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PubMed1989

Membrane potential dependence of Fe(III) uptake by mouse duodenum.

Intestinal iron uptake by mouse duodenal fragments is inhibited in the absence of oxygen and glucose from the incubation medium and by a variety of metabolic inhibitors. The mechanism of energy coupling to iron uptake is, however, unclear. In vitro experiments using duodenal fragments showed Fe3+ uptake to be markedly inhibited, in a reversible fashion, by the replacement of incubation medium Na+ by K+. Addition of phloridzin to the medium failed to affect iron uptake, suggesting that the above effect was not a consequence of reduced glucose uptake. Substitution of Na+ by Rb+ also potently reduced duodenal iron uptake. Replacement of medium NaCl by either mannitol or choline chloride had no significant effect on Fe3+ uptake, thus excluding the possibility of the Fe3+ uptake process being Na+-dependent. Similar observations were made with duodenal fragments from animals with enhanced Fe3+ absorption, due to chronic hypoxia. Valinomycin (1-5 microM) increased the uptake of both glucose and Fe3+. Higher concentrations (22.5 microM) of the ionophore were inhibitory. In vivo studies (tied-off segments) using Rb+-containing medium confirmed the inhibitory effects of univalent cations on Fe3+ absorption. Enhanced absorption of Fe3+ was also demonstrable in vivo, with low concentrations of valinomycin and nigericin added to the luminal medium. These observations suggest that the Fe3+ uptake process may be dependent on the brush-border membrane potential.

Animals↗
PubMed1993

The transport mechanisms of organic cations and their zwitterionic derivatives across rat intestinal brush-border membrane. II. Comparison of the membrane potential effect on the uptake by membrane vesicles.

Further investigation of organic cation transport mechanisms were continued using rat intestinal brush-border membranes following our previous report. The net uptake of organic cations was superior to that of their zwitterionic derivatives. This result agreed with the absorption behaviour of these compounds from rat intestinal loop. The uptake of tyramine and 5-benzyloxytryptamine was significantly stimulated by the valinomycin-generated K(+)-diffusion potential (inside-negative). On the other hand, the uptake of zwitterionic derivatives was not affected by the valinomycin-induced K(+)-diffusion potential. The voltage-clamped brush-border membrane vesicles exhibited a complete disappearance of the overshoot-uptake of organic cations. Therefore, this permeation mechanism across the intestinal brush-border membrane seems to be different from the well-known H(+)-antiport system of organic cation found in other organs such as kidney and liver, and depends upon an inside-negative H(+)- or K(+)-diffusion potential.

Animals↗
PubMed1993

Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes.

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.

Arylsulfonates↗
PubMed1981

Effect of ionophores and metabolic inhibitors on protein synthesis in rabbit reticulocytes.

The effect of some ionophores and metabolic inhibitors on reticulocytes protein synthesis was examined. At microM concentrations, valinomycin, nigericin and CCCP rapidly inhibit protein synthesis, while with antimycin-A or DCCD the inhibition is rather slow. The onset of the arrest of protein synthesis coincides with a 20--30% drop in the intracellular ATP content. No inhibition in protein synthesis or drop in ATP was found after 1 h incubation without glucose or in the presence of 2-deoxyglucose. It is shown that the inhibition by valinomycin, nigericin or CCCP is not due to their effect on K+ and/or H+ fluxes through the plasma membrane. Reticulocytes incubated at pH 8.2 show much lower inhibition of protein synthesis by nigericin, CCCP, DCCD or antimycin-A. On the other hand, at this alkaline pH, starvation to glucose causes high inhibition of protein synthesis. It is concluded that the ionophores inhibit protein synthesis due to their uncoupling effect on mitochondrial ATP synthesis. At high pH, the glycolytic activity is relatively high, and the ATP generated by the glycolysis can compensate to some degree for the ATP loss in the oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1983

Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation.

The reverse phase evaporation procedure was used to prepare large unilamellar liposomes containing bacteriorhodopsin. Electron microscopy showed that proteoliposomes were unilamellar and fairly uniform in size provided the preparation was extruded through calibrated nucleopore membranes : the vesicles have diameters around 200 nm. The spectral properties of the bacteriorhodopsin in the large liposomes resembled those of bacteriorhodopsin in purple membrane. Furthermore, the chromoprotein in the reconstituted vesicles had an inside-out orientation and on illumination, translocated protons efficiently from the external medium into the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, a light-independent transmembrane potential of about 60 mV was measured from thiocyanate distribution. In the presence of valinomycin, this transmembrane electrical potential was abolished and then a light-dependent transmembrane pH gradient of about 2 pH units could be generated.

