Báo cáo khoa học: Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts doc

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 concentrat

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 demonstrate that NIH3T3 mouse

fibro-blasts express a saturable, high affinity taurine transporter

(TauT, Km¼ 18 lM), and that taurine uptake via TauT is

a Na+- and Cl–-dependent process with an apparent

2.5 : 1 : 1 Na+/Cl–/taurine stoichiometry Transport

activ-ity is reduced following acute administration of H2O2 or

activators of protein kinases A or C TauT transport activity,

expression and nuclear localization are significantly

increased upon serum starvation (24 h), exposure to tumour

necrosis factor alpha (TNFa; 16 h), or hyperosmotic

med-ium (24 h); conditions that are also associated 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 concentration In contrast to active taurine uptake, swelling-induced taurine release is significantly reduced following preincubation with TNFa (16 h) but unaffected by high extracellular taurine concentration (24 h) Thus, in NIH3T3 cells, (a) active taurine uptake reflects TauT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of TauT is regu-lated; and (d) volume-sensitive taurine release is not medi-ated by TauT

Keywords: TNFa; creatine; microtubules; reactive oxygen species; volume-sensitive taurine leak pathway

Taurine, amino ethane sulfonic acid, plays an essential role

not only as an organic osmolyte and substrate for the

formation of bile salt, but also in the modulation of the

cellular, free Ca2+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 reaction with cellular hypochlorous acid

(HOCl), produces the less toxic taurine chloramine (TauCl)

and thus serves a tissue protective role against oxidative

injury [5,6] Hence, changes in the net cellular content of

taurine may have a dramatic impact on cell function

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 selectivity 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 is 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 12 times, with the N- and C-terminal ends exposed to the cytosolic compartment The cytosolic domains contain several serines, threonines, 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 of 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 renal inner medullary collecting 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-jB is typically modulated by interleukines (IL) and 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 response element binding protein; IL, interleukines;

PKA/C, protein kinase A or C; ROS, reactive oxygen species.

(Received 29 July 2004, revised 30 September 2004,

accepted 6 October 2004)

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 in rat astrocytes [18] and CaCo-2 cells

[17] Ton-EBP is phosphorylated under hypertonic

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 of TauT in NIH3T3

fibroblasts It is demonstrated that: (a) the saturable taurine

transporter TauT is present in NIH3T3 cells and taurine

uptake is a Na+- and Cl–-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 the TauT expression

level; and (c) TauT does not promote volume-sensitive

taurine release

Materials and methods

Chemicals

Bovine serum albumin (BSA), H2O2 (1.03M), mouse

TNFa, (5 lgÆmL)1 in 1 mgÆmL)1 BSA), N-methyl-D

-glutamine (NMDG), theophylline (40 mM, ddH20),

forsk-olin (50 mM, 96% ethanol), 4b-phorbol 12-myristate

13-acetate (PMA, 40 lM, 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 Inc (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 488 donkey

anti-rabbit IgG, Alexa Fluor 488 mouse anti-chicken IgG and

Alexa Fluor 568 goat anti-rabbit IgG were from

Molecu-lar Probes (Leiden, the Netherlands) [14C]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/Pi) contained 137 mM

NaCl, 2.6 mM KCl, 6.5 mM Na2HPO4, and 1.5 mM

KH2PO4 Iso-osmotic NaCl medium contained 143 mM

NaCl, 5 mM KCl, 1 mMNa2HPO4, 1 mM CaCl2, 0.1 mM

MgSO4, 5 mMglucose and 10 mM N-2-hydroxyethyl

pip-erazine-N¢-2-ethanesulfonic acid (Hepes) Iso-osmotic

NMDGCl solution was similar to NaCl with NMDG

being substituted for sodium Iso-osmotic KCl medium

contained 150 mMKCl, 1.3 mMCaCl2, 0.5 mMMgCl2, and

10 m Hepes Hypo-osmotic NaCl/KCl solution were

prepared by reduction of the NaCl/KCl concentration in the iso-osmotic solutions to 95 mM, with reducing the other components Hypertonic NaCl medium was prepared from isotonic NaCl medium by supplementation with 100 mM sucrose In all solutions, pH was adjusted at 7.40 Unless otherwise noted, experiments were carried out room tem-perature (typically 18–22C)

