Influx Of Anions Research Articles

Acidification and Ion Permeabilities of Highly Purified Rat Liver Endosomes

While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport. Acidification was monitored using hepatocyte and total liver endosomes selectively labeled with pH-sensitive markers of receptor-mediated endocytosis (fluorescein isothiocyanate asialoorosomucoid) or fluid-phase endocytosis (fluorescein isothiocyanate-dextran). In addition, changes in membrane potential accompanying ATP-dependent acidification were directly measured using the voltage-sensitive fluorescent dye Di-S-C3(5). Our results indicate that ATP-dependent acidification of liver endosomes is electrogenic, with proton transport being accompanied by the generation of an interior-positive membrane potential opposing further acidification. The membrane potential can be dissipated by the influx of permeant external anions or efflux of internal alkali cations. Replacement externally of permeable anions with less permeable anions (e.g. replacing Cl- with gluconate) diminished acidification, as did replacement internally of a more permeant cation K+ with less permeant species (such as Na+ or tetramethylammonium). ATP-dependent H+ transport was not coupled to any specific anion or cation, however. The endosomal membrane was found to be extremely permeable to protons, with protons able to leak out almost as fast as they are pumped in. Thus, the internal pH of endosomes is likely to reflect a dynamic equilibrium of protons regulated by the intrinsic ion permeabilities of the endosomal membrane, in addition to the activity of an ATP-driven proton pump.

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro. To investigate the possible basis for pH regulation in endosomes, we have studied the transport properties and ion permeabilities of early and late endosomes isolated from Chinese hamster ovary cells. Using endosomes selectively labeled with pH-sensitive endocytic tracers, we found that ATP-dependent acidification is electrogenic, being accompanied by the generation of an interior-positive membrane potential which opposes further acidification. While membrane potential and, consequently, acidification was controlled by the influx of permeant anions and efflux of protons and alkali cations, acidification was further modulated in Na+ and K+-containing buffers by the ouabain- and vanadate-sensitive Na+,K+-ATPase, which appears to be a functional component of the endosomal membrane. The data suggest that electrogenic Na+ transport due to Na+,K+-ATPase activity contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport. Importantly, inhibition of acidification by Na+,K+-ATPase activity was found only in early endosomes, consistent with their limited acidification capacity relative to late endosomes and lysosomes.

Insulin release has no absolute requirement for extracellular monovalent ions

Current chemiosmotic models of exocytosis ascribe an essential role to the influx of extracellular monovalent anions or cations into the secretory granules apposed to the plasma membrane. These hypotheses were tested by measuring insulin release in sucrose media devoid of monovalent ions. A small response to glucose (25% of controls) was still observed, which could be potentiated by isobutylmethylxanthine and suppressed by cobalt or low temperature. Substitution of Ba 2+ for Ca 2+ triggered a practically normal release of insulin that was inhibited by blockers of Ca 2+ channels (cobalt or D 600) and abolished by low temperature. These results show that insulin release remains possible in the absence of extracellular monovalent ions and, therefore, that the chemiosmotic models of exocytosis do not entirely apply to insulin release.

Mechanism for electrosilent Ca 2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.

Mode of transport and possible mechanism of action of l-phenylalanine benzyl ester as an anti-sickling agent

l-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5–3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM l-phenylalanine for 30 min at 37°C results in accumulation of 2.0 mmol l-phenyalanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol l-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both l-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for l-phenylalanine, uridine and sulphate ions, as well as the Na + pump and the Na +/K + cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37°C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.

Ionic mechanism of water receptors in the laryngeal mucosa of the rabbit.

The effects of various ions on water response was studied by recording unitary discharges in single superior laryngeal nerve fibers of the rabbit. The water response was strongly influenced by anions. The inorganic anions used were divided into two groups. One group consisted of anions which depress the water response (depressing anions), the depressing action sequence being SCN- greater than Br- greater than than Cl- greater than I- greater than NO-3. Another one consisted of anions which facilitate the water response (facilitating anions), the facilitating action sequence being citrate greater than SO34- greater than IO-3 greater than F- greater than BrO-3 greater than acetate. Physicochemically, facilitating anions belong to "the water structure-making ions" group; on the other hand, depressing anions belong to "the water structure-breaking ions" group. SO34- weakened the depressing effect of Cl- when the concentration of Cl- was below 60 mM, and enhanced it when the concentration was above 60 mM. Na+ and Li+ had no appreciable effect on the water unit activity, but K+ had a weakly stimulating effect. It is concluded that an outflux of anions through the membrane stimulates the water receptor while an influx of anions depresses it, and SO34- facilitates Cl- permeability. The mechanism of the facilitating effect of SO24- on Cl- permeation is discussed in terms of the interaction between the water structure surrounding the ions and the receptor membrane.

Anion influx, ATP level and CO2 fixation in Limnophila gratioloides and Chara foetida

Influx of anions (5x10−4 M Cl− or SO42−) across the plasmalemma, ATP levels and CO2 fixation in Limnophila and Chara have been measured in a comparative study.

Ion transport and energy conservation in submitochondrial particles

The combination of valinomycin and nigericin in the presence of K(+) uncouples submitochondrial particles (SMP) as evidenced by: 1) loss and release of the oligomycin-induced respiratory control; 2) inhibition of the P/0 ratio; 3) inhibition of three energy-linked reactions - pyridine-nucleotide transhydrogenation, reversal of electron-transfer, and bromthymol blue and 8-anilino-1-naphtalenesulfonate responses; and 4) change of redox state of cytochromes to the same extent obtained with conventional uncouplers. Neither antibiotic alone, in the presence of K(+), markedly affected the energized state of the system. Direct measurements of K(+) and H(+) movements showed that SMP did indeed translocate these ions in a predictable manner, i. e., a nigericin-stimulated influx of K(+) to SMP, followed by a valinomycin-mediated efflux of the K(+) taken up. The NH 4 (+) -dependent uncoupling is demonstrated to be associated with the uptake of NH 4 (+) by SMP with a consequent collapse of the pH gradient established during respiration, followed by a valinomycin-mediated efflux of the NH 4 (+) taken up. The effects of cations and antibiotics can be mimicked by suitable combinations of cations and anions, suggesting that the valinomycin-mediated efflux of cations from SMP is electrophoretic in nature and can be replaced by an electrophoretic influx of appropriate anions. Analogies are drawn with observations reported on bacterial chromatophores and chloroplasts, and a general scheme is suggested. The implications of these results are discussed in terms of the current hypotheses of energy coupling in oxidative and photosynthetic phosphorylation.

Mechanism of cis- and trans-stimulation of anion fluxes across the plasma membrane of plant cells

Trans-stimulation by salts of the passive efflux of Cl⊖ across the plasma membrane of plant cells was established previously. In this paper the trans-effect of salts is compared with the effect of nystatin on ion efflux. It is further shown that the influx of anions is also stimulated by external salts. Influx of Cl⊖ was stimulated by K2SO4 (>~1 mM), influx of SO4 2⊖ was stimulated by KCl (>~lmM). This suggests that with increasing external salt concentration not only the electrical potential across the plasmalemma is lowered (due to preferential permeability to monovalent cations) but alsoth e permeability (i. e. the permeability coefficient) of the plasmalemma to anions is increased. According to the model proposed for the salt-stimulated decrease in the resistance to passive anion permeation the plasmalemma may be considered a lipid lattice-electrofilter. The nature of the coupling of the counter fluxes is discussed.

Ion Transport in Liver Mitochondria: IV. THE RELATIONSHIP BETWEEN ION TRANSLOCATION AND ELECTRON TRANSPORT

Abstract Addition of small amounts of Ca++ to liver mitochondria incubated in the absence of phosphate or acetate results in an inhibition of respiration. The inhibition of respiration is reversed by increasing the concentration of substrate anions in the medium, and is enhanced by increasing the ionic strength or the pH of the medium. The H+:O and Ca++:O ratios are increased severalfold when measured under conditions of inhibited respiration. Furthermore, mitochondria which have entered a state of inhibited respiration continue to take up Ca++ and to release H+. It is concluded that the above stoichiometries in the absence of phosphate or acetate are determined by both uninhibited and inhibited respirations. The higher the concentration of Ca++, the larger is the contribution of the inhibited respiration. In the presence of acetate, the Ca++ to total oxygen ratio is in the region of 2.0 to 2.5 with succinate as substrate. The inhibition of respiration is related to the increase of the intramitochondrial pH as followed kinetically with the bromthymol blue technique. The bromthymol blue response is dependent on the rate of influx of anions. Increasing the ionic strength and pH enhances the bromthymol blue response, presumably by restraining the influx of anions. The mechanism by which the inhibition of respiration causes the superstoichiometric ratios is discussed.

Effect of pH change upon influx of anions and cations into mitochondria.

SummaryChange in the pH of the incubation medium has been shown to have opposite effects upon influx rates of cations and of anions into mitochondrial substance. Alkalinization was associated with increase in influx of Na+ and K+; and conversely, acidification resulted in more rapid influx of Br- and Cl-. Increased sensitivity to pH change was observed in the presence of the buffer ion imidazole. At pH 7 imidazole carries a positive charge and inhibition to influx of Na+ was observed. The inhibition was absent at pH 8.5 with conversion to the uncharged free base.