Human placental brush-border membrane Na(+)-pantothenate cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin pantothenate were investigated by assessing the possible presence of a Na(+)-pantothenate cotransport mechanism in the maternal facing membrane of human placental epithelial cells. The presence of Na(+)-pantothenate cotransport was determined from radiolabeled tracer flux measurements of pantothenate uptake using preparations of purified brush-border membrane vesicles. Compared with other cations the imposition of an inward Na+ gradient stimulated vesicle uptake of pantothenate to levels approximately 40-fold greater than those observed at equilibrium. The observed stimulation of pantothenate uptake was not the result of indirect electrostatic coupling to an inside positive Na+ diffusion potential. In the absence of Na+ and pantothenate concentration gradients an inside negative voltage difference induced a Na(+)-dependent net influx of pantothenate, suggesting the presence of an electrogenic Na(+)-pantothenate cotransport mechanism. The effect of biotin on the kinetics of Na(+)-dependent pantothenate uptake and the effect of pantothenate on the kinetics of Na(+)-dependent biotin uptake suggested that placental absorption of biotin and pantothenate from the maternal circulation occurs by a common Na+ cotransport mechanism in apical brush-border membrane.     

Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.     

Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.     

Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms

The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5'-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5'-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding.     

Pantothenate-sodium cotransport in renal brush-border membranes

The mechanism of pantothenate transport into rabbit renal brush-border membrane vesicles was studied. Under voltage-clamped conditions, an inward NaCl gradient induced the transient accumulation of pantothenate against its concentration gradient, indicating Na+/pantothenate cotransport. K+, Rb+, Li+, NH4+, and choline+ were ineffective in replacing Na+. Pantothenate analogs, D-glucose, and various carboxylic acids did not inhibit Na+-dependent pantothenate transport, suggesting that this system is specific for pantothenate. Kinetic analysis of the Na+-dependent pantothenate uptake revealed a single transport system which obeyed Michaelis-Menten kinetics (Km = 16 microM and Vmax = 6.7 pmol X mg-1 X 10 s-1). Imposition of an inside-negative membrane potential caused net uphill pantothenate accumulation in the presence of Na+ but absence of a Na+ gradient, indicating that Na+/pantothenate cotransport is electrogenic. The relationship between extravesicular Na+ concentration and pantothenate transport measured under voltage-clamped conditions was sigmoidal: a Hill coefficient (napp) of 2 and a [Na+]0.5 of 55 mM were calculated. It is suggested that an anionic pantothenate1- molecule is cotransported with two Na+ to give a net charge of +1. The coupling of pantothenate transport to the Na+ electrochemical gradient may provide an efficient mechanism for reabsorption of pantothenate in the kidney.     

Na(+)-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex

The presence of a Na(+)/D-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na(+) gradient, but not in its absence, D-mannose was transiently accumulated by the BBMV. D-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na(+) chemical gradient. D-Mannose transport vs. external D-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 microM D-mannose. D-Mannose uptake was inhibited by D-mannose > D-glucose>phlorizin, whereas for alpha-methyl glucopyranoside the order was D-glucose=phlorizin > D-mannose. The initial rate of D-mannose uptake increased as the extravesicular Na(+) concentration increased, with a Hill coefficient of 1, suggesting that the Na(+):D-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na(+)-dependent D-mannose transport mechanism, which is different from SGLT1.     

Biotin transport in rat intestinal brush-border membrane vesicles

Transport of biotin across rat intestinal brush-border membrane was examined using the brush-border membrane vesicle (BBMV) technique. Uptake of biotin by BBMV is the result of transport of the substrate into the intravesicular space with negligible binding to membrane surfaces. In the presence of a Na+ gradient (out greater than in), transport of biotin was higher with a transient 'overshoot' phenomenon. In comparison, transport of biotin in the presence of a choline gradient (out greater than in) was lower with no 'overshoot' phenomenon. In both jejunal and ileal BBMV, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear in the presence of a choline gradient (out greater than in). Vmax of the Na+-dependent transport system was 0.88 and 0.37 pmol/mg protein per s and apparent Kt was 7.57 and 7.85 microM in jejunal and ileal BBMV, respectively. Structural analogues inhibited the transport process of biotin. Unlike the electrogenic transport of D-glucose, the transport of the anionic biotin was not affected by imposing a relatively positive intravesicular potential with the use of valinomycin and an inwardly-directed K+ gradient, suggesting that biotin transport is most probably an electroneutral process. This suggestion was further supported by studies on biotin transport in the presence of anions of different lipid permeability. The results of this study demonstrate that biotin transport across rat intestinal brush-border membrane is by a carrier-mediated, Na+-dependent and electroneutral process. Furthermore, transport of biotin is higher in the jejunum than the ileum.     

