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 more rapid in newborn, but for high concentrations the rate is greater in adult tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ than those of the adult for high affinity system but adult synaptosomes have a higher $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ 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 $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system for lysine uptake.     

DEVELOPMENTAL AND OTHER ASPECTS OF [35S]CYSTEINE TRANSPORT BY RAT BRAIN SYNAPTOSOMES

Abstract— Cysteine uptake by rat brain synaptosomes occurs by active transport. The uptake by synaptosomes isolated from newborn brain is slower and the concentration gradient achieved is lower than that observed in adult tissue. Synaptosomal fractions from both adult and newborn rat brains accumulate cysteine by two saturable systems. The calculated parameters show that the maximum rates of cysteine uptake in adult synaptosomes are approximately twice that observed in newborn synaptosomes for both the high and low affinity systems. The uptake by the high affinity system is sodium dependent and is inhibited by glycine and dibasic amino acids. Uptake by synaptosomes from 14-day-old animals is close to that observed in adult tissue. The uptake of cysteine differs greatly from that of cystine since the oxidized form, cystine, is taken up more slowly by systems with low affinities which are sodium independent, do not interact with dibasic amino acids and are independent of age.     

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.     

l-[35S]CYSTINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of [³⁵S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent K _m values of 0.14 and 1.4 m m . The low K _m system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.     

A Comparative Study and Partial Characterization of Multi-Uptake Systems for γ-Aminobutyric Acid

Previous work by the authors had indicated that synaptosome-enriched preparations from the cerebral cortex of the rat contained a high-, a medium-, and a low-affinity uptake system for gamma-aminobutyric acid (GABA). The present study demonstrated that this phenomenon also prevailed in synaptosomes from rat diencephalon, mesencephalon, and cerebellum, although the Vmax values for the high- and medium-affinity systems in the cerebellum were very low relative to those of the other regions. When a different type of preparation containing nerve endings (glomeruli) was obtained from the cerebellum, it possessed a Vmax value for the high-affinity system that was more similar to that for the corresponding system in synaptosomes from the other brain regions. In contrast to the above situation, synaptosomes from rat olfactory bulb lacked the low-affinity uptake system, as did synaptosomes from dog olfactory bulb. The aspartate/glutamate uptake systems, as measured with D-aspartate, provided a regional pattern quite different from those of GABA uptake. Only two uptake systems, a high- and low-affinity system, were observed in all regions tested. All three GABA uptake systems were present in cortical synaptosomes from the mouse, hamster, and guinea pig, and all three systems were sodium dependent, energy dependent, temperature sensitive, and totally inhibited by nipecotic acid.     

Synaptosomal Uptake and Release of Dopamine in Rat Striatum After Hypoxia

Hypoxia induces alterations of central monoaminergic transmission and of behavior. We studied the effect of hypoxia on adult and newborn rats to obtain more information about long-lasting changes of dopamine (DA) transmission caused by neonatal hypoxia. One single exposure of adult rats to hypoxia leads to short-term alterations of DA uptake: decreased affinity of the uptake carrier to DA (Km, 269.5% versus control) and a sharp increase of Vmax up to 301.4% resulting in an increase of total uptake of DA into the striatum synaptosomes. The K+-evoked DA release decreased to 69.5%. After 1 week of recovery all parameters are normalized. Chronic postnatal hypoxia (postnatal day 2-11) caused long-lasting changes of DA release and uptake opposite to those observed in adult rats. Three months after hypoxia, the K+-stimulated DA release was enhanced (132% of control), and the uptake was reduced due to decreased affinity of the uptake carrier system for the substrate (Km, 187% of control value). In conclusion, the alterations observed after chronic postnatal hypoxia reflect special adaptive processes that are related to the high plasticity of the immature neonatal brain and contribute to an increased DA function in the nigrostriatal system.     

SELECTIVE LOCALIZATION OF A HIGH AFFINITY CHOLINE UPTAKE SYSTEM AND ITS ROLE IN ACh FORMATTON IN CHOLINERGIC NERVE TERMINALS

The avian iris-ciliary nerve preparation exhibits two distinct choline uptake systems. One component, a sodium dependent, high affinity system Km-2 am and Vmax - 0.5 pmolpin per preparation is confined to nerve terminals. The other component is localized in muscle cells. It is sodium independent and low affinity system (Km - 200 am and Vmax - 16 pmol/min per muscle). The high affinity uptake of choline and the synthesis of ACh in the nerve terminals are coupled. Vmax Ach formation -0.5 pmol/min. is the same as Vmax for choline transport; however. with the external choline concentration equal to that of avian plasma only -50% of choline taken up is converted to ACh. In contrast to the nerve terminals, the cell bodies of the same neurons are deficient in the high affinity uptake-ACh synthesis coupled system. This indicates a nerve terminal membrane specialization related to neuro-transmitter synthesis.     

