Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts Jesper W. Voss, Stine F. Pedersen, Søren T. Christensen and Ian H. Lambert The August Krogh Institute, Biochemical Department, Universitetsparken 13, Copenhagen, Denmark The cellular level of the organic osmolyte taurine is a balance between active uptake and passive leak via a volume sensitive pathway. Here, we d emonstrate t hat NIH3T3 mouse fibro- blasts express a saturable, high affinity taurine transporter (TauT, K m ¼ 18 l M ), and that taurine uptake via TauT is aNa + -andCl – -dependent process with an apparent 2.5 : 1 : 1 Na + /Cl – /taurine stoichiometry. Transport activ- ity is reduced following acute administration of H 2 O 2 or activators of p rotein kinases A or C. TauT transport a ctivity, expression and nuclear localization are significantly increased upon serum starvation (24 h), exposure to tumour necrosis factor alpha (TNFa; 16 h), or hyperosmotic med- ium (24 h); conditions that are a lso a ssociated with increased localization of TauT to the cytosolic network of micro- tubules. Conversely, transport activity, expression and nuclear localization of TauT are reduced in a reversible manner following long-term exposure (24 h ) to high extra- cellular taurine con centration. In contrast to active taurine uptake, swelling-induced taurine release is significantly reduced fo llowing preincub ation w ith T NFa (16 h) but unaffected by high extracellular taurine concentration (24 h). Thus, in NIH3T3 cells, (a) active taurine uptake reflects T auT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of T auT is regu- lated; and (d) volume -sensitive taurine release is not medi- ated by TauT. Keywords:TNFa; creatine; microtubules; reactive oxygen species; v olume-sensitive t aurine leak pathway. Taurine, amino ethane s ulfonic a cid, plays an essential role not only as an organic os molyte and substrate for the formation of bile salt, but also in the mo dulation of the cellular, free Ca 2+ concentration and regulation of neuro- transmission through interaction with GABA- and glycine- gated Cl – channels [1–4]. More recently it has been shown that taurine, via its r eaction with cellular hypochlorous acid (HOCl), produces the less toxic taurine chloramine (TauCl) and thus serves a tissue protective role against oxidative injury [5,6]. He nce, changes i n the net cellular content of taurine may have a dramatic impact on cell f unction. The cellular taurine content is a balance between synthesis from methionine/cysteine, active uptake via the saturable, taurine transporter TauT, and release via a volume-sensitive taurine leak pathway [7]. TauT has a high affinity and s electivity towards taurine but a low transport capacity, and active uptake of one molecule of taurine has been demonstrated to require two to three Na + ions and one Cl – ion [7]. TauT i s a member of the neurotransmitter transporter family that includes the transporters for serotonin (SEROT), c-amino butyric acid ( GAT1-3) as well as the creatine transporter (CREAT) [8]. All members of this family span the membrane 1 2 times, with t he N- and C-terminal ends exposed to the c ytosolic compartment. The cytosolic domains contain several serines, threo nines, and tyrosines positioned in motifs highly conserved for phos- phorylation. It was previously shown that NIH3T3 cells release taurine via a volume-sensitive osmolyte transport pathway. This pathway differs pharmacologically and functionally from the volume-sensitive Cl – channel by its sensitivity towards anion channel blockers and kinase inhibitors, time course for activation and inactivation following hypotonic exposure, as well as sensitivity towards expression of constitutively active RhoA [7,9]. However, little is known about the molecular identity o f the taurine transporter responsible for the swelling-induced taurine release. The promoter region of rat and human taurine transporter genes, TauT, contain consensus binding sites for transcription factors p53 and NF-jB and for tonicity response element binding protein (Ton-EBP) [10–12]. The activity of p53 is regulated by a series of protein phosphorylations, acetylations and glycosylations of par- ticular regulatory domains of p53 [13], and p53 is up-regulated by moderate hypertonicity that protects re nal inner medullary co llecting duct cells from apoptosis [14]. TauT expression is down-regulated after activation of p53 in renal cells [15] but up-regulated by p53 in MCF-7 human breast cancer cells [16]. The activity of NF- jBis typically modulated by interleukines (IL) a nd the cellular Correspondence to I. H. Lambert, The August Krogh Institute, Biochemical Department, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark. E-mail: ihlambert@aki.ku.dk Abbreviations: TauCl, taurine chloramine; HOCl, hypochlorous acid; SEROT, transporters for serotonin; CREAT, creatine transporter; Ton-EBP, tonicity respo n se elem en t b inding protein; IL, interleukines; PKA/C, protein kinase A or C; ROS, reactive oxygen species. (Received 2 9 July 2 004, revised 30 September 2004, accepted 6 October 2004) Eur. J. Biochem. 271, 4646–4658 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04420.x antioxidant TauCl [5]. The tumour necrosis factor-a (TNFa) is reported to increase the mRNA level of TauT in rat brain capillary endothelial cells (TR-BBB13) [12] and human intestinal epithelial CaCo-2 cells [17] as well as the taurine uptake i n rat astrocytes [18] and CaCo-2 cells [17]. Ton-EBP is phosphorylated under hyperton ic condi- tions, whereupon it translocates to the nucleus, binds to the tonicity response element (Ton-E) and initiates transcription of hypertonicity-induced proteins such as TauT [12]. The present work was initiated to characterize activity, expression and subcellular localization o f TauT i n N IH3T3 fibroblasts. It i s demonstrated that: ( a) the saturable taurine transporter TauT is present in NIH3T3 cells and taurine uptake i s a Na + -andCl – -dependent process that correlates with Taut expression; (b) it is possible to modulate the expression of TauT and taurine transport activity by multiple stimuli both acutely and at t he TauT expression level; and (c) TauT does not promote volume-sensitive taurine release. Materials and methods Chemicals Bovine serum albumin (BSA), H 2 O 2 (1.03 M ), mouse TNFa,(5lgÆmL )1 in 1 m gÆmL )1 BSA), N-methyl- D - glutamine (NMDG), theophylline (40 m M , ddH 2 0), forsk- olin (50 m M , 96% ethanol), 4b-phorbol 