Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.     

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.     

Non-catalytic role of carbonic anhydrase in rat intestinal absorption

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.     

Electrogenic Na(+)-dependent L-alanine transport in the lizard duodenum. Involvement of systems A and ASC

L-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net L-alanine fluxes, transepithelial potential difference (PD), and short-circuit current (Isc) showed concentration-dependent relationships. Na(+)-dependent L-alanine transport was substantially inhibited by the analog alpha-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited by L-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K(m) values. Na(+)-dependent L-alanine transport, but not MeAIB transport, was partially inhibited by L-serine and L-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for L-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the L-alanine-induced electrogenicity. It is concluded from the present study that the active Na(+)-dependent L-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.     

Cyclooxygenase blockade and exogenous glutamine enhance sodium absorption in infected bovine ileum

We have previously shown that prostanoids inhibit electroneutral sodium absorption in Cryptosporidium parvum-infected porcine ileum, whereas glutamine stimulates electroneutral sodium absorption. We postulated that glutamine would stimulate sodium absorption via a cyclooxygenase (COX)-dependent pathway. We tested this hypothesis in C. parvum-infected calves, which are the natural hosts of cryptosporidiosis. Tissues from healthy and infected calves were studied in Ussing chambers and analyzed via immunohistochemistry and Western blots. Treatment of infected tissue with selective COX inhibitors revealed that COX-1 and -2 must be blocked to restore electroneutral sodium absorption, although the transporter involved did not appear to be the expected Na(+)/H(+) exchanger 3 isoform. Glutamine addition also stimulated sodium absorption in calf tissue, but although this transport was electroneutral in healthy tissue, sodium absorption was electrogenic in infected tissue and was additive to sodium transport uncovered by COX inhibition. Blockade of both COX isoforms is necessary to release the prostaglandin-mediated inhibition of electroneutral sodium uptake in C. parvum-infected calf ileal tissue, whereas glutamine increases sodium uptake by an electrogenic mechanism in this same tissue.     

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

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.     

The preferential interaction of L-threonine with transport system ASC in cultured human fibroblasts.

The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts.     

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Topographical similarities between harmaline inhibition sites on Na+-dependent amino acid transport system ASC in human erythrocytes and Na+-independent system asc in horse erythrocytes

Na+-dependent system ASC and Na+-independent system asc are characterized by a common selectivity for neutral amino acids of intermediate size such as L-alanine and by their interactions with dibasic amino acids. For system ASC, the positive charge on the dibasic amino acid side chain is considered to occupy the Na+-binding site on the transporter. We report here the use of harmaline (a Na+-site inhibitor in some systems) as a probe of possible structural homology between these two classes of amino acid transporter. Harmaline was found to inhibit human erythrocyte system ASC noncompetitively with respect to L-alanine concentration, but approximated competitive inhibition with respect to Na+ concentration (apparent Ki = 2.0 and 0.9 mM, respectively). Similarly, harmaline noncompetitively inhibited L-alanine uptake by horse erythrocyte systems asc1 and asc2 (apparent Ki = 2.0 and 1.9 mM, respectively). In contrast, harmaline functioned as a competitive inhibitor of L-lysine uptake by system asc1 (apparent Ki = 2.6 mM). It is concluded that harmaline competes with Na+ for binding to system ASC and that a topographically similar harmaline inhibition site is present on system asc. This site does not however bind Na+, the asc1 transporter exhibiting normal L-alanine and L-lysine influx kinetics in the total absence of extracellular cations.     

Characterisation and cloning of a Na(+)-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B(0) transporter family

Na(+)-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B(0). This transporter is shown to differ in specificity from the B(0) transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B(0) transporter family we have isolated a cDNA encoding the NBL-1 cell System B(0) transporter. When expressed in Xenopus oocytes the clone catalysed Na(+)-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na(+)-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B(0)/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B(0) transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.     

