Expression Of TauT Research Articles

Involvement of TauT/SLC6A6 in Taurine Transport at the Blood-Testis Barrier.

Taurine transport was investigated at the blood–testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [3H]taurine (27.7 μL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [3H]taurine uptake with a Km of 13.5 μM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [3H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as β-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [3H]taurine uptake revealed an IC50 of 378 μM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [3H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.

Observation of Acupuncture Effects on the Expression of Taurine Transporter and Taurine in the Senescence-Accelerated Mouse Brain: A Pilot Study.

In living organisms, taurine is important for homeostasis and is known to have preventive and inhibitory effects on various diseases. Taurine has been reported to play an important role in the development of the brain in newborns and in reducing the damage caused by cerebral ischemia. Previous studies indicated that acupuncture on a pair of acupoints of "DU16" and "DU20" can increase brain taurine transporter (TauT) expression in mice with penicillin-induced epilepsy and enhanced taurine anti-epilepsy effects. Our previous study reported that manual acupuncture stimulation of the head acupuncture points "Bai-Hui" (GV 20) and "Yintang" (Ex-HN3) could produce antidepressant effects. In this study, we investigated whether acupuncture stimulation of the head acupuncture points GV 20 and Ex-HN3 affects the expression of TauT and taurine in the cerebellum and hippocampus in senescence-accelerated mice. Acupuncture stimulation significantly increased the mRNA expression of TauT in the cerebellum and hippocampus. Immunohistochemical staining confirmed that the expression of TauT and taurine in the cerebellum and hippocampus increased. These results indicate that acupuncture stimulation might improve cognitive and behavioral abilities by increasing the expression of TauT and taurine.

Molecular Characterization and Taurine Regulation of Taurine Transporter in Goldfish

AbstractTaurine transporter (TauT) is a sodium‐ and chloride‐dependent transport carrier, and it plays a key role in the transportation of taurine. At present, the many functions of TauT in fish remain obscure. In this study, a cDNA clone for encoding a taurine transporter was isolated from Goldfish Carassius auratus. The nucleotide sequence predicted 625 amino acids with significant homology to the TauT gene of Zebrafish Danio rerio. Hydropathic analysis revealed stretches of hydrophobic amino acids that were indicative of 12 transmembrane domains that were similar to those proposed for other cloned TauT genes. The results of a reverse transcription polymerase chain reaction (RT‐PCR) analysis demonstrated that TauT broadly expressed in all of the selected tissues and developmental stages in Goldfish but with prominent differential expression levels. The expression of TauT in Goldfish has diurnal changes. The highest expression was at 1200 hours, and the lowest was at 2100 hours. Moreover, taurine treatments showed similar stimulatory effects on the expression of TauT in the intestines of Goldfish both in vitro and in vivo. Overall, these results may provide new insights into the regulation of TauT expression in fish, which has potential applications for improving fish dietary formulations.

Taurine metabolism and effects of inclusion levels in rotifer (Brachionus rotundiformis, Tschugunoff, 1921) on Atlantic bluefin tuna (Thunnus thynnus, L.) larvae