12-myristate 13-acetate (PMA, 40 l M , 96% ethanol), Ortho-phtaldehyde (OPA), creatine, taurine, b-alanine, sucrose, mouse anti- acetylated a-tubulin, gentamycin, amphotericin were from Sigma Chemical (St. Louis, MO, USA). Rabbit anti-rat TauT against the C-terminal TauT sequence was from Alpha Diagnostics I nc (San Antonio, TX, USA). Rabbit anti-human TauT against the N-terminal sequence was donated by J. Mollerup (Institute of Molecular Biology, University of Copenhagen). Goat anti-rabbit lactate dehy- drogenase was from Abcam, Ltd (Cambridge, UK). Alka- line phosphatase conjugated goat anti-rabbit IgG and donkey anti-goat IgG were from Jackson Laboratories (Bar Harbor, ME, USA). Alexa Fluor Ò 488 donkey anti- rabbit I gG, Alexa Fluor Ò 488 mouse anti-chicken IgG and Alexa F luorÒ 568 g oat anti-rabbit IgG were from Molecu- lar Probes (Leiden, the Netherlands). [ 14 C]Taurine was from NEN Life Science Products (Boston, MA, USA). Penicillin, streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum and trypsin were from Invitrogen (Taastrup, Denmark). Media The phosphate buffered saline ( NaCl/P i ) contained 137 m M NaCl, 2.6 m M KCl, 6.5 m M Na 2 HPO 4 ,and1.5m M KH 2 PO 4 . Iso-osmotic NaCl medium contained 143 m M NaCl, 5 m M KCl, 1 m M Na 2 HPO 4 ,1m M CaCl 2 ,0.1m M MgSO 4 ,5m M glucose and 10 m M N-2-hydroxyethyl pip- erazine-N¢-2-ethanesulfonic acid ( Hepes). Iso-osmotic NMDGCl solution was similar to NaCl with NMDG being s ubstituted for sodium. Iso-osmotic KCl m edium contained 150 m M KCl, 1.3 m M CaCl 2 ,0.5m M MgCl 2 ,and 10 m M Hepes. Hypo-osmotic NaCl/KCl solution were prepared by reduction of the NaCl/KCl concentration in the iso-osmotic solutions to 95 m M , with reducing t he other components. Hypertonic NaCl medium w as prepared from isotonic NaCl medium by supplementation with 100 m M sucrose. In all solutions, pH was adjusted at 7.40. Unless otherwise noted, experiments were carried out room tem- perature (typically 18–22 °C). Cell culture Measurements were performed on Swiss NIH3T3 mouse fibroblasts (clone 7 obtained from B. M. Willumsen, Institute of Molecular Biology, University of Copenhagen, Denmark) and mouse myoblast (C2C12, American Type Culture Collection, Manassas, VA, USA). F ibroblasts were cultured in DMEM supplemented with 10% (v/v) heat inactivated fetal bovine serum and 1% (w/v) penicillin/ streptomycin. C2C12 were cultured in DMEM with 10% foetal bovine serum supplemented with 2 0 lgÆmL )1 genta- mycin, 3 lgÆmL )1 amphotericin B, 100 lgÆmL )1 strepto- mycin sulfate and 100 IUÆmL )1 penicillin. Cells were grown at 37 °C, 5% CO 2and 95% humidity. Cell cultures were passaged every 3–4 days by trypsinatio n (0.5%), and only passages 10–30 were used for experiments. Estimation of the taurine influx NIH3T3 cells were grown to 80% confluence in six-well polyethylene dishes (9.6 cm 2 per well). The c ells were washed three times by gentle aspiration/addition of 1 mL experimental solution. Following the final w ash, the cells in five of the wells were exposed to isotonic NaCl medium containing [ 14 C]taurine ( 2.54 · 10 11 c.p.m.Æmmol )1 , 1.4 l M ). The sixth well had added isotope-free NaCl medium and was used for estimation of the average protein content (g protein per well) by the Lowry method [19] using BSA as standard. At g iven time points (4–20 min) taurine uptake was terminated by removal of the extracellular medium, r apid add ition/aspiration of 1 mL i ce-cold M gCl 2 (100 m M ), followed by cell lysis with 500 lL 96% (v/v) ethanol. The ethanol was blown off and the cellular [ 14 C]taurine activity extracted by addition of 1 ml ddH 2 O (1 h ), which was transferre d to a scintillation vial for estimation of 14 C activity (b-scintillation counting, Ultima Gold TM ). The wells were washed twice with ddH 2 O. The total [ 14 C]taurine (c.p.m.) taken up by the cells in well one to five w as in each case estimated a s the sum o f 14 C a ctivity in the cell extract and water washouts. The taurine uptake (nmolÆgprotein )1 ) at a given time point is calculated from the ce llular [ 14 C]taurine activity, the extra ce llular specific activity and the protein content. The taurine influx (nmolÆ gprotein )1 Æmin )1 ) w as estimated as the slop e o f the cellular taurine uptake plotted vs. time. Estimation of the taurine efflux Taurine efflux measurements were performed at room temperature as described previously [20]. Briefly, NIH3T3 cells were grown to 80% confluence in six-well polyethy- lene dishes (9.6 cm 2 per well) and loaded for 2 h in 1 ml DMEM containing [ 14 C]taurine (80 nCiÆmL )1 ). The pre- incubation solution was aspirated and the cells washed Ó FEBS 2004 TauT expression, activity and subcellular localization (Eur. J. Biochem. 271) 4647 five times with 1 ml isosmotic solution in order to remove exce ss extracellular [ 14 C]taurine a nd cellular debris. The efflux was initiated after the final wash by addition of one ml of experimental solution. The cells were left for 2 min and then the medium was transferred to scintillation vial and rapidly substituted by 1 mL fresh medium. This procedure was carried out for 20 min. Cells were lysed at the end of the experiment by addition of 1 ml NaOH (0.5 m M ). The total 14 C activity in the cell system was estimated a s the sum of 14 C activity (b-scintillation counting, Ultima Gold TM ) in all the efflux samples, the NaOH lysate plus the two final w ash outs with ddH 2 O. The natural logarithm to the fraction of 14 C activity remaining in the fibroblast was plotted vs. time, and the rate constant for the taurine e fflux (min )1 ) at each time point was estimated as the negative slope of the graph between the time point and the proceeding time point. The taurine efflux at a g iven time point can be estimated as t he product o f the rate constant and the cellular taurine pool. I n the case where T auT activity w as down-regulated and preloading of the cells was imposs- ible, th e activity of the swelling-induced taurine transport was followed as influx in isotonic or hypotonic N a + -free, KCl media over 20 min. Estimation of the cellular amino acid content The amino acid content was estimated by OPA deriva- tization followed by reversed-phase HPLC separation (Gilson: 322-Pump, 2 34-Autoinjector, 155-UV/VIS detec- tion, BioLab, Aarhus, Denmark). Cells, grown at 80% confluence (75 cm 2 flasks) were washed three times with NaCl/P i medium, the medium was a spirated and the cells lyzed/deproteinized