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Aim Serotonin is crucial for proper foetal development, and the placenta has been described as a 'donor' of serotonin for the embryo/foetus. However, in later stages of gestation the foetus produces its own serotonin from maternally-derived tryptophan and placental supply is no longer needed. We propose a novel model of serotonin homeostasis in the term placenta with special focus on the protective role of organic cation transporter 3 (OCT3/SLC22A3). Methods Dually perfused rat term placenta was employed to quantify serotonin/tryptophan transport and metabolism. Placental membrane vesicles isolated from human term placenta were used to characterize serotonin transporters on both sides of the syncytiotrophoblast. Results We obtained the first evidence that serotonin is massively taken up from the foetal circulation by OCT3. This uptake is concentration-dependent and inhibitable by OCT3 blockers of endogenous (glucocorticoids) or exogenous (pharmaceuticals) origin. Population analyses in rat placenta revealed that foetal sex influences placental extraction of serotonin from foetal circulation. Negligible foetal serotonin levels were detected in maternal-to-foetal serotonin/tryptophan transport and metabolic studies. Conclusion We demonstrate that OCT3, localized on the foetus-facing membrane of syncytiotrophoblast, is an essential component of foeto-placental homeostasis of serotonin. Together with serotonin degrading enzyme, monoamine oxidase-A, this offers a protective mechanism against local vasoconstriction effects of serotonin in the placenta. However, this system may be compromised by OCT3 inhibitory molecules, such as glucocorticoids or antidepressants. Our findings open new avenues to explore previously unsuspected/unexplained complications during pregnancy including prenatal glucocorticoid excess and pharmacotherapeutic risks of treating pregnant women with OCT3 inhibitors.

Vitamin B-6 deficiency is associated with impaired tryptophan metabolism because of the coenzyme role of pyridoxal 5′-phosphate (PLP) for kynureninase and kynurenine aminotransferase. To investigate the underlying mechanism, we developed a mathematical model of tryptophan metabolism via the kynurenine pathway. The model includes mammalian data on enzyme kinetics and tryptophan transport from the intestinal lumen to liver, muscle, and brain. Regulatory mechanisms and inhibition of relevant enzymes were included. We simulated the effects of graded reduction in cellular PLP concentration, tryptophan loads and induction of tryptophan 2,3-dioxygenase (TDO) on metabolite profiles and urinary excretion. The model predictions matched experimental data and provided clarification of the response of metabolites in various extents of vitamin B-6 deficiency. We found that moderate deficiency yielded increased 3-hydroxykynurenine and a decrease in kynurenic acid and anthranilic acid. More severe deficiency also yielded an increase in kynurenine and xanthurenic acid and more pronounced effects on the other metabolites. Tryptophan load simulations with and without vitamin B-6 deficiency showed altered metabolite concentrations consistent with published data. Induction of TDO caused an increase in all metabolites, and TDO induction together with a simulated vitamin B-6 deficiency, as has been reported in oral contraceptive users, yielded increases in kynurenine, 3-hydroxykynurenine, and xanthurenic acid and decreases in kynurenic acid and anthranilic acid. These results show that the model successfully simulated tryptophan metabolism via the kynurenine pathway and can be used to complement experimental investigations.

Exercise raises brain serotonin release and is postulated to cause fatigue in athletes; ingestion of branched-chain amino acids (BCAA), by competitively inhibiting tryptophan transport into brain, lowers brain tryptophan uptake and serotonin synthesis and release in rats, and reputedly in humans prevents exercise-induced increases in serotonin and fatigue. This latter effect in humans is disputed. But BCAA also competitively inhibit tyrosine uptake into brain, and thus catecholamine synthesis and release. Since increasing brain catecholamines enhances physical performance, BCAA ingestion could lower catecholamines, reduce performance and thus negate any serotonin-linked benefit. We therefore examined in rats whether BCAA would reduce both brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Sedentary and exercising rats received BCAA or vehicle orally; tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis rates were measured 1 h later in brain. BCAA reduced brain tryptophan and tyrosine concentrations, and serotonin and catecholamine synthesis. These reductions in tyrosine concentrations and catecholamine synthesis, but not tryptophan or serotonin synthesis, could be prevented by co-administering tyrosine with BCAA. Complete essential amino acid mixtures, used to maintain or build muscle mass, were also studied, and produced different effects on brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Since pharmacologically increasing brain catecholamine function improves physical performance, the finding that BCAA reduce catecholamine synthesis may explain why this treatment does not enhance physical performance in humans, despite reducing serotonin synthesis. If so, adding tyrosine to BCAA supplements might allow a positive action on performance to emerge.

Purpose:Serotonin, a neurotransmitter synthesized from tryptophan, has been proposed to play a key role in central fatigue. In this study, we examined whether tryptophan itself and/or its two metabolites, kynurenic acid (KYNA) and quinolinic acid (QUIN), are involved in central fatigue.Materials and Methods:Experiments were conducted using Sprague-Dawley rats (SDR) and Nagase analbuminemic rats (NAR). Central fatigue was assessed by treadmill running and a Morris water maze test. Microdialysis was used to collect samples for measurement of extracellular concentration of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) and to infuse test agents. To examine the kinetics of release, synaptosomes in the striatum were prepared in vitro to measure intra- and extrasynaptosomal concentration of tryptophan, serotonin and 5-HIAA.Results:The concentration of tryptophan secreted into the extracellular space of the striatum was higher during fatigue only, and quickly returned to basal levels with recovery from fatigue. Running time to exhaustion was reduced by activation of tryptophan receptors. Time to exhaustion was shorter in NAR, which maintain a higher extracellular level of striatum tryptophan than SDR. Impaired memory performance in a water maze task after tryptophan treatment was attributable to high levels of KYNA and QUIN in the hippocampus acting synergistically on N-methyl-D-aspartic acid receptors. When branched-chain amino acids were administered, tryptophan transport to the extracellular space of the striatum was drastically inhibited.Conclusion:Our findings demonstrate that the increase in fatigue which occurs because of excessively elevated brain tryptophan can be further amplified by the use of synthetic KYNA and QUIN.

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.

