Polarized nature of taurine transport in LLC-PK1 and MDCK cells: Further characterization of divergent transport models

Summary Taurine transport was measured in cultured epithelial cells-LLC-PK1 and MDCK-grown on permeable membrane supports. Taurine transport by LLC-PK1 cells was greater on the apical surface compared to the basolateral surface. MDCK cells exhibited greater taurine uptake from the basolateral side. Transepithelial taurine flux was in the direction of apical to basolateral in the LLC-PK1 monolayers. There was no net transepithelial movement of taurine in the MDCK monolayers. Efflux of taurine from the apical and the basolateral membrane surfaces of LLC-PK1 cell monolayers was stimulated by external-alanine but not L-alanine. Efflux of taurine from MDCK cell monolayers was stimulated by-alanine on the basolateral surface. While the competitive inhibitor guainidinoeithane sulfonate (GES) competitively inhibited taurine uptake to a similar degree on the apical and basolateral surface of LLC-PK1 cell monolayers, GES had a more potent inhibitory effect on the basolateral taurine uptake in MDCK cells when compared to its inhibition of apical taurine transport. We conclude that there are characteristic differences in transport of taurine by apical and basolateral surfaces of LLC-PK1 and MDCK cells which may be the consequence of asymmetric distribution or unique structural properties of the taurine transporter.Supported by a grant from the National Institutes of Health (DK 37223), the American Heart Association (92-004470).     

Secondary Transport of Amino Acids in Prokaryotes

Amino acid transport is a ubiquitous phenomenon and serves a variety of functions in prokaryotes, including supply of carbon and nitrogen for catabolic and anabolic processes, pH homeostasis, osmoprotection, virulence, detoxification, signal transduction and generation of electrochemical ion gradients. Many of the participating proteins have eukaryotic relatives and are successfully used as model systems for exploration of transporter structure and function. Distribution, physiological roles, functional properties, and structure-function relationships of prokaryotic α-amino acid transporters are discussed.     

Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na⁺-independent transport of L-glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst-stage embryos. In contrast, the Na⁺-dependent transport of L-aspartate is not detectable in oocytes, but it is detectable in eight-cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x^?_c and X^?_AG, respectively. Here we report the detection of Na⁺-dependent L-aspartate transport, which increased as pig blastocysts developed, although Na⁺-dependent aspartate transport was not detected in pig oocytes. Mediated Na⁺-independent L-glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X^?_AG is similar in both the pig and the mouse, system x^?_c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species. © 1993 Wiley-Liss, Inc.     

Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone in amino acid loaded rats

Summary In adult female anaestetized rats, the influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in leucine (20mg/100g b.wt.) or glutamine (45mg/100g b.wt.) loaded animals. Bolus injections of both amino acids were followed by temporary increase in fractional excretion of the administered amino acids as well of the endogenous amino acids which were not administered.Under load conditions (leucine and glutamine), dexamethasone treatment (60 g/100 g b.wt. for 3 days, i.p. once daily) was followed by a stimulation of renal amino acid reabsorption. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. The effect of triiodothyronine (20,g/1008 b.wt. for 3 days, i.p. once daily) was different in leucine and glutamine loaded animals: after leucine bolus injection a comparable stimulatory effect as shown for dexamethasone could be demonstrated, but after glutamine administration the stimulatory action of T3 was masked. T3 even increases fractional amino acid excretion in glutamine loaded rats as a sign of enhanced house-keeping in the renal tubular cells. These results confirm previous findings and indicate different effects of both hormones on the renal handling of amino acids.     

Influence of triiodothyronine and dexamethasone on renal amino acid handling in rats loaded with various amino acid mixtures

Summary In adult female rats, the influence of dexamethasone or triiodothyronine on renal amino acid handling was investigated in amino acid loaded animals. Amino acids were administered intravenously as two mixtures, each containing four amino acids to overload amino acid reabsorption capacity. Bolus injections of both mixtures were followed by temporary increase in fractional excretion of the administered amino acids as well of the amino acids which were not covered in the mixtures. The administration of the two mixtures was followed by different interactions between various amino acid carriers.After dexamethasone pretreatment (60µg/100g b.wt. for 3 days, once daily) a stimulation of the renal amino acid handling could be shown. Triiodothyronine (20µg/100g b.wt. for 3 days, once daily) did not increase tubular reabsorption capacity for amino acids. It even increased fractional amino acid excretion in amino acid loaded rats as a sign of enhanced amino acid metabolism in the kidney and/or increased amino acid uptake into the tubular cells from the luminal site.     

Energetics of membrane transport in protoplasts

Examples are given to illustrate the recent use of isolated protoplasts in the study of membrane transport with the emphasis on the energetics of solute transport. A model is also presented for the mechanism of active solute transport at the plasmalemma.     

