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2Cl− cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells
Archives of Biochemistry and Biophysics 401 (2002) 187–197 www.academicpress.com
Expression and regulation of the Naþ=Kþ =2Cl cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells Freimut Schliess, Christine Sch€ afer, Stephan vom Dahl, Richard Fischer, Mohammad R. Lordnejad, and Dieter H€ aussinger* Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, D-40225 D€usseldorf, Germany Received 6 December 2001, and in revised form 7 March 2002
Abstract The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased a1 -smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory Kþ uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sodium potassium chloride cotransporter; Betaine; Cell volume; Hepatic stellate cells; Liver; Proliferation
The sodium potassium chloride cotransporter (NK CC) 1 accomplishes for coupled electroneutral transport of Naþ , Kþ , and Cl ions into and out of epithelial and nonepithelial cells. NKCC is sensitive to the 5-sulfamoyl benzoic acid loop diuretics bumetanide, benzmetanide, and furosemide (reviewed by Russel [1]). In various cell types Naþ =Kþ =Cl cotransport is involved in epithelial absorptive and secretory processes [1], cell volume regulation [2], progression through the cell cycle [3], and growth factor signaling [4]. Two isoforms of the NKCC *
Corresponding author. Fax: +49-211-811-8838. E-mail address: [email protected] (D. H€aussinger). 1 Abbreviations used: NKCC, sodium potassium chloride cotransporter; HSC, hepatic stellate cells; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; SEC, sinusoidal endothelial cells; HPRT, hypoxanthine guanine phosphoribosyltransferase; BrdU, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay; DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; KC, Kupffer cell; SMA, smooth muscle actin; PDGF, platelet-derived growth factor; RVI, regulatory volume increase.
(NKCC1 and NKCC2) were cloned, which belong to the superfamily of the cation chloride cotransporters including NCC, KCC1, and KCC2 [5]. NKCC1 shares 58% amino acid identity with NKCC2 and is expressed in many tissues, whereas NKCC2 expression is restricted to the kidney [6]. Like NKCC2, the NKCC1 forms 12 membrane-spanning domains [1]. Recent studies identified the specific binding sites of the cations and chloride as well as bumetanide (e.g., Isenring and Forbush [7]) and provided evidence for a cooperative and ordered ion binding to the NKCC [8]. NKCC activity is regulated by phosphorylation, the cytoskeleton, and the intracellular chloride concentration and probably depends on homooligomerization; however, the precise mechanisms are still under discussion [1]. Mice bearing a NKCC1 knockout suffer from deafness, imbalance, and severe impairment of salivation [9,10]. Pharmacological evidence suggested the expression of NKCC transport activity in the liver. Thus, furosemide antagonized regulatory volume increase induced by hyperosmotic exposure of cultured hepatocytes [11].
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 0 4 7 - 4
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Inhibition of hepatic proteolysis by insulin and ethanol is sensitive to bumetanide, which impairs also cellular net Kþ uptake and associated cell swelling in response to insulin and ethanol [12,13]. Bumetanide-induced insulin resistance was also observed at the level of MAP-kinases [14], confirming the idea that cell swelling due to Naþ =Kþ =2Cl cotransport activation represents a critical step of insulin signal transduction in liver parenchymal cells [15–17]. Bumetanide was shown to prevent pinacidil-induced apoptosis in HepG2 human hepatoma cells [18] and to provide hepatic tolerance against ischemia/reperfusion [19]. Little is known about hepatic expression and regulation of the NKCC1 transport protein. Northern blot analysis unraveled a weak expression of the NKCC1 mRNA in human liver [20] but not in liver from rabbit [20] and mouse [21]. Although the NKCC1 has recently been cloned from rat parotid [22], its expression at the mRNA or protein level in rat liver is unknown. Moreover, nothing is known about the presence or function of Naþ =Kþ =2Cl cotransport in nonparenchymal liver cells. In the present study NKCC1 mRNA and protein were detected in different rat liver cell types. NKCC1 expression in hepatic stellate cells increased upon their transformation into myofibroblasts during culture. NKCC1 activity may delay initiation of HSC transformation by culture of the cells but did not contribute to proliferation of 14-day-cultured HSCs. Short-term hyperosmotic perfusion of rat liver induced a volumeregulatory potassium uptake, which was partially blunted by bumetanide. Long-term dehydration of HuH-7 cells by solutions made hyperosmotic by NaCl and raffinose stimulated NKCC1 expression, which was counteracted by the compatible organic osmolytes betaine and taurine. The data suggest a role of NKCC1 in short-term and long-term adaption of the liver to dehydrating conditions.
Materials and methods Materials RPMI 1640 and DMEM, glutamine, and fetal calf serum were from Biochrom (Berlin, Germany). Nycodenz was from Nycomed (Oslo, Norway). Pronase was from Merck (Darmstadt, Germany). L -Lactic acid was from Roth (Karlsruhe, Germany). Radiochemicals were from Amersham (Braunschweig, Germany). DNase I and collagenase were from Boehringer Mannheim. Horseradish peroxidase conjugated anti-mouse IgG antibody was from Bio-Rad Labs (Hercules, CA). RNeasy total RNA kit was obtained from Qiagen (Hilden, Germany). Betaine and taurine were from Sigma (Deisenhofen, Germany). Nitrocellulose membranes were purchased from Schleicher & Schuell
(Dassel, Germany). [a-32 P]dCTP was from Amersham. Polybead fluorescent microspheres (2.5% solids latex, 1.1 lm diameter) were obtained from Polysciences Ltd. (St. Goar, Germany). All other chemicals were from Merck (Darmstadt, Germany). Cell preparation and culture Treatments of rats. All animals received human care in compliance with the German Animal Protection Act, which is in accordance with the National Research Council’s criteria. Isolation and culture of parenchymal cells from rat liver. Isolated hepatocytes were prepared from livers of male Wistar rats by a collagenase perfusion technique as described [23]. A total of 1:5 106 cells were plated on collagen-coated culture dishes (36 mm diameter) and maintained in Krebs–Henseleit medium (115 mmol/L NaCl/25 mmol/L NaHCO3 /5.9 mmol/L KCl/1.18 mmol/ L MgCl2 /1.23 mmol/L NaH2 PO4 /1.2 mmol/L Na2 SO4 / 1.25 mmol/L CaCl2 ), supplemented with 6 mmol/L glucose at 37 °C and a 5% CO2 atmosphere. After 2 h, cells were washed twice and culture was continued for 24 h in 1.5 mL Williams medium E (Sigma, Munich, Germany), supplemented with glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (0.1 mg/mL), insulin (107 mol/L), dexamethasone (107 mol/L), and 5% fetal-calf serum (FCS). Hepatocyte viability was more than 95% as assessed by trypan blue exclusion. Isolation and culture of rat hepatic stellate cells. HSC from 1- to 3-year-old male Wistar rats were prepared as previously described [24] by collagenase/pronase perfusion and isolated by Nycodenz gradient. The cells were seeded at a density of 0:15 106 =cm2 on glass coverslips in a 24-well culture plate (Falcon) or 60-mm well (Falcon, Heidelberg, Germany) and maintained in DMEM containing 10% (v/v) heat-inactivated FCS and 1% (w/v) penicillin/streptomycin. Culture was performed in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The culture medium was changed after 24 h and thereafter every 2 days. The purity of HSC was >95%, as assessed 24 h after seeding by their typical light-microscopic appearance and vitamin-A-specific autofluorescence, their inability to phagocytose fluorescent 1.1-lm latex particles, and their positive immunofluorescent staining for desmin, GFAP, and, at the seventh day of culture, for a1 -smooth muscle actin. Isolation and culture of rat Kupffer cells and rat sinusoidal endothelial cells. Cells were isolated from 1-yearold male Wistar rats by collagenase–pronase perfusion and separated by a single Nycodenz gradient and centrifugal elutriation [25]. Kupffer cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS for up to 24 h. Sinusoidal endothelial cells (SEC) were plated on collagen 1-coated culture dishes and maintained in RPMI 1640 medium supplemented
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with 10% heat-inactivated FCS for 24 h. Culture was performed in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Purity of Kupffer cells (KC) was P98% as assessed after seeding by their typical light microscopic appearance, by immunological staining for ED2 (specific macrophages marker protein) [26], and by their ability to phagocytose fluorescent 1.1-lm latex particles. Purity of SEC was P80% as assessed 24 h after seeding by their typical light microscopic appearance and by their inability to phagocytose fluorescent 1.1-lm latex particles. HuH-7 and H4IIE cell culture. HuH-7 human hepatoma cells [27] and H4IIE-C3 rat hepatoma cells (ATCC CRL 1600) were maintained in DMEM–F12/ 5% CO2 =5 mM glucose at 37 °C, pH 7.4, supplemented with 10% FCS. When cells had reached 90% confluency, they were washed with Dulbecco’s PBS and the culture was continued in serum-free medium for an additional 24 h. Extracellular osmolarity was adjusted by dilution of the media with the appropriate volume of the respective NaCl-free medium leading to hypoosmolarity (205 mosmol/L) or with medium of elevated NaCl content leading to hyperosmolarity (405 mosmol/ L), respectively. In the normoosmotic control (305 mos mol/L), the same volume of normoosmotic medium was added.
