The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.     

Renal transport of taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by two Na(+)-dependent (Km1 = 0.086 mM, Km2 = 5.41 mM) systems, and one Na(+)-independent (Km = 2.87 mM) system, which in the presence of an inwardly directed H(+)-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system (Km1 = 0.012 mM, Km2 = 5.62 mM), which was strictly dependent on Na+. At acidic pH with or without a H(+)-gradient, the Na(+)-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na(+)-dependent high-affinity taurine transporter in the presence of beta-alanine, whereas there was no significant inhibition with alpha-amino acids, indicating a beta-amino acid specific transport system. Addition of beta-alanine, L-alanine, L-proline and glycine, but not L-serine reduced the H(+)-dependent uptake of taurine to approx. 50%. Moreover, only the Na(+)-dependent high-affinity transport systems in both segments specifically required Cl-. Investigation of the stoichiometry indicated 1.8 Na+: 1 Cl-: 1 taurine (high affinity), 1 Na+: 1 taurine (low affinity) and 1 H+: 1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na+: 1 Cl-: 1 taurine (high affinity) and 1 Na+: 1 taurine (low affinity).     

Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation

Transplacental transfer is the fetus' primary source of taurine, an essential amino acid during fetal life. In intrauterine growth restriction (IUGR), placental transport capacity of taurine is reduced and fetal taurine levels are decreased. We characterized the protein expression of the taurine transporter (TAUT) in human placenta using immunocytochemistry and Western blotting, tested the hypothesis that placental protein expression of TAUT is reduced in IUGR, and investigated TAUT regulation by measuring the Na(+)-dependent taurine uptake in primary villous fragments after 1 h of incubation with different effectors. TAUT was primarily localized in the syncytiotrophoblast microvillous plasma membrane (MVM). TAUT was detected as a single 70-kDa band, and MVM TAUT expression was unaltered in IUGR. The PKC activator PMA and the nitric oxide (NO) donor 3-morpholinosydnonimine decreased TAUT activity (P < 0.05, n = 7-15). However, none of the tested hormones, e.g., leptin and growth hormone, altered TAUT activity significantly. PKC activity measured in MVM from control and IUGR placentas was not different. In conclusion, syncytiotrophoblast TAUT is strongly polarized to the maternal-facing plasma membrane. MVM TAUT expression is unaltered in IUGR, suggesting that the reduced MVM taurine transport in IUGR is due to changes in transporter activity. NO release downregulates placental TAUT activity, and it has previously been shown that IUGR is associated with increased fetoplacental NO levels. NO may therefore play an important role in downregulating MVM TAUT activity in IUGR.     

A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-)-dependent high affinity taurine transporter

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.     

Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts.

The cellular level of the organic osmolyte taurine is a balance between active uptake and passive leak via a volume sensitive pathway. Here, we demonstrate that NIH3T3 mouse fibroblasts express a saturable, high affinity taurine transporter (TauT, Km = 18 microm), and that taurine uptake via TauT is a Na+- and Cl(-)-dependent process with an apparent 2.5 : 1 : 1 Na+/Cl-/taurine stoichiometry. Transport activity is reduced following acute administration of H2O2 or activators of protein kinases A or C. TauT transport activity, expression and nuclear localization are significantly increased upon serum starvation (24 h), exposure to tumour necrosis factor alpha (TNFalpha; 16 h), or hyperosmotic medium (24 h); conditions that are also associated with increased localization of TauT to the cytosolic network of microtubules. Conversely, transport activity, expression and nuclear localization of TauT are reduced in a reversible manner following long-term exposure (24 h) to high extracellular taurine concentration. In contrast to active taurine uptake, swelling-induced taurine release is significantly reduced following preincubation with TNFalpha (16 h) but unaffected by high extracellular taurine concentration (24 h). Thus, in NIH3T3 cells, (a) active taurine uptake reflects TauT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of TauT is regulated; and (d) volume-sensitive taurine release is not mediated by TauT.     

