Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.     

Two-compartment behavior during transport of folate compounds in L1210 cell plasma membrane vesicles

Summary The transport of [³H] 1,l 5-formyltetrahydrofolate, [³H] folic acid, and [³H]methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

Studies on isolated subcellular components of cat pancreas

Summary Transport of alanine was studied in isolated plasma membrane vesicles from cat pancreas using a rapid filtration technique. The uptake is osmotically sensitive and the kinetics ofl-alanine transport are biphasic showing a saturable and a nonsaturable component. The saturable component is seen only when a sodium gradient directed from the medium to the vesicular space is present. Under this condition an overshooting uptake ofl-but not ofd-alanine occurs. The Na⁺ gradient stimulated uptake ofl-alanine is inhibited byl-serine andl-leucine and stimulated when the membrane vesicles had been preloaded withl-alanine,l-serine orl-leucine.The ionophore monensin inhibits stimulation of uptake caused by a sodium gradient. In the presence of valinomycin or carbonyl cyanidep-trifluoromethoxyphenylhydrazone (CFCCP), the sodium-dependent transport is augmented in vesicles preloaded with K₂SO₄ or H⁺ ions (intravesicular pH 5.5), respectively. In the presence of different anions, the Na⁺-dependent transport is stimulated according to increasing anionic penetration through membranes (lipid solubility). We conclude that a sodium dependent electrogenic amino acid transport system is present in pancreatic plasma membranes.     

Sodium-cotransport systems in intestine and kidney of the winter flounder

Summary Brush-border membrane vesicles were isolated from the intestine and kidney of the winter flounder,Pseudopleuronectes americanus, and the transport ofd-glucose,l-alanine and sodium was examined by a rapid filtration technique.d-glucose,l-alanine, and sodium entered the same osmotically reactive space suggesting that uptake into vesicles represents transport across rather than binding to the membrane. d-glucose andl-alanine uptake by intestinal and renal brush-border membrane vesicles was stimulated by sodium as compared to potassium or choline. In the presence of a sodium chloride gradient, overshooting uptake was observed indicating a transient intravesicular accumulation ofd-glucose andl-alanine. The sodium-dependentd-glucose uptake was inhibited by phlorizin andd-galactose while the transport ofl-alanine was inhibited byl-phenylalanine. The sodium-dependent transport ofd-glucose andl-alanine was affected by the electrical potential difference across the vesicle membrane; the addition of valinomycin in the presence of an inwardly directed potassium chloride gradient inhibited sodium-dependent solute uptake, whereas replacing chloride or gluconate with more permeant anions, such as SCN^–, stimulated uptake. Similar results were obtained with intestinal and renal membranes; they document the presence of sodium/d-glucose and sodium/l-alanine cotransport systems in the brush-border membrane of intestine and kidney.Sodium uptake into brush border membrane vesicles from the flounder intestine and kidney was saturable (tracer replacement) and trans-stimulated (tracer coupling), indicating transport via facilitated diffusion systems. Additionally, sodium uptake was only slightly affected by superimposing diffusion potentials demonstrating that the majority of sodium transport was by electroneutral coupled processes. In both the intestinal and kidney brush-border membrane vesicles sodium uptake was inhibited by an inwardly directed proton gradient suggesting the presence of a sodium/proton exchange mechanism. In intestinal, but not in renal membrane preparations, sodium uptake was stimulated by chloride. Chloride stimulation was abolished after preincubation with furosemide indicating the presence of an additional coupled sodium-chloride transport in the intestinal brush-border membranes.The experiments were carried out at the Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USAAddress effective February 1, 1980: Albert Einstein College of Medicine, Department of Physiology, 1300 Morris Park Avenue, Bronx, New York 10461, USA     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

Obtaining functional membrane vesicles from Leuconostoc oenos to study l-malate transport mechanisms

Membrane vesicles from the malolactic bacterium Leuconostoc oenos were obtained by a modified version of the procedure of Kaback [Methods Enzymol 22:99–120 (1971)]. Protoplasts were produced at frequencies greater than 95% by a method entailing mutanolysis digestion and osmotic shock. Glycerol or polyethyleneglycol 600 was required as an osmotic stabilizer while the use of sucrose prevented closed vesicle formation during osmotic shock. The membrane vesicles retained their functional properties and accumulated l-malic acid in response to an ATPase-induced proton gradient across the membrane of ATP-loaded vesicles. l-Malate uptake was strongly inhibited by dicyclohexylcarbodiimide, a specific inhibitor of membrane-bound ATPase. These data support the possibility of a pH-dependent transport of l-malate. Vesicles not loaded with ATP were slightly permeable to malic acid with an initial uptake rate (0.5 nmol·l^–1·s^–1) similar to the diffusion rate obtained previously in a L. oenos malate-transport-deficient strain. These results confirm two simultaneous uptake mechanisms in L. oenos, a permease-mediated transport and a passive diffusion for the anionic and the undissociated forms of l-malic acid respectively.     

