Glycine Transport System Research Articles

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Preclinical Reproductive and Developmental Toxicity Profile of a Glycine Transporter Type 1 (Glyt1) Inhibitor

Bitopertin is a glycine type 1 (GlyT1) inhibitor intended for the treatment of psychiatric disorders. The principle adverse effect in the regulatory reproductive toxicity studies was peri-natal pup death when rat dams were treated during parturition at a dose resulting in five-times the human therapeutic exposure (AUC). Cessation of dosing two days before parturition prevented the pup deaths. Investigatory experiments and pharmacokinetic modelling suggested that the neonatal mortality was related to transplacental passage of bitopertin leading to high systemic levels in the newborn pups. Brain levels of bitopertin in the rat fetus and neonate were two-fold higher than in the mother. As illustrated by knock-out mice models, GlyT1 function is essential for neonatal pup survival in rodents, but is not necessary for normal prenatal morphological development. The glycine transport systems are immature at birth in the rat, but are functionally well-developed in the human newborn. While the relevance to humans of the neonatal mortality seen in rats following late gestational exposure is unknown, bitopertin would not be recommended for use during late pregnancy unless the anticipated benefit for the mother outweighs the potential risk to the newborn.

Analysis of the Role of Satiety of the Parents during Irradiation in the Assimilation of Substrates of the Protein Origin in two Generations of their Posterity

Thanks to the use of nuclear energy a society lives under the conditions of increased radiation background. Malformations and other inherited diseases caused by damage to the genetic apparatus by low doses of irradiation appear even in the remote generations. It determines the need for research of the remote effects of irradiation of parents on their posterity. One of the most radiosensitive tissues is small intestine which is responsible for the last stages of digestion and absorption of nutrients. Therefore the aim of our work was the analysis of the role of satiety of the parents during irradiation in the assimilation of substrates of the protein origin in two generations of their posterity. Materials and methods: In vitro were studied parameters of transport of free glycine and “peptide” glycine formed from glycyl-glycine in the small intestine of the 2nd generation of posterity of male rats, that were irradiated by a dose 0,5 Gy after taking meal, and intact females. Results: Parameters of the transport of the free glycine and “peptide” glycine in the small intestine of the 2nd generation of posterity of irradiated satiety male rats are lower than corresponding parameters in the 1st generation and in the intact groups but are in the borders of active component of transport. Stability of transport of the free glycine and “peptide” glycine are higher than in the corresponding intact groups. Conclusions: In comparison with the corresponding data for 2nd generation of irradiated fasting male rats, satiety of the male-predecessors lead to the stabilization of the activity of transport system as for free glycine, so for the “peptide” glycine both in the small intestine of their posterity. Apparently, the protective effect of the presence of food in the gastrointestinal tract of precursor at exposure is realized by stabilizing of the activity of transport system for free glycine and peptide glycine both in the small intestine of two generations of posterity, and maintaining its work at the level necessary and sufficient for normal body functioning.

Impacts of the Osmolality and the Lumenal Ionic Strength on Osmosensory Transporter ProP in Proteoliposomes

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(1/2)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(1/2)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.

Renal imino acid and glycine transport system ontogeny and involvement in developmental iminoglycinuria

Renal maturation occurs post-natally in many species and reabsorption capacity at birth can vary substantially from the mature kidney. However, little is known regarding the maturation of amino acid transport mechanisms, despite the well-known physiological state of developmental iminoglycinuria. Commonly seen during early infancy, developmental iminoglycinuria is a transient version of the persistent inherited form of the disorder, referred to as iminoglycinuria, and manifests as a urinary hyperexcretion of proline, hydroxyproline and glycine. The transporters involved in developmental iminoglycinuria and their involvement in the improvement of renal reabsorption capacity remain unknown. qPCR (quantitative real-time PCR) and Western blot analysis in developing mouse kidney revealed that the expression of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 was lower at birth (approx. 3.4-, 5.0-, 2.4- and 3.0-fold less than adult kidney by qPCR respectively) and increased during development. Furthermore, immunofluorescence confocal microscopy demonstrated the absence of apical expression of Slc6a18, Slc6a19, Slc6a20a and the auxiliary protein collectrin in kidneys of mice at birth. This correlated with the detection of iminoglycinuria during the first week of life. Iminoglycinuria subsided (proline reduction preceded glycine) in the second week of life, which correlated with an increase in the expression of Slc6a19 and Slc6a20a. Mice achieved an adult imino acid and glycine excretion profile by the fourth week, at which time the expression level of all transporters was comparable with adult mice. In conclusion, these results demonstrate the delayed expression and maturation of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 in neonatal mice and thus the molecular mechanism of developmental iminoglycinuria.

