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PubMed1992

Utilization of ATP-depleted cells in the analysis of taurocholate uptake by isolated rat hepatocytes.

The usefulness of ATP-depleted rat hepatocytes in transport studies was examined. ATP-depleted hepatocytes were prepared by incubating cell suspensions with 30 microM rotenone. In ATP-depleted hepatocytes, plasma membrane permeability was increased and mitochondrial membrane potential decreased, while both intracellular volume and pH remained normal. Furthermore, in the presence of valinomycin, the initial uptake rates of 3H-tetraphenyl phosphonium (TPP+) with varied medium concentrations of potassium were predicted according to the Goldman-Hodgkin-Katz equation, which demonstrated that a potassium diffusion potential could be produced in this system. Using the thus-characterized ATP-depleted cells, the uptake mechanism of taurocholate was investigated. In the presence of an inwardly directed Na gradient, the taurocholate uptake was markedly stimulated and bile acid was transiently accumulated at a concentration 3-times higher than at equilibrium ('overshoot') in ATP-depleted cells. No overshoot was observed in viable cells, however, which suggests that in ATP-depleted cells the Na gradient, a driving force for taurocholate uptake, decreased with time. In both viable and ATP-depleted cells, the relationship between medium concentrations of Na and the Na-dependent initial uptake rate were sigmoidal, and the Hill coefficients were close to 2. The Na-dependent initial uptake rate of taurocholate was stimulated by a valinomycin-induced inside negative potassium-diffusion potential in ATP-depleted cells, and the movement of a 'one plus' (as a net) charge was revealed by fitting the data to the Goldman-Hodgkin-Katz equation. These results support the hypothesis that sodium-coupled hepatic uptake of taurocholate occuthrough an electrogenic process with the stoichiometry of 2 Na: 1 taurocholate, although this issue is controversial. In the presence of an outwardly directed sodium gradient, efflux of taurocholate from ATP-depleted cells was not stimulated. Consequently, the physiological transport vector of taurocholate from blood to cell is not only due to the direction of the sodium gradient (blood to cell) but also to membraneous orientation of transport carriers. In conclusion, kinetic analysis using ATP-depleted hepatocytes allowed the formulation of a new approach to clarify the as yet unresolved issues concerning transport stoichiometry and the mechanism for vectorial transport of taurocholate.

Adenosine Triphosphate↗

PubMed1992

Synaptosomal plasma and mitochondrial membrane potentials during anoxia.

The precise mechanism by which altered oxidative metabolism impairs neuronal function is unknown. Previous indirect studies suggest that anoxia's effects on the mitochondrial membrane potentials may underlie anoxia's actions. Twenty minutes of anoxia reduced the mitochondrial membrane potential of intact synaptosomes by 38-59 mV, but diminished the plasma membrane potential by only 4-10 mV. Anoxia did not alter the response of the plasma or mitochondrial membrane potentials to K+, nor did anoxia affect the reaction of the plasma membrane potential to valinomycin. However, anoxia diminished the response of the mitochondrial membrane potential to valinomycin by 50%. Thus, partial collapse of the mitochondrial membrane potential may be an important mediator of hypoxia-or anoxia-induced changes in neuronal function.

Anaerobiosis↗

PubMed1976

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria. The role of potassium.

In previous studies it was found that change in the concentrations of Ca2+, H+, and HPO2-4 in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)2D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K+ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)2D-3 synthesis by isolated chick mitochondria; that the magnitnitude of this K+-dependent stimulation is enhaced by optimal concentrations of calcium (pCa = 5) and phosphate (pPi = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na+ concentration; and that the stimulatory effect of K+ is not blocked by ruthenium red, and inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH) 2D-3 synthesis. It was also found that valinomycin, a K+-specific ionophore, enhanced the sensitivity of the mitochondrial 1 alpha-hydroxylase activity to K+. In the presence of valinomycin, an increase of pK+ to 3 was sufficient to cause a significant stimulation of 1,25(OH)2D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulated the activity of the 1 alpha-hydroxylase.

