HIGH AFFINITY UPTAKE OF l-GLUTAMINE IN RAT BRAIN SLICES

—l -Glutamine is taken up into rat brain slices by a specific‘high affinity’uptake system (K_m 52 μm ) which is not influenced by high concentrations of l -glutamate and l -asparagine. The uptake system appears to be associated with cellular structures that do not survive homogenization under conditions which yield synaptosomes. The‘high affinity’uptake of glutamine is dependent on the external sodium ion concentration and can be inhibited by p-chloromercuriphenylsulphonate, amino-oxyacetic acid, ouabain, dibenamine and allylglycine. The effects of several inhibitors indicate that l -asparagine uptake is mediated by a system different from the‘high affinity’system mediating l -glutamine uptake.     

Ion Uptake Efficiency of Sunflower Roots

The term ion uptake efficiency is used for the rate of uptake of a particular ion from nutrient solutions holding a standard concentration of that ion (0.5 mM sulphate, 1.5 mM phosphate or 2.0 mM rubidium). The uptake efficiency for rubidium and phosphate in roots of intact sunflower plants depended on the salt status of the plants and on the concentration of the ion under investigation in pretreatment solutions. The effect of pretreatment was a rapid process causing differences of more than 300% in ion uptake efficiency within 1 h, depending on the composition of the pretreatment solution. At concentrations above 0.1 mM the rate of uptake of rubidium in the root was higher than the net potassium uptake necessary for adequate growth. The rate of sulphate uptake was related to potassium uptake but not to phosphate uptake. It is suggested that ion uptake of the roots is regulated by internal factors as well as by direct interactions between the medium and the absorbing surfaces.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

HIGH AFFINITY CHOLINE UPTAKE: IONIC AND ENERGY REQUIREMENTS

Abstract— High affinity choline uptake into rat hippocampal synaptosomes was examined at 37°C when various ions were deleted from normal Kreb's-Ringer media. When sodium chloride was replaced by sucrose, lithium chloride, cesium chloride or rubidium chloride, choline uptake was markedly reduced. When the sodium concentrations of the Kreb's media were gradually reduced to zero, the uptake was gradually reduced in parallel. A kinetic analysis performed at low and normal sodium concentrations revealed changes in K_m and V^max values. When several non-chloride sodium salts were utilized, the uptake was reduced in all cases suggesting also a chloride-dependence in addition to the sodium-dependence. Omission of calcium chloride or magnesium sulfate from the media did not alter uptake. Sodium-dependent choline uptake was examined over a range of potassium concentrations (0–35 DIM). It was found that uptake was maximal between potassium concentrations of 0.35–4.8 mm but was reduced at both lower and higher potassium concentrations. The kinetics of uptake were examined under varying potassium concentrations, and at low potassium, only a change in V_max was observed while at high potassium concentrations, there were changes in both K^m and V^max values. Preincubation and incubation of synaptosomes with 0.1 m -ouabain, 0.1 mm -2,4-dinitrophenol and 1 mm -KCN caused a reduction in sodium-dependent uptake. When dextrose was omitted from the preincubation and incubation media there was also a reduction in sodium-dependent uptake. By contrast, the sodium-independent uptake was unaffected by the metabolic inhibitors or omission of dextrose, and had a very low Q₁₀. When various incubation temperatures were utilized in uptake experiments, the Q¹⁰ for the interval 37-27°C was 2.7 and the activation energy was 22.7 kcal/mol. Slightly different ionic dependences were observed when animals pretreated with pentobarbital of oentylenetetrazol were utilized as the source of synaptosomes.     

