Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

星形神经胶质细胞摄取谷氨酸与环境因子和离子运输的关系

本文以星形神经胶质细胞为对象,用同位素示踪技术较详细地研究了介质中Na、、K~+和CL~-、不同浓度的卡因酸以及几种抑制剂对L-谷氨酸摄取的影响;并观察了L-谷氨酸对星形神经胶质细胞膜运输Na~+、K~+、Cl~-和Ca~(2+)等的作用.结果表明:L-谷氨酸的摄取依赖于介质中是否存在Na~+ ,在缺Na~+介质中对Cl~-的依赖性也较明显,但在正常Na~+浓度下,含Cl~_和缺Cl~_没有明显差别.当增加介质中K~+浓度引起膜的去极化时,则能降低L~_谷氨酸的摄取.反过来,L-谷氨酸的摄取也对Na~+、K~+、Cl~-等的运输起刺激作用.此外,卡因酸及所用的几种抑制剂对谷氨酸的摄取办有明显抑制作用.     

Proline Metabolism in Leishmania donovani Promastigotes*

Oxygen uptake of Leishmania donovani culture promastigotes was stimulated by L-proline and to a lesser extent by L-glutamate and L-arginine. L-proline reversed partially KCN-induced inhibition of respiration and completely, inhibition caused by malonate. Labeled proline, glutamate, alanine, and arginine were detected by thin layer chromatography in the free amino acid pool from cells incubated with L-proline-¹⁴C. Labeled tricarboxylic acid cycle intermediates, α-ketoglutarate, succinate, fumarate, malate, and oxaloacetate, also were found by this method in extracts from organisms incubated with L-proline-¹⁴C which contained also pyruvate. Cells incubated with malic acid-¹⁴C contained labeled alanine, glutamate, and arginine. Labeled L-proline was not found in promastigotes incubated with D-glucose-¹⁴C, although arginine, glutamate, and alanine were detected in extracts from these organisms. Indirect evidence for the presence of a NADP-dependent malic enzyme was obtained by Ochoa's method. All results suggest the presence of a proline-glutamate interconversion pathway in L. donovani promastigote culture forms.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.     

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.     

Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.     

Characterization of an inducible citrate uptake system in Penicillium simplicissimum

When citrate was used as a sole source of carbon, citrate uptake by Penicillium simplicissimum increased 267-fold (if glucose-grown mycelium was adapted to citrate) or 1400-fold (if the fungus was grown on citrate) compared to glucose-grown mycelium. Inhibition of macromolecular synthesis prevented this stimulation of citrate uptake. Citrate uptake by glucose-grown mycelium was low (0.0015 nmol min(-1) (mg DW)(-1)) and most probably due to diffusion of undissociated citric acid. Citrate-adapted mycelium had a K(M) of 65 micromol l(-1) and a V(max) of 0.34 nmol min(-1) (mg DW)(-1). In citrate-grown mycelium K(M) was 318 micromol l(-1) and V(max) was 8.5 nmol min(-1) (mg DW)(-1). Citrate uptake was inhibited by sodium azide and uncouplers (TCS, 3,3',4',5-tetrachlorosalicylanilide; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone). Because of this we postulate that the induced citrate uptake must be an active transport process. The pH optimum of citrate uptake was between pH 6 and 7. EDTA and Mg2+, Mn2+, Cu2+, Zn2+, Fe2+, Ca2+ only weakly influenced the induced citrate uptake. The properties of citrate uptake by Aspergillus niger and P. simplicissimum are compared.     

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

L-Proline inhibits glutamate-based spreading depressions (SDs) at low concentrations (2--2.5 mM) and promotes K+-based SDs at higher concentrations (5 mM). The inhibition of glutamate-based SDs was postulated to be due to competition of L-glutamate and L-proline for glutamate receptors on somatic and dendritic plasma membranes. The binding of proline to glutamate receptors was furthermore postulated to 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 C atom in the ring structure than proline had similar effects as the latter amino acid, but an analog, L-azetidine-2-carboxylic acid, with one less C atom in the ring had little effect on SD in the retina.     

The acidic amino acid transport system of the baby hamster kidney cell line BHK21-C13

The uptake of L-glutamate into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported via a relatively high affinity, low capacity, Na+-dependent transport system capable of the rapid accumulation of substrate amino acids. Kinetic studies of the inhibition of L-glutamate uptake has provided information as to the substrate and the molecular configuration required for transport via the glutamate transport system. This system exhibited marked substrate specificity and was only capable of transporting L-glutamate and aspartate and certain closely related acidic amino acid analogues.     

Electrogenicity of sodium/L-glutamate cotransport in rabbit renal brush-border membranes: a reevaluation

In order to clarify contradictory reports on the electrogenicity of sodium/L-glutamate cotransport, this cotransport was studied using brush-border membrane vesicles isolated from rabbit renal cortex. Beforehand, the claim that the symport of L-glutamate with Na+ is linked to simultaneous antiport with K+ has been confirmed by the demonstration that equilibrium exchange of L-glutamate is inhibited by potassium. Concerning the electrogenicity of the system, the following results are reported: net uptake of sodium-dependent L-glutamate uptake was stimulated when the transmembranal electrical potential difference was increased by replacing a sodium sulfate gradient by a sodium nitrate gradient. At 100 mM Na+ the 'relative electrogenicity' of the initial uptake in the presence of intravesicular potassium was 2-times higher than in its absence. At a sodium concentration of 20 mM, when overall uptake was reduced, the relative electrogenicity in the presence of K+ was even 3-fold higher than in K+-free media. The relative electrogenicity of sodium/D-glucose cotransport measured under the same experimental conditions was not affected by K+. These results are discussed in terms of a model where the apparent electrogenicity of a cotransport system is dependent on the extent to which the charge translocating step is rate limiting ('rate limitancy'). It is proposed that potassium antiport, while decreasing charge stoichiometry of Na+/glutamate transport, increases the relative rate limitancy of the transport step translocating three cations (probably two Na+, one H+) together with one glutamate. Thereby the positive electrogenicity of glutamate uptake increases, in complete contrast to what would be expected from simple considerations of charge stoichiometry.     