Animals↗

PubMed1982

The inhibition of the serum-stimulated increase of ornithine decarboxylase by ionophores and local anesthetics.

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchicine, vinblastine) or cations (5-10 mM K+, Na+ or Mg2+) abolishes this increase of ornithine decarboxylase activity. (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212-219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six ionophores tested, valinomycin was the most potent one, with I50 value (concentration that gives 50% inhibition of ornithine decarboxylase activity) of 6.10(-9) M. Dibucaine and tetracaine were also effective inhibitors at 10(-4)-10(-5) M. The I50 values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1.10(-6) M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.

Anesthetics, Local↗

PubMed1992

Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae.

We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.

Adenosine Triphosphatases↗

PubMed1998

Voltage clamp studies on S-layer-supported tetraether lipid membranes.

Isolated subunits from the cell surface proteins (S-layer) of Bacillus coagulans E38-66 have been recrystallized on a glycerol dialkyl nonitol tetraether lipid (GDNT)-monolayer and the electrophysical features of this biomimetic membrane have been investigated in comparison to unsupported GDNT-monolayers. The GDNT-monolayer, spread on a Langmuir-Blodgett trough, was clamped with the tip of a glass patch pipette. In order to investigate the barrier function and potential to incorporate functional molecules, voltage-clamp examinations on plain and S-layer-supported GDNT-monolayers were per-formed. Our results indicate the formation of a tight GDNT-monolayer sealing the tip of the glass pipette, and a decrease in conductance of the GDNT-monolayer upon recrystallization of the S-layer protein. Thus, the S-layer protein, apparently, did not penetrate or rupture the lipid monolayer. The valinomycin-mediated increase in conductance was less pronounced for the S-layer-supported than for the plain GDNT-monolayer, indicating differences in the accessibility and/or in the fluidity of the lipid membranes. Furthermore. in contrast to plain GDNT-monolayers. S-layer supported GDNT-monolayers with high valinomycin-mediated conductance persisted over long, periods of time, indicating enhanced stability. These composite S-layer/lipid films may constitute a new tool for electrophysical and electrophysiological studies on membrane-associated and membrane-integrated biomolecules.

Anti-Bacterial Agents↗

PubMed1983

Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli.

An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios. We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment. The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi. (b) An increase in [K+] from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity. (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM [K+], causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV. From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM. (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution. (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors. (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.

Cell Membrane↗

PubMed1987

Effects of double-layer polarization on ion transport.

It has been proposed that changes in ionic strength will alter the shape of current-voltage relations for ion transport across a lipid membrane. To investigate this effect, we measured currents across glyceryl monooleate membranes at applied potentials between 10 and 300 mV using either gramicidin and 1 mM NaCl or valinomycin and 1 mM KCl. A bridge circuit with an integrator as null detector was used to separate the capacitative and ionic components of the current. The changes in the current-voltage relations when ionic strength is varied between 1 and 100 mM are compared with predictions of Gouy-Chapman theory for the effects of these variations on polarization of the electrical diffuse double-layer. Double-layer polarization accounts adequately for the changes observed using membranes made permeable by either gramicidin or valinomycin.

Biological Transport↗

PubMed1989

Proton flux in large unilamellar vesicles in response to membrane potentials and pH gradients.

The transport of protons across liposomes composed of phosphatidylcholine in response to electrical potentials or pH gradients has been investigated. The results support three major conclusions. The first of these concerns the need for reliable measurements of electrical potentials and pH gradients. It is shown that the potential probe tetraphenylphosphonium and the pH probe methylamine provide accurate and self consistent measures of electrical potentials and pH gradients respectively in these systems. Second, it is shown by two independent techniques that the pH gradients induced in response to valinomycin and potassium dependent electrical potentials are significantly smaller than would be expected for electrochemical equilibrium. The pH gradients observed are stable over an 8 h time course and are sensitive to the ionic composition of the buffers employed, where the presence of external sodium results in the smallest induced pH gradients. These results are discussed in terms of current models of proton conductance across membranes. In a final area of investigation, it is shown that valinomycin and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) can transport sodium ions in a synergistic manner.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗

PubMed2001

Activation of ion-conducting pathways in the inner mitochondrial membrane - an unrecognized activity of fatty acid?