Modification of the salinity response of wheat by the genome ofElytrigia elongatum

Summary Triticum aestivum cv. Chinese Spring wheat,Elytrigia elongatum (tall wheatgrass), and theTriticum-Elytrigia amphiploid were grown in complete nutrient culture containing, in addition, 0, 40, 80 and 120 mM NaCl. The 3 genotypes responded quite differently to increasing salinity; the Na concentration of wheat shoots increased in direct proportion to the increase in salinity of the external medium whereas the Elytrigia response was interpreted as showing high affinity for Na at low external Na (40 mM) but comparative exclusion of Na at high salinities (120 mM). In contrast, Na levels of the amphiploid were less than those of either wheat or Elytrigia under both low and high salinities. Thus the amphiploid behaved like wheat at 40 mM NaCl but more like Elytrigia at 120 mM NaCl because Na transport to the amphiploid shoot was restricted over the whole salinity range. The K concentration of the amphiploid shoot at high salinities was significantly greater than the K concentrations of either wheat or Elytrigia.     

The Kinetics of Ouabain Inhibition and the Partition of Rubidium Influx in Human Red Blood Cells

In the development of ouabain inhibition of rubidium influx in human red blood cells a time lag can be detected which is a function of at least three variables: the concentrations of external sodium, rubidium, and ouabain. The inhibition is antagonized by rubidium and favored by sodium. Similar considerations could be applied to the binding of ouabain to membrane sites. The total influx of rubidium as a function of external rubidium concentration can be separated into two components: (a) a linear uptake not affected by external sodium or ouabain and not requiring an energy supply, and (b) a saturable component. The latter component, on the basis of the different effects of the aforementioned factors, can be divided into three fractions. The first is ouabain-sensitive, inhibited by external sodium at low rubidium, and requires an energy supply; this represents about 70–80% of the total uptake and is related to the active sodium extrusion mechanism. The second is ouabain-insensitive, activated by external sodium over the entire range of rubidium concentrations studied, and dependent on internal ATP; this represents about 15% of the total influx; it could be coupled to an active sodium extrusion or belong to a rubidium-potassium exchange. The third, which can be called residual influx, is ouabain-insensitive, unaffected by external sodium, and independent of internal ATP; this represents about 10–20% of the total influx.     

Effect of CaSO4 and NaCl on mineral content ofLeucaena leucocephala

The effects of varying CaSO₄ and NaCl levels on the nutrient content ofLeucaena leucocephala were established by examining the concentrations of Na, Ca, Cl, K and Mg in leucaena roots, stems and leaves. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM), in randomized blocks with five replications. Leucaena excluded sodium from stems and leaves when NaCl concentration was 50 mM or less. Sodium uptake decreased as CaSO₄ concentration increased. Calcium uptake was affected by NaCl concentration when substrate CaSO₄ concentration was 0.5 mM. At this level, 100 mM NaCl caused a marked decrease in leaf calcium and a marked increase in leaf Cl. In all other treatments, Cl uptake was not affected by CaSO₄ concentration. Potassium uptake was strongly depressed as NaCl concentration increased at low Ca concentration, but this effect was offset at high Ca. Magnesium uptake decreased as CaSO₄ levels increased.     

Transmembrane effects in the sodium pump system. II. The ratio of the sodium efflux to the sodium concentration in frog muscle in media with various sodium substitutes

The dependence of sodium efflux on the internal sodium concentration on sodium-free magnesium, Tris, coline and lithium media was investigated on frog striated muscle. In all the sodium-substituted media, the efflux concentration curve was found to be dependent on the external rubidium concentration, being S-shaped at the saturating external rubidium (potassium) concentration and becoming close to linear at the low external rubidium concentration (0.5-1.0 microM). The maximal sodium efflux at saturating levels of internal sodium concentrations remains unchanged with various sodium substitutes in the medium, whereas the affinity constant of internal sodium sites is dependent on the external cations.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Utilization of Carbohydrates by Rhynchosporium secalis II. Absorption and Metabolism of Glucose, Galactose and Galacturonic acid

