Dependent Taurine Transporter Research Articles

Significance of taurine transporter (TauT) in homeostasis and its layers of regulation (Review).

Taurine (2-aminoethanesulfonic acid) contributes to homeostasis, mainly through its antioxidant and osmoregulatory properties. Taurine's influx and efflux are mainly mediated through the ubiquitous expression of the sodium/chloride-dependent taurine transporter, located on the plasma membrane. The significance of the taurine transporter has been shown in various organ malfunctions in taurine-transporter-null mice. The taurine transporter differentially responds to various cellular stimuli including ionic environment, electrochemical charge, and pH changes. The renal system has been used as a model to evaluate the factors that significantly determine the regulation of taurine transporter regulation.

Identification of competitive inhibitors of the human taurine transporter TauT in a human kidney cell line

The osmolyte and antioxidant taurine plays an important role in regulation of cellular volume, oxidative status and Ca2+-homeostasis. Taurine uptake in human cells is regulated by the Na+- and Cl--dependent taurine transporter TauT. In order to gain deeper structural insights about the substrate binding pocket of TauT, a HEK293 cell line producing a GFP-TauT fusion protein was generated. Transport activity was validated using cell-based [3H]-taurine transport assays. We determined the Km and IC50 values of taurine, β-alanine and γ-aminobutyrate. Additionally we were able to identify structurally similar compounds as potential new substrates or inhibitors of the TauT transporter. Substrate induced cytotoxicity was analyzed using a cell viability assay. In this study we show competitive effects of the 3-pyridinesulfonate, 2-aminoethylhydrogen sulfate, 5-aminovalerate, β-aminobutyrate, piperidine-4-sulfonate, 2-aminoethylphosphate and homotaurine. We demonstrate that taurine uptake can be inhibited by a phosphate. Furthermore our studies revealed that piperidine-4-sulfonate interacts with TauT with a higher affinity than γ-aminobutyrate and imidazole-4-acetate. We propose that piperidine-4-sulfonate may serve as a potential lead structure for the design of novel drug candidates required for specific modulation of the TauT transporter in therapy of neurodegenerative diseases.

Na+‐dependent transport of taurine is found only on the abluminal membrane of the blood‐brain barrier

Luminal and abluminal plasma membranes, isolated from bovine brain microvessels, were used to characterize Na+‐dependent and facilitative taurine transport. Initial transmembrane potential was − 59 mV 0, external Na+ was 126 mM, and T= 25°C. Apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 μM to 11.4 μM. Abluminal membranes had Na+‐dependent taurine transport as well as facilitative transport; luminal membranes only had facilitative transport. Apparent Km's for facilitative and Na+‐dependent taurine transports were 0.06 ± 0.02 μM and 0.7 ± 0.1 μM, respectively. Na+‐dependent transport of taurine was voltage dependent between −25 to −101 mV. Transport was over 5 times greater at −101 mV compared with Vm was −25 mV. Sensitivity to external osmolality of Na+‐dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H2O) using mannitol as the osmotic agent to adjust osmolality. For these experiments the concentration of Na+wasmaintained constant at 50 mM; the calculated transmembrane potential was −59 mV. Na+‐dependent transport was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H2O.

Na+-dependent transport of taurine is found only on the abluminal membrane of the blood–brain barrier

Luminal and abluminal plasma membranes were isolated from bovine brain microvessels and used to identify and characterize Na+-dependent and facilitative taurine transport. The calculated transmembrane potential was −59mV at time 0; external Na+ (or choline under putative zero-trans conditions) was 126mM (T=25°C). The apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24μM to 11.4μM. Abluminal membranes had both Na+-dependent taurine transport as well as facilitative transport while luminal membranes only had facilitative transport. The apparent Km for facilitative and Na+-dependent taurine transport were 0.06±0.02μM and 0.7±0.1μM, respectively. The Na+-dependent transport of taurine was voltage dependent over the range of voltages studied (−25 to −101mV). The transport was over 5 times greater at −101mV compared to when Vm was −25mV. The sensitivity to external osmolality of Na+-dependent transport was studied over a range of osmolalities (229 to 398mOsm/kg H2O) using mannitol as the osmotic agent to adjust the osmolality. For these experiments the concentration of Na+ was maintained constant at 50mM, and the calculated transmembrane potential was −59mV. The Na+-dependent transport system was sensitive to osmolality with the greatest rate observed at 229mOsm/kg H2O.