RT-PCR Total RNA was extracted from guanidinium thiocyanate solutions [28] using the RNeasy TM total RNA kit (Qiagen) according to the manufacturer’s recommendations. cDNA was obtained from 1 lg total RNA with the kit from Roche Molecular Biochemicals (Mannheim, Germany), using 20 U reverse transcriptase from the avian myoblastosis virus and 1.6 lg oligop(dT)15 primer. The reaction mixture was incubated at 25 °C for 1 h and then at 42 °C for an additional 1 h. The reverse transcriptase was inactivated by a 5-min incubation at 99 °C. One-hundred nanograms of cDNA was PCR amplified by using the following primers: NKCC1 sense, 50 CCA GAT GTT TGC GAA AGG TT-30 ; NKCC1 antisense, 50 -CCC AGT TCA CAT CTG GCT TT-30 ; NKCC2 sense, 50 -CCG AGT TCG GTG GGT CAA TAG GCT-30 ; NKCC2 antisense, 50 -CTC CAG AGA TGT TGG CAC CAG CAA-30 , yielding 362- and 435-kb cDNA fragments representing NKCC1 and NKCC2, respectively. Sequence analysis with the Abi Prism 310 genetic analyzer (Perkin Elmer Applied Biosystems, Weiterstadt, Germany) confirmed the specificity of the PCR amplifications. The PCR reactions were performed in 50 lL of the following reaction mixture: 34.75 lL water, 5 lL reaction buffer ð10Þ5 lL 25 mmol=L MgCl2 , 1 lL 10 mmol/L dNTP mix, 1 lL 10 lmol/L primer sense, 1 lL 10 lmol/L primer anti-
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sense, 0.25 lL 5 U/lL Goldstar DNA polymerase (Eurogentec, Seraing, Belgium) and 2 lL cDNA. Regulation of the NKCC1 mRNA levels in HuH-7 cells was analyzed by means of semiquantitative PCR analysis according to Kinoshita et al. [29]. As an internal control a 415-kb fragment of the of the hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA was synthesized using the primers 50 -TGGT CGT CGT GAT TAG TGA TG-30 (sense) and 50 -CTG CAT TGT TTT GCC AGT GT-30 (antisense). Primers were chosen using the Primer3 program (http://www-genome.wi.mit.edu/ cgi-bin/primer/primer3.cgi) and purchased from the MWG Biotech AG (Ebersberg, Germany). For each primer set, an increasing number of PCR cycles with otherwise fixed conditions was performed to determine the optimal number of cycles to be used. The optimal number of cycles was determined as the midpoint of the exponential phase. The numbers of cycles were 35 for NKCC1 and NKCC2 and 30 for HPRT. Determination of hepatic stellate cell proliferation Hepatic stellate cell proliferation was addressed by measuring BrdU incorporation into DNA using the colorimetric BrdU cell proliferation ELISA kit (Roche Molecular Biochemicals) according to the manufacturer’s suggestions. Hepatic stellate cells plated on 96well dishes (50,000 cells per well) were cultured for 2 or 14 days in DMEM containing 1 or 10% FCS. For experimental treatment medium was removed and cells were incubated for an additional 48 h within the same medium plus 100 lmol=L BrdU in presence or absence of PDGF (25 ng/mL). In parallel experiments cells amiloride (100 lmol=L) or bumetanide (5 lmol=L) were present during the 48-h incubation period. Incorporation of BrdU into DNA was monitored at 450 nm using an peroxidase-coupled antibody and tetramethylbenzidine as a substrate with an ELISA reader (MRX microplate reader, Dynatec Laboratories, Denkendorf, Germany). Rat liver perfusion Livers from male Wistar rats (160–230 g), who were fed on a standard chow, were perfused in an open nonrecirculating manner as described previously [30] with bicarbonate-buffered Krebs–Henseleit saline as perfusion medium plus lactate (2.1 mM) and pyruvate (0.3 mM), gassed with O2 =CO2 (19/1, v/v). The average flow rate was kept constant throughout the individual perfusion experiment and was 3.5–4.5 mL/min/g. For changing the osmolarity, the NaCl concentration was varied, resulting in corresponding changes of osmolarity. Additions were made by dissolution in DMSO, which was present throughout control and inhibitor experiments at an infusion rate of 20 lL= min. Viability
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of the livers was assessed by monitoring effluent oxygen concentration and measurement of LDH leakage from livers, which did not exceed 15–25 mU/min/g liver. Effluent perfusate pH was monitored continuously with a pH-sensitive electrode and effluent perfusate oxygen concentration was monitored with a Clark-type oxygen electrode (Biolytik, Bochum, Germany). The perfusion pressure was detected by a pressure transducer (Hugo Sachs Electronics, Hugstetten, Germany). Basal portal pressure was 3–5 cm H2 O and was not affected by the compounds used in this study. Liver mass was recorded by placing the excised perfused liver on a specially constructed balance pan [31]. Changes of liver mass were expressed on a percentage basis and referred to liver wet weight at the end of the experiment, a time point when normoosmotic conditions had been installed for at least 30 min. The intracellular water space was calculated from the difference of washout profiles of simultaneously infused [14 C]urea and [3 H]inulin as described previously [32]. In fed animals, the cell water under control conditions was 547 11 ll=g ðn ¼ 16Þ. Effluent Kþ concentration was registered continuously with Kþ -sensitive electrodes (Radiometer, Munich, Germany) and calculations were made by planimetry. When present, a baseline drift was taken into account. Western blot At the end of the experimental treatment, the medium was removed and the cells were immediately lysed at 4 °C using 50 mmol/L Tris/HCl buffer (pH 7.2) containing 150 mmol/L NaCl, 40 mmol/L NaF, 5 mmol/L EDTA, 5 mmol/L EGTA, 1 mmol/L vanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.1% aprotinine, 1% Nonidet P-40, 0.1% sodium deoxycholate, and 0.1% SDS. The homogenized lysates were centrifuged at 20,000g at 4 °C and the supernatant was added to an identical volume of gel loading buffer containing 200 mmol/L dithiothreitol (pH 6.8). After heating to 95 °C for 5 min, the proteins were subjected to gel electrophoresis (50 lg protein/lane, 9% gel). Following electrophoresis, gels were equilibrated with transfer buffer (39 mmol/L glycine, 48 mmol/L Tris/HCl, 0.03% SDS, 20% methanol). Proteins were transferred to nitrocellulose membranes using a semidry transfer apparatus (Pharmacia, Freiburg, Germany). Blots were blocked in 5% bovine serum albumin containing TBST (20 mmol/L Tris/HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween 20) and then incubated overnight with the 1:1000-diluted T4 monoclonal antibody raised against the 310 C-terminal residues of the human colonic NKCC1 [33]. Following washing with TBST and incubation with horseradish peroxidase-coupled anti-mouseIgG antibody diluted 1:10,000 at room temperature for
1 h, the blots were washed three times and developed using enhanced chemiluminescent detection (Amersham). Statistics Results from n independent experiments are expressed as means SE. Results were compared using the Student’s t test: P < 0:05 was considered statistically significant.
Results NKCC1 expression in liver cells NKCC1 mRNA expression in isolated liver cell types was studied by RT-PCR as described under Materials and methods. As shown in Fig. 1A, NKCC1 mRNA expression was detected in parenchymal cells, macrophages (KCs), and SECs isolated from rat liver. NKCC1 mRNA was also expressed in HuH-7 human hepatoma cells and to a less degree in H4IIE rat hepatoma cells (Fig. 1A). NKCC2 mRNA was not detectable in the liver cell types investigated but, as expected, was expressed in rat kidney tissue (Fig. 1A). Western blot analysis (Fig. 1B) unraveled NKCC1 protein expression in rat liver tissue as well as in isolated rat liver parenchymal and sinusoidal endothelial cells. NKCC1 protein was barely detectable in Kupffer cells and in 1-day-cultured hepatic stellate cells. In addition, NKCC1 was expressed in HuH-7 human but not in H4IIE rat hepatoma cells. NKCC1 expression increases during transformation of rat hepatic stellate cells in culture Hepatic stellate cells transform from a ‘‘quiescent’’ to a myofibroblast-like ‘‘activated’’ phenotype during culture on uncoated plastic dishes (e.g., Peters-Regehr et al. 34]). This is accompanied by morphological changes and the expression of a1 -smooth muscle actin (aSMA) from day 3–15 of cultivation of culture (Fig. 1 in PetersRegehr et al. [34], Fig. 3A, this paper). As demonstrated in Fig. 2 by semiquantitative PCR and Western blot analysis, respectively, the NKCC1 mRNA and protein expression levels progressively increased from day 1 to day 9 of hepatic stellate cell culture. To examine a potential contribution of NKCC1 activity to aSMA expression, hepatic stellate cells were cultured for 1, 2, 3, 7, and 14 days in presence or absence of 5 lmol=L bumetanide. As shown in Fig. 3A, bumetanide accelerated the appearance of aSMA, which was present already at the second day of culture, whereas aSMA expression at later time points was largely independent from the presence or absence of bumetanide. This suggests a role
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Fig. 1. NKCC1 and NKCC2 expression in different liver cell types. Abbreviations: PC, parenchymal cells; SEC, sinusoidal endothelial cells; KC, Kupffer cells; HSC, hepatic stellate cells; L, liver; M, standard. Cells were lysed 24 h after isolation for mRNA or protein extraction, respectively. (A) NKCC1- and NKCC2 mRNA expression was addressed by means of RT-PCR in parenchymal, sinusoidal, Kupffer, and stellate cells from rat liver as well as in HuH-7 human hepatoma cells, H4IIE rat hepatoma cells, and rat kidney. (B) NKCC1 protein expression in rat liver and isolated rat liver parenchymal, siunusoidal, Kupffer, and stellate cells as well as HuH-7 human and H4IIE rat hepatoma cells was determined by Western blot analysis using the T4 monoclonal antibody. Variable posttranslational modifications may account for multiple immunoreactive bands. Representatives of three independent experiments are shown.