Glucose, galactose and galacturonic acid were taken up at different rates by the fungus Rhynchosporium secalis and were intracellularly converted to other forms of carbohydrate at different rates. These differences explain why, when there is only a single source of nutrient carbon in the growth medium, development of the fungus is greatest when glucose is present and least when galactose is present. Glucose and galactose were taken up by the same mechanism for their uptake showed a reciprocal competitive inhibition. Uptake mechanisms had a high affinity for glucose (apparent K_m 2.76 mM) and galacturonic acid (apparent K_m 3.10 mM) and a low affinity for galactose (apparent K_m 29.67 mM). After uptake, galactose accumulated in the mycelium, whereas glucose and galacturonic acid were rapidly converted to other soluble carbohydrates, principally trehalose and mannitol. The insoluble carbohydrates within the mycelium were little affected by the type of carbohydrate that was supplied to the fungus.     

Plant growth and ion relations in lucerne (Medicago sativa L.) in response to the combined effects of NaCl and P

The combined effect of NaCl and P on the growth of lucerne was studied in two hydroponic greenhouse experiments. NaCl concentrations were identical in each experiment (0, 50 and 100 mM NaCl) while external P concentrations were low (viz. 0.002, 0.02 and 0.2 mM measured as 0.006, 0.026 and 0.2 mM, respectively) in one experiment and higher (0.5 and 5.0 mM) in the second. Plant biomass was reduced more by the low P levels than by high concentrations of NaCl. A significant NaCl^*P effect was found where external P concentrations were low (0.006–0.2 mM) but there was no difference in plant production between the two P concentrations of 0.5 and 5.0 mM. Shoot and root concentrations of Na and Cl increased significantly with increasing NaCl concentration in both experiments and there were some differences in the concentrations of these ions at different external P levels. At low P, NaCl had no significant effect on shoot concentrations of P; however, root P concentrations tended to decrease with increasing NaCl level. Increasing external P from 0.006 to 0.2 mM led to significant increases in P concentrations in both roots and shoots. At higher P, concentrations of P in both the shoots and the roots did not differ with external NaCl or P conditions. Our results illustrate the complex relationship that exists between NaCl and P at low P levels. We conclude that high or non-limiting concentrations of P (0.2 – 5.0 mM) do not affect lucerne's response to NaCl.     

Kinetics of ionic transport across frog skin: Two concentration-dependent processes

Summary Sodium and chloride influxes across the nonshort-circuited isolated skin ofRana esculenta were measured at widely varying external ionic concentrations.The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed.At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na _ext ⁺ /H _int ⁺ ; Cl _ext ^– /HCO _3int ^– ) functioning at low external concentrations.At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components.The physiological significance of these results is discussed.     

Studies on Arabidopsis athak5, atakt1 double mutants disclose the range of concentrations at which AtHAK5, AtAKT1 and unknown systems mediate K+ uptake

The high‐affinity K⁺ transporter AtHAK5 and the inward‐rectifier K⁺ channel AtAKT1 have been described to contribute to K⁺ uptake in Arabidopsis thaliana. Studies with T‐DNA insertion lines showed that both systems participate in the high‐affinity range of concentrations and only AtAKT1 in the low‐affinity range. However the contribution of other systems could not be excluded with the information and plant material available. The results presented here with a double knock‐out athak5, atakt1 mutant show that AtHAK5 is the only system mediating K⁺ uptake at concentrations below 0.01 mM. In the range between 0.01 and 0.05 mM K⁺ AtHAK5 and AtAKT1 are the only contributors to K⁺ acquisition. At higher K⁺ concentrations, unknown systems come into operation and participate together with AtAKT1 in low‐affinity K⁺ uptake. These systems can supply sufficient K⁺ to promote plant growth even in the absence of AtAKT1 or in the presence of 10 mM K⁺ where AtAKT1 is not essential.     

