Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.     

A PVC-based electrode sensitive to DDA+ as a device for monitoring the membrane potential in biological systems

A polyvinyl chloride (PVC)-based membrane electrode sensitive to dibenzyldimethyl ammonium cation (DDA⁺) was constructed, and operational parameters such as the selectivity coefficients, the detection limit, and the response time were obtained. In comparison with the selectivity coefficients obtained with the previous liquid-membrane electrode, significant improvement was not obtained, but the response time became pronouncedly shorter. Furthermore, the electrode lifetime was remarkably prolonged. With the electrode developed, the change in the membrane potential of liposomes containing dibutyl ferrocene which separated oxidizing and reducing agent solutions was measured. The DDA⁺ uptake, U, and the membrane potential estimated from U changed in accordance with the redox potential in the medium when the concentration of internal ferricyanide was kept constant. The membrane potential collapsed when the uncoupler of oxidative phosphorylation was added. The ANS fluorescence measurement indicated that negative charges appeared on energization with oxidizing-reducing agent. The change in membrane potential of mitochondria during energization was also measured. It was found that the liposome described above is a good model for the generation of membrane potentials in mitochondria.     

Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator.

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.     

Effect of ruthenium red upon Ca2+ and Mn2+ uptake in Saccharomyces cerevisiae. Comparison with the effect of La3+

The initial rate of both Ca2+ and Mn2+ uptake is inhibited by ruthenium red to about the same extent as by equivalent concentrations of La3+. The inhibition of Ca2+ uptake, however, is relieved during further incubation with ruthenium red. On preincubating the cells with ruthenium red even a stimulation of divalent cation uptake can be found. Relieve of the inhibition of divalent cation uptake is accompanied by K+ efflux. Both ruthenium red and La3+ displace Ca2+ very effectively from binding sites at the cell surface. The inhibition of initial Ca2+ uptake is accompanied by a reduction in the binding of Ca2+.     

Induction of mitochondrial Ca2+ uptake by mersalyl

1. The addition of mersalyl to aged mitochondria from rat kidneys, is followed by induction of an ATP-driven Ca2+ uptake which is sensitive to Ruthenium Red. 2. This Ca2+ influx requires Mg2+, albumin, and is accomplished by membrane energization. 3. The activation of Ca2+ uptake by the mercurial in the presence of ATP can be explained if it is assumed that the inorganic phosphate generated by ATPase activity, and trapped in the matrix by the thiol reagent, provides the negative potential which results in an electrophoresis cation influx.     

OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis

For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.     

Localized energization of the mitochondrial inner membrane by ATP.

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane. The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F1 . F0) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211-222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F1), reflecting the localized energization of the membrane by ATP.     

10-N nonyl-acridine orange: a fluorescent probe which stains mitochondria independently of their energetic state

The specificity of binding of 10-N Nonyl Acridine Orange to mitochondria, and more precisely to inner membranes, is demonstrated by subcellular fractionation of hepatocytes. Unlike Rhodamine 123, which is a preferential marker of the transmembrane potential, Nonyl Acridine Orange binding is essentially independent of the mitochondria energization state although a low uptake of this dye, in response to the potential, may be measured. So 10-N Nonyl acridine orange is an appropriate marker of the mitochondial membrane surface per unit of cell mass.     

The effects of ionophores on the fluorescence of the cation 3,3''-dipropyloxadicarbocyanine in the presence of pigeon erythrocytes, erythrocyte ''ghosts'' or liposomes.

1. Pigeon erythrocytes, resealed lysed erythrocytes or liposomes derived from erythrocyte lipids were suspended in solutions containing up to 2 micrometer-3,3'-dipropyloxadicarbocyanine iodide. Gramicidin, valinomycin, nigericin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or combinations of these, were used to induce electrical diffusion potentials dependent on Na+, K+ or protons. In each instance hyperpolarization of the cell membrane lowered the fluorescence of the cell suspension, a process that was completed in about 1 min. Subsequent depolarization caused an increase in fluorescence. 2. Quenching of the fluorescence of the cell suspension appeared to be due to the reversible binding of the dye to the cells. Much larger amounts of dye were bound, both to the intact and to the resealed erythrocytes, than would be expected if partitioning of the dye cation followed the Nernst equation. The dependence of the binding on the extracellular dye concentration was studied in the presence and absence of valinomycin. The results were consistent with the suggestion of Sims, Waggoner, Wang & Hoffman [(1974) Biochemistry 13, 3315-3330] that the dye was bound at both membrane surfaces and that, at low dye concentrations, hyperpolarizing the cells promoted dye binding at the inner membrane surface. 3. The applications of the technique are limited by the circumstance that the direct effect of the electric field on the uptake of the dye into the cells is amplified by a binding process that may be affected by other physiological variables.     