of NKCC1 in modulating the initiation phase of HSC transformation. PDGF represents a powerful mitogen for HSCs [34] and PDGF-induced activation of Naþ =Kþ =2Cl cotransport is associated with proliferation of vascular smooth muscle cells from hypertensive rats [35]. In order to investigate a potential involvement of the NKCC1 in hepatic stellate cell proliferation, the effect of bumetanide on basal and PDGF-induced proliferation of 14-day-old HSCs was examined. As shown in Fig. 3B bumetanide at a concentration of 5 lmol=L, which inhibits insulin-induced Kþ uptake in perfused rat liver [13], was without effect on both basal and PDGF-induced proliferation of HSCs being 14 days in culture. Similar results were obtained with proliferation studies in the presence of 1% FCS or with HSCs cultured for 4 days (not shown). Inhibition of HSC proliferation was also not found at higher bumetanide concentrations (not shown). However, in line with a recent report [36] inhibition of the Naþ =Hþ exchange with amiloride significantly reduced HSC proliferation in presence and absence of PDGF, respectively (Fig. 3B). This argues against a major role
of NKCC1 for proliferation of 14-day-old HSCs. Similar results were obtained for 2-day-cultured HSCs (data not shown). NKCC1-mediated K þ uptake contributes to the hyperosmolarity-induced regulatory volume increase in perfused rat liver The involvement of the NKCC1 in hyperosmolarityinduced regulatory volume increase (RVI) was examined in the perfused rat liver. Perfusion medium was switched from normo- to hyperosmolarity (305–385 mosmol/L) in presence or absence of bumetanide. Institution of hyperosmolarity by addition of NaCl led to a volumeregulatory net Kþ uptake, which is terminated within 30 min [37]. Thereafter, liver cells remained in a slightly shrunken state as shown by measurements of liver mass and cell water (Table 1). In presence of bumetanide (5 lmol=L), the volume-regulatory net Kþ uptake was inhibited by about 40%, and consequently the extent of cell shrinkage after completion of RVI was markedly enhanced (Table 1). These data suggest a significant contribution of the NKCC1-mediated Kþ uptake to
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Fig. 2. Transformation-dependent increase of NKCC1 expression in rat hepatic stellate cells HSCs were lysed at days 1, 4, and 9 after isolation. These time points represent progressive transformation of the stellate cells into myofibroblasts [33]. Data from three cell preparations are summarized within this figure. *Expression differs significantly ðP < 0:05Þ from that found in 1-day-cultured cells. (A) Dependence of NKCC1 mRNA in rat hepatic stellate cells on the duration of culture was estimated by semiquantitative PCR. The top panel shows results of a representative PCR experiment. (B) NKCC1 protein expression in hepatic stellate cells increased during culture of the cells was monitored by Western blot analysis. A representative Western blot is shown in the top panel.
regulatory volume increase induced by hyperosmolarity in perfused rat liver. Regulation of NKCC1 expression by osmolarity and osmolytes in HuH-7 cells Clinical settings such as sepsis, burn injury, and diabetes mellitus are often associated with hyperosmotic tissue dehydration [17,38,39]. HuH-7 cells were used to study NKCC1 expression under conditions of long-term hyperosmotic challenge. Hyperosmolarity was adjusted by elevation of the medium NaCl concentration. As shown in Fig. 4 a hyperosmotic (405 mosmol/L) exposure of HuH-7 cells induced an increase in NKCC1 mRNA expression, which was maximal between 8 and 16 h and declined thereafter. No significant changes in NKCC1 mRNA expression were observed under the control condition (305 mosmol/L). The osmolarity dependence of NKCC1 mRNA expression was studied at the 8-h time point (Fig. 4B). Hypoosmolarity was lar-
gely without effect on NKCC1 mRNA expression. However, NKCC1 mRNA expression was significantly elevated already at an osmolarity increase of 20 mosmol/ L and further increased by elevating the osmolarity up to 445 mosmol/L. Like NaCl, the impermeable osmolyte raffinose led to induction of NKCC1 mRNA expression (Fig. 5), indicating that the hyperosmotic effect on NKCC1 mRNA expression is triggered by cell shrinkage rather than by increased activities of extracellular Naþ and Cl . Increasing medium osmolarity to 405 mosmol/ L by mannitol, which slowly equilibrates in rat liver between extra- and intracellular compartments [40], induced a delayed NKCC1 mRNA expression after 24 h. Urea, a solute rapidly penetrating liver cells [32], was ineffective at modifying NKCC1 mRNA expression levels in HuH-7 cells. This indicates that cell shrinkage but not the osmolarity increase per se is the trigger for NKCC1 expression. Taurine and betaine are compatible organic osmolytes, which do not disturb protein function even at high concentrations and are involved in the long-term adaption to hyperosmolarity [41]. These osmolytes accumulate in liver cells due to cumulative uptake by specific transport systems [42]. When HuH-7 cells were allowed to accumulate taurine or betaine for a period of 12 h before switching to 405 mosmol/L with NaCl-enriched medium, the hyperosmotic increase in NKCC1 mRNA expression was suppressed and did not significantly differ from the control situation (305 mosmol/L in presence or absence of betaine or taurine, respectively; Fig. 6A). Under normoosmotic conditions betaine or taurine were ineffective to modulate NKCC1 mRNA expression. As shown in Fig. 6B the regulation of the NKCC1 by hyperosmolarity was roughly mirrored at the protein level. Taurine largely abolished hyperosmotic NKCC1 protein accumulation, whereas a delayed induction of NKCC1 expression occurred in presence of betaine. This may be due to the protein stabilizing property of this osmolyte on the one hand [43,44] and on the smaller intracellular accumulation of betaine compared to taurine in hepatocytes on the other hand [45]. The data suggest an involvement of the NKCC1 in long-term regulation of cell volume and intracellular osmolyte balance.
Discussion The present study demonstrates the expression of the Naþ =Kþ =2Cl cotransporter NKCC1 in rat liver parenchymal cells, sinusoidal endothelial and stellate cells. The NKCC1 was also found in human HuH-7 but not in rat H4IIE hepatoma cells. HSCs represent the principal fibrogenic cell type of the liver. Following liver injury or during culture they undergo activation, i.e., the transition of resting cells into proliferative, fibrogenic, and contractile myofibro-
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Fig. 3. Pharmacological characterization of NKCC1 function in rat hepatic stellate cells. (A) Culture-induced a1 SMA expression in HSCs exposed to bumetanide. Freshly isolated HSCs were cultured in presence or absence of 5 lmol=L bumetanide. After the indicated time periods cells were lysed. Cell lysates were analyzed for the presence of a1 SMA in Western blot. (B) Basal and PDGF-induced HSC proliferation in presence of bumetanide or amiloride. Proliferation of 14-day-old HSCs was measured by immunoperoxidase staining of nuclei that incorporated BrdU for 48 h in presence or absence of 25 ng/mL PDGF. In parallel experiments bumetanide (5 lmol=L) or amiloride (100 lmol=L) were present during the 48-h incubation period. Data from four different cell preparations are summarized. Control, bumetanide, amiloride, and PDGF absent; Bu, bumetanide; Am, amiloride. *Proliferation significantly ðP < 0:05Þ different from the control; # proliferation significantly P < 0:05 different from that observed in presence of PDGF in absence of amiloride and bumetanide.
Table 1 Hyperosmolarity-induced changes of liver mass and intracellular water space and cell-volume-regulatory Kþ uptake in perfused rat in absence and presence of bumetanide Parameter Change of intracellular water space (%) Change of liver mass (%) Cell-volume-regulatory Kþ uptake (lmol=g)
Bumetanide absent
Bumetanide present
8:1 0:9 ð4Þ
18:9 3:9 ð5Þ
6:9 0:5 ð6Þ
10:4 0:7 ð7Þ
þ13:0 0:6 ð8Þ
7:7 0:6 ð7Þ
Note. Livers from male, fed Wistar rats were perfused for 150 min. After a 90-min period of isoomotic equilibration (305 mosmol/L), livers were exposed to hyperosmotic perfusion medium (385 mosmol/L) for 30 min due to the addition of 40 mmol/L NaCl. If present, bumetanide (5 lM) was infused 30 min prior to installation of hyperosmolarity. The changes in intracellular water space, liver mass, and Kþ fluxes were determined as described under materials and methods. Numbers in brackets denote the number of different perfusion experiments. Data are shown as means SE. ; Significant difference from control in absence of bumetanide, P < 0:01, P < 0:05. Cell water under control conditions was 547 11 ll=g ðn ¼ 16Þ.