subsequently by addition of 1.2 mL 4% (v/v) sulfosalisylic acid. The cell homogenate was transferred to eppendorf tubes and sonicated (2· 10 s) on ice. Some suspension (200 lL) was d enaturated overnight after dilution with 200 lLNaOH(2 M ) and used for estimation of the protein content using the Lowry procedure [19] and BSA as p rotein standard. The residual 1000 lL of the suspension was centrifuged (20 000 g, 10 min) and the supernatant filtered (Milex-GV, 0.45 lm). The amino acids in the filtered sample were separated on a Nucleosil column (Macherey-Nagel, D € uren, Germany, C18, 250/4, 5 l M ) using a 1 ml per min flow rate and increasing the acetonitrile fraction in a 1 2.5 m M phosphate buffer (pH 7.2) from 0% to 25% within the initial 24 min and from 25% to 50% within the subsequent five min. UV absorption (330 nm) of the samples and taurine standard (0.1 m M ) were used for estimation of the taurine content in the samples. The cellular amino acid content (lmolÆgprotein )1 ) was estimated from t he amino acid and protein content in the flask. Fractionation, SDS/PAGE and Western blotting NIH3T3 cells grown to 80% confluence i n Petri dishes were washed quickly in ice-cold NaCl/P i ,treatedwith100lL lysis buffer [150 m M NaCl, 20 m M Hepes, 10% (v/v) glycerine, 1% (v/v) Triton X-100,1 m M EDTA, 1 m M NaF and 1 m M Na 3 VO 4 ] and 1% (w/v) SDS, scraped off with a rubber policeman and processed 10 times through a 27 gauge needle. For cell fractionation, fibroblasts were lysed in lysis buffer containing 0.5% (v/v) T riton X-100 and no SDS. The cell lysate was then centrifuged at 600 g fo r 10 min (4 °C) to give the nuclear fraction (pellet) and the supernatant was centrifuged at 40 000 g for 1 h (4 °C) to give the membrane fraction (pellet) and the cytosolic fraction (supernatant). The two p ellets were washed once in lysis buffer containing 0.5% (v/v) Triton X-100 and resuspended in 60 lL lysis buffer containing 1% (v/v) Triton X-100 and SDS. The protein concentrations were estimated using a BCA protein k it (Pierce, BB Gruppen, Denmark). Proteins were resolved by gel electrophoresis under denaturing and reducing conditions and electropho- retically transferred to a nitrocellulose membrane (Invitro- gen) as previously described [21]. The membranes were incubated with either rabbit anti-rat TauT (1 : 250), rabbit anti-human TauT (1 : 250) or goat anti-rabbit lactate dehydrogenase (1 : 800) Igs at room temperature for 2 h or overnight a t 4 °C followed b y identification wi th species- specific alkaline phosphatase-coupled secondary antibodies [1 : 1200 (TauT); 1 : 900 (lactate dehydrogenase)] and development with BCIP/NBT (Kirkegaard and Perry Laboratories, Gaithersburg, MA, USA). Immunocytochemistry NIH 3T3 cells grown on glass coverslips in six-well test plates (Nunc, Rosklide, Denmark) were fixed in 4% (v/v) paraformaldehyde, permeabilized in 0.2% (v/v) Triton X- 100, quenched in NaCl/P i with 2% (w/v) BSA and incubated with primary antibodies for 2 h at room temper- ature or overnight at 4 °C: anti-acetylated a-tubulin (1 : 400), rabbit anti-rat TauT (1 : 100) or rabbit anti- human TauT (1 : 100) Igs. Cells were washed in NaCl/P i and incubated with 4,6-diamidino-2-phenylindole (DAPI) (1 : 100), Alexa FluorÒ 488 donkey anti-rabbit IgG (1 : 200), Alexa FluorÒ 488 mouse anti-chicken IgG (1 : 600), and/or Alexa FluorÒ 568 goat anti-rabbit IgG (1 : 600). For epifluorescence studies, fluorescence was visualized on either a Microphot-FXA microscope with EPI-FL3 filter (Nikon, DFA A/s, Copenhagen, Denmark). Confocal microcopy was performed using a Leica DM IRB/E microscope coupled to a Leica TSC NT confocal laser scanning unit (Leica Lasertechnik GmbH, Heidelberg, Germany). Excitation of DAPI and Alexa F luorÒ 488 was carried out using the 364 nm UV laser-line and the 488 nm argon/krypton laser-line, respectively. Emission wavelengths, Photo Multiplyer Tube (PMT) and laser intensity settings were optimized to minimize bleed-through, and to set fluorescence detected from preparations labelled with secondary antibody only to zero. Images were taken using a 40·/ 1.25 NA planapochromat objective, a 0.75 Airy disc pinhole, and an optical slice thickness of 0.25 lm. Images (512 2 pixels) were frame averaged and presented in pseudocolour. Digital images were enhanced by Adobe PHOTOSHOP Ò 6.0. Data and statistical analysis The neural network algorithm, NetPhos 2.0 [22], manual homology searches as well a s searches in the ELM database 4648 J. W. Voss et al. (Eur. J. Biochem. 271) Ó FEBS 2004 (http://elm.eu.org/) were used to predict serines, threonines and tyrosines in the intracellular domains of TauT suitable for phosphorylation. Data are presented either as individual experiments, representative of at least three independent experiments, or as mean values ± SEM, n indicates the number of i ndependent experiments. Statis tical s ignificance was estimated by the Student’s t-test. For all statistical evaluations, P-values < 0.05 were taken to indicate a significant difference. Results TauT in NIH3T3 fibroblasts – affinity, substrate specificity and ion dependency TauT in most cell systems has a high affinity towards taurine but a l ow transport capacity [7]. T he traces in Fig. 1 demonstrate that active taurine uptake in NIH3T3 mouse fibroblasts is linear within the initial 20 min following the addition of 14 C-labelled taurine. Using taurine uptake within the initial 20 min in NaCl medium containing extracellular taurine (0–50 l M ) and fitting the uptake data to a M ichaelis–Me nten equation revealed that the K m value in NIH3T3 cells, i.e. t he extracellular taurine concentration required for half maximal taurine uptake, is 18 ± 1 l M (n ¼ 3). It is also recognized that active taurine uptake via TauT is Na + -andCl – -dependent in various cell systems and that only close analogues t o taurine (such a s b-alanine) are potential inhibitors of the active uptake [7]. The data shown in Fig. 1 and summarized in Table 1 indicate that the i nitial taurine uptake is reduced in the presence of b-alanine (Fig. 1A) and following substitution of extracellular K + for Na + or extracellular N O 3 – for Cl – (Fig. 1B). From Fig. 1A and T able 1 it i s also seen t hat t aurine uptake is r educed to about 75% in the presence of 5 m M creatine. Creatine (a-methylguanido acetic acid) is accumulated by muscle cells via the active creatine transporter CreaT, which has a structure similar to that o f TauT and exhibits