Bacteriorhodopsins↗
PubMed1992

Ionophore-induced Cl- transport in human erythrocyte suspensions: a multinuclear magnetic resonance study.

To investigate the effect of ionophores on Cl- distribution in human erythrocyte suspensions, we measured the membrane potential by using 19F and 31P NMR methods. Incubation of human erythrocytes with 0.005 mM of the neutral ionophores valinomycin and nonactin resulted in membrane potentials of -21.2 and -17.8 mV in the presence and absence of DIDS. However, 0.020 mM of the carboxylic ionophores lasalocid, monensin, and nigericin yielded membrane potentials similar to those measured in the absence of ionophore (-9.4 mV). In methanol, the 35Cl- NMR linewidth in the presence of valinomycin was twice as broad as those observed in the presence of carboxylic ionophores, suggesting that neutral ionophores induce Cl- efflux in part via ion pairing.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Evidence that a ryanodine receptor triggers signal transduction in the osteoclast.

We have investigated the effect of the alkaloid ryanodine on the release of intracellularly stored Ca2+ in response to activation of the osteoclast Ca2+ receptor by the surrogate agonist, Ni2+, Ni2+ (6 mM) in the presence of ethylene-glycol bis-(aminoethyl ether) tetraacetic acid (EGTA) (1.2 mM) and valinomycin (5 microM) induced a transient elevation of cytosolic [Ca2+] in fura 2-loaded osteoclasts. This transient was superimposed upon a small steady elevation of cytosolic [Ca2+] induced by the initial application of valinomycin alone. Ryanodine (10 microM) completely abolished such responsiveness. However, cytosolic [Ca2+] transients were restored when osteoclasts were depolarized by the extracellular inclusion of 100 mM-[K+] in the same solution. Thus, we demonstrate a sensitivity of the osteoclast signal transduction system to ryanodine for the first time to our knowledge.

Animals↗
PubMed1989

Role of ions and membrane potential in uptake of serotonin into plasma membrane vesicles from mouse brain.

Plasma membrane vesicle preparations from mouse cerebral cortex actively accumulated [3H]serotonin upon the imposition of a K+ gradient (in greater than out), a Na+ gradient (out greater than in), and the presence of external Cl-. Maximal stimulation of uptake by internal K+ occurred at 15 mM and half-maximal stimulation at 2 mM. Internal K+ did not enhance uptake merely via generation of a membrane potential because simultaneous parallel increases in internal and external K+ concentration also stimulated uptake. External Cl- increased serotonin uptake with a Km of 18 mM and a Hill number of 1.0, suggesting a requirement for one chloride ion for transport. Uptake could not be driven by internal H+ instead of K+. Estimation of the membrane potential by the distribution of triphenylmethylphosphonium ion showed a modest effect of valinomycin (1-20 microM) in increasing the potential from -19 to -31 mV accompanied by an increase in serotonin uptake. Proton ionophores prevented this effect of valinomycin and, by themselves, reduced the potential to -6 mV, but did not affect serotonin transport. A model is proposed for serotonin transport in brain plasma membrane vesicles that is similar to the model for porcine blood platelet vesicles as far as electroneutrality and stimulation by K+, Na+, and Cl- are concerned, but that is different in substitution of internal H+ for K+.

Animals↗
PubMed1993

Differential effects of metabolic inhibitors on cellular and mitochondrial uptake of organic cations in rat liver.