The effect of non-esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large-amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non-ionic detergent Triton X-100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K+ and Cl- in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate-induced Cl- -permeation (measured with KCl medium plus valinomycin) was inhibited by N,N'-dicyclohexylcarbodiimide, quinine or ATP. Myristate- or dodecyl sulfate-initiated swelling was paralleled by the lowering of endogenous Mg2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187-induced swelling has identical inhibiting characteristics on Cl- -permeation with respect to N,N'-dicyclohexylcarbodiimide, quinine and ATP as the myristate-stimulated swelling. Therefore, we conclude that non-esterified fatty acids increase the permeability of mitochondria to K+ and Cl- at alkaline pH by activating Mg2+-dependent ion-conducting pathways in IMM.

Animals↗

PubMed1998

Detection of the local H+ gradients on the internal mitochondrial membrane.

Respiration-dependent responses of a pH probe (fluorescein isothiocyanate, FITC), covalently bound to the membrane proteins of mitochondria and submitochondrial particles (SMP) have been studied. A spectral shift indicating FITC deprotonation was observed when respiration was activated in coupled mitochondria. Such a response was increased by valinomycin and reduced by uncoupler. Some FITC deprotonation was detected in the presence of excess of an uncoupler, but the response was smaller and insensitive to valinomycin. FITC deprotonation was also observed in submitochondrial particles after succinate addition. In this case it was not affected by uncoupler. Increase in the buffer concentration was found to (i) decrease the FITC response and (ii) increase the rate of uncoupled respiration in both mitochondria and submitochondrial particles. The results are consistent with the assumption that respiration initiates appearance of local H+ activity gradients on the inner side of the internal mitochondrial membrane during the steady-state H+ pumping. We suggest that the formation of this gradient is due to kinetic barrier to proton transfer from the bulk phase to the respiratory proton pump vicinity.

Acetone↗

PubMed1984

Ionophore-induced functional changes in macrophages.

Ca ionophore A 23187 and valinomycin known to induce Ca2+ influx, i.e. K+ efflux, appeared to induce activation-related changes in rat peritoneal macrophages (PM) at 10(-5)-10(-7) M concentrations as revealed by aggregation, nitro blue tetrazolium (NBT) reduction, superoxide anion production as well as beta glucuronidase release; however, lactate dehydrogenase (LDH) activity was measured in supernatants only after treatment with 10(-5) M of A 23187. The PM-activating effect of A 23187 was abolished in the presence of 5 mM EGTA, whereas the activating effect of K ionophore did not depend on the extracellular Ca2+. Fc receptor (FcR) mediated incorporation, intracellular killing as well as the antibody dependent cytotoxicity (ADCC) were markedly inhibited upon incubation with Ca ionophore or valinomycin. C3bR mediated phagocytosis was found to be more resistant to the cation transport affecting drugs. The impairment of effector functions was elicited by nontoxic concentrations of ionophores, however, their inhibiting effects did not require extracellular Ca2+ at the time of ionophore-exposition.

Animals↗

PubMed1976

[Permeability of yeast mitochondrial internal membrane: structure-activity relationship].