the same requirement for Na + and Cl – for initiation of active uptake of creatine [23]. However, Western blotting using a polyclonal antibody raised against the human creatine transporter (Research Diagnostics Inc, Flanders, NJ USA) and C2C12 myoblasts as a positive c ontrol, indicated that CreaT is apparently ab sent in NIH3T3 cells (n ¼ 3, data not shown). C reatine resembles GABA (c-amino butyric acid), which reduces the active taurine uptake in, e.g. Ehrlich ascites tumour cells [24], and it is possible that creatine binds to TauT with low affinity and competitively reduces active taurine uptake. This was not investigated further. From Fig. 2 it is seen that active taurine uptake in NIH3T3 cells is a sigmoidal function of the extracellular Na + concentration ([Na + ] o ) and a hyperbolic function of the extracellular Cl – concentration ([Cl – ] o ). Fitting these uptake data to Hill type equations it is estimated that 2.5 Na + and 1 Cl – ions are required for initiation of the uptake of one taurine molecule (Fig. 2). From the data presented in Figs 1–2 and Table 1 it is suggested that taurine uptake in NIH3T3 cells is mediated by a system that exhibits characteristics typical for TauT in other cell types, i.e. NIH3T3 TauT has a high specificity and a ffinity towards taurine a nd 2.5 Na + and 1 Cl – are i nvolved in the uptake of one taurine. Acute regulation of TauT transport activity by PKC, PKA and H 2 O 2 TauT possesses several cytosolic serine and threonine residues that are potential targets for protein kinase A and C (PKA, PKC) mediated phosphorylation [7,25]. From Fig. 3 it is seen that acute exposure e ither to PMA to stimulate PKC, or t o forskolin plus theophylline t o increase cellular cAMP level and thus presumably stimulate PKA, results in a reduced taurine uptake. Exposing the NIH3T3 cells to H 2 O 2 also reduces the taurine uptake (Fig. 3). Reactive oxygen species (ROS) exhibits a variety of physiological effects [7] and H 2 O 2 , which is highly cell permeable, is reported to elevate the phosphorylation of tyrosine residues, most probably via inhibition of a p rotein Fig. 1. Substrate specificity and ion requirement of TauT. NIH3T3 cells, grown at 80% confluence, were exposed to isotonic N aCl medium containing [ 14 C]taurine (2.54 · 10 11 c.p.m.Æmmo l )1 ,1.4l M ). At time points 4–20 min taurine uptake was terminated by removal of the extra- cellular medium and the cellular [ 14 C]taurine was extracted. The cellular taurine uptake (nmolÆgprotein )1 )atagiventimepointwascalculatedfrom the cellular [ 14 C]taurine activity, the extra cellular specific activity and the protein content. (A) Taurine uptake was followed in the abse nce (control cells) and in t he presence of 5 m M creatine or 5 m M b-alanine. ( B) Taurine uptake w as fo llowed i n control cells e xposed to NaCl medium and in cells incubated with N a + -free KCl m edium or Cl – free NaNO 3 medium. T he cu rve s in A and B are all r e presentative o f three inde penden t s ets o f experiments. Ó FEBS 2004 TauT expression, activity and subcellular localization (Eur. J. Biochem. 271) 4649 tyrosine phosphatase [26]. Thus, t hese data could indicate that an increased phosphorylation of TauT or a putative regulator of TauT could be involved in the H 2 O 2 -induced reduction in the active taurine uptake in NIH3T3 cells. Expression and subcellular localization of TauT The p romoter r egion of the TauT gene in human [10] and rat [11] contains consensus binding sites for the transcription factors p 53 and NF-jB. The cellular expression of p 53 is reported to be increased following serum-starvation [27] or exposure to hypertonic conditions [14], whereas NF-jB activity is typically modulated by IL (TNFa) . Active taurine uptake in NIH3T3 cells is significantly increased by 11% and 17% following exposure to TNFa (2 0 ngÆmL )1 )for 16 h or serum starvation for 24 h, respectively (Fig. 4). Epifluorescence microscopy analysis shows that a poly- clonal TauT antibody, raised against the C-terminal sequence of the rat TauT, localizes to a region of NIH3T3 control cells, which appears to be within the nucleus, as well as in the cytosol and at the plasma membrane (Fig. 5A, row 1; TauT red colour, microtubules green colour). Following TNFa treatment, TauT immunolocalization is augmented, particularly in the nucleus and the perinuclear area (Fig. 5A, row 2). Thus, the data in Figs 4 and 5 indicate a correlation between taurine transport and total cellular TauT expression. To verify the apparent cytoplasmic and nuclear localization of TauT, we employed confocal laser scanning microscopy of TauT and DAPI-stained NIH3T3 cells. From t he confocal visualization s tudies in Fig. 5B it is seen that TauT (green colour) appears throughout the nuclear compartment. It is emphasized that the p inhole size was kept s mall enough to exclude that staining observed in these c ompartments is caused by out-of-focus fluorescence from transporters localized, e.g. to the p lasma m embrane. As controls, a similar pattern of TauT localization was observed with an antibody raised against the N-terminal part of TauT, and immunolocalization of the antibody raised against the C-terminal part of TauT was abolished by a specific blocking peptide to t his antibody (data not shown). Besides being regulated by p53 and NF-jB, TauT expression and TauT activity are also sensitive to exogenous taurine [28]. From Fig. 6A it can be seen that polyclonal antibody raised against the C-terminal sequence of the rat TauT, recognizes a m ajor protein band at 67 kDa in lysates of whole NIH3T3 cells, corresponding to the molecular mass of TauT. The expression of this protein is reduced following 24 h exposure to DMEM supplemented with 1m M or 100 m M exogenous taurine, and i ncreased follow- Fig. 2. N a + /Cl – /taurine stoichiometry for taurine uptake v ia TauT . N IH3T3 cells, grown at 80% co nfluen ce, were exposed to isotonic mediu m containing [ 14 C]taurine ( 2.54 · 10 11 c.p.m.