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.     

An electron microscopic study of structural changes during the large amplitude swelling and contraction of isolated beef-heart mitochondria

Summary The structural changes of isolated beef-heart mitochondria undergoing hypotonic swelling and ATP-induced contraction are described. During swelling the mitochondria take up water, the matrix space enlarges, the inner membrane becomes greatly extended with material which is apparently derived from the cristae (the rest of which fragment) and the outer membrane is ruptured. On addition of ATP, Mn⁺⁺ and Ca⁺⁺ water is extruded as the extended inner membrane contracts around the remnants of the cristae to give compact bundles of vesicles which bear little resemblance to the original organized structure.On leave of absence from Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, England.     

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.     

Ultrastructural Changes during Swelling and Contraction of Mitochondria from Cold-hardened and Non-hardened Winter Wheat

Mitochondria isolated from both 2 and 24 C grown winter wheat (Triticum aestivum L.) undergo spontaneous swelling in isomolar KCI solutions, but only 24 C mitochondria exhibit a substrate-induced contraction response. Electron microscopic examination revealed that 24 C mitochondria have more clearly defined cristae, less matrix material, and are generally more electron-dense than 2 C mitochondria. During swelling, the matrix material of both 2 and 24 C mitochondria expands and the mitochondria become less electron-dense. After partial swelling, 24 C mitochondria contract upon addition of succinate, and regain structural characteristics similar to those of untreated mitochondria. In contrast, mitochondria from 2 C seedlings continue to swell after addition of substrate, and many of the mitochondria become irregular in shape and lose much of their matrix material. A comparison of results obtained from absorbancy measurements, electron microscopy, and a Coulter Counter indicate that swelling and contraction involve changes both in over-all volume, and internal structural characteristics of mitochondria from 2 and 24 C grown seedlings. Electron microscopic examination of shoot cells showed that mitochondria in 24 C grown seedlings possessed more recognizable cristae and greater internal organization than mitochondria in 2 C seedlings.     

A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.

The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.     

Studies on ultrastructure and purification of isolated plant mitochondria

Sweetpotato mitochondria, that showed respiratory control, were studied with respect to ultrastructure. If fixed in media containing sucrose at 0.4 M, the cristae were dilated and the matrix was highly condensed. A more orthodox ultrastructural form was observed when the mitochondria were fixed in a medium containing sucrose at 0.25 M, i.e., the matrix was more expanded, the cristae were less dilated, and peripherally, the inner membrane element lay adjacent to the outer membrane element. These results are discussed in terms of a sucrose-accessible space (space between outer and inner membrane elements including intracristal space), and a space relatively inaccessible to sucrose (matrix). Ultrastructural shifts were not observed with change in metabolic steady state of the mitochondria. High resolution electron micrographs showed that the ultrastructure of sweetpotato mitochondria is very similar to that of animal mitochondria.

Purity and homogeneity of mitochondrial fractions were followed both by phase-contrast and electron microscopy. Preparations from sweetpotato, using older methods, were relatively homogeneous with respect to particle type and size, whereas avocado preparations contained a high proportion of chloroplasts and cellular debris. A method of purification involving sucrose-density-gradient centrifugation was developed. Purified mitochondria exhibited respiratory control and appeared similar to unpurified mitochondria under the electron microscope.

     

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.     

大鼠心肌线粒体缺血再灌损伤及SOD抗缺血再灌损伤作用的形态计量研究

研究了在体大鼠心肌缺血再灌模型心肌线粒体再灌损伤和SOD抗缺血再灌损伤的作用。实验分为:A组(缺血再灌组)B组(SOD组)和C组(假手术组)。电镜见A组线粒体高度肿胀、外膜缺损、嵴断裂溶解,出现无定形致密体、四膜嵴和杆状嵴等改变。B组线粒体轻度或中度肿胀、少数有轻度嵴溶解。个别线粒体内出现无定形致密体。三组线粒体超微结构立体计量数据比较,A组线粒体密度、线粒体体密度与肌原纤维体密度比率增高((P<0.01),线粒体比表面和比膜面降低(P<0.01)。自身动图像分析仪检测SDH反应灰度值A组呈强损伤反应,B组反应较轻,二者差别有显著性(P<0.05)。结果表明,心肌缺血再灌可致线粒体发生不可逆性损伤,SOD能减轻线粒体缺血再灌性损伤。     

Detection of new double-membrane structures in native mitochondria by the method of small-angle neutron scattering

The structure of mitochondrial cristae has been studied for the first time by the method of small-angle neutron scattering. Experiments were performed on intact (functioning) mitochondria from rat liver. Mitochondrial cristae are usually considered to be folds of the inner membrane with arbitrary variable intermembrane distances. Under conditions of low-amplitude swelling, mitochondrial cristae transformed into double-membrane structures with a distance of 190 Å between the central planes of the membranes. The formation of double-membrane structures and their structural parameters did not depend on the method for inducing swelling which was accomplished either by placing the mitochondria into a hypotonic medium or through the opening of nonspecific pores.     

Structural changes during swelling of isolated rat mitochondria

Summary Isolated rat liver and kidney mitochondria were swollen in hypotonie sucrose solutions and then examined in the electron microscope. The results were consistent with the hypothesis that unfolding of the cristae plays some part in swelling since cristae of swollen mitochondria appeared less abundant and with increased intercristal distances. The matrix became more electron-transparent. The fate of the outer membrane is uncertain.This investigation was supported by Public Health Service Research Grants GM-08900, NB-02145, Interdisciplinary Grant 5T1-MH-6418 and a Lederle Medical Faculty Award.     