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.     

Inhibition of succinate production during yeast fermentation by deenergization of the plasma membrane

Succinate production by a respiratory-deficient yeast was inibited by substances known to depolarize the plasma membrane. These substances include high concentrations of the permeable cation potassium, the ATPase inhibitor diethylstilbestrol, the polyene antibiotic amphotericin B, and the uncouplers carbonyl cyanidem-chlorophenylhydrazone and dinitrophenol. Results suggest that succinate effux from yeast cells is driven by membrane energization in the form of an electrical potential. As sucinate is one of the major by-products of alcoholic fermentation, deenergization of yeast plasma membrane may be a useful approach to increasing the yield of ethanol in industrial fermentations.     

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.     

Ion selectivity of the Vibrio alginolyticus flagellar motor.

The marine bacterium, Vibrio alginolyticus, normally requires sodium for motility. We found that lithium will substitute for sodium. In neutral pH buffers, the membrane potential and swimming speed of glycolyzing bacteria reached maximal values as sodium or lithium concentration was increased. While the maximal potentials obtained in the two cations were comparable, the maximal swimming speed was substantially lower in lithium. Over a wide range of sodium concentration, the bacteria maintained an invariant sodium electrochemical potential as determined by membrane potential and intracellular sodium measurements. Over this range the increase of swimming speed took Michaelis-Menten form. Artificial energization of swimming motility required imposition of a voltage difference in concert with a sodium pulse. The cation selectivity and concentration dependence exhibited by the motile apparatus depended on the viscosity of the medium. In high-viscosity media, swimming speeds were relatively independent of either ion type or concentration. These facts parallel and extend observations of the swimming behavior of bacteria propelled by proton-powered flagella. In particular, they show that ion transfers limit unloaded motor speed in this bacterium and imply that the coupling between ion transfers and force generation must be fairly tight.     

Glutathione reduces cytoplasmic vanadate. Mechanism and physiological implications

The mechanism by which cells reduce cytoplasmic vanadium(V) (vanadate) to vanadium(IV) was investigated using the human red cell as a model system. Vanadate uptake by red cells occurs with a rapid phase involving chemical equilibration across the plasma membrane and a slower phase resulting in a high concentration of bound vanadium(IV). The slow phase was inhibited in glucose-starved cells and restored upon addition of glucose indicating an energy requirement for this process. The time course of vanadium(IV) appearance (monitored by EPR spectroscopy of intact cells) paralleled the slow phase of uptake indicating that this phase involves vanadium reduction. The reduction of intracellular vanadate to vanadium(IV) was nearly quantitative after 23 h. The intracellular reduction is not enzymatic, since a similar time course of vanadium reduction and binding to hemoglobin was observed when glutathione was added to a hemoglobin + vanadate solution in vitro. Vanadium(IV) binding to hemoglobin was reduced by addition of ATP, 2,3-diphosphoglycerate or EDTA, probably through chelation of the cation. The stability constant of the ATP-vanadium (IV) complex was determined to be 150 M-1 at pH 4.9. The time course of red cell vanadate uptake and reduction was followed in the concentration range in which approximately 60% inhibition of the (Na+ + K+)-ATPase is observed. It is concluded that vanadate is reduced by cytoplasmic glutathione in this concentration range and that the reduction explains the resistance of the (Na+ + K+)-ATPase to vanadium in intact cells.     

The H+-ATPase from chloroplasts: effect of different reconstitution procedures on ATP synthesis activity and on phosphate dependence of ATP synthesis

The H+-ATP synthase from chloroplasts, CF0F1, was isolated, reconstituted into liposomes and ATP synthesis activity was measured after energization of the proteoliposomes with an acid-base transition. The ATP yield was measured as a function of the reaction time after energization, the data were fitted by an exponential function and the initial rate was calculated from the fit parameters. CF0F1 was reconstituted by detergent dialysis in asolectin liposomes and phosphatidylcholine/phosphatidic acid (PtdCho/PtdAc from egg yolk) liposomes. In asolectin liposomes, high initial rates of ATP synthesis (up to 400 s(-1)) were observed with a rapid decline of the rate; in PtdCho/PtdAc liposomes the initial rate is smaller (up to 200 s(-1)), but the decline of the activity is slower. CF0F1 was reconstituted into PtdCho/PtdAc liposomes either by detergent dialysis or into reverse phase liposomes. The dependence of the rate of ATP synthesis on the phosphate concentration was measured with both types of proteoliposomes. The data can be described by Michaelis-Menten kinetics with a K(M) value of 350 microM for reverse phase liposomes and a K(M) value of 970 microM for dialysis liposomes. Both K(M) values depend neither on the magnitude of DeltapH nor on the electric potential difference, whereas V(max) decreases strongly with decreasing energization. At low phosphate concentration, there are small deviations from Michaelis-Menten kinetics. The measured rates are higher than those calculated from the fitted Michaelis-Menten parameters. This effect is interpreted as evidence that more than one phosphate binding site is involved in ATP synthesis.     