blasts [46]. In addition to a1 SMA and collagen the expression of the betaine transporter BGT-1 was identified as another marker of HSC transformation [34]. Similar to the BGT-1, NKCC1 mRNA and protein expression increased during the transformation process (Fig. 2), suggesting, that alterations of the intracellular osmolyte balance accompany HSC transformation. Bumetanide induced a1 SMA expression in HSCs at the second day of culture but did not further increase a1 SMA on later time points (Fig. 3A). Thus Naþ =Kþ =2Cl cotransport may antagonize the initiation phase of the transformation process. The underlying mechanism is currently unknown. One speculation is that NKCC1-mediated cell swelling may impair the generation of reactive oxygen intermediates, which triggers HSC activation. In this respect it is interesting to note that hypoosmotic swelling was shown to protect perfused liver from oxidative injury and enhances the generation of reducing equivalents via the pentose phosphate shunt [47], whereas hyperosmotic shrinkage produces oxidative stress [47,48]. Inhibition of NKCC1 by bumetanide may
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Fig. 4. Hyperosmotic stimulation of NKCC1 mRNA expression in HuH-7 human hepatoma cells NKCC1 mRNA expression levels were determined by semiquantitative RT-PCR. (A) Time course of hyperosmotic NKCC1 mRNA induction. HuH-7 cells were exposed to hyperosmolarity (405 mosmol/L) for the indicated time periods. Alternatively, the cells were maintained under the normoosmotic control condition (305 mosmol/L). Cells were lysed at the time points indicated. (B) Osmolarity dependence of NKCC1 mRNA expression. HuH-7 cells were exposed for 8 h to normoosmotic (305 mosmol/L) or anisoosmotic solutions. Then cells were lysed for RNA preparation. Results show means SE from three independent experiments. *Relative mRNA expression significantly different from the respective normoosmotic control. The top panels show the results of representative RT-PCR determinations.
shrink the HSCs leading to an amplification of the generation and action of reactive oxygen intermediates thereby accelerating the initiation of transformation. Interestingly in other cell types, cell shrinkage augments differentiation (reviewed in Lang et al. [16]).
NKCC1 activity seems not to stimulate basal or PDGF-induced proliferation of 14-day-old HSCs (Fig. 3B). However, as reported [36], inhibition of the Naþ =Hþ exchanger markedly interfered with HSC proliferation (Fig. 3B). Although a mitogen-stimulated
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Fig. 5. Differential effects of hyperosmolarity adjusted by raffinose, mannitol, urea, or NaCl on NKCC1 mRNA induction in HuH-7 human hepatoma cells HUH7 human hepatoma cells were exposed to hyperosmotic solutions adjusted to 405 mosmol/L by raffinose, mannitol, urea, or NaCl, respectively. Alternatively cells were maintained under the normoosmotic control condition. After 8, 16, or 24 h cells were lysed for RNA preparation. NKCC1 mRNA expression was determined by semiquantitative RT-PCR. The figure summarizes data from three different experiments. *Relative mRNA expression significant different from the respective normoosmotic control. The top panels show the results of representative RTPCR determinations. Control, normoosmotic control condition (305 mosmol/L).
rapid increase in intracellular Naþ or Kþ was recognized as an essential trigger for progression through the cell cycle [3], the relative contributions of Naþ =Kþ =2Cl transport and Naþ =Hþ exchange to generate this signal may depend on the expression levels of the respective transport systems, intracellular pH, and Kþ content and the mitogen-induced signals generated upstream of ion uptake [49]. Acutely hyperosmotically challenged liver cells shrink but simultaneously activate electrolyte uptake thereby preventing excessive shrinkage and performing a regulatory volume increase [16]. Bumetanide markedly increased hyperosmotic shrinkage, probably due to an impairment of the hyperosmolarity-induced volumeregulatory Kþ uptake, thereby reducing regulatory cell volume increase (Table 1). In perfused liver, however, no major contribution of Naþ =Kþ =2Cl cotransport to a ‘‘secondary’’ regulatory volume increase (i.e., the volume-regulatory response induced by normoosmotic exposure of hypoosmotically preperfused livers) could be shown [50,51]. These findings indicate a significant contribution of the NKCC1 in mediating a hyperosmolarity-induced ‘‘primary’’ regulatory volume increase in perfused liver, whereas in isolated hepatocytes only a minor contribution was reported [11]. The activation of
volume-regulatory ion transport depends on cellular interactions with the extracellular matrix and the cytoskeleton [52], which may explain the differences between perfused liver and cultured hepatocytes. The different roles of NKCC1 in ‘‘secondary’’ and ‘‘primary’’ regulatory volume increase can be explained by differences in the Naþ and Cl transmembrane gradients, which determine the activity and direction of NKCC1-mediated transport. Clinical settings such as sepsis, burn injury, and diabetes mellitus are often associated with hyperosmotic tissue dehydration, which contributes to catabolic protein metabolism and insulin resistance [17,38]. Induction of NKCC1 expression by chronic hyperosmolarity (Figs. 4 and 5) may play a role in long-term adaption to hyperosmotic stress. Hyperosmotic NKCC1 expression was also observed in Ehrlich ascites tumor cells [53] and corneal epithelial cells [54]. On the other hand, regulatory volume increase by ion uptake rises the intracellular ionic strength, which interferes with native protein structure and function [41]. Another cellular strategy to adapt to long-term hyperosmolarity is the isoosmotic exchange of ions against organic osmolytes, which are compatible with protein function even at high millimolar concentrations [42]. In the presence of betaine and
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Fig. 6. Modulation of hyperosmotic NKCC1 expression by betaine and taurine in HuH-7 human hepatoma cells. HuH-7 cells were exposed to 10 mmol/L betaine or taurine for 12 h. Then, medium osmolarity was increased to 405 mosmol/L by elevation of NaCl concentration for the indicated time periods. Alternatively, the normoosmotic control condition (305 mosmol/L) was adjusted for identical time periods. Thereby, the presence of betaine and taurine was maintained. Then, cells were lysed for RNA isolation (A) or protein extracts were prepared (B). NKCC1 transcript levels were determined by semiquantitative RT-PCR and NKCC1 protein expression was evaluated in Western blot. Three independent experiments were performed. *RT-PCR amplification significantly ðP < 0:05Þ different from the normoosmotic control; **significantly ðP < 0:05Þ different from the relative NKCC1 mRNA expression under hyperosmotic conditions in absence of betaine or taurine, respectively. C, control (betaine and taurine absent); B, betaine; T, taurine.
taurine this seems to represent a predominant adaption mechanism in HuH-7 cells as suggested by suppression of the hyperosmotic NKCC1 mRNA expression by these osmolytes (Fig. 5). Betaine and taurine abolish the hyperosmotic induction of cyclooxygenase-2 expression in Kupffer cells [55,56] and betaine accumulation substitutes for HSP70 expression in high-salt-exposed Madin–Darby canine kidney cells [57]. Preventing an increase in intracellular ionic strength and ameliorating proteotoxicity may blunt the trigger for stress gene expression induced by hyperosmotic shrinkage. In addition to its role in cell volume regulation NKCC1 is involved in the regulation of hepatic metabolism by insulin and ethanol [12,58]. Inhibition of insulin-induced Kþ -uptake by bumetanide or short-term hyperosmotic treatment induce insulin resistance in
perfused rat liver [13,14]. It seems attractive to speculate that the hyperosmotic stimulation of NKCC1-mediated volume-regulatory Kþ uptake (this paper) prevents further stimulation by insulin and this may account for the insulin resistance under these conditions. Increased NKCC1 expression levels may help to improve hepatic sensitivity to hormones and substrates under conditions of chronic dehydration. Further studies are required to define the functional importance of hepatic NKCC1 expression in parenchymal and nonparenchymal cells.
Acknowledgments This work was supported by the Sonderforschungsbereich 575 ‘‘Experimentelle Hepatologie’’ and the
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Fonds der Chemischen Industrie, Frankfurt, Germany. The monoclonal antibody T4 raised against the 310 Cterminal residues of the human colonic NKCC1 was developed by Christian Lytle and Bliss Forbush III and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa. The technical assistance of Nicole Beyen and Markus Mroz is greatly acknowledged. References [1] J.M. Russel, Physiol. Rev. 80 (2000) 211–276. [2] B. Sarkadi, J.C. Parker, Biochim. Biophys. Acta 1071 (1991) 407– 427. [3] E. Berman, I. Sharon, H. Atlan, Biochim. Biophys. Acta 1239 (1995) 177–185. [4] E. Rozengurt, Science 234 (1986) 161–166. [5] M. Haas, B. Forbush, J. Bioenerg. Biomembr. 30 (1998) 161– 172. [6] G. Gamba, A. Miyanoshita, M. Lombardi, J. Lytton, W.S. Lee, M.A. Hediger, S.C. Hebert, J. Biol. Chem. 269 (1994) 17713– 17722. [7] P. Isenring, B. Forbush, J. Biol. Chem. 272 (1997) 24556–24562. [8] C. Lytle, T.J. McManus, M. Haas, Am. J. Physiol. 274 (1998) C299–C309. [9] E. Delpire, J. Lu, R. England, C. Dull, T. Thorne, Nature Genet. 22 (1999) 192–195. [10] R.L. Evans, K. Park, R.J. Turner, G.E. Watson, H.V. Nguyen, M.R. Dennett, A.R. Hand, M. Flagella, G.E. Shull, J.E. Melvin, J. Biol. Chem. 275 (2000) 26720–26726. [11] F. Wehner, H. Tinel, J. Physiol. 506 (1998) 127–142. [12] S. vom Dahl, D. H€ aussinger, Gastroenterology 114 (1998) 1046– 1053. [13] C. Hallbrucker, S. vom Dahl, F. Lang, W. Gerok, D. H€aussinger, Eur. J. Biochem. 199 (1991) 467–474. [14] F. Schliess, S. vom Dahl, D. H€aussinger, Biol. Chem. 382 (2001) 1063–1069. [15] D. H€ aussinger, F. Lang, Trends Pharmacol. Sci. 13 (1992) 371– 373. [16] F. Lang, G.L. Busch, M. Ritter, H. V€ olkl, S. Waldegger, E. Gulbins, D. H€ aussinger, Physiol. Rev. 78 (1998) 247–306. [17] F. Schliess, D. H€ aussinger, Cell Physiol. Biochem. 10 (2000) 403– 409. [18] J.A. Kim, Y.Y. Kang, Y.S. Lee, Biochem. Biophys. Res. Commun. 281 (2001) 511–519. [19] R.J. Fiegen, U. Rauen, M. Hartmann, U.K. Decking, H. de Groot, Hepatology 25 (1997) 1425–1431. [20] J.A. Payne, J.C. Xu, M. Haas, C.Y. Lytle, D. Ward, B. Forbush, J. Biol. Chem. 270 (1995) 17977–17985. [21] E. Delpire, M.I. Rauchman, D.R. Beier, S.C. Hebert, S.R. Gullans, J. Biol. Chem. 269 (1994) 25677–25683. [22] M.L. Moore-Hoon, R.J. Turner, Biochim. Biophys. Acta 1373 (1998) 261–269. [23] A.J. Meijer, J.A. Gimpel, G.A. Deleeuw, J.M. Tager, J.R. Williamson, J. Biol. Chem. 250 (1975) 7728–7738. [24] R.M. Reinehr, R. Kubitz, R.T. Peters, J.G. Bode, D. H€aussinger, Hepatology 28 (1998) 1566–1577.