Glutamate-induced inhibition of D-aspartate uptake in Müller glia from the retina.

Müller glial cells from the retina "in situ" and in primary culture, mainly express the high-affinity sodium-coupled glutamate/aspartate transporter GLAST-1, which dominates total retinal glutamate (Glu) uptake, suggesting a major role for these cells in the modulation of excitatory transmission. The possible involvement of ionotropic and metabotropic Glu receptors in the regulation of Glu uptake was studied in primary cultures of Müller glia. We demonstrate that exposure to 1 mM L-Glu induces a time-dependent inhibition of D-aspartate (D-Asp) uptake in a Na+-dependent manner, as a result of a reduction in the number of transporters at the plasma membrane. The inhibition of D-Asp uptake by Glu was not mimicked by agonists or modified by antagonists of ionotropic and metabotropic Glu receptors. In contrast, transport was inhibited by GLAST-1 transportable substrates threo-hydroxyaspartate and aspartate-beta-hydroxamate, but not by the nontransportable inhibitors trans-pyrrolidine dicarboxylate or DL-threo-beta-benzyloxyaspartic acid. Under the same experimental conditions, L-Glu did not affect the sodium-dependent transport systems for glycine or GABA. The present results demonstrate that the specific downregulation of glutamate/aspartate transport by L-Glu is unrelated to Glu receptor activation, and results from the internalization of transporter proteins triggered by the transport process itself. Such negative feedback of Glu on Glu transport, could contribute to retinal toxicity under pathological conditions in which high extracellular concentrations of Glu are reached.

Osmoregulation and cell volume regulation in the preimplantation embryo

The early preimplantation mammalian embryo possesses mechanisms that regulate intracellular osmolarity and cell volume. While transport of osmotically active inorganic ions might play a role in this process in embryos, the major mechanisms that have been identified and studied are those that employ organic osmolytes. Organic osmolytes provide a substantial portion of intracellular osmotic support in embryos and are required for their development under in vivo conditions. The main osmolytes that have been identified in cleavage stage embryos are accumulated via two transport systems of the neurotransmitter transporter family active in early preimplantation embryos--the glycine transport system (GLY) and the beta-amino acid transport system (system beta). While system beta has been established to have a similar role in many other cells, this is a novel function for the GLY transport system. The intracellular concentration of organic osmolytes such as glycine in early preimplantation embryos is regulated by tonicity, allowing the embryo to regulate its volume against shrinkage and to control its internal osmolarity. In addition, the cells of the embryo can regulate against an increase in volume via controlled release of osmolytes from the cytoplasm. This is mediated by a swelling-activated anion channel that is also highly permeable to a range of organic osmolytes, and which closely resembles similar channels found in many other cell types (VSOAC channels). Together, these mechanisms appear to regulate cell volume in the egg through the early cleavage stages of embryogenesis, after which there are indications that the mechanisms of osmoregulation change.

L-proline and glycine transport system from brain to blood across the blood-brain barrier

L-proline and glycine transport system from brain to blood across the blood-brain barrier

The adenylate cyclase inhibitor MDL-12330A has a non-specific effect on glycine transport in Müller cells from the retina

Müller glial cells express two transport systems for glycine (Gly): one with low affinity and another identified as GLYT1 with high affinity. The latter colocalizes with NMDA receptors in the CNS. Gly is considered as an obligatory coagonist at NMDA receptors, and, hence, the Gly transport system could contribute to the modulation of glutamate (Glu) excitatory transmission in the vertical pathways of the retina. For this reason, the regulation of Gly transport by cAMP was studied. We report here a non-specific effect of MDL-12330A, a compound reported to inhibit adenylate cyclase (AC), on Gly transport in Müller glia. This effect might be due to a toxic action on the cells, decreasing cell viability, and not to a specific inhibition of the adenylate cyclase. Non-specific effects of this drug should be considered when the participation of cAMP in any biological process is studied. We have clearly demonstrated that cAMP does not participate in the regulation of Gly transport in Müller glia.

