A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.     

Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

L-alanine transport by systems A and ASC in plasma membrane vesicles isolated from Ehrlich cells.

Plasma membrane vesicles were prepared from Ehrlich cells using two-phase system compartmentation. These vesicles accumulated L-alanine mainly by means of Na(+)-dependent transport systems A and ASC. The kinetic of both transport systems could be elucidated by specific inhibition of system A with methyl-aminoisobutyric acid.     

Amino acid-dependent increase in hepatic system N activity is linked to cell swelling

Treatment of cultured rat hepatocytes with certain amino acids stimulates the activity of the System N transporter. The present report investigates the mechanism by which the stimulatory amino acids elicit their effect. Activation of System N-mediated transport by amino acids is rapid, cycloheximide-insensitive, and involves neither trans-stimulation nor recruitment of additional carriers to the plasma membrane. In addition, the activation is Na(+)-dependent, supporting the related observation that the most effective stimulatory amino acids are substrates of Na(+)-dependent transport Systems A, ASC, and N whereas substrates of Na(+)-independent System L and non-amino acid metabolites are ineffective. The data suggest that active accumulation of amino acids via Na(+)-dependent carriers is necessary for the activation to occur. The amino acid-dependent stimulation is blocked in a concentration-dependent manner by increasing extracellular K+. Treatment of hepatocytes with an amino acid such as asparagine causes cell swelling and stimulation of System N activity; both of these effects are reduced by hypertonic media. Furthermore, swelling of rat hepatocytes with hypotonic media mimics the System N-stimulatory effects of asparagine. Among the Na(+)-dependent amino acid transport systems present in rat hepatocytes, System N is stimulated preferentially by amino acid-containing or hypotonic media. Collectively, these results demonstrate that cell swelling is a prerequisite for the amino acid-dependent activation of the hepatic System N transporter.     

Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes

The expression of the basolateral Na+/bile acid (taurocholate) cotransport system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA into the oocytes resulted in the functional expression of Na+ gradient stimulated taurocholate uptake within 3-5 days. This Na(+)-dependent portion of taurocholate uptake exhibited saturation kinetics (apparent Km approximately 91 microM) and could be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. Furthermore, the expressed taurocholate transport activity demonstrated similar substrate inhibition and stimulation by low concentrations of bovine serum albumin as the basolateral Na+/bile acid cotransport system previously characterized in intact liver, isolated hepatocytes, and isolated plasma membrane vesicles. Finally, a 1.5- to 3.0-kilobase size-class of mRNA could be identified that was sufficient to express the basolateral Na+/taurocholate uptake system in oocytes. These results demonstrate that "expression cloning" represents a promising approach to ultimately clone the gene and to further characterize the molecular properties of this important hepatocellular membrane transport system.     

Regulation of calcium content in bovine spermatozoa

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm. Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity. It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm (Rufo, G. A., Jr., Singh, J. P., Babcock, D. F., and Lardy, H. A. (1982) J. Biol. Chem. 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.     

Short-term stimulation of Na+-dependent amino acid transport by dibutyryl cyclic AMP in hepatocytes. Characteristics and partial mechanism.

The short-term protein-synthesis-independent stimulation of alanine transport in hepatocytes was further investigated. Cyclic AMP increased the Vmax. of alanine transport. Amino acid transport via systems A, ASC and N was stimulated. A good correlation was found between the initial rate of transport and the cell membrane potential as calculated from the distribution of Cl-. Cyclic AMP increased the rate of alanine transport, stimulated Na+/K+ ATPase (Na+/K+-transporting ATPase) activity and caused membrane hyperpolarization. The time courses and cyclic AMP dose-dependencies of all three effects were similar. Ouabain abolished the effect of cyclic AMP on Cl- distribution and on transport of alanine. The effect of cyclic AMP on alanine transport and Cl- distribution was mimicked by the antibiotic nigericin; the effect of nigericin was also abolished by ouabain. It is concluded that the effect of cyclic AMP on transport is mediated via membrane hyperpolarization. It is suggested that the primary action of cyclic AMP is to increase the activity of an electroneutral Na+/K+-exchange system in the liver cell plasma membrane, thus hyperpolarizing the membrane by stimulating the electrogenic Na+/K+ ATPase.     