Æmmol )1 ,1.4l M ) f or 20 min. Taurine uptake w as terminated and the cellular [ 14 C]taurine (c.p.m. per 20 min) estimated. (A) The extracellular Na + concentration was varied between 0 and 150 m M adjusting the concentration to 150 m M with NMDG. The influx w as plotted vs. the extracellular Na + concentration ([Na + ]) and fitted to the Hill type equation: Y ¼ (V max [Na + ] n )/ ((K Na ) n +[Na + ] n ), where V max is the maximal uptake, K Na is the Na + concentration required f or half maximal u ptake a nd n is the number of ions required for initiation of uptake of one taurine. (B) The extracellular Cl – concentration was varied between 0 and 150 m M adjusting the con- centration to 150 m M with NaNO 3 . The influx was plotted vs. the extracellular Cl – concentration ([Cl – ]) and fitted to the Hill type equation: Y ¼ Y o +(V max [Cl – ] n )/((K Cl ) n +[Cl – ] n ), where Y o is taurine uptake in the absence of Cl – ,andK Cl is the Cl – concentration required for half maximal uptake. The stoichiometry values i ndic ated on the fi gure were estimated for N a + and Cl – in five a nd three sets of experiments, respectively. Table 1. TauT substrate specificity a nd ion d ependency. Taurine u ptake was estimat ed a s in dicated i n F ig. 1 in the absence or presence of 5 m M creatine/b-alanin e and in Na + -free NMDG-medium, KCl medium or Cl – free NaNO 3 medium. The absolute tau rine influx (nmolÆgpro- tein )1 Æmin )1 ) w as estimated by linear regression, u sing values in the time frame 4–20 min . Taurine uptake from three sets of paired experiments is given relative to control values ± SEM. P indicates the level of significance in a paired Student’s t-test against the control value. Taurine influx P Substrate specificity Control 1 Creatine 0.730 ± 0.027 0.005 b-alanine 0.007 ± 0.001 <0.001 Ion dependency Control, NaCl 1 NaNO 3 0.116 ± 0.018 0.01 KCl 0.003 ± 0.001 <0.001 NMDG 0.001 ± 0.001 <0.001 4650 J. W. Voss et al. (Eur. J. Biochem. 271) Ó FEBS 2004 ing exposure to DMEM supplemented with 100 m M sucrose. Similar data where obtained using the antibody raised against the N-terminal sequence of T auT, confirming antibody specificity (data not shown). Pre-exposure to exogenous taurine for 24 h is accompanied by a reduction in the initial taurine uptake, whereas the taurine uptake is significantly increased following pre-exposure to sucrose (Fig. 6B). It is noted that the reduced taurine uptake following exposure to 100 m M taurine is not secondary to cell shrinkage induced by the hypertonic conditions, as evidenced by the effec t of addition of 100 m M sucrose to increase extracellular osmolarity to the same extent. The taurine-induced down regulation of the taurine uptake in NIH3T3 cells is reversible. This is seen from Fig. 6C where it is shown that in NIH3T3 cells, pre-exposed to 100 m M taurine for 24 h normal transport capacity is g radually regained and has returned to control values within 24 h. It is noted that TauT activity in cells exposed to DMEM supplemented with 100 m M sucrose for 48 h is slightly increased compared to that in control cells exposed to DMEM, indicating that TauT activity in NIH3T3 cells is not affected by long-term hypertonic exposure. Figure 7A shows that the immunofluorescence intensity of a ntibody against the C-terminal sequence of the rat TauT is dramatically reduced following long-term exposure to high concentrations of taurine (compare 1st and 2nd row) and increased following long-term exposure to high s ucrose (3rd row), indicating that the variations in the TauT transport activity in Fig. 6B reflect TauT expression. The fluorescence intensity of the cells shown in Fig. 7 is enhanced by electronic manipulation (to the same extent for all p anels, i.e. maintaining the same relative intensity) in comparison to those in Fig. 5. This was done in order to facilitate the visualization of the inhibitory effect of the taurine, which was so marked, that there was no or very little visible labelling in the taurine-treated cells in the nonenhanced images. The fluorescence images presented in Fig. 7B (frames 0 –12 h ) show the t ime-dependent effect of sucrose on the level of TauT expression in cells pre-exposed to h igh concentrations of taurine for 24 h. It is seen that the level of TauT increases at about 4 h after sucrose was su bstituted for taurine in the incubation medium. Thus, in accordance with the data in Figs 4 and 5 the active taurine uptake correlates with TauT expression. In order to confirm changes in subcellular level of TauT expression and TauT localization upon supplementation of taurine and sucrose, we performed Western blotting analysis of TauT expression in cytosolic, nuclear and membrane fractions of NIH3T3 cells. The cytosolic protein lactate dehydrogenase w as used as a c ontrol to ensure that nuclear and membrane fractions were not contaminated with cytosolic TauT. The cell fractionation experiment presen ted in Fig. 8 confirms the presence of a major 67 kDa TauT protein in whole NIH3T3 fibroblasts, a nd that this pro tein is mainly localized to the cytosolic fraction. In contrast, we find that TauT in the nuclear and membrane fractions mainly appears as a 90 kDa protein. The intensity of both protein bands is reduced in NIH3T3 cells exposed to 100 m M extracellular taurine compared to cells exposed to 100 m M sucrose. In particular, the protein level of the nuclear and membrane-associated 90 kDa forms of TauT is down-regulated at least eight and three times, respectively, in the taurine supplemented cells. Thus, the variation of nuclear TauT expression, observed by immunofluorescence microscopy analysis upon taurine and sucrose sup plemen- tation (Fig. 7), probably reflects variation in the level of the 90 kDa TauT protein. Three TauT protein bands in the range 50–70 kDa have been previously demonstrated by immune blotting in Ehrlich ascites tumour cells and it has been suggested that the band in the Ehrlich cells with the highest apparent molecular mass represents a phosphoryl- ated form of TauT [29]. Whether the variance in the Fig. 4. Augmentation of the taurine uptake by long-term exposure to TNFa or serum-free conditions. NIH3T3cellsweregrowninDMEM (control), DMEM containing TNFa (20 ng ÆmL )1 , 16 h) or serum-free DMEM (24 h, serum-starved) before initiation o f the influx e xperi- ment. Taurine up take was followed with time using [ 14 C]taurine as indicated in Fig. 1. The influx, estimated from the slope of the uptake curves, was estimated and in ea ch case giv en r elative to t he influx i n control cells ± SEM (n ¼ 3). # , Significantly different f rom t he control (P <0.05). Fig. 3. Modulation of taurine uptake by phosphorylation. NIH3T3 cells were grown to 80% confluence. The cellular taurine uptake was fol- lowed with t ime in isotonic N aCl m edium using [ 14 C]taurine as indi- cated in Fig. 1. PM A (50 n M ), forskolin (10 l M ) plus theoph ylline (0.5 m M ), an d H 2 O 2 (2 m M ) w ere i nclude d i n t he experimental medium from the time of the initiation of the influx experiment. The influx was estimated f rom the slop e of t he uptake cu rves and i n each case given relative to the infl ux in c ontrol cells ± SEM (n ¼ 3). #, Significantly different from the c ontrol (P < 0.05). Ó FEBS 2004 TauT expression, activity and subcellular localization (Eur. J. Biochem. 