The binding of L-triiodothyronine (T3) to rat erythrocyte membranes (ghosts and peripheral protein-depleted vesicles) was studied under equilibrium conditions. Ghosts contained high-affinity T3 binding sites whose dissociation constant (21 nM) was similar to the equilibrium-exchange Michaelis constant of T3 transport measured in ghosts. Each ghost contained about 8.10(3) high-affinity binding sites. The high-affinity T3 binding was stereospecific and was inhibited by L-tryptophan (Trp) but not by L-leucine. The iodothyronine and amino acid specificity of binding is therefore similar to that of System T, the erythrocyte T3/Trp transporter. These Trp-inhibitable high-affinity T3-binding sites were also present in peripheral protein-depleted membrane vesicles, indicating that they are integral part of the membrane. Ghosts prepared from human erythrocytes, which have very low System T transport activities, contained no detectable Trp-inhibitable high-affinity T3-binding sites. In rat erythrocyte ghosts, N-ethylmaleimide inactivated both the binding and the transport of T3. This inactivation was blocked by T3 and Trp with similar efficiencies. Phenylglyoxal, an arginine residue modifier, also inhibited both high-affinity T3 binding and System T transport activity. It is concluded that the Trp-inhibitable high-affinity T3-binding sites in the rat erythrocyte membrane are likely to be associated with System T.

Mammalian cells rely on cellular uptake of the essential amino acid tryptophan. Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation, indoleamine 2,3-dioxygenase (IDO), e.g., in cancer and inflammation, is thought to suppress the immune response via T cell starvation. Additionally, the excreted tryptophan catabolites (kynurenines) induce apoptosis of lymphocytes. Whereas tryptophan transport systems have been identified, the molecular nature of kynurenine export remains unknown. To measure cytosolic tryptophan steady-state levels and flux in real time, we developed genetically encoded fluorescence resonance energy transfer nanosensors (FLIPW). The transport properties detected by FLIPW in KB cells, a human oral cancer cell line, and COS-7 cells implicate LAT1, a transporter that is present in proliferative tissues like cancer, in tryptophan uptake. Importantly, we found that this transport system mediates tryptophan/kynurenine exchange. The tryptophan influx/kynurenine efflux cycle couples tryptophan starvation to elevation of kynurenine serum levels, providing a two-pronged induction of apoptosis in neighboring cells. The strict coupling protects cells that overproduce IDO from kynurenine accumulation. Consequently, this mechanism may contribute to immunosuppression involved in autoimmunity and tumor immune escape., Author Summary Although regulated suppression of the immune system prevents autoimmunity and is important during pregnancy to protect the fetus or after organ transplant to block graft rejection, it can be harmful if co-opted by tumors to escape detection. T cells of the immune system normally recognize and destroy abnormal cells, including cancerous and grafted tissues. This process requires the amino acid tryptophan. Foreign grafts and cancer cells can dampen the immune response by starving T cells of tryptophan through a mechanism involving uptake and conversion to kynurenines using an enzyme called indoleamine-2,3-dioxygenase (IDO). Independent of tryptophan starvation, kynurenines induce T cell death when they are excreted through an unknown mechanism. We constructed fluorescent nanosensors that report tryptophan levels in two immortalized cell lines, COS-7 cells and human oral cancer KB cells. Using single-cell, real-time imaging analysis, we demonstrate that an L-amino acid transporter protein (LAT1) exchanges tryptophan for its kynurenine degradation products. LAT1 together with IDO forms a metabolic minicycle that couples tryptophan starvation with kynurenine-induced cell death, providing a two-pronged inactivation of neighboring cells. The strict coupling protects the cells that overproduce IDO from accumulating kynurenine. This cycle may contribute to suppression of the immune response in autoimmune diseases and cancer., Genetically encoded FRET nanosensors were developed to measure cytosolic tryptophan steady-state levels and flux in real time.

Tryptophan catabolism via the enzyme indoleamine 2,3-dioxygenase (IDO) allows human monocyte-derived macrophages (MDM) and other APC to suppress T cell proliferation. IDO helps protect murine fetuses from rejection by the maternal immune system and can promote tolerance and immunosuppression. For tryptophan to be catabolized by IDO, it must first enter the APC via transmembrane transport. It has been shown that MDM in vitro readily deplete tryptophan present in the extracellular medium to nanomolar levels via IDO activity; yet, no currently known amino acid transport system displays high affinity and specificity sufficiently to permit efficient uptake of tryptophan at these low concentrations. Here, we provide biochemical characterization of a novel transport system with nanomolar affinity and high selectivity for tryptophan. Tryptophan transport in MDM was predominantly sodium-independent and occurred via two distinct systems: one consistent with the known system L transporter and a second system with 100-fold higher affinity for tryptophan (Km

Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [(3)H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced.

Gamma-hydroxybutyrate (GHB) is both a therapeutic agent and a recreative drug. It has sedative, anxiolytic and euphoric effects. These effects are believed to be due to GHB-induced potentiation of cerebral GABAergic and dopaminergic activities, but the serotonergic system might also be involved. In this study, we examine the effects of pharmacological doses of GHB on the serotonergic activity in rat brain. Administration of 4.0 mmol/kg i.p. GHB to rats induces an accumulation of tryptophan and 5-HIAA (5-hydroxyindole acetic acid) in the frontal cortex, striatum and hippocampus without causing significant change in the tissue serotonin content. In the extracellular space, GHB induced a slight decrease in serotonin release. The tryptophan and 5-HIAA accumulation induced by GHB is mimicked by the GHB receptor agonist para-chlorophenyl-transhydroxycrotonate (NCS-356) and blocked by NCS-382 (6,7,8,9-tetrahydro-5-[H]-benzocycloheptene-5-ol-4-ylidene acetic acid) a selective GHB receptor antagonist. GHB induces the accumulation of either a derivative of or [3H]-tryptophan itself in the extracellular space, possibly by increasing tryptophan transport across the blood-brain barrier. The blood content of certain neutral amino-acids, including tryptophan, is also increased by peripheral GHB administration. Some of the effect of GHB could be reproduced by baclofen and reduced by the GABAB antagonist CGP 35348. Taken together, these results indicate that the GHB-induced stimulation of tissue serotonin turnover may be due to an increase in tryptophan transport to the brain and in its uptake by serotonergic cells. As the serotonergic system may be involved in the regulation of sleep, mood and anxiety, the stimulation of this system by high doses of GHB may be involved in certain neuropharmacological events induced by GHB administration.

