INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

TRANSPORT OF TYROSINE, PHENYLALANINE, TRYPTOPHAN AND GLYCINE IN NEUROBLASTOMA CLONES

Amino acid transport was studied in three neuroblastoma clones, N-TD6, which synthesizes norepinephrine, N-T16, which synthesizes small amounts of serotonin, and N-S20Y, which synthesizes acetylcholine. All three clones exhibited high-affinity saturable transport systems for tyrosine, phenylalanine, tryptophan and glycine as well as systems unsaturated at amino acid concentrations of 1 mM in the external medium. Tyrosine, phenylalanine and tryptophan enter all three clones by rapidly exchanging transport systems which appear to be relatively insensitive to lowered external [Na⁺] or to the presence of 2,4-dinitrophenol (DNP). Glycine uptake was slower and was much more sensitive to lowered external [Na⁺] and to the presence of DNP in the medium. Glycine transport in N-T16 cells was decreased more markedly at low temperature than was transport of the three aromatic amino acids. K_m and V_max values found for saturable transport of tyrosine, phenylalanine and tryptophan were sufficiently low to suggest that, if similar amino acid transport systems exist in neuronal membranes, and if amino acid levels in brain extracellular fluid are similar to levels in plasma, such systems may serve, in conjunction with transport systems in cerebral capillaries, to limit the entry of amino acids into brain cells when blood amino levels are near the normal physiological range.     

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.     

EXCHANGE OF TAURINE IN BRAIN SLICES OF ADULT and 7-DAY-OLD RATS

Abstract— The influx or efflux of taurine in brain slices prepared from adult and 7-day-old rats was studied in Krebs-Ringer bicarbonate-glucose medium with 0,2 and 10 m m -taurine. The exchange of taurine between the slices and the medium was slow, and no steady-state concentration was reached within the experimental period of 150 min. In both experimental groups there was a net influx of taurine into the slices from 10 m m -taurine and a slight net efflux from the slices into 2 m m -taurine. The rate of influx from 10 m m -taurine was about the same in the two groups after an initial period of faster influx into the slices of adult rats. There was some rapid initial efflux into 0 and 2 m m -taurine solutions from the slices from 7-day-old rats, but with prolonged incubation these slices were better able to maintain their intracellular taurine than the slices from adult rats. The reasons and significance of the high cerebral concentration of taurine in immature brain in vivo are briefly discussed in the light of the present and earlier studies.     

Mechanism of l-histidine-induced stimulation of amino acid transport in slices of rat cerebral cortex

Effects of l-histidine on the transport of other amino acids were studied with slices of rat cerebral cortex. Histidine (0.5 mM) significantly increased the 60-min accumulation of large neutral and basic amino acids (0.5 mM). The effect was dependent on sodium ions and could be demonstrated in slices from both adult and newborn rats. Other amino acids tested were either ineffective or inhibitory; in particular, l-phenylalanine was strongly inhibitory. The 5-min influx of amino acids into slices was also enhanced by preincubation with histidine. This effect was stereospecific for l-histidine, sodium-independent and not produced by histidine metabolites or activation of histamine H₁ and H₂ receptors. Kinetic analysis of leucine influx showed that the maximal velocity of transport (V) increased relatively more than the other transport parameters. The results could be explained by stimulation of amino acid exchange by intracellular l-histidine. The opposite effects of histidine and phenylalanine on the accumulation of other amino acids are in keeping with the generally less severe impairment of cerebral functions in clinical histidinemia as compared to that in phenylketonuria.     