271) 4651 molecular mass of TauT from the nuclear/membrane and the cytosolic fractions in NIH3T3 fibroblasts reflects differences in post-transcriptional modifications such as glycosylation and/or phosphorylation or expression of different TauT isoforms was not investigated further. We fu rther observed that TauT in sucrose-treated cells strongly localizes in a punctuate pattern along the c ytosolic network o f acetylated microtubules (Fig. 9), indicating that localization and functional targeting of TauT to subcellular domains, such a s the plasma membrane, may be coupled to microtubules-associated carrier vesicles. Effect of long-term exposure to TNFa and exogenous taurine on the volume-sensitive taurine leak pathway The experiments in Fig. 10 were performed in order to evaluate whether up/down r egulation of T auT transport activity is paralleled by a similar up/down regulation of t he activity of the volume s ensitive taurine efflux pathway. It is seen that the rate constant for taurine release is increased transiently following hypotonic exposure and that the maximal rate constant is reduced following pre-exposure to TNFa (Fig. 10A). It is estimated that pre-exposure to 20 ng TNFaÆmL )1 for 16 and 48 h reduces the maximal rate constant for the swelling-induced taurine release from NIH3T3 cells to 75% and 60% of the control value, respectively (Fig. 10B). A 16 h exposure to TNFa also reduces the rate constant for taurine release from NIH3T3 cells under isotonic conditions by 30%, i.e. the rate constant in three sets of experiments was e stimated at 0.0020 ± 0.00005 min )1 (control cells) and 0.0014 ± 0.00005 min )1 (20 ng TNFaÆmL )1 ,16h;P ¼ 0.005). T he cellular taurine content was, in three sets of separate experiments, estimated at 0.031 ± 0.003 and 0.025 ± 0.003 lmolÆgprotein )1 in control cells and cells treated with TNFa, respectively. Pasantes-Morales and coworkers [30] have similarly estimated the cellular taurine content in NIH3T3 cells under control conditions at 0.052 lmolÆg protein )1 . As the taurine efflux at a given time point is equal to the r ate constant times the cellular pool it is estimated Fig. 5. Modulation of subcellular localization and expression of TauT. (A) NIH3T3 cells, grown on cover slips at 60–70% confluence, were grown in DMEM (control cells, 1st row) or DMEM plus TNFa (20 n gÆmL )1 , 16 h , 2nd ro w). Cells were fi xed in paraformaldehyde (4%, 15 min) and permeabilized with Triton X-100 ( 0.2%, 10 min ). T he nucleus (blue) was visualized wit h DAPI. T he microtubules (green) were marked with a primary antibody against acetylated a-tubuline and visualized with Alexa 488. TauT (red) was marked with a primary antibody raised against the C-terminus of TauT and visualized with Alexa 568. The merged column (3rd column) is an overlay of the 1st column (nucleus, microtubules) and the 2nd column (TauT). Each image is representative of at least t hree images from separate experiments. (B) Cells were incubated in isotonic medium und er co ntrol c o nditio ns, fixed , a nd TauT and nuclei w ere l abelled a s described in Materials and methods. Cells were vi ewed using a 40·/1.25 NA planapochromat objective on a Leica DM IRB/E microscope coupled to a Leica TSC NT confocal laser scanning unit. Visualization of TauT (green) was carried out by excitation of DAPI and Alexa FluorÒ 488 using t he 364 nm UV laser-line and the 4 88 nm ar gon/krypton laser- line, respectively. Emission wavelengths, PMT and laser intensity se ttings were optim ized to minimize bleed-through, and to set fluore scence detected from preparations labelled with secondary antibody only to zero. Images were taken at a 0.75 Airy disc pinhole, and an o ptical slic e thickness of 0.25 lm, frame averaged and prese nte d i n p seudocolo ur. Im ages shown w ith a re o ptical s lices taken at 0 .75 lm intervals, moving towards the bottom of t he cell from left to right. Th e experiment shown is r epresen tative of three independent experiments. 4652 J. W. Voss et al. (Eur. J. Biochem. 271) Ó FEBS 2004 that 16 h e xposure to TNFa reduces the maximal taurine efflux under hypotonic conditions by 40%. Thus, TNFa exposure has opposing effects on the activity of TauT and the v olume-sensitive taurine leak pathway i n N IH3T3 cells. Serum starvation increased the maximal rate constant for the s welling-induced taurine influx in hypoton ic media (200 mOsm) by 20 ± 7% (n ¼ 4). Figure 10C shows that a n increase in taurine transport via the volume-sensitive taurine leak pathway following reduction in the extracellular tonicity can be demonstrated as an increase in taurine uptake in Na + -free hypotonic KCl medium. This technique was used as down regulation of TauT prevents the [ 14 C]taurine preloading of the cells required for the standard efflux procedure. Exposing NIH3T3 cells, preincubated for 24 h with DMEM medium containing 100 m M sucrose or 100 m M taurine to hypotonic KCl medium, resulted in an increase in the taurine uptake which is similar o r slightly larger than the influx seen in NIH3T3 cells pre-exposed to DMEM alone (Fig. 10D). The l atter is most probably a consequence of the fact that cells pre-exposed to hypertonic conditions experience a more dramatic reduction in the extracellular osmolarity when exposed to the hypotonic conditions. Thus, the volume-sensitive taurine r elease seems to not be affected b y long-term hypertonic e xposure o r t o l ong-term exposure t o high extracellular taurine concentrations. Discussion The organic osmolyte, taurine, is present in high concen- trations in heart- and skeletal m uscles, brain, kidney and retina. The cellular level of taurine is a balance between active uptake via TauT and passive leak via a volume sensitive pathway. Within recent years it has become evident that taurine is a multifunctional molecule th at r egulates cell volume, cellular free Ca 2+ concentration, cellular oxidative status and interferes w ith cell survival [7,31,32]. The data presented in Figs 1–3 and 5,6 and Table 1 indicate that TauT is present in the NIH3T3 mouse fibroblasts and exhibits typical