Characterization of the glycine transport system GLYT 1 in human placental choriocarcinoma cells (JAR)

Transport of glycine in confluent monolayer cultures of JAR human placental choriocarcinoma cells was investigated. Glycine uptake in these cells was made up of two components, one being Na +-dependent with no requirement for Cl − and the other being dependent on Na + as well as Cl −. Substrate specificity studies indicated that distinct transport systems were responsible for these two components. Alanine inhibited the Na +-dependent glycine uptake preferentially and the Na +- and Cl −-dependent glycine uptake represented > 95% of total uptake in the presence of 5 mM alanine. Competition experiments revealed that the Na +- and Cl −-dependent transport system exhibited a very narrow substrate specificity with affinity toward only glycine and its derivatives such as sarcosine, glycine methyl ester and glycine ethyl ester. These characteristics identify the transport system as GLYT 1. This system showed high affinity for glycine, with a Michaelis-Menten constant of 15 μM. The Na +:Cl −: glycine stoichiometry appeared to be 2:1:1. Treatment of JAR cells with calmodulin antagonists resulted in the inhibition of the transport function of GLYT 1 and this inhibition was solely due to a decrease in the maximal velocity of the system with no change in the substrate affinity. It is concluded that the placental choriocarcinoma cell line JAR expresses robust activity of the glycine transporter GLYT 1 and that the activity of this transporter is under the regulation of calmodulin-dependent cellular processes.

Na(+)‐ and H(+)‐gradient‐dependent transport of alpha‐aminoisobutyrate by luminal membrane vesicles from rabbit proximal tubule.

1. The characteristics of renal transport of alpha-aminoisobutyrate (AIB) by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubule were investigated. 2. Transport of AIB in vesicles from pars convoluta was mediated by both Na(+)-dependent and Na(+)-independent systems, which in the presence of an inwardly directed H+ gradient can drive the uphill transport of AIB into these vesicles. 3. By contrast, in luminal membrane vesicles from pars recta, transient accumulation of AIB was only dependent on Na+. Lowering pH without a H+ gradient (pHi = pH0 = 5.5) completely abolished the Na(+)-dependent transient accumulation of AIB in these vesicle preparations. 4. Attempts to determine the stoichiometry of both the Na(+)-AIB and H(+)-AIB transporters located in these two segments of proximal tubule suggested that one Na+ and one H+ ion may be involved in the transport of AIB. 5. Sodium-dependent uptake of AIB in vesicles from pars convoluta was competitively inhibited by L-serine and L-phenylalanine, whereas the presence of L-proline, L-alanine and glycine had no significant effect. By contrast, the H(+)-gradient-dependent uptake of AIB was drastically reduced (30% of the control value) by L-proline, L-alanine and glycine, while L-serine and L-phenylalanine had no significant effect. 6. On the other hand, pars recta vesicles exhibited a different transport specificity. L-Phenylalanine, L-serine, L-alanine and glycine, but not L-proline competitively inhibited the uptake of AIB, providing evidence for the existence of a common transport system for AIB, L-phenylalanine, L-serine, L-alanine and glycine in this segment of rabbit proximal tubule.

Photorespiratory Mutants of the Mitochondrial Conversion of Glycine to Serine

Two mutants of barley (Hordeum vulgare L.), LaPr 85/55 and LaPr 87/30, have been isolated that accumulate glycine, with a concomitant reduction in the aminodonors glutamate and alanine, when transferred to air. Studies have shown that these plants have wild-type levels of serine transhydroxymethylase (EC 2.1.2.1) activity. When supplied (14)CO(2), 48 and 66% of the supplied carbon was retained as glycine in LaPr 85/55 and LaPr 87/30, respectively, compared with a value of 11% for the wild type. In the short-term, both mutant plants are unable to metabolize [(14)C] glycine, but when fed the isotope for 2 hours, LaPr 85/55 was able to metabolize most (70%) of the supplied carbon into sugars with only 15% remaining in glycine. LaPr 87/30, however, was unable to metabolize more than 4% of the supplied carbon into sugars even after 2 hours. Measurement of glycine decarboxylase (EC 2.1.2.10) activity via the glycine-bicarbonate exchange reaction showed LaPr 85/55 to have approximately 70% wild-type activity with LaPr 87/30 having only 14% wild-type activity. The approximation of LaPr 85/55 to wild-type activities was maintained for (14)CO(2) release from [(14)C]glycine feeding and ammonia accumulation in the presence of methionine sulphoximine with the equivalent rates for LaPr 87/30 being less than 40% and 10%, respectively. CO(2) fixation rates for the mutants fell to between 35 and 40% of wild-type rates within 10 min of transfer to air. This was shown to be partly due to a run down of aminodonors, because when 40 millimolar serine was supplied through the xylem stream these rates recovered for both mutants to 70% of the wild-type rate. These data suggested a mutation in a glycine transport system for LaPr 85/55 and in the proteins of glycine decarboxylase for LaPr 87/30. Western blotting with antisera to the P, H, T, and L proteins of glycine decarboxylase showed cross-reaction against all four proteins for LaPr 85/55 but little cross-reaction against P or H protein for LaPr 87/30, reaffirming the possibility of a transport mutation in LaPr 85/55. We also suggest that genes for P and H proteins could be either coordinately regulated or that one protein is undetectable or unstable in the absence of the other.