1. A study was conducted to characterise the development of amino acid transport in broiler chicks, using L-tryptophan as a model. The chicks were maintained on a broiler starter diet between hatch and 21 d of age. 2. There was a significant reduction in the rate of uptake of 0.04 mM L-tryptophan with age in both the jejunum and ileum. Uptake was enhanced in the presence of 50 mM sodium chloride to different degrees depending on age and intestinal site. At both intestinal sites, uptake capacity increased with age while there was a reduction in uptake efficiency with age. 3. At a concentration of 25 mM, both sodium chloride and potasium chloride increased uptake by ileal brush-border membrane vesicles (BBMV) of 7-d-old chicks but uptake was reduced when potassium chloride was included at a concentration of 50 mM. In the presence of valinomycin, uptake by jejunal BBMV was stimulated by 25 mM sodium chloride. In the presence of both sodium chloride and potassium chloride and in the absence of valinomycin, uptake was increased by 42.6% but this was reduced to 23.4% when the ionophore was included in the buffer. 4. The Na+-independent uptake of L-tryptophan into jejunal vesicles of 21-d-old chicks was lower in the presence of D-tryptophan than in the presence of 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH). The inclusion of BCH in the incubation medium at low concentrations significantly enhanced the uptake of 0.04 mM L-tryptophan by jejunal BBMV. 5. At similar concentration (0.04 mM) to L-tryptophan, lysine, methionine and alanine in the presence of Na+ also stimulated L-tryptophan uptake. The uptake of L-tryptophan was reduced at a higher concentration, 25 mM, of these amino acids. 6. The study revealed a decline in rate of amino acid uptake and an increase in total uptake capacity with age. Tryptophan uptake was both Na+-independent and dependent, and occurred more in the ileum than in the jejunum. The uptake of L-tryptophan depended on the concentration of other amino acids and other factors in the diet and digesta.

Amino acid supply in brain is regulated by the activity of the large neutral amino acid transporter (LAT) at the brain capillary endothelial cell, which forms the blood–brain barrier (BBB) in vivo . Bovine BBB poly(A) + RNA was isolated from 2.0 kg of fresh bovine brain and size fractionated on a sucrose density gradient, and a size-fractionated bovine BBB cDNA library in the pSPORT vector was prepared. The full-length cDNA encoding the bovine BBB LAT was isolated from this library, and the predicted amino acid sequence was 89–92% identical to the LAT1 isoform. The bovine BBB LAT1 mRNA produced a 10-fold enhancement in tryptophan transport into frog oocytes coinjected with bovine BBB LAT1 mRNA and the mRNA for 4F2hc, which encodes the heavy chain of the heterodimer. Tryptophan transport into the mRNA-injected oocytes was sodium independent and was specifically inhibited by other large neutral amino acids, and the K m of tryptophan transport was 31.5 ± 5.5 μM. Northern blotting with the bovine BBB LAT1 cDNA showed that the LAT1 mRNA is 100-fold higher in isolated bovine brain capillaries compared with C6 rat glioma cells or rat brain, and the LAT1 mRNA was not detected in rat liver, heart, lung, or kidney. These studies show that the LAT1 transcript is selectively expressed at the BBB compared with other tissues, and the abundance of the LAT1 mRNA at the BBB is manyfold higher than that of transcripts such as the 4F2hc antigen, actin, or the Glut1 glucose transporter.

We have identified both N-ethylmaleimide (NEM)-resistant (system T) and NEM-sensitive (system L1) L-[3H]tryptophan transporters in sinusoidal membrane vesicles (SMVs) from euthyroid, hypothyroid (propylthiouracil-treated), and hyperthyroid [L-3,5,3'-triiodothyronine (L-T3)-injected] rats. L-[125I]T3 associates with SMVs largely by surface binding. Kinetic characteristics of tryptophan uptake and T3 binding (transporter or receptor abundance and substrate affinity) are not significantly affected by thyroid status. T3 and thyroxine (T4) inhibit NEM-resistant tryptophan uptake in SMVs to an extent dependent on the thyroid status of the donor rat, increasing in the order hypothyroid < euthyroid < hyperthyroid; the inhibitor constant for this inhibition (0.3 microM T3) is equal to the dissociation constant for T3 binding. Both T3 binding and T3 inhibition of tryptophan transport in SMVs are markedly reduced by treatments (Triton X-100 or trypsin) that do not significantly affect vesicle integrity or transport of tryptophan and glucose. T3 and/or T4 transport at the liver-plasma interface may be facilitated by direct interactions between hormone receptors and system T transporter proteins. Modulation of such interactions may be important for control of hepatic T4 and/or T3 turnover and aromatic amino acid metabolism during altered thyroid status.

Liver failure, or shunting of intestinal blood around the liver, results in hyperammonemia and cerebral dysfunction. Recently it was shown that ammonia caused some of the metabolic signs of hepatic encephalopathy only after it was metabolized by glutamine synthetase in the brain. In the present study, small doses of methionine sulfoximine, an inhibitor of cerebral glutamine synthetase, were given to rats either at the time of portacaval shunting or 3-4 weeks later. The effects on several characteristic cerebral metabolic abnormalities produced by portacaval shunting were measured 1-3 days after injection of the inhibitor. All untreated portacaval-shunted rats had elevated plasma and brain ammonia concentrations, increased brain glutamine and tryptophan content, decreased brain glucose consumption, and increased permeability of the blood-brain barrier to tryptophan. All treated rats had high ammonia concentrations, but the brain glutamine content was normal, indicating inhibition of glutamine synthesis. One day after shunting and methionine sulfoximine administration, glucose consumption, tryptophan transport, and tryptophan brain content remained near control values. In the 3-4-week-shunted rats, which were studied 1-3 days after methionine sulfoximine administration, the effect was less pronounced. Brain glucose consumption and tryptophan content were partially normalized, but tryptophan transport was unaffected. The results agree with our earlier conclusion that glutamine synthesis is an essential step in the development of cerebral metabolic abnormalities in hyperammonemic states.

