Am J Physiol Cell Physiol Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 295: C791-C799, 2008. First published July 23, 2008; doi:10.1152/ajpcell.00390.2007
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/3/C791    most recent
00390.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bissonnette, P.
Right arrow Articles by Lapointe, J.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bissonnette, P.
Right arrow Articles by Lapointe, J.-Y.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Effects of hyperosmolarity on the Na+-myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line

Pierre Bissonnette,1,2 Karim Lahjouji,1,2 Michael J. Coady,2 and Jean-Yves Lapointe2

Groupe d'étude des protéines membranaires (GÉPROM), Departments of 1Physiology and 2Physics, University of Montreal, Montreal, Quebec, Canada

Submitted 29 August 2007 ; accepted in final form 17 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral Na+-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter (Na+-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment.

myo-inositol transport; regulation; hyperosmolarity; Madin-Darby canine kidney cells

MYO-INOSITOL (MI), ALONG WITH other compatible osmolytes, such as betaine and taurine, is accumulated intracellularly in response to increases in surrounding milieu tonicity (7, 14, 16). This compensatory behavior is of the utmost importance in organs in which cell volume is tightly regulated, such as brain and kidney (8, 22). This physiological response has been well demonstrated in cell cultures, such as the Madin-Darby canine kidney (MDCK) and inner medullary collecting duct (IMCD) cell lines, which are derived from kidney. When submitted to hyperosmotic stress, elicited by addition of osmolytes, such as raffinose or NaCl, MDCK cells upregulate many specific proteins, including heat shock protein (HSP70) (13), HSP27, F-actin (38), and key transport systems, such as aquaporins (AQP) (35), amino acid system A (11, 21), Na+-MI cotransporter (SMIT1), betaine transporter (BGT1), and taurine transporter (15, 16, 40). These systems are directly involved in a compensatory response that prevents nonphysiological increases in intracellular ionic strength that could ultimately lead to apoptosis (20).

This adaptive response has been shown essentially to rely on transcriptional modulation of target genes (49, 53). Previous studies, focusing on cell response to hyperosmotic treatment, established the involvement of mitogen-activated protein kinases (MAPK) such as ERK, p38 kinase, and JNK (3, 48, 50, 54). In kidney cell models, such as IMCD and MDCK, the hyperosmotic induction of these MAPK can be achieved using nonpermeant osmolytes, such as mannitol, raffinose, or NaCl, while permeant osmolytes, such as urea, are usually without significant effect (3, 41, 50, 54). More recently, the transcriptional activator, tonicity-responsive enhancer binding protein (TonEBP), expressed in kidney (24, 37) among other tissues, has been shown to be a crucial mediator involved in the activation of many of these transporters (23, 42, 46). In response to hyperosmotic challenge, TonEBP levels are increased, and the protein is mobilized to the nucleus to increase synthesis of target proteins, such as those mentioned above (10, 17, 18, 32, 51). Furthermore, by means of specific inhibitors, modulation of TonEBP has been linked to p38 (27, 44). MDCK cells have been of great use in the study of TonEBP (36, 37, 40, 41, 51), as well as for the cloning and characterization of the SMIT1 protein, which mediates MI transport (2, 29, 39, 51). Still, it should be noted that the cellular response to hypertonic shock, as determined in cell culture studies by induction of various transport systems, displays a biphasic time response (11, 18, 25, 26, 45), which suggests the existence of at least one other regulatory pathway aside from TonEBP.

