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J Physiol Volume 558, Number 3, 759-768, August 1, 2004 DOI: 10.1113/jphysiol.2004.064311
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Expression of the sodium–myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells

Pierre Bissonnette, Michael J. Coady and Jean-Yves Lapointe

Groupe d'étude des protéines membranaires, département de physique, Université de Montréal, Canada


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Myo-inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na+-dependent transporters such as SMIT1. We have recently identified a second Na+–myo-inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin-Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo-inositol by this cell line is 20-fold larger than that seen in native MDCK cells. The affinity for myo-inositol of MDCK cells transfected with SMIT2 is slightly lower (Km= 334 µM) than that found in voltage-clamped Xenopus laevis oocytes expressing SMIT2 (Km= 120 µM). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2-transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5-fold when submitted to 24 h hypertonic treatment (+200 mosmol l–1). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.

(Received 15 March 2004; accepted after revision 2 June 2004; first published online 4 June 2004)
Corresponding author P. Bissonnette: Dép. physiologie, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, Canada, H3C3J7. Email: pierre.bissonnette{at}umontreal.ca


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Myo-inositol (MI) is the basic component of the inositol phosphates, which form major pathways in cell signalling. MI also acts as a compatible osmolyte when cells are challenged by variations in extracellular tonicity (Burg et al. 1997). Although this activity is of the greatest importance in the renal medulla, where the tonicity of the interstitial milieu fluctuates widely and constantly, other tissues such as brain, liver, fibroblasts and retinal endothelium also show a similar use of MI as an osmolyte when cell volume homeostasis is challenged (Berry et al. 1994; Weise et al. 1996; Karihaloo et al. 1997). It is believed that intracellular MI levels are controlled by regulation of MI transport across the plasma membrane (Strange et al. 1994; Weise et al. 1996; Burg et al. 1997). Net MI transport must also occur through the apical membranes of intestinal epithelia and renal proximal tubules since it is both absorbed from dietary sources and reabsorbed from the renal tubule lumen.

Plasma levels of MI are low (50 µM) compared to those reached within cells (up to 30 mM: see Dolhofer & Weiland, 1987; Schmolke et al. 1990). To achieve such high intracellular MI concentrations, cells rely on Na+-dependent cotransporters. Over the past decade, considerable effort has focused on the study of SMIT1, a MI cotransporter whose cDNA was originally cloned from the renal MDCK cell line (Kwon et al. 1992). This cell line has been useful in characterizing the basic features of this transport system and its regulation by tonicity. mRNA encoding this transporter has been detected in many tissues, including kidney (Kwon et al. 1992), and transport assays in MDCK cell cultures indicate that MI uptake occurs at the basolateral membrane (Yamauchi et al. 1991; Burg et al. 1997). Within both renal tissue and cell lines, the level of SMIT1 transcription is largely controlled by extracellular tonicity (Burg et al. 1997); there is also evidence of post-translational regulation of SMIT1 activity (Karihaloo et al. 1997; Yorek et al. 2000). In addition, longstanding data has indicated the existence of at least one other transport pathway in the kidney. Early reports identified MI transport in purified brush border membranes from kidney (Takenawa et al. 1977; Hammerman et al. 1980), which were substantiated by recent data showing MI transport in different tubule segments, including the proximal tubule (Silbernagl et al. 2003). Also, the TALH cell line, derived from Henle's loop, presents apical MI transport (Grunewald et al. 2001) while the liver cell line HepG2 displays MI transport with stereospecificity for D-chiro-inositol (DCI), which is not recognized by SMIT1 (Ostlund et al. 1996). It should be noted that altered transport and metabolism of DCI and MI are associated with several pathological conditions including type II diabetes (Mato et al. 1987; Larner, 2001; Barker et al. 2002), polycystic ovary syndrome (Nestler et al. 1999) and several psychiatric disorders such as panic disorders, obsessive compulsive disorder and manic depression, for which clinical trials based on inositol treatments are being pursued (Van Calker & Belmaker, 2000; Einat & Belmaker, 2001).

Recently, our laboratory determined that one orphan member of the Na+-dependent cotransporter family (SLC5) specifically transports MI (Coady et al. 2002). We have studied the expression of this protein (SMIT2) in Xenopus laevis oocytes, delineating the basic biophysical characteristics of this transport system. Since SMIT2 is strongly expressed in the kidney, amongst other tissues (brain, heart and liver: see Hitomi & Tsukagoshi, 1994; Roll et al. 2002), we decided to continue the study of this transporter using a kidney cell line transfected with the SMIT2 transporter as a more appropriate physiological environment.