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Expression and regulation of the Naþ=Kþ =2Cl cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells Freimut Schliess, Christine Sch€ afer, Stephan vom Dahl, Richard Fischer, Mohammad R. Lordnejad, and Dieter H€ aussinger* Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, D-40225 D€usseldorf, Germany Received 6 December 2001, and in revised form 7 March 2002
Abstract The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased a1 -smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory Kþ uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sodium potassium chloride cotransporter; Betaine; Cell volume; Hepatic stellate cells; Liver; Proliferation
The sodium potassium chloride cotransporter (NK CC) 1 accomplishes for coupled electroneutral transport of Naþ , Kþ , and Cl ions into and out of epithelial and nonepithelial cells. NKCC is sensitive to the 5-sulfamoyl benzoic acid loop diuretics bumetanide, benzmetanide, and furosemide (reviewed by Russel [1]). In various cell types Naþ =Kþ =Cl cotransport is involved in epithelial absorptive and secretory processes [1], cell volume regulation [2], progression through the cell cycle [3], and growth factor signaling [4]. Two isoforms of the NKCC *
Corresponding author. Fax: +49-211-811-8838. E-mail address: [email protected] (D. H€aussinger). 1 Abbreviations used: NKCC, sodium potassium chloride cotransporter; HSC, hepatic stellate cells; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; SEC, sinusoidal endothelial cells; HPRT, hypoxanthine guanine phosphoribosyltransferase; BrdU, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay; DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; KC, Kupffer cell; SMA, smooth muscle actin; PDGF, platelet-derived growth factor; RVI, regulatory volume increase.
(NKCC1 and NKCC2) were cloned, which belong to the superfamily of the cation chloride cotransporters including NCC, KCC1, and KCC2 [5]. NKCC1 shares 58% amino acid identity with NKCC2 and is expressed in many tissues, whereas NKCC2 expression is restricted to the kidney [6]. Like NKCC2, the NKCC1 forms 12 membrane-spanning domains [1]. Recent studies identified the specific binding sites of the cations and chloride as well as bumetanide (e.g., Isenring and Forbush [7]) and provided evidence for a cooperative and ordered ion binding to the NKCC [8]. NKCC activity is regulated by phosphorylation, the cytoskeleton, and the intracellular chloride concentration and probably depends on homooligomerization; however, the precise mechanisms are still under discussion [1]. Mice bearing a NKCC1 knockout suffer from deafness, imbalance, and severe impairment of salivation [9,10]. Pharmacological evidence suggested the expression of NKCC transport activity in the liver. Thus, furosemide antagonized regulatory volume increase induced by hyperosmotic exposure of cultured hepatocytes [11].
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 0 4 7 - 4
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Inhibition of hepatic proteolysis by insulin and ethanol is sensitive to bumetanide, which impairs also cellular net Kþ uptake and associated cell swelling in response to insulin and ethanol [12,13]. Bumetanide-induced insulin resistance was also observed at the level of MAP-kinases [14], confirming the idea that cell swelling due to Naþ =Kþ =2Cl cotransport activation represents a critical step of insulin signal transduction in liver parenchymal cells [15–17]. Bumetanide was shown to prevent pinacidil-induced apoptosis in HepG2 human hepatoma cells [18] and to provide hepatic tolerance against ischemia/reperfusion [19]. Little is known about hepatic expression and regulation of the NKCC1 transport protein. Northern blot analysis unraveled a weak expression of the NKCC1 mRNA in human liver [20] but not in liver from rabbit [20] and mouse [21]. Although the NKCC1 has recently been cloned from rat parotid [22], its expression at the mRNA or protein level in rat liver is unknown. Moreover, nothing is known about the presence or function of Naþ =Kþ =2Cl cotransport in nonparenchymal liver cells. In the present study NKCC1 mRNA and protein were detected in different rat liver cell types. NKCC1 expression in hepatic stellate cells increased upon their transformation into myofibroblasts during culture. NKCC1 activity may delay initiation of HSC transformation by culture of the cells but did not contribute to proliferation of 14-day-cultured HSCs. Short-term hyperosmotic perfusion of rat liver induced a volumeregulatory potassium uptake, which was partially blunted by bumetanide. Long-term dehydration of HuH-7 cells by solutions made hyperosmotic by NaCl and raffinose stimulated NKCC1 expression, which was counteracted by the compatible organic osmolytes betaine and taurine. The data suggest a role of NKCC1 in short-term and long-term adaption of the liver to dehydrating conditions.
Materials and methods Materials RPMI 1640 and DMEM, glutamine, and fetal calf serum were from Biochrom (Berlin, Germany). Nycodenz was from Nycomed (Oslo, Norway). Pronase was from Merck (Darmstadt, Germany). L -Lactic acid was from Roth (Karlsruhe, Germany). Radiochemicals were from Amersham (Braunschweig, Germany). DNase I and collagenase were from Boehringer Mannheim. Horseradish peroxidase conjugated anti-mouse IgG antibody was from Bio-Rad Labs (Hercules, CA). RNeasy total RNA kit was obtained from Qiagen (Hilden, Germany). Betaine and taurine were from Sigma (Deisenhofen, Germany). Nitrocellulose membranes were purchased from Schleicher & Schuell
(Dassel, Germany). [a-32 P]dCTP was from Amersham. Polybead fluorescent microspheres (2.5% solids latex, 1.1 lm diameter) were obtained from Polysciences Ltd. (St. Goar, Germany). All other chemicals were from Merck (Darmstadt, Germany). Cell preparation and culture Treatments of rats. All animals received human care in compliance with the German Animal Protection Act, which is in accordance with the National Research Council’s criteria. Isolation and culture of parenchymal cells from rat liver. Isolated hepatocytes were prepared from livers of male Wistar rats by a collagenase perfusion technique as described [23]. A total of 1:5 106 cells were plated on collagen-coated culture dishes (36 mm diameter) and maintained in Krebs–Henseleit medium (115 mmol/L NaCl/25 mmol/L NaHCO3 /5.9 mmol/L KCl/1.18 mmol/ L MgCl2 /1.23 mmol/L NaH2 PO4 /1.2 mmol/L Na2 SO4 / 1.25 mmol/L CaCl2 ), supplemented with 6 mmol/L glucose at 37 °C and a 5% CO2 atmosphere. After 2 h, cells were washed twice and culture was continued for 24 h in 1.5 mL Williams medium E (Sigma, Munich, Germany), supplemented with glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (0.1 mg/mL), insulin (107 mol/L), dexamethasone (107 mol/L), and 5% fetal-calf serum (FCS). Hepatocyte viability was more than 95% as assessed by trypan blue exclusion. Isolation and culture of rat hepatic stellate cells. HSC from 1- to 3-year-old male Wistar rats were prepared as previously described [24] by collagenase/pronase perfusion and isolated by Nycodenz gradient. The cells were seeded at a density of 0:15 106 =cm2 on glass coverslips in a 24-well culture plate (Falcon) or 60-mm well (Falcon, Heidelberg, Germany) and maintained in DMEM containing 10% (v/v) heat-inactivated FCS and 1% (w/v) penicillin/streptomycin. Culture was performed in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The culture medium was changed after 24 h and thereafter every 2 days. The purity of HSC was >95%, as assessed 24 h after seeding by their typical light-microscopic appearance and vitamin-A-specific autofluorescence, their inability to phagocytose fluorescent 1.1-lm latex particles, and their positive immunofluorescent staining for desmin, GFAP, and, at the seventh day of culture, for a1 -smooth muscle actin. Isolation and culture of rat Kupffer cells and rat sinusoidal endothelial cells. Cells were isolated from 1-yearold male Wistar rats by collagenase–pronase perfusion and separated by a single Nycodenz gradient and centrifugal elutriation [25]. Kupffer cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS for up to 24 h. Sinusoidal endothelial cells (SEC) were plated on collagen 1-coated culture dishes and maintained in RPMI 1640 medium supplemented
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with 10% heat-inactivated FCS for 24 h. Culture was performed in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Purity of Kupffer cells (KC) was P98% as assessed after seeding by their typical light microscopic appearance, by immunological staining for ED2 (specific macrophages marker protein) [26], and by their ability to phagocytose fluorescent 1.1-lm latex particles. Purity of SEC was P80% as assessed 24 h after seeding by their typical light microscopic appearance and by their inability to phagocytose fluorescent 1.1-lm latex particles. HuH-7 and H4IIE cell culture. HuH-7 human hepatoma cells [27] and H4IIE-C3 rat hepatoma cells (ATCC CRL 1600) were maintained in DMEM–F12/ 5% CO2 =5 mM glucose at 37 °C, pH 7.4, supplemented with 10% FCS. When cells had reached 90% confluency, they were washed with Dulbecco’s PBS and the culture was continued in serum-free medium for an additional 24 h. Extracellular osmolarity was adjusted by dilution of the media with the appropriate volume of the respective NaCl-free medium leading to hypoosmolarity (205 mosmol/L) or with medium of elevated NaCl content leading to hyperosmolarity (405 mosmol/ L), respectively. In the normoosmotic control (305 mos mol/L), the same volume of normoosmotic medium was added.
