Na+ and pH dependence of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes

The dependence of the high-affinity transport systems for 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) and methotrexate on sodium ions and on pH was examined in freshly isolated rat hepatocytes. Previous studies indicated that transport of these folate derivatives was sodium-dependent. Experiments to determine the Km for sodium of 5-CH3-H4PteGlu transport showed no dependence on extracellular sodium. However, uptake was sodium-dependent when hepatocytes were preincubated for 30 min in sodium-free medium, a treatment which resulted in an increase in the transmembrane pH gradient (delta pH = pH out-pH in) and a decrease in the uptake of 5-CH3-H4PteGlu. Uptake of methotrexate displayed a linear dependence on extracellular sodium ions. Uptake of 5-CH3-H4PteGlu increased linearly as the transmembrane pH gradient decreased; i.e., as the medium became more acid with respect to the cytosol. Lineweaver-Burk and Scatchard plots of 5-CH3-H4PteGlu uptake indicated an apparent Km for H+ of about 24 nM, equivalent to a pH of 7.6. Hill-plots suggested a stoichiometry of 1:1 for the interaction of protons with the 5-CH3-H4PteGlu transport system. Both the Km and Vmax for 5-CH3-H4PteGlu transport were increased at pH 5.5 compared to pH 7.4, suggesting that extracellular protons increased the number of and/or the activity of the membrane carrier. In contrast, methotrexate transport was maximal at pH 7 where the transmembrane pH gradient was zero. These results suggest the possibility that 5-CH3-H4PteGlu may be cotransported along with H+ ions in hepatocytes, although they do not rule out a 'catalytic coupling' whereby protons interact with the carrier to stimulate substrate flux without concomitant H+ transport.     

Transport of cations and anions across forestomach epithelia: conclusions from in vitro studies

Secretion of saliva as well as absorptive and secretory processes across forestomach epithelia ensures an optimal environment for microbial digestion in the forestomachs. Daily salivary secretion of sodium (Na+) exceeds the amount found in plasma by a factor of 2 to 3, while the secretion of bicarbonate (HCO3-) is 6 to 8 times higher than the amount of HCO3- in the total extracellular space. This implies a need for efficient absorptive mechanisms across forestomach epithelia to allow for an early recycling. While Na+ is absorbed from all forestomachs via Na+/H+ exchange and a non-selective cation channel that shows increased conductance at low concentrations of Mg2+, Ca2+ or H+ in the luminal microclima and at low intracellular Mg2+, HCO3- is secreted by the rumen for the buffering of ingesta but absorbed by the omasum to prevent liberation of CO2 in the abomasum. Fermentation provides short chain fatty acids and ammonia (NH3) that have to be absorbed both to meet nutrient requirements and maintain ruminal homeostasis of pH and osmolarity. The rumen is an important location for the absorption of essential minerals such as Mg2+ from the diet. Other ions can be absorbed, if delivered in sufficient amounts (Ca2+, Pi, K+, Cl- and NH4+). Although the presence of transport mechanisms for these electrolytes has been described earlier, our knowledge about their nature, regulation and crosstalk has increased greatly in the last years. New transport pathways have recently been added to our picture of epithelial transport across rumen and omasum, including an apical non-selective cation conductance, a basolateral anion conductance, an apical H+-ATPase, differently expressed anion exchangers and monocarboxylate transporters.     

Zn(2+) inhibits the anion conductance of the glutamate transporter EEAT4

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+) ions and 1 H(+) and the counter-transport of 1 K(+) ion, which ensures that extracellular glutamate concentrations are maintained in the submicromolar range. In addition to the coupled ion fluxes, glutamate transport activates an uncoupled anion conductance that does not influence the rate or direction of transport but may have the capacity to influence the excitability of the cell. Free Zn(2+) ions are often co-localized with glutamate in the central nervous system and have the capacity to modulate the dynamics of excitatory neurotransmission. In this study we demonstrate that Zn(2+) ions inhibit the uncoupled anion conductance and also reduce the affinity of L-aspartate for EAAT4. The molecular basis for this effect was investigated using site-directed mutagenesis. Two histidine residues in the extracellular loop between transmembrane domains three and four of EAAT4 appear to confer Zn(2+) inhibition of the anion conductance.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate

The permeabilities of sarcoplasmic reticulum vesicle membrane for various ions and neutral molecules were measured by following the change in light scattering intensity due to the osmotic volume change of the vesicles. 4-Acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS), which is a potent inhibitor for the anion permeability of red blood cells membrane, inhibited the permeability of sarcoplasmic reticulum for anions such as Cl-, Pi and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na+, K+, choline and glycerol. Binding of 5 mumol SITS/g protein was necessary for the inhibition of anion permeability. These results suggest the existence of a similar anion transport system in sarcoplasmic reticulum membrane as revealed in red blood cell membrane.     