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Na(+)-dependent transport of alanine and serine by liver plasma-membrane vesicles from rats fed a low-protein or a high-protein diet

Plasma-membrane vesicles prepared from the liver of rats fed either a low-(LP) or a high-protein (HP) diet exhibited Na(+)-dependent active transport of alanine and serine. The process gave apparent kinetic parameters compatible with a single saturable component for both amino acids. Na,K-ATPase (EC 3.6.1.37), marker of the basolateral domain of the hepatocyte plasma-membrane, was chosen as reference for the expression of amino acid transport in vesicle preparations. The high-protein diet induced a significant increase in liver Na,K-ATPase activity also found in corresponding plasma-membrane preparations, in parallel with an increase in the capacity towards amino acid transport. This suggests that in rats fed the high protein diet, transcellular Na+ exchange, although increased, remains well balanced. N-Methylaminoisobutyric acid (MeAIB), due to its poor velocity, proved unsuitable to distinguish between systems A and ASC in the experimental model. Comparing Na(+)- and Li(+)-driven transport, a family of carriers with strict Na(+)-dependency (A-like) was evidenced in LP vesicles but not in HP vesicles. The sensitivity to the lowering of the pH from 7.5 to 6.5 in the external medium was similar in both type of vesicles when Na+ was the driving ion. In the HP vesicles the Li(+)-tolerant, pH-insensitive component (ASC-like) was increased in parallel with overall Na(+)-dependent transport. These functional properties suggest that the carriers involved in the stimulation of transport in HP vesicles are composite in nature. Increasing concentrations of an amino acid mixture mimicking the changes of portal aminoacidemia inhibited the transport of alanine and of serine. The degree of inhibition was correlated with the relative concentration of substrate and was independent of the nutritional treatment.     

Na+-dependent transport of alpha-aminoisobutyrate in isolated basolateral membrane vesicles from rat parotid glands

Basolateral plasma membranes were prepared from rat parotid gland after centrifugation in a self-orienting Percoll gradient. K+-dependent phosphatase [Na+ + K+)-ATPase), a marker enzyme for basolateral membranes, was enriched 10-fold from tissue homogenates. Using this preparation, the transport of alpha-aminoisobutyrate was studied. The uptake of alpha-aminoisobutyrate was Na+-dependent, osmotically sensitive, and temperature-dependent. In the presence of a Na+ gradient between the extra- and intravesicular solutions, vesicles showed an 'overshoot' accumulation of alpha-aminoisobutyrate. Sodium-dependent alpha-aminoisobutyrate uptake was saturable, exhibiting an apparent Km of 1.28 +/- 0.35 mM and Vmax of 780 +/- 170 pmol/min per mg protein. alpha-Aminoisobutyrate transport was inhibited considerably by monensin, but incubating with ouabain was without effect. These results suggest that basolateral membrane vesicles, which possess an active amino acid transport system (system A), can be prepared from the rat parotid gland.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

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.     

Modification of system A amino acid carrier by diethyl pyrocarbonate

Sodium-dependent alanine transport in plasma membrane vesicles from rat liver was inactivated in a time- and concentration-dependent fashion by prior treatment of membranes with the acylating reagent diethyl pyrocarbonate (DEPC). Both components of Na+/alanine cotransport (systems A and ASC) were inhibited. Exposure of vesicles to p-bromophenacyl bromide and methyl p-nitrobenzenesulfonate, which share with DEPC reactivity against histidine residues, also led to inhibition of alanine transport through systems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure to vesicles to DEPC protected against inactivation of system A (but not system ASC) transport activity. This protective effect was specific and required the presence of L-alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) did not cause any detectable protection. This overall pattern of protection is opposite to that previously found against specific sulfhydryl reagents (i.e. N-ethylmaleimide), where protection of system ASC was nearly maximal. The pH profile for DEPC-dependent inhibition of system A transport activity suggests modification of amino acid residue(s) with a pKr of approximately 7, most likely histidine(s), in close parallel with the pH dependence of system A transport activity. Our results suggest the presence of critical histidine residues on the system A carrier that may be responsible for the pH dependence of system A transport activity.     

Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth

Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0) and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Amino acid transport in kidney epithelial cell line (MDCK): characteristics of Na+/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Translocation of fatty acids across the basolateral rat liver plasma membrane is driven by an active potential-sensitive sodium-dependent transport system

In previous studies it was shown that hepatocellular uptake of fatty acids is mediated by a specific fatty acid binding membrane protein. To determine now directly the driving forces for their entry into hepatocytes, the uptake of a representative long chain fatty acid, [3H]oleate, by basolateral rat liver plasma membrane vesicles was examined. Influx of oleate was stimulated by increasing the Na+ concentration of the medium. In the presence of an inwardly directed Na+ gradient (NaSCN, NaNO3, NaCl) oleate was accumulated during the initial uptake phase (20 s) at a concentration of 1.4-1.9-fold that at equilibrium (overshoot). This activation of influx was not observed after replacement of Na+ by Li+, K+, or choline+. Na+-dependent oleate uptake was significantly stimulated by creation of a negative intravesicular potential, either by altering the accompanying anions or by valinomycin-induced K+ diffusion potentials, suggesting an electrogenic transport mechanism. Na+-dependent fatty acid uptake was temperature dependent, with maximal overshoots occurring at 37 degrees C, and revealed saturation kinetics with a Km of 83.1 nM and Vmax of 2.9 nmol X min-1 X mg protein-1. These studies demonstrate that the carrier-mediated hepatocellular uptake of fatty acids represents an active potential-sensitive Na+-fatty acid cotransport system.     

Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.