Taurine appears to be a crucial nutrient for teleosts, especially top predator species such as Atlantic bluefin tuna (Thunnus thynnus, L.; ABT). While dietary taurine supplementation has been highly recommended, there is a lack of studies on taurine assimilation and biosynthesis for this iconic species. The present study aims to provide insight into the molecular mechanisms involved in taurine biosynthesis and transport in ABT by studying tissue distribution and ontogenetic development of expression of cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), 2-aminoethanethiol dioxygenase (ado) and taurine transporter (tauT) in response to graded levels of dietary taurine supplementation. The full open reading frame (ORF) for cdo and partial sequences for csad, ado and tauT were obtained, with the translated polypeptides being 202, 176, 166 and 324 amino acids, respectively. All three showed characteristics such as cupin motifs in Cdo and predicted N-glycosylation sites in Taut that are common to these genes in other species. Phylogenetic analysis showed that the ABT sequences clustered with sequences of other teleosts, and separately from mammals and molluscs. Tissue distribution varied, with adipose tissue, kidney, white muscle and testis/brain showing highest expression of cdo, csad, ado and tauT, respectively. Whole larvae expression of csad peaked at 15 dah, whereas the other genes generally increased throughout development to show highest expression at 25 dah. The nutritional trial was carried out by feeding ABT larvae from mouth opening to 14 days after hatching (dah) with rotifers (Brachionus rotundiformis) enriched with 4 different levels of taurine: 0.0 (tau0), 0.5 (tau0.5), 1.0 (tau1), and 2.0 g taurine per 106 rotifers (tau2). Rotifers effectively accumulated taurine with ABT larvae fed on treatment tau2 attaining the highest concentration of taurine. However, ABT larvae fed tau1 displayed higher growth and survival, and flexion index at 14 dah, than larvae fed the other taurine levels. Larvae fed tau1 also showed generally higher expression of tauT and cdo and digestive and antioxidant enzyme genes. While this study showed that larval ABT express taurine metabolism genes, suggesting possible synthesis that could contribute to the taurine pool in the fish, larval performance was enhanced by a level of dietary taurine (3.7 mg taurine g−1 rotifer) supplied by enrichment of rotifers at 1 g taurine per 106 rotifers.

Targeting Taurine Transporter (TauT) for Cancer Immunotherapy of p53 Mutation Mediated Cancers - Molecular Basis and Preclinical Implication.

Taurine transporter (TauT) has been identified as a target gene of p53 tumor suppressor. TauT is also found to be overexpressed in variety type of human cancers, such as leukemia. This study showed that expression of TauT was upregulated by c-Myc and c-Jun oncogenes. To explore whether blocking of TauT inhibits tumor development, the RNA interference (RNAi) and immune targeting approaches were tested in tumor cells in vitro and in p53 mutant mice in vivo. Knockdown of TauT expression by RNAi resulted in cell cycle G2 arrest and suppressed human breast cancer MCF-7 cells proliferation determined by colonies production and cell migration assays. Knockdown of TauT also rendered MCF-7 cells more susceptible to chemotherapeutic drug-induced apoptosis. An antibody specifically against TauT blocked taurine uptake and induced cell cycle G2 arrest leading to cell death of variety type of tumor cells without affecting the viability of normal mammalian cells. TauT peptide vaccination significantly increased median lifespan (1.5-fold) of the p53 null mice and rescued p53+/- mice by extending the median lifespan from 315days to 621days. Furthermore, single dose treatment of tumor-bearing (thymic lymphoma) p53 null mice with TauT peptide reduced tumor size by about 50% and significantly prolonged survival of these mice from average 7days (after observing the thymic lymphoma) to 21days. This finding demonstrates that a novel TauT peptide vaccine can delay, inhibit, and/or treat p53 mutation related spontaneous tumorigenesis in vivo. Therefore, TauT peptide may be used as a universal cancer vaccine to prevent and/or treat patients with p53 mutation-mediated cancers.

Protein malnutrition blunts the increment of taurine transporter expression by a high-fat diet and impairs taurine reestablishment of insulin secretion.

Taurine (Tau) restores β-cell function in obesity; however, its action is lost in malnourished obese rodents. Here, we investigated the mechanisms involved in the lack of effects of Tau in this model. C57BL/6 mice were fed a control diet (CD) (14% protein) or a protein-restricted diet (RD) (6% protein) for 6 wk. Afterward, mice received a high-fat diet (HFD) for 8 wk [CD + HFD (CH) and RD + HFD (RH)] with or without 5% Tau supplementation after weaning on their drinking water [CH + Tau (CHT) and RH + Tau (RHT)]. The HFD increased insulin secretion through mitochondrial metabolism in CH and RH. Tau prevented all those alterations in CHT only. The expression of the taurine transporter (Tau-T), as well as Tau content in pancreatic islets, was increased in CH but had no effect on RH. Protein malnutrition programs β cells and impairs Tau-induced restoration of mitochondrial metabolism and biogenesis. This may be associated with modulation of the expression of Tau-T in pancreatic islets, which may be responsible for the absence of effect of Tau in protein-malnourished obese mice.-Branco, R. C. S., Camargo, R. L., Batista, T. M., Vettorazzi, J. F., Borck, P. C., dos Santos-Silva, J. C. R., Boschero, A. C., Zoppi, C. C., Carneiro, E. M. Protein malnutrition blunts the increment of taurine transporter expression by a high-fat diet and impairs taurine reestablishment of insulin secretion.

