Proline uptake in Streptomyces clavuligerus

Streptomyces clavuligerus was able to accumulate proline intracellularly throughout a wide range of external proline concentrations. Kinetic analysis of proline uptake indicated that this phenomenon is mediated by two saturable systems. One of them was a high-affinity system (Km = 11 microM), with low uptake capacity and specific for proline. The second system had lower affinity for proline (Km = 10.2 mM), higher uptake capacity, and was inhibited by several amino acids. Proline was not required as an inducer of the systems, which were neither repressed nor inhibited by ammonium.     

Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea pig jejunal brush border membrane vesicles

D-Glucose transport was studied with isolated brush border membrane vesicles from guinea pig jejunum. Saturation curves were carried out at either 25 or 35 degrees C in buffers containing Na+, Li+, K+ (100 mM chloride salt), or sorbitol (200 mM). Uncorrected uptake rates were fitted by nonlinear regression analysis to an equation involving one diffusional and two saturable terms. In the presence of Na+ at 35 degrees C, two saturable systems (Km = 0.4 and 24 mM, respectively) were evident, as well as a diffusion component quantitatively identical with that measured with L-glucose in separate experiments. In contrast, at 25 degrees C only one saturable system was apparent (Km = 1.2 mM): the second exhibited diffusion-like kinetics. In the presence of Na+ at 35 degrees C, D-glucose uptake was fully inhibited by both D-glucose and D-galactose, whereas alpha-methylglucoside gave kinetics of partial inhibition. We conclude that in the presence of Na+ there are at least two distinct D-glucose transport systems: 1) System I, a low temperature-sensitive system, fully inhibited by D-glucose, D-galactose, and alpha-methylglucoside; we identify it as the "classical" D-glucose/Na+ cotransport system, insensitive to inhibition by cytochalasin B and obligatorily dependent on Na+; and 2) System II, a high temperature-sensitive system where D-glucose and D-galactose inhibit but alpha-methylglucoside is inert. Its cation specificity is unclear but it appears to be sensitive to cytochalasin B inhibition. When Li+ or K+ substituted for Na+, only one transport system was apparent. The Li+-activated transport was: independent of the incubation temperature; inhibited by D-glucose and D-galactose but not by alpha-methylglucoside, 2-deoxy-D-glucose, D-mannose, and D-xylose; and sensitive to cytochalasin B inhibition. The exact nature of the system (or systems) involved in D-glucose transport in the absence of sodium remains to be established.     

Amine uptake into intact mast cell granules in vitro

Histamine, the principal amine of rat peritoneal mast cells, is taken up into isolated granules with intact membranes. Uptake is pH- and concentration-dependent and is not stimulated by the addition of Mg2+-ATP. The saturable uptake has a Km of 91.1 microM and a Vmax of 95.4 pmol (mg of protein)-1 min-1. Uptake is abolished by 5 mM ammonium ion. 5-HT, the other endogenous amine of the granules, and dopamine and tyramine, which do not occur naturally in rat mast cells, each competitively inhibits [3H]-histamine uptake with Ki's close to 1 microM. Reserpine, a putative amine carrier blocker, inhibits uptake at nanomolar concentrations. At high concentrations, uptake of [3H]-5-HT is nonsaturable; at low concentrations, a saturable component is observed with a Km of 1.6 microM. Uptake of [3H]-5-HT is not enhanced by Mg2+-ATP. It is pH-dependent but with a lower apparent pKa than that of histamine. [3H]-5-HT uptake can be completely inhibited by ammonium ions. Amine inhibition of [3H]-5-HT gives nonlinear Dixon plots, and high concentrations of the competing amines or reserpine cannot completely block uptake. We propose a model consistent with these results in which amine uptake occurs by several distinct saturable transport systems. According to the model, histamine is transported by a single system, which also transports 5-HT and dopamine. 5-HT and dopamine are transported by one or more other systems.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Independent blood-brain barrier transport systems for nucleic acid precursors.

The blood-brain barrier permeability to certain 14-C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15s later. The amount of 14-C-labelled base or nucleoside remaining in brain was expressed in relation to 3-H2O (a highly diffusible internal standard) and 113m-In-labelled EDTA (an essentially non-diffusible internal standard). Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine. Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (Km equals 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (Km equals 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [14-D]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.     

alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.     

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.     

Characterization of basolateral membrane Na/H antiport in rat jejunum

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.     