Ethanol intake and 3H-serotonin uptake I: A study in Fawn-Hooded rats

Ethanol intake and synaptosomal 3H-serotonin uptake were studied in male Fawn-Hooded and Sprague-Dawley rats. Fawn-Hooded rats consumed more alcohol and more water than Sprague-Dawley rats. Plasma alcohol levels of Sprague-Dawley rats were not detectable but were about 5 mg/dl in Fawn-Hooded rats. Ethanol intake increased the Vmax of serotonin uptake in Fawn-Hooded rats in hippocampus and cortex, but not in thalamus. In Fawn-Hooded rats, serotonin uptake (Vmax) was higher than in Sprague-Dawley rats cortex. Ethanol intake reduced the Vmax of serotonin uptake in Fawn-Hooded rats in hippocampus and cortex. In cortex, the carrier affinity for serotonin was increased in alcoholized Fawn-Hooded rats. These results indicate that synaptosomal 3H-serotonin uptake is affected by ethanol intake. In Fawn-Hooded rats, high ethanol consumption is associated with high serotonin uptake. In rats presenting high serotonin uptake, alcoholisation reduces 3H-serotonin internalisation in synaptosomes, indicating a specific sensitivity to alcohol intake of serotonin uptake system.     

On the development of glycine transport systems by rat renal cortex

The initial uptake of glycine by renal cortical slices from newborn Sprague-Dawley and Long-Evans rats is the same as that observed in adult tissues. Both newborn and adult tissue possess similar high and low affinity glycine transport systems which require an examination of velocity measurements over a wide range of concentration (0.02--50.0 mM) for their discernment. Initial rates of glycine uptake by isolated renal tubule fragments from newborn and adults are similar at a physiological substrate concentration but at high glycine levels there appears to be a decrease in velocity of uptake (V) associated with the high Km system in the young. Whatever preparation of renal cortex is studied, there is a consistent finding that immature tissue is able to accumulate much higher intracellular levels of glycine than the adult, a finding consistent with slower efflux from the cell. An interpretation of the etiology of physiologic aminoaciduria in young animals should take this into account.     

5-fluorotryptophan resistant mutants affecting the A and L transport systems in the mouse L cell line A9

Tryptophan transport has been examined in A9 and in mutants resistant to 5-fluorotryptophan (5-FT). Evidence indicates that in A9 cells two systems are present for tryptophan transport, which are analogous to the A and L systems found in Ehrlich ascites cells differing, however, in terms of amino acid specificity. Tryptophan uptake via the L system, a high affinity, low capacity system, is Na⁺ independent and occurs by a counter transport mechanism, while uptake via the A system, a low affinity, high capacity system, is Na⁺ dependent. Alanine, arginine, lysine, proline, asparagine, and aspartate (listed in order of decreasing inhibitory effect) inhibit tryptophan uptake via the A system from approximately 80-50% while having no inhibitory effect on the L system. In addition, glutamine which inhibits tryptophan uptake by 80% via the L system only inhibits to the extent of 20% via the A system. Previous kinetic studies of 5FT resistant clone FT^r37 indicated system A was altered while the analysis of the effects of the mutation on system L was inconclusive. However, in these studies Na⁺ independent uptake was not altered in FT^r 37 indicating system L was not affected. Amino acid competition studies confirmed this observation and suggested that a change in the specificity of system A had occurred in FT^r 37. The amino acid competition studies in FT^r 23, indicated that the specificities of both systems differed from A9. The possibility that this change may be due to a single mutational event is discussed.     

Evolvement of systems of serotonin uptake in rat hypothalamus during its development]

Kinetics of 3H serotonin accumulation into slices from hypothalamus have been compared in adult, puppy and foetus rat. In 15 days-old, as in adult rat, there are two components of 5 HT accumulation corresponding to the low and high affinity transport systems. For this latter, Km and Vmax values are much higher in adult than in 15 days old rat (in adult, Km=1,3 X 10(-7) and Vmax=0,33 X 10(-10); in 15 days old rat, Km=0,5 X 10(-7) and Vmax=0,125 X 10(-10)). On the opposite, in the 7 days old rat and in the 21 days old foetus, it is only possible to arbitrarely define one uptake system corresponding to the following apparent values: in the 7 days old rat, Km= 5 X 10(-7) and Vmax=2 X 10(-10), in the foetus, Km=0,2 X 10(-7) and Vmax=0,15 X 10(-10). These results showed important developmental differences in affinity of 3H serotonin to hypothalamus. The low and high affinity uptake systems existing in adult are only individualized in the 15 days old little rat.     