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) Fibroblasts 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 20 lgÆmL)1 genta-mycin, 3 lgÆmL)1 amphotericin B, 100 lgÆmL)1 strepto-mycin sulfate and 100 IUÆmL)1penicillin Cells were grown

at 37C, 5% CO2 and 95% humidity Cell cultures were passaged every 3–4 days by trypsination (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 cm2 per well) The cells were washed three times by gentle aspiration/addition of 1 mL experimental solution Following the final wash, the cells

in five of the wells were exposed to isotonic NaCl medium containing [14C]taurine (2.54· 1011c.p.m.Æmmol)1, 1.4 lM) 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 given time points (4–20 min) taurine uptake was terminated by removal of the extracellular medium, rapid addition/aspiration of 1 mL ice-cold MgCl2 (100 mM), followed by cell lysis with 500 lL 96% (v/v) ethanol The ethanol was blown off and the cellular [14C]taurine activity extracted by addition of 1 ml ddH2O (1 h), which was transferred to a scintillation vial for estimation of14C activity (b-scintillation counting, Ultima GoldTM) The wells were washed twice with ddH2O The total [14C]taurine (c.p.m.) taken up by the cells in well one to five was in each case estimated as the sum of14C activity in the cell extract and water washouts The taurine uptake (nmolÆg protein)1) at a given time point is calculated from the cellular [14C]taurine activity, the extra cellular specific activity and the protein content The taurine influx (nmolÆ

g protein)1Æmin)1) was estimated as the slope of 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 cm2per well) and loaded for 2 h in 1 ml DMEM containing [14C]taurine (80 nCiÆmL)1) The pre-incubation solution was aspirated and the cells washed

five times with 1 ml isosmotic solution in order to

remove excess extracellular [14C]taurine and 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 mM) The total 14C activity in the

cell system was estimated as the sum of 14C activity

(b-scintillation counting, Ultima GoldTM) in all the efflux

samples, the NaOH lysate plus the two final wash outs

with ddH2O The natural logarithm to the fraction of

14C activity remaining in the fibroblast was plotted vs

time, and the rate constant for the taurine efflux (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 given time point can

be estimated as the product of the rate constant and the

cellular taurine pool In the case where TauT activity was

down-regulated and preloading of the cells was

imposs-ible, the activity of the swelling-induced taurine transport

was followed as influx in isotonic or hypotonic Na+-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, 234-Autoinjector, 155-UV/VIS

detec-tion, BioLab, Aarhus, Denmark) Cells, grown at 80%

confluence (75 cm2 flasks) were washed three times with

NaCl/Pimedium, the medium was aspirated 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 denaturated overnight

after dilution with 200 lL NaOH (2M) and used for

estimation of the protein content using the Lowry

procedure [19] and BSA as protein 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 lM) using a 1 ml per min flow rate and

increasing the acetonitrile fraction in a 12.5 mMphosphate

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 mM) were used for estimation of the taurine

content in the samples The cellular amino acid content

(lmolÆg protein)1) was estimated from the amino acid and

protein content in the flask

Fractionation, SDS/PAGE and Western blotting

NIH3T3 cells grown to 80% confluence in Petri dishes were

washed quickly in ice-cold NaCl/Pi, treated with 100 lL

lysis buffer [150 mM NaCl, 20 mM Hepes, 10% (v/v)

glycerine, 1% (v/v) Triton X-100,1 mM EDTA, 1 mM

NaF and 1 mMNa3VO4] 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) Triton X-100 and

no SDS The cell lysate was then centrifuged at 600 g for

10 min (4C) to give the nuclear fraction (pellet) and the supernatant was centrifuged at 40 000 g for 1 h (4C) to give the membrane fraction (pellet) and the cytosolic fraction (supernatant) The two pellets 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 kit (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 at 4C followed by identification with 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/Pi with 2% (w/v) BSA and incubated with primary antibodies for 2 h at room temper-ature or overnight at 4C: anti-acetylated a-tubulin (1 : 400), rabbit rat TauT (1 : 100) or rabbit anti-human TauT (1 : 100) Igs Cells were washed in NaCl/Pi and incubated with 4,6-diamidino-2-phenylindole (DAPI) (1 : 100), Alexa Fluor 488 donkey anti-rabbit IgG (1 : 200), Alexa Fluor 488 mouse anti-chicken IgG (1 : 600), and/or Alexa Fluor 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 Fluor 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 (5122 pixels) were frame averaged and presented in pseudocolour Digital images were enhanced by Adobe PHOTOSHOP 6.0