The effects of several metabolic inhibitors on the uptake of tri-n-butylmethylammonium (TBuMA) were studied in isolated rat liver mitochondria, isolated rat hepatocytes and isolated perfused rat livers, in order to characterize further the mechanisms for carrier-mediated uptake and cellular accumulation of organic cations in the liver. Treatment of isolated hepatocytes with valinomycin, carbonylcyanide-m-chlorophenyl-hydrazone (CCCP), dinitrophenol, oligomycin or antimycin resulted in a rapid decrease in cellular ATP within 3 min of addition. The initial uptake rate of TBuMA was generally largely affected by these treatments. However, fructose at 10 mM had no effect at all on the uptake rate of the cation whereas cellular ATP was decreased to an extent comparable to that after treatment with the metabolic inhibitors. Consequently it was hypothesized that the metabolic inhibitors affected the initial cellular uptake rate of organic cations due to either altered intracellular sequestration (e.g. mitochondria) or alternatively to direct effects on the plasma membrane rather than by decreasing cellular ATP. Isolated rat mitochondria were shown to take up organic cations very efficiently. Accumulation in this organelle is probably driven by the negative membrane potential as measured by the uptake of the lipophilic cation [3H]tetraphenylphosphonium. Treatment of the isolated mitochondria with various metabolic inhibitors decreased the membrane potential in parallel to the effects on the uptake of TBuMA. Since mitochondria constitute a considerable intracellular volume, they may contribute largely to the storage of the organic cation in the hepatocyte. In isolated perfused livers, preloaded with either TBuMA or tetraphenylphosphonium (TPP+), the addition of valinomycin or CCCP leads to a marked backflux of the cations from the liver into the perfusion medium. This suggests strongly that a large part of the intracellular storage capacity is lost after metabolic inhibitor treatment, probably as the consequence of dissipation of the mitochondrial membrane potential. Since the metabolic inhibitors in contrast to TBuMA uptake did not decrease the initial uptake rate of TPP+ into isolated hepatocytes, it was concluded that mitochondrial uptake (mitochondria are the major storage sites for TPP+) is not an essential determinant of the initial uptake rate in intact hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1984

Interference by DDT and cyclodiene types of insecticides with chloroplast-associated reactions.

The effects of DDT, some of its analogs, and selected cyclodiene insecticides on isolated spinach (Spinacea oleracea L.) thylakoids were identified, characterized, and compared to responses induced by selected herbicides. Except for endrin, the insecticides inhibited light-induced electron transport, altered chlorophyll fluorescence transients, and competitively displaced [14C]atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], a known photosystem II inhibitor, from the membranes. The insecticides appeared to act at, or near B, the secondary electron acceptor of photo-system II. Binding of DDT and dieldrin was estimated at 900 and 2200 molecules, respectively, per photosynthetic unit (490 chlorophyll molecules). The insecticides also inhibited valinomycin-induced swelling of the thylakoid membrane. Whereas inhibition of electron transport can be attributed to interaction by the insecticides with a proteinaceous component of the thylakoid membrane, interference with the action of valinomycin may involve interaction with lipoidal constituents of the membrane.

Binding, Competitive↗
PubMed1989

Interactive effects of Na and K in killing by human natural killer cells.

Contact-mediated lysis by human natural killer cells is inhibited by a number of drugs that block the predominant K channel. In this study we have further examined the role of the K channel and the interactions between passive K and Na transport in killing. Low external Na-inhibited killing and inhibition were not due to reduce inward current through the Na channels in the target cell. A role for the Na/H antiport is suggested since amiloride inhibited killing in a dose-dependent manner that was competitive with external Na. Depolarizing the killer cell with elevated external K did not inhibit killing. On the contrary, high K0 reduced the inhibition caused by low Na0 and by the K-channel blockers quinidine, verapamil, and retinoic acid. Hyperpolarizing the killer cell with low K0 or valinomycin inhibited killing. Valinomycin, which should prevent the depolarization caused by K-channel block, did not reverse the effect of the blockers quinidine, verapamil, and 4-aminopyridine. Hence, the primary role of the K channels during killing is not maintain the negative membrane potential. On the contrary, depolarization may promote killing under conditions where killing is submaximal.

Amiloride↗
PubMed1991

Ionophore-induced apoptosis: role of DNA fragmentation and calcium fluxes.

Two ionophores specific for K+, valinomycin and beauvericin, induce a type of cell death very similar to apoptosis due to tumor necrosis factor (TNF alpha). Both ionophores cause cytolysis accompanied by internucleosomal DNA fragmentation of the dying cell into units of 200 base pairs. Morphologically, the cell death appears to consist of a mixture of nuclear apoptotic changes and cytoplasmic necrotic changes. As in the case for TNF alpha-mediated death, metabolic inhibitors have no effect on the course of cell death, but DNA fragmentation and cytolysis are decreased by the endonuclease inhibitor, zinc. Beauvericin and valinomycin trigger an increase in the cytoplasmic calcium concentration, most likely due to release of calcium from intracellular stores, and chelation of cytoplasmic calcium with quin-2 inhibits DNA fragmentation. Thus, these ionophores set off apoptosis through a calcium-activatable endonuclease, suggesting that other nonphysiological toxins might also cause apoptosis through their ability to indirectly elevate the cytoplasmic calcium concentration, without the need to invoke specific surface receptors.

Animals↗
PubMed1992

Ca2+/Na+ exchanger and Na+,K+ 2Cl- cotransporter in lens fiber plasma membrane vesicles.

