Tracking of proton flow during transition from anaerobiosis to steady state in rat liver mitochondria

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H+/K+ exchanger, nigericin, to anaerobic mitochondria. Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9-19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa3 and the rise of delta mu H+. With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H+ displacement of about 0.8-1.2 ngatom/mg on the basis of the H+/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2-3 ngatom H+/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445-502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the delta mu H+ rise. The fact that H+ extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr2(+)-supplemented anaerobic mitochondria results in an H+ extrusion whose amount and rate is proportional to the Sr2+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin

The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M omega, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K+ concentration of the medium and magnitude of the K+ diffusion potentials was consistent with the presence of a high internal concentration of K+, (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay of membrane leakiness. Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication.     

Giant mitochondria distinct from enlarged mitochondria in secretory and ciliated cells of gerbil trachea and bronchioles

Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.     

The effect of hormones on proton compartmentation in hepatocytes

Liver mitochondria isolated from rats treated acutely with glucagon exhibit higher respiration-dependent H+ ion gradients across the mitochondrial inner membrane than mitochondria from control rats. It has been suggested that similar increases in mitochondrial delta pH in situ could stimulate gluconeogenesis, chiefly because the transport of pyruvate into mitochondria would increase in response to the increase in mitochondrial matrix pH. In order to determine whether the increased delta pH observed in vitro in isolated mitochondria also occurs in situ, the effect of glucagon on the pH in the cytosol and mitochondria matrix spaces of isolated hepatocytes was determined. For qualitative results, the spectral responses of intracellularly trapped 6-carboxyfluorescein was used to monitor cytosol pH, while fluorescein-loaded hepatocytes were used to monitor the mitochondrial pH. Hepatocytes were incubated with the diacetate ester derivatives of these dyes. The esters are permeable to the cell membranes, but are rapidly hydrolyzed in the cells. The free unesterified dyes are relatively impermeable to the cell membranes. After being trapped in the cell, 6-carboxyfluorescein remains localized in the cell cytosol, whereas fluorescein is taken up by the mitochondria as a function of the mitochondrial delta pH. In order to quantitate the actual pH in these compartments, the spectral responses (490-465 nm) of 6-carboxyfluorescein-loaded hepatocytes were used to determine the cytosolic pH. Calibration of these responses was obtained within the cell by determination of the dye's differential absorption coefficient (epsilon 490-465 nm) in various high K+ buffers after equilibration of the internal and external pH with valinomycin and the uncoupler 1799. All absorbance values were corrected for dye leakage. Equal hematocrits of unloaded cells were used to correct for absorbance contributions from cellular constituents. The mitochondrial pH was determined by a combination of the indicator dye and [14C]5,5'-demethyloxazolidine-2, 4-dione (DMO) distribution ratio methods. The weak acid DMO freely distributes across the plasma membrane and mitochondrial membrane in whole cells according to the pH gradient across each membrane. Knowledge of the cytoplasmic pH from the 6-carboxyfluorescein data allows the expected distribution of DMO across the plasma membrane to be calculated. The excess accumulation of DMO in intact hepatocytes over that predicted from the plasma membrane pH gradient alone was then used to calculate the pH gradient across the mitochondrial inner membrane. The effects of valinomycin, uncouplers, and hormones on the pH in cytosolic and mitochondrial compartm     

Na+/H+ exchange in mitochondria as monitored by BCECF fluorescence

The recently developed method of loading isolated heart mitochondria with the fluorescent pH indicator, BCECF, was applied to monitor the Na+o/H+i exchange process from the matrix side of the membrane. The Na+-induced changes in the pH of the matrix (pHm) showed that: (i) the Na+o/H+i exchange followed Michaelis-Menten kinetics with respect to external Na+ with a Km of approx. 20 mM; (ii) in contrast to this, the dependence of the exchange rate on the matrix [H+] did not obey the Michaelian model. No Na+-induced alkalinization occurred above a pHm of 7.45 +/- 0.09 (n = 4). Below this value the reciprocal of the transport rate and that of the matrix [H+] deviated upwardly from the straight line. The results suggest that internal H+ might exert allosteric control on the mitochondrial Na+/H+ exchange process.     