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na+-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na+. Although taurine uptake was reduced in Cl- free medium a significant portion of taurine uptake persisted in the presence of NO3 -. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the Vmax of influx was increased without affecting the Km. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na+. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na+-dependent taurine uptake may not be strictly dependent upon extracellular Cl-.

Functional Expression of Taurine Transporter and its Up-Regulation in Developing Neurons from Mouse Cerebral Cortex

In the present study, we investigate the characteristics of taurine transport in primary cultures of neurons from mouse cerebral cortex to understand the possibility that taurine might attenuate the effects of central nervous system drugs. Primary cultured neurons from mouse cerebral cortex were used to determine the transport characteristics of taurine. The expression of taurine transporter (TAUT) in mouse neurons was determined by use of reverse transcriptase-polymerase chain reaction and Western blotting. In vitro transport of taurine in mouse cerebrocortical neurons at day 9 was Na+-dependent and saturable with a Michaelis-Menten constant (Kt) of 10.6 +/- 4.1 microM and a maximum velocity (Vmax) of 6.68 +/- 0.85 nmol/mg protein/10 min. Na+ and Cl- activation kinetics revealed that the Na+-to-Cl(-)-to-taurine stoichiometry was 2:1:1. Na+-dependent [3H]-taurine transport was competitively inhibited by beta-alanine with an inhibitory constant (Ki) of 47.4 +/- 6.5 microM. Gamma-aminobutyric acid also inhibited Na+-dependent [3H]-taurine transport with relatively low affinity (Ki = 273 +/- 71 microM). TAUT mRNA was detected in mouse primary cultured neurons, and TAUT protein was also expressed at approximately 70 kDa. Na+-dependent taurine transport activity was increased with developing neurons and corresponded with the increasing mRNA and protein level of TAUT. The present study revealed that Na+/Cl(-)-coupled taurine transporter TAUT is responsible for taurine uptake in mouse cerebrocortical neurons, and that the expression of TAUT is increased with developing cerebrocortical neurons.

High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na(+)-dependent taurine transporter TauT, and passive release via a volume-sensitive leak pathway. Here we demonstrate that: (i) TauT localizes to the primary cilium of growth-arrested NIH3T3 fibroblasts, (ii) long-term exposure to TNF(alpha) or hypertonic sucrose medium, i.e., growth medium supplemented with 100 mM sucrose, increases ciliary TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium.

Characterisation of long term cat placental explant cultures: uptake of taurine by system β

Dietary taurine is essential for cats and deficiency during pregnancy may lead to abortion, growth restriction or impaired neurological function of kittens. We previously described Na(+)- and Cl(-)-dependent taurine transport by system beta in fragments of freshly isolated cat placenta [Champion EE, Bailey SJ, Glazier JD, Jones CJP, Mann SJ, Rawlings JM, et al. Taurine uptake into cat placental tissue fragments. Placenta 2001;22:A.42]. Here we evaluate long term culture of cat placental explants as a model for the future study of chronic nutrient regulation of amino acid transport in this species. The cat placental explants displayed (i) Na(+)-dependent [(3)H]taurine uptake and (ii) taurine transporter protein on day 7 of culture, as observed in fresh cat placental fragments. The explants had preserved the ability to secrete PGF(2alpha) hormone until day 11 of culture and remained morphologically largely intact until day 7 of culture. This model of placental explant culture will provide an important in vitro method for the study of chronic regulation of amino acid transport in the cat.

マウス大脳皮質初代培養神経細胞を用いた Na+ 依存性タウリントランスポーターの機能解析

マウス大脳皮質初代培養神経細胞を用いた Na+ 依存性タウリントランスポーターの機能解析

Electrophysiological properties of the mouse Na+/Cl(-)-dependent taurine transporter (mTauT-1): steady-state kinetics: stoichiometry of taurine transport.