Transport of L-proline and its regulation in Leishmania tropica promastigotes.

Leishmania tropica promastigotes transport L-proline through an active uptake system that has saturation kinetics, temperature dependence, a requirement for metabolic energy and transport against a concentration gradient. In experiments lasting 10 min, less than 10% of the proline transported is incorporated into macromolecules. The remainder is largely unaltered proline with an intracellular concentration nearly 60 times that in the reaction mixture. The uptake system has a relatively broad specificty; it is competitively inhibited by D-proline as well as by alanine, methionine, valine, azetidine-2-carboxylate, thioproline, 3,4-dehydropoline, hydroxyproline and alpha-aminoisobutyric acid. Pre-established intracellular proline pools exchange with external proline as well as compounds that compete with it for uptake. Evidence is presented that feedback inhibition and transinhibition may regulate proline uptake in this organism.     

Ferrous-iron-dependent uptake of L-glutamate by a mesophilic,mixotrophic iron-oxidizing bacterium strain OKM-9

Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.     

Osmotic regulation of intracellular solute pools in Lactobacillus plantarum.

Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor pressure. The cytoplasmic pools of K+, proline, glutamate, alanine, and glycine of Lactobacillus plantarum ATCC 14917 increased when the osmolarity of the growth media was raised from 0.20 to 1.51 osmol/kg by KCL. When glycine-betaine was present in a high-osmolarity chemically defined medium, it was accumulated to a high cytoplasmic concentration, while the concentrations of most other osmotically important solutes decreased. These observations, together with the effects of glycine-betaine on the specific growth rate under high-osmolarity conditions, suggest that glycine-betaine is preferentially accumulated in L. plantarum. Uptake of glycine-betaine, proline, glutamate, and alanine was studied in cells that were alternately exposed to hyper- and hypo-osmotic stresses. The rate of uptake of proline and glycine-betaine increased instantaneously upon increasing the osmolarity, whereas that of other amino acids did not. This activation occurred also under conditions in which protein synthesis was inhibited was most pronounced when cells were pregrown at high osmolarity. The duration of net transport was a function of the osmotic strength of the assay medium. Glutamate uptake was not activated by an osmotic upshock, and the uptake of alanine was low under all conditions tested. When cells were subjected to osmotic downshock, a rapid efflux of accumulated glycine-betaine, proline, and alanine occurred whereas the pools of other amin acids remained unaffected. The results indicate that osmolyte efflux is, at least to some extent, mediated via specific osmotically regulated efflux systems and not via nonspecific mechanisms as has been suggested previously.     

Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.     

Na+ -dependent proline transport in isolated membrane vesicles from the L6 muscle cell line. Stimulation of uptake by intravesicular proline

Membrane vesicles of L6 myoblasts were prepared in order to study the amino acid transport system A. The role of the membrane in the adaptive response of transport to amino acid-supplementation was assessed. The membranes, prepared by N2 cavitation, displayed Na+ (but not K+)-dependent L-proline uptake. An overshoot of L-[3H]proline uptake was observed after exposure of the vesicles to an inward Na+ gradient. Isolated membrane vesicles loaded with 50 microM proline displayed countertransport (stimulation of proline uptake). It is concluded that the adaptive decrease of proline uptake observed in amino acid-supplemented cells cannot be accounted for by trans-inhibition of transport.     

Uptake of proline by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up 1 millimolar l-[¹⁴C]proline at an initial rate of about 6.5 micromoles gram⁻¹ fresh weight hour⁻¹ (pH 5, 30°C). The uptake had a pH optimum at 5. The bulk of the uptake (93%) was via carrier-mediated active transport. All of the 19 l-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. d-Proline inhibited this system as strongly as l-proline. Nine of the 16 l-amino acids tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain.     

Transport of L-Proline and its Regulation in Leishmania tropica Promastigotes*

Leishmania tropica promastigotes transport L-proline through an active uptake system that has saturation kinetics, temperature dependence, a requirement for metabolic energy and transport against a concentration gradient. In experiments lasting 10 min, less than 10% of the proline transported is incorporated into macromolecules. The remainder is largely unaltered proline with an intracellular concentration nearly 60 times that in the reaction mixture. The uptake system has a relatively broad specificity; it is competitively inhibited by D-proline as well as by alanine, methionine, valine, azetidine-2–carboxylate, thioproline, 3,4–dehydroproline, hydroxyproline and α-aminoisobutyric acid. Pre-established intracellular proline pools exchange with external proline as well as compounds that compete with it for uptake. Evidence is presented that feedback inhibition and transinhibition may regulate proline uptake in this organism.     

Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na⁺-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min^–1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min^–1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.Abbreviations MeAIB methyl aminoisobutyric acid