Effects of Na+, H+, and Cl− on alanine transport by lobster hepatopancreatic brush border membrane vesicles

Summary Epithelial brush border membrane vesicles (BBMV) of lobster hepatopancreas were formed by a magnesium precipitation technique previously described (Ahearn et al. 1985).³H-l-alanine transport by these vesicles was sodium and potassium insensitive, in contrast to a strong Na-dependency exhibited by³H-d-glucose transport. Initial alanine entry rates (15 s uptake) were stimulated and transient alanine uptake overshoots were observed when external pH was acidic (e. g. pH 4.0, 5.0 or 6.0) and a Cl gradient was imposed across the vesicular wall; at pH_o=7.4 alanine uptake was reduced in rate and hyperbolic in character. Alanine uptake from an acidic extravesicular environment in the absence of Cl responded to a transmembrane electrical potential difference created by an outwardly-directed, valinomycin-induced, potassium diffusion potential, suggesting that the alanine molecule alone carried sufficient charge under these conditions to respond to the electrical gradient. External 5.0 mMl-lysine andl-serine similarly inhibited the influx and overshoot properties of 0.05 mM³H-l-alanine uptake, whereas 5.0 mMl-leucine had virtually no effect. Trans-stimulation of alanine initial uptake rates and an enhancement of alanine accumulation against a concentration gradient were observed by vesicles preloaded with 1 mMl-lysine, but not by vesicles lacking amino acids or those containing 1 mMl-leucine orl-serine.³H-l-alanine influx from acidic external environments in the presence of a Cl gradient occurred by a combination of carrier-mediated transfer and apparent diffusion. Decreasing pH_o from 6.0 to 4.0 elevated alanineK _t from 0.55 to 2.64 mM, while alanineJ _M increased from 55 to 550 pmol/mg protein· 15 s. Apparent diffusional permeability of the membranes to alanine under these conditions increased slightly. These results suggest, but do not conclusively prove, that alanine transport across BBMV of lobster hepatopancreas may occur by way of a classical y+ transprot protein at acidic pH. The extent of this transport is determined by the magnitude of the transmembrane chloride gradient which serves as a powerful driving force for cationic amino acids in this tissue.     

Cation-dependent nutrient transport in shrimp digestive tract

Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. ³H-d-glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. ³H-l-histidine transport was only stimulated by a transmembrane potassium gradient, while ³H-l-leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to l-leucine. Uptake of ³H-l-leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn²⁺, Cu²⁺, Mn²⁺, Cd²⁺, or Co²⁺) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. ³H-l-histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis–Menten kinetics. The apparent affinity constant (e.g., K _m) for manganese was an order of magnitude smaller (K _m = 0.22 μM Mn) than that for zinc (K _m = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J _max). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Differential expression of Na<Superscript>+</Superscript>/<Emphasis Type="SmallCaps">d</Emphasis>-glucose cotransport in isolated cells of<Emphasis Type="Italic"> Marsupenaeus japonicus</Emphasis> hepatopancreas

d-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na⁺-dependent d-glucose transport (Na⁺/d-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na⁺/d-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na⁺/d-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na⁺/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na⁺/d-glucose cotransport system is still to be established.Communicated by: G. Heldmaier     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

In vitro ethanol effects on the transport properties of isolated renal brush-border membrane vesicles

Summary Thein vitro effect of ethanol on membrane structure and transport properties was studied in isolated renal brush border membrane vesicles.³¹P-NMR studies showed a dose-dependent increase in the quantity of an isotropic, possibly inverted-micellar component of the renal brush-border membrane as a result of treatment with ethanol. Such structures have been shown to be instrumental in the translocation of material across membrane bilayers. A²³Na-NMR study of Na⁺ exchange in artificial phosphatidylcholine liposomes indicated that ethanol (0.1%) was capable of rending the otherwise inert vesicles permeable to sodium, supporting the idea that ethanol may exert its action via a direct effect on the structure of the phospholipid bilayer. In the isolated renal brush-border membrane vesicles, like in the artificial liposomes, amiloride-insensitive pathways of Na⁺ transport were shown to be markedly activated by ethanol. These results were consistent with the inhibitory effect ethanol had on Na⁺ gradient-dependent transport systems such as the Na⁺ gradient-dependentd-glucose transport and Na⁺/H⁺ exchange. In conclusion, our results indicate that ethanol exerts its effect on the renal brush-border membrane by causing a structural change in the phospholipid bilayer which activates sodium intake. The inhibitory effect of ethanol on glucose uptake and Na⁺/H⁺ exchange is secondary, as a result of the dissipation of the energy-producing Na⁺ gradient.     

Interactions between valproate,glutamate, aspartate,and GABA with respect to uptake in astroglial primary cultures

Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na⁺-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and -aminobutyric acid (GABA) into astrocytes were examined. The [³H]valproate transport into the astrocytes was increased after exposure tol-glutamate but notl-aspartate. On the other hand, after acute exposure for the drug, the transport of [³H]l-glutamate and [³H]l-aspartate decreased, as also did the affinity but not the transport capacity for the [³H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics ofl-glutamate orl-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes.     

Functional characteristics ofl-glutamate,N-methyl-d-aspartate and kainate receptors in isolated brain synaptic membranes

l-Glutamic acid (l-Glu) and other excitatory amino acids and amino acid analogs enhanced [³⁵S]thiocyanate (SCN^–) uptake in isolated-resealed synaptic membrane vesicles. The SCN^– uptake was used as a measure of membrane depolarization to evaluate the characteristics of functional excitatory amino acid receptors in the synaptic membranes.N-Methyl-d-aspartate (NMDA) andl-Glu produced additive effects on SCN^– accumulation indicating the presence of distinctl-Glu and NMDA receptors. On the other hand, kainic acid (KA) andl-Glu shared either common receptor sites or ion channels. The effects of antagonists on NMDA,l-Glu, and KA stimulation of SCN^– influx were consistent with previously reported electrophysiologic observations in intact neurons.     

Substrate regulation of ascorbate transport activity in astrocytes

Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na⁺-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[¹⁴C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V _max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[¹⁴C]ascorbate. The increase inV _max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[³H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.