The Na(+)-independent neutral amino acid transporter (NAA-Tr) that we had previously cloned from rat kidney has been investigated with respect to its distribution in mammalian tissues and cells. By Northern blot analysis and RNase protection assay, a 2.4-kilobase (kb) mRNA in rat intestine was found to be identical to that in rat kidney. Of the other rat tissues examined, only brain and heart were found to contain mRNAs related to kidney NAA-Tr by Northern assay. However, these were larger (approximately 5 and approximately 7 kb). Mouse and rabbit kidney also contain mRNAs of 2.4 kb that exhibited a high degree of homology with rat kidney NAA-Tr. Of the several cultured cells investigated that demonstrated considerable Na(+)-independent neutral amino acid transport activity, only human colon carcinoma (Caco) cells were positive by Northern assay. The failure to detect NAA-Tr mRNA in many cells and tissues that carry out Na(+)-independent transport indicates that unrelated transporters must also exist. Cells and tissues that were negative with respect to rat kidney NAA-Tr as well as those that were positive transported leucine and tryptophan equally well. However, when mRNA from the same cells and tissues was expressed in oocytes, in all cases tryptophan was transported far less efficiently than leucine. This defect in tryptophan transport is apparently due to aberrant expression of neutral amino acid transporters in general in Xenopus oocytes.

1. Tegumental transport of choline at concentrations ranging from 0.005–5.0 mM provided no evidence for saturable, carrier-mediated entry of this amine in the tegument of the rat tapeworm (Hymenolepis diminuta). 2. In contrast, the large neutral amino acid tryptophan appears to be taken up via a high-affinity transporter. In the 1st quartile of 17-day-old tapeworms (Km = 0.033 mM, Vmax = 0.7 nmol·min−1 · g−1), in the 2nd quartile (Km = 0.015 mM, Vmax = 0.3 nmol·min−1 · g−1), in the 3rd quartile (Km = 0.022 mM, Vmax = 0.5 nmol · min−1 · g−1) and in the 4th quartile (Km = 0.025 mM, Vmax = 0.5 nmol · min−1 · g−1) saturable tryptophan transport was kinetically characterized. 3. The non-saturable diffusion component (Kd) for tryptophan transport ranged from 3.8–10.2 μl · min−1 · g−1. 4. These studies suggest choline does not appear to be transported across the tapeworm tegument. Saturable transport of tryptophan via a high-affinity carrier is reported, and no regional variations in indole amino acid uptake were detected.

The kinetic properties of transport system T, which is specific for uptake of aromatic amino acids, were studied in rat erythrocytes in the presence of leucine in order to block the neutral amino acid transport system L. Since the triiodothyronine (T3) transport system and system T are closely related, the trans effect of T3 and tryptophan on [3H]tryptophan transport and the trans effects of aromatic amino acids on [125I]T3 transport were studied. Equilibrium-exchange, zero-trans and infinite-trans studies of [3H]tryptophan transport indicated that system T in rat erythrocytes is a simple carrier with exchanging properties resulting in trans-acceleration of influx and trans-inhibition of efflux when tryptophan was present at the trans side of the membrane. In erythrocytes preloaded with unlabelled tryptophan, countertransport resulted in a 7-fold accumulation of labelled substrate inside the cells. T3 on the trans side of the membrane inhibited both influx and efflux of tryptophan, with Ki values similar to the Km values of the T3 transport system. Extracellular tryptophan trans-inhibited [125I]T3 efflux in a manner similar to [3H]tryptophan efflux. Preloading erythrocytes with tryptophan resulted in trans-acceleration of T3 uptake and a transient 5-fold accumulation of free T3 into erythrocytes. Phenylalanine and tyrosine (but not the D-isomer of tryptophan or non-aromatic amino acids) also produced trans-acceleration for T3 uptake and T3 countertransport. These results are compatible with a kinetic model assuming a common simple carrier of T3 and tryptophan transport and point to a countertransport pathway driving the uphill uptake of T3 by hetero-exchange with intracellular aromatic amino acids.

We have studied transport of L-tryptophan, L-tyrosine and L-phenylalanine as factors contributing to homeostasis of these amino acids in preimplantation mouse conceptuses. Benzenoid amino acids were transported by the Na(+)-independent systems L and b0,+ in 1-cell conceptuses, and by these systems plus the Na(+)-dependent systems B0,+ and B in blastocysts. In addition, a component of Na(+)-independent tryptophan, tyrosine and phenylalanine transport in 1-cell and 2-cell conceptuses and in blastocysts resisted inhibition by L-leucine. The latter component of transport not only preferred benzenoid amino acids and in particular tryptophan as substrates, but it also was inhibited strongly and competitively by alpha-N-methyl-L-tryptophan. The leucine-resistant component of tryptophan transport also was inhibited strongly by N-ethylmaleimide and D-tryptophan, and it appeared to be inhibited weakly by 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid (BCO) but not by other amino acids tested as inhibitors. By these criteria, the leucine-resistant component of transport of benzenoid amino acids resembled system T in human red blood cells and rat hepatocytes. It is not entirely clear why preimplantation blastocysts have five good systems for transport of tryptophan. It is possible, however, that tryptophan homeostasis is particularly important during preimplantation development since it has been shown elsewhere that tryptophan availability in blood increases within one day after rat eggs are fertilized.

Portacaval shunting in rats results in brain dysfunction, as indicated by reduced energy metabolism and behavioral abnormalities, as well as many biochemical changes in plasma and brain. No etiological connections have been made between these findings, which have been studied mainly 2 wk or more after shunting. To determine how soon the various abnormalities occur and which are associated temporally with the decrease in brain glucose use, we studied shunted and sham-operated rats between 6 h and 11 days after surgery. Six hours after portacaval shunting, plasma aromatic amino acids, brain glutamine, aromatic amino acids, 5-hydroxyindoleacetic acid, and tryptophan transport into the brain were all significantly higher than normal. Brain glucose use showed a downward trend and was fully depressed within 1 day. Plasma branched-chain amino acids and threonine were decreased, and brain serotonin and norepinephrine content increased only after 2 days; these changes were therefore dissociated from the other abnormalities that developed in a shorter period. The results showed that the cerebral dysfunction characteristic of portacaval shunting began within hours and was fully established by 2 days.