characteristics of mammalian TauT, i.e. TauT has a high affinity/specificity for taurine, the uptake of one molecule of taurine via TauT involves 2 Na + and 1 Cl – , and the TauT transport activity is reduced following acute stimulation of PKC and PKA. Stimulation of PKC and PKA has n o detectable effect on the e xpression of TauT in NIH3T3 fibroblasts (data not shown) and most probably does not involve modulation of the transcriptional rate of TauT. Taurine release from NIH3T3 cells is increased Fig. 6. Reversibility of substrate-induced down regulation of TauT activity. (A) NIH3T3 fibroblasts were grown in DMEM (control), DMEM plus 1m M /100 m M taurine or DMEM plus 100 m M sucrose for 24 h. The cells were lyzed, sonicated and proteins separated by SDS/PAGE (10%) and visualized with Western blotting using a primary antibody raised against th e C-terminus of TauT and a secondary, alkaline, phosphatase conjugated antibody. The bands represent a 67 kDa protein. The blot is representative three se ts of paired experiments. (B) Cells were pretreated with taurine and sucrose as indicated in panel A. Cells were washed five times prior to the initiation of the influx experiments, in order to remove excess unlabelled taurine, and t aurine uptake was f ollowed w ith t ime using [ 14 C]taurine as outlined in Fig. 1. In o rder to p reserve the tonicity during the washing procedure and the influx experiment we used isotonic standard NaCl medium for control cells and cells pretreated with 1 m M taurine, and standard N aCl medium supplemented with 100 m M sucrose for cells treated with 1 00 m M taurine or 100 m M sucrose. The i n flux, estimated from the slope of the uptake curves, is in e ach case given relative to the influx in control cells ± SEM (n ¼ 3). (C) Cells, grown in DMEM supplemented with 100 m M taurine for 24 h, were washed and incubated for a nother 2, 4, 8, 12, 2 4 h in DMEM supplemented with 100 m M sucrose. Cells were grown 48 h in DMEM (control cells) and DMEM supplemented with 100 m M sucrose, respective ly. Taurine uptake was followed with time (4.20 min) in isoton ic NaCl me dium (control) or s tandard NaCl m edium s upplemented with 100 m M sucrose (Tau/Suc, Sucrose) and the influx estimated by linear regression as indicated above. Values for 24 h taurine plus 24 h suc rose and for 48 h sucrose treatment are given relative to the isotonic c ontrol ± S EM, represe nting five and t hree in depende nt sets of e x perimen ts. Valu es for c ells incub ated f or 24 h with taurine followed by 2 to 12 h incubation in NaCl medium supplemented with sucrose are given relative to the isotonic control and represents the mean of two sets o f experiments. #, S ignific antly different from c ontrol (P <0.05). Ó FEBS 2004 TauT expression, activity and subcellular localization (Eur. J. Biochem. 271) 4653 following osmotic cell swelling (Fig. 10) and it has previ- ously been demonstrated that the swelling-induced taurine efflux is via a volume-sensitive taurine leak pathway which is sensitive to various anion ch annel blockers but different from the swelling-induced Cl – efflux pathway [7,25]. Role of reactive species in the regulation of taurine transport From the data in Figs 3 and 4 it is seen that the active taurine t ransport in NIH3T3 i s reduced following exposure to H 2 O 2 and increased following preincubation with TNFa. In the case of TNFa we also observed an increased expression of TauT in NIH3T3 cells (Fig. 5). It has been demonstrated recently that that e xposure to H 2 O 2 increases taurine release from NIH3T3 cells following hypotonic incubation and it w as suggested that this effect reflected an inhibition of a p rotein tyrosine phosphatase [20]. Several of the serines, t hreonines and tyrosin es in the intracellular domains of TauT are situated in motifs highly suitable as targets for protein kinases. It is possible that a shift of TauT to a m ore tyrosine phosphorylated state i n congruence with Fig. 7. Reversibility of substrate-induced down regulation of TauT expression. (A) NIH3T3 cells, grown on cover slips at 60–70% confluence, were exposed to DM EM (control cells, 1st ro w), DMEM p lus 100 m M taurine (24 h, 2nd row) or DM EM s upplemen ted w ith 100 m M sucrose (24 h, 3rd row) as indicat ed i n F ig. 6 A,B. C ells were fixed in paraformaldehyde (4%, 15 min) and permeabilized with Triton X-100 ( 0.2%, 10 mi n). T he nucleus (blue), the microtubu les (green) and TauT (red) were visualized as indicated in the legend to Fig. 5 . The 3rd column is the merge of the 1st column (nucleus, microtubules)andthe2ndcolumn(TauT).Eachimageisrepresentative of at least 5 images from separate experimental setups. (B) Cells, grown in DMEM plus 100 m M taurine for 24 h, were washed and exposed to DMEM plus 100 m M sucroseforthetimeperiodindicated (Frame 2… 12 h). Cells were fixed and TauT ( red ) detected as indic ated above. Images are re presentative of two s e ts of experiments. 4654 J. W. Voss et al. (Eur. J. Biochem. 271) Ó FEBS 2004 increased serine/threonine phosphorylation of TauT redu- ces the active taurine uptake following exposure to H 2 O 2 . Kang and coworkers [12] found that exposing the blood- barrier TR)BBB13 cells to TNFa (2 0 ngÆmL )1 )resultedin an increased mRNA level of the TauT and a concomitant 1.7-fold increase in taurine uptake. The promoter to TauT contains a b inding site for the transcription factor NF-jB andactivationofNF-jB involves phosphorylation of the inhibitory complex IjB, dissociation of the heterodimer NF-jB(p50,RelA)fromIjB, translocation of NF-jBto the nucleus and subsequent initiation of gene expression. HOCl which is a highly reactive oxidant species generated from H 2 O 2 and Cl – , is converted to the less reactive TauCl by combination with t aurine. I t has been proposed recently by M iyamoto and coworkers [5] that TauCl is involved in oxidation of IjB which prevents phosphorylation of IjB and a ctivation o f NF-jB. Thus, the effect of longterm exposure to TNFa, and most probably t o H 2 O 2 ,ontaurine uptake could in volve modulation o f the NF- jBmediated regulation of the transcription of TauT.TNFa is a well- known inducer of apoptosis. However, Lang and coworkers [33] have demonstrated previously that taurine uptake in Jurkat lymphocytes is increased following stimulation with the apoptosis inducing ligand Fas (CD95) which presum- ably relieves the apoptotic process. Whether an increased taurine content in TNFa-treated cells actually counteracts