Expression of organic osmolyte transporters in cultured rat astrocytes and rat and human cerebral cortex

This study characterizes the expression of the osmolyte transporters betaine/γ-amino-n-butyric acid (GABA) transporter (BGT-1), the taurine transporter (TauT) and the sodium-dependent myo-inositol transporter (SMIT) in various rat brain cells in culture and in rat and human cerebral cortex in situ. Osmolyte transporter expression greatly differed between cultured brain cells with highest mRNA expression levels for SMIT in astrocytes and TauT in neurons. BGT-1 mRNA and protein were expressed in microglia but not in astrocytes and neurons. In rat and human cerebral cortex, SMIT was expressed in astrocytes and TauT was found in neurons. Osmolyte transporter expression was subject to regulation by factors relevant for hepatic encephalopathy (HE). Hypoosmolarity, NH4Cl (0.5–5mmol/l), diazepam (10μmol/l) and TNFα (10ng/ml) time-dependently decreased mRNA expression of SMIT and/or TauT in cultured astrocytes. NH4Cl-induced SMIT/TauT mRNA expression changes were sensitive to inhibitors of glutamine synthetase and NADPH oxidase. In rat cerebral cortex, SMIT mRNA expression decreased after portal vein ligation or ammonium acetate injection probably due to astrocyte swelling in these HE animal models. It is concluded that osmolyte transporters are heterogeneously expressed in brain and are subject to regulation by HE-relevant factors.

Development of a Novel Cysteine Sulfinic Acid Decarboxylase Knockout Mouse: Dietary Taurine Reduces Neonatal Mortality

We engineered a CSAD KO mouse to investigate the physiological roles of taurine. The disruption of the CSAD gene was verified by Southern, Northern, and Western blotting. HPLC indicated an 83% decrease of taurine concentration in the plasma of CSAD−/−. Although CSAD−/− generation (G)1 and G2 survived, offspring from G2 CSAD−/− had low brain and liver taurine concentrations and most died within 24 hrs of birth. Taurine concentrations in G3 CSAD−/− born from G2 CSAD−/− treated with taurine in the drinking water were restored and survival rates of G3 CSAD−/− increased from 15% to 92%. The mRNA expression of CDO, ADO, and TauT was not different in CSAD−/− compared to WT and CSAD mRNA was not expressed in CSAD−/−. Expression of Gpx 1 and 3 was increased significantly in CSAD−/− and restored to normal levels with taurine supplementation. Lactoferrin and the prolactin receptor were significantly decreased in CSAD−/−. The prolactin receptor was restored with taurine supplementation. These data indicated that CSAD KO is a good model for studying the effects of taurine deficiency and its treatment with taurine supplementation.

Amino acid residues involved in the substrate specificity of TauT/SLC6A6 for taurine and γ-aminobutyric acid.

Taurine transporter (TauT/SLC6A6) is an "honorary" γ-aminobutyric acid (GABA) transporter because of its low affinity for GABA. The sequence analysis of TauT implied the role of Gly57, Phe58, Leu306 and Glu406 in the substrate recognition of TauT, and amino acid-substitutions were performed. Immunocytochemistry supported no marked effect of mutations on the expression of TauT. TauT-expressing oocytes showed a reduction in [(3)H]taurine uptake by G57E, F58I, L306Q and E406C, and change in [(3)H]GABA uptake by G57E and E406C, suggesting their significant roles in the function of TauT. G57E lost the activity of [(3)H]taurine and [(3)H]GABA uptake, suggesting that Gly57 is involved in the determination of substrate pocket volume and in the interaction with substrates. E406C exhibited a decrease and an increase in the affinity for taurine and GABA, respectively, suggesting the involvement of Glu406 in the substrate specificity of TauT. The inhibition study supported the role of Glu406 in the substrate specificity since [(3)H]taurine and [(3)H]GABA uptake by E406C was less sensitive to taurine and β-alanine, and more sensitive to GABA and nipecotic acid than was the case with wild type of TauT. F58I had an increased affinity for GABA, suggesting the involvement of Phe58 in the substrate accessibility. The kinetic parameters showed the decreased and increased affinities of L306Q for taurine and GABA, respectively, supporting that substrate recognition of TauT is conformationally regulated by the branched-side chain of Leu306. In conclusion, the present results suggest that these residues play important roles in the transport function and substrate specificity of TauT.