The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na(+)-dependent and highly selective. The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.

The uptake of tryptophan at various stages of development was examined in plasma membrane vesicles derived from rat brain. The total uptake has two components Na+-dependent and Na+-independent respectively. The Na+-dependent component of the transport system appears around the 5th postnatal day and increases with the age. The Km value of the system does not vary during development. The Vmax increases five-fold between 14 and 35 day of postnatal life. Plasma membrane vesicles derived from T3-treated rats are able to accumulate nearly three-fold more tryptophan than nontreated rats. The results support the idea that thyroid hormones at the earlier stages of life, promote the establishment of neurotransmission in the developing nervous system.

1. l. In in vitro experiments with accumulating mucosal preparations (AMP) and everted intestinal sacs, as well as in in vivo experiments with isolated loops of the small intestine the stimulating effect of a number of amino acids on l -tryptophan uptake was investigated. 2. 2. Under “switched off” active transport (anoxia, 2,4-DNF treatment, sodium ion replacement by lithium ions in the mucosal solution) an expressed stimulation of l -tryptophan transport was observed within the mucosa and across the wall of the small intestine in the presence of l -proline, glycine, l -α-alanine, l -histidine and l -lysine. 3. 3. Preincubation of AMP in the solutions of glycine, l -α-alanine and l -lysine was characterized by a stimulation of l -tryptophan transport, and the increase of its concentration in tissue was accompanied by the exit of an equivalent amount of glycine from it. 4. 4. These observations show the participation of exchange transport in the uptake of l -tryptophan in the small intestine of chicks. 5. 5. The mechanism of exchange transport in chicks starts to function on the 25th day after hatching and its intensity depends on the character of amino acid-modifier participating in the process. 6. 6. Maximum activity of the exchange transport of l -tryptophan is demonstrated in the middle ileum. 7. 7. l -α-Alanine stimulates the absorption of l -tryptophan from the isolated intestinal loop proving the existence of an exchange transport mechanism in a living organism. 8. 8. An increased intensity of exchange transport is observed when feeding chicks with diets deficient and enriched in tryptophan.

Phenylalanine is accumulated in the genetically linked deficiency phenylketonuria. The effect ofl-phenylalanine on the transport of tryptophan was studied using membrane vesicles from rat-brain synaptosomes. Phenylalanine at similar concentrations to those found in phenylketonuric patients competitively inhibits tryptophan uptake, with aK i of the same order as theK m for tryptophan. This inhibition could be responsible for the depletion of serotonin found in phenylketonuria.

— Tryptophan transport across the blood-brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis-Menten kinetics (normal: Vmax= 19.5 nmol.min−1.g−1. Km= 113 μM; hepatic failure: Vmax, = 33.8 nmol.min−1.g−1, Km= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min−1.g−1; hepatic failure: K= 0.067 ml.min−1.g−1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual Kms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood-brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood-brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.

Alcoholics were found to have decreased plasma levels of tryptophan, the serotonin precursor, and a decreased ratio of tryptophan over amino acids competing for transport into the brain. Studies conducted in the plasma of rats and baboons with carefully controlled alcohol and dietary intake showed a decreased in the ratio of tryptophan over competing amino acids resulting mostly from increases in valine in the rat and in valine, leucine and isoleucine in the baboon. In the rat concomitant decreases in brain tryptophan and serotonin were noted. Central serotonin dificiency may contribute to the depressive states frequently seen in alcoholics.

— 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 (Tewset 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.

Rats with portacaval shunts were used as a model of hepatic encephalopathy and compared to shamoperated controls. First, the changes in intermediary metabolites and amino acids in blood and whole brain were characterized and found to be similar at 4 and 7 weeks after shunting. Second, the effects of nutritional therapy on selected metabolites and tryptophan transport into brain were assessed in rats 5 weeks after surgery. Ordinary food was removed and the rats were treated with glucose given either by mouth or intravenously, or intravenous glucose plus branched chain amino acids. Several abnormalities in plasma amino acid concentrations were reversed by treatment. The abnormally high brain uptake index of tryptophan, a consequence of portacaval shunting, was not lowered by any of the treatment regimens; it was even higher in the groups given glucose by mouth and glucose plus amino acids. Calculated competition for entry of tryptophan, phenylalanine, and tyrosine into brain was unchanged (glucose plus amino acids), or reduced (glucose alone). Brain glutamine content was brought to near normal by all treatments. Infusion of glucose plus branched chain amino acids normalized brain content of tryptophan, phenylalanine, and tyrosine, even though the brain uptake index of tryptophan was higher in this group. Thus, partial or complete reversal of several abnormalities found after portacaval shunting was achieved by removal of oral food and administration of glucose. The addition of branched chain amino acids to the glucose infusion restored brain content of three aromatic amino acids to near normal, by a mechanism which appeared to be unrelated to transport across the blood-brain barrier.

Tryptophan transport through the blood-brain barrier of lithium-treated animals did not differ from transport by controls. Lithium ion, itself, did not appear to alter such transport, nor did lithium treatment alter serum constituents so as to modify tryptophan passage through the blood-brain barrier. These results suggest that the accumulation of tryptophan in brain during lithium treatment probably results from the reported lithium-induced increase in the high-affinity, neuronal uptake system.

The response to increased blood NH4+ of three blood-brain barrier transport systems, which are altered after portacaval anastomosis, was studied. NH4+ acetate was infused for 4 or 22 h to raise blood and brain NH4+, and brain glutamine, to levels similar to those observed after portacaval anastomosis. While brain glutamine content was much higher (16–20 mumol/g) than normal (6 mumol/g) at both times, the permeability of the blood-brain barrier to the neutral amino acid [14C]tryptophan was greater only after 22 h of infusion. After discontinuing the infusion for 5 h, tryptophan transport returned to normal, whereas brain glutamine remained elevated (13 mumol/g). Thus there seemed to be no relationship between the rate of transport and glutamine content. The permeability to [14C]sucrose was unaltered, showing that the integrity of the blood-brain barrier was maintained. Other changes that are characteristic of portacaval shunting, such as decreased basic amino acid ([14C]lysine) and monocarboxylic acid (3-[14C]hydroxybutyrate) transport, were not reproduced by 22 h of infusion. The results demonstrated that the continued presence of NH4+ could be responsible for the change in at least one of the transport systems that are affected by portacaval shunting.