Transport of threonine and tryptophan by rat brain slices: relation to other amino acids at concentrations found in plasma

Threonine content of brain decreases in young rats fed a threonine-limiting, low protein diet containing a supplement of small neutral amino acids (serine, glycine and alanine), which are competitors of threonine transport in other systems (Tews et al., 1977). Threonine transport by brain slices was inhibited more by a complex amino acid mixture resembling plasma from rats fed the small neutral amino acid supplement than by mixtures resembling plasma from control rats or from rats fed a supplement of large neutral amino acids. Greater inhibition was seen with mixtures containing only the small neutral amino acids than with mixtures containing only large neutral amino acids. On an equimolar basis, serine and alanine were the most inhibitory; large neutrals were moderately so; and glycine and lysine were without effect. Threonine transport was also strongly inhibited by α-amino-n-butyric acid and homoserine, less so by α-aminoisobutyric acid, and not at all by GABA. The complex amino acid mixtures strongly inhibited α-aminoisobutyric acid transport by brain or liver slices but, in contrast to effects in brain, the extent of the inhibition in liver was not much affected by altering the composition of the mixture. Tryptophan accumulation by brain slices was effectively inhibited by other large neutral amino acids in physiologically occurring concentrations. Threonine, or a mixture of serine, glycine and alanine only slightly inhibited tryptophan uptake; basic amino acids were without effect and histidine stimulated tryptophan transport slightly. These results support the conclusion that a diet-induced decrease in the concentration in brain of a specific amino acid may be related to increased inhibition of its transport into brain by increases in the concentrations of transport-related, plasma amino acids.     

DEVELOPMENTAL CHANGES IN Na+, K+ AND ATP AND IN THE LEVELS AND TRANSPORT OF AMINO ACIDS IN INCUBATED SLICES OF RAT BRAIN

Abstract—

• 1 Upon incubation, slices of brain tissue took up fluid; the degree of swelling increased with increasing age. No sweiling occurred in slices from foetal brain. Since this swelling was associated with increases in the inulin space, the percentage of inulin space in slices at the end of incubation increased during brain development.
• 2 Most of the capacity for ion transport seemed to be absent from foetal brain. In vivo and in slices, Na⁺ was very high and K⁺ was very low in comparison to levels at other ages. There was a rapid change around birth, but no significant change at later ages. Upon incubation, Na⁺ levels increased in other slices, but not in slices of foetal brain.
• 3 Upon incubation of the slices, ATP levels were restored to levels close to those in the living brain; there were no significant alterations in available energy during development to explain changes in amino acid transport.
• 4 The composition of the free pool of cerebral amino acids in vivo changed with development, with some compounds (glutamic acid and related compounds) increasing, others (mostly‘essential’amino acids) decreasing, with age. These changes were not linear with time, and the level of a compound might exhibit several peaks during development.
• 5 The uptake (influx) of taurine, glutamate and glycine into brain slices increased rapidly during the foetal and early neonatal periods, reached a maximum between 2 and 3 weeks of postnatal age and then declined to adult levels. The levels of steady-state uptake with glycine also exhibited a maximal peak at 2-3 weeks of postnatal age. Steady-state uptake of taurine and glutamate reached adult levels by about 3 weeks of age.
• 6 The pattern of inhibition of amino acid transport by two specific amino acid analogues changed during development for some amino acids (GABA, glycine and glutamate), indicating an alteration in substrate specificity.
• 7 The results demonstrate complex changes in cerebral amino acid transport during development, with several maxima or minima and with changes in specificity for at least some compounds.

     

Hypotaurine Uptake by Brain Slices from Adult and 8-Day-Old Mice

Abstract: Uptake of [³⁵S]hypotaurine by brain slices prepared from adult and 8-day-old mice was studied at varying temperatures, under O₂ and N₂ atmospheres, and in the presence of metabolic inhibitors and varying concentrations of hypotaurine in the incubation medium. The tissue/medium concentration gradients generated were exceptionally high for an amino acid. Hypotaurine uptake was energy- and temperature-dependent, more strictly in adult mice. Uptake was saturable, containing a high-affinity and a low-affinity component. The estimated transport constants for the high-affinity uptake of hypotaurine (8-day-old mice, 17.2 μ mol/liter; adults, 35.3 μ mol/liter) were of the same order of magnitude as the reported transport constants of putative amino acid transmitters, but the total transport capacity appears to be greatest for hypotaurine.     