Data and statistical analysis The neural network algorithm, NetPhos 2.0 [22], manual homology searches as well as searches in the ELM database

(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 independent experiments Statistical significance

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 low transport capacity [7] The traces in Fig 1

demonstrate that active taurine uptake in NIH3T3 mouse

fibroblasts is linear within the initial 20 min following the

addition of 14C-labelled taurine Using taurine uptake

within the initial 20 min in NaCl medium containing

extracellular taurine (0–50 lM) and fitting the uptake data

to a Michaelis–Menten equation revealed that the Kmvalue

in NIH3T3 cells, i.e the extracellular taurine concentration

required for half maximal taurine uptake, is 18 ± 1 lM

(n¼ 3) It is also recognized that active taurine uptake via

TauT is Na+- and Cl–-dependent in various cell systems and

that only close analogues to taurine (such as b-alanine) are

potential inhibitors of the active uptake [7] The data shown

in Fig 1 and summarized in Table 1 indicate that the initial

taurine uptake is reduced in the presence of b-alanine

(Fig 1A) and following substitution of extracellular K+for

Na+or extracellular NO3 for Cl–(Fig 1B) From Fig 1A

and Table 1 it is also seen that taurine uptake is reduced to

about 75% in the presence of 5 mM creatine Creatine

(a-methylguanido acetic acid) is accumulated by muscle

cells via the active creatine transporter CreaT, which has a

structure similar to that of 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 control, indicated that CreaT is apparently absent in NIH3T3 cells (n¼ 3, data not shown) Creatine 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 affinity towards taurine and 2.5 Na+and 1 Cl–are involved in the uptake

of one taurine

Acute regulation of TauT transport activity

by PKC, PKA and H2O2 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 either to PMA to stimulate PKC, or to forskolin plus theophylline to increase cellular cAMP level and thus presumably stimulate PKA, results in a reduced taurine uptake Exposing the NIH3T3 cells to H2O2 also reduces the taurine uptake (Fig 3) Reactive oxygen species (ROS) exhibits a variety of physiological effects [7] and H2O2, which is highly cell permeable, is reported to elevate the phosphorylation of tyrosine residues, most probably via inhibition of a protein

Fig 1 Substrate specificity and ion requirement of TauT NIH3T3 cells, grown at 80% confluence, were exposed to isotonic NaCl medium containing [ 14 C]taurine (2.54 · 10 11 c.p.m.Æmmol)1, 1.4 l M ) At time points 4–20 min taurine uptake was terminated by removal of the extra-cellular medium and the extra-cellular [14C]taurine was extracted The cellular taurine uptake (nmolÆg protein)1) at a given time point was calculated from the cellular [14C]taurine activity, the extra cellular specific activity and the protein content (A) Taurine uptake was followed in the absence (control cells) and in the presence of 5 m M creatine or 5 m M b-alanine (B) Taurine uptake was followed in control cells exposed to NaCl medium and in cells incubated with Na+-free KCl medium or Cl–free NaNO 3 medium The curves in A and B are all representative of three independent sets of experiments.

tyrosine phosphatase [26] Thus, these data could indicate

that an increased phosphorylation of TauT or a putative

regulator of TauT could be involved in the H2O2-induced

reduction in the active taurine uptake in NIH3T3 cells

Expression and subcellular localization of TauT

The promoter region of the TauT gene in human [10] and

rat [11] contains consensus binding sites for the transcription

factors p53 and NF-jB The cellular expression of p53 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 (20 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 the confocal visualization studies in Fig 5B it is seen that TauT (green colour) appears throughout the nuclear compartment It is emphasized that the pinhole size was kept small enough to exclude that staining observed in these compartments is caused by out-of-focus fluorescence from transporters localized, e.g to the plasma membrane

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 this 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 major 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

1 mMor 100 mMexogenous taurine, and increased

follow-Fig 2 Na+/Cl–/taurine stoichiometry for taurine uptake via TauT NIH3T3 cells, grown at 80% confluence, were exposed to isotonic medium containing [14C]taurine (2.54 · 10 11

c.p.m.Æmmol)1, 1.4 l M ) for 20 min Taurine uptake was terminated and the cellular [14C]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 was 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 for half maximal uptake and 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–, and K Cl is the Cl–concentration required for half maximal uptake The stoichiometry values indicated on the figure were estimated for Na+and Cl–in five and three sets of experiments, respectively.