Three cases of placental mesenchymal dysplasia in our hospital

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3 ± 0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Expression of Taurine Transporter (TauT) is Modulated by Heat Shock Factor 1 (HSF1) in Motor Neurons of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive paralysis caused by the degeneration of motor neurons throughout the central nervous system. Mutations of the free radical scavenging enzyme Cu/Zn superoxide dismutase 1 (SOD1) are a cause of familial ALS. In the present study, we demonstrated an age-dependent increase in taurine transporter (TauT) immunoreactivity in spinal cord motor neurons of ALS transgenic mice (mutant SOD1 (G93A)) and a similar increase in TauT in spinal motor neurons of patients with ALS. Chromatin immunoprecipitation analysis verified that heat shock factor 1 (HSF1) preferentially occupies the HSF1 binding element in the promoter of TauT under oxidative stress conditions. Knockdown of HSF1 by small interfering RNA reduced the transcriptional activity of TauT. Using [(3)H] taurine, we confirmed that an elevated expression of TauT directly contributes to increased taurine uptake in ALS motor neurons. In addition, we showed that taurine plays an antioxidant role and may prevent motor neuron loss due to oxidative stress in ALS. Our findings suggest that HSF1-induced TauT expression partially protects motor neurons by compensating for constitutive oxidative stress, which is thought to be a key mechanism contributing to the pathogenesis of ALS. Taken together, our results suggest that TauT is a novel pathological marker for stressed motor neurons in ALS and that modulation of TauT and taurine may slow neuronal degeneration in ALS.

Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [(3)H]taurine. Furthermore, most of the uptake of [(3)H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

Function and regulation of taurine transport in Müller cells under osmotic stress

In the retina, taurine works as an osmolyte to exert a neuroprotective function, and it has been proposed that Müller cells, a major type of retinal glial cells, are involved in the osmolarity regulation of retinal neural cells by controlling the taurine concentration in retinal extracellular fluid (ECF). However, the detailed mechanism of taurine transport in Müller cells has not fully examined, and we investigated this using a conditionally immortalized rat Müller cell line (TR-MUL5 cells). In the uptake study, TR-MUL5 cells exhibited Na+-, Cl−-dependent [3H]taurine uptake with a Km of 37.9μM. The [3H]taurine uptake by TR-MUL5 cells was strongly inhibited by β-alanine and hypotaurine, substrates of taurine transporter TAUT (SLC6A6), and RT-PCR and immunoblot analyses demonstrated the expression of TAUT in Müller cells, suggesting the involvement of TAUT in taurine uptake by Müller cells. In the efflux study, [3H]taurine efflux by TR-MUL5 cells under hypotonic conditions was significantly greater than that under isotonic conditions, and significantly enhanced by sphingosine 1-phosphate (S1P), suggesting that the volume-sensitive taurine release is enhanced via G protein-coupled receptors (GPCRs) in Müller cells. Furthermore, [3H]taurine efflux by TR-MUL5 cells under hypotonic conditions was significantly inhibited in the presence of the volume-sensitive organic osmolyte and anion channel (VSOAC) inhibitor, suggesting a major contribution of VSOAC to the volume-sensitive taurine release by Müller cells. This is the first description of the detailed mechanism of taurine transport in Müller cells, indicating a possible function of Müller cells in retinal neuroprotection by regulating osmolarity of retinal ECF.