In the past, we have successfully used the MDCK cell line to express the Na+–glucose cotransporter SGLT1 (Bissonnette et al. 1999), a protein closely related to SMIT2. This epithelial model is a well-established expression system which performs appropriate targeting of plasma membrane proteins, a prerequisite for the present study. Although MDCK displays SMIT1 activity, as do all other kidney-derived cell lines we have tested so far, this well-characterized cell line enabled expression of transfected SMIT2. We have selected a highly expressing stable SMIT2-MDCK transfectant (20-fold more activity than SMIT1) and assessed the functionality of this transporter, looking at its affinity for MI and at its polarity when cultured on semipermeable filters.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Oocyte preparation, injection and maintenance

Gravid Xenopus laevis (Connecticut Valley Biological Supply Co., Southampton, MA, USA) were anaesthetized with 2-aminobenzoic acid ethyl ester. Ovarian nodes were surgically removed and oocytes dissected by hand as previously described (Bissonnette et al. 1999). All manipulations (anaesthesia, surgery and killing of the animals after the final collection of oocytes) were performed in accordance with the Canadian guidelines and ethics committee from the Université de Montréal. After defolliculation with collagenase, the oocytes were rinsed and kept at 18°C in Barth's solution (mM: 88 NaCl, 3 KCl, 0.82 MgSO4, 0.4 CaCl2, 0.33 Ca(NO3)2 and 5 Hepes, pH 7.6) supplemented with horse serum (5%), sodium pyruvate (2.5 mM) and antibiotics (100 U ml–1 penicillin, 0.1 mg ml–1 streptomycin, 0.1 mg ml–1 kanamycin). After a 24 h recovery period, the oocytes were injected with mRNA encoding either canine SMIT1 (46 ng oocyte–1; kindly provided by Dr Moo Kwon, Johns Hopkins University) or rabbit SMIT2 (9 ng oocyte–1; Coady et al. 2002) and further incubated at 18°C until use (5–7 days).

MDCK culture and transfection

MDCK cells (strain I) were routinely grown in 100 mm Petri dishes using high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (Invitrogen) at 37°C in a 95% air–5% CO2 atmosphere. The media was changed every other day. Transfection was performed on 1-day-old MDCK cultures in 100 mm Petri dishes seeded at 5 x 105 cells per dish, which gives a coverage of about 40% of the Petri dish surface. Transfection was performed by calcium phosphate precipitation using the pMT21-SMIT2 vector (4 µg per Petri dish), encoding the rabbit SMIT2 protein, and the pcDNA3.1/neo vector (1 µg per Petri dish) to confer geneticin resistance as a selection marker. Two days after transfection, cells were incubated in low serum (0.5%) media supplemented with geneticin (Gibco, G-418 at 0.5 mg ml–1) to allow selection of transfectants. Four days after transfection, media serum was restored to 5% and geneticin was maintained thereafter. Control MDCK cells were transfected with the pcDNA3.1/neo vector alone to generate a cell population resistant to geneticin. After 1 passage, the SMIT2-transfected cells were subcloned in order to isolate high-expressing cells. Seventeen clones were selected, propagated and tested for the expression of SMIT2 activity. Two of these clones displayed SMIT2 transport activity, one with high expression and one with a modest activity level. The high-expression clone was used throughout the present study. For transport studies, cells were seeded either at 3 x 105 cells per 35 mm Petri dishes or at 2 x 104 cells per 12 mm filter (Costar). All uptakes were performed on 5- to 7-day-old cultures.

Isotopic uptakes

Oocytes.  SMIT1- and SMIT2-expressing oocytes (5–7 days incubation) were rinsed twice with substrate-free Barth's solution and transport was initiated by replacement with transport solution containing 10 µM MI along with radiolabelled MI (1 µCi ml–1[3H]MI, specific activity, 20 Ci mmol–1; ICN Biomedicals, Montreal, QC, Canada). The non-specific component of uptake was determined by similar incubation in media containing 10 mM cold substrate. Since 100 mML-fucose was used in one uptake condition, all other uptake media included a compensatory 100 mM mannitol. The oocytes were incubated in batches (8–10 oocytes) at room temperature for 30 min in 1 ml media. The incubation was stopped by rapid removal of radiotracer followed by addition of 2 ml ice-cold substrate-free media. The oocytes were further rinsed three times and transferred individually to scintillation vials. Digestion of the oocytes were performed by addition of 0.2 ml 10% SDS for 2 h prior to addition of scintillation cocktail (BetaBlend, ICN Biomedicals). The vials were measured for tritium content using an LS6000 SC scintillation counter (Beckman).