Mitochondrial permeability transition and cytochrome c release induced by selenite

Cytochrome c release and mitochondrial permeability transition (MPT) play important roles in apoptosis. In this study, we found that selenium, an essential trace element, induced mitochondrial membrane potential (Delta psi(m)) loss, swelling, and cytochrome c release in isolated mitochondria. All of the above observations were blocked by cyclosporin A (CsA), which is a specific inhibitor to permeability transition pore (PTP), indicating selenite-induced mitochondrial changes were mediated through the opening of PTP. In physiological concentration, selenite could induce mitochondria at low-conductance PTP 'open' probability, which is correlated to regulate the physiological function, whereas in toxic concentration, induce mitochondria at high-conductance PTP 'open' probability and rapidly undergo a process of osmotic swelling following diffusion toward matrix as for inducer (Ca(2+)/P(i)). Selenite also induced other mitochondrial marker enzymes including monoamine oxidase (MAO) and mitochondria aspartate aminotransferase (mAST). Oligomycin inhibited the selenite-induced cytochrome c release and Delta psi(m) loss, showing that F(0)F(1)-ATPase was important in selenite or Ca(2+)/P(i)-induced MPT.     

Intersection between Mitochondrial Permeability Pores and Mitochondrial Fusion/Fission

The goal of this review is to highlight recent developments in the field of mitochondrial membrane processes, which provide new insights into the relation between mitochondrial fission/fusion events and the mitochondrial permeability transition (MPT). First, we distinguish between pore opening events at the inner and outer mitochondrial membranes. Inner membrane pore opening, or iMPT, leads to membrane depolarization, release of low molecular weight compounds, cristae reorganization and matrix swelling. Outer membrane pore opening, or oMPT, allows partial release of apoptotic proteins, while complete release requires additional remodeling of inner membrane cristae. Second, we summarize recent data that supports a similar temporal and physical separation between inner and outer mitochondrial membrane fusion events. Finally, we focus on cristae remodeling, which may be the intersection between oMPT and iMPT events. Interestingly, components of fusion machinery, such as mitofusin 2 and OPA1, appear to play a role in cristae remodeling as well. Special issue dedicated to John P. Blass.     

EFFECTS OF POTASSIUM ON INDICATOR SPACES AND FLUXES IN SLICES OF BRAIN CORTEX FROM ADULT AND NEW-BORN RATS

—Inulin, sucrose and chloride spaces were measured in slices of brain cortex from adult and from new-born rats incubated in‘balanced', potassium-rich and sodium-rich media. The efflux of the radioactive markers was followed in the two first media and the following results were obtained: (1) In brain slices from new-born rats inulin and sucrose spaces were of identical magnitude (35 per cent). The space magnitude was essentially unaffected by excess potassium. The chloride space was somewhat larger than the inulin (sucrose) space, and the difference increased continuously but relatively slightly with the external potassium concentration. By far the largest amount (i.e. about 90 per cent) of the efflux of radioactive inulin, sucrose and chloride occurred from a rapidly exchanging compartment during incubation in both ‘balanced’ and potassium-rich media. (2) In brain slices from adult rats the inulin space (35 per cent) was significantly smaller than that of sucrose (50 per cent) and of chloride (65 per cent); it seemed to represent the extracellular space relatively well although 10 per cent of the efflux occurred from a slowly exchanging (probably intracellular) compartment. High concentrations of potassium led to a reduction of the inulin space which was probably a result of the concomitant intracellular swelling. The hyperosmolarity per se did not affect the space magnitude, but an increase of the sodium concentration exerted a competitive inhibition of potassium effects on the inulin space. Of the sucrose efflux, 20 per cent occurred from a slowly exchanging compartment in both ‘balanced’ and potassium-rich media, and 30 per cent of the chloride exchanged with this compartment when the tissue was incubated in a ‘balanced’ medium. An increase of the external potassium concentration caused a drastic increase of the chloride space and a reduction of the slowly exhanging fraction of chloride efflux to less than 10 per cent.     

An open issue: the inner mitochondrial membrane (IMM) as a free boundary problem

The inner mitochondrial membrane (IMM) is structured in cristae, which contributes to the best functioning of ions and adenylates exchange between the matrix and the intermembrane space. The central hypothesis of this paper is that the cristae structure favours a minimal mean free path of adenylates between translocation sites (translocase/ANT sites) and metabolic sites (ATPase sites). We propose a mathematical model and then give simulations. Based on simple hypotheses about cristae growth, they show that we can account for the major features of the IMM organization and functioning by minimizing the mean interdistance between ADP/ATP translocation and transformation sites.     

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.     

Hyperplastic processes in cardiac muscle mitochondria exposed to toxic doses of adrenaline]

Experiments were conducted on albino rats; a study was made of hyperplastic processes in the mitochondria of the myocytes of the heart with the action of toxic adrenaline doses. A solution of adrenaline chloride was injested intramuscularly (3 mg/kg). Three types of mitochondria were revealed in electron microscopic study. Mitochondria of the first type were of the size and structure characteristic of the muscle cells of the myocardium. Mitochondria of the second type had a very dense, finegrained matrix and a great number of cristae per unit of the area. Mitochondria of the third type had two "sections" under the common external membrane, differing from one another by the matrix density, distribution and number of cristae. It is supposed that the ultrastructural peculiarities of each of the types reflected their functional condition.     

Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis

During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.     

Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.     

Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.