Characteristics of lysine uptake by isolated renal cortical tubule fragments from mature and immature dogs

The uptake of L-lysine was examined in isolated renal cortical tubule fragments from adult and 1-week-old dogs. Lysine uptake by adult tubules was initially more rapid than that by the immature tubules. This uptake by mature tubules reached a steady state after 30 min of incubation, while the newborn tubules still had not reached a steady state by 90 min of incubation. Because a steady state of lysine uptake was not attained with the immature tubules, their uptake of lysine exceeded that of the adult after 60 min of incubation. Kinetic studies revealed that lysine was taken up by one saturable transport system with a Km of 0.56 mM and Vmax of 6.18 mmol/liter intercellular fluid per 5 min in the adult and one saturable transport system in the 1-week-old with a Km of 0.38 mM and Vmax of 3.66 mmol/l intracellular fluid per 5 min. Lysine also entered the renal tubule cells in both age groups via a diffusional pathway with a kd of 0.35 min-1 in the adult and 0.30 min-1 in the newborn. Cystine competitively inhibited lysine uptake by adult dog tubules with a Ki of 0.61 mM. The other dibasic amino acids, ornithine and arginine, also inhibited lysine uptake in both the adult and the newborn.     

Neutral-sugar transport by rat liver lysosomes.

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.     

myo-Inositol Transport in Mouse Astroglia-Rich Primary Cultures

Uptake of radiolabeled myo-inositol was studied in astroglia-rich primary cultures derived from neonatal mouse brains. The uptake was saturable in the presence of Na+ with a Km of 25 microM and a Vmax of 60 pmol.min-1.(mg protein)-1, suggesting a high-affinity transport system for myo-inositol in astroglial cells. In addition, a Na(+)-independent, nonsaturable component was found. Carrier-mediated uptake was not inhibited by cytochalasin B (50 microM), but was reduced by depolarizing concentrations of K+ and, to different extents, in the presence of phloretin, ouabain, or amiloride (1 mM each). scyllo-Inositol, glucose, and galactose also reduced myo-inositol uptake; inhibition by the two hexoses was not reversed in the presence of 0.4 mM sorbinil. On the other hand, uptake of 2-deoxyglucose was not inhibited by high concentrations of myo-inositol. Preincubation of the cells with glucose-free or inositol-free medium stimulated uptake of myo-inositol and preincubation with 25 mM glucose in the presence of 0.4 mM sorbinil had no effect on the rate of uptake. The results suggest that myo-inositol is taken up into the astroglial cells by a transport mechanism that is distinct from that of glucose and probably is an active one. Sorbitol pathway activity does not interfere with myo-inositol uptake.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

Developmental and other characteristics of lysine uptake by rat brain synaptosomes.

Synaptosomes isolated from adult or newborn rat cerebrum take up L-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is mort tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher Vmax than those of the adult for high affinity system but adult for high affinity system but adult synaptosomes have a higher Vmax than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low Km system for lysine uptake.     

Ornithine uptake by isolated hepatocytes and distribution within the cell

Uptake of ornithine by isolated hepatocytes and its distribution within the cell was investigated. Ornithine uptake was energy independent and exhibited a saturable and a nonsaturable component. The Km value of the saturable component was 1.3 mM. At an external ornithine concentration of 0.5 mM the rate of ornithine uptake was 127 +/- 19 nmol/g. Lysine inhibited ornithine uptake, indicating the existence of an ornithine transport system. It was concluded that ornithine transport can limit urea synthesis in the state of transition from a low ammonia to a high ammonia supply.     

Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

Characterization of D-Aspartic Acid Uptake by Rat Hippocampal Slices and Effect of Ischemic Conditions

The cellular uptake of D-aspartic acid (D-Asp) as a model compound for glutamic acid transport was studied in rat hippocampal slices. D-Asp is accumulated by both Na(+)-dependent and Na(+)-independent processes in hippocampal slices, and both processes are dependent on temperature. The Na(+)-dependent uptake is assumed to be high in affinity (apparent Km = 0.17 mM), but low in capacity, whereas the Na(+)-independent uptake is much lower in affinity (Km = 2.86 mM), but higher in capacity. L-Aspartic acid, L-glutamic acid, dihydrokainic acid, and threo-3-hydroxy-DL-aspartic acid markedly inhibited the uptake of D-Asp with Na+ in the medium, whereas D-glutamic acid, glycine, and L-lysine had no significant effect. The Na(+)-dependent uptake of D-Asp was significantly reduced under "hypoglycemic," "anoxic," and "ischemic" conditions, whereas the Na(+)-independent uptake was unaffected. Metabolic inhibitors such as NaCN and ICH2COOH significantly inhibited the Na(+)-dependent uptake, but not the Na(+)-independent uptake. These results suggest that the Na(+)-dependent component of D-Asp transport in rat hippocampal cells is inactivated under ischemic conditions, whereas the Na(+)-independent component is unaffected.