Mitochondrial behaviour during the yeast-hypha transition of Candida albicans

Yeast cells of Candida albicans were brought to germ tube formation and hyphal growth in liquid synthetic medium. The behaviour of mitochondria and mitochondrial nucleoids (mt-nucleoids) during morphological conversion was examined by fluorescence staining with 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) and 4',6-diamidino-2-phenylindole (DAPI). Parent yeast cells possessed one or very few branched giant mitochondria which were stained intensely with DASPMI. When a germ tube emerged from the parent cell, one end of a giant mitochondrion extended into the germ tube and developed into the elongated form. In mycelia, apical hyphal cells contained giant mitochondria, whereas older hyphal compartments near the parent cells were vacuolated and possessed small, peripherally located mitochondria. The vacuolated hyphal compartments resynthesized cytoplasm before producing branches and contained giant mitochondria. The cytological model for germ tube formation and hyphal growth proposed by Gow and Gooday (1984) is discussed.     

Membrane potential and surface potential in mitochondria. Binding of a cationic spin probe

The interaction of the cationic spin probe 4-(N,N-dimethyl-N-dodecyl)-ammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (Cat12) with intact mitochondria and submitochondrial particles was investigated as a function of salt concentration, pH and energization by ATP. In the presence of 1 mM Fe(CN)-36, which inhibits the probe reduction by the mitochondria, the probe signal is stable and shows both bound and free forms. The partition of the probe into mitochondrial membranes is decreased by various salts depending on the cation valency, indicating that the membrane is negatively charged (-10 to -15 mV at pH 7.0). The surface potential increases with pH from -3 mV at pH 5.0 to -18 mV at pH 8.0. Energization of intact mitochondria by ATP reduces the magnitude of both bound and free signals by more than 50%; the signal of the bound form slowly disappears on further incubation. The ATP effect is inhibited and also reversed by either oligomycin or CCCP. Similar effects of ATP were observed in mitoplasts but not in submitochondrial particles. In submitochondrial particles ATP has no effect on the probe signal or binding. These results suggest that the formation of membrane potential in mitochondria induces uptake and internal binding of the probe which results in broadening of the EPR signal of the internally bound probe. It is concluded that Cat12 is not a suitable probe for measurement of surface potential in energized mitochondria.     

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.     

Do mitochondria in Dendrobium petal mesophyll cells form vacuole-like vesicles?

Using transmission electron microscopy, we investigated the ultrastructure of mitochondria in petal mesophyll cells of the orchid Dendrobium cv. Lucky Duan, from the time of floral opening to visible petal senescence. Cells close to the vascular bundle contained many mitochondria, some of which showed internal degeneration. This inner mitochondrial breakdown was accompanied by an eightfold increase in mitochondrial volume. Small electron-dense granules (approximately 0.04 μm in diameter) at the periphery of the mitochondrial matrix remained. These granules were used as an indicator of still later stages of mitochondrial development in these cells. The apparent final stage of mitochondrial degeneration was a single-membrane-bound vesicle, resembling a vacuole. No evidence was found for the idea that mitochondria became transferred (intact or degenerated) into a lytic vacuole. Taken together, the data suggest the hypotheses that (a) mitochondria in cells close to the vascular bundle in petals of open Dendrobium cv. Lucky Duan flowers undergo large-scale internal degeneration and that (b) such degenerating mitochondria form vacuole-like vesicles.     

pKa Values and Partition Coefficients of Nitroxide Spin Probes for Membrane Bioenergetics Measurements

Knowledge of pK_a's is necessary to calculate intracellular/intravesicular pH values from nitroxide accumulation in cells or vesicles as detected with electron spin resonance (ESR) spectroscopy. pK_a values were confirmed in lipid vesicles of known internal pH. To help select probes that do not accumulate in lipid membranes, octanol/buffer partition coefficients of uncharged nitroxides were determined. As an application of selected probes, pH gradients and internal aqueous volumes were analyzed in mitochondria (one internal compartment) and in the cyanobacterium Synechococcus 6311 (two internal compartments). The combination of 3-carboxy-, 3-amino- and 3-aminocarbonyl-2,2,5,5-tetramethylpyrrolidin-1-yloxyl was found to be most satisfactory for determinations of internal pH and volumes.     

Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (～7.6) and ΔpH(m) (～0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (～5 mm, pH 7.8) compared with the cytosol (～20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons

Simultaneous measurements of electrical activity and light absorbance have been made on nerve cell bodies from Archidoris monteryensis injected with indicator dyes. pH indicators, phenol red and bromocresol purple, and arsenazo III, which under normal conditions is primarily a calcium indicator have been employed. Voltage clamp pulses which induced calcium influx caused an absorbance decrease of the pH dyes indicating an internal acidification. The onset of the pH drop lagged the onset of Ca2+ influx by 200-400 ms, and pH continued to decrease for several seconds after pulse termination which shut off Ca2+ influx. Trains of action potentials also produced an internal pH decrease. Recovery of the pH change required periods greater than 10 min. The magnitude of the pH change was largely unaffected by external pH in the range 6.8-8.4. The voltage dependence of the internal p/ change was similar to the voltage dependence of calcium influx determined by arsenazo III, and removal of calcium from the bathing saline eliminated the pH signal. In neurons injected with EGTA (1-5 mM), the activity- induced internal Ca2+ changes were reduced or eliminated, but the internal pH drop was increased severalfold in magnitude. After the injection of EGTA, voltage clamp pulses produced a decrease in arsenazo III absorbance instead of the normal increase. Under these conditions the dye was responding primarily to changes in internal pH. Injection of H+ caused a rise in internal free calcium. The pH buffering capacity of the neurons was measured using three different techniques: H+ injection, depressing intrinsic pH changes with a pH buffer, and a method employing the EGTA-calcium reaction. The first two methods gave similar measurements: 4-9 meq/unit pH per liter for pleural ganglion cells and 13-26 meq/unit pH per liter for pedal ganglion cells. The EGTA method gave significantly higher values (20-60 meq/unit pH per liter) and showed no difference between pleural and pedal neurons.     

Formation of elongated giant mitochondria in DFO-induced cellular senescence: involvement of enhanced fusion process through modulation of Fis1

Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H2O2 (200 microM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF beta1- or H2O2-induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DeltaTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence.     

On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons

The inner membrane of mitochondria possesses a pH-regulated anion uniporter which is activated by depletion of matrix divalent cations with A23187 (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). It is now shown that Cl- transport through this pathway is inhibited by Mg2+ and Ca2+. There appear to be two sites for inhibition by Mg2+. One has an IC50 = 38 microM at pH 7.4 and appears to be on the inside since it is only observed in the presence of A23187 (10 nmol/mg). The other has an IC50 = 440 microM at pH 7.4 and appears to be on the outside since it is observed in mitochondria pretreated with very low doses of A23187 (0.25 nmol/mg or less) and in A23187-pretreated mitochondria washed to remove A23187. Ca2+ is found to inhibit anion uniport in the presence or absence of A23187 with an IC50 of about 17 microM. In contrast to these findings Cl- uniport, activated by addition of valinomycin to respiring mitochondria without depleting endogenous Mg2+ is found to be very insensitive to exogenous Mg2+, being inhibited with an IC50 of 3.2 mM. This is explained by examination of the pH dependence of the Mg2+ IC50 in non-respiring mitochondria. The internal IC50 is found to be pH-dependent, rising to about 250 microM at pH 8.4. The external IC50 is also pH-dependent, rising to 2.5 mM or above at pH 8.4. These data are consistent with a model in which Mg2+ can only bind to the protein when it is protonated at a site with a pK of about 6.8 located in the matrix. Thus, both the intrinsic activity of the uniporter and its inhibition by Mg2+ appear to be regulated by matrix protons. This makes the rate of anion uniport much more sensitive to changes in matrix pH which is physiologically advantageous for its proposed role in volume homeostasis.     