Amino acid transport and rubidium-ion uptake in monolayer cultures of hepatocytes from neonatal rats

Amino acid and K(+) transport during development has been investigated in hepatocyte monolayer cultures with either alpha-amino[1-(14)C]isobutyrate or (86)Rb(+) used as a tracer for K(+). Parenchymal cells from neo- and post-natal rat livers have been isolated by an improved non-perfusion technique [Bellemann, Gebhardt & Mecke (1977)Anal.Biochem.81, 408-415], and the resulting hepatocyte suspensions purified from non-hepatocytes before inoculation. In the presence of Na(+) (Na(+)-dependent component), the rates of amino acid uptake in neonatal hepatocytes were markedly enhanced compared with cells from 30-day-old rats. When Na(+) was replaced by choline (Na(+)-independent component) the accumulation of alpha-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na(+)-dependent alpha-aminoisobutyrate transport revealed the existence of a high-affinity low-K(m) component (K(m)0.91mm) with a V(max.) of 2.44nmol/mg of protein per 4min, which later declined gradually with progressive development. Rates of Rb(+) transport were concomitantly enhanced in neonatal hepatocytes and thereafter declined with postnatal age. The increased Rb(+) influx was effectively inhibited by ouabain and reflected elevated activity of the electrogenic Na(+)/K(+)-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb(+) uptake indicates multiple and co-operative binding sites of the enzyme involved in the Rb(+) uptake, and the transport system is positively co-operative (the Hill coefficient h is >1.0). In short, amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-K(m) component for the Na(+)-dependent alpha-aminoisobutyrate uptake, which endows the hepatocytes with a high capability for concentrating amino acids at low ambient values. The concomitant enhancement of K(+) transport reflects changes in the electrochemical gradient for Na(+) across the hepatocellular membrane and, along with this, presumably alterations in the membrane potential; the latter might be the driving force for the enhanced alpha-aminoisobutyrate transport in the alanine-preferring system during postnatal age.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Na(+)-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.     

Characteristics of L-glutamine transport during Caco-2 cell differentiation

Glutamine is the main fuel of intestinal epithelial cells, as well as a precursor for the intense nucleotide biosynthesis which arises with the rapid turnover of enterocytes. In order to determine whether glutamine uptake may vary as a function of metabolic demand, glutamine transport across the brush-border membrane of differentiating Caco-2 cells has been investigated. The uptake of L-[(3)H]glutamine was measured between day 7 and day 21 post-seeding. Kinetic analysis with glutamine concentrations ranging from 6.25 microM to 12.8 mM revealed the involvement of high affinity Na(+)-dependent (K(t)=110 microM) and low affinity Na(+)-independent (K(t)=900 microM) transport components at day 7. Both components were partially inhibited by L-lysine in a competitive fashion, suggesting that four different systems were responsible for glutamine uptake: B(0), B(0,+), b(0,+) and L. All four systems were present during the differentiation process, with systems L and B(0) being responsible for up to 80% of glutamine uptake. Caco-2 cell differentiation was associated with a marked decrease in L-glutamine uptake, which affected both the Na(+)-dependent and the Na(+)-independent components. In contrast to glucose uptake, the development of L-glutamine uptake across the brush-border membrane of Caco-2 cells may reflect an adjustment to cell metabolic demand rather than the progressive appearance of a vectorial transport process.     

Increased colonic sodium absorption in rats with chronic renal failure is partially mediated by AT1 receptor agonism

To test the hypothesis that colonic Na(+) transport is altered in the 5/6 nephrectomized rat model of chronic renal failure (CRF), we measured Na(+) fluxes across distal colon from control (CON), CRF, and CRF rats treated with the angiotensin II (ANG II) receptor antagonist losartan (+LOS). We also evaluated overall fluid and Na(+) balance and compared colonic protein and mRNA expression profiles for electroneutral [sodium-hydrogen exchanger (NHE)] and electrogenic Na(+) transport [epithelial sodium channel (ENaC)] in these groups. Consistent with a 60% enhancement in colonic Na(+) absorption in CRF, urinary Na(+) excretion increased by about 50% while serum Na(+) homeostasis was maintained. These CRF-induced changes in Na(+) handling were normalized by treatment with LOS. Net Na(+) absorption was also stimulated in in vitro tissues from CON rats following acute serosal addition of ANG II (10(-7) M), and this increase was blocked by AT(1) antagonism but not by an AT(2) antagonist. In CRF, colonic protein and mRNA expression variably increased for apical NHE2, NHE3, and ENaC alpha-, beta-, gamma-subunits, whereas expression of basolateral NHE1 and Na(+)-K(+)-ATPase (alpha-isoform) remained unaltered. Upregulation of the ENaC subunit mRNA was attenuated somewhat by LOS treatment. Previously, we showed that colonic AT(1) receptor protein is upregulated twofold in CRF, and here we find that AT(1) and AT(2) mRNA and AT(2) protein abundance is unchanged in CRF. We conclude that Na(+) absorption in CRF rat distal colon is increased due to elevated expression of proteins mediating electroneutral and electrogenic uptake and that it is partially mediated by AT(1) receptors.     

Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts

The effects of hyperthermia (41-43 degrees C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 degrees C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37 degrees C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+:H+ exchange. Shifting temperature to 41 degrees C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43 degrees C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41 degrees C only in the presence of ouabain. At 43 degrees C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43 degrees C, 0.25 mM amiloride inhibited heat-stimulated Na+ uptake. Na+ efflux was enhanced at 41 degrees C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41 degrees C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures greater than or equal to 43 degrees C, the Na+ pump is inhibited.     

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na+-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convoluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na+-dependent, but electrically silent transport of propionate is saturable (Km = 10.9 +/- 1.1 mM and Vmax = 3.6 +/- 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of L- and D-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na+-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K+ diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na+-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of L- and D-lactate in this region of rabbit kidney proximal tubule.     

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.     

Stereoselective transport of histidine in rat lung microvascular endothelial cells

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na(+)-K(+)-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na(+) with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma-monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na(+). Moreover, the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na(+) removal, indicating facilitated diffusion by a Na(+)-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na(+)-dependent system N and Na(+)-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na(+) replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma-monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.