The effect of lithium on the sodium-dependent high-affinity system for tryptophan uptake was examined in plasma membrane vesicles derived from rat brain. We demonstrated that Na+ could be replaced by lithium in the external medium and the presence of lithium produced an increase in the Vmax of the tryptophan transport whereas it had no significant effect on the Km for the substrate. Plasma membrane vesicles derived from synaptosomes obtained from long-term lithium-treated rats are able to accumulate tryptophan to a greater extent than normal rats and maintain a more negative membrane potential than controls. Our data support the idea that the stimulation by lithium of the high-affinity uptake system for tryptophan by maintaining adequate membrane potentials across the membrane, could lead to the stabilization of serotonin production, as has been demonstrated in long term-lithium treatment.

Efflux of preloaded [3H]tryptophan from rat cerebral cortex slices has been monitored into superfusion media that were altered in their sodium content. Total replacement of sodium with choline greatly increased the release of tryptophan. This release could be cancelled by re-introducing sodium into the slices. A brief exposure to ouabain, an efficient inhibitor of Na+, K+-ATPase activity, only slightly increased tryptophan efflux at the concentration of 0.1 mM, whereas at 1.0 mM it produced a similar effect as the sodium-free medium. Accordingly, when the slices were superfused in the presence of ouabain and sodium, the change of medium to sodium-free caused much greater relative enhancement of tryptophan efflux with 0.1 than 1.0 mM ouabain. Tryptophan efflux was modified by changes in sodium fluxes also in slices initially depleted of sodium ions and treated with ouabain. The results suggest that the sodium-free medium and ouabain have a similar mechanism of action in modifying the tryptophan transport, and that the cation gradients across the cell membranes are more crucial for normal amino acid transport than the functional Na+, K+-ATPase.

A comparative study of the intestinal transport of DL-tryptophan and its 1-methylindole (tryptophan-l-Me) and benzo[b]thiophene (tryptophan-S) analogs has been carried out in vitro, using the everted intestinal sac of the rat and hamster. Both tryptophan and tryptophan-S are actively transported across the intestine, while tryptophan-l-Me is not actively transported. The active transport of tryptophan is competitively inhibited by tryptophan-S, suggesting a similar carrier, while tryptophan-l-Me is not an inhibitor of tryptophan transport, suggesting little or no interaction with the carrier. The transport of tryptophan and tryptophan-S is depressed at concentrations (10 mM), and all three amino acids produce subtle alterations in the barrier properties of the sacs, as evidenced by increased tetraethylammonium bromide-14C diffusion.

Dissected Malpighian tubules from wild type and the eye color mutant white of Drosophila were compared with respect to their abilities to transport tryptophan and kynurenine into tubule cells. It was determined that mutation at white greatly impairs the ability of Malpighian tubule cells to take up tryptophan. Functional studies on the extracellular spaces and ultrastructural observations indicated no differences in these respects between wild type and white tubules. It is consistent with several observations that much of the tryptophan associated with white exists in the intercellular spaces. Furthermore, the uptake of tryptophan by the w+ system of wild type tubules is inhibited by the analogue 5-methyl-tryptophan. However, the incorporation of radioactive tryptophan into protein in tubule cells from wild type and white occurs at the same rates and is not affected by 5-methyl-tryptophan. Therefore, it is apparent that Malpighian tubules have a transport system that enables entry of tryptophan into a cellular pool and that this cellular pool is initially independent of the tryptophan pool used for protein synthesis. The mutant white lacks this transport system. From these studies and others it appears that compartmentalization of cellular pools may be brought about via the utilization of specific membrane transport systems.

Severe hyperphenylalaninemia induced in infant rats by 3 days of treatment with p-chlorophenylalanine (p-cl phe) plus phenylalanine (phe) did not lower the tryptophan concentration of the brain, and the cerebral serotonin (5-HT) deficiency was attributable entirely to the known suppression to tryptophan hydroxylase (TPH) by p-cl phe. The decrease in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) was thus no more pronounced than in rats which, treated with p-cl phe alone, were devoid of hyperphenylalaninemia. Suppression of TPH was found to also underlie the decrease in cerebral 5-HT caused by treatment with alpha-methylphenylalanine (alpha-mephe) alone: a 22% loss of midbrain TPH activity was detectable 24 hr after an injection only, reverted toward the normal during the next 2 days, and was clearly unrelated to the weak competitive inhibition of the enzyme by alpha-mephe in vitro. However, alpha-mephe (unlike p-cl phe), when administered together with phe, did not suppress TPH, nor did it counterbalance the reduction of cerebral tryptophan uptake by excess phe. Thus the 5-HT diminution in the rat model of phenylketonuria produced by treatment with alpha-mephe plus phe was attributable to hyperphenylalaninemia and the inhibition of tryptophan transport to the brain. Injection of tryptophan was found to restore the cerebral 5-HT level in the face of persistently severe hyperphenylalaninemia.