Taurine, one of the most abundant free amino acids found in many cells, is found in millimolar concentrations in the brain, heart, eye and liver1,2. Among the many postulated roles of taurine (such as a neurotransmitter, a cytoprotector and an essential nutrient for the fetal and neonatal brain development), the best understood role is as an osmolyte in the kidney and brain1,2. Na+ and Cl- -dependent transport of taurine have been described in a variety of tissues including kidney, heart, retina, brain and choroid plexus2. Over the last several years, the cDNA encoding the Na+/Cl--dependent taurine transporter has been cloned from rat brain3, mouse brain4, dog MDCK epithelial cells5, human thyroid6, human placental JAR cells7, mouse retina8 and bovine aortic endothelial cells9. The proteins encoded by these clones show high degree of sequence homology at the amino acid level9. The Na+/Cl--dependent taurine transporter belongs to the Na+/Cl-GABA cotransporter family of proteins10. The hallmark of this family of proteins is that they all predicted to contain twelve transmembrane helices crossing the membrane in a zigzag fashion, and both the N- and C-terminal ends are predicted to face the cytoplasm milieu.

Protein kinase C and cAMP mediated regulation of taurine transport in human colon carcinoma cell lines (HT-29 & Caco-2).

Taurine is one of the most abundant free amino acids comprising 38% and 18% of the total free amino acid pool in human duodenal and colonic mucosa, respectively1. Recent observations have demonstrated that serum and intestinal taurine concentrations were lower in patients with cancer2, trauma3, surgery4 and critical illnesses5 compared to healthy controls. These results postulate the possibility that intestinal taurine absorption might be impaired in such stressed conditions. Taurine transport across the intestinal epithelium plays an important role in the regulation of taurine homeostasis in neonates and adults particularly under diverse stressed conditions6. The brush border membrane of intestinal mucosal cells express the Na+- and Cl--dependent taurine transporter (TAUT) responsible for the active absorption of taurine across the epithelium. TAUT activity in the intestinal epithelium is known to be regulated in response to changes in the availability of dietary sulfur amino acids6,7.

Disruption of the taurine transporter gene (taut) leads to retinal degeneration in mice.

Taurine is involved in cell volume homeostasis, antioxidant defense, protein stabilization, and stress responses. High levels of intracellular taurine are maintained by a Na+-dependent taurine transporter (TAUT) in the plasma membrane. In view of the immunomodulatory and cytoprotective effects of taurine, a mouse model with a disrupted gene coding for the taurine transporter (taut-/- mice) was generated. These mice show markedly decreased taurine levels in a variety of tissues, a reduced fertility, and loss of vision due to severe retinal degeneration. In particular, the retinal involvement identifies the taurine transporter as an important factor for the development and maintenance of normal retinal functions and morphology.

Gene expression analysis of EpiDerm ™ following exposure to SLS using cDNA microarrays

There is a need to investigate the mechanistic basis of the human skin irritation response if relevant in vitro test systems for the predictive identification of skin irritation hazards are to be developed. Recent progress in genomics technologies mean that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available. The aim of this work was to identify genes (for further mechanistic investigation) which may be regulated in response to skin irritation, following exposure of the EpiDerm™ skin model to the known skin irritant sodium lauryl sulphate (SLS). EpiDerm cultures were treated in triplicate with a non-cytotoxic dose of SLS (0.1 mg/ml, as determined by the MTT assay and histological examination) for 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h. Total RNA was extracted from the pooled EpiDerm cultures and used to probe Atlas™ human arrays (Clontech) covering approximately 3600 genes. Preliminary data indicated an up-regulation at early time points (15–30 min) of a number of genes involved in transportation (e.g. the sodium and chloride dependent taurine transporter) and receptors (e.g. ZAP70 and protocadherin 42 precursor). The gene encoding the UV excision repair protein and other DNA repair genes (e.g. DNA-directed RNA polymerase II) were up-regulated after 1–3 h, along with TGFβ3 and other tumour suppressors, which play a role in cellular development and wound healing. At the later time points of 4–24 h, genes involved in protein translation (e.g. Cathepsin D receptor) and metabolism (e.g. CYP27A) were up-regulated. In addition, a number of genes were down-regulated in response to treatment with SLS, although these followed less of a time dependent pattern. These results indicate the differential regulation of a number of genes in response to treatment with SLS, some of which may provide additional clues to the molecular events underpinning the irritation response to this particular surfactant and possibly to other chemical irritants.