When the Ca(2+)-sensitive fluorescent probe, Fura-2, or the Na(+)-sensitive probe, SBFI, in their cell permeable forms or the Cl(-)-specific probe, SPQ, were incubated with plasma membrane vesicles prepared from dogfish and bovine lenses fibers, there was a selective accumulation of the ion-specific probes within the vesicles. The SBFI and Fura-2 fluorescent excitation ratios of 340 nm to 380 nm (em: 505 nm) in the presence of an outwardly-directed Na+ gradient across the vesicles membrane, indicate that the influx of Ca2+ is increased by 152.5% and 147.4% for dogfish and bovine vesicles, respectively. The Na+ influx into the vesicles is also enhanced by 154.1% for dogfish and 149.1% for bovine lens when an outwardly-directed Ca2+ was present. This stimulation is not affected when either 50 microM valinomycin, or 50 mM K+ is present. The activity of this bidirectional Ca2+/Na+ exchanger could be inhibited by 100 microM bepridil or 200 microM La3+. The entrance behaviour of Cl- as monitored by the SPQ fluorescent signal indicates that, the Cl- influx is Na(+)-dependent. The Cl- influx is stimulated 152.8% and 187.6% for dogfish and bovine lens, respectively, when an inwardly-directed Na+ gradient is present, and is further enhanced when a K+ gradient is also present. The stoichiometry of Na+ to Cl- entering the vesicles was 1:2. This Na+,K+ 2Cl- cotransporter is not affected by 20 microM valinomycin or 50 mM K+. However, the transporter is completely inhibited by 50 microM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Compartmentation of inorganic phosphate in perfused rat liver. Can cytosol be distinguished from mitochondria by 31P NMR?

Compartmentation of inorganic phosphate was studied in intact perfused rat liver at 4 degrees C by 31P NMR. It was shown that decreases in cytosolic pH or cytosolic Pi concentration induced the appearance of an additional Pi resonance at low field which was assigned to Pi from an alkaline compartment. Valinomycin (K+ ionophore) induced a further splitting of the lines whereas nigericin (K+/H+ antiport) or potassium cyanide (inhibitor of cytochrome oxidase) had opposite effects. As valinomycin acts mainly on the cytosolic/mitochondrial K+ gradient and KCN on the mitochondrial respiratory chain, it was deduced that the alkaline compartment as revealed by the second Pi resonance was probably mitochondria. Single Pi lines observed on perchloric extracts of livers exhibiting two resonances during cold perfusion confirmed that the split peaks in the intact liver indeed arose from the same molecular species.

Animals↗
PubMed1987

Ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus: the effect of metals and metabolic inhibitors.

Ferrous iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out in a well-defined salt solution termed "modified Hanks solution" at both high iron concentrations (LAFIUS conditions) and low concentrations (HAFIUS conditions). Various divalent metals, Mn2+, Zn2+, Ni2+ and Cu2+, inhibited iron uptake under HAFIUS conditions in a non-competitive manner, and in a pseudo-competitive manner under LAFIUS conditions. Cr2+ had no effect. Co2+ inhibited iron uptake competitively under HAFIUS conditions. Metabolic affectors that inhibited iron uptake both under HAFIUS and LAFIUS conditions were: tetraphenylphosphonium chloride, diethylstilbesterol, vanadate, carbonylcyanide-m-chlorophenyl-hydrazone, and a mixture of valinomycin and nigericin. Substances that stimulated iron uptake were KCl, valinomycin, and nigericin. Iron uptake under LAFIUS conditions in piperazine-buffered modified Hanks solution was higher than that in the acetate-buffered solution, and acetate inhibited iron uptake in the piperazine buffer. HAFIUS showed no difference. It is concluded that iron uptake in bifidobacteria is driven by an ATPase-dependent proton-motive force and that both the pH gradient and membrane potential are involved in this process. Mn2+, Zn2+, Ni2+, and Cu2+ may be transported via LAFIUS, but not HAFIUS. HAFIUS may transport only Co2+ in addition to Fe2+.

Bifidobacterium↗
PubMed1983

Effects of chlordecone and its alteration products on isolated rat liver mitochondria.