Characteristics and adaptive regulation of glycine transport in cultured glial cells.

The transport of glycine in C6 glioma cells takes place mainly in a heterogeneous Na+-dependent manner which can be resolved into different components. A Na+- and Cl(-)-dependent component with high affinity for glycine is pH-sensitive and inhibited by sarcosine, all these characteristics corresponding to System Gly. The low-affinity component of the transport of glycine can be discriminated as two components, namely System A and System ASC. The main proportion of glycine transport through the low-affinity system is carried out by the ASC System, which appears to be constitutively expressed by the cells. The adaptive response of the low-affinity Na+-dependent transport of glycine to amino acid deprivation was identified with System A on the basis of its ion-dependency, pH-sensitivity and by inhibition analysis. The possible physiological role of the high- and low-affinity components of the transport system for glycine in glial cells is discussed.

The role of chloride ions on the transport of glycine in plasma membrane vesicles from glial cells

The high-affinity transport system for glycine in plasma membrane vesicles from C6 glioma cells is dependent on Na + and also on the presence of Cl − in the incubation medium. This anion requirement is relatively specific for Cl −, since other anions are also capable of stimulating the glycine transport in the following order of decreasing efficacy: Cl − >Br − >SCN − ≅I − >NO 3 − >F −. Chloride ions raise the V max for transport and, to a lesser extent, act on the K m. The data provided by direct measurements of the coupling of sodium and chloride to the transport of glycine by using a kinetic approach suggest a stoichiometry for the translocation cycle catalyzed by the glycine transporter of two sodium ions and one chloride ion per glycine zwitterion.

Characteristics of acidic, basic and neutral amino acid transport in the perfused rat hindlimb.

1. We have employed a paired-tracer isotope dilution technique in a perfused rat hindlimb preparation to obtain information on the kinetics of transport across the sarcolemmal membranes of acidic, neutral and basic amino acids. 2. We have defined the characteristics of the saturable transport of amino acids normally regarded as paradigm substrates for the A, ASC, L, y+(basic) and the dicarboxylic amino acid transport systems. Their maximal transport capacities (Vmax, nmol min-1 (g muscle)-1 and substrate concentrations for half-maximal transport (Km, mM) of representative amino acid substrates are as follows: 2-aminoisobutyrate (AIB), Vmax = 15 +/- 7, Km = 1.26 +/- 0.6; alanine, Vmax = 332 +/- 53, Km = 3.9 +/- 0.9; serine, Vmax = 410 +/- 61, Km 3.4 +/- 0.5; leucine, Vmax = 2800 +/- 420, Km = 20 +/- 2; lysine, Vmax = 136 +/- 46, Km = 2.1 +/- 1.3; glutamate, Vmax = 86 +/- 6, Km = 1.05 +/- 0.05; proline, Vmax = 196 +/- 48, Km = 4.1 +/- 0.6. 3. Glycine uptake was faster than expected on the basis of diffusion but was not saturable and showed uptake that could be best described by a first-order rate constant of 0.07 +/- 0.003 min-1. 4. We have attempted to discriminate kinetically between possible routes of entry for an amino acid on the basis of competitive and non-competitive interaction between substrates potentially sharing common routes. On this basis, the major routes of alanine entry appear to be via the ASC and L systems with the A system playing a quantitatively minor role. Glutamate and aspartate appear to be transported exclusively by a dicarboxylate amino acid carrier. The branched-chain amino acids (BCAA) and the aromatic amino acid, phenylalanine, are almost equivalent substrates for an L-like system. 5. Insulin had no detectable effect on the uptake of paradigm substrates for ASC, L, y+, the dicarboxylic amino acid or glycine transport systems. 6. Transport of serine and lysine was Na+ dependent. Lysine transport apparently occurred with a stoichiometry of 2 Na+: 1 lysine. With the exception of alanine, whose transport was partially Na+ dependent, all other amino acids examined in the present study were transported in a Na+-independent manner.

