Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

Cation movements in the high sodium erythrocyte of the cat

The uptake of ⁴²K and ²⁴Na by cat erythrocytes was investigated. Under steady-state conditions, the nontransient component of ⁴²K influx was found to be 0.18 ± 0.01 meq/liter RBC/hr and insensitive to ouabain (100 µM); the corresponding value for ²⁴Na was 17 ± meq/liter RBC/hr. A study was made of the effects of anions upon cation movements in these and other mammalian red cells. Iodide was found to inhibit markedly (>50%) Na inward movements in cat and dog but not in the other erythrocytes. An increase (15–30%) in K uptake in the presence of iodide was noted in all the mammalian cells studied.     

Sodium movement in high sodium feline red cells

The transport of Na in the cat red cells has been studied under various experimental conditions. The unidirectional radioactive Na influx increased with increasing temperature until it reached a maximum value at 37°C ± 2°C and then decreased with a further increase in temperature. Errors stated in this paper represent 1.0 standard errors of the mean. The apparent activation energy was calculated in the region between 25 and 37°C and was found to be 4.9 ± 0.5 kcal/mole. Copper at a concentration of 0.04 mM inhibited this influx by 65%. When cells were suspended in isosmotic KCl buffer, cell volume was found to decrease initially with time. This unusual behavior is discussed in terms of Na to K preference of the cell membrane. In cat red cells, Na influx was found to increase about 13-fold when cell volume was decreased from 1.16 normal to 0.87. This effect could not be reproduced when the medium osmolarity was changed only by the addition of urea, a permeating molecule. On the other hand, K influx was found to decrease from 0.24 ± 0.03 mEq/liters RBC, hr at a relative cellular volume equal to 1.0 to 0.11 ± 0.01 mEq/liters RBC, hr at a cell volume of 0.75. Na influx in human red cells did not show any significant dependence on cell volume. The properties of Na movement in the cat red cells are compared to those of human red cells.     

Sulphated polysaccharide synthesis in brown algae

Summary Histochemical and autoradiographic techniques have been used to investigate the sites of synthesis, transport and location of sulphated polysaccharides in some larger brown seaweeds.The most rapid uptake of ³⁵SO₄ occurred when material was incubated in medium with 10^-4M carrier sulphate, negligable uptake occurring from seawater.Autoradiography using ³⁵SO₄ has shown that in Pelvetia sulphated material is synthesised by all cell types, particularly epidermal cells. In Laminaria spp. this activity is confined to specialized secretory cells which discharge into mucilage canals. In both instances the process of carbohydrate sulphation appears to occur in the Golgi-rich perinuclear region.     

Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter

Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [³⁵S]SO₄²⁻ accumulation. Maximum uptake is secured in the presence of Mg²⁺ and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [³⁵S]SO₄²⁻ uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter.     

Some aspects of zinc transport across eel (Anguilla anguilla) red blood cell membranes.

Zinc movement across eel and human red blood cell membranes was measured by atomic absorption spectrophotometry. It was observed that:

• 1) In human red blood cells zinc uptake is twice as rapid as in fish red blood cells over a temperature range of 10-40°C. The low rate of zinc uptake in eel red blood cell may be simply the side effect of different surface area to volume ratios for the differences in cell size or, it may be due to the low permeability of bicarbonate through the red blood cell membranes.
• 2) Zinc uptake measured in eel and human red blood cells treated and untreated with external trypsin shows different features. The zinc uptake was reduced by about 40% in treated eel red blood cells with respect to the total uptake of untreated red blood cells. Human red blood cells treated and untreated with trypsin do not show any differences in the amount of zinc transported.
• 3) In fish red blood cells, zinc uptake in NANO₃ medium is markedly reduced, compared with that measured in NaCl medium. The [Zn²⁺_i slightly increases in the presence of bicarbonate. In human red blood cells in NANO₃ medium the zinc uptake is strongly reduced and the presence of bicarbonate marginally increases the zinc influx.
• 4) In eel red blood cells there seem to be two independent pathways for zinc uptake: one DIDS-sensitive and the other DIDS-insensitive. DIDS 10 μM inhibits only 64% of the total zinc transported. Iincreasing the DIDS concentration did not give more inhibition. In human red blood cells only one DIDS-sensitive pathway for zinc transported seems to exist, because, 2,5 μM DIDS inhibits 97% of zinc uptake.