Partitioning of chlordecone, chlordecone alcohol, monohydrochlordecone, and dihydrochlordecone (2 to 100 microM) into isolated rat liver mitochondria altered the permeability properties of the inner membrane as evidenced by: inhibition of valinomycin-induced swelling, induction of passive swelling, oxidation of exogenous NADH, and induction of lysis. Associated with the increase in permeability were stimulation of state 4 and inhibition of state 3 respiration for the oxidation of both succinate and glutamate. Except for the inhibition of valinomycin-induced swelling, the following order of potency for all assays was obtained: chlordecone alcohol greater than or equal to chlordecone greater than monohydrochlordecone much greater than dihydrochlordecone. Mirex, a 12-chlorine analog of chlordecone, and a dioxolane-chlordecone adduct did not affect any of the reactions at saturating concentrations of 40 and 100 microM, respectively. The hydrated ketone, or a hydroxyl moiety, and a high chlorine content appear to be responsible for lytic and inhibitory activity. Lysis resulted in the leakage of water-soluble matrix enzymes from the mitochondria, but not in the solubilization of integral proteins.

Animals↗
PubMed1987

Interactions of mitochondrial inhibitors with methylmercury on spontaneous quantal release of acetylcholine.

The interaction of methylmercury (MeHg) with various inhibitors of mitochondrial function (dinitrophenol, 50 microM; dicoumarol, 100 microM; valinomycin, 20 microM; and ruthenium red, 20 microM) on spontaneous quantal release of acetylcholine was tested at the neuromuscular junction of the rat. The objective was to determine whether these mitochondrial inhibitors blocked the MeHg-induced increase of spontaneous release of acetylcholine, an effect measured electrophysiologically as increased miniature endplate potential (MEPP) frequency. MEPPs were recorded from myofibers of the rat hemidiaphragm using conventional, intracellular microelectrode recording techniques. When given alone, all four inhibitors increased MEPP frequency from resting levels of 1-2/sec (Hz) to approximately 10-60 Hz after a latency which ranged from 5 to 30 min. MEPP frequency subsequently returned to control levels. Subsequent concomitant application of MeHg (100 microM) with dinitrophenol, dicoumarol, or valinomycin increased MEPP frequency sharply to peak values of 40-60 Hz after 15-20 min. MEPP frequency subsided to pre-MeHg levels 10 min later. The time course and peak MEPP frequency elicited by MeHg after pretreatment with these uncouplers were similar to results obtained in preparations treated with MeHg alone. Ruthenium red, a putative specific inhibitor of the Ca2+ uptake uniporter in mitochondria, increased MEPP frequency to 12 Hz after 8.5 min when given alone. MEPP frequency returned to control levels approximately 10 min later. Subsequent application of MeHg and ruthenium red for up to 80 min failed to increase MEPP frequency. The inability of MeHg to increase MEPP frequency in ruthenium red-treated preparations was not due to depletion of acetylcholine nor to block of postjunctional receptors by ruthenium red since subsequent treatment with La3+ (2 mM) increased MEPP frequency to 12.5 Hz within 10 min. Thus, ruthenium red blocked the stimulatory effect of MeHg on MEPP frequency while uncouplers of oxidative phosphorylation and a K+ ionophore did not. The results with ruthenium red are consistent with the proposal that MeHg may block mitochondrial uptake of Ca2+ or promote its release, leading to an increased free cytosolic Ca2+ concentration which in turn stimulates spontaneous release of acetylcholine.

2,4-Dinitrophenol↗
PubMed1987

Measurement of sulfobromophthalein uptake in isolated rat hepatocytes by a direct spectrophotometric method.

A spectrophotometric technique is described for the continuous recording of sulfobromophthalein uptake by isolated hepatocytes. The technique is based on the principle that sulfobromophthalein behaves as a pH-indicator and may be followed photometrically when moving from the medium at pH 7.8 into the interior of the cell. Data show that upon addition of cells to a sulfobromophthalein solution, an absorbance change can be recorded. The kinetics of the process is biphasic and the initial rate is linearly related to the amount of cells added. By this technique it was confirmed that the substrate dependence of the initial velocity of transport is a compound function including a saturable portion with an apparent Km in the mu molar region. Experiments carried out either in the presence of valinomycin or of high concentrations of potassium chloride indicate that the presence of a membrane potential opposes the entry of sulfobromophthalein into isolated hepatocytes. This finding is in agreement with previous observations in isolated plasma membrane vesicles and in liposomes reconstituted with purified bilitranslocase which indicate a rheogenic type of transport for the dye. Low concentrations of nicotinate (1.6 microM) efficiently inhibit the saturable transport. It is suggested, in addition, that the sensitivity of the transport to valinomycin could be used as an early indication of the functional integrity of cell preparations.

Animals↗