RT-PCR Total RNA was extracted from guanidinium thiocyanate solutions [28] using the RNeasy TM total RNA kit (Qiagen) according to the manufacturer’s recommendations. cDNA was obtained from 1 lg total RNA with the kit from Roche Molecular Biochemicals (Mannheim, Germany), using 20 U reverse transcriptase from the avian myoblastosis virus and 1.6 lg oligop(dT)15 primer. The reaction mixture was incubated at 25 °C for 1 h and then at 42 °C for an additional 1 h. The reverse transcriptase was inactivated by a 5-min incubation at 99 °C. One-hundred nanograms of cDNA was PCR amplified by using the following primers: NKCC1 sense, 50 CCA GAT GTT TGC GAA AGG TT-30 ; NKCC1 antisense, 50 -CCC AGT TCA CAT CTG GCT TT-30 ; NKCC2 sense, 50 -CCG AGT TCG GTG GGT CAA TAG GCT-30 ; NKCC2 antisense, 50 -CTC CAG AGA TGT TGG CAC CAG CAA-30 , yielding 362- and 435-kb cDNA fragments representing NKCC1 and NKCC2, respectively. Sequence analysis with the Abi Prism 310 genetic analyzer (Perkin Elmer Applied Biosystems, Weiterstadt, Germany) confirmed the specificity of the PCR amplifications. The PCR reactions were performed in 50 lL of the following reaction mixture: 34.75 lL water, 5 lL reaction buffer ð10Þ5 lL 25 mmol=L MgCl2 , 1 lL 10 mmol/L dNTP mix, 1 lL 10 lmol/L primer sense, 1 lL 10 lmol/L primer anti-
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sense, 0.25 lL 5 U/lL Goldstar DNA polymerase (Eurogentec, Seraing, Belgium) and 2 lL cDNA. Regulation of the NKCC1 mRNA levels in HuH-7 cells was analyzed by means of semiquantitative PCR analysis according to Kinoshita et al. [29]. As an internal control a 415-kb fragment of the of the hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA was synthesized using the primers 50 -TGGT CGT CGT GAT TAG TGA TG-30 (sense) and 50 -CTG CAT TGT TTT GCC AGT GT-30 (antisense). Primers were chosen using the Primer3 program (http://www-genome.wi.mit.edu/ cgi-bin/primer/primer3.cgi) and purchased from the MWG Biotech AG (Ebersberg, Germany). For each primer set, an increasing number of PCR cycles with otherwise fixed conditions was performed to determine the optimal number of cycles to be used. The optimal number of cycles was determined as the midpoint of the exponential phase. The numbers of cycles were 35 for NKCC1 and NKCC2 and 30 for HPRT. Determination of hepatic stellate cell proliferation Hepatic stellate cell proliferation was addressed by measuring BrdU incorporation into DNA using the colorimetric BrdU cell proliferation ELISA kit (Roche Molecular Biochemicals) according to the manufacturer’s suggestions. Hepatic stellate cells plated on 96well dishes (50,000 cells per well) were cultured for 2 or 14 days in DMEM containing 1 or 10% FCS. For experimental treatment medium was removed and cells were incubated for an additional 48 h within the same medium plus 100 lmol=L BrdU in presence or absence of PDGF (25 ng/mL). In parallel experiments cells amiloride (100 lmol=L) or bumetanide (5 lmol=L) were present during the 48-h incubation period. Incorporation of BrdU into DNA was monitored at 450 nm using an peroxidase-coupled antibody and tetramethylbenzidine as a substrate with an ELISA reader (MRX microplate reader, Dynatec Laboratories, Denkendorf, Germany). Rat liver perfusion Livers from male Wistar rats (160–230 g), who were fed on a standard chow, were perfused in an open nonrecirculating manner as described previously [30] with bicarbonate-buffered Krebs–Henseleit saline as perfusion medium plus lactate (2.1 mM) and pyruvate (0.3 mM), gassed with O2 =CO2 (19/1, v/v). The average flow rate was kept constant throughout the individual perfusion experiment and was 3.5–4.5 mL/min/g. For changing the osmolarity, the NaCl concentration was varied, resulting in corresponding changes of osmolarity. Additions were made by dissolution in DMSO, which was present throughout control and inhibitor experiments at an infusion rate of 20 lL= min. Viability
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of the livers was assessed by monitoring effluent oxygen concentration and measurement of LDH leakage from livers, which did not exceed 15–25 mU/min/g liver. Effluent perfusate pH was monitored continuously with a pH-sensitive electrode and effluent perfusate oxygen concentration was monitored with a Clark-type oxygen electrode (Biolytik, Bochum, Germany). The perfusion pressure was detected by a pressure transducer (Hugo Sachs Electronics, Hugstetten, Germany). Basal portal pressure was 3–5 cm H2 O and was not affected by the compounds used in this study. Liver mass was recorded by placing the excised perfused liver on a specially constructed balance pan [31]. Changes of liver mass were expressed on a percentage basis and referred to liver wet weight at the end of the experiment, a time point when normoosmotic conditions had been installed for at least 30 min. The intracellular water space was calculated from the difference of washout profiles of simultaneously infused [14 C]urea and [3 H]inulin as described previously [32]. In fed animals, the cell water under control conditions was 547 11 ll=g ðn ¼ 16Þ. Effluent Kþ concentration was registered continuously with Kþ -sensitive electrodes (Radiometer, Munich, Germany) and calculations were made by planimetry. When present, a baseline drift was taken into account. Western blot At the end of the experimental treatment, the medium was removed and the cells were immediately lysed at 4 °C using 50 mmol/L Tris/HCl buffer (pH 7.2) containing 150 mmol/L NaCl, 40 mmol/L NaF, 5 mmol/L EDTA, 5 mmol/L EGTA, 1 mmol/L vanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.1% aprotinine, 1% Nonidet P-40, 0.1% sodium deoxycholate, and 0.1% SDS. The homogenized lysates were centrifuged at 20,000g at 4 °C and the supernatant was added to an identical volume of gel loading buffer containing 200 mmol/L dithiothreitol (pH 6.8). After heating to 95 °C for 5 min, the proteins were subjected to gel electrophoresis (50 lg protein/lane, 9% gel). Following electrophoresis, gels were equilibrated with transfer buffer (39 mmol/L glycine, 48 mmol/L Tris/HCl, 0.03% SDS, 20% methanol). Proteins were transferred to nitrocellulose membranes using a semidry transfer apparatus (Pharmacia, Freiburg, Germany). Blots were blocked in 5% bovine serum albumin containing TBST (20 mmol/L Tris/HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween 20) and then incubated overnight with the 1:1000-diluted T4 monoclonal antibody raised against the 310 C-terminal residues of the human colonic NKCC1 [33]. Following washing with TBST and incubation with horseradish peroxidase-coupled anti-mouseIgG antibody diluted 1:10,000 at room temperature for
1 h, the blots were washed three times and developed using enhanced chemiluminescent detection (Amersham). Statistics Results from n independent experiments are expressed as means SE. Results were compared using the Student’s t test: P < 0:05 was considered statistically significant.