MDCK cells.  MI transport was tested using 5- to 7-day-old MDCK cultures (control or SMIT2-expressing cells) in 35 mm Petri dishes or filters. The uptake procedure was as previously described (Bissonnette et al. 1996). Briefly, Petri dishes were rinsed three times with Krebs solution (mM: 137 NaCl, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 10 Hepes, pH 7.2) and transport was initiated by replacement with Krebs solution containing substrate (1 µCi ml–1[3H]MI with 10 µM cold substrate, except for selection of transfectants where only radiolabelled tracer was used). Unless otherwise mentioned, all uptakes were performed in the presence of either 100 mML-fucose to minimize the SMIT1 component of transport or in the presence of 100 mM mannitol. The non-specific component of transport was measured in the presence of 10 mM cold substrate (+ 90 mM mannitol). All uptakes were performed at 37°C and, unless otherwise specified in the figure legends, for periods of 30 min. Uptakes were stopped by removal of substrate followed by rinsing four times with ice-cold Krebs solution. Tissues were digested for 2 h in 0.5 ml 1M NaOH and counted using BetaBlend as mentioned above.

Kidney BBMVs.  Rabbit brush border membrane vesicles (BBMVs) were prepared using a calcium precipitation technique (Bissonnette et al. 1996). The intravesicular composition was 400 mM mannitol, 50 mM Hepes, pH 7.5; BBMVs were either used immediately or maintained at –80°C until use. Transport media (150 mM NaCl or KCl, 100 mM mannitol or L-fucose, and 50 mM Hepes, pH 7.5) contained tracer quantities of [3H]MI (50 nM). As with uptake experiments on MDCK cells, L-fucose was added to the transport media in order to identify the SMIT2 specific activity. Transport was initiated by the addition of 50 µl BBMVs to 950 µl transport medium. At given times, aliquots of 100 µl were filtered on nitrocellulose filters (0.65 µm, Millipore) and rinsed with 5 ml ice-cold substrate-free transport media. Filters were dissolved in BetaBlend and tritium activity measured.

Antibody production and affinity purification

Anti-SMIT2 antibody was raised in rabbits (Sigma Genosys, The Woodlands, TX, USA) against a consensus amino acid sequence from the N-terminus of the SMIT2 protein of several species (rabbit, mouse and human) aligned using ClustalX with default settings (Thompson et al. 1997). The peptide epitope contains 18 residues (MESSTSSPQPPQSDP) and care was taken to avoid any significant similarity to related transporters such as SMIT1 or to SGLT sequences obtained from diverse species. Following three inoculations, the rabbit was exsanguinated and total serum was purified by immunoaffinity against the antigenic peptide using Affi-Gel-15 as support (Biorad, Mississauga, ON, Canada). The purified antibody was tested for positive labelling using transfected MDCK cells along with non-transfected MDCK cells as well as by peptide displacement assay.

Purification of membranes from MDCK cells and Western blot detection

Western blots were performed on BBMVs purified from MDCK cells. MDCK cells, 7 days old, grown on 550 cm2 plates, were rinsed three times with cold phosphate-buffered saline solution (PBS), and scraped and processed for BBMV purification in PBS in the presence of protease inhibitors (Sigma) as mentioned above for kidney. The final BBMVs were resuspended in PBS with protease inhibitors and kept at –20°C until use. Samples (25 µg protein) were tested by Western blot detection using methods already described (Bissonnette et al. 1999) and for all assays, the validity of electrophoresis and transfer procedures were monitored using Ponceau S staining of the nitrocellulose membrane (Klein et al. 1995). Briefly, non-specific binding to nitrocellulose transfer membranes was blocked by incubation in TBS-T (Tris-buffered saline + Tween 1%) with 5% non-fat milk (30 min) and incubated overnight at 4°C in the same solution containing anti-SMIT2 antibody (1: 100) (see below). After 4 rinses (4 x 15 min), the membranes were again blocked with TBS-T–milk and further incubated for 1 h at room temperature with secondary antibody (donkey anti-rabbit–horseradish peroxidase (HRP) coupled, 1: 25 000). The membranes were rinsed another four times and HRP activity was detected using enhanced chemiluminescence detection (Phototope-HRP, New England Biolabs).