Sodium Movement across Single Perfused Proximal Tubules of Rat Kidneys

Using perfusion techniques in single proximal tubule segments of rat kidney, the relationship between net sodium movement and active transport of ions, as measured by the short-circuit method, has been studied. In addition, the role of the colloid-osmotic pressure gradient in proximal transtubular fluid and sodium movement has been considered. Furthermore, the limiting concentration gradient against which sodium movement can occur and the relationship between intratubular sodium concentration and fluid transfer have been investigated. Comparison of the short-circuit current with the reabsorptive movement of sodium ions indicates that this process is largely, perhaps exclusively, active in nature. No measurable contribution of the normally existing colloid-osmotic pressure gradient to transtubular water movement was detected. On the other hand, fluid movement across the proximal tubular epithelium is dependent upon the transtubular sodium gradient and is abolished when a mean concentration difference of 50 mEq/liter is exceeded.     

Cation Dependence of Hypotaurine Uptake in Mouse Brain Slices

Abstract: Mouse brain slices take up hypotaurine (2-aminoethanesulphinic acid) from medium by means of two concentrative low- and high-affinity transport systems. [³⁵S]Hypotaurine uptake by the slices was significantly reduced in the absence of external potassium, calcium, or magnesium ions. An excess of potassium ions also inhibited hypotaurine uptake by one-half. Uptake was almost completely abolished on removal of sodium ions. The K _m constants for both low- and high-affinity transport components increased in a low-sodium medium, suggesting that sodium ions are required when hypotaurine is attached to its possible carrier sites in plasma membranes. Sodium ions also mimicked allosteric effectors of hypotaurine transport, showing positive cooperativity. More than two sodium ions may be involved in the transport of one hypotaurine molecule across the cell membrane. The calculated activation energies of transport were fairly similar in normal and sodium-deficient media and thus sodium ions may not participate in the activation mechanisms of the transport. With respect to cation dependence, hypotaurine transport in brain slices exhibits features characteristic of neurotransmitter amino acids.     

Sulfate transport in toad skin: evidence for mitochondria-rich cell pathways in common with halide ions

1. In short-circuited toad skin preparations exposed bilaterally to NaCl-Ringer's containing 1 mM SO2(-4), influx of sulfate was larger than efflux showing that the skin is capable of transporting sulfate actively in an inward direction. 2. This active transport was not abolished by substituting apical Na+ for K+. 3. Following voltage activation of the passive Cl- permeability of the mitochondria-rich (m.r.) cells sulfate flux-ratio increased to a value predicted from the Ussing flux-ratio equation for a monovalent anion. 4. In such skins, which were shown to exhibit vanishingly small leakage conductances, the variation of the rate coefficient for sulfate influx (y) was positively correlated with the rate coefficient for Cl- influx (x), y = 0.035 x - 0.0077 cm/sec (r = 0.9935, n = 15). 5. Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine to the serosal bath of short-circuited preparations resulted in a significant stimulation of the passive Cl- and SO2(-4) permeabilities. 6. It is suggested that SO2(-4) and Cl- ions are transported along the same pathway of the m.r. cells. Depending on the transport mode of the apical Cl- transport system, electro-diffusion, active transport (sulfate:bicarbonate exchange) and self-exchange diffusion take place. Irrespective of the mechanism of transport, sulfate is probably transported as a monovalent anion species.     