In order to investigate the possible relations between the anionic permeability and the functions (or the structure ) of the inner mitochondrial membrane, three types of organelles isolated from S. cerevisiae were tested: mitochondria (aerobic culture), promitochondria (anaerobic culture) and CAP-mitochondria (aerobic culture with chloramphenicol added). By using the technique of swelling in isoosmotic potassium salts, after a derermination of the isotonic conditions, it was possible to discriminate between an electrogenic (valinomycin induced) or an electroneutral (both valinomycin and uncoupler induced) translocation. 1) Mitochondria: The permeability properties of mitochondria are energy dependent: a) Respiring mitochondria are permeable to Cl-; Mg2+, however, inhibits this translocation. Phosphate transport seems to be exclusively electrogenic and mersalyl sensitive, but swelling inhibition by that thiol reagent is restored by Mg2+. b) Non respiring mitochondria are impermeable to Cl-, but ATP addition restores the permeability. Thiocyanate permeates as the anionic form and acetate as the undissociated form. The phosphate transport, sensitive to mersalyl, seems to be partially electrogenic. 2) Promitochondria: Deficient of respiratory enzymes but containing an oligomycin sensitive ATPase, they are impermeable to Cl- only when Mg2+ is added. In these conditions, an electrogenic phosphate transport, sensitive to mersalyl, is observed. 3) CAP-mitochondria: Although CAP-mitochondria are cytochrome deficient and contain an oligomycin insensitive ATPase, they are also impermeable to Cl- in presence of Mg2+. As in fully differenciated mitochondria, an electroneutral phosphate entry is observed; Mg2+ is required for mersalyl sensitivity.

Adenosine Triphosphatases↗

PubMedDate not supplied

Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein.

The steroidogenic acute regulatory (StAR) protein, which mediates cholesterol delivery to the inner mitochondrial membrane and the P450scc enzyme, has been shown to require a mitochondrial electrochemical gradient for its activity in vitro. To characterize the role of this gradient in cholesterol transfer, investigations were conducted in whole cells, utilizing the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) and the potassium ionophore valinomycin. These reagents, respectively, dissipate the mitochondrial electrochemical gradient and inner mitochondrial membrane potential. Both MA-10 Leydig tumor cell steroidogenesis and mitochondrial import of StAR were inhibited by m-CCCP or valinomycin at concentrations which had only minimal effects on P450scc activity. m-CCCP also inhibited import and processing of both StAR and the truncated StAR mutants, N-19 and C-28, in transfected COS-1 cells. Steroidogenesis induced by StAR and N-47, an active N-terminally truncated StAR mutant, was reduced in transfected COS-1 cells when treated with m-CCCP. This study shows that StAR action requires a membrane potential, which may reflect a functional requirement for import of StAR into the mitochondria, or more likely, an unidentified factor which is sensitive to ionophore treatment. Furthermore, the ability of N-47 to stimulate steroidogenesis in nonsteroidogenic HepG2 liver tumor cells, suggests that the mechanism by which StAR acts may be common to many cell types.

Animals↗

PubMed2002

Potassium-selective atomic force microscopy on ion-releasing substrates and living cells.

A new method for simultaneous mapping of cell topography and ion fluxes was developed. A highly sensitive ion sensor system was generated by coating atomic force microscopy tips with a PVC layer containing valinomycin, an ionophore for potassium. The activity of specific ions was traced on artificial ion-releasing PVC substrates. A boundary potential was generated owing to the selective exchange of a specific ion between coated tip and ion-releasing substrate. The boundary potential was detectable as a force induced by ion-selective electrostatic interactions. The selectivity coefficient of valinomycin for potassium against sodium (K(K,Na)f) was -2.5 +/- 0.5. Potassium efflux was measured on living MDCK-F1 cells expressing BK(Ca) channels. We could demonstrate localized areas of high potassium concentrations at the cell surface. The potassium efflux could be reversibly inhibited by thapsigargin, which is known to inhibit the efflux of potassium from BK(Ca) channels by suppression of calcium ATPase.

Biosensing Techniques↗

PubMed1982

Proline transport by synaptosomal membrane vesicles isolated from rat brain: energetics and inhibition by free fatty acids.