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 μM. The Na +/Cl −/taurine coupling ratio for cloned transporter was estimated as 2:1:1. Uptake of radiolabeled taurine was inhibited by excessive cold taurine, hypotaurine, β-alanine and γ-aminobutyric acid but not by α-alanine, glycine, leucine or glycinebetaine. Taurine transporter mRNA is ubiquitously distributed in carp tissues, and its level decreases in the order: kidney>spleen>heart>fin>eye≈intestine≈gill>skin>brain>muscle>hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH 2O. These data suggest the significant role of taurine as an osmolyte in carp cells.

Intrauterine Growth Restriction Is Associated with a Reduced Activity of Placental Taurine Transporters

Taurine is an essential amino acid during fetal life and appears to be vital for the growth of the fetus and for the development of the CNS. In intrauterine growth restriction (IUGR), fetal plasma concentrations of taurine are reduced, and we tested the hypothesis that this is caused by altered placental transport of taurine. Syncytiotrophoblast microvillous membrane (MVM) and basal membrane (BM) vesicles were isolated from control (fetal weight, 3068+/-191 g; gestational age, 37.0+/-0.7 wk; n=13) and IUGR pregnancies (fetal weight, 1724+/-118 g; gestational age, 35.8+/-0.7 wk; n=11). Uptake of [3H]taurine (0.5 microM) was studied at 22 degrees C using rapid filtration techniques. Sodium stimulated taurine uptake 35-fold in MVM, confirming Na+-dependent transport in this membrane. A Na+-dependent taurine transport could also be demonstrated in BM; however, the activity was only 6% of that in MVM. Na+-independent transport activities were similar in MVM and BM. In IUGR, MVM Na+-dependent taurine transport was reduced by 34% (p < 0.05), whereas Na+-independent uptake was unaltered. In contrast to MVM, Na+-dependent taurine uptake in BM was unaffected by IUGR, whereas Na+-independent transport was decreased by 33% (p < 0.05). The highly polarized distribution of the Na+/taurine cotransporter to the MVM in conjunction with similar Na+-independent transport rates for taurine in MVM and BM provides the basis for net taurine flux from the mother to the fetus. These data suggest that the low plasma concentrations of taurine in IUGR fetuses are caused by a reduced activity of placental taurine transporters.

Calyculin A modulates the kinetic constants for the Na +-coupled taurine transport in Ehrlich ascites tumour cells

The effect of the phosphatase inhibitor calyculin A (cal A) on the kinetic parameters of the Na +-coupled taurine uptake via the taurine transporter in the Ehrlich ascites tumour cells has been investigated. Preincubation with cal A (100 nM) reduces the initial taurine influx by about 20%, but has no effect on the diffusional component of the taurine influx or on the taurine release from cells suspended in isotonic or in hypotonic medium. Thus, cal A-sensitive phosphatases only affect taurine transport mediated by the Na +-dependent taurine transporter. Cal A increases the Michaelis–Menten constant for binding of taurine to the transporter from 31±6 to 45±4 μM and reduces the taurine transport capacity from 210±20 to 170±10 pmol×g dry wt −1×min −1. The Michaelis–Menten constant for binding of Na + to the taurine transporter is concomitantly increased from 96±11 to 129±8 mM and the Na +:taurine coupling ratio for activation of the transport cycle is reduced from 3.3±0.6 to 2.4±0.2. This suggests that cal A-sensitive phosphatases maintain a high affinity of the taurine transporter towards Na + and taurine as well as a high taurine transport capacity in unpertubated Ehrlich cells.

The effect of taurine on the activation osmolality of the osmosensitive current in single ventricular myocytes of rabbits.