C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake

Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C₆-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4 °C and 37 °C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox.SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4 °C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.     

The release of membrane-bound calcium by radiation and sulfhydryl reagents

Effects of ionizing radiation and of sulfhydryl reagents on the ⁴⁵Ca binding of red cell membranes were studied. Corresponding effects of these agents on potassium leak from intact red cells were also determined. Essentially all the ⁴⁵Ca associated with the ghosts appeared to be bound. Calcium binding could be described by assuming two independent groups of binding sites with dissociation constants of about 6 × 10^?4 m and 2 × 10^?4 m. The total binding capacity was about 2.5 × 10^?4 moles/g ghost protein. Membrane calcium was decreased by radiation and by the two sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and N-ethyl maleimide (NEM). The tightly bound calcium fraction appeared to be most affected by these agents. Changes in potassium leak evoked by varying doses of agents appeared to parallel effects on membrane calcium. These investigations suggest that the increased cation permeability observed after exposure or red cells to radiation or sulfhydryl reagents may be related to alterations in the calcium-binding properties of the cell membrane.     

Anion transport during maturation of erythroblastic cells

Bromide uptake was measured in single maturing erythroblastic cells of rabbits by means of X-ray microanalysis. Increase in bromide uptake as the cells matured was observed. The order of cells from low to high bromide uptake was: early erythroblast less than late erythroblast less than marrow red cells less than peripheral red blood cells. The transition from low to high bromide uptake is correlated to the accumulation of iron which begins in the late erythroblast. A decrease in rubidium uptake also occurs as iron accumulates in the cell. These results indicate that the anion and cation transport changes during maturation are parallel in time course but opposite in direction. In addition, the increase in bromide uptake can be accounted for by the increase in surface-to-volume ratios of the cells. Surface-to-volume ratios were estimated by morphometric techniques.     

Turbulent flow of red cells in dilute suspensions. Effect on kinetics of O2 uptake.

The turbulent flow properties of dilute (0.06% by volume) suspensions of human red blood cells in 4-mm-bore glass tubing were estimated by laser anemometry. The flow properties of the dilute red cell suspension were similar to those of a dilute suspension of polystyrene spheres (0.5 micron diameter) in isotonic NaCl solution. Flow was found to be laminar when the Reynolds number was below 2,000, transitional in the range of Reynolds numbers from 2,000 to 3,000, and fully turbulent above Reynolds number 3,000. These results differ from previous studies of more concentrated red cell suspensions. The length scales of the turbulence were also estimated: at a Reynolds number near 4,000 the macroscale is about 1.25 mm, the Taylor microscale is about 0.85 mm, and the Kolmogoroff scale is near 0.075 mm. The results are discussed in relation to previous measurements of the rate of oxygen uptake by dilute red cell suspensions in the flow-type rapid reaction apparatus. Our results suggest that under the conditions of most of these oxygen uptake measurements, the turbulent flow is characterized by eddies about 1 mm across, mixing with each other on a time scale of about 45 ms. Since most of the reported oxygen uptake measurements involve a similar time scale, it is possible that an effective "unstirred layer" influenced the reported rate of oxygen uptake.     

Glucose transport of Haloferax volcanii requires the Na(+)-electrochemical potential gradient and inhibitors for the mammalian glucose transporter inhibit the transport.

The uptake of glucose and its non-metabolizing analogues by Haloferax volcanii, one of the glucose-utilizing Halobacteria, was examined using intact cells and envelope vesicles. Results obtained were: (1) The transport system is inducible. (2) The uptake requires the gradient of Na(+)-electrochemical potential. (3) Inhibitors for mammalian glucose transport also have an effect on this system, implying that the transporters resemble each other. (4) It is suggested that the mobility of the transporter is regulated by the membrane energization.