Depolarization-induced release of glycine and β-alanine from plasma membrane vesicles derived from rat brain synaptosomes

Glycine and β-alanine actively loaded into brain synaptic plasma membrane vesicles were released into the external medium by using the classical depolarization agents high K + and veratridine. This release occurs via a Ca +-independent process. Measurements of membrane depolarization using tetraphenylphosphonium uptake show a close correlation between changes in the membrane potential and stimulation of the efflux process. Results shown herein and previously reported by our group (Aragón, M.C. and Giménez, C. (1986) Biochim. Biophys. Acta 855, 257–264; Agulló, L., Jiménez, B., Aragón, M.C. and Giménez, C. (1986) Eur. J. Biochem. 159, 611–617), suggest that the glycine and β-alanine transport systems in synaptic plasma membranes are susceptible of modulation by changes in ionic fluxes and hence in the membrane potential, similar to those occurring during depolarization and repolarization.

Inhibition of phototrophically growing Chromatium vinosum D by glycine

The effect of glycine on phototrophically growing Chromatium vinosum D was investigated in the presence and absence of various amino acids. Inhibition of autotrophic cells was more pronounced than that of heterotrophic cells. Cells of growth-inhibited cultures were misshaped, showing vacuolation and partial protoplast formation. Cells exposed to glycine in the presence of equimolar amounts of either d- or l-alanine did not show any inhibition of growth. Neither serine, leucine, valine, isoleucine nor α-aminoisobutyric acid could counteract the inhibition of growth by glycine. The inhibitory effect of glycine on growth is thus likely to be caused by a competition between glycine and alanine during cell wall synthesis. The possible functions of glycine transport system(s) in the natural habitat of C. vinosum are discussed.

Chloride dependence of the sodium-dependent glycine transport in pig kidney cortex brush-border membrane vesicles

The Na +-dependent glycine uptake in pig kidney cortex brush-border membrane vesicles is specifically enhanced by the presence of Cl −. The Na +-independent glycine uptake is not affected by Cl −. Various anions tested could not substitute Cl − in the activation of the Na +-dependent glycine transport. Cl − is specifically required on the outer membrane side. The Na +-dependent glycine uptake is higher in the presence of an inwardly directed Cl − gradient than the one measured in the presence of equilibrated Cl −. The Na +-dependent glycine uptake depends on, and is saturable at increasing Cl − concentrations. By studying the activation of glycine uptake by Na + in the presence and in the absence of Cl −, evidence was found that two different Na +-dependent glycine transport pathways are present in pig kidney cortex brush-border membrane vesicles. The kinetics of the glycine uptake measured in the presence of an inwardly directed NaCl gradient show the presence of two glycine transport systems, a low-affinity, high-capacity one and a high-affinity, low capacity one. In the absence of Cl − the high-affinity, low-capacity transport is almost suppressed, thus indicating the presence of a high-affinity glycine transport system simultaneously dependent on both Na + and Cl − ions.

Criteria for discrimination among glycine-transport systems

Criteria for discrimination among glycine-transport systems

The roles of insulin and glucagon in the regulation of amino acid turnover rate and pool size: In vivo study with [ 15N]glycine and gas chromatography—mass spectrometry

Gas chromatography—mass spectrometry analysis of plasma amino acid derivatives has been used to determine the 15N enrichment time decay curves of plasma glycine following a single dose administration of [ 15N]glycine in untreated and insulins-, glucagon-, and cycloheximide-treated rabbits. The present study indicated the following: (a) Increases of 80 and 50% in plasma glycine disappearance rate constants occurred in insulin- and glucagon-treated rabbits as compared with control postabsorptive rabbits; (b) The hormones in the intact rabbits caused a significant depletion in glycine pool size, which led to a moderate reduction in the fluxes of glycine. (c) A significant reduction in glycine turnover rate constants and pool size was noted at 3 and 24 hr following the administration of a sublethal dose of cycloheximide and a restoration towards control post-absorptive values was observed 48 hr after cycloheximide administration. (d) Sublethal doses of cycloheximide inhibited by 60 and 90% the stimulatory action of insulin and glucagon on plasma glycine disappearance, respectively. The present data suggest that both insulin and glucagon may act directly on plasma glycine disappearance rates. The stimulatory action of insulin differs from the action of glucagon in that it is not completely blocked by cycloheximide. Presumably glucagon and insulin modify the glycine transport system at different sites or by a different mechanism.