CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

A COMPARATIVE STUDY ON THE SYNTHESIS OF THE NATRIURETIC HORMONE DOPAMINE IN OK AND LLC-PK1CELLS

TheV_maxvalues (in nmol/mg protein/15 min) for AAAD in OK cells (0.94±0.08) were found to be significantly (P<0.01) lower than those observed in LLC-PK₁cells (4.37±0.08). However, in both cell lines decarboxylation reaction was a saturable process with similarK_mvalues (OK cells=1.1 mm (0.3, 1.8); LLC-PK₁cells=1.8 mm (1.6, 2.1)). Contrariwise to OK cells, decarboxylation ofl -DOPA to dopamine in LLC-PK₁cells followed a linear (7.6±0.1 pmol/mg protein/min) non-saturable kinetics till 120 min of incubation. The formation of dopamine from increasing concentrations ofl -DOPA (10 to 500 μm ) followed a non-linear kinetics in both cell lines; the process ofl -DOPA decarboxylation was saturated at low concentrations ofl -DOPA with an apparentK_mvalue of 11 μm (0.2, 22.6) in OK cells and 27.4 μm (11.1, 43.7) in LLC-PK₁cells. The formation of dopamine in LLC-PK₁cells (V_max=2097±113 pmol/mg protein/6 min) was 13.7-fold that occurred in OK cells (V_max=153±10 pmol/mg protein/6 min). In conclusion, LLC-PK₁cells appear to be endowed with a greater ability to form dopamine from exogenousl -DOPA when compared to OK cells.     

Relationships between ethylenediamine and GABA transport systems in rat brain slices

[¹⁴C]EDA was accumulated by slices of adult rat cerebral cortex, although the tissue:medium ratios achieved were very much lower than those for GABA. EDA uptake was temperature dependent and appeared to take place by both sodium dependent and sodium independent mechanisms. Kinetic analysis of the uptake revealed a major low affinity component with an apparent K_m of 1.11 ± 0.05 mM and a V_max of 9.8 ± 0.2 μmol/hg wet wt, with a second site of K_m about 20 μM but a 50 fold lower V_max. Inhibition studies indicate that EDA may be transported in part by the ‘small basic’ amino acid transport system and in part by polyamine systems shown to be present in CNS tissue. High levels of displaceable binding of radioactive EDA to glass-fibre filters were observed; studies using [¹⁴C]EDA may be complicated by binding to tissue macromolecules. Potassium stimulated, calcium dependent release of radioactivity from brain slices labelled with [¹⁴C]EDA in the presence of sodium ions was observed. Extracellular EDA stimulated the release of [³H]GABA and [³H]beta-alanine from preloaded slices, although GABA and beta-alanine did not stimulate [¹⁴C]EDA release. It appears that extracellular EDA can counterexchange with intracellular GABA or beta-alanine, but that EDA which is accumulated by the tissue may then be bound or move to pools not directly accessible to these amino acids. Ouabain released radioactivity from slices labelled by [¹⁴C]EDA in the presence of sodium but not from slices labelled in the absence of sodium. These results suggests that EDA is not acting simply as a substrate for GABA transport sites.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