The effects of sodium and potassium on ouabain binding by human erythrocytes

Ouabain binding by the human erythrocyte membrane is reversible, exhibits a high degree of chemical specificity, and can be detected at ouabain concentrations as low as 1 x 10^-10 M. The relation between ouabain binding and ouabain concentration can be described by a rectangular hyperbola permitting determination of the maximal binding (B _max) and the ouabain concentration at which ouabain binding is half-maximal (K_B). Reducing the external sodium concentration increased K_B, while reducing the external potassium concentration decreased K_B. Neither cation altered B _max The reciprocal of K_B was a linear function of the sodium concentration at sodium concentrations ranging from 0 to 150 mM. Conversely, the relation between the reciprocal of K_B and the external potassium concentration was nonlinear, and raising the potassium concentration above 4 mM produced no further increase in K_B. These results are compatible with a model which postulates that the erythrocyte membrane contains a finite number of receptors each composed of a glycoside-binding site and a cation-binding site. When sodium occupies the cation-binding site, the affinity of the glycoside site for ouabain is increased; when potassium occupies the cation-binding site the affinity of the glycoside site for ouabain is decreased.     

Transport of ferrous iron and lactate production inBifidobacterium bifidum var.pennsylvanicus

Initial rates of ferrous iron transport intoBifidobacterium bifidum var.pennsylvanicus were measured at low and high iron concentrations. The low affinity system (LAFIUS) had an apparent K_m of 167 μM, the high affinity system (HAFIUS) had a K_m of 50 μM. Iron removal from preloaded bifidobacteria revealed the existence of a labile and an inert iron pool in the bacterial cells. Iron uptake by the bifidobacteria was associated with lactate production, though lactate production could continue without iron uptake. Cessation of iron uptake and lactate production was not because of an exhaustion of any nutrient nor the accumulation of fermentation end products in the medium. It was apparently the result of an inactivation of the cellular enzyme machinery without replacing it through normal biosynthetic processes.     

Uptake of Some Quaternary Ammonium Ions by Human Erythrocytes

In many biophysical studies on erythrocytes some quaternary ammonium ions are used as replacements for Na⁺ and K⁺ of the physiological solutions. The object of this work was to study the possible uptake of quaternary ammonium ions by erythrocytes. Uptake of C¹⁴–choline chloride and C¹⁴–tetramethylammonium chloride by human erythrocytes was proved. It was shown that the compounds were neither incorporated into phospholipids of the cell nor converted to any other metabolites. Studies of uptake as a function of time, at several external concentrations of choline and tetramethylammonium, showed that within the first 4 hours uptake was a linear function of time regardless of the external concentration of the quaternary ammonium ions. The effects of various external concentrations of choline and tetramethylammonium ions on the rate of uptake by the cells were studied. The results showed the presence of two distinct mechanisms for the uptake of choline: one, a facilitated uptake mechanism which becomes saturated at low external concentrations of the ion; the other, a simple diffusion mechanism in which the rate of uptake is proportional to concentration. For the facilitated part of the uptake the external choline concentration at which half-maximum rate was obtained was found to be 0.02 mm. Although the kinetic studies with tetramethylammonium ion were not as extensive as those with choline, they did suggest the presence of similar mechanisms for the uptake of both ions. Tetramethylammonium and tetraethylammonium ions were shown to be competitive inhibitors of the facilitated choline uptake.     

Effects of aluminium on growth and nutrient uptake of small Picea abies and Pinus sylvestris plants

Summary The effects of aluminium concentrations between 0.2 and 30 mM at pH 3.8 ±0.2 on small plants of Norway spruce [(Picea abies (L.) Karst], Scots pine (Pinus sylvestris L.), and Scots pine infected with the ectomycorrhizal fungus Suillus bovinus (L. ex Fr.) O. Kuntze were investigated. The plants were grown at maximum relative growth rate (R_G % day^–1) with free access but very low external concentrations of nutrients. Steady-state conditions with respect to relative growth rate (R_G) and internal nutrient concentrations were achieved before addition of aluminium, which was added as AlCl₃ and/or Al(NO₃)₃. There were reductions in rg at aluminium concentrations of 0.3 mM in spruce, 6 mM in pine and 10 mM in ectomycorrhizal pine, i. e. at aluminium concentrations considerably higher than those normally occurring in the top layer of the mineral soil where most fine roots are found. Nutrient uptake rate per unit root growth rate was calculated for different nutrient elements. The uptake rate of calcium and magnesium was reduced at aluminium concentrations of 0.2 mM (spruce), 1 mM (pine) and 3 mM (ectomycorrhizal pine), without influencing R_g. The results question the validity of the hypothesis of aluminium toxicity to forest tree species at low external concentrations.     

