Measurements of membrane potentials in plant mitochondria with the safranine method

The positively charged dye, safranine, has been used as an indicator of membrane potentials in mung bean (Phaseolus aureus) and Voodoo lily (Sauromatum guttatum) mitochondria under a variety of metabolic conditions. The spectral response of safranine has been calibrated with respect to a K⁺ diffusion potential and was found to be linearly related to the developed potential within the range of 50 to 160 millivolts. Both respiration and ATP hydrolysis gave rise to a membrane potential of approximately 135 millivolts. Respiratory inhibitors such as cyanide and antimycin depolarized the potential, whereas rotenone has little effect. No potentials were developed during NADH supported cyanide insensitive respiration. It is concluded that safranine may be a useful spectrophotometric probe of the mitochondrial membrane potential.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

Fluorescamine-induced membrane permeability in mitochondria.

1. Addition of fluorescamine (75 microM) to mitochondria induced an increase in membrane permeability. 2. The leakiness of the inner mitochondrial membrane is characterized by extensive release of accumulated Ca2+, collapse of the transmembrane potential, mitochondrial swelling and efflux of matrix proteins, among them, malate dehydrogenase. 3. These effects were diminished by supplementing the media with 1 mM phosphate, and partially prevented by Mg2+. 4. These results indicate that the primary amino groups of membrane components contribute, partially, to the maintenance of the permeability barrier in mitochondria.     

Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.     

Imaging the permeability pore transition in single mitochondria.

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.     

The respiratory chain of plant mitochondria: x. Oxidation-reduction potentials of the flavoproteins of skunk cabbage mitochondria

The oxidation-reduction potentials of the flavoproteins of skunk cabbage (Symplocarpus foetidus) mitochondria have been measured under anaerobic conditions by means of a combined spectrophotometric or fluorimetric-potentiometric method. Five components were resolved whose oxidation-reduction reactions corresponded to two-electron changes, as expected for flavoproteins. The midpoint potentials at pH 7.2 are as follows, listed in order of increasingly negative potential: +170 millivolts, +110 millivolts, +20 millivolts, −70 millivolts, and −155 millivolts. The most negative component was highly fluorescent; the other components could only be identified by their characteristic absorbance changes. In addition to these components, which are mitochondrial, variable amounts of a very highly fluorescent flavoprotein with a midpoint potential of −215 millivolts was found. This component appears to be extra-mitochondrial. The same midpoint potential values at pH 7.2 were obtained with mitochondria in the uncoupled state as in mitochondria energized with ATP in the absence of phosphate.     

Electrostatic potentials in membrane systems

A membrane with an arbitrary distribution of fixed charges inside and on its surfaces is considered. A procedure for calculating the local electrostatic potential at an arbitrary point of the system is described and its validity discussed. This procedure is based on the linearization of the 3-dimensional Poisson-Boltzmann equation around an exact 1-dimensional solution.     

Monamine oxidase in outer membrane of skeletal muscle mitochondria.

(1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.     

Imaging axonal transport of mitochondria in vivo

Neuronal mitochondria regulate synaptic physiology and cellular survival, and disruption of their function or transport causes neurological disease. We present a fluorescence method to selectively image mitochondrial dynamics in the mouse nervous system, in both live mice and acute explants. We show that axon damage and recovery lead to early and sustained changes in anterograde and retrograde transport. In vivo imaging of mitochondria will be a useful tool to analyze this essential organelle.     

Energetics of Ehrlich ascites mitochondria: membrane potential of isolated mitochondria and mitochondria within digitonin-permeabilized cells

Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.     

Amniotic membrane graft: histopathological findings in five cases

Amniotic membrane transplantation (AMT) is an effective treatment for ocular surface reconstruction; however, the mechanisms through which amniotic membrane (AM) exerts its effects as well as its fate after transplantation have not been entirely elucidated and have been investigated only in part. We evaluate the integration of AM in the host cornea in five patients who underwent AMT as the result of Bowen's disease, band keratopathy, radio- or cryotherapy-induced keratopathy, chemical burn or post-herpetic deep corneal ulcer with descemetocele. Due to persistent opacification in four cases and a progressing tumor in one case, penetrating keratoplasty (PK) and enucleation were performed as early as 2 months and up to 20 months after AMT. The corneas were analyzed histopathologically. To evaluate AM remnants, corneas were stained with periodic acid Schiff's reaction (PAS), Alcian blue, and Gomory and Masson trichrome; immunostaining including collagens III and IV antibodies was also performed. None of the corneas showed remnants of AM. In all cases, we observed discontinuity of Bowman's membrane. In three cases, the corneal epithelium was completely restored, ranging from three to six cell layers. In the other two cases, we detected an intense inflammatory reaction with rich neovascularization; the epithelial surface of the central cornea was completely restored, while at the periphery of the cornea goblet mucus-producing cells were present. Although clinically useful in all cases, restoration of a stable corneal epithelium through AMT is limited by the extent and severity of limbal stem cell deficiency (LSCD). The lack of histologically documented AM remnants in our cases seems to explain the efficacy of AMT more through its biological properties than through its mechanical properties.