SODIUM-DEPENDENT EFFLUX AND EXCHANGE OF GABA IN SYNAPTOSOMES

Abstract— The influx and efflux of [³H]GABA were investigated in synaptosomes. Two efflux components were detected. The first, termed spontaneous efflux, was not affected by the external sodium chloride concentration. The second, termed GABA-stimulated efflux, was observed when low levels of GABA were added to the incubation medium and was found to require external sodium chloride. The rate of spontaneous efflux at 0°C was about 37 per cent of the rate at 27°C but both GABA-stimulated efflux and GABA influx were completely inhibited at 0°C. The stimulation of efflux by external GABA followed simple Michaelis–Menten kinetics with respect to external GABA. The concentration of external GABA required for half-maximal stimulation was 4·9 ± 1·4 μm and the V_max for efflux was 1·0 ± 0·6 nmol. min^-1.mg^-1 of protein. A similar stimulation of efflux was observed with GABA analogue l -2,4-diamino-butyric acid which is a competitive inhibitor of influx. The concentration of external l -2,4-diaminobutyric acid required for half-maximal stimulation of efflux was 51 ± 12 μm and the V_max for efflux was 0·8 ± 0·5 nmol.min^-1.mg^-1 of protein. Since the sodium-dependency, temperature sensitivity, and kinetic properties of the GABA-stimulated efflux system were similar to the influx system, GABA-stimulated efflux was attributed to carrier-mediated exchange diffusion. Measurement of efflux and influx in the same preparation showed there was a net efflux when total fluxes were considered and that the exchange ratio (influx to GABA-stimulated efflux) was 0·9 when carrier-mediated fluxes were considered. The effect of the temperature of the fluid used to rinse synaptosomes collected on filters in influx experiments was investigated. There was no detectable difference in measured values of influx between samples rinsed with cold fluid (0°C) and warm fluid (27°C). The endogenous GABA content of synaptosomes was found to be 20·3 ± 2·5 nmol GABA per mg of protein. From this value, the cytoplasmic concentration of GABA in synaptosomes was estimated to be a maximum of 40 mm . About 5 per cent of total cerebral cortical GABA was found in the synaptosomal fraction.     

Oxidative Metabolism of 4-Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched-chain Fatty Acid

Abstract: The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2, 2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O₂ uptake, was determined in medium containing either 5 or 100 mM-[K⁺]. The apparent K_m for GABA was 1.16 ± 0.19 mM and the V_max in state 3 was 23.8 ± 5.5 ng-atoms O₂. min^?1. mg protein^?1 in 5 mM-[K⁺]. In a medium with 100 mM-[K⁺] the apparent K_m was 1.11 ± 0.17 mM and V_max was 47.4 ± 5.7 ng-atoms O₂. min^?1. mg protein^?1. The K_m for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM-[K⁺], respectively. For DMV, the K_i was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM-[K⁺] medium, respectively. The O₂ uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K²] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V_max for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

Characterization of mannosyl transferases during the pupal instar of Stomoxys calcitrans (L)

Particulate fractions (10,000g) from pupae of Stomoxys calcitrans transfer [¹⁴C]-mannose from GDP-[¹⁴C]-mannose to dolichol monophosphate and proteins. Production of the mannosyl lipid was inhibited by Mn²⁺, UDP, GMP, GDP, and EDTA. The insect growth regulator diflubenzuron had no effect on mannosyl transferase activity. Dolichol monophosphate and Mg²⁺ stimulated mannosyl transferase activity. The mannosyl lipid product was identified as mannosyl-phosphoryl-dolichol (Man-P-Dol). The apparent K_m and V_max values for the formation of Man-P-Dol using GDP-[¹⁴C]-Man while holding dolichol phosphate constant were 2.4 ± 0.9 μM and 9.4 ± 2.3 pmol Man-P-Dol·min^?1·mg^?1 protein, respectively. The apparent K_m and V_max values using dólichol phosphate while holding GDP-Man constant were 2.2 ± 1.2 μM and 18.5 ± 1.7 pmol Man-P-Dol·min^?1·mg^?1 protein.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

Ibuprofen Inhibits Arylamine N-Acetyltransferase Activity in the Bacteria Klebsiella pneumoniae

Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 ± 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.506 ± 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 ± 0.016 nmol/min/10¹⁰ colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.228 ± 0.008 nmol/min/10¹⁰ CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h. For in vitro examination, the values of apparent K _m and V _max were 1.08 ± 0.05 mM and 9.17 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent K _m and V _max were 1.19 ± 0.01 mM and 6.67 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the values of apparent K _m and V _max were 1.24 ± 0.48 mM and 4.18 ± 1.06 nmol/min/10 × 10¹⁰ CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent K _m and V _max were 0.95 ± 0.29 mM and 2.77 ± 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae. Received: 28 January 1997 / Accepted: 12 February 1997     

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.     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide

The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10–200 μM) stimulation of 5-HT transport that was rapid and sustained over 0–10 minutes of incubation. When platelets were incubated with diamide (10–200 μM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 μM, while washed platelets incubated with diamide (10–200 μM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (V_max control = 28.4 ± 1.4 vs. V_max diamide = 60.9 ± 4.1 pM/10⁸ platelets/4 min) but did not significantly alter the K_m values. Ouabain, an inhibitor of platelet Na⁺-K⁺ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.     