Inhibition of transport system b0,+ in blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor site

The most conspicuous, Na(+)-independent amino acid transport process in preimplantation mouse blastocysts was provisionally designated system b0,+ because it accepts some cationic and zwitterionic amino acids about equally well as substrates. Although system b0,+ is not Na(+)-stimulated, it has not been determined if it is inhibited by Na+, or if its activity is affected by most other ions. Therefore, we measured uptake of amino acids by blastocysts in isotonic solutions of different ionic and nonionic osmolites. Na(+)-independent L-leucine uptake was unaffected by the ion concentration, but L-lysine transport was several-fold faster in isotonic solutions of non-electrolytes than in similar solutions of inorganic and organic salts or zwitterions. The Km value for 'Na(+)-independent' L-lysine transport was about 10-fold higher in isotonic salt solutions than in solutions of nonionic osmolites, whereas the Km value for L-leucine transport was about the same in either type of solution. Therefore, inorganic and organic cations and the cationic portions of zwitterions appear to compete with cationic but not zwitterionic amino acids for system b0,+ receptor sites. The cation, harmaline, was a particularly strong competitive inhibitor of 'Na(+)-independent' L-lysine uptake by system b0,+, even in isotonic salt solutions, whereas it inhibited L-leucine uptake noncompetitively. Moreover, harmaline appeared to compete with inorganic cations for the lysine receptor sites of system b0,+. Harmaline also has been found by other investigators to competitively inhibit L-lysine uptake by the Na(+)-independent system asc1 in horse erythrocytes, whereas it noncompetitively inhibits alanine uptake by the same system. Similarly, harmaline noncompetitively inhibits L-alanine uptake by the Na(+)-dependent system ASC in human erythrocytes, but it appears to compete for binding with L-alanine's cosubstrate, Na+. In addition, others have found that the positively-charged side chains of cationic amino acids seem to take the place of Na+ needed near side chains in order for zwitterionic amino acids to be transported by systems ASC and y+. We conclude that system b0,+ may be similar to systems asc1, ASC and y+, and that each of these systems may be a variant of the same ancestral transport process. We speculate that since it appears to accept a broader scope of substrates and to interact with a wider variety of cations than do systems asc1, ASC or y+, system b0,+ may more closely resemble the proposed ancestral transport process than any of the other contemporary systems.     

Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells

We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.     

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.     

Transport system ASC for neutral amino acids. An electroneutral sodium/amino acid cotransport sensitive to the membrane potential.

The influx of L-threonine through system ASC does not influence the membrane potential in cultured human fibroblasts although comparable fluxes of amino acids through another Na(+)-dependent agency, system A, effectively depolarize the cells. The membrane potential, however, stimulates the influx of amino acids through system ASC with a maximal effect at -50 mV. The sensitivity of amino acid influx through system ASC to the membrane potential is not constant, but rather, is dependent on intracellular and extracellular concentrations of the substrates, Na+ and amino acids, of the system. Conditions which favor the loading of the ASC carrier at the external surface reduce the sensitivity of ASC-mediated amino acid influx to the membrane potential; in contrast, the sensitivity of this amino acid influx increases under conditions which favor loading of the carrier at the internal surface. Trans-stimulation, a well-known characteristic of system ASC, also varies with the concentrations of the substrates of the system and, in fact, this characteristic is not observed when external Na+ is low. These data may be accommodated by a model in which an electrically silent mode of operation of the transporter is dominant. The influence of the membrane potential on the transport system is dependent on the extent to which a charge-translocating step in the cycling of the carrier is rate limiting (relative rate limitance).     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.     

Proximal-distal absorptive gradients in the in vivo intestine of normal and infected (Hymenolepis diminuta: Cestoda) rats.

Using a single-pass perfusion technique, H2O, Na+, Cl-, HCO3-, and glucose absorption were studied in the jejunum and proximal and distal ileum of rats either uninfected or infected with a tapeworm parasite (Hymenolepis diminuta). The effect of parasitization, region of the intestine, type of buffer, and concentration of glucose in the perfusion fluids on the transport data were analyzed by univariate and multivariate techniques. Proximal-distal flux gradients were observed for water and all the solute species studied, as well as for glucose- and bicarbonate-stimulated salt and water transport; there was a decreasing sensitivity to low pH proceeding distally. The major regional differences occurred between the proximal and distal ileum, with the fluxes in the jejunum being similar to those in the proximal ileum. Na+, H2O, and glucose transport decreased, while Cl- absorption increased, proceeding distally. The parasites diminished the rates of absorption of glucose, salt, and water, and altered the flux gradients, particularly the Na+ and HCO3- transport gradients. The differences in the gradients between control and infected animals were related to differential sensitivity of the different transport systems in the various regions of the gut to parasitism.