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.     

Tyrosine residues are essential for the activity of the human placental taurine transporter

Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane greater than NBD-Cl greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. The dependence of taurine uptake rate on extravesicular Na+ concentration was sigmoidal and analysis of the data revealed that two Na+ ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na+-binding site of the transporter.     

Molecular Characterization and In Situ Localization of a Mouse Retinal Taurine Transporter

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na⁺- and Cl^?-dependent [³H]taurine transport activity in Xenopus laevis oocytes that saturated with an average K_m of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl^? was varied, a single saturation process with an average apparent equilibrium constant (K_Cl?) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na⁺ was varied (apparent equilibrium constant, K_Na+～54.8 mM). Analyses of the Na⁺- and Cl^?-concentration dependence data suggest that at least two Na⁺ and one Cl^? are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β-alanine > l -diaminopropionic acid > guanidinoethane sulfonate > β-guanidinopropionic acid > chloroquine > γ-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.     

System beta and system A amino acid transporters in the feline endotheliochorial placenta

There is no knowledge of the transport mechanisms by which solutes cross the cat placenta or any other endotheliochorial placenta. Here, we investigated whether the amino acid transport systems beta and A are present in the cat placenta using a placental fragment uptake technique. Data were compared with studies in the human placenta, in which the presence of these two transport systems has been well established. A time course of [(3)H]taurine (substrate for system beta) and [(14)C]MeAIB (nonmetabolizable substrate for system A) uptake was determined in the term cat and human placental fragments in the presence and absence (choline substituted) of Na(+), and further studies were carried out over 15 min. Taurine uptake into both cat and human placenta fragments was found to be Na(+) and Cl(-) dependent, and Na(+)-dependent taurine uptake was blocked by excess beta-alanine. MeAIB uptake was found to be Na(+) dependent, and Na(+)-dependent MeAIB uptake was blocked by excess MeAIB or glycine. Western blotting and immunohistochemistry performed on cat and human placenta showed expression of TAUT and ATA2 (SNAT2), proteins associated with system beta and system A activity, respectively. This study therefore provides the first evidence of the presence of amino acid transport systems beta and A in the cat placenta.     

Characterization of the defect in the Na(+)-phosphate transporter in vitamin D-resistant hypophosphatemic mice.

Hypophosphatemic vitamin D-resistant rickets is the most common form of vitamin D-resistant rickets in man. The hypophosphatemic mouse model (Hyp) is phenotypically and biochemically similar to the human disease. Biochemically, hypophosphatemia is the hallmark of this disorder. The cause of the hypophosphatemia is thought to be secondary to a defect in the renal and/or intestinal Na(+)-phosphate transporter. The current studies were designed to investigate and characterize the localization of the defect in the Na(+)-phosphate transporter in this disorder. Phosphate uptake by renal brush border membrane vesicles (BBMV) showed a significant decrease in the slope of the initial rate of phosphate uptake in (Hyp) compared with control mice (0.009 versus 0.013, respectively). The slopes representing initial rates of phosphate uptake by jejunal BBMV were similar in (Hyp) and control mice (0.004 and 0.004, respectively). Kinetics of jejunal Na(+)-dependent phosphate uptake showed a Vmax of 0.63 +/- 0.12 and 0.64 +/- 0.12 nmol/mg protein/15 s in (Hyp) and control mice, respectively, whereas Km values were 0.12 +/- 0.08 and 0.2 +/- 0.11 mM, respectively. Similar kinetic analysis in the kidney showed a Vmax of 0.32 +/- 0.06 and 1.6 +/- 0.1 (p less than 0.01) and Km of 0.07 +/- 0.06 and 0.39 +/- 0.05 (p less than 0.02) in (Hyp) and control mice, respectively. Na(+)-dependent D-glucose uptake by BBMVs of intestine and kidney showed typical overshoot phenomena in (Hyp) and control mice. In order to explore these findings further, Na(+)-phosphate transporter expression from intestine and kidney was accomplished by microinjection of 50 ng of poly(A)+ RNA into Xenopus laevis oocytes. Na(+)-dependent phosphate uptake was expressed 6 days after the microinjection of intestinal and kidney poly(A)+ RNA from control mice. However, expression of the transporter from (Hyp) mice occurred only from the intestine, and not from the kidney. The decrease in the expression of the Na(+)-dependent phosphate transporter was not secondary to accelerated efflux of phosphate or decreased metabolism in oocytes injected with poly(A)+ RNA from (Hyp) mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.     

Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells.

The cDNA clone GAT-1, which encodes a Na(+)- and Cl(-)-coupled GABA transporter from rat brain, has been expressed in mammalian cells using three different systems: (1) transient expression upon transfection of mouse Ltk- cells with a eukaryotic expression vector containing GAT-1; (2) stable expression in L-cells transfected with the same vector; (3) transfection of HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Similar results both qualitatively and quantitatively were obtained with all systems. The GABA transporter expressed in HeLa and L-cells retains all the properties described previously for GABA transport into synaptosomes and synaptic plasma membrane vesicles. It was fully inhibited by cis-3-aminocyclohexanecarboxylic acid (ACHC) and not by beta-alanine. The KM for GABA transport and the IC50 for ACHC inhibition were similar to the presynaptic transporter. Accumulated [3H]GABA was released from transfected cells by dissipating the transmembrane Na+ gradient with nigericin or by exchange with unlabeled external GABA. Accumulation was stimulated by both Na+ and Cl- in the external medium. However, in the absence of external Cl-, a small amount of GABA transport remained which was dependent on GAT-1 transfection. Functional expression of the GABA transporter was abolished by tunicamycin. An antitransporter antibody specifically immunoprecipitates a polypeptide with an apparent molecular mass of about 70 kDa from GAT-1-transfected cells. When cells were grown in the presence of tunicamycin, only a faint band of apparent mass of about 60 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.     

Regulation of the Na(+)-dependent and the Na(+)-independent polyamine transporters in renal epithelial cells (LLC-PK1)

We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.     

High affinity transport of taurine by the Drosophila aspartate transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.     

Expression of Na(+) / D-glucose cotransport in Xenopus laevis oocytes by injection of poly(A)(+) RNA isolated from lobster (Homarus americanus) hepatopancreas

Xenopus laevis oocytes were used for expression and characterization of lobster (Homarus americanus) hepatopancreas Na(+)-dependent D-glucose transport activity. Poly(A)(+) RNA from the whole hepatopancreatic tissue was injected and transport activity was assayed by alpha-D-[2-(3)H] glucose. Injection of lobster hepatopancreatic poly(A)(+) RNA resulted in a dose (1-20 ng) and time (1-5 days) dependent increase of Na(+)-dependent D-glucose uptake. Kinetics of Na(+)-dependent glucose transport was a hyperbolic function (K(m)=0.47+/-0.04 mM) of external D-glucose concentration and a sigmoidal function (K(Na)=68.32+/-1.57 mM; Hill coefficient=2.22+/-0.09) of external Na(+) concentration. In addition, Na(+)-dependent D-glucose uptake was significantly inhibited by both (0.1-0.5 mM) phloridzin and (0.1-0.5 mM) methyl-alpha-D-glucopyranoside. After size fractionation through a sucrose density gradient, poly(A)(+) RNA fractions with an average length of 2-4 kb induced a twofold increase in Na(+)-dependent phloridzin-inhibited D-glucose uptake as compared to total poly(A)(+) RNA-induced uptake. The results of this study provide the functional basis to screen lobster hepatopancreatic cDNA libraries for clones encoding putative and still not known crustacean SGLT-type Na(+)/glucose co-transporter(s).     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.