     

Vasopressin stimulates sulfate incorporation into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture

The effects of lysine vasopressin (1–100 ng/ml) on the 24 h incorporation of [³⁵SO₄⁻] into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture has been investigated. The hormone enhances sulfate incorporation into proteoglycans released in the medium and those associated with the cell layer. This enhancement was independent of cell density or stimulation of cell division by the hormone or calf serum. These observations provide evidence that the hormone stimulation of sulfate incorporation is not directly linked to hormone stimulation of cell division.     

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺), identity. The Q* decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ ∼ I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO₄²⁻ anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.     

Synthesis of glycosaminoglycans by human embryonic lung fibroblasts. Different distribution of heparan sulphate, chondroitin sulphate and dermatan sulphate in various fractions of the cell culture

Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate ³⁵SO₄²⁻ for various periods of time. ³⁵S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO₂ and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various ³⁵S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The ³⁵S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the ³⁵S-labelled galactosaminoglycans were extensively degraded by periodate oxidation–alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Åberg & Fransson (1975) Biochem. J. 151, 477–489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected.     

Separate effects of exogenous hyaluronic acid on proteoglycan synthesis and deposition in pericellular matrix by cultured chick embryo limb chondrocytes

The effects of exogenous hyaluronic acid on cell cultures of chick embryo limb chondrocytes are reported in this paper. The evidence shows that exogenous hyaluronic acid (HA) can both depress the incorporation of ³⁵SO₄ into glycosaminoglycans and cause a displacement of newly synthesized proteoglycan from the cell layer to the culture medium. The results demonstrate that these two effects are mediated by distinct mechanisms. The displacement effect has a rapid onset (by 2 hr) while the effect of exogenous HA on ³⁵SO₄ incorporation has a long latency (12 hr). The displacement effect is produced by a lower concentration (5 μg/ml) of hyaluronate oligomers than the effect on ³⁵SO₄ incorporation (50 μg/ml). In addition, displacement is produced only by hyaluronate oligomers that are decasaccharides or larger. The depression of ³⁵SO₄ incorporation is produced by tetrasaccharides as well as high molecular weight HA. In fact tetrasaccharides can depress ³⁵SO₄ incorporation without causing the displacement effect.     

Sulphate uptake and metabolism in the chrysomonad,Monochrysis lutheri

The intracellular concentration of inorganic ³⁵SO₄ in Monochrysis lutheri cells exposed to 0.513 mM Na₂ ³⁵SO₄ for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this ³⁵SO₄ with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 μmoles/g cells/hr). The flux of free ³⁵SO₄ to organic ³⁵S was 0.029 μmoles/g cells/hr. Assuming an internal electrical potential in the cells of-70 mV, this intracellular concentration of inorganic ³⁵SO₄ was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a K _m of 3.2×10^-5 M and a V _max of 7.9×10^-5 μmoles sulphate/hr/10⁵ cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the proteins(s) associated with sulphate transport.     

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl^? and SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl^? and SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl^? and SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Ion and water shifts produced by ammonium chloride in high K and low K red cells

The effect of ammonium chloride on the cellular Na⁺, K⁺ and water has been examined in human and horse (high K), cow (medium K) and cat (low K) red cells. It was found that high K red cells, especially those of the horse, gained water an Na⁺, whereas the net movement of K⁺ was negligible. There was a correlation between the increase of cellular Na⁺ concentration and of the packed red cell volume. In contrast, the packed cell volume of low K red cells increased slightly or not at all, and Na⁺ ions leaked out from the cells. The high K cells had a lower Cl^? concentration and higher buffer capacity than the low K cells. The results obtained with the medium K (cow) cells usually lay between those of the other two cell types. In all the cases both the plasma and cell pH decreased resulting from the addition of ammonium chloride. The mechanism of movements of water and Na⁺ ions in high K cells remained unsolved, but the response of low K cells to ammonium chloride was near that of a cation exchange resin.     

Relationship of Cell Sap pH to Organic Acid Change During Ion Uptake

Excised roots of barley (Hordeum vulgare, var. Campana) were incubated in KCl, K₂SO₄, CaCl₂, and NaCl solutions at concentrations of 10⁻⁵ to 10⁻² n. Changes in substrate solution pH, cell sap pH, and organic acid content of the roots were related to differences in cation and anion absorption. The pH of expressed sap of roots increased when cations were absorbed in excess of anions and decreased when anions were absorbed in excess of cations. The pH of the cell sap shifted in response to imbalances in cation and anion uptake in salt solutions as dilute as 10⁻⁵ n. Changes in cell sap pH were detectable within 15 minutes after the roots were placed in 10⁻³ n K₂SO₄. Organic acid changes in the roots were proportional to expressed sap pH changes induced by unbalanced ion uptake. Changes in organic acid content in response to differential cation and anion uptake appear to be associated with the low-salt component of ion uptake.     