Results NKCC1 expression in liver cells NKCC1 mRNA expression in isolated liver cell types was studied by RT-PCR as described under Materials and methods. As shown in Fig. 1A, NKCC1 mRNA expression was detected in parenchymal cells, macrophages (KCs), and SECs isolated from rat liver. NKCC1 mRNA was also expressed in HuH-7 human hepatoma cells and to a less degree in H4IIE rat hepatoma cells (Fig. 1A). NKCC2 mRNA was not detectable in the liver cell types investigated but, as expected, was expressed in rat kidney tissue (Fig. 1A). Western blot analysis (Fig. 1B) unraveled NKCC1 protein expression in rat liver tissue as well as in isolated rat liver parenchymal and sinusoidal endothelial cells. NKCC1 protein was barely detectable in Kupffer cells and in 1-day-cultured hepatic stellate cells. In addition, NKCC1 was expressed in HuH-7 human but not in H4IIE rat hepatoma cells. NKCC1 expression increases during transformation of rat hepatic stellate cells in culture Hepatic stellate cells transform from a ‘‘quiescent’’ to a myofibroblast-like ‘‘activated’’ phenotype during culture on uncoated plastic dishes (e.g., Peters-Regehr et al. 34]). This is accompanied by morphological changes and the expression of a1 -smooth muscle actin (aSMA) from day 3–15 of cultivation of culture (Fig. 1 in PetersRegehr et al. [34], Fig. 3A, this paper). As demonstrated in Fig. 2 by semiquantitative PCR and Western blot analysis, respectively, the NKCC1 mRNA and protein expression levels progressively increased from day 1 to day 9 of hepatic stellate cell culture. To examine a potential contribution of NKCC1 activity to aSMA expression, hepatic stellate cells were cultured for 1, 2, 3, 7, and 14 days in presence or absence of 5 lmol=L bumetanide. As shown in Fig. 3A, bumetanide accelerated the appearance of aSMA, which was present already at the second day of culture, whereas aSMA expression at later time points was largely independent from the presence or absence of bumetanide. This suggests a role
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Fig. 1. NKCC1 and NKCC2 expression in different liver cell types. Abbreviations: PC, parenchymal cells; SEC, sinusoidal endothelial cells; KC, Kupffer cells; HSC, hepatic stellate cells; L, liver; M, standard. Cells were lysed 24 h after isolation for mRNA or protein extraction, respectively. (A) NKCC1- and NKCC2 mRNA expression was addressed by means of RT-PCR in parenchymal, sinusoidal, Kupffer, and stellate cells from rat liver as well as in HuH-7 human hepatoma cells, H4IIE rat hepatoma cells, and rat kidney. (B) NKCC1 protein expression in rat liver and isolated rat liver parenchymal, siunusoidal, Kupffer, and stellate cells as well as HuH-7 human and H4IIE rat hepatoma cells was determined by Western blot analysis using the T4 monoclonal antibody. Variable posttranslational modifications may account for multiple immunoreactive bands. Representatives of three independent experiments are shown.
of NKCC1 in modulating the initiation phase of HSC transformation. PDGF represents a powerful mitogen for HSCs [34] and PDGF-induced activation of Naþ =Kþ =2Cl cotransport is associated with proliferation of vascular smooth muscle cells from hypertensive rats [35]. In order to investigate a potential involvement of the NKCC1 in hepatic stellate cell proliferation, the effect of bumetanide on basal and PDGF-induced proliferation of 14-day-old HSCs was examined. As shown in Fig. 3B bumetanide at a concentration of 5 lmol=L, which inhibits insulin-induced Kþ uptake in perfused rat liver [13], was without effect on both basal and PDGF-induced proliferation of HSCs being 14 days in culture. Similar results were obtained with proliferation studies in the presence of 1% FCS or with HSCs cultured for 4 days (not shown). Inhibition of HSC proliferation was also not found at higher bumetanide concentrations (not shown). However, in line with a recent report [36] inhibition of the Naþ =Hþ exchange with amiloride significantly reduced HSC proliferation in presence and absence of PDGF, respectively (Fig. 3B). This argues against a major role
of NKCC1 for proliferation of 14-day-old HSCs. Similar results were obtained for 2-day-cultured HSCs (data not shown). NKCC1-mediated K þ uptake contributes to the hyperosmolarity-induced regulatory volume increase in perfused rat liver The involvement of the NKCC1 in hyperosmolarityinduced regulatory volume increase (RVI) was examined in the perfused rat liver. Perfusion medium was switched from normo- to hyperosmolarity (305–385 mosmol/L) in presence or absence of bumetanide. Institution of hyperosmolarity by addition of NaCl led to a volumeregulatory net Kþ uptake, which is terminated within 30 min [37]. Thereafter, liver cells remained in a slightly shrunken state as shown by measurements of liver mass and cell water (Table 1). In presence of bumetanide (5 lmol=L), the volume-regulatory net Kþ uptake was inhibited by about 40%, and consequently the extent of cell shrinkage after completion of RVI was markedly enhanced (Table 1). These data suggest a significant contribution of the NKCC1-mediated Kþ uptake to
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Fig. 2. Transformation-dependent increase of NKCC1 expression in rat hepatic stellate cells HSCs were lysed at days 1, 4, and 9 after isolation. These time points represent progressive transformation of the stellate cells into myofibroblasts [33]. Data from three cell preparations are summarized within this figure. *Expression differs significantly ðP < 0:05Þ from that found in 1-day-cultured cells. (A) Dependence of NKCC1 mRNA in rat hepatic stellate cells on the duration of culture was estimated by semiquantitative PCR. The top panel shows results of a representative PCR experiment. (B) NKCC1 protein expression in hepatic stellate cells increased during culture of the cells was monitored by Western blot analysis. A representative Western blot is shown in the top panel.
regulatory volume increase induced by hyperosmolarity in perfused rat liver. Regulation of NKCC1 expression by osmolarity and osmolytes in HuH-7 cells Clinical settings such as sepsis, burn injury, and diabetes mellitus are often associated with hyperosmotic tissue dehydration [17,38,39]. HuH-7 cells were used to study NKCC1 expression under conditions of long-term hyperosmotic challenge. Hyperosmolarity was adjusted by elevation of the medium NaCl concentration. As shown in Fig. 4 a hyperosmotic (405 mosmol/L) exposure of HuH-7 cells induced an increase in NKCC1 mRNA expression, which was maximal between 8 and 16 h and declined thereafter. No significant changes in NKCC1 mRNA expression were observed under the control condition (305 mosmol/L). The osmolarity dependence of NKCC1 mRNA expression was studied at the 8-h time point (Fig. 4B). Hypoosmolarity was lar-
gely without effect on NKCC1 mRNA expression. However, NKCC1 mRNA expression was significantly elevated already at an osmolarity increase of 20 mosmol/ L and further increased by elevating the osmolarity up to 445 mosmol/L. Like NaCl, the impermeable osmolyte raffinose led to induction of NKCC1 mRNA expression (Fig. 5), indicating that the hyperosmotic effect on NKCC1 mRNA expression is triggered by cell shrinkage rather than by increased activities of extracellular Naþ and Cl . Increasing medium osmolarity to 405 mosmol/ L by mannitol, which slowly equilibrates in rat liver between extra- and intracellular compartments [40], induced a delayed NKCC1 mRNA expression after 24 h. Urea, a solute rapidly penetrating liver cells [32], was ineffective at modifying NKCC1 mRNA expression levels in HuH-7 cells. This indicates that cell shrinkage but not the osmolarity increase per se is the trigger for NKCC1 expression. Taurine and betaine are compatible organic osmolytes, which do not disturb protein function even at high concentrations and are involved in the long-term adaption to hyperosmolarity [41]. These osmolytes accumulate in liver cells due to cumulative uptake by specific transport systems [42]. When HuH-7 cells were allowed to accumulate taurine or betaine for a period of 12 h before switching to 405 mosmol/L with NaCl-enriched medium, the hyperosmotic increase in NKCC1 mRNA expression was suppressed and did not significantly differ from the control situation (305 mosmol/L in presence or absence of betaine or taurine, respectively; Fig. 6A). Under normoosmotic conditions betaine or taurine were ineffective to modulate NKCC1 mRNA expression. As shown in Fig. 6B the regulation of the NKCC1 by hyperosmolarity was roughly mirrored at the protein level. Taurine largely abolished hyperosmotic NKCC1 protein accumulation, whereas a delayed induction of NKCC1 expression occurred in presence of betaine. This may be due to the protein stabilizing property of this osmolyte on the one hand [43,44] and on the smaller intracellular accumulation of betaine compared to taurine in hepatocytes on the other hand [45]. The data suggest an involvement of the NKCC1 in long-term regulation of cell volume and intracellular osmolyte balance.
Discussion The present study demonstrates the expression of the Naþ =Kþ =2Cl cotransporter NKCC1 in rat liver parenchymal cells, sinusoidal endothelial and stellate cells. The NKCC1 was also found in human HuH-7 but not in rat H4IIE hepatoma cells. HSCs represent the principal fibrogenic cell type of the liver. Following liver injury or during culture they undergo activation, i.e., the transition of resting cells into proliferative, fibrogenic, and contractile myofibro-
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Fig. 3. Pharmacological characterization of NKCC1 function in rat hepatic stellate cells. (A) Culture-induced a1 SMA expression in HSCs exposed to bumetanide. Freshly isolated HSCs were cultured in presence or absence of 5 lmol=L bumetanide. After the indicated time periods cells were lysed. Cell lysates were analyzed for the presence of a1 SMA in Western blot. (B) Basal and PDGF-induced HSC proliferation in presence of bumetanide or amiloride. Proliferation of 14-day-old HSCs was measured by immunoperoxidase staining of nuclei that incorporated BrdU for 48 h in presence or absence of 25 ng/mL PDGF. In parallel experiments bumetanide (5 lmol=L) or amiloride (100 lmol=L) were present during the 48-h incubation period. Data from four different cell preparations are summarized. Control, bumetanide, amiloride, and PDGF absent; Bu, bumetanide; Am, amiloride. *Proliferation significantly ðP < 0:05Þ different from the control; # proliferation significantly P < 0:05 different from that observed in presence of PDGF in absence of amiloride and bumetanide.