Synaptosomal membrane vesicles have been employed to study the energetics of proline transport and the inhibition of proline transport by unsaturated free fatty acids. Active uptake of proline into synaptosomal membrane vesicles requires extravesicular Na+ and is primarily driven by a Na+ gradient created by diluting K+-loaded vesicles into Na+-containing buffers. Uptake of proline under these conditions is enhanced up to 2-fold by a valinomycin-induced diffusion potential (interior negative). Proline transport is reduced in the absence of external C1- or internal K+. Strong (40-90%) inhibition of proline uptake occurs upon collapse of the Na+ gradient by ionophores such as gramicidin D or activation of the action potential Na+ channel by veratridine or Tityus serrulatus venom. Less (15-25%) inhibition is obtained with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, which also prevents the stimulation of proline uptake by the valinomycin-induced diffusion potential. Unsaturated free fatty acids inhibit proline uptake. The inhibition is greatest for arachidonic acid and was somewhat less for oleic acid. The saturated fatty acids palmitic and stearic have little or no inhibitory capacity. Endogenous unsaturated free fatty acids may exert similar inhibitory effects on the reuptake systems for neuroactive amino acids and thus modulate their action in the central nervous system.

Animals↗

PubMed1986

Transmembrane electrical potential affects the lipid composition of Acholeplasma laidlawii.

In membranes of Acholeplasma laidlawii, lipid composition is regulated as a function of several stimuli affecting the volume and length of the hydrocarbon chains and the hydrocarbon-water interfacial area. This regulation is vizualized as changes in the relative amounts of the major polar lipids monoglucosyl diglyceride and diglucosyl diglyceride. These lipids form reversed hexagonal and lamellar phases with water, respectively. However, mixtures of the two lipids, in the molar proportions found in the A. laidlawii membrane, form a lamellar phase. By adjustment of the glycolipid ratio as a response to environmental stimuli, a certain stability of the lamellar membrane is maintained. In growing cells with oleoyl membrane lipids, a transmembrane electrical potential of approximately -50 mV (inside negative), but no transmembrane pH difference, was found. Addition of the K+ ionophore valinomycin caused a rapid and dose-dependent hyperpolarization remaining for at least 7 h. Simultaneously, a rapid and lasting metabolic decrease in the ratio monoglucosyl diglyceride/diglucosyl diglyceride occurred. The increase in potential and the decrease in the lipid ratio were both reversed in a dose-dependent manner by extracellular KCl. Likewise, the lipophilic cation tetraphenylphosphonium caused a dose-dependent decrease in membrane potential and an increase in the monoglucosyl diglyceride/diglucosyl diglyceride ratio, respectively. The ionophores monensin and particularly nigericin had similar but less pronounced effects on the potential and lipid ratios as valinomycin. The uncoupler carbonyl cyanide m-chlorophenylhydrazone had no effect on cell growth, membrane potential, or lipid regulation at 10 microM. These dissimilar structures and the low concentrations used make a direct disturbance of drug molecules on lipid packing in membranes less likely.(ABSTRACT TRUNCATED AT 250 WORDS)

Acholeplasma laidlawii↗

PubMed1975

Energy dependent hydrogen ion accumulation in submitochondrial particles.

The fluorescence quenching of 9-aminoacridine (9AA) in suspension of beef heart EDTA submitochondrial particles was studied and was used to calculate the pH gradient between these particles and the medium. This pH gradient, which is energy dependent, is also dependent strongly on the presence of anion species in the medium. It is 2.2 pH units in acetate medium and can be as high as 3.6 units in the presence of other highly lyophilic anions. The anions tested were found to be effective in the following order: SCN- greater than I- greater than NO3- greater than Br- greater than Cl-. The validity of the deltapH calculations was confirmed by comparison with deltapH values calculated from NH4+ uptake. In contrast, calculations based on quinacrine (QA) fluorescence quenching under the same assumption used for 9AA did not agree with NH4+ measurements and show quantitative and in some cases even qualitative differences. Both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NH4+ decreased deltapH significantly. When the rate of electron transport is slow, i.e., with succinate as substrate or with NADH and low concentration of rotenone, very low concentration of nigericin (less than 20 ng/ml) decreased deltapH. Under these conditions, valinomycin antagonized the nigericin effect and restored deltapH to its original value. Upon increasing nigericin concentration (greater than 100 ng/ml) the valinomycin effect is gradually replaced by a slower response of further reduction of deltapH.

Acetates↗