Effects of cytoplasmic sodium concentration on the electrogenicity of the sodium pump

Inside-out membrane vesicles were prepared from human red blood cells pretreated with diisothiocyano-2,2'-disulfonic stilbene to inhibit anion fluxes. The pH-sensitive probe fluorescein isothiocyanate-dextran was incorporated inside the vesicles. Formation of pH gradients due to proton transport by the sodium pump was distinguished from pH gradients formed in response to transmembrane electrical potentials generated by the pump by virtue of their insensitivity and sensitivity, respectively, to dissipation by lipophilic cations. Under the conditions used (pH 6.6), proton transport by the Na,K-ATPase was minimized, and the formation of pH gradients in response to electrical potentials was detected. Thus, the generation of a strophanthidin-sensitive, ATP-dependent electrical potential, inside positive (approximately 1 mV) upon addition of 4 meq of sodium to potassium-filled inside-out vesicles is consistent with the well documented stoichiometry of three sodium ions exchanging with two potassium ions. In contrast, when the cytoplasmic sodium concentration is reduced to less than or equal to 0.4 mM, the potential generated is of the opposite sign, i.e. inside negative, consistent with the decreased Na:K coupling ratio reported previously, i.e. Na:K(Rb) coupling ratios of approximating 1:2 when the sodium concentration is reduced to 0.2 mM (Blostein, R. (1983) J. Biol. Chem. 258, 12228-12232).     

Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2Cl] Co-transport

The transient increase in cation permeability observed in duck red cells incubated with norepinephrine has been shown to be a linked, bidirectional, co-transport of sodium plus potassium. This pathway, sensitive to loop diuretics such as furosemide, was found to have a [Na + K] stoichiometry of 1:1 under all conditions tested. Net sodium efflux was inhibited by increasing external potassium, and net potassium efflux was inhibited by increasing external sodium. Thus, the movement of either cation is coupled to, and can be driven by, the gradient of its co-ion. There is no evidence of trans stimulation of co- transport by either cation. The system also has a specific anion requirement satisfied only by chloride or bromide. Shifting the membrane potential by varying either external chloride (at constant internal chloride) or external potassium (at constant internal potassium in the presence of valinomycin and DIDs [4,4'-diisothiocyano- 2,2'-disulfonic acid stilbene]), has no effect on nor-epinephrine- stimulated net sodium transport. Thus, this co-transport system is unaffected by membrane potential and is therefore electrically neutral. Finally, under the latter conditions-when Em was held constant near EK and chloride was not at equilibrium-net sodium extrusion against a substantial electrochemical gradient could be produced by lowering external chloride at high internal concentrations, thereby demonstrating that the anion gradient can also drive co-transport. We conclude, therefore, that chloride participates directly in the co- transport of [Na + K + 2Cl].     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by 4-acetoamido-4′-isothiocyanostilbene-2,2′-disulfonate

The permeabilities of sarcoplasmic reticulum vesicle membrane for various ions and neutral molecules were measured by following the change in light scattering intensity due to the osmotic volume change of the vesicles. 4-Acetoamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS), which is a potent inhibitor for the anion permeability of red blood cells membrane, inhibited the permeability of sarcoplasmic reticulum for anions such as Cl^?, P_i and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na⁺, K⁺, choline and glycerol. Binding of 5μmol SITS/g protein was necessary for the inhibition of anion permeability. These results suggest the existence of a similar anion transport system in sarcoplasmic reticulum membrane as revealed in red blood cell membrane.     

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

A two-site model for sodium transport in human erythrocytes

A kinetic model has been proposed for human erythrocytes to account for the responses of sodium transport to alterations in the extracellular sodium concentration in the presence and absence of potassium. The proposed model characterizes the movement of sodium from the outer surface of the erythrocyte membrane to the inner surface in terms of a carrier which has two sites with differing affinities for sodium ions.     

Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon.

The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14. Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615. The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E. coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM. After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+. The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions. The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+. External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions. Sodium ions appear to facilitate mercury transport. We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E. coli and that the Na+ gradient may energize the transport of Hg2+.     

Ion transport across transmembrane pores

To study the pore-mediated transport of ionic species across a lipid membrane, a series of molecular dynamics simulations have been performed of a dipalmitoyl-phosphatidyl-choline bilayer containing a preformed water pore in the presence of sodium and chloride ions. It is found that the stability of the transient water pores is greatly reduced in the presence of the ions. Specifically, the binding of sodium cations at the lipid/water interface increases the pore line tension, resulting in a destabilization of the pore. However, the application of mechanical stress opposes this effect. The flux of ions through these mechanically stabilized pores has been analyzed. Simulations indicate that the transport of the ions through the pores depends strongly on the size of the water channel. In the presence of small pores (radius <1.5 nm) permeation is slow, with both sodium and chloride permeating at similar rates. In the case in which the pores are larger (radius >1.5 nm), a crossover is observed to a regime where the anion flux is greatly enhanced. Based on these observations, a mechanism for the basal membrane permeability of ions is discussed.     

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.     