Mutants of A9 mouse fibroblast, resistant to the killing effect of 0.4 mM 5-flurotryptophan (5-FT), have altered L-tryptophan transport properties. The resistant phenotype is stable for at least 90 generations of growth in MEM. A fluctuation test indicated that clones resistant to 0.4 mM 5-FT occurred spontaneously. An average mutation rate was estimated at 1.6 X 10(-6). Treatment with N-methyl-N'-nitro-N-nitrosoguanidine increased the frequency of these clones by at least 100-fold. These results indicate that the resistant clones arose as a result of a mutation. All the resistant mutant tested accumulate less 5-FT at near steady-state conditions than the wild type. Lineweaver-Burk plots of initial rates of tryptophan uptake yield a biphasic curve suggesting that tryptophan is transported by two transport systems. Kinetic constants determined by a computer program indicate that both proposed transport systems were modified in each of two 5-FT resistant mutants.

l -Glutamine at the concentration present in cerebrospinal fluid decreases the steady-state accumulation of the aromatic amino acids tryptophan, tyrosine and dihydroxyphenylalanine (DOPA) in rat striatal synaptosomes. Glutamine significantly inhibits synaptosomal tryptophan hydroxylase activity; it has less marked effects on tyrosine hydroxylase and DOPA decarboxylase activities. Thus, interaction between glutamine and tryptophan transport into nerve terminals may be one of the factors regulating the rate of serotonin synthesis in vivo.

— 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 Km (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 Km values are computed for the essential neutral amino acids from known data on amino acid transport Km and plasma concentrations. The apparent Km 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.

The ability of low protein diets containing small neutral, dispensable amino acids to induce threonine imbalance has been examined. Diets containing amino acids which compete for threonine transport in vitro (serine, alanine, alpha-amino-n-butyrate) caused depressions of growth and food intake which could be corrected to varying degrees by adding threonine to the diet. Large neutral, indispensable amino acids, moderately inhibitory of threonine transport, also induced the imbalance. Some amino acids that had little or no effect on threonine transport in vitro (acidic amino acids and proline) did not cause growth and food intake depressions. Other non-inhibitory amino acids (arginine and lysine) caused growth depressions which were not satisfactorily corrected by additional threonine alone, but were prevented by supplements of all the indispensable amino acids including threonine. Ornithine which was also not inhibitory of threonine transport was an exception. It induced a moderate growth depression which was corrected by additional threonine. Similar studies showed that histidine or tryptophan imbalance could be induced by feeding diets containing only those large neutral amino acids which compete for histidine or tryptophan transport in vitro. These experiments show that, based on the results of transport competition experiments, it is generally possible to devise amino acid supplements which can induce a dietary imbalance of a given amino acid.

—Slices of rat cerebral cortex, when incubated aerobically at 37°C in Krebs-Ringer-phosphate solution (pH 7.0) containing 10 mm glucose and 1.0 mm l-tryptophan [1-14C], accumulated tryptophan. Within the first 15 min of incubation the ratio of the concentration of the amino acid in the tissue to that in the medium reached 3.5:1. Uptake of tryptophan was linear for the first 30 min and attained a maximum concentration ratio (tissue:medium) of 6.5:1 within 60 min. The transport mechanism became saturated at 1.0-3.0 mm tryptophan. Entry of the amino acid into the cortical cells was thereafter directly proportional to its initial concentration in the medium. The tissue: medium ratio at 15 min decreased significantly under the following experimental conditions: (1) lowering the incubation temperature to 0°C; (2) incubating under N2; (3) omitting glucose; (4) decreasing the Na+ concentration below 50 mm; (5) removing K+ from the medium; or (6) adding 1.0 mm NaCN or 0.1 mm protoveratrine B to the medium. These results provided evidence that the accumulation of tryptophan against its concentration gradient was an active process. The effects of a number of amino acids on the uptake of tryptophan were studied: of these, l-phenylalanine, dl-p-chlorophenylalanine, l-tyrosine, l-DOPA, the branched chain aliphatic amino acids (l-leucine, l-isoleucine, l-valine) and l-glutamic acid were found to be the most potent inhibitors of tryptophan transport. Several tryptophan metabolites were tested; only l-kynurenine inhibited the uptake of tryptophan.

The intestinal transport of l- and d-isomers of tryptophan and ten derivatives were studied in everted intestinal sacs of golden hamsters. It was found that l-tryptophan, 4-CH3-, 5-CH3-, and 6-CH3-dl-tryptophan, and N-chloroacetyl-l-tryptophan were transported against a concentration gradient and the d-isomer, 5-OH- and 5-benzyloxyl-dl-tryptophan, N-acetyl-l- and N-2,4-dinitrophenyl-dl-tryptophan, tryptamine and 5-OH-tryptamine were not transported. The rate of l-tryptophan transport was much greater than that of the methyl derivatives and N-chloroacetyl-derivative. The transport reached saturation at 3 mm concentration and was inhibited in presence of 6 mm of l-tyrosine or l-phenylalanine. Kinetic study showed that this inhibition was a competitive one. Studies with H3-dl-tryptophan have shown that the transport mechanism was mainly located in the mucosal, not the serosal side. Replacement of Na+ ion either by Li+, K+, or choline in either mucosal or both sides, inhibited the tryptophan transport. Replacement of chloride by sulfate did not show any inhibitory effect.

Hyperammonemia was provoked in rats by urease injection over three days. Tryptophan transport into the forebrain measured by the bolus injection technique was increased in hyperammonemic rats in comparison with pairfed controls. The concentration of the large neutral aminoacids, of tryptophan and of 5-hydroxyindole acetic acid were increased in the forebrain and brainstem. Probenecid administration led to a significantly higher accumulation of 5-hydroxyindole acetic acid in the forebrain of hyperammonemic rats. Since liver function was not impaired the data indicate that hyperammonemia in absence of hepatic insufficiency alters the carrier function for large neutral aminoacids at the blood brain barrier.

The principles of the competitive ligand binding assay have been extended to the in vivo state to study the competition for tryptophan binding between albumin and the tryptophan transport system localized in the brain capillary wall, i.e., the blood-brain barrier (BBB). Based on the concentration of albumin (1.4 mM) which yields 50% inhibition of BBB tryptophan transport, the dissociation constant of tryptophan binding to albumin (KD = 0.13 mM), and the affinity constant of the BBB tryptophan transport system (KM = 0.19 mM), the apparent binding capacity of the BBB (1.9 mM), may be calculated. The high apparent binding capacity of the BBB enables the capillary transport system to compete with albumin for tryptophan binding; the inhibition of albumin binding by the carrier increases the fraction of tryptophan that is free in vivo to values greater than the fraction that is free in vitro. Depending on physiological changes in KM (e.g., due to plasma amino acid competition) or KD (due to plasma free fatty acid), the apparent free fraction of tryptophan in vivo may approximate either one of two extremes, i.e., the in vitro free fraction or the total tryptophan.

Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain bamer (BBB) of the newborn rabbit. The K,, V,,,, and KD of the transport of eight amino acids were determined by a nonlinear regres- sion analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K,, V,,,, and KD of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro, but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentra- tions of N-methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain. Key Words: Blood-brain barrier-Amino acid transport-Developing brain. Pardridge W. M. and Mietus L. J. Kinetics of neutral amino acid transport through the blood-brain barrier of the newborn rabbit. J. Neurochem. 38, 955-962 (1982).

We studied the transport of tryptophan through transport system T in the human red cell, the Ehrlich ascites-tumour cell and in everted sacs of rat intestine. In red cells we confirmed earlier results on Na+-independence and aromatic amino acid specificity (Rosenberg, R., Young, J.D. and Ellory, J.C. (1980) Biochim. Biophys. Acta 598, 375-384). In addition we observed that N-methylation or N-acetylation did not reduce the affinity of the substrates for system T, hydroxylation could increase or decrease substrate affinity, and system T was insensitive to pH changes in the medium. These results characterized reactive differences between system T and other known amino acid transport systems. We also found that D-isomers were about 1/3 as effective as L-isomers to inhibit L-tryptophan uptake. D-Tryptophan competitively inhibited L-tryptophan uptake, but was not taken up by system T. L-Tryptophan produced trans-stimulation of the uptake (influx) and trans-inhibition of the release (efflux) of L-[3H]tryptophan; D-tryptophan produced trans-inhibition of the efflux but did not affect significantly the uptake. These results show that in red cells the transport properties of transport system T are asymmetric. Transport system T seems to be absent in the other two preparations studied, the Ehrlich ascites-tumour cell and the rat intestine.

In congenital urea cycle disorders, detoxification of ammonia is impaired, leading to hyperammonemia. Ammonia is the major component causing the acute neurological disturbances. It may influence the supply of substrate and its transport at the blood-brain barrier (BBB) which results in alterations in the synthesis and catabolism of neurotransmitters in the brain. In hyperammonemic rats, the uptake of tryptophan into the brain is increased with an augmented flux through the serotonin pathway. In the forebrain, glutamine as well as amino acids transported with the same L-carrier system, such as phenylalanine, tyrosine and tryptophan, are elevated. It is postulated that the increased transport of tryptophan at the BBB occurs in exchange with glutamine. Methionine sulfoximine (MSO) inhibits glutamine synthetase in the cerebral cortex. The activity drops from 5.85 +/- 0.38 to 1.07 +/- 0.37 mumol/min/g wet weight. Under MSO, the brain tryptophan uptake also decreased to 64.2 +/- 4.5% in hyperammonemic rats, to 54.1 +/- 8.0% in untreated hyperammonemic rats, whereas without MSO an increase of tryptophan uptake was observed. An effect of glutamine on tryptophan transport could also be demonstrated using brain microvessel preparations as a model for the BBB. Our findings indicate that preloading isolated microvessels with L-glutamine increases tryptophan uptake into the endothelia when L-glutamine is at concentrations found in brain homogenates under hyperammonemia. Since brain microvessels do not contain glutamine synthetase activity, enzymes from the gamma-glutamyl cycle may be involved in the glutamine-mediated tryptophan transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Segments of newborn or adult rabbit jejunum were mounted as flat sheets in flux chambers. Mucosal-to-serosal (Jm leads to s) and serosal-to-mucosal (Js leads to m) fluxes of tryptophan (Try), phenylalanine (Phe), and alanine (Ala) were measured in the absence of electrochemical gradients. In the presence of Na, J Try s leads to m increased linearly with concentration, whereas the relationship of J Try m leads to s and concentration contained both linear and saturating components. The relationship of net Try flux and concentration was in accord with Michaelis-Menten kinetics. Ouabain (10(-3) M) abolished net Na fluxes in both newborn and adult rabbit jejunum and abolished net Try flux in the adult, but did not reduce net Try flux to zero in the newborn. In the presence of Na-free solutions small net fluxes of Try, Phe, and Ala occurred in the newborn. Addition of 10(-4) M 2,4-dinitrophenol abolished net Try flux in ouabain-treated newborn rabbit jejunum that was bathed by either Na-free or Na-containing solutions. In the presence of Na, Phe and Ala reduced J Try m leads to s, and Try reduced the fluxes of Phe and Ala. Phe inhibited J Try m leads to s in Na-free solutions, and Try reduced J Phe m leads to s. These results suggest that the newborn rabbit jejunum resembles the adult in that Try is absorbed primarily by a Na-dependent system. These results show that the newborn differs from the adult in that Try is absorbed by a system that is independent of Na but requires metabolic energy.

The transintestinal transport of tryptophan by everted sacs of the rat smal intestine was studied. It was shown that: 1. 1. The transport of trytophan was enhanced by methionine, leucine and sarcocine; 2. 2. Trytophan was a potent inhibitor of the transport of leucine; 3. 3. The transport of trytophan was inhibited by higher concentrations of methionine; 4. 4. Tryptophan was a potent competitive inhibitor of the transport of lysine; 5. 5. Lysine inhibited the transport of tryptophan when a part of the intestinal capacity for transport of neutral amino acids was occupied by methionine; 6. 6. The effect of increasing the initial mucosal concentrations of tryptophan or lysine, keeping the initial serosal fluids free of amino acids, was to saturate the capacity for transport of these amino acids; 7. 7. The net transport of tryptophan and lysine from the mucosal to the serosal fluids was reduced if these amino acids were initially present in the serosal fluids. It is concluded that: 8. (i) tryptophan is a substrate for the carrier of the diamino acids as well as for the carrier of the neutral amino acids; 9. (ii) The enhancement by methionine, leucine and sarcosine of the transintestinal tryptophan transport represents a counterflow effect of these amino acids on the transport of tryptophan by diamino acid carrier; 10. (iii) The reduction caused to the transport of tryptophan and lysine by their presence in the initial serosal fluids may be explained as a kinetic consequence of the transport of these amino acids by a mobile carrier.