Potential benefits of taurine in the prevention of skeletal muscle impairment induced by disuse in the hindlimb-unloaded rat

Hindlimb unloading (HU) in rats induces severe atrophy and a slow-to-fast phenotype transition in postural slow-twitch muscles, as occurs in human disuse conditions, such as spaceflight or bed rest. In rats, a reduction of soleus muscle weight and a decrease of cross-sectional area (CSA) were observed as signs of atrophy. An increased expression of the fast-isoform of myosin heavy chain (MHC) showed the phenotype transition. In parallel the resting cytosolic calcium concentration (restCa) was decreased and the resting chloride conductance (gCl), which regulates muscle excitability, was increased toward the values of the fast-twitch muscles. Here, we investigated the possible role of taurine, which is known to modulate calcium homeostasis and gCl, in the restoration of muscle impairment due to 14-days-HU. We found elevated taurine content and higher expression of the taurine transporter TauT in the soleus muscle as compared to the fast-twitch extensor digitorum longus (EDL) muscle of control rats. Taurine level was reduced in the HU soleus muscle, although, TauT expression was not modified. Taurine oral supplementation (5g/kg) fully prevented this loss, and preserved resting gCl and restCa together with the slow MHC phenotype. Taurine supplementation did not prevent the HU-induced drop of muscle weight or fiber CSA, but it restored the expression of MURF-1, an atrophy-related gene, suggesting a possible early protective effect of taurine. In conclusion, taurine prevented the HU-induced phenotypic transition of soleus muscle and might attenuate the atrophic process. These findings argue for the beneficial use of taurine in the treatment of disuse-induced muscle dysfunction.

Effects of Taurine on Glial Cells Apoptosis and Taurine Transporter Expression in Retina Under Diabetic Conditions

Taurine, a ß-aminosulfonic acid, has been reported to reduce the risk of a number of diseases, including cardiovascular disease, diabetes, and also perhaps to reduce neurodegeneration in the elderly. The transport of taurine is known to be mediated by taurine transporter (TauT). The purpose of this study is to examine the effects of taurine on glial cells apoptosis and on TauT expression in retina of diabetic rats and retinal glial cells cultured with high glucose. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining analysis showed that the number of TUNEL-positive cells in taurine treated diabetic rats was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses, respectively (all P<0.001). No TUNEL-positive cells were observed in retina of control groups and taurine treated control groups. In cultured retinal glial cells, the apoptosis in high glucose-treated cells was significantly increased vs the control. When the cells were incubated with high glucose and taurine at 0.1, 1.0 and 10mmol/l, the percentage of apoptosis was significantly decreased to 16.4, 5.7 and 7.6% respectively (all P<0.05). With supplementation of taurine in diet and culture medium, higher expression of TauT in retina of diabetic rats and cultured retinal glial cells under diabetic conditions were detected by western-blotting (P<0.05). Taken together, our data suggest that diabetes or high glucose induced retinal glial cells apoptosis can be inhibited by taurine, and that taurine reverses the diabetes-induced or high glucose-induced decrease in TauT expression.

Stress-responsive gene TauT and acute kidney injury

BackgroundCisplatin is a commonly used chemotherapeutic agent that has a major limitation because of its nephrotoxicity. We have demonstrated that cisplatin down-regulates the expression of the taurine transporter gene (TauT) in renal cells and that forced overexpression of TauT protects against cisplatin-induced apoptosis in renal cells in vitro and in vivo. In the present study, we have investigated how TauT is regulated by p53 and c-Jun and its role during acute kidney injury (AKI).MethodsRegulation of TauT by p53 and c-Jun was determined by reporter gene assay, DNA binding, Western blot analysis, and immunohistochemistry.ResultsTauT was down-regulated by p53 and up-regulated by c-Jun. Two potential binding sites for c-Jun were identified in the promoter region of TauT. Inhibition of c-Jun N-terminal kinase (JNK) enhanced TauT promoter activity. Overexpression of TauT protects against cisplatin-induced kidney injury in a TauT transgenic mouse model.ConclusionsOur findings suggest that TauT plays a critical role in renal function. Expression of TauT is negatively regulated by p53 and positively regulated by c-Jun, which is mediated by the JNK signaling pathway. The outcome level of TauT may determine the fate of renal cells during stress-induced AKI.