Data analysis

Values for MI uptakes are given as pmoles or nmoles per milligram of protein. Protein content was determined using the BCA assay (Pierce). Determination of kinetic parameters was performed by fitting data to the Michaelis-Menten equation containing a non-specific component of uptake using Origin 6.1 software (OriginLab Corp., Northampton, MA, USA). Evaluation of the Ki value for L-fucose was performed by using a competitive inhibition equation containing a non-specific component of uptake, also using Origin software. The uncertainties described for the calculated parameters represent the accuracy of the fitting procedure determined by the software. Evaluation of Western blot intensities was performed by spot densitometry analysis using an Alpha Imager 2200 (Alpha Innotech Corp, San Leandro, CA, USA) and values represent integrated density units (IDU). All experiments were performed at least three times on different cultures.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Discrimination between SMIT1 and SMIT2 activities

MI transport is found in nearly all cell lines and is particularly well characterized in MDCK cells; discrimination between the activities of the endogenous SMIT1 and heterologously expressed SMIT2 is thus required for this study. Previous studies have shown that L-fucose may be used to distinguish between the two MI transporters since this sugar effectively inhibits SMIT1 without significantly affecting SMIT2 (Hager et al. 1995; Coady et al. 2002). This discriminative effect of L-fucose is illustrated in Fig. 1 where Xenopus laevis oocytes injected with either SMIT1 or SMIT2 cRNA were tested for MI uptake in the presence or absence of 100 mML-fucose. The non-specific uptake is determined by assay in the presence of excess cold substrate (10 mM MI: equivalent to 100–200 times the Km value determined for MI on oocytes; see Hager et al. 1995; Coady et al. 2002). While 100 mML-fucose abolishes nearly all specific MI uptake in SMIT1-expressing oocytes, the sugar does not significantly alter MI uptake in SMIT2-expressing oocytes. This sensitivity of SMIT1 to L-fucose inhibition can also be observed in MDCK cells. L-Fucose inhibition of MI uptake was measured with non-transfected MDCK cells. As shown in Fig. 2, the specific uptake of 10 µM MI is blocked by increasing concentrations of L-fucose (up to 100 mM). The analysis of MI uptake inhibition by L-fucose was performed using the competitive inhibition equation, which gave an estimated Ki value of 4.8 ± 1.0 mM. For this evaluation, a Km value of 130 µM for MI was used (Kitamura et al. 1997). The presence of 100 mML-fucose was consequently used routinely to permit the specific measurement of SMIT2-mediated MI uptake both for the selection of stable transfectants and for further characterization of SMIT2 transport in this cell line. Saturation with this competitive inhibitor enables strong SMIT1 blockade in routine assays (10 µM MI). When high MI concentrations are needed, as for measuring SMIT2 kinetics, the L-fucose inhibition of SMIT1 activity is less effective (70% inhibition at 1 mM MI and 31% at 5 mM MI). Nevertheless, in this cellular system where SMIT1 only accounts for 5% of total MI uptake activity, the contamination of SMIT2 uptake by residual SMIT1 uptake is negligible and thus does not significantly affect the kinetic evaluation.



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  		Figure 1.  Myo-inositol (MI) uptakes in SMIT1- or SMIT2-expressing oocytes
Oocytes were injected with mRNA encoding either dog SMIT1 (46 ng) or rabbit SMIT2 (9 ng) and were tested for MI (10 µM) accumulation 5–7 days after injection; the uptakes were for 30 min in Barth's solution in the presence or absence of 100 mML-fucose (to discriminate between SMIT1 and SMIT2) or with 10 mM MI for evaluation of non-specific background. Mannitol was used to replace L-fucose. Uptake through SMIT1 was determined by subtraction of the uptake in the presence of L-fucose from that in the absence of L-fucose. SMIT2 activity was determined by subtraction of the uptake in the presence of 10 mM MI from that in the presence of L-fucose. Data are mean ±S.E.M. from 5–8 oocytes.

 


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  		Figure 2.  Inhibition of SMIT1 by L-fucose in MDCK cells
Confluent MDCK cells (days 5–7) were tested for uptake of MI (10 µM) in the presence of increasing concentrations of L-fucose (0–100 mM). Cells were incubated for 30 min at 37°C in a modified Krebs solution containing L-fucose or mannitol in order to maintain equivalent osmotic conditions for all assays (+ 100 mM). Data are mean ±S.E.M. of 5 replicates. The Ki value for L-fucose of 4.8 ± 1.0 mM was determined by fitting the data to a competitive inhibition equation including a non-specific component of uptake.