The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential

The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.     

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.     

Developmental pattern of cystine transport in isolated rat renal tubules

Isolated renal cortical tubule fragments from rats ranging in age from less than 48 h to 15 weeks were used to examine the pattern of cystine uptake with development. Immature tubules took up cystine with a faster initial rate than mature tubules and did not reach a steady state by 60 min. By eight weeks of age, the timed uptake of cystine began to approach a steady state and between 8 and 11 weeks the uptake pattern achieved its adult form of reaching a steady state by 30 min of incubation. Analysis of the intracellular metabolism of the cystine taken up by the newborn tubules revealed that the majority had been reduced to cysteine with the formation of small amounts of reduced glutathione. Cystine entered the renal cortical tubule cell from the newborn via two saturable transport systems similar to the mature animal. The kinetic parameters of initial uptake of these two transport systems were similar in the mature and newborn animal except for a higher maximum transport velocity for the low K_m, low capacity system in the newborn. Lysine inhibited cystine uptake by newborn tubules and this inhibition appeared to occur on the low K_m, low capacity transport system similar to the adult. Cystine uptake was sodium dependent with an apparent affinity for sodium of 36 mequiv./l. From this data, the physiologic cystinuria of the immature animal does not appear to be referrable to a lower rate of influx as previously observed with the cortical slice. Other mechanisms should be sought to explain this phenomenon of immaturity.     

GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (K_m = 300 μ m ) and predominates in cerebral cortex, cerebellum and mid-brain, the other has a higher affinity for glycine (K_m = 40 μ m ) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro , and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p -chloro-mercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.     

ON THE UPTAKE OF INOSITOL BY RAT BRAIN SYNAPTOSOMES

The uptake of inositol by rat brain synaptosomes occurs via an unsaturable process that even at substrate concentrations as low as 1 μM is unable to achieve a concentration gradient indicative of active transport. Dinitrophenol, ouabain and cytochalasin B did not affect uptake of the cyclitol. The data indicate that inositol uptake by rat synaptosomes occurs by diffusion or by a system with an affinity so low it is difficult to discern. The low capacity, saturable inositol uptake system observed in rabbit brain slices may reflect a species difference or uptake by elements of the slice other than neuronal membranes.     

COMPARATIVE STUDIES ON SYNAPTOSOMES: UPTAKE OF [N-Me-3H]CHOLINE BY SYNAPTOSOMES FROM SQUID OPTIC LOBES

Abstract— The uptake of [ N -Me-³H]choline into synaptosomes from squid optic lobes was studied using a Millipore filtration technique. When incubated in an artificial sea water medium at 26°C, but not at 0°C, the synaptosomes rapidly accumulated choline, most of which could be recovered as unchanged free choline. The accumulated choline was readily released by treatment of the synaptosomes with Triton X-100 or exposing them to hypo-osmotic conditions. The influx of choline increased with increasing concentrations of choline and could be resolved into saturable and non-saturable components. Kinetic analysis revealed the presence of two saturable components one of high affinity ( K _m about 2 μ m ) and one of lower affinity ( K _m 25 μ m ). The rate of choline uptake by these synaptosomes was considerably greater than by mammalian brain synaptosomes. Both high and low affinity systems were Na⁺-requiring and inhibited by hemicholinium no. 3, levorphanol and dextrorphan. NaCN, 2,4-dinitrophenol and ouabain also inhibited choline uptake, the high affinity system being particularly sensitive to these agents. It is suggested that the high affinity system is specific for cholinergic terminals.     

Adaptive changes in phosphate uptake by the fungus Neurospora crassa in response to phosphate supply.

The phosphate uptake rate of Neurospora crassa germlings growing exponentially in media containing phosphate at concentrations between 10 mM and 50 micronM was virtually constant. The uptake characteristics of these germlings were studied in detail assuming the simultaneous operation of two uptake systems, one of low affinity and one of high affinity. The Km of the low-affinity system was constant after growth at phosphate concentrations greater than 1 mM but became progressively lower as the concentration was reduced below 1 mM. In contrast, the Km of the high-affinity system was independent of the phosphate concentration of the growth medium. The Vmax of each system was highest after growth at low phosphate concentrations. As the phosphate concentration was increased to a maximum of 100 mM, the Vmax of the low-affinity system fell gradually, whereas that of the high-affinity system at first fell rapidly but then reached a constant minimum value at concentrations of 2.5 mM and higher. The differences in the kinetic parameters fully account for the constancy of uptake rate shown by the germlings.     

Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.