Cysteine-induced H2S emission, ATP depletion, and membrane electrical responses in oat coleoptiles

Incubation of oat ( Avena sativa L. cv. Victory) coleoptile segments in 4 m M L-cysteine reduced the tissue ATP level to 42 nmol ( g fresh weight)⁻¹ (35% of normal) over a 2 h period. Emissions of H₂S accompanied this depletion in ATP suggesting an H₂S production by desulfhydration of cysteine similar to that reported in other plants. Additions of exogenous H₂S to the sections also caused ATP depletion. Aminooxyacetate, an inhibitor of cysteine desulfhydrase (EC 4.4.1.1), eliminated the cysteine-induced H₂S emission and the ATP depletion. Prolonged exposure to cysteine depressed the electrical polarity of the cell membrane from – 116 mV to –85 mV. That and other electrical responses appear to reflect a reduced capacity for ATP-dependent H⁺ extrusion. These effects should be taken into account whenever cysteine is used in physiological experiments.     

The Lens Circulation

The lens is the largest organ in the body that lacks a vasculature. The reason is simple: blood vessels scatter and absorb light while the physiological role of the lens is to be transparent so it can assist the cornea in focusing light on the retina. We hypothesize this lack of blood supply has led the lens to evolve an internal circulation of ions that is coupled to fluid movement, thus creating an internal micro-circulatory system, which makes up for the lack of vasculature. This review covers the membrane transport systems that are believed to generate and direct this internal circulatory system.     

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.     

Uptake of Amino Acids by Brain Microvessels Isolated from Rats after Portacaval Anastomosis

Abstract: The uptake of amino acids by microvessels isolated from brains of rats was studied. Previous studies have demonstrated alterations in blood-brain amino acid transport after portacaval shunt in rats. In order to elucidate whether such changes in the blood-brain barrier were located in the microvessels, brain microvessels were isolated from both rats with portacaval shunt and controls. Brain microvessels from rats 2 weeks after shunt operations took up significantly greater amounts of ¹⁴C-labeled neutral amino acids, but not of glutamic acid. lysine, or α-methylaminoisobutyric acid than microvessels from sham-operated controls. Measurement of uptake kinetics showed a higher V _max for phenylalanine and leucine uptake and a lower V _max for lysine uptake in microvessels from shunted rats compared with control, whereas the respective K _m's of uptake were similar in both preparations. The results suggest that changes in brain microvessel transport activity account for altered brain neutral amino acid concentrations after portacaval shunt and that such changes can be studied in vitro in isolated microvessels.     

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H⁺/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5′ and 3′ untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na⁺-independent uptake of proline, glycine, l-alanine and α-(methylamino)isobutyric acid. Proline uptake was maximal at pH?5.0 (K_m?2.2±0.7?mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na⁺ but in the presence of an inwardly directed H⁺ gradient. In the presence of Na⁺ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

BAT1, a bidirectional amino acid transporter in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Arabidopsis thaliana At2g01170 gene is annotated as a putative gamma amino butyric acid (GABA) permease based on its sequence similarity to a yeast GABA transporting gene (UGA4). A cDNA of At2g01170 was expressed in yeast and analyzed for amino acid transport activity. Both direct measurement of amino acid transport and yeast growth experiments demonstrated that the At2g01170 encoded-protein exhibits transport activity for alanine, arginine, glutamate and lysine, but not for GABA or proline. Significantly, unlike other amino acid transporters described in plants to date, At2g01170 displayed both export and import activity. Based on that observation, it was named bidirectional amino acid transporter 1 (BAT1). Sequence comparisons show BAT1 is not a member of any previously defined amino acid transporter family. It does share, however, several conserved protein domains found in a variety of prokaryotic and eukaryotic amino acid transporters, suggesting membership in an ancient family of transporters. BAT1 is a single copy gene in the Arabidopsis genome, and its mRNA is ubiquitously expressed in all organs. A transposon—GUS gene-trap insert in the BAT1 gene displays GUS localization in the vascular tissues (Dundar in Ann Appl Biol, 2009) suggesting BAT1 may function in amino acid export from the phloem into sink tissues.     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Uptake of Amino Acids by Brain Micro vessels Isolated from Rats with Experimental Chronic Renal Failure

The neurological disorders seen in patients with chronic renal failure and liver cirrhosis are analogous. Previous in vivo studies have shown that the impaired blood-brain amino acid transport seen in rats with chronic renal failure is similar to that of rats with portocaval anastomosis. To elucidate whether a comparable underlying pathogenic mechanism plays a role in both pathological conditions, blood and brain amino acid levels together with amino acid transport by isolated brain microvessels have been studied in rats with chronic renal failure and in sham-operated rats. Brain microvessels isolated from rats with experimental chronic renal failure showed that the uptake of labeled large neutral amino acid, i.e., leucine or phenylalanine, but not of lysine or alpha-methylaminoisobutyric acid, was significantly increased with respect to sham-operated rats; conversely, the uptake of glutamic acid in rats with chronic renal failure was significantly lower compared with values in controls. Kinetic analysis indicated that this was mainly due to increased exchange transport activity (Vmax) of the L-system, rather than to changes in the affinity (Km) of the carrier system for the relative substrate. These data, together with the significant rise of brain glutamine levels and an increased brain-to-plasma ratio of the sum of large neutral amino acids, are analogous to what was previously observed in rats with portocaval anastomosis.(ABSTRACT TRUNCATED AT 250 WORDS)