Effects of L-proline and some of its analogs on retinal spreading depression

L-Protine inhibits glumate-based spreading depressions (SDs) at low concentrations (2–2.5mM) and promotes K⁺-based SDs at higher concentrations (5 mM). The inhibition of glutamate-based SDs was postulate receptors on somatic and dendritic plasma membranes. The binding of proline to glutamate receptors was furthermore postulated to result in a result in a release of K⁺ from the intracellular compartment, enhancing the extracellular K⁺ concentration sufficiently to promote K⁺ based SDs. A proline analog, L-baikiain, containing a double bond and one more based A atom in the ring structure than proline had similar affects as the latter amino acid, but an analog, L-azetidine-2-hydroxylic acid, with one less C atom in the ring had little effect on SD in the retina.     

The nature of the chick's magnesium-sensitive retinal spreading depression

Spreading depression (SD) in the chick retina is completely suppressed by 10 mM MgCl2 in the bathing solution (Mg-sensitive SD). However, after increasing the KCl concentration in the Mg solution to values between 10 and 20 mM the retina can again exhibit SDs (Mg-insensitive SD). It has been postulated that the Mg-sensitive SD is a glutamatergic phenomenon. This is supported by the effect of four gl(utamate)-antagonists--L-proline, glutamic acid diethyl ester (GDEE), D-alpha-aminoadipate (D-AA), and 2-amino-4-phosphonobutyrate (APB)--which all suppressed this type of SD. It was suggested that this effect is due to competitive binding of glutamate involved in the Mg-sensitive SD and the gl-antagonist to glutamate receptors. The suppression of SD could be reversed by washing the preparation in a physiologic salt solution. The gl-antagonists in relatively high concentrations had a cytotoxic effect which, when severe, suppressed SD and prevented the recovery of this phenomenon by washing the compound out of the tissue. The compounds examined had, in addition to their gl-antagonistic properties, a gl-agonistic effect, which was postulated to enhance the Na+ permeability of neural membranes resulting in a release of K+ into the extracellular space. In preparations bathed in 10 mM MgCl2 (which suppresses Mg-sensitive SDs) the four compounds investigated promoted Mg-insensitive SDs supposedly when the extracellular K+ concentration reached values between 10 and 20 mequiv.     

Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes

Quinolinic acid (QA) is an endogenous neurotoxin involved in various neurological diseases, whose action seems to be exerted via glutamatergic receptors. However, the exact mechanism responsible for the neurotoxicity of QA is far from being understood. We have previously reported that QA inhibits vesicular glutamate uptake. In this work, investigating the effects of QA on the glutamatergic system from rat brain, we have demonstrated that QA (from 0.1 to 10mM) had no effect on synaptosomal L-[3H]glutamate uptake. The effect of QA on glutamate release in basal (physiological K+ concentration) or depolarized (40 mM KCl) conditions was evaluated. QA did not alter K+-stimulated glutamate release, but 5 and 10mM QA significantly increased basal glutamate release. The effect of dizolcipine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor on glutamate release was investigated. MK-801 (5 microM) did not alter glutamate release per se, but completely abolished the QA-induced glutamate release. NMDA (50 microM) also stimulated glutamate release, without altering QA-induced glutamate release, suggesting that QA effects were exerted via NMDA receptors. QA (5 and 10mM) decreased glutamate uptake into astrocyte cell cultures. Enhanced synaptosomal glutamate release, associated with inhibition of glutamate uptake into astrocytes induced by QA could contribute to increase extracellular glutamate concentrations which ultimately lead to overstimulation of the glutamatergic system. These data provide additional evidence that neurotoxicity of QA may be also related to disturbances on the glutamatergic transport system, which could result in the neurological manifestations observed when this organic acid accumulates in the brain.     

Two mechanisms for spreading depression in the chicken retina

Two mechanisms have been proposed to explain spreading depression (SD): one based on a release of glutamate (Van Harreveld, 1959), and the other on a release of potassium (Grafstein, 1956) from neuronal elements. Both glutamate and KCl cause transparency changes in the retina, comparable to those occurring in this tissue during SD. The glutamate effect is inhibited by MgCl₂ (10 mM), in contrast to the transparency change due to KCl which is not affected by Mg⁺⁺. Also SD is usually inhibited by MgCl₂ which suggests that such SDs are based on a glutamate release. Impairment of the tissue metabolism promotes SDs which are insensitive to MgCl₂. The resulting failure of the mechanisms that transport K⁺ and glutamate which leak out of the intracellular compartment back into the cells and fibers, seems to be involved in the generation of Mg⁺⁺ insensitive SDs. This may facilitate either K-based SDs or glutamate-based SDs since the inhibitory effect of Mg⁺⁺ is counteracted by an enhanced glutamate concentration. Both proposed mechanisms for SD seem to be possible under special circumstances.     

Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.     

Proline promotes decrease in glutamate uptake in slices of cerebral cortex and hippocampus of rats

In the present study we first investigated the in vitro and in vivo effects of proline on glutamate uptake in the cerebral cortex and hippocampus slices of rats. The action of alpha-tocopherol and/or ascorbic acid on the effects elicited by administration of proline was also evaluated. For in vitro studies, proline (30.0 microM and 1.0 mM) was added to the incubation medium. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or saline (control) and were sacrificed 1 h later. Results showed that addition of proline in the assay (in vitro studies) reduces glutamate uptake in both cerebral structures. Administration of proline (in vivo studies) reduces glutamate uptake in the cerebral cortex, but not in the hippocampal slices of rats. In another set of experiments, 22-day-old rats were pretreated for one week with daily administration of alpha-tocopherol (40 mg/kg) or ascorbic acid (100 mg/kg) or with both vitamins. Twelve hours after the last vitamins injection, rats received a single injection of proline or saline and were killed 1 h later. Pretreatment with alpha-tocopherol and/or ascorbic acid did not prevent the effect of proline administration on glutamate uptake. alpha-Tocopherol plus ascorbic acid prevented the inhibitory effect of acute hyperprolinemia on Na(+),K(+) -ATPase activity in the cerebral cortex of 29-day-old rats. The data indicate that the effect of proline on reduction of glutamate uptake and Na(+),K(+) -ATPase activity may be, at least in part, involved in the brain dysfunction observed in hyperprolinemic patients.     

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

The sites of high affinity binding of L-[3H]glutamic acid in human platelets. A new type of platelet receptor?

The total membrane fraction of human platelets was found to contain high affinity sites of L-[3H]glutamic acid binding (Kd = 100 nM, Bmax = 1.06 pmol/mg protein). The pH optimum for binding is at pH approximately 6.9 Na+ (1-150 mM) inhibit glutamate binding by platelet membranes (IC50 = 12 mM). Ca2+ (50-100 microM) stimulate the binding by 10-20% and inhibit it by 20-30% at concentrations of 1-5 mM. Monoclonal antibodies to the glutamate receptor strongly suppress the L-[3H]glutamate binding by platelet membranes (IC50 = 300 nm). The presence in human platelets of a glutamate-sensitive receptor complex similar to the central nervous system glutamate receptor is postulated.     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.     

Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.     

ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells.

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.     

The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium.

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.     

Analysis of the kefA2 mutation suggests that KefA is a cation-specific channel involved in osmotic adaptation in Escherichia coli

Mechanosensitive channels play an essential role in the regulation of turgor pressure in bacteria. In Escherichia coli, there are multiple mechanosensitive channels that have been characterized genetically: MscL, YggB and KefA. In this report, we describe the cloning of the kefA gene, the organization of the KefA protein and the phenotype of a missense mutation, kefA, which affects the KefA mechanosensitive channel. The altered function of the channel is manifest through increased sensitivity to K+ during growth at low osmolarity and complete inhibition of growth in media containing high K+ concentrations (0.6 M) in the presence of betaine or proline. Growth in high Na+ medium (0.6 M NaCl plus 20 mM K+) is normal. Analysis of the cytoplasmic pools shows that the mutant cannot regulate the K+ content of the cytoplasm when grown in high K+ medium. However, regulation of pools of amino acids is essentially normal and the mutant can accumulate high pools of proline during growth inhibition. The mutant shows increased sensitivity to acid hypo-osmotic shock (transition from neutral to acid pH combined with a reduction in osmolarity). The data are consistent with abnormal regulation of KefA in the presence of high K+ concentrations and either betaine or proline.     

Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis.

gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline.     

gltB gene and regulation of nitrogen metabolism by glutamine synthetase in Escherichia coli.