An osmosensitive current was identified in rabbit ventricular cells and the effects of taurine on osmotic stress were examined. The osmosensitive current was measured with the whole-cell patch clamp technique and all known currents were blocked using Ba2+, Cd2+, Ni2+, diltiazem, ouabain, Cs+ and tetraethyl ammonium. When hyposmotic solution (200 mosmol/kg) was applied, the background current increased gradually. This osmosensitive current was dependent on Cl-. When the conductance of the activated current reached three times the control conductance, the osmolality was arbitrarily named the activation osmolality. The activation osmolality was regarded as an indication of cell volume expansion. With 20 mM taurine in the pipette solution, the activation osmolality was lowered significantly. With high [Na+] (32.5 mM) in the pipette solution, the effect of taurine in reducing the activation osmolality was larger than that in low [Na+] (2.5 mM). From these results, we conclude that taurine reduces the activation osmolality and this effect is more pronounced in the presence of high [Na+] in the pipette solution. It is suggested that Na+-dependent taurine transport could be involved in reducing osmotic stress.

Identification of a high affinity taurine transporter which is not dependent on chloride.

Taurine transport by lactating gerbil mammary tissue has been examined. Taurine uptake is mediated by a high-affinity system which is specific for beta-amino acids. The uptake of taurine is Na(+)-dependent but appears not to be obligatory dependent upon Cl-. Thus, replacing Na+ with choline almost abolished taurine uptake. Substituting Cl- with NO3- had no effect whereas SCN- induced a small but significant increase in taurine influx. Taurine uptake was Na(+)-dependent under conditions where Cl- had been replaced with NO3-. However, it is apparent that the Na(+)-dependent taurine transport system requires the presence of a permeable anion because replacing Cl- with gluconate markedly reduced taurine uptake. Cell-swelling, induced by a hyposmotic challenge, increased the efflux of taurine from gerbil mammary tissue via a pathway sensitive to niflumic acid.

Fasting enhances taurine transport by rat liver plasma membrane vesicles

Hepatic taurine stores are maintained by biosynthesis from the sulfur-containing amino acids, methionine and cysteine, and by uptake via a Na(+)- and Cl(-)-dependent transport system, which is specific for beta-amino acids. We hypothesized that liver stores of taurine are maintained by enhanced hepatic transport during fasting when dietary sources for taurine and its precursors are diminished. Liver plasma membrane vesicles, enriched for the basolateral domain, were prepared from adult male rats fasted for 72 h and from control rats. The maximum velocity for Na(+)-dependent taurine uptake was twofold greater for the fasted group compared with the control group (0.87 +/- 0.09 vs. 0.31 +/- 0.03 nmol.mg protein-1.min-1). The apparent Michaelis constant for taurine was also greater for fasted compared with control (154.0 +/- 0.5 vs. 80.0 +/- 2.0 microM). gamma-Aminobutyric acid, but not alanine or glutamine, abolished the effect of fasting on hepatic taurine transport. To determine the effect of fasting independent of changes in the lipid microenvironment, taurine uptake was measured in proteoliposomes reconstituted by inserting detergent-solubilized membrane proteins into asolectin vesicles. Taurine uptake by proteoliposomes reconstituted from membranes prepared from the fasted group was significantly greater than from the control group. We conclude that Na(+)-dependent taurine transport is enhanced in liver plasma membranes prepared from fasted rats. Our findings imply that enhanced taurine uptake with fasting is due to either an increased number of functional carriers or activation of existing transporters.

High affinity (Na(+) + Cl-)-dependent taurine transport by lactating mammary tissue.

The transport of taurine by lactating rat mammary tissue has been examined. Taurine uptake was found to be dependent upon the presence of extracellular Na+ and Cl-, which is consistent with (Na(+) + Cl(-) + taurine) cotransport. The Ka and Vmax of taurine influx were respectively 43 microM and 37.5 mumol/kg cell water per 15 min. It is apparent that the mechanism responsible for taurine uptake is highly selective for beta-amino acids. Taurine efflux consisted of a fast extracellular component and a slow membrane-limited component. The slow component was relatively insensitive to temperature, suggesting that it may represent simple diffusion. Mammary tissue was found to contain a high level of intracellular taurine: 7.29-7.44 mmol/kg cell water. We suggest that taurine is taken up and concentrated by the mammary gland by a (Na(+) + Cl(-) + taurine) cotransport mechanism situated in the blood-facing aspect of the secretory epithelium and that a low outward permeability to taurine allows a high intra-to-extracellular concentration gradient to be maintained. Milk taurine may be derived from taurine diffusing from the cell cytosol across the apical membrane.