A re-evaluation of conditions required for an accurate estimation of the extramitochondrial ATP/ADP ratio in isolated rat-liver mitochondria

The values reported in the literature for the extramitochondrial ATP/ADP ratio in resting rat-liver mitochondria (State 4) vary widely. The conditions required for an accurate determination of this parameter were therefore investigated. In experiments with rat-liver mitochondria incubated under State-4 conditions, it was found that the extramitochondrial ATP/ADP ratio, as calculated from the values measured in neutralised perchloric acid extracts, was lower than that estimated from the concentrations of creatine and creatine phosphate, using the metabolite indicator method. The discrepancy is due to hydrolysis of ATP occurring in the presence of perchloric acid. Conditions are described for minimising ATP hydrolysis in the presence of perchloric acid, and include the use of low concentrations of perchloric acid, short times of exposure to the acid before neutralisation, low temperatures and the presence of excess EDTA. Under these conditions, the values obtained for the extramitochondrial ATP/ADP ratio agreed with those calculated by the metabolite indicator method, provided ratios do not exceed the value of 100. In cases where the extramitochondrial ATP/ADP does exceed 100, phenol/chloroform/isoamyl alcohol must be used to quench the reactions, as described by Slater et al. (Slater, E.C., Rosing, J. and Mol, A. (1973) Biochim. Biophys. Acta 292, 534-553). With this method, the extramitochondrial ATP/ADP ratio was found to have a value of more than 1000 in rat-liver mitochondria incubated with succinate + rotenone in the resting state (pH 7.0; T = 37 degrees C), in agreement with Slater et al.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

Titration of mitochondrial buffer by accumulated anions

Summary Acidimetric titration of intact rat liver mitochondria discloses a buffer power of about 45 mEquiv per g between pH 7 and 8, the value rises to 60 mEquiv per g per pH unit after lysis using Triton X-100. The existence and properties of this buffer system have been related to mitochondrial anion accumulation. The uptake of permeant anions by mitochondria occurs to a charge-dependent extent and they are in electrochemical equilibrium with each other and the protons as in a Donnan system. Adding permeant anion causes the intramitochondrial anion content to rise towards a saturation level, the inside to outside concentration ratio falls and concomitantly the transmembrane proton gradient diminishes, making the interior less alkaline. The falling internal pH is associated with protonation of the internal buffer, thus providing a second method for measuring the buffer power, a method which also tests the arguments used in the calculations. The titration curve is constructed by relating the internal pH (deduced from the permeant anion ratio) to the total internal anion equivalents which in turn determines the ionization state of the buffer because the sum of the internal anion equivalents, including the buffer anion, equals the equivalents of internal cation. The buffer power so measured agrees with the acidimetric method applied to lysed mitochondria. The disparity between the acidimetric data from lysed and unlysed mitochondria follows theoretical predictions.     

The internal concentration of K+ in isolated rat liver mitochondria

A simple osmotic method has been developed to determine the internal K+ concentration of mitochondria by determining the concentration of external K+ at constant osmotic pressure at which metabolically inhibited mitochondria neither shrink nor swell. This concentration has been found to correspond to approx. 80-85 mM in freshly isolated mitochondria and considerably lower after additional centrifugation procedures. Since mitochondria are in osmotic equilibrium with the suspending medium (in this case, 0.32 osmolal), and K+ is the primary exchangeable internal ion, a significant proportion of the internal osmotic pressure must be exerted by the sucrose. Results for experiments determining internal K+ after centrifuging mitochondria at various G values confirm the reports of Sitaramam et al. (Sitaraman, V. and Sarma, M.K.J. (1981) Proc. Natl. Acad. Sci. USA 78, 3441-3445 and Sambasivarao, D. and Sitaramam, V. (1983) Biochim. Biophys. Acta 722, 256-270) that centrifugation induces the entry of sucrose in mitochondria isolated in a sucrose medium.     

Giant mitochondria do not fuse and exchange their contents with normal mitochondria

Giant mitochondria accumulate within aged or diseased postmitotic cells as a consequence of insufficient autophagy, which is normally responsible for mitochondrial degradation. We report that giant mitochondria accumulating in cultured rat myoblasts due to inhibition of autophagy have low inner membrane potential and do not fuse with each other or with normal mitochondria. In addition to the low inner mitochondrial membrane potential in giant mitochondria, the quantity of the OPA1 mitochondrial fusion protein in these mitochondria was low, but the abundance of mitofusin-2 (Mfn2) remained unchanged. The combination of these factors may explain the lack of mitochondrial fusion in giant mitochondria and imply that the dysfunctional giant mitochondria cannot restore their function by fusing and exchanging their contents with fully functional mitochondria. These findings have important implications for understanding the mechanisms of accumulation of age-related mitochondrial damage in postmitotic cells.