A mutant (gltB) of Escherichia coli lacking glutamate synthase (GOGAT) was unable to utilize a wide variety of compounds as sole nitrogen source (e.g., arginine, proline, gamma-aminobutyrate, and glycine). Among revertants of these Asm- strains selected on one of these compounds (e.g., arginine, proline, or gamma-aminobutyrate) were those that produce glutamine synthetase (GS) constitutively (GlnC phenotype). These revertants had a pleiotropically restored ability to grow on compounds that are metabolized to glutamate. This suggested that the expression of the genes responsible for the metabolism of these nitrogen sources was regulated by GS. An examination of the regulation of proline oxidase confirmed this hypothesis. The differential sensitivities of GlnC and wild-type strains to low concentrations (0.1 mM) of the glutamine analog L-methionine-DL-sulfoximine supported the conclusion that the synthesis of a glutamine permease was also positively controlled by GS. During the course of this study we found that the reported position of the locus (gltB) for glutamate synthase is incorrect. We have relocated this gene to be 44% linked to the argG locus by P1 transduction. Further mapping has shown that the locus previously called aspB is in reality the gltB locus and that the "suppressor" of the aspB mutation (A. M. Reiner, J. Bacteriol. 97:1431-1436, 1969) is the locus for glutamate dehydrogenase (gdhA).     

Mutant strains (nit) of Salmonella typhimurium with a pleiotropic defect in nitrogen metabolism.

We have isolated mutant strains (nit) of Salmonella typhimurium that are defective in nitrogen metabolism. They have a reduced ability to use a variety of compounds including glutamate, proline, arginine, N-acetyl-glucosamine, alanine, and adenosine as sole nitrogen source. In addition, although they grow normally on high concentrations of ammonium chloride (greater than 1 mM) as nitrogen source, they grow substantially more slowly than wild type at low concentrations (less than 1 mM). We postulated that the inability of these strains to utilize low concentrations of ammonium chloride accounts for their poor growth on other nitrogen sources. The specific biochemical lesion in strains with a nit mutation is not known; however, mutant strains have no detectable alteration in the activities of glutamine synthetase, glutamate synthetase, or glutamate dehydrogenase, the enzymes known to be involved in assimilation of ammonia. A nit mutation is suppressed by second-site mutations in the structural gene for glutamine synthetase (glnA) that decrease glutamine synthetase activity.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate

A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-[3H]glutamate. Effective labeling of glutamate receptors in slide-mounted 10-microns sections was obtained using a low incubation volume (0.15 ml) and rapid washing: a procedure where high ligand concentrations were achieved with minimal waste. Saturation experiments using [3H]glutamate revealed a single binding site of micromolar affinity. The Bmax was trebled in the presence of Ca2+ (2.5 mM) and Cl- (20 mM) with no change in the Kd. Binding was rapid, saturable, stereospecific, and sensitive to glutamate receptor agonists. The proportions of [3H]glutamate binding sensitive to N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were 34, 54, and 51%, respectively. NMDA inhibited binding at a distinct subset of L-[3H]glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-[3H]glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue.     

Cell volume regulatory responses of isolated perfused rat liver. The effect of amino acids

1) Addition of glutamine, glycine, alanine, serine, phenylalanine, proline at a concentration of 3mM, each, or of an amino-acid mixture resembling the physiological amino-acid composition of portal venous blood, to influent perfusate of isolated perfused rat liver led to a 4-6% increase of liver mass without increase of the [3H]inulin space, and biphasic K+ movements across the plasma membrane. These K+ movements consisted of an initial net K+ uptake (0.4-0.9 mumol X g-1 liver) for about 2 min, being followed by a net K+ release (1.0-2.8 mumol X g-1 liver) during the next 10 min. Withdrawal of the amino acids from influent perfusate caused a slow net K+ reuptake by the liver and restored the initial liver mass. No effects on liver mass and K+ fluxes were observed following addition of glutamate or glucose at a concentration of 3mM, each. 2) Aminooxyacetate did not affect the alanine (3 mM) induced increase in liver mass. However, in presence of aminooxyacetate the alanine-induced net K+ release from the liver (i.e. K+ release from 2-10 min minus initial K+ uptake) increased from 0.1 to 2.2 mumol X g-1 liver, whereby simultaneously the alanine tissue level rose from 6.8 to 13.3 mumol X g-1 (corresponding to an increase of the intracellular alanine concentration from about 12 to 25 mM) in presence of aminooxyacetate. 3) When livers were perfused with different glutamine concentrations, a maximal increase in liver mass of 5-6% was observed at glutamine concentrations above 1.5-2mM. A halfmaximal increase in liver mass was observed at 0.6-1.0mM glutamine in influent, i.e. at the physiological portal glutamine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.