In 2002, our laboratory identified a second SMIT, SMIT2 (12), which is expressed in the brain and kidney (19, 43), as is SMIT1. Basic transport features have been determined for SMIT2 using both Xenopus laevis oocytes and stably transfected MDCK cells as expression systems (4, 6, 12). The SMIT2 protein shares many functional similarities to SMIT1, including comparable affinities for MI (12) and a 2 Na+-1 MI transport stoichiometry (6). Still, some notable differences do exist, which include plasma membrane targeting: SMIT1 is basolateral and involved in the osmolarity-dependent MI uptake, whereas SMIT2 is apical in both transfected MDCK cells and rabbit proximal convoluted tubules (4) and is believed to mediate MI uptake from the ultrafiltrate (31). Substrate specificities [L-fucose for SMIT1 and D-chiro-inositol for SMIT2 (12)] enable us to functionally identify each transporter in systems where they coexist (4). MDCK were first considered for SMIT2 transfection, since this cell line offers a kidney phenotype, including cell polarity, as well as very weak levels of endogenous SMIT2 activity (4). The stably transfected MDCK-SMIT2 cell line was primarily designed to characterize SMIT2's functionality within a mammalian renal cell system. Since SMIT2 is essentially expressed through an exogenous vector in these cells, its relevance in regulation studies should concern posttranscriptional events, such as targeting, protein recycling and stability, and protein-protein interactions. A recent study has shown the impact of hyperosmotic conditions in the plasma membrane targeting of the BGT using a stably transfected MDCK cell (26). We thus sought to determine whether a similar regulatory pathway could also affect SMIT2 transfected in the same cell line. Surprisingly, the response of SMIT2 to hyperosmotic stress in this MDCK-SMIT2 cell essentially relied on transcriptional regulation. We further characterizes the response of exogenous SMIT2 to hyperosmotic stress, looking both at mRNA and protein content changes, compared with those of the endogenous SMIT1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. MDCK cells type I [control, SMIT2 transfected (using pcDNA3.1 vector) and Na-glucose cotransporter (SGLT) 1 transfected (using pG3M vector)] were cultured as described earlier (4). Briefly, cells were seeded at a density of 3 x 105 in 35-mm Petri dishes and cultured for 7 days before uptake assays. Culture media consisted of high-glucose DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and geneticin (only in transfected cells: 500 µg/ml). Cultures were maintained at 37°C in a 95% air–5% CO2 atmosphere, and media were changed every other day. For hyperosmotic treatment (+200 mosM), normal media was supplemented with 200 mM raffinose, urea, or 100 mM NaCl, and Petri dishes were incubated for 24 h before assay, unless otherwise specified. Care was taken to confirm confluency of cell monolayers before imposing hyperosmotic conditions, since it was noted that insufficient cell density under these conditions resulted in cell death. Addition of specific inhibitors (3 µM MG-132 against TonEBP, 2.5 µM U-0126 against ERK, 2.5 µM SB-203580 against p38, and 10 µM SP-600125 against JNK, all from Calbiochem, San Diego, CA) were made using x1,000 stock solutions in DMSO.

Uptake assays. Uptake of radiolabeled substrates [MI, {alpha}-methyl-D-glucose ({alpha}-MG), 2-deoxyglucose (2-DG), and lactate] was performed as described previously (4). Briefly, 3H-labeled substrate (0.5 µCi/ml) was mixed with cold substrate at low (10 nM) or saturating (10 mM) concentrations in Krebs solution (in mM: 137 NaCl, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 10 HEPES, pH 7.2) to identify saturable transport processes. Petri dishes were rinsed in substrate-free Krebs, and uptake was initiated by replacement with transport media. All uptakes were performed at 37°C and were stopped using ice-cold Krebs (3 x 2 ml). Monolayers were then dissolved with 1 N NaOH, scintillation cocktail was added, and radioactivity was measured using a scintillation counter. Preliminary assays have shown that linearity of uptake was maintained for ~60 min under all conditions used throughout this study (tracer or saturating conditions, isotonic and hypertonic media), which validated the chosen uptake time of 30 min. Discrimination between SMIT1 and SMIT2 activities was performed, as presented elsewhere (1, 4, 31), using specific uptake conditions. This strategy, which compares activities in the absence of inhibitor (total uptake) to those in the presence of either 100 mM L-fucose (blocking condition for SMIT1) or saturating MI (10 mM, blocking both SMIT1 and SMIT2), enables a functional isolation of both SMIT1 (subtracting L-fucose condition from total) and SMIT2 (subtracting saturating condition from L-fucose). The specific, transporter-mediated uptake was evaluated by subtraction of the nontransporter-mediated fraction of uptake [as estimated by measuring in the presence of a saturating level (10 mM) of cold substrate] from total uptake values.

Western blot. Western blot detection of SMIT2 was performed as previously described (4). Briefly, cell monolayers were rinsed in cold PBS containing protease inhibitor (Sigma) and homogenized in the same solution (1 ml per dish) using a Potter-Elvehjem homogenizer (Wheaton). For Western blot assay, 100 µg protein were electrophoresed on a 7.5% SDS-PAGE, then transferred onto nitrocellulose, and probed using a custom-made anti-SMIT2 polyclonal antibody (Biotechnology Research Institute, Montreal, QC, Canada; http://www.irb.cnrc.gc.ca), at a dilution of 1:500. This antibody was raised against an amino acid sequence specific to rabbit SMIT2 (FLALASNRSENSSCGL at position 238–253) that shares no significant similarities to related proteins, such as SMIT1, HMIT, or the SGLT cotransporters. Blots were then incubