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

Archives of Biochemistry and Biophysics 560 (2014) 59–72

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi

Expression of organic osmolyte transporters in cultured rat astrocytes and rat and human cerebral cortex Jessica Oenarto a, Boris Görg a, Michael Moos a, Hans-Jürgen Bidmon b, Dieter Häussinger a,⇑ a b

Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine University, Düsseldorf, Germany C. & O. Vogt Institute for Brain Research, Heinrich Heine University, Düsseldorf, Germany

a r t i c l e

i n f o

Article history: Received 24 April 2014 and in revised form 18 June 2014 Available online 5 July 2014 Keywords: Osmolyte transporter Hepatic encephalopathy Astrocytes Ammonia

a b s t r a c t This study characterizes the expression of the osmolyte transporters betaine/c-amino-n-butyric acid (GABA) transporter (BGT-1), the taurine transporter (TauT) and the sodium-dependent myo-inositol transporter (SMIT) in various rat brain cells in culture and in rat and human cerebral cortex in situ. Osmolyte transporter expression greatly differed between cultured brain cells with highest mRNA expression levels for SMIT in astrocytes and TauT in neurons. BGT-1 mRNA and protein were expressed in microglia but not in astrocytes and neurons. In rat and human cerebral cortex, SMIT was expressed in astrocytes and TauT was found in neurons. Osmolyte transporter expression was subject to regulation by factors relevant for hepatic encephalopathy (HE). Hypoosmolarity, NH4Cl (0.5–5 mmol/l), diazepam (10 lmol/l) and TNFa (10 ng/ml) time-dependently decreased mRNA expression of SMIT and/or TauT in cultured astrocytes. NH4Cl-induced SMIT/TauT mRNA expression changes were sensitive to inhibitors of glutamine synthetase and NADPH oxidase. In rat cerebral cortex, SMIT mRNA expression decreased after portal vein ligation or ammonium acetate injection probably due to astrocyte swelling in these HE animal models. It is concluded that osmolyte transporters are heterogeneously expressed in brain and are subject to regulation by HE-relevant factors. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Due to its encapsulation into rigid bone and the necessity to maintain the size of extracellular functional compartments such as the synaptic cleft, the brain requires potent mechanisms for cell volume regulation. These mechanisms comprise transport systems which facilitate the release or uptake of osmotically active compounds which are present at millimolar concentrations inside cells such as ions or organic osmolytes. During transient episodes of osmotic stress, cell volume homeostasis is maintained primarily through activation of ion transport systems [1], whereas prolonged osmotic challenges lead to adaptations of the expression of organic osmolyte transporters such as the myo-inositol transporter (SMIT),1 ⇑ Corresponding author. Address: Universitätsklinikum Düsseldorf, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-40225 Düsseldorf, Germany. Fax: +49 211 811 8838. E-mail address: [email protected] (D. Häussinger). 1 Abbreviations used: BGT-1, betaine/GABA transporter-1; GABA, gamma amino butyric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; HE, hepatic encephalopathy; HPRT, hypoxanthine phosphoribosyltransferase; IB4, isolectin B4; MAP2, microtubule associated protein 2; MSO, methionine sulfoximine; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; NF-jB, nuclear factor kappa B; NKCC1, sodium-dependent potassium chloride co-transporter 1; PA, parvalbumin; PTN, protein tyrosine nitration; PVL, portal vein ligation; RNOS, reactive nitrogen and oxygen species; SMIT, sodium-dependent myo-inositol co-transporter; TauT, taurine transporter; TNFa, tumor necrosis factor alpha. http://dx.doi.org/10.1016/j.abb.2014.06.024 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

the taurine transporter (TauT) or the betaine/GABA transporter (BGT1). Organic osmolytes not only serve to maintain cell volume, but can also act as antioxidants and molecular chaperones in order to counteract proteotoxicity [2,3]. Furthermore, small changes of cell hydration, as induced by hormones, oxidative stress or cumulative substrate uptake are a physiological signal for regulation of cell function (for reviews see [4–6]). Hepatic encephalopathy (HE) is a neuropsychiatric disorder frequently accompanying acute or chronic liver failure. Symptoms of HE comprise a wide spectrum of neuropsychiatric abnormalities, such as impaired cognitive functions and motor disturbances. In liver cirrhosis, HE represents the clinical manifestations of a low-grade cerebral edema [7–9] and an accompanying oxidative/nitrosative stress response [10,11]. Astrocyte swelling is central in the pathogenesis of HE in patients with liver cirrhosis and ammonia detoxification by glutamine synthetase in astrocytes is accompanied by glutamine accumulation and a compensatory depletion of other organic osmolytes such as myo-inositol [7]. This exhausts the volume regulatory capacity and renders the astrocyte volume susceptible towards further osmotic challenges which are introduced by heterogeneous precipitating factors such as benzodiazepines, hyponatremia or inflammatory cytokines [7,8]. In line with this, hyponatremia in patients with liver cirrhosis and hepatic encephalopathy increases morbidity and mortality [12].

60

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

HE-precipitating factors such as ammonia, benzodiazepines, hypoosmolarity and inflammatory cytokines also induce astrocyte swelling in vitro [13–15]. Whereas osmotic stress induces oxidative/nitrosative stress in astrocytes, reactive oxygen and nitrogen species (RNOS) in turn promote cell swelling [13] thereby triggering a self-amplifying cycle between osmotic and oxidative stress [16], which results in posttranslational modifications of proteins [17], RNA oxidation [18] and alterations of gene expression [19– 21]. These phenomena are thought to trigger alterations of synaptic plasticity and disturbances of oscillatory networks, which finally account for HE symptoms [22]. Given the central role of astrocyte swelling in the pathogenesis of HE, we studied the expression of the osmolyte transporters SMIT, TauT and BGT-1 and the osmolyte taurine in different rat brain cells in vitro and rat brain in vivo and the expression of SMIT in human cerebral cortex. The data show that osmolyte transporter expression differs amongst various brain cells and is affected by HE-precipitating factors.