Isolation of Plasma Membrane Vesicles from Mouse Placenta at Term and Measurement of System A and System β Amino Acid Transporter Activity

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3±0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Effect of taurine on GFAP and TauT expressions in rat retinal Müller cells in high glucose culture

Objective To detect the expression of glial fibrillary acid protein (GFAP) and taurine transporter (TauT) in the retinal Müller cells in high glucose culture with taurine and to explore the influence of glucose on the taurine transporting, and the possible protective effects of taurine on Müller cells in early diabetic retinopathy. Methods The Müller cells from the rat retina were cultured in high glucose, and GFAP and TauT expressions were detected in the cells treated with different doses of taurine by immuocy-tochemical fluorescein staining and Western blotting. Results High glucose enhanced the expression of GFAP and decreased the expression of TauT in Müller cells. Taurine decreased the up-regulation of GFAP in the cells which was induced by high glucose; 0. 1–10 mmol/L taurine increased the expression of TauT in Müller cells. Conclusion Taurine can inhibit the changes in Muller cell resulted from high glucose.

The taurine transporter: mechanisms of regulation

Taurine transport undergoes an adaptive response to changes in taurine availability. Unlike most amino acids, taurine is not metabolized or incorporated into protein but remains free in the intracellular water. Most amino acids are reabsorbed at rates of 98-99%, but reabsorption of taurine may range from 40% to 99.5%. Factors that influence taurine accumulation include ionic environment, electrochemical charge, and post-translational and transcriptional factors. Among these are protein kinase C (PKC) activation and transactivation or repression by proto-oncogenes such as WT1, c-Jun, c-Myb and p53. Renal adaptive regulation of the taurine transporter (TauT) was studied in vivo and in vitro. Site-directed mutagenesis and the oocyte expression system were used to study post-translational regulation of the TauT by PKC. Reporter genes and Northern and Western blots were used to study transcriptional regulation of the taurine transporter gene (TauT). We demonstrated that (i) the body pool of taurine is controlled through renal adaptive regulation of TauT in response to taurine availability; (ii) ionic environment, electrochemical charge, pH, and developmental ontogeny influence renal taurine accumulation; (iii) the fourth segment of TauT is involved in the gating of taurine across the cell membrane, which is controlled by PKC phosphorylation of serine 322 at the post-translational level; (iv) expression of TauT is repressed by the p53 tumour suppressor gene and is transactivated by proto-oncogenes such as WT1, c-Jun, and c-Myb; and (v) over-expression of TauT protects renal cells from cisplatin-induced nephrotoxicity.

Regulation of TauT by cisplatin in LLC-PK1 renal cells

Cisplatin is a commonly used chemotherapeutic agent that has a major limitation because of its nephrotoxicity. Since cisplatin-induced renal injury is mainly confined to the S3 segment of renal proximal tubules-the primary site for renal adaptive regulation of TauT-we hypothesize that TauT functions as an anti-apoptotic gene and plays a role in protecting renal cells from drug-induced nephrotoxicity. In the present study we demonstrated that expression of TauT was significantly reduced by cisplatin (50 muM) in LLC-PK1 cells. Down-regulation of TauT by cisplatin occurs at the transcriptional level in a dose-dependent manner, as demonstrated through a reporter gene driven by the TauT promoter. It appears that cisplatin down-regulates TauT expression, at least in part, through the p53-dependent pathway, since cisplatin induces the p53 expression, which, in turn, represses TauT. Cisplatin induces apoptosis of LLC-PK1 cells in a dose-dependent manner. However, forced over-expression of TauT by stable transfection of a taurine transporter cDNA (pNCT) in LLC-PK1 cells was able to attenuate cisplatin-induced down-regulation of taurine uptake by LLC-PK1 cells and protect renal tubular cells from apoptosis. The mechanism by which TauT serves as an anti-apoptotic gene in cisplatin-induced renal injury remains to be determined, but could relate to taurine-dependent cell volume regulation.