Mechanism of l-histidine-induced stimulation of amino acid transport in slices of rat cerebral cortex

Effects of l-histidine on the transport of other amino acids were studied with slices of rat cerebral cortex. Histidine (0.5 mM) significantly increased the 60-min accumulation of large neutral and basic amino acids (0.5 mM). The effect was dependent on sodium ions and could be demonstrated in slices from both adult and newborn rats. Other amino acids tested were either ineffective or inhibitory; in particular, l-phenylalanine was strongly inhibitory. The 5-min influx of amino acids into slices was also enhanced by preincubation with histidine. This effect was stereospecific for l-histidine, sodium-independent and not produced by histidine metabolites or activation of histamine H₁ and H₂ receptors. Kinetic analysis of leucine influx showed that the maximal velocity of transport (V) increased relatively more than the other transport parameters. The results could be explained by stimulation of amino acid exchange by intracellular l-histidine. The opposite effects of histidine and phenylalanine on the accumulation of other amino acids are in keeping with the generally less severe impairment of cerebral functions in clinical histidinemia as compared to that in phenylketonuria.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

Thyroxine-Induced Changes in the Development of Neutral α-Amino Acid Transport Systems of Rat Brain

Abstract: Transport of representative neutral α-amino acids was measured in brain slices after injecting thy-roxine into donor rats of various ages from 1 to 23 days old. The hormone did not alter uptake in slices from 1-day-old rats even when treatment was begun on pregnant rats as much as 10 days before delivery. Injecting thy-roxine until age 6 days, however, decreased the activity of transport system A (the major sodium-dependent system in most mammalian cells) and caused appearance of a new transport system used by the model amino acids, 2-aminoisobutyric acid and 2-(methylamino)isobutyric acid. Uptake at 6 days was similar to that found in slices from older, untreated rats (e.g., those 14 days old). These results strongly suggest that one action of thyroxine is to accelerate the development of neutral α-amino acid transport systems of brain over the first six days after birth. Thyroxine treatment of rats from birth to age 14 days also appears to increase the activities of both system A and the second transport system used by the two model amino acids in brains from 14-day-old rats.     

Blood-Brain Barrier Transport of 1-Aminocyclohexanecarboxylic Acid, a Nonmetabolizable Amino Acid for In Vivo Studies of Brain Transport

Regional transport of 1-aminocyclohexanecarboxylic acid (ACHC), a nonmetabolizable amino acid, across the blood-brain barrier was studied in pentobarbital-anesthetized rats using an in situ brain perfusion technique. The concentration dependence of influx was best described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants of the frontal cortex equaled 9.7 X 10(-4) mumol/s/g for Vmax, 0.054 mumol/ml for Km, and 1.0 X 10(-4) ml/s/g for KD in the absence of competing amino acids. Saturable influx could be reduced by greater than 85% by either L-phenylalanine or 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with transport by the cerebrovascular neutral amino acid transport system. The transport Km for ACHC was one-fifth that for the more commonly used homologue, 1-aminocyclopentanecarboxylic acid, and was similar to values for several natural amino acids, such as L-methionine, L-isoleucine, and L-tyrosine. The results indicate that ACHC may be a useful probe for in vivo studies of amino acid transport into brain.     

Uptake of leucine,lysine, aspartic acid,and glycine into isolated neurons and astrocytes

The uptake of tritium-labeledl-leucine,l-lysine,l-aspartic acid, and glycine by neurons and astrocytes isolated from the cerebral cortex of 3-week-old rats was followed for varying periods up to 40 min at amino acid concentrations from 1 to 2000 mol/liter in medium. The effects of a low-sodium (15.5 mmol/liter) medium on the uptake were also studied. The influx of the amino acids was faster into astrocytes than into neurons. Leucine penetrated into the cells faster than the other amino acids. Amino acid transport was mainly saturable at the lowest amino acid concentrations studied, whereas nonsaturable penetration into the cells dominated in the millimolar concentration range. The saturable transport comprised only one transport system with relatively small transport constants, resembling in nature the so-called high-affinity transport. The maximal velocities of transport were about two times higher in astrocytes than in neurons. In neurons the partial substitution of sodium by choline in medium had the most effect in reducing the influx of glycine and aspartic acid. In astrocytes the effects were generally less pronounced. The results suggest that extracellular amino acids generally penetrate more readily into astrocytes than into neurons. Both cell types transport essential amino acids more effectively than other amino acids.     