Calcium and Salt Toleration by Bean Plants

The role of calcium in the salt relations of the bean plant, Phaseolus vulgaris, was examined. Brittle wax bush bean plants were cultured in nutrient solutions containing 50 mM NaCl. In the absence of added calcium the plants showed a general breakdown of the roots. A low concentration of calcium in the nutrient solution (0.1 mM) prevented this. Without added calcium the plants absorbed and translocated sodium at such a rate that high concentrations of it built up in the leaves within two days. With increasing concentrations of calcium in the nutrient solution the leaves contained progressively less sodium, and at 3 mM CaSO₄ the concentrations of sodium in the leaves was equal to that of the control plants grown without addition of salt. Even after both roots and stems had reached a high concentration of sodium, the leaves of plants grown in the presence of adequate concentrations of calcium contained little sodium.     

HIGH AFFINITY AMINO ACID TRANSPORT BY FROG SPINAL CORD SLICES

The characteristics of amino acid uptake by frog spinal cord slices was studied by in vitro incubations in appropriate media. The uptake mechanisms exhibited saturation; kinetic analysis demonstrated 2 distinct systems for the influx of the possible neurotransmitters: GABA, glycine, L-glutamic acid and L-aspartic acid. One system showed a comparatively high substrate affinity (K_m values, 10-26 μM) while the other system had a lower affinity (K_m, 0.4-1.6 mM).-Leucine, an amino acid presumably not a transmitter, was accumulated only by a low affinity mechanism (K_m 1.6 mM). The process responsible for high affinity uptake had many of the properties of an active transport mechanism. These included temperature sensitivity, energy dependence, requirement for Na⁺ ions and inhibition by ouabain. GABA and glycine uptake was inhibited only by closely related amino acids or structural analogues. The influx of L-glutamic acid was competitively inhibited by the presence of L-aspartic acid in the medium; the converse was also demonstrated. Thus, the high affinity uptake system for possible transmitter amino acids in the frog spinal cord closely resembles that described for mammalian CNS tissue. These results are compatible with the assumption that GABA, glycine, L-glutamic acid and L-aspartic acid are neurotransmitters in the amphibian spinal cord.     

Multiphasic uptake of potassium by barley roots of low and high potassium content: Separate sites for uptake and transitions

In the range 10^?6M - 5 × 10^?2M uptake of K⁺ in excised roots of barley (Hordeum vulgare L. cv. Herta) with low and high K content could in both cases be represented by an isotherm with four phases. Uptake, especially in the range of the lower phases, was reduced in high K roots through decreases in V_max and increases in K_m. Similar data for other plants are also shown to be consistent with multiphasic kinetics. The concentrations at which transitions occurred were not affected by the K status, indicating the existence of separate uptake and transition sites. Uptake was markedly reduced in the presence of 10^?5M 2,4-dinitrophenol, especially at low K⁺ concentrations, but the isotherms remained multiphasic. This contraindicates major contributions from a non-carrier-mediated, passive flux. A tentative hypothesis for multiphasic ion uptake envisions a structure which changes conformation as a result of all-or-none changes in a separate transition site. The structure is “tight” at low external ion concentrations (low V_max. low K_m. active uptake, allosteric regulation) and “loose” at high concentrations (high V_max- high K_m- facilitated diffusion, no regulation).