Purity of the astrocyte culture was determined by immunofluorescence analysis of glial fibrillary acidic protein (GFAP). Preparation and culture of rat brain microglia Cells were prepared from cerebral hemispheres of newborn male Wistar rats (P1–P3) as described recently [24]. Isolated cortices were trypsinized, triturated and plated onto tissue culture flasks in DMEM (Gibco, Invitrogen, Carlsbad, CA) containing 20% heat-inactivated FCS. Mixed glial cultures were maintained for 14 days before microglia were removed by rotation on a shaker at 200 rpm for 12 h at 37 °C. Detached microglia were plated on plastic dishes (Greiner, Solingen, Germany) and allowed to adhere for 30 min at 37 °C and 10% CO2 before removing the unbound cells by repetitive changing of the culture medium. Viability of isolated cells was routinely checked and always >98% as assessed by trypan blue exclusion. Preparation of rat brain neurons

Materials and methods Materials L-Methionine-sulfoximine (MSO), phalloidin-FITC, isolectin B4FITC and diazepam were from Sigma-Aldrich (Deisenhofen, Germany). NH4Cl and CH3NH3Cl were from Merck (Bad Soden, Germany). Tumor-necrosis factor alpha (TNFa) was from Roche (Mannheim, Germany). Cell culture media and Hoechst 34580 were from Gibco Life Technologies (Invitrogen, Karlsruhe, Germany), fetal calf serum was from PAA Laboratories GmbH (Linz, Austria). Detailed information on primary and secondary antibodies used in the present study is provided in Tables 1 and 2.

Preparation of rat brain astrocytes Primary astrocytes were prepared from cerebral hemispheres of newborn Wistar rats and cultured for 6–8 weeks as described [23].

Primary neocortical neurons were prepared from Wistar rats on embryonic day 15 (E15) and cultured on 60 mm tissue culture dishes coated with (1 mg/ml) poly-D-lysine and (13 lg/ml) laminin at a density of 6  106 cells [25]. Preparation of rat brain fibroblasts Primary meningeal fibroblasts were prepared from newborn Wistar rats according to a protocol established by [26]. Portal vein ligation and acute ammonium acetate treatment of rats For portal vein ligation a surgical procedure established by Chojkier et al. [27] was employed. In brief, the abdomen of anesthetized and ventilated rats (160 g) was opened by a midline incision followed by intraperitoneal injection of heparin (125 IE) and the portal vein was extracorporally displaced from the intestine.

Table 1 Characteristics of primary antibodies used in the present study. Target

Company

Host

Isotype

Clonality

Reactivity

Dilution

Form

SMIT TauT TauT BGT-1 Taurine GFAP GS GS MAP2 SMI311 PA GFAP

Santa Cruz (sc-23142) a Diagnostics (Tau11-A) Santa Cruz (sc-47450) a Diagnostics (BGT11-A) Abcam (ab9448) Millipore (AB5541) Millipore (610517) Sigma (G2781) Novus Biologicals (NB300-213) Covance (SMI-311R) Sigma (P3088) Sigma (G3893)

g r g r r c m r c m m m

IgG IgG IgG IgG IgG IgY IgG2a IgG IgY IgG1,IgM IgG1 IgG1

pAb pAb pAb pAb pAb pAb mAb pAb pAb mAb mAb mAb

r, r h r r r, r, r, r, r, r, r,

1:50 (h) 1:100 (r) 1:200 1:25 1:200 1:50 1:200 1:200 1:200 1:200 1:200 1:1000 1:200

ap ap ap ap pure IgG pure IgY ap pure IgG (NH4)2S2O8 precipitation af af af

h

h h h h h h h

g: goat; r: rabbit; c: chicken; m: mouse; h: human; ap: affinity purified; af: ascites fluid; Ig: immunoglobulin; mAb: monoclonal antibody; PA: parvalbumin; pAb: polyclonal antibody.

Table 2 Characteristics of secondary antibodies used in the present study.

1

Host

Reactivity

Company

Isotype

Specificity

Min. react.1

Conjugate

Dilution

d g d d d

g r r m c

Dianova Dianova Dianova Dianova Dianova

IgG IgG IgG IgG IgG

IgG(H + L) IgG(H + L) IgG(H + L) IgG(H + L) IgY(H + L)

h, h, h, h h,

Cy3 Cy3 FITC FITC FITC

1:200 1:200 1:200 1:200 1:200

(705-165-147) (111-165-144) (711-095-152) (715-096-150) (703-096-155)

ra, m ra ra, m ra

Minimal cross-reactivity with the indicated species due to immunoglobulin pre-absorbtion. d: donkey; g: goat; r: rabbit; c: chicken; m: mouse; h: human; ra: rat; Ig: immunoglobulin; FITC: fluorescein-isothiocyanate; Cy3: cyanine 3; H + L: heavy and light chain.

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

61

Fig. 1. Osmolyte transporter mRNA expression in cultured rat brain cells in vitro. Total RNA was extracted from lysates of cultured rat astrocytes, microglia, neurons or fibroblasts and mRNA expression level of BGT-1, TauT or SMIT were analyzed using real time-PCR. Relative osmolyte transporter mRNA expression levels are given in relation to HPRT expression and termed ‘‘relative mRNA units’’ (RRU) (n = 3–5).

In the sham-operated animal group the portal vein was transiently pinched off for 60 s. For portal vein ligation a sterile 2–0 silk fiber was placed around the portal vein and tied to a 20-gage obturator (Braun, Melsungen, Germany) which was placed along the vein. A defined stenosis of the portal vein was obtained after carefully removing the obturator. After intraperitoneal application of carprofen (1 mg/kg BW, Rimadyl, Pfitzer, Berlin, Germany) the abdomen was occluded using a 2–0 vicryl silk (Ethicon, Johnson & Johnson, Norderstedt, Germany). 16 days after surgery rats were anesthetized (pentobarbital, 50 mg/kg) and transcardially perfused with 100 mL physiological saline containing heparin (10,000 iU/L, Liquemin, Hoffmann La Roche, Basel, Switzerland) before the cerebral cortex was dissected from the brain and used for RNA analysis. Acute ammonium acetate (NH4Ac) intoxication was performed in rats (220–250 g) by intraperitoneal injection of 4.5 mmol/kg body mass ammonia acetate solved in saline (0.9%, sterile, for injection). As for control animals received vehicle (0.9% NaCl) only. 6 h after injection animals were anesthetized (pentobarbital, 50 mg/kg) and transcardially perfused with physiological saline containing heparin (10,000 iU/L, Liquemin, Hoffmann La Roche, Basel, Switzerland). The cerebral cortex was dissected from the brain and taken for RNA extraction as described above. All experiments were approved by the National Animal Welfare Legislation.