Table 1 TauT substrate specificity and ion dependency Taurine uptake

was estimated as indicated in Fig 1 in the absence or presence of 5 m M

creatine/b-alanine and in Na + -free NMDG-medium, KCl medium or

Cl–free NaNO 3 medium The absolute taurine influx (nmolÆg

pro-tein)1Æmin)1) was estimated by linear regression, using 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

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

ing exposure to DMEM supplemented with 100 mM

sucrose Similar data where obtained using the antibody

raised against the N-terminal sequence of TauT, 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 mMtaurine is not secondary to

cell shrinkage induced by the hypertonic conditions, as

evidenced by the effect of addition of 100 mMsucrose 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 mM taurine for 24 h normal transport capacity is gradually regained and has returned to control values within 24 h It is noted that TauT activity in cells exposed to DMEM supplemented with 100 mM 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 antibody 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 sucrose (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 panels, 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 time-dependent effect of sucrose

on the level of TauT expression in cells pre-exposed to high concentrations of taurine for 24 h It is seen that the level of TauT increases at about 4 h after sucrose was substituted 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 was used as a control to ensure that nuclear and membrane fractions were not contaminated with cytosolic TauT The cell fractionation experiment presented

in Fig 8 confirms the presence of a major 67 kDa TauT protein in whole NIH3T3 fibroblasts, and that this protein

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 mMextracellular taurine compared to cells exposed to

100 mM 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 supplemen-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 NIH3T3 cells were grown in DMEM

(control), DMEM containing TNFa (20 ngÆmL)1, 16 h) or serum-free

DMEM (24 h, serum-starved) before initiation of the influx

experi-ment Taurine uptake was followed with time using [14C]taurine as

indicated in Fig 1 The influx, estimated from the slope of the uptake

curves, was estimated and in each case given relative to the influx in

control cells ± SEM (n ¼ 3) #, Significantly different from the 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 time in isotonic NaCl medium using [ 14 C]taurine as

indi-cated in Fig 1 PMA (50 n M ), forskolin (10 l M ) plus theophylline

(0.5 m M ), and H 2 O 2 (2 m M ) were included in the experimental medium

from the time of the initiation of the influx experiment The influx was

estimated from the slope of the uptake curves and in each case given

relative to the influx in control cells ± SEM (n ¼ 3) #, Significantly

different from the control (P < 0.05).

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 further observed that TauT in sucrose-treated cells

strongly localizes in a punctuate pattern along the cytosolic

network of acetylated microtubules (Fig 9), indicating that

localization and functional targeting of TauT to subcellular

domains, such as 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 regulation of TauT transport

activity is paralleled by a similar up/down regulation of the

activity of the volume sensitive 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)1for 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 estimated

at 0.0020 ± 0.00005 min)1 (control cells) and 0.0014 ± 0.00005 min)1(20 ng TNFaÆmL)1, 16 h; P¼ 0.005) The cellular taurine content was, in three sets of separate experiments, estimated at 0.031 ± 0.003 and 0.025 ± 0.003 lmolÆg protein)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 rate 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 ngÆmL)1, 16 h, 2nd row) Cells were fixed in paraformaldehyde (4%, 15 min) and permeabilized with Triton X-100 (0.2%, 10 min) The nucleus (blue) was visualized with DAPI The 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 three images from separate experiments (B) Cells were incubated in isotonic medium under control conditions, fixed, and TauT and nuclei were labelled as described in Materials and methods Cells were viewed 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 Fluor 488 using the 364 nm UV line and the 488 nm argon/krypton laser-line, respectively Emission wavelengths, 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 at a 0.75 Airy disc pinhole, and an optical slice thickness of 0.25 lm, frame averaged and presented in pseudocolour Images shown with are optical slices taken at 0.75 lm intervals, moving towards the bottom of the cell from left to right The experiment shown is representative of three independent experiments.

that 16 h exposure 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 volume-sensitive taurine leak pathway in NIH3T3 cells

Serum starvation increased the maximal rate constant for

the swelling-induced taurine influx in hypotonic media (200

mOsm) by 20 ± 7% (n¼ 4)