EFFECT OF ELECTRICAL STIMULATION AND CHLORPROMAZINE ON THE UPTAKE AND RELEASE OF TAURINE, γ-AMINOBUTYRIC ACID AND GLUTAMIC ACID IN MOUSE BRAIN SYNAPTOSOMES

—The concentrations of taurine and GABA were determined in isolated mouse brain synaptosomes incubated in Krebs-Ringer phosphate medium (pH 7·4). The concentration of GABA gradually decreased during incubation, but that of taurine remained approximately unchanged. In the presence of chlorpromazine the amount of GABA in the synaptosomes increased, but the efflux and influx of GABA were slightly reduced. The content and efflux of both taurine and GABA increased in electrically stimulated synaptosomes, and the influx of taurine, GABA and glutamate into the synaptosomes similarly increased. All three amino acids are taken up by the synaptosomes through at least two mechanisms: low-affinity and high-affinity. In the high-affinity system the K_m values were 33 μm for taurine, 24 μm for GABA and 68 μm for glutamate, and in the low-affinity one 1·1 mil, 0·9 mm and 1·2mm , respectively. The influx capacity (V_max) was highest for glutamate, second highest for GABA and lowest for taurine.     

A KINETIC ANALYSIS OF SODIUM DEPENDENT GLUTAMIC ACID TRANSPORT IN PERIPHERAL NERVE

—The kinetics of sodium dependent glutamic acid transport have been studied in desheathed frog sciatic nerve. Initial velocities have been measured as a function of both glulamic acid and sodium concentration. Lineweaver–Burk plots are constructed from these data, and the kinetic constants describing uptake are estimated. V_max is unaffected by sodium concentration, which implies that translocation is not directly affected by sodium. K₁ is sodium dependent, which implies that sodium affects the affinity of the carrier for glutamic acid. Reciprocal plots of velocity vs [Na] or K₁ vs 1/[Na] are linear, suggesting that glutamic acid and sodium are co-transported on a one-to-one basis. _t, the sodium concentration giving half maximal velocity of uptake, was found to vary from about 57 mm to 48 mm at glutamic acid concentrations of 1.0–10.0 ± 10^?6m . A model of a mechanism by which sodium and glutamate could be co-transported is presented; the model is in very good agreement with the experimental data.     

l-Proline transport into renal OK epithelial cells: a second renal proline transport system is induced by amino acid deprivation

Influx of [³H]-l-proline into renal OK cells revealed that basal transport was mediated by the transporter SIT1. When cells were submitted for 8 h to amino acid deprivation, uptake of l-proline was now dominated by a low-affinity system with an apparent K _m of 4.4 ± 0.6 mM and a V _max of 10.2 ± 0.6 nmol/mg of protein/min operating in addition to the high-affinity SIT1 system with a K _m of 0.12 ± 0.01 mM and a V _max of 0.28 ± 0.04 nmol/mg of protein/min. The low- and high-affinity proline transporting systems were sensitive to inhibitors of JNK and PI-3 kinases, whereas a GSK-3 inhibitor affected only the upregulated transport system. Ion-replacement studies and experiments assessing substrate specificities for both systems provided strong evidence that SNAT2, that showed two- to threefold increased mRNA levels, is the responsible transporter mediating the increased proline influx under conditions of amino acid deprivation.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

Kinetics of urea uptake by Melosira italica (Ehr.) Kütz at different luminosity conditions

The kinetic parameters K_m and V_max for urea uptake by Melosira italica were determined at 160 μeinsteins m⁻² s⁻¹ and in the dark. The transport systems showed an affinity for the substrate and a storing capacity in the dark (K_m = 65.07 μM; V_max = 2.18 nmoles 10⁵ cells ⁻¹ h⁻¹) greater than under 160 μE m⁻² s ⁻¹ (K_m = 111.2 μM; V_max = 1.11 nmoles 105 cells⁻¹ h⁻¹). Similarly, a reduction in consumption rate of urea under increasing photon flux densities was observed. The use of an inhibitor (potassium cyanide) indicated that the uptake process requires metabolic energy. That urea transport is more important in darkness, may constitute a survival strategy in which this compound is utilized by cells mainly during heterotrophic growth.