apopthosis in NIH3T3 cells is under investigation. High extracellular taurine down-regulates TauT expression and transport activity From Figs 6 and 7 i t is seen that exposure to growth medium supplemented with 100 m M taurine reduces the expression as well as transport a ctivity o f TauT in NIH3T3 Fig. 8. N uc lear localizatio n o f TauT. Cells, g rown in DMEM supplemented with 100 m M taurine or 100 m M sucrose for 24 h were l yzed , so nicated, fractionated and proteins f rom the different fractions were separated by SDS/PAGE (10%). TauT was v isualized with a primary antibody raised against the C-terminus of TauT and a secondary, alkaline, phosphatase conjugated antibody. The cytosolic protein lactate dehydrogenase, used to exclude c ytosolic contamination of nuclear and membrane fractions, was visualized with goat anti-rabbit lactate dehydrogenase and alkaline phosphatase conjugated donkey anti-goat. C, whole cell homogenate; Nu, nuclear fraction; Cyt, cytosolic fraction and Mem, membrane fraction. The gel is representative of three set of experiments. Fig. 9. M icro tubule association of TauT. (A) NIH 3T3 cells, grown on cover slips at 60–70% con fluence, were wash ed, fixed, pe rmeabiliz ed and preceded for d etection of TauT and c ytosolic m icrtubules a s i ndicated i n F ig. 7 . TauT w as visualized using a C -terminal antibody raised against the rat TauT. Images are representative of at least three sets of experiments. Frames A–D represent tubuline system (A: green), TauT (B: red), merged image ( C), a nd enlargement of the framed section from C (D) with the microtubules and TauT images slightly shifted in o rder to facilitate visualization of t he TauT/microtubules colocalization. Ó FEBS 2004 TauT expression, activity and subcellular localization (Eur. J. Biochem. 271) 4655 [...]... oxidation of IjB and consequently impairment of the NF-jB-mediated regulation of the transcription of TauT From Fig 6C it is seen that the taurine- induced downregulation of TauT is reversible and that TauT in NIH3T3 cells regain 50% of their transport capacity within 12 h and full capacity 24 h after removal of extracellular taurine This reversibility is reflected in a concomitant increased expression of TauT. .. 2004 Fig 10 Effect of long-term exposure to TNFa and high extracellular taurine on the swelling-induced taurine transport (A) Cells, grown to 80% confluence, were incubated for 16 h in DMEM in the absence (control) or presence of 20 ngÆmL)1 TNFa [14C ]Taurine was included in the preincubation medium during the last 2 h The cells were washed and the release of [14C ]taurine followed with time in NaCl medium... biochemistry and insights on the biological functions of taurine Adv Exp Med 483, 1–25 4 Schaffer, S., Takahashi, K & Azuma, J (2000) Role of osmoregulation in the actions of taurine Amino Acids 19, 527–546 5 Miyamoto, Y., Kanayama, A., Inoue, J.I., Konishi, Y.S & Shimizu, M (2003) Taurine is involved in oxidation of I kappa B alpha at Met45 – N-halogenated taurine and anti -in ammatory action In Taurine in the. .. extracellular taurine concentration on the active taurine uptake via TauT and the passive taurine release via the volumesensitive leak pathway (Fig 4 vs 10A,B; Fig 6B vs 10D) clearly indicate that TauT is not involved in the swellinginduced taurine release in NIH3T3 The molecular identity of the volume-sensitive taurine leak pathway is unresolved It has been demonstrated recently that phospholipase A2 (iPLA2) and. .. translocate to the nucleus upon TNFa treatment, and proposed to play a role in the cellular stress response following apoptotic stimuli [40] Is the swelling-induced taurine efflux via TauT? Release of taurine is a characteristic feature of ischemia and it has been suggested that the efflux of taurine involves both osmotic stress and a Na+-dependent reversal of TauT [41] The opposing effect of TNFa and high... represent immature TauT present in the ER/Golgi compartments The nuclear localization of TauT, the high molecular mass of nuclear TauT, and its apparent increase upon TNF-a treatment, is puzzling, and more studies are needed to determine the phosphorylation state and the potential role of TauT in the nucleus However, it is interesting to note that another ion transport protein, the organellular chloride... activity The effect of high extracellular taurine concentration has been reported to involve a Ca2+-sensitive interaction of taurine with at least one unidentified cis-element in the 5¢-flanking region of the TauT promoter region [36,37] An alternative explanation to the effect of high extracellular taurine on TauT expression and activity could be a concomitant increased cellular level of taurine and TauCl... with 100 mM sucrose increases the expression as well as transport activity of TauT Kang and coworkes [12] demonstrated accordingly that hypertonic treatment of TR-BBB13 cells increased the level of TauT mRNA as well as active taurine uptake, whereas excess taurine conditions resulted in a reduced level of TauT mRNA and a concomitant reduction in taurine uptake Furthermore, Bitoun and Tappaz [28] have... that increasing the cellular taurine content prevented osmolarity-induced up -regulation of TauT mRNA expression in astrocyte primary cultures and they suggested that TauT expression is controlled by a taurine- induced down -regulation and an osmolarity-induced up -regulation In this context it is noted that Ton-EBP, which transactivates osmo-protective genes, is expressed and up-regulated following hypertonic... activity is required for the swelling-induced activation of the volume-sensitive taurine leak pathway in NIH3T3 cells and that ROS, produced by a NAD(P)H oxidase complex, could inhibit protein tyrosine phosphase activity, reduce Src kinase-activity and thereby potentiate the swelling-induced taurine release [20,42] Liu and McHowat [43] have demonstrated that TNFa interferes with two types of PLA2 with different . within the initial 20 min following the addition of 14 C-labelled taurine. Using taurine uptake within the initial 20 min in NaCl medium containing extracellular taurine (0–50 l M ) and fitting the. specific activity and the protein content. The taurine in ux (nmolÆ gprotein )1 Æmin )1 ) w as estimated as the slop e o f the cellular taurine uptake plotted vs. time. Estimation of the taurine efflux Taurine. Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts Jesper W. Voss, Stine F. Pedersen, Søren T. Christensen and Ian H.