Figure 10C shows that an 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 [14C]taurine preloading of the cells

required for the standard efflux procedure Exposing

NIH3T3 cells, preincubated for 24 h with DMEM medium

containing 100 mMsucrose or 100 mMtaurine to hypotonic

KCl medium, resulted in an increase in the taurine uptake

which is similar or slightly larger than the influx seen in

NIH3T3 cells pre-exposed to DMEM alone (Fig 10D)

The latter 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 release seems to not be affected by

long-term hypertonic exposure or to long-term exposure to high extracellular taurine concentrations

Discussion The organic osmolyte, taurine, is present in high concen-trations in heart- and skeletal muscles, 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 that regulates cell volume, cellular free Ca2+concentration, cellular oxidative status and interferes with 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 no detectable effect on the expression 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

1 m 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 the C-terminus of TauT and a secondary, alkaline, phosphatase conjugated antibody The bands represent a 67 kDa protein The blot is representative three sets 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 taurine uptake was followed with time using [ 14 C]taurine as outlined in Fig 1 In order to preserve 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 NaCl medium supplemented with 100 m M sucrose for cells treated with 100 m M taurine or 100 m M sucrose The influx, estimated from the slope of the uptake curves, is in each 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 another 2, 4, 8, 12, 24 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, respectively Taurine uptake was followed with time (4.20 min) in isotonic NaCl medium (control) or standard NaCl medium supplemented 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 sucrose and for 48 h sucrose treatment are given relative to the isotonic control ± SEM, representing five and three independent sets of experiments Values for cells incubated for 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 of experiments #, Significantly different from control (P < 0.05).

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 channel 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 transport in NIH3T3 is reduced following exposure

to H2O2and 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 exposure to H2O2increases taurine release from NIH3T3 cells following hypotonic incubation and it was suggested that this effect reflected an inhibition of a protein tyrosine phosphatase [20] Several of the serines, threonines and tyrosines 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 more tyrosine phosphorylated state in 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 DMEM (control cells, 1st row), DMEM plus 100 m M taurine (24 h, 2nd row) or DMEM supplemented with 100 m M sucrose (24 h, 3rd row) as indicated in Fig 6A,B Cells were fixed in paraformaldehyde (4%, 15 min) and permeabilized with Triton X-100 (0.2%, 10 min) The nucleus (blue), the microtubules (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) and the 2nd column (TauT) Each image is representative 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 sucrose for the time period indicated (Frame 2… 12 h) Cells were fixed and TauT (red) detected as indicated above Images are representative of two sets of experiments.

increased serine/threonine phosphorylation of TauT

redu-ces the active taurine uptake following exposure to H2O2

Kang and coworkers [12] found that exposing the

blood-barrier TR)BBB13 cells to TNFa (20 ngÆmL)1) resulted in

an increased mRNA level of the TauT and a concomitant

1.7-fold increase in taurine uptake The promoter to TauT

contains a binding site for the transcription factor NF-jB

and activation of NF-jB involves phosphorylation of the

inhibitory complex IjB, dissociation of the heterodimer

NF-jB (p50, RelA) from IjB, translocation of NF-jB to

the nucleus and subsequent initiation of gene expression

HOCl which is a highly reactive oxidant species generated

from H2O2and Cl–, is converted to the less reactive TauCl

by combination with taurine It has been proposed recently

by Miyamoto and coworkers [5] that TauCl is involved in

oxidation of IjB which prevents phosphorylation of IjB

and activation of NF-jB Thus, the effect of longterm

exposure to TNFa, and most probably to H2O2, on taurine uptake could involve modulation of the NF-jB mediated 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 it is seen that exposure to growth medium supplemented with 100 mM taurine reduces the expression as well as transport activity of TauT in NIH3T3

Fig 8 Nuclear localization of TauT Cells, grown in DMEM supplemented with 100 m M taurine or 100 m M sucrose for 24 h were lyzed, sonicated, fractionated and proteins from the different fractions were separated by SDS/PAGE (10%) TauT was visualized 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 cytosolic 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 Microtubule association of TauT (A) NIH3T3 cells, grown on cover slips at 60–70% confluence, were washed, fixed, permeabilized and preceded for detection of TauT and cytosolic micrtubules as indicated in Fig 7 TauT was 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), and enlargement of the framed section from C (D) with the microtubules and TauT images slightly shifted in order to facilitate visualization of the TauT/microtubules colocalization.