small slices of isolated rat hearts on respiration, internucleosomal DNA fragmentation, and mitochondrial ultrastructure was investigated. Anoxic incubation for 72 h induced apoptosis accompanied by internucleosomal DNA fragmentation and changes in respiration and mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In a significant part of the mitochondrial population there were signs of mitochondrial swelling and appearance of electron-dense mitochondria. Anoxia also induced the appearance of an atypical (and previously unknown) population of small electron-dense mitochondria. They were characterized by unusual localization inside electron-light mitochondria. Under anoxic conditions the inner mitochondrial membrane formed electron-dense ordered structures. All changes described here reflect two opposing processes occurring in mitochondria: apoptotic destruction and compensatory processes responsible for maintenance of mitochondria.     

Swelling of Phaseolus Mitochondria in Relation to Free Fatty Acid Levels

Freshly isolated Phaseolus vulgaris mitochondria contain, 1.8 micromoles of long chain, primarily unsaturated, free fatty acids per milligram of nitrogen. Although there is no measurable increase in free fatty acid content as a result of spontaneous swelling in buffered KCl, bovine serum albumin strongly inhibits the degree of swelling. The mitochondria only swell slowly in sucrose, but rapid swelling can be induced by the addition of oleic acid in a process inhibited by bovine serum albumin. The possible participation of the endogenous free fatty acids of Phaseolus mitochondria in spontaneous swelling is discussed.     

Calcium-dependent spontaneously reversible remodeling of brain mitochondria

An exposure of cultured hippocampal neurons expressing mitochondrially targeted enhanced yellow fluorescent protein to excitotoxic glutamate resulted in reversible mitochondrial remodeling that in many instances could be interpreted as swelling. Remodeling was not evident if glutamate receptors were blocked with MK801, if Ca(2+) was omitted or substituted for Sr(2+) in the bath solution, if neurons were treated with carbonylcyanide p-trifluoromethoxyphenylhydrazone to depolarize mitochondria, or if neurons were pretreated with cyclosporin A or N-methyl-4-isoleucine-cyclosporin (NIM811) to inhibit the mitochondrial permeability transition. In the experiments with isolated brain synaptic or nonsynaptic mitochondria, Ca(2+) triggered transient, spontaneously reversible cyclosporin A-sensitive swelling closely resembling remodeling of organelles in cultured neurons. The swelling was accompanied by the release of cytochrome c, Smac/DIABLO, Omi/HtrA2, and AIF but not endonuclease G. Depolarization with carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the Ca(2+) uniporter with Ru360 prevented rapid onset of the swelling. Sr(2+) depolarized mitochondria but failed to induce swelling. Neither inhibitors of the large conductance Ca(2+)-activated K(+) channel (charybdotoxin, iberiotoxin, quinine, and Ba(2+)) nor inhibitors of the mitochondrial ATP-sensitive K(+) channel (5-hydroxydecanoate and glibenclamide) suppressed swelling. Quinine, dicyclohexylcarbodiimide, and Mg(2+), inhibitors of the mitochondrial K(+)/H(+) exchanger, as well as external alkalization inhibited a recovery phase of the reversible swelling. In contrast to brain mitochondria, liver and heart mitochondria challenged with Ca(2+) experienced sustained swelling without spontaneous recovery. The proposed model suggests an involvement of the Ca(2+)-dependent transient K(+) influx into the matrix causing mitochondrial swelling followed by activation of the K(+)/H(+) exchanger leading to spontaneous mitochondrial contraction both in situ and in vitro.     

Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half-maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane. It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short-term hormonally induced activation of the enzyme observed in isolated hepatocytes.     

Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences

Mitochondrial swelling is a hallmark of mitochondrial dysfunction, and is an indicator of the opening of the mitochondrial permeability transition pore. We introduce here a novel quantitative in situ single-cell assay of mitochondrial swelling based on standard wide-field or confocal fluorescence microscopy. This morphometric technique quantifies the relative diameter of mitochondria labeled by targeted fluorescent proteins. Fluorescence micrographs are spatial bandpass filtered transmitting either high or low spatial frequencies. Mitochondrial swelling is measured by the fluorescence intensity ratio of the high- to low-frequency filtered copy of the same image. We have termed this fraction the “thinness ratio”. The filters are designed by numeric optimization for sensitivity. We characterized the thinness ratio technique by modeling microscopic image formation and by experimentation in cultured cortical neurons and astrocytes. The frequency domain image processing endows robustness and subresolution sensitivity to the thinness ratio technique, overcoming the limitations of shape measurement approaches. The thinness ratio proved to be highly sensitive to mitochondrial swelling, but insensitive to fission or fusion of mitochondria. We found that in situ astrocytic mitochondria swell upon short-term uncoupling or inhibition of oxidative phosphorylation, whereas such responses are absent in cultured cortical neurons.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

Swelling and Contraction of Mitochondria from Cold-hardened and Nonhardened Wheat and Rye Seedlings

A comparison of mitochondria isolated from 2 and 24 C grown winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) seedlings revealed no correlation between changes in swelling and contraction characteristics and extent of cold hardiness. The swelling response changed markedly due to growth at low temperature, but the change was similar for the four cultivars examined. The swelling response was also observed to change rapidly during aging of isolated mitochondria, either at 2 or 24 C. Spontaneously swollen mitochondria, isolated from 24 C grown seedlings, contracted abruptly upon addition of certain oxidizable substrates, but this response was lost when seedlings were transferred from 24 to 2 C. Studies on the effect of various substrates and respiratory inhibitors on the swelling and contraction responses indicate that inhibitors which reduce or stop electron flow through the electron transport chain also inhibit substrate induced mitochondrial contraction.     

Effect of ozone on swelling of tobacco mitochondria

The swelling of mitochondria isolated from leaves, roots, and callus tissues of Nicotiana tabacum, L, var. White Gold, was measured by following changes in optical density at 520 mμ in buffered 0.25 m sucrose or 0.125 m KCl. Ozone induced rapid swelling of the isolated mitochondria and increased the permeability of mitochondrial membranes. The extent of mitochondrial swelling and the amount of soluble proteins and other substances absorbing at 260 and 280 mμ released from mitochondria into the suspending medium were positively correlated with the length of exposure to O₃. The correlation between the extent of mitochondrial swelling and the loss of intramitochondrial materials was also highly significant.     

Inhibition by secalonic acid D of oxidative phosphorylation and Ca2+-induced swelling in mitochondria isolated from rat livers

The in vitro biological activity of secalonic acid D, a mycotoxin from Aspergillus ochraceus, was studied to assess its cytotoxicity for isolated rat liver mitochondria. Secalonic acid D uncoupled the oxidative phosphorylation of mitochondria and caused a mild inhibition of state 3 respiration. Secalonic acid D weakly enhanced latent ATPase activity in mitochondria but suppressed 2,4-dinitrophenol-stimulated ATPase activity. Secalonic acid D did not induce pseudoenergized swelling of mitochondria and markedly inhibited the Ca2+-induced swelling of mitochondria in KCl isotonic solution.     

Inhibition by secalonic acid D of oxidative phosphorylation and Ca2+-induced swelling in mitochondria isolated from rat livers.

The in vitro biological activity of secalonic acid D, a mycotoxin from Aspergillus ochraceus, was studied to assess its cytotoxicity for isolated rat liver mitochondria. Secalonic acid D uncoupled the oxidative phosphorylation of mitochondria and caused a mild inhibition of state 3 respiration. Secalonic acid D weakly enhanced latent ATPase activity in mitochondria but suppressed 2,4-dinitrophenol-stimulated ATPase activity. Secalonic acid D did not induce pseudoenergized swelling of mitochondria and markedly inhibited the Ca2+-induced swelling of mitochondria in KCl isotonic solution.     

Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation

Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria.     

Energetics of swelling in isolated hepatocytes: A comprehensive study

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

Ultrastructural changes in adrenergic neurons following chemical sympathectomy

The paper describes the ultrastructural changes of the sympathetic neurons following guanethidine administration in mice. The main characteristics changes, after prolonged administration (1-10 weeks) of guanethidine (Ismelin, Ciba-Geigy, Basel, Switzerland) at doses of 30-50 mg/kg/day i.p. and i.m. were: (1) Mitochondrial damages: swelling and deformation of the mitochondria, with disruption and dispersion of the cristae and condensation or dissolution of the matrix. Membranolysis of the outer and inner mitochondrial membranes. (2) Neural processes: swelling and disorganization of the internal architecture in the postganglionic axonal and dendritic synaptic varicosities. (3) Adrenergic vesicles: disappearance of the granular vesicles in the early stage of the experiment, increase of the coated vesicles in the later stage of treatment. The effect of guanethidine is specific for the postganglionic adrenergic neurons, but the observed destruction is not caused solely by guanethidine; other chemical substances induce the same or similar degeneration in the sympathetic ganglion.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation

Proper distribution of mitochondria within axons and at synapses is critical for neuronal function. While one-third of axonal mitochondria are mobile, a large proportion remains in a stationary phase. However, the mechanisms controlling mitochondrial docking within axons remain elusive. Here, we report a role for axon-targeted syntaphilin (SNPH) in mitochondrial docking through its interaction with microtubules. Axonal mitochondria that contain exogenously or endogenously expressed SNPH lose mobility. Deletion of the mouse snph gene results in a substantially higher proportion of axonal mitochondria in the mobile state and reduces the density of mitochondria in axons. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation, probably by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. These findings demonstrate a molecular mechanism for controlling mitochondrial docking in axons that has a physiological impact on synaptic function.     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Chick embryo heart fragments in primary hanging-drop culture were treated with sodium fluoroacetate to induce inhibition of aconitate hydratase, a mitochondrial enzyme of the tricarboxylic acid cycle. The mitochondria were analyzed in the living myoblasts by phase-contrast time-lapse cinemicrography. The results were recorded in a 16 mm film. After 20–30 minutes contact of the cells with the inhibitor some mitochondria became thickened and swollen. The swelling was polymorphous, asynchronous and reversible; the same mitochondrion could swell and shrink many times. Some mitochondria seemed not to respond to fluoroacetate and remained rod-like. Mitochondria appeared the only cell components to be morphologically affected by fluoroacetate and the changes were specifically caused by the inhibitor. The type of mitochondrial swelling differed from the large-amplitude respiration-dependent swelling of the isolated mitochondria in vitro and from the configurational changes of isolated mitochondria associated with the respiratory states. The evidence pointed to a specific connection between the biochemical lesion caused by fluoroacetate and the configurational changes of the mitochondria. The mitochondrial swelling was to a large extent reversed by washing the cultures with Tyrode physiological saline solution and the reversal was further accentuated by incubation of the cultures in fresh nutrient medium.This work was supported by grants of the Consiglio Nazionale delle Richerce of Italy to both Institutes.