The interaction of chloride with the sulfate transport system of ehrlich ascites tumor cells

The effect of Cl^? on SO₄^?2 efflux was studied in both Cl^?-containing and Cl^?-free ascites tumor cells loaded with ³⁵SO₄^?2 to test the hypothesis that Cl^?-SO₄^?2 exchange is mediated by the same mechanism responsible for SO₄^?2-self exchange. The addition of Cl^?-free, ³⁵SO₄^?2 loaded cells to a SO₄^?2-free, Cl^? medium results in: (1) SO₄^?2 efflux that is dependent on the extracellular Cl^? concentration (Km = 4.85 mM; k_e = 0.048 min^?1 at 50 mM Cl^?) and (2) net Cl^?-uptake that exceeds SO₄^?2 loss. Both SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate) and ANS (1-anilino-8-napthalene sulfonate) inhibit SO₄^?2 efflux but are without effect on Cl^? uptake. The addition of Cl^?-containing, ³⁵SO₄^?2 loaded cells to a SO₄^?2-free, C1^? medium results in: (1) a slight gain in cellular Cl^? and (2) k efor SO₄^?2 efflux identical to that for Cl^?-free cells. The results are compatible with the suggestion that: (1) Cl^? interacts with a membrane component responsible for transmembrane SO₄^?2 movement; (2) Cl^? interaction stimulates the rate of unidirectional SO₄^?2 efflux from cells initially free of Cl^? as well as the rate of SO₄^?2 turnover in cells maintained in the steady state with respect to Cl^? and SO₄^?2; and (3) in the case of cells initially free of Cl^?, the Cl^?-SO₄^?2 pathway represents only a small fraction of the total unidirectional Cl^?-influx the remainder being compatible with the electroneutral accumulation of NaCl and KCl.     

Studies on isolated subcellular components of cat pancreas

Summary Transport of alanine was studied in isolated plasma membrane vesicles from cat pancreas using a rapid filtration technique. The uptake is osmotically sensitive and the kinetics ofl-alanine transport are biphasic showing a saturable and a nonsaturable component. The saturable component is seen only when a sodium gradient directed from the medium to the vesicular space is present. Under this condition an overshooting uptake ofl-but not ofd-alanine occurs. The Na⁺ gradient stimulated uptake ofl-alanine is inhibited byl-serine andl-leucine and stimulated when the membrane vesicles had been preloaded withl-alanine,l-serine orl-leucine.The ionophore monensin inhibits stimulation of uptake caused by a sodium gradient. In the presence of valinomycin or carbonyl cyanidep-trifluoromethoxyphenylhydrazone (CFCCP), the sodium-dependent transport is augmented in vesicles preloaded with K₂SO₄ or H⁺ ions (intravesicular pH 5.5), respectively. In the presence of different anions, the Na⁺-dependent transport is stimulated according to increasing anionic penetration through membranes (lipid solubility). We conclude that a sodium dependent electrogenic amino acid transport system is present in pancreatic plasma membranes.     

Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42− uptake and expression of a band 3 like protein in the marine sponge,Microciona prolifera

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of ³⁵SO₄^2? uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in ³⁵SO₄^2? uptake and incorporation which could be greatly augmented in the presence of Ca²⁺/Mg²⁺. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa ³⁵SO₄^2? sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

Growth of the Peritrich Opercularia coarctata in Axenic Culture*

A synthetic medium for Opercularia coarctata was developed that contains 20 amino acids, 10 vitamins, an 8-component balanced salt solution, Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O, Tween 80, stigmasterol, a 7-component nucleic acid mixture, phenol red as an indicator, and 2,500 U.S.P. units/ml penicillin to maintain sterility. This medium supported axenic survival for 96 hr. Multiple supplements of thioctic acid, niacin, niacinamide, inositol, PABA, oleic acid, and Fe(NO₃)₂·9H₂O instead of Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O coverted the survival medium into a growth medium, which permitted 36–45 days continuous cultivation of populations in excess of 4 × 10³ cells/3.0 ml final volume. Five generations were produced during the 48 hr logarithmic growth period. Serial transfers at 72 hr and during periods of greatest cell density produced a maximum of 8 generations 96 hr after initiation but the medium failed to sustain growth through more than 6 serial transfers. Extension of this investigation to formulating a minimal axenic medium is discussed.