 
Characterization of SMIT2 activity in transfected MDCK cells

Evaluations of SMIT2 transport activities in transfected MDCK cells were determined in the presence of 100 mML-fucose, as mentioned above. As shown in Fig. 3, we were able to isolate (from 17 clones) one stable transfectant expressing a high level of SMIT2 activity (clone 2). The level of SMIT2-mediated MI uptake by this clone exceeds that of resident SMIT1 activity 20-fold, which identifies it as a very good candidate with which to study SMIT2. A second clone with a more modest expression of SMIT2 (equal activity levels for SMIT1 and SMIT2) was also isolated but was not used further in this study (clone 1). The level for MI transport in the SMIT2-MDCK clone 2 was shown to be stable throughout successive passages (from passages 1 to 15).



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  		Figure 3.  MI uptakes in SMIT2-MDCK clones and parental cell line
SMIT2-transfected MDCK cells were subcloned and 17 colonies were isolated and tested for their SMIT1 and SMIT2 activities using the L-fucose criteria (as shown in Fig. 2 and Methods). Confluent monolayers were incubated with radiolabelled MI (50 nM) for 30 min at 37°C. The SMIT2-specific uptake was determined by incubation in the presence of 100 mML-fucose (defined as L-fucose-resistant system) while the SMIT1 fraction was determined by subtraction of uptake in the presence of 100 mML-fucose from that of total uptake (thus representing the L-fucose-sensitive system). This figure presents data for the parental cell line (Ctrl) as well as for the two positive clones identified. Clone 1 gave similar SMIT1 and SMIT2 activities while clone 2 presents 20 times more SMIT2 activity than SMIT1. Data are mean ±S.E.M. of 5 replicates.

 
The time course of MI accumulation into SMIT2-MDCK cells is presented in Fig. 4A in the presence of 100 mML-fucose and 10 µM MI. The curve displays linearity of uptake for at least 60 min. In comparison, control MDCK cells accumulated only 5% of the amount of MI found in SMIT2-MDCK cells under similar conditions. Kinetic studies were thus performed using a 30 min incubation period. Determination of kinetic parameters for SMIT2 was performed using MI concentrations varying from 0.05 to 5000 µM. As shown in Fig. 4B, the data describe a typical Michaelis-Menten curve with a Km value of 334 ± 6 µM and a Vmax of 17.4 ± 0.2 nmol (mg protein)–1 (30 min)–1 (one other Km determination performed on a different subculture gave 241 ± 26 µM). The Eadie-Hofstee transformation presented in the inset clearly shows linearity of data, compatible with the presence of a single transport system.



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  		Figure 4.  SMIT2 kinetics in transfected MDCK cells
A, accumulation over time of MI via SMIT2 in control and in SMIT2-MDCK cells. Confluent cultures of both control ({blacktriangleup}) and SMIT2-MDCK ({blacksquare}) cells were incubated with 10 µM MI, in the presence of 100 mML-fucose to inhibit SMIT1, for periods up to 180 min. MI uptake into SMIT2-MDCK cells was found to be linear for at least the first 60 min of incubation (dashed line). At 60 min, the accumulation of substrate in SMIT2-MDCK was 19-fold that of control cells. B, determination of kinetic parameters for SMIT2 in transfected MDCK cells. SMIT2-transfected MDCK cells were incubated for 30 min at 37°C with 25 nM tritiated MI along with increasing concentrations of cold MI (0–5000 µM). Media also contained 100 mML-fucose to eliminate SMIT1 transport. The data were fitted with the Michaelis-Menten equation including a non-specific component of uptake. Kinetic parameters are Km= 334 ± 7 µM and Vmax= 17.4 ± 0.2 nmol (mg protein)–1 (30 min)–1. The inset illustrates an Eadie-Hofstee transformation of data corrected for the non-specific component of uptake. Data for both experiments are mean ±S.E.M. of 5 replicates.

 
Control and SMIT2-MDCK cells were also used to evaluate the polarity of SMIT2 expression by culturing these epithelial cells on filters. As shown in Fig. 5, no L-fucose-resistant transport of MI is found at the apical domain of control cells while SMIT2-transfected cells present a large MI uptake at this surface. MI transport through the basolateral surface displays a modest L-fucose-insensitive uptake both in control and in SMIT2-MDCK cells, which corresponds to about 6% of the corresponding transport activity found at the apical surface of transfected cells.