Foster City, USA). Each gene in each sample was analyzed in duplicate and mean values of cycle numbers for the target amplification were subtracted from the mean of cycle numbers of the housekeeping genes hypoxanthine phosphoribosyltransferase (HPRT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the respective sample and taken to the power of 2. This value represents the mRNA expression of the target gene in relation to HPRT/GAPDH expression and is termed ‘‘relative mRNA unit’’ (RRU). Long term treatment (>24 h) with NH4Cl reduced HPRT mRNA expression levels but had no effect on GAPDH mRNA expression in cultured microglia. Therefore GAPDH was used as a housekeeping gene. The following sequences were used for PCR primer design. BGT1 for: 50 -GCC TTC TTC ATC CCC TAC TTC-30 ; BGT-1 rev: 50 -CCT CCA AGC AGT AAC ACT CC-30 ; SMIT for: 50 -TGA TCT ACA CAG ACA CTC TCC-30 ; SMIT rev: 50 -CTT AAC TTC CTC AAA CCC TCC-30 ; TauT for 50 GAG GTC ATC ATA GGC CAG TAC AC-30 ; TauT rev 50 -TTG CGC TCC CAG AAC TCG ATC A-30 ; HPRT for 50 -TGC TCG AGA TGT CAT GAA GGA-30 ; HPRT rev 50 -CAG AGG GCC ACA ATG TGA TG-30 . GAPDH for 50 -TGC ACC ACC AAC TGC TTA GC-30 ; GAPDH rev 50 -TGG TCA TGA GCC CTT CCA C-30 . Sequences for TauT real time-PCR were derived from Nishimura et al. [28].

Immunofluorescence analysis Post mortem human brain tissue Post mortem human cerebrocortical brain tissue was obtained from autopsy of a 77 year old male patient without liver and neurological disease. A written consent for tissue analysis for scientific purposes was included in the body donor program of the Department of Anatomy of the University of Düsseldorf, Germany. Analysis of TauT, SMIT and BGT-1 mRNA expression Total RNA was isolated from cultured rat astrocytes, microglia, neurons or fibroblasts using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturers´ guidelines. Purified RNA was quantified using the NanoDrop 1000 System (Thermo Scientific, Wilmington, USA). First strand cDNA was synthesized from RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) which includes DNAse-dependent digestion of DNA. Gene expression levels were quantified by real-time SYBR Green PCR on a 7500 real-time PCR system (Applied Biosystems,

Immunofluorescence analysis was performed by epifluorescence or confocal laser scanning microscopy employing the Cell Observer Z1 (Carl Zeiss AG, Oberkochen, Germany) or LSM510META (Carl Zeiss AG, Oberkochen, Germany), respectively. Cultured rat brain cells were seeded on IbidiÒ (Ibidi, Martinsried, Germany) or MaTekÒ dishes (MatTek Corporation, Ashland, USA). Cells were washed twice using ice cold PBS before fixation with paraformaldehyde (4%) for 10 min at RT. Thereafter, cells were extensively washed with ice-cold PBS and incubated in PBS containing 10% BSA for 15 min. Immunostaining was performed for 2 h at RT using primary antibodies as indicated. Cells were washed extensively and incubated again for 2 h with fluorochrome-conjugated secondary antibodies at room temperature and washed as described previously. Cell nuclei were counterstained using Hoechst 34580 (Invitrogen, Darmstadt, Germany). For immunofluorescence analysis of rat and human cerebral cortex, tissue was fixed with Zamboni-fixative or paraformaldehyde, respectively as described [17].

62

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

Fig. 2. Osmolyte transporter expression and taurine immunoreactivity in cultured rat brain cells. Detection of TauT, SMIT, BGT-1 and taurine in cultured rat astrocytes (A), neurons (B), microglia (C) or fibroblasts (D) by immunofluorescence analysis using confocal laser scanning microscopy. Astrocytes, neurons, microglia and fibroblasts were identified by immunostaining of GFAP, MAP2, isolectin-B4 (IB4) and phalloidin, respectively. Nuclei were counterstained using Hoechst 34580. One representative immunofluorescence analysis out of 3 independent experiments is shown.

For immunofluorescence staining of TauT in human cerebral cortex, tissue was incubated with primary anti-TauT antibodies for 120 h at 4 °C. Following immunofluorescence staining, autofluorescence of human brain tissue was eliminated using Autofluorescence Eliminator Reagent (Merck Chemicals, Schwalbach, Germany) according to the manufacturers´ instructions. Acquired z-Stacks were used for 3-dimensional reconstruction using Volocity Software (Improvision, Perkin Elmer, Rodgau, Germany). Analysis of results Each experiment was carried out with at least three independent cell preparations. Results from n-independent experiments are expressed as means ± standard error of the mean (SEM). For each experimental treatment and time point analyzed, a separate control experiment was carried out. Statistical analysis was performed using two-sided Student’s t-test or one way analysis of variance (ANOVA) followed by Dunett’s multiple comparison post hoc tests were appropriate. A p-value 60.05 was considered statistically significant.

Results Cell type-specific osmolyte transporter expression in cultured rat brain cells Osmolyte transporter expression was analyzed in cultures of different rat brain cell types using real time-PCR or immunofluorescence analysis. When compared to cultured cerebrocortical neurons, microglia or fibroblasts, sodium-dependent myo-inositol co-transporter (SMIT) mRNA expression levels were strongest in cultured astrocytes, whereas taurine transporter (TauT) mRNA levels where highest in cultured neurons. Whereas considerable amounts of TauT mRNA were present in cultured astrocytes, microglia or fibroblasts, betaine/GABA transporter 1 (BGT-1) mRNA expression was virtually absent in all examined cultured cell types with the exception of microglia (Fig. 1). SMIT protein was detected by immunofluorescence analysis in cultured astrocytes, neurons and microglia but not in cultured fibroblasts, whereas anti-TauT immunoreactivity was detected in

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

63

Fig. 2 (continued)

all cell types examined (Fig. 2A–D). However, cellular localization of TauT greatly differed in astrocytes and fibroblasts when compared to neurons and microglia. Whereas in microglia TauT was predominantly localized in the plasma membrane, astrocytes, neurons and fibroblasts showed a more diffuse staining pattern throughout the entire cell. In astrocytes, strong anti-TauT immunoreactivity was also detected in vicinity to the nucleus (Fig. 2A). In line with the expression of TauT, immunoreactive taurine was found in astrocytes, microglia and neurons, but was almost undetectable in cultured fibroblasts. Anti-taurine immunoreactivity was validated in astrocytes exposed to hypoosmotic culture media (205 mosmol/l, 10 min), which induces taurine release from astrocytes [29]. Hypoosmolarity strongly reduced intracellular antitaurine immunoreactivity as compared to astrocytes incubated in iso-osmotic media (320 mosmol/l) (data not shown). Anti-BGT-1 immunoreactivity was only found in cultured microglia in close vicinity to the plasma membrane (Fig. 2C), but was absent in astrocytes, neurons or fibroblasts (Fig. 2A, B, and D). The results show cell type-specific osmolyte transporter expression and a strong correlation between mRNA and protein expression levels in cultured rat brain cells, which may suggest that osmolyte transporters are specifically detected by immunofluorescence.

Effects of HE-relevant factors on osmolyte transporter mRNA expression in cultured astrocytes Astrocyte swelling as induced by hypoosmotic media activates the release of taurine from cultured astrocytes [29] and decreases the intracellular concentrations of taurine and myo-inositol [30,31]. As shown in the present study, hypoosmolarity (205 mosmol/l) also time-dependently decreased mRNA expression of TauT and SMIT within 6 h of treatment by about 50% (Fig. 3A). Astrocyte shrinkage as induced by hyperosmotic exposure increases intracellular taurine- and myo-inositol concentrations [32,33] and upregulates TauT and SMIT mRNA as shown by Northern-blot or RT-PCR [34,33]. This was confirmed in the present study by means of quantitative real time-PCR, which showed upregulation of SMIT- and TauT mRNA in cultured astrocytes, which was maximal after about 9 h of hyperosmotic exposure (Fig. 3B). Exposure of cultured rat astrocytes to NH4Cl decreases the intracellular myo-inositol content and activates the release of taurine [35,36]. As shown in Fig. 3C, NH4Cl (5 mmol/l) treatment decreased SMIT and TauT mRNA expression in a time-dependent manner. A significant downregulation of TauT and SMIT mRNA was first observed 48 h and 72 h after NH4Cl (5 mmol/l)-treatment, respectively (Fig. 3C). Inhibition of glutamine synthetase by

64

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

Fig. 3. Effects of HE-relevant factors on osmolyte transporter expression in cultured rat astrocytes. Astrocytes were either treated with (A) hypoosmotic- (205 mosmol/l) or (B) hyperosmotic- (405 mosmol/l) media, (C) NH4Cl (5 mmol/l), (E) CH3NH3Cl (5 mmol/l), (F) TNFa (10 ng/ml) or (G) diazepam (10 lmol/l) for the time periods indicated. For control, cells were either left untreated or were treated with DMSO for the respective time period. mRNA expression levels of TauT and SMIT were quantified by real time-PCR. (D) Pharmacological characterization of NH4Cl (5 mmol/l, 72 h)-mediated downregulation of SMIT and TauT mRNA expression. Astrocytes were either left untreated or treated with NH4Cl (5 mmol/l) for 72 h in the absence or presence of L-methionine-sulfoximine (MSO, 3 mmol/l, 30 min pretreatment), apocynin (300 lmol/l, 30 min pretreatment) or L-NAME (1 mmol/l, 30 min pre-treatment). SMIT and TauT mRNA expression levels in NH4Cl-treated astrocytes are given relative to the respective control (untreated, apocynin or L-NAME, respectively). ⁄: statistically significantly different as compared to the respected control condition (p < 0.05, n = 3–13). n.s.: not statistically significantly different as compared to the respective control condition.

L-methionine

sulfoximine (MSO, 3 mmol/l) prevented the NH4Cl (5 mmol/l, 72 h)-induced downregulation of TauT and SMIT mRNA levels (Fig. 3D) suggesting a regulatory role of glutamine-induced astrocyte swelling on TauT and SMIT mRNA expression. Downregulation of SMIT and TauT mRNA levels in NH4Cl

(5 mmol/l, 72 h)-treated astrocytes was completely blunted by apocynin (300 lmol/l), an inhibitor of NADPH-oxidase, whereas the nitric oxide synthase inhibitor L-NAME (1 mmol/l) was ineffective (Fig. 3D). These results suggest a role of NADPH-oxidase derived reactive oxygen species (ROS), but not of nitric oxide in

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

65

Fig. 3 (continued)

the ammonia-induced downregulation of SMIT and TauT mRNA in cultured astrocytes. CH3NH3Cl (5 mmol/l), which affects intracellular pH similar to NH4Cl [37], significantly increased osmolyte transporter mRNA expression in cultured astrocytes up to 48 h after treatment (Fig. 3E). An increased taurine uptake was shown in TNFa (20 ng/ml)treated astrocytes [38]. As shown in the present study, TNFa (10 ng/ml) increased TauT mRNA expression, however without reaching statistical significance (Fig. 3F). As opposed to TauT, SMIT mRNA expression was rapidly downregulated by TNFa within 9 h by about 65% (Fig. 3F).

Diazepam (10 lmol/l)-treatment for up to 72 h did not significantly alter SMIT mRNA expression in astrocytes. However, TauT mRNA expression levels decreased in a biphasic manner in response to diazepam. A first decrease to about 40% of vehicle-treated (DMSO) controls was noted after 6 h, with transient recovery to control levels after 24 h followed by a 50%-decrease after 72 h (Fig. 3G). Osmolyte transporter mRNA expression changes as observed in astrocytes treated with NH4Cl (5 mmol/l), CH3NH3Cl (5 mmol/l), TNFa (10 ng/ml), DMSO (1:1000), diazepam (10 lmol/l), hypoosmotic (205 mosmol/l)- or hyperosmotic (405 mosmol/l)-cell

66

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

Fig. 4. Osmolyte transporter expression and taurine immunoreactivity in rat liver and brain. (A) Quantification of osmolyte transporter mRNA expression levels in rat brain and liver by real time-PCR. mRNA expression levels of SMIT, TauT and BGT-1 are given relative to HPRT expression levels and termed RRU (RRU: relative mRNA units) (n = 5– 8). (B-F) Detection of osmolyte transporters TauT, SMIT, BGT-1 or taurine in rat brain by immunofluorescence staining and confocal laser scanning microscopy. Astrocytes were counterstained using antibodies against glutamine synthetase (GS) or glial fibrillary acidic protein (GFAP), neurons were co-stained with microtubule associated protein 2 (MAP2) or pan-neuronal neurofilament marker (SMI311) and interneurons were co-stained with parvalbumin.

culture media were not accompanied by increased astrocyte apoptosis as shown by TUNEL-labeling (Suppl. Fig. 1). Osmolyte transporter expression in rat brain in vivo As detected by real time-PCR, strong SMIT and TauT but only weak BGT-1 mRNA expression was found in rat cerebral cortex (Fig. 4A). As shown in Fig. 4B and C, TauT protein expression and taurine immunoreactivity in rat cerebral cortex were mainly confined to neuronal somata, but were also present in MAP2 positive dendrites. Whereas most astrocytes stained negative for TauT, weak TauT immunoreactivity was detected only occasionally in astrocytes close to the pial surface in the vicinity to blood vessels

(Fig. 4B and C). Likewise, strong immunoreactivity against taurine was present in only few protoplasmic subpial astrocytes (Fig. 4C). Strong SMIT immunoreactivity was found in GFAP- and GSexpressing astrocytes (Fig. 4D and E), but not in SMI311 positive neurons or parvalbumin positive interneurons (Fig. 4F) in rat cerebral cortex. Intensity of anti-SMIT immunoreactivity in SMI311 expressing neurons was similar to the surrounding environment (Fig. 4F). Betaine/GABA transporter 1 (BGT-1) was strongly expressed at the meningeal surface of the cerebral cortex which is constituted by endothelial cells and fibroblasts, but was only weakly detected in neuronal somata and MAP2 positive dendrites (Fig. 4G). No BGT1 immunofluorescence above background staining intensity was

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

67

Fig. 4 (continued)

detected in glutamine-synthetase expressing astrocytes in rat cerebral cortex (Fig. 4G). The results show a cell type-specific osmolyte transporter expression pattern in rat cerebral cortex. Effect of acute ammonia intoxication and portal vein ligation on osmolyte transporter mRNA expression in rat brain in vivo

kg BW, 6 h), but were not affected in rats with portal vein ligation. No significant expression level changes were noted for the housekeeping gene HPRT1. The results indicate that osmolyte transporter expression changes in animal models for acute and chronic liver failure and hepatic encephalopathy. Osmolyte transporter expression in human cerebral cortex in vivo

As shown in Fig. 5, both, acute ammonium acetate intoxication (4.5 mmol/kg BW, 6 h), as well as portal vein ligation (16 days) significantly decreased SMIT but not TauT mRNA expression levels in rat cerebral cortex. BGT-1 mRNA expression levels decreased in cerebral cortex after ammonium acetate-intoxication (4.5 mmol/

As shown by immunofluorescence analysis, SMIT is expressed in glutamine-synthetase-positive astrocytes in human cerebral cortex (Fig. 6A/B). In contrast to astrocytes (Fig. 6C), TauT immunoreactivity was clearly present in SMI311 positive neurons (Fig. 6D).

68

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

Fig. 4 (continued)

Discussion The present study identified cell type-specific osmolyte transporter expression patterns in rat brain indicating that the different brain cell types pursue different osmolyte strategies to cope with osmotic stress and proteotoxicity. The reason why different cells preferentially use different organic osmolytes is unclear, however, recent data suggest that organic osmolytes differ in their proteinstabilizing characteristics [39]. In astrocytes, an important role for myo-inositol is indicated by high expression levels of the myo-inositol transporter SMIT (Figs. 1, 2A and 4D/E). SMIT was expressed in astrocytes in human brain

(Fig. 6A/B) and both, hypoosmolarity- and ammonia-mediated astrocyte swelling induced a downregulation of SMIT mRNA in cultured rat astrocytes (Table 1, Fig. 3A/C) and triggered myo-inositol release [36], depleted the intracellular myo-inositol pool [31] and inhibited myo-inositol uptake [40]. Consistent with a role of glutamine for astrocyte swelling in human brain in hepatic encephalopathy [7], downregulation of SMIT mRNA by ammonia in cultured astrocytes was sensitive to inhibition of glutamine synthetase by MSO (Fig. 3D). Given the important interplay between osmotic and oxidative stress in astrocytes in the pathogenesis of HE [10,19], glutaminedependent formation of reactive nitrogen and oxygen species

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

69

Fig. 4 (continued)

Fig. 5. Effect of portal vein ligation or acute ammonium acetate intoxication on osmolyte transporter mRNA expression in rat brain in vivo. Total RNA was prepared from the cerebral cortex of (A) NaCl- (0.9%) treated or ammonia acetate- (4.5 mmol/ kg BW, 6 h) intoxicated rats or (B) sham-operated or portal vein ligated (16 days) rats and used for quantification of osmolyte transporter mRNA expression levels by real time-PCR. Expression of the respective osmolyte transporter gene in relation to HPRT expression is given as fold of (A) vehicle- (NaCl, 0.9%) or (B) sham-operated controls. ⁄: statistically significantly different as compared to the respected control condition (p < 0.05, n = 4–7).

(RNOS) [41] might contribute to downregulation of SMIT mRNA expression. This is supported by the present finding, that ammonia

lowered SMIT and TauT mRNA levels in a glutamine synthesis- and NADPH oxidase-dependent manner (Fig. 3D). Currently, little is known about the molecular mechanisms involved in the downregulation of SMIT mRNA levels in response to osmotic stress. However, downregulation of SMIT mRNA levels following hyperosmolarity–isotonicity transition in endothelial and kidney cells depends on the transcription and translation of currently unknown NF-jB responsive genes which might destabilize SMIT mRNA [42]. In line with this, ammonia was shown to activate NF-jB in an oxidative stress dependent manner [17,43]. As opposed to astrocytes, ammonia had no significant effect on SMIT mRNA expression levels in cultured rat microglia (Table 4). In line with the reported glutamine synthesis-dependent astrocyte swelling in acutely ammonia-challenged rats [44], acute ammonium acetate treatment significantly reduced SMIT mRNA expression levels in rat cerebral cortex (Fig. 5A). Cerebrocortical SMIT mRNA expression was also downregulated after portal vein ligation (Fig. 5B). Reduced brain myo-inositol levels were also observed in rats with portacaval anastomosis, another animal model for chronic liver failure [45]. Although both, TNFa and diazepam were reported to induce astrocyte swelling [13,14], only TNFa, but not diazepam reduced SMIT mRNA levels in cultured astrocytes. On the other hand, diazepam, but not TNFa reduced TauT mRNA levels. The reasons for these differences are unclear, but may relate to different signaling events triggered by TNFa and diazepam, respectively. Interestingly, both, decreased brain myo-inositol levels as well as increased expression levels of the myo-inositol transporter are associated with neuropsychiatric diseases such as mood disorders or Down’s syndrome [46,47]. Whether deranged astrocyte hydration contributes to the pathogenesis of these diseases is currently unclear. However and as shown in the present study, SMIT is also expressed in interneurons in human brain (Fig. 6D). Therefore, disturbed inositol phosphate-dependent intracellular second messenger signaling in neurons may contribute to the pathogenesis of these diseases [46,47]. Taurine transporter (TauT) mRNA and protein were strongly expressed in cultured astrocytes, but in the cerebral cortex TauT

70

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

Fig. 6. SMIT and TauT expression in human brain. 3-dimensional reconstruction of sodium-dependent myo-inositol transporter (SMIT) and taurine transporter (TauT) in human cerebral cortex as detected by immunofluorescence analysis and confocal laser scanning microscopy. (A) Co-staining of SMIT and the astrocytic marker glutaminesynthetase (GS). Colocalized SMIT and TauT immunoreactivity was extracted and is shown in yellow. Nuclei were counterstained using Hoechst 34580. (B) Electronic magnification of the boxed area in (A). (C) Co-staining of TauT and the astrocytic marker glutamine-synthetase (GS). I-III: electronic magnification of the boxed areas in (C). (D) Co-staining of SMIT and the interneuron marker parvalbumin (left) or TauT and the neuronal neurofilament marker (SMI311) (right).

immunoreactivity was observed only in very few astrocytes in the subpial region close to the subarachnoidal space and extracerebral liquor (Figs. 1, 2A and 4B). This finding corroborates a recent report showing absence of TauT isoform 1 which was analyzed in the

present study and low expression of TauT isoform 2 in astrocytes in rat brain [48]. The discrepancy regarding TauT expression in astrocytes in culture versus astrocytes in situ, suggests that TauT expression in

71

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

Fig. 6 (continued)

Table 3 Concentration-dependence of NH4Cl-mediated SMIT- and TauT-mRNA downregulation in cultured rat astrocytes. Astrocytes were either left untreated or treated with NH4Cl for 72 h. RNA was isolated and SMIT or TauT mRNA expression levels were quantified by real time-PCR analysis. Relative mRNA levels after NH4Cl-treatment are expressed as multiple of untreated controls (n = 5–8; ⁄p < 0.05; n.s. not significant). mmol NH4Cl (72 h)

0

0.1

0.5

1

2.5

5

SMIT TauT

1.0 1.0

0.94 ± 0.13n.s. 0.87 ± 0.10n.s.

0.99 ± 0.10n.s. 0.71 ± 0.06⁄

0.79 ± 0.09n.s. 0.65 ± 0.08⁄

0.54 ± 0.08⁄ 0.27 ± 0.07⁄

0.61 ± 0.12⁄ 0.41 ± 0.14⁄

cultured astrocytes may reflect a cell culture phenomenon. This may explain why ammonia induces the release of taurine from cultured astrocytes [35] and downregulates TauT mRNA 72 h after ammonia treatment (Table 3, Fig. 3C). Recently, it was shown that microglia in culture can express glutamine synthetase and rapidly swell upon ammonia exposure in a glutamine synthesis-dependent manner [13]. This may be reflected by a strong tendency (p = 0.09) towards downregulation of TauT mRNA in cultured microglia 24 h after ammonia treatment (Table 4). As opposed to astrocytes, strong anti-TauT immunoreactivity was consistently found in neurons in both, cell culture and rat and human cerebral cortex in vivo (Figs. 2B, 4B and 6D), suggesting that taurine is an important organic osmolyte in neurons. In cultured rat astrocytes and neurons and in neurons in rat cerebral cortex TauT immunoreactivity was also present in the nucleus (Fig. 2A/B, 4B). Presently, a role for nuclear TauT is unclear. However, taurine was suggested to protect nuclear DNA against oxidative stress [49]. TauT mRNA expression in rat cerebral cortex in vivo was not affected by acute ammonium intoxication or portal vein ligation (Fig. 5A/B). This finding is not surprising, since TauT expression in the cerebral cortex is largely confined to neurons (Fig. 4B), which were recently shown not to swell in response to ammonia [13]. In contrast to SMIT and TauT, BGT-1 mRNA expression levels were very low in cultured astrocytes and neurons (Fig. 1) and rat cerebral cortex (Fig. 4A). In line with a recent report in mice [50], anti-BGT-1 immunoreactivity was absent in astrocytes in rat cerebral cortex, but was found in cerebrocortical meninges (Fig. 4G). In contrast to Zhou et al. [50] who reported lack of neuronal BGT-1 expression in mouse brain, in rat cerebral cortex BGT-1 was weakly detected in neuronal somata and dendrites (Fig. 4G). This discrepancy may be due to species differences. However, given the high cerebral GABA expression levels and its abundant role as a major inhibitory neurotransmitter, both, very low BGT-1 mRNA levels (Fig. 4A) and weak BGT-1 immunoreactivity (Fig. 4G) in rat cerebral cortex may question a central role for BGT-1 for GABA re-uptake in rat brain [50]. Unfortunately, it was not possible to analyze BGT-1 expression in human brain, since BGT-1 antibodies used in the present study do not bind to human BGT-1 (Table 1).

BGT-1 was expressed in cultured microglia, as it was described for other macrophages, such as rat Kupffer cells, mouse RAW 264.7 cells and human peripheral blood-derived macrophages and monocytes [51–54]. In these cell types betaine is involved in the regulation of immune functions, such as phagocytosis and eicosanoid formation [52,53]. Interestingly, treating microglia with NH4Cl (5 mmol/l) for 48 and 72 h strongly elevated BGT-1 mRNA levels in cultured microglia (Table 4). However, it remains to be established, whether betaine has a similar immunoregulatory role in microglia, which is known to be activated, but not reactive in response to ammonia or in human HE [55]. Unfortunately, it was not possible to colocalize BGT-1 with microglia markers in rat brain due to antibody incompatibilities. BGT-1 mRNA expression was reduced in rat cerebral cortex in vivo after ammonium acetate treatment, but not after portal vein ligation (Fig. 5A/B). These findings are difficult to interpret with regard to osmotic disturbances, because BGT-1 is only weakly expressed in rat brain (Fig. 4A/G) and may not only transport the osmolyte betaine, but also the neurotransmitter GABA. Taken together, the results of the present study show cell typespecific osmolyte transporter expression profiles in cultured brain cells and rat brain in situ with differential regulation by HE-relevant factors. In vitro and in vivo data point to an important volume-regulatory role of myo-inositol during astrocyte swelling in hepatic encephalopathy [7]. Whereas these findings strengthen the significance of osmotic stress in astrocytes in the pathogenesis

Table 4 Effect of NH4Cl on SMIT, TauT and BGT-1 mRNA expression levels in cultured rat microglia. Microglia were either left untreated or treated with NH4Cl (5 mmol/l) for the indicated time period. RNA was isolated and SMIT, TauT or BGT-1 mRNA expression levels were quantified by real time-PCR analysis. Expression of the respective osmolyte transporter gene in relation to GAPDH expression is given relative to untreated controls (n = 4–5; ⁄p 6 0.05; n.s. not significant). NH4Cl (5 mmol/l)

24 h

48 h

72 h

SMIT TauT BGT-1

1.96 ± 0.58n.s. 0.76 ± 0.10n.s. 0.89 ± 0.09n.s.

1.37 ± 0.64n.s. 0.78 ± 0.14n.s. 2.49 ± 0.29⁄

1.51 ± 0.64n.s. 1.74 ± 0.18⁄ 5.35 ± 0.62⁄

72

J. Oenarto et al. / Archives of Biochemistry and Biophysics 560 (2014) 59–72

of HE, further studies using astrocyte-specific conditional deletion of SMIT are needed to clarify the role of myo-inositol in the pathogenesis of HE and ammonia toxicity. Acknowledgments This study was supported by Deutsche Forschungsgemeinschaft through Collaborative Research Center SFB 974 ‘‘Communication and Systems Relevance in Liver Injury and Regeneration’’ (Düsseldorf). Expert technical assistance of Lilia Igdalova, Torsten Janssen, Ursula Kristek and Brigida Ziegler is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2014.06.024. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]

A.A. Mongin, S.N. Orlov, Pathophysiology 8 (2001) 77–88. R. Kumar, Arch. Biochem. Biophys. 491 (2009) 1–6. P.H. Yancey, J. Exp. Biol. 208 (2005) 2819–2830. F. Lang, G.L. Busch, M. Ritter, H. Völkl, S. Waldegger, E. Gulbins, D. Häussinger, Physiol. Rev. 78 (1998) 247–306. D. Häussinger, H. Sies (Eds.), Osmosensing and Osmosignaling, Methods Enzymol., 428, Academic Press, San Diego, 2007. F. Schliess, R. Reinehr, D. Häussinger, FEBS J. 274 (2007) 5799–5803. D. Häussinger, J. Laubenberger, J. Hennig Gastroenterol. 107 (1994) 1475– 1480. D. Häussinger, G. Kircheis, R. Fischer, F. Schliess, J. Hepatol. 32 (2000) 1035– 1038. N.J. Shah, H. Neeb, G. Kircheis, P. Engels, D. Häussinger, K. Zilles, Neuroimage 41 (2008) 706–717. D. Häussinger, F. Schliess, Gut 57 (2008) 1156–1165. B. Görg, F. Schliess, D. Häusssinger, Arch. Biochem. Biophys. 536 (2013) 158– 163. P. Ginès, M. Guevara, Hepatology 48 (2008) 1002–1010. V. Lachmann, B. Görg, H.J. Bidmon, V. Keitel, D. Häussinger, Arch. Biochem. Biophys. 536 (2013) 143–151. K.V. Rama Rao, A.R. Jayakumar, X. Tong, V.M. Alvarez, M.D. Norenberg, J. Neuroinflam. 7 (2010) 66. R. Reinehr, B. Görg, S. Becker, N. Qvartskhava, H.J. Bidmon, O. Selbach, H.L. Haas, F. Schliess, D. Häussinger, Glia 55 (2007) 758–771. F. Schliess, B. Görg, D. Häussinger, Biol. Chem. 387 (2006) 1363–1370. F. Schliess, B. Görg, R. Fischer, P. Desjardins, H.J. Bidmon, A. Herrmann, R.F. Butterworth, K. Zilles, D. Häussinger, FASEB J. 16 (2002) 739–741. B. Görg, N. Qvartskhava, V. Keitel, H.J. Bidmon, O. Selbach, F. Schliess, D. Häussinger, Hepatology 48 (2008) 567–579. B. Görg, H.J. Bidmon, D. Häussinger, Hepatology 57 (2013) 2436–2447.

[20] C. Kruczek, B. Görg, V. Keitel, E. Pirev, K.D. Kröncke, F. Schliess, D. Häussinger, Glia 57 (2009) 79–92. [21] U. Warskulat, B. Görg, H.J. Bidmon, H.W. Müller, F. Schliess, Glia 40 (2002) 324–336. [22] M. Butz, E.S. May, D. Häussinger, A. Schnitzler, Arch. Biochem. Biophys. 536 (2013) 197–203. [23] F. Schliess, R. Sinning, R. Fischer, C. Schmalenbach, D. Häussinger, Biochem. J. 320 (1996) 167–171. [24] H.P. Matthiessen, C. Schmalenbach, H.W. Müller, Glia 2 (1989) 177–188. [25] H.W. Müller, S. Beckh, W. Seifert, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 1248– 1252. [26] A. Koops, J. Kappler, U. Junghans, G. Kuhn, H. Kresse, H.W. Müller, Brain Res. Mol. Brain Res. 41 (1996) 65–73. [27] M. Chojkier, R.J. Groszmann, Am. J. Physiol. 240 (1981) G371–G375. [28] T. Nishimura, Y. Sai, J. Fujii, M. Muta, H. Iizasa, M. Tomi, M. Deureh, N. Kose, E. Nakashima, Placenta 31 (2010) 1003–1009. [29] H. Pasantes Morales, A. Schousboe, J. Neurosci. Res. 20 (1988) 503–509. [30] J. Morán, T.E. Maar, H. Pasantes-Morales, Neurochem. Res. 19 (1994) 415–420. [31] C. Zwingmann, D. Leibfritz, Neurochem. Int. 47 (2005) 39–50. [32] J.W. Beetsch, J.E. Olson, Biochim. Biophys. Acta 1290 (1996) 141–148. [33] K. Strange, F. Emma, A. Paredes, R. Morrison, Glia 12 (1994) 35–43. [34] M. Bitoun, M. Tappaz, Glia 32 (2000) 165–176. [35] J. Albrecht, A.S. Bender, M.D. Norenberg, Brain Res. 660 (1994) 288–292. [36] R.E. Isaacks, A.S. Bender, C.Y. Kim, Y.F. Shi, M.D. Norenberg, Neurochem. Res. 24 (1999) 51–59. [37] T.N. Nagaraja, N. Brookes, Am. J. Physiol. 274 (1998) C883–C891. [38] R.C. Chang, A. Stadlin, D. Tsang, Neurochem. Int. 38 (2001) 249–254. [39] A. Roychoudhury, A. Bieker, D. Häussinger, F. Oesterhelt, Biol. Chem. 394 (2013) 1465–1474. [40] R.E. Isaacks, A.S. Bender, C.Y. Kim, N.M. Prieto, M.D. Norenberg, Neurochem. Res. 19 (1994) 331–338. [41] A.R. Jayakumar, K.V. Rama, Glia 46 (2004) 296–301. [42] M.A. Yorek, J.A. Dunlap, W. Liu, W.L. Lowe Jr., Am. J. Physiol. Cell Physiol. 278 (2000) C1011–C1018. [43] A.P. Sinke, A.R. Jayakumar, K.S. Panickar, M. Moriyama, P.V. Reddy, M.D. Norenberg, J. Neurochem. 106 (2008) 2302–2311. [44] H. Takahashi, R.C. Koehler, S.W. Brusilow, R.J. Traystman, Am. J. Physiol. 261 (1991) H825–H829. [45] J. Cordoba, J. Gottstein, A.T. Blei, Hepatology 24 (1996) 919–923. [46] N.J. Coupland, C.J. Ogilvie, K.M. Hegadoren, P. Seres, C.C. Hanstock, P.S. Allen, Biol. Psychiatry 57 (2005) 1526–1534. [47] G.T. Berry, J.J. Mallee, H.M. Kwon, J.S. Rim, W.R. Mulla, M. Muenke, N.B. Spinner, Genomics 25 (1995) 507–513. [48] D.V. Pow, R. Sullivan, P. Reye, S. Hermanussen, Glia 37 (2002) 153–168. [49] S.H. Cheong, S.H. Moon, S.J. Lee, S.H. Kim, K.J. Chang, Adv. Exp. Med. Biol. 776 (2013) 167–177. [50] Y. Zhou, S. Holmseth, R. Hua, A.C. Lehre, A.M. Olofsson, I. Poblete-Naredo, S.A. Kempson, N.C. Danbolt, Am. J. Physiol. Renal. Physiol. 302 (2012) F316–F328. [51] U. Warskulat, M. Wettstein, D. Häussinger, FEBS Lett. 377 (1995) 47–50. [52] U. Warskulat, F. Zhang, D. Häussinger, FEBS Lett. 391 (1996) 287–292. [53] F. Zhang, U. Warskulat, M. Wettstein, D. Häussinger, Gastroenterology 110 (1996) 1543–1552. [54] C. Denkert, U. Warskulat, F. Hensel, D. Häussinger, Arch. Biochem. Biophys. 354 (1998) 172–180. [55] I. Zemtsova, B. Görg, V. Keitel, H.J. Bidmon, K. Schrör, D. Häussinger, Hepatology 54 (2011) 204–215.