Sodium, potassium, calcium, magnesium, zinc, citrate and chloride content of human prostatic and seminal fluid

The ionic composition of human prostatic fluid varied greatly between individuals, reflecting the secretory activity of the gland and the presence or absence of prostatic inflammatory disease. In normal prostatic fluid the major anion was citrate, while chloride concentrations were lower. Their counterions were mainly sodium and potassium, together with calcium, magnesium and zinc. Prostatic secretions from men with prostatitis comprised mainly sodium and chloride. The electrolytes were closely correlated to each other (except for sodium, which was essentially invariant at about 145 nm). The molar changes per mole of citrate were about 0.52, potassium; -0.53, chloride; 0.17, calcium; 0.14, magnesium; and 0.09, zinc. The pH was also associated with citrate, decreasing from 8.0 to 6.2 as the citrate increased. These various ionic changes can be explained as responses to citrate secretion, without the need to propose specific transport mechanisms for the other ions measured. The marked effect of prostatic inflammation on the composition of prostatic fluid can be seen as being due mainly to decreased secretion rather than active modification.     

Aldosterone as a factor of extracellular volume regulation: mechanism of action

The key role assumed by aldosterone in the regulation of extracellular volumes, requires that chloride moves along with sodium when active transport of the later ion is stimulated. In the mammalian nephron, aldosterone promotes reabsorption of sodium by the principal cells of the cortical portion of the collecting ducts. This results from hormone-induced increase in conductance for sodium at the apical pole of the target cells, and later on, from associated increased density of the sodium "pump" units at the basolateral pole. Studies carried out on amphibian epithelia indicate that chloride permeability - of mitochondria-rich cells, in all likelihood - is concurrently increased by aldosterone. It therefore looks as though this steroid hormone influences in a concerted way 2 cell populations, one being involved in transepithelial sodium transport, the other one representing the route of passage for the accompanying anion.     

Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS.

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.     

Effects of cholecalciferol on the translocation of calcium by non-everted ileum in vitro

An apparatus is described that allows perfusion of a non-everted segment of intestine in vitro and the study of the accumulation of substances within the mucosal cells. The translocation of Ca(2+) by rachitic-chick ileum and the effect of pretreatment with cholecalciferol was investigated, with the following conclusions. (1) Entry of Ca(2+) across the microvilli into mucosal cells is by diffusion; it does not require metabolic energy or the presence of any other inorganic ions. (2) Pretreatment of the chick with cholecalciferol causes increased permeability of the microvillus to Ca(2+) in both directions (lumen to cell, cell to lumen). The increased transport brought about by cholecalciferol in vivo can be partially mimicked by sodium dodecyl sulphate added in vitro. (3) The sign and the magnitude of the electrical potential difference prevailing across the ileum does not influence Ca(2+) transport. (4) Exit of Ca(2+) from the mucosal cell is temperature-sensitive, requires metabolic energy and Na(+). (5) Pretreatment with cholecalciferol caused increased movement of Ca(2+) out of the cell across the basement membranes. This effect of cholecalciferol given in vivo could be markedly increased by the presence of dicyclohexylcarbodi-imide in the perfusion fluid. These observations suggested that cholecalciferol increased Ca(2+) entry (and exit) at the mucosal surface and also caused Ca(2+) to be more available to the pump at the serosal surface.     

Ionic transfer across the isolated frog large intestine

The unidirectional fluxes of sodium, chloride, and of the bicarbonate and CO(2) pair were determined across the isolated large intestine of the bullfrog, Rana catesbiana. The isolated large intestine of the frog is characterized by a mean transmembrane potential of 45 mv., serosal surface positive with respect to mucosal. The unidirectional sodium flux from mucosal to serosal surface was found to be equal to the short-circuit current, thus the net flux was less than the simultaneous short-circuit current. This discrepancy between active sodium transport and short-circuit current can be attributed to the active transport of cation in the same direction as sodium and/or the active transport of anion in the opposite direction. The unidirectional fluxes of chloride and the bicarbonate and CO(2) pair revealed no evidence for active transport of either anion. A quantitative study of chloride fluxes at 45 mv. revealed a flux ratio of 1.8 which is considerably less than a ratio of 6 expected for free passive diffusion. It was concluded that a considerable proportion of the isotopic transfer of chloride could be attributed to "exchange diffusion." Study of the electrical properties of the isolated frog colon reveals that it can be treated as a simple D. C. resistance over the range of -20 to +95 mv.