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  		Figure 5.  Polarization of MI transport in control and in SMIT2-transfected MDCK cells
Control and transfected cells cultured on semipermeable filters until confluence (7 days) were incubated for 30 min at 37°C with 10 µM radiolabelled MI at either the apical or basolateral side. Media contained 100 mML-fucose to eliminate SMIT1 activity and non-specific uptake measured in the presence of saturating MI (10 mM) was subtracted from total uptake values. Data are presented as picomoles per filter per 30 min and represent mean ±S.E.M. of 4 replicates.

 
In accordance with the apical localization of SMIT2 in transfected MDCK cells, Western blot detection of this transporter in purified brush border membrane vesicles from these cells shows a unique band at 66 kDa (Fig. 6, lane 2). Interestingly, a much fainter band of similar molecular mass is also found in the control cells (lane 1). Signals from both cell types are completely displaced when incubated with antibody preadsorbed with peptide.



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  		Figure 6.  Detection of SMIT2 on Western blot
Western blot detection was performed on purified brush border membranes (25 µg protein per lane) of both control (lanes 1 and 3) and SMIT2-transfected (lanes 2 and 4) MDCK cells. After SDS-PAGE and transfer onto nitrocellulose (see Methods), the membrane was blotted with anti-SMIT2 antibody 1/100 in the presence (Ab + peptide) or absence (Ab) of peptide. A single band was found in SMIT2-MDCK cells at 66 kDa (lane 2), which was completely displaced when the antibody was incubated in the presence of peptide. A similar but faint signal was often found in control cells (lane 1).

 
Hypertonicity is a key regulator of MI transport. Consequently, we tested SMIT2 activity in cells that had previously been challenged (24 h) by incubation in the presence of hypertonic media (+ 200 mM raffinose). SMIT2-mediated MI transport (L-fucose resistant) was found to be very low in control MDCK cells (12 ± 2 pmol (30 min)–1) but was stimulated by hypertonicity (87 ± 2 pmol (30 min)–1). In SMIT2-MDCK cells, the SMIT2-specific signal is stimulated 5-fold when submitted to hypertonic media (from 441 ± 23 to 2144 ± 159 pmol (30 min)–1; Fig. 7). We also examined whether the increased transport activity was reflected in the amount of SMIT2 protein present in the apical membrane. Figure 8 shows a Western blot performed on BBMVs purified from both control and SMIT2-MDCK cells which were treated as described in Fig. 7. Evaluation of bands by densitometry revealed that treatment in hypertonic media increases the amount of SMIT2 by 70 ± 25% in transfected cells while in control cells a faint signal can only sometimes be detected under hypertonic conditions (values are mean ±S.D. of three determinations).



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  		Figure 7.  Effect of hypertonicity on SMIT2 activity
Control and SMIT2-transfected MDCK cells were incubated for 24 h in hypertonic culture media (+ 200 mM raffinose) prior to transport assay. MI uptakes were performed using normal transport media in the presence of 100 mML-fucose to isolate SMIT2 activity or in the presence of 10 mM MI to evaluate the non-specific fraction of uptake. Data are mean ±S.E.M. of 5 replicates.

 


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  		Figure 8.  Evaluation of SMIT2 in MDCK cells challenged by tonicity
Western blot detection was performed on purified brush border membranes (25 µg protein per lane) of both control (lanes 1 and 2) and SMIT2-transfected (lanes 3 and 4) MDCK cells. Cells were incubated for 24 h in isosmotic (lanes 1 and 3) or hyperosmotic (lanes 2 and 4) conditions prior to purification of membranes. After SDS-PAGE and transfer onto nitrocellulose (see Methods), the membrane was blotted with anti-SMIT2 antibody 1/100. Evaluation of bands was performed by spot densitometry and for lanes 1–4, respectively, were: not detectable, 4000, 67 000 and 113 000.

 
MI transport in purified kidney brush border membrane vesicles

Since SMIT2 is located apically in MDCK cells, we sought to determine whether this transporter could be found in the apical membrane of renal proximal tubules by measuring MI transport in purified brush border membranes of rabbit kidney. As shown in Fig. 9A, Na+-dependent MI transport is achieved with a typical overshoot profile ({blacksquare}). When sodium is replaced by potassium, MI uptake is dramatically reduced and the overshoot is completely abolished ({blacktriangleup}). In the presence of 100 mML-fucose, the specific accumulation of tracer amounts of MI (50 nM) is reduced by 45% after a 1 min uptake, indicating the possible coexistence of two MI transport pathways, one compatible with SMIT1 and the other with SMIT2 (Fig. 9B).