Table 1 Hyperosmolarity-induced changes of liver mass and intracellular water space and cell-volume-regulatory Kþ uptake in perfused rat in absence and presence of bumetanide Parameter Change of intracellular water space (%) Change of liver mass (%) Cell-volume-regulatory Kþ uptake (lmol=g)
Bumetanide absent
Bumetanide present
8:1 0:9 ð4Þ
18:9 3:9 ð5Þ
6:9 0:5 ð6Þ
10:4 0:7 ð7Þ
þ13:0 0:6 ð8Þ
7:7 0:6 ð7Þ
Note. Livers from male, fed Wistar rats were perfused for 150 min. After a 90-min period of isoomotic equilibration (305 mosmol/L), livers were exposed to hyperosmotic perfusion medium (385 mosmol/L) for 30 min due to the addition of 40 mmol/L NaCl. If present, bumetanide (5 lM) was infused 30 min prior to installation of hyperosmolarity. The changes in intracellular water space, liver mass, and Kþ fluxes were determined as described under materials and methods. Numbers in brackets denote the number of different perfusion experiments. Data are shown as means SE. ; Significant difference from control in absence of bumetanide, P < 0:01, P < 0:05. Cell water under control conditions was 547 11 ll=g ðn ¼ 16Þ.
blasts [46]. In addition to a1 SMA and collagen the expression of the betaine transporter BGT-1 was identified as another marker of HSC transformation [34]. Similar to the BGT-1, NKCC1 mRNA and protein expression increased during the transformation process (Fig. 2), suggesting, that alterations of the intracellular osmolyte balance accompany HSC transformation. Bumetanide induced a1 SMA expression in HSCs at the second day of culture but did not further increase a1 SMA on later time points (Fig. 3A). Thus Naþ =Kþ =2Cl cotransport may antagonize the initiation phase of the transformation process. The underlying mechanism is currently unknown. One speculation is that NKCC1-mediated cell swelling may impair the generation of reactive oxygen intermediates, which triggers HSC activation. In this respect it is interesting to note that hypoosmotic swelling was shown to protect perfused liver from oxidative injury and enhances the generation of reducing equivalents via the pentose phosphate shunt [47], whereas hyperosmotic shrinkage produces oxidative stress [47,48]. Inhibition of NKCC1 by bumetanide may
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Fig. 4. Hyperosmotic stimulation of NKCC1 mRNA expression in HuH-7 human hepatoma cells NKCC1 mRNA expression levels were determined by semiquantitative RT-PCR. (A) Time course of hyperosmotic NKCC1 mRNA induction. HuH-7 cells were exposed to hyperosmolarity (405 mosmol/L) for the indicated time periods. Alternatively, the cells were maintained under the normoosmotic control condition (305 mosmol/L). Cells were lysed at the time points indicated. (B) Osmolarity dependence of NKCC1 mRNA expression. HuH-7 cells were exposed for 8 h to normoosmotic (305 mosmol/L) or anisoosmotic solutions. Then cells were lysed for RNA preparation. Results show means SE from three independent experiments. *Relative mRNA expression significantly different from the respective normoosmotic control. The top panels show the results of representative RT-PCR determinations.
shrink the HSCs leading to an amplification of the generation and action of reactive oxygen intermediates thereby accelerating the initiation of transformation. Interestingly in other cell types, cell shrinkage augments differentiation (reviewed in Lang et al. [16]).
NKCC1 activity seems not to stimulate basal or PDGF-induced proliferation of 14-day-old HSCs (Fig. 3B). However, as reported [36], inhibition of the Naþ =Hþ exchanger markedly interfered with HSC proliferation (Fig. 3B). Although a mitogen-stimulated
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Fig. 5. Differential effects of hyperosmolarity adjusted by raffinose, mannitol, urea, or NaCl on NKCC1 mRNA induction in HuH-7 human hepatoma cells HUH7 human hepatoma cells were exposed to hyperosmotic solutions adjusted to 405 mosmol/L by raffinose, mannitol, urea, or NaCl, respectively. Alternatively cells were maintained under the normoosmotic control condition. After 8, 16, or 24 h cells were lysed for RNA preparation. NKCC1 mRNA expression was determined by semiquantitative RT-PCR. The figure summarizes data from three different experiments. *Relative mRNA expression significant different from the respective normoosmotic control. The top panels show the results of representative RTPCR determinations. Control, normoosmotic control condition (305 mosmol/L).
rapid increase in intracellular Naþ or Kþ was recognized as an essential trigger for progression through the cell cycle [3], the relative contributions of Naþ =Kþ =2Cl transport and Naþ =Hþ exchange to generate this signal may depend on the expression levels of the respective transport systems, intracellular pH, and Kþ content and the mitogen-induced signals generated upstream of ion uptake [49]. Acutely hyperosmotically challenged liver cells shrink but simultaneously activate electrolyte uptake thereby preventing excessive shrinkage and performing a regulatory volume increase [16]. Bumetanide markedly increased hyperosmotic shrinkage, probably due to an impairment of the hyperosmolarity-induced volumeregulatory Kþ uptake, thereby reducing regulatory cell volume increase (Table 1). In perfused liver, however, no major contribution of Naþ =Kþ =2Cl cotransport to a ‘‘secondary’’ regulatory volume increase (i.e., the volume-regulatory response induced by normoosmotic exposure of hypoosmotically preperfused livers) could be shown [50,51]. These findings indicate a significant contribution of the NKCC1 in mediating a hyperosmolarity-induced ‘‘primary’’ regulatory volume increase in perfused liver, whereas in isolated hepatocytes only a minor contribution was reported [11]. The activation of
volume-regulatory ion transport depends on cellular interactions with the extracellular matrix and the cytoskeleton [52], which may explain the differences between perfused liver and cultured hepatocytes. The different roles of NKCC1 in ‘‘secondary’’ and ‘‘primary’’ regulatory volume increase can be explained by differences in the Naþ and Cl transmembrane gradients, which determine the activity and direction of NKCC1-mediated transport. Clinical settings such as sepsis, burn injury, and diabetes mellitus are often associated with hyperosmotic tissue dehydration, which contributes to catabolic protein metabolism and insulin resistance [17,38]. Induction of NKCC1 expression by chronic hyperosmolarity (Figs. 4 and 5) may play a role in long-term adaption to hyperosmotic stress. Hyperosmotic NKCC1 expression was also observed in Ehrlich ascites tumor cells [53] and corneal epithelial cells [54]. On the other hand, regulatory volume increase by ion uptake rises the intracellular ionic strength, which interferes with native protein structure and function [41]. Another cellular strategy to adapt to long-term hyperosmolarity is the isoosmotic exchange of ions against organic osmolytes, which are compatible with protein function even at high millimolar concentrations [42]. In the presence of betaine and
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Fig. 6. Modulation of hyperosmotic NKCC1 expression by betaine and taurine in HuH-7 human hepatoma cells. HuH-7 cells were exposed to 10 mmol/L betaine or taurine for 12 h. Then, medium osmolarity was increased to 405 mosmol/L by elevation of NaCl concentration for the indicated time periods. Alternatively, the normoosmotic control condition (305 mosmol/L) was adjusted for identical time periods. Thereby, the presence of betaine and taurine was maintained. Then, cells were lysed for RNA isolation (A) or protein extracts were prepared (B). NKCC1 transcript levels were determined by semiquantitative RT-PCR and NKCC1 protein expression was evaluated in Western blot. Three independent experiments were performed. *RT-PCR amplification significantly ðP < 0:05Þ different from the normoosmotic control; **significantly ðP < 0:05Þ different from the relative NKCC1 mRNA expression under hyperosmotic conditions in absence of betaine or taurine, respectively. C, control (betaine and taurine absent); B, betaine; T, taurine.
taurine this seems to represent a predominant adaption mechanism in HuH-7 cells as suggested by suppression of the hyperosmotic NKCC1 mRNA expression by these osmolytes (Fig. 5). Betaine and taurine abolish the hyperosmotic induction of cyclooxygenase-2 expression in Kupffer cells [55,56] and betaine accumulation substitutes for HSP70 expression in high-salt-exposed Madin–Darby canine kidney cells [57]. Preventing an increase in intracellular ionic strength and ameliorating proteotoxicity may blunt the trigger for stress gene expression induced by hyperosmotic shrinkage. In addition to its role in cell volume regulation NKCC1 is involved in the regulation of hepatic metabolism by insulin and ethanol [12,58]. Inhibition of insulin-induced Kþ -uptake by bumetanide or short-term hyperosmotic treatment induce insulin resistance in
perfused rat liver [13,14]. It seems attractive to speculate that the hyperosmotic stimulation of NKCC1-mediated volume-regulatory Kþ uptake (this paper) prevents further stimulation by insulin and this may account for the insulin resistance under these conditions. Increased NKCC1 expression levels may help to improve hepatic sensitivity to hormones and substrates under conditions of chronic dehydration. Further studies are required to define the functional importance of hepatic NKCC1 expression in parenchymal and nonparenchymal cells.
Acknowledgments This work was supported by the Sonderforschungsbereich 575 ‘‘Experimentelle Hepatologie’’ and the
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