Tryptophan and phenylalanine transport in rat cerebral cortex slices as influenced by sodium ions

Tryptophan and phenylalanine transport in rat cerebral cortex slices was studied in sodium-free media and during influx and efflux of sodium ions. Choline as a substitute for sodium in incubation media increased efflux and decreased influx of tryptophan and phenylalanine. Exchange of intracellular [³H]tryptophan and [³H]phenylalanine with extracellular unlabeled histidine, phenylalanine, and tryptophan was sodium-independent. Efflux of sodium ions from the slices had no immediate effects on phenylalanine and tryptophan efflux, but influx decreased. Influx of sodium into the sodium-depleted slices provoked a transient increase in tryptophan and phenylalanine efflux and also enhanced influx. The results are interpreted to indicate that sodium ions may possibly affect the function of the primary transport sites for aromatic amino acids at cerebral membranes by controlling the orientation of their reactive sites towards the intracellular and extracellular sides, rather than by being directly involved in the binding of amino acids to the carriers.     

Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

COMPARTMENTATION OF GLUTAMATE METABOLISM IN THE DEVELOPING BRAIN: EXPERIMENTS WITH LABELLED GLUCOSE, ACETATE, PHENYLALANINE, TYROSINE AND PROLINE

—(1) Phenylalanine, proline and presumably tyrosine are precursors of the small glutamate pool in brain. This follows from the finding that with these precursors the specific radioactivity of glutamine is higher than the specific radioactivity of glutamate. (2) Glucose is not as efficient a precursor of glutamate and related amino acids in the brain of 10-day-old mice as it is in the adult brain. (3) Acetate, phenylalanine, tyrosine and proline are incorporated to about the same extent in glutamate, aspartate and glutamine in the brains of 10-day-old and adult mice. (4) The results suggest that the brain of the immature animal uses substrates other than glucose, relative to glucose better than the brain of adult animals.     

Effect of acute cyanide intoxication on the active transport of amino acids in brain slices

(1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices. (2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used. (3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals. (4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals. (5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices. (6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.     

EVIDENCE FOR SEPARATE SYSTEMS FOR THE TRANSPORT OF NEUTRAL AND BASIC AMINO ACIDS ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The effects of high circulating concentrations of several amino acids on the free amino acids of rat brain were measured, to see whether or not the results followed any consistent pattern. High circulating concentrations of large, neutral amino acids (phenylalanine, valine or isoleucine) caused significantly decreased values only of other large, neutral amino acids in the brains. High circulating concentrations of the basic amino acids lysine or arginine caused significantly decreased values only of each other. The data suggest that there are separate systems for the transport of neutral and basic amino acids across the blood-brain barrier. The effects of valine and lysine on the uptake by brain and the con-vulsant action of allylglycine (a neutral amino acid) were consistent with the concept of separate systems for the transport of amino acids across the blood-brain barrier. Valine inhibited the uptake by brain and the convulsant action of allylglycine in mice, but lysine did not. The data suggest that allylglycine and valine are transported into the brain by a common mechanism that does not transport lysine.     

HIGH AFFINITY UPTAKE OF TRANSMITTERS: STUDIES ON THE UPTAKE OF l-ASPARTATE, GABA, l-GLUTAMATE AND GLYCINE IN CAT SPINAL CORD

Abstract— The uptake of l -aspartate, l -glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent K_m's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically-sensitive particles of equilibrium density equivalent to that of 1.0 m -sucrose were at least in part responsible for the uptake of these amino acids. Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids:

• 1 specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord;
• 2 specific for GABA—not inhibited by glycine, taurine, l -aspartate or l -glutamate and (3) specific for l -aspartate and l -glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l -cysteate and l -cysteine sulphinate.

The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l -glutamate diethyl ester (l -aspartate and l -glutamate). p-Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l -aspartate/l -glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l -aspartate or l -glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.