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  		Figure 9.  Uptake of MI into rabbit brush border membrane vesicles (BBMVs)
A, prolonged uptake of MI into rabbit BBMVs. Rabbit BBMVs (50 µl) were incubated with 950 µl media containing either NaCl ({blacksquare}) or KCl ({blacktriangleup}) along with tracer MI (50 nM). Aliquots of 100 µl were taken at various times, filtered and rinsed using 5 ml ice-cold substrate-free media. Data are mean ±S.E.M. of triplicates. B, brief uptakes of MI into rabbit BBMVs. Rabbit BBMVs (50 µl) were incubated with 950 µl media containing either 100 mM mannitol ({blacksquare}, total uptake), 100 mML-fucose (•, SMIT2-specific fraction of uptake) or 10 mM MI with 90 mM mannitol ({blacktriangleup}, showing the non-specific uptake) along with tracer MI (50 nM). Aliquots of 100 µl were removed, filtered and rinsed using 5 ml ice-cold substrate-free media. Data are mean ±S.E.M. of triplicates.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Most mammalian cells accumulate MI via Na+-dependent transport systems. Many studies, generally using cell lines, have measured this transport but most have solely associated it with SMIT1 activity (Strange et al. 1994; Weise et al. 1996; Kitamura et al. 1997). As there are now known to be at least two different Na+–MI cotransporters, it is necessary to distinguish between their biological activities by relying on specific inhibitors or substrates. Previous studies in oocytes have shown the existence of one specific substrate for SMIT1 (L-fucose: see Hager et al. 1995) and one for SMIT2 (D-chiro-inositol (DCI): see Coady et al. 2002). Measuring the uptake of individual substrates is an ideal situation for dual monitoring of SMIT1 and SMIT2. Unfortunately, the absence of a reliable source of radiolabelled DCI necessitated the use of an alternate strategy for studying SMIT2, consisting of blocking SMIT1 through saturation by L-fucose. As shown in Fig. 2, L-fucose efficiently inhibits the uptake of MI (10 µM) through SMIT1 in MDCK cells (Ki= 4.8 mM) and the presence of 100 mM of this sugar eliminates practically all of the MI transport through SMIT1. More precisely, for most of the studies described here on MDCK cells, 10 µM MI is used as a standard substrate concentration and the residual SMIT1 uptake found in the presence of L-fucose represents 5% of the SMIT1 uptake measured in the absence of L-fucose (evaluated with a MI Km value for SMIT1 of 130 µM in MDCK cells, and an L-fucose Ki value of 4.8 mM). L-Fucose does not seem to affect SMIT2 function, as shown in oocytes (Fig. 1). The presence of 100 mML-fucose was thus established as a standard condition for measuring the activity of SMIT2 in all studies. The effectiveness of this approach is presented in Fig. 3 where the uptake of MI into two SMIT2-MDCK clones is compared to uptake into control cells. In control MDCK cells, a basal activity level is found for SMIT1 that is unaltered in the two clones selected. As already mentioned, only one of the 17 clones tested displayed a high expression level of SMIT2, reaching 20-fold that of SMIT1. In this clone, using a medium containing 100 mML-fucose, nearly all of the MI accumulation is mediated though SMIT2 since SMIT1 represents less than 5% of total MI transport activity and L-fucose inhibits more than 95% of this activity. Under these conditions, the transport of MI is virtually all mediated through SMIT2.

The SMIT2 Km value for MI obtained in MDCK cells is slightly higher than that previously determined in voltage-clamped oocytes (Coady et al. 2002). This discrepancy may be explained by the differences in expression systems and experimental procedures. Km values for a single transport system may fluctuate due to parameters such as unstirred layers, geometry of tissue and variation of transport velocity (Winne, 1973). Furthermore, electrophysiological studies are generally performed at a constant membrane potential whereas isotopic uptakes are not. The electrogenicity of SMIT2 will induce membrane depolarization which, in turn, has been shown to increase the Km value for MI (Coady et al. 2002). Thus, the discrepancy found for Km values between expression in oocytes and transfected MDCK cells may well be explained by such factors and should not be interpreted a priori as a difference in substrate affinity of the SMIT2 protein in oocytes in comparison to MDCK cells.

One very important question about the function of SMIT2 concerns its targeting in polarized cells. As mentioned earlier, SMIT1 has been shown to be located at the basolateral domain of epithelial cells (Yamauchi et al. 1991) but some reports have described the existence of an apical transport system for MI (Takenawa et al. 1977; Grunewald et al. 2001) which matches the location of transfected SMIT2, as demonstrated in Fig. 6. Recently, Silbernagl et al. (2003) presented clear evidence, using micropuncture in rat kidney, of a MI transport system insensitive to luminal L-fucose in the proximal tubule. This is inconsistent with SMIT1 expression, based on Northern blots, which locates SMIT1 in renal medulla but not in renal cortex (Kwon et al. 1992). Our studies using rabbit BBMVs corroborate the finding that an L-fucose-resistant transport system is present at the apical side of proximal tubules. Na+-dependent transport is observed in this purified preparation (Fig. 9A), which displays both a fucose-sensitive and a fucose-insensitive MI transport, consistent with the coexistence of SMIT1 and SMIT2 in the apical membrane with similar levels of activity (Fig. 9B).

Measurements of L-fucose-independent MI uptake into MDCK cells on semipermeable filters show that SMIT2 is targeted to the apical membrane (Fig. 5). The modest basolateral MI uptake found in both the control and SMIT2-transfected MDCK cells may suggest basolateral targeting of SMIT2, but most probably represents SMIT1 activity that is not completely inhibited by 100 mML-fucose. It should be noted that this activity is detected only when MI is present at the basolateral side of the monolayer cultured on filters and is never seen when uptake is measured on regular Petri dishes. MI has only poor access to the basolateral surface of the monolayer in such cultures while the free access permitted with filters greatly increases the uptake of MI through basolateral SMIT1.

Western blot detection of SMIT2 using purified brush border membranes of transfected MDCK cells shows a single band of 66 kDa (Fig. 6). It is interesting that a faint band of identical molecular mass is sometimes but not always found in control cells, which is unexpected since no MI uptake characteristic of SMIT2 is detected with these cells. It is not currently possible to explain the function of this endogenous SMIT2. It is not known whether this SMIT2 signal actually originates from BBMVs or if it consists of contaminating intracellular structures that are present in the purified brush border membrane fraction. Further studies are needed in order to clarify the issue of native SMIT2 expression in MDCK cells.

As MI is an important metabolite in osmotic regulation, many studies have examined the regulation of MI transport in cells challenged by anisotonic extracellular solutions. We submitted both control and SMIT2-MDCK cells to hypertonic shock by adding 200 mM raffinose to the culture media for 24 h prior to transport assay. Under these conditions, SMIT2-mediated MI transport was found to be stimulated about 5-fold over isotonic media while the very weak SMIT2 uptake found in control cells was also increased. The low, residual SMIT1 activity, which remains in the presence of 100 mML-fucose and contaminates that of SMIT2, is negligible under isotonic conditions but becomes more substantial when hypertonic conditions are imposed. Under hypertonic conditions, we evaluate that about half of the MI uptake in control cells attributed to SMIT2 is actually mediated by SMIT1. On the other hand, such contamination is minimal when testing SMIT2-MDCK cells. Although such an increase in activity might be expected in a cell endogenously expressing SMIT2, we were surprised to detect it in a transfected cell. By evaluating the amount of SMIT2 protein present at the apical membrane, using densitometry of Western blots on purified MDCK BBMVs, we have only found a 70 ± 25% increase (average of three determinations), which only partially explains the 5-fold increase in transport activity. It is thus expected that the regulation of SMIT2, at least in this transfected cell line, will be a somewhat complex feature. Further studies are needed to delineate the mechanism(s) responsible for the regulation of SMIT2. While transcriptional aspects of SMIT2 regulation may not be studied with the SMIT2-transfected MDCK cells, this system will be useful for examining post-translational events related to the protein's activity. The existence of an apical MI transporter was expected based on published reports (Takenawa et al. 1977; Grunewald et al. 2001; Eladari et al. 2002; Silbernagl et al. 2003) and future studies will have to examine the expression topology of this new SMIT2 system along the nephron.

In summary, we find that SMIT2 is apically targeted in MDCK cells, which is consistent with studies in BBMVs where L-fucose-resistant MI transport is observed. The SMIT2 activity is shown to be stimulated by 24 h exposure to hypertonic conditions. We conclude that SMIT2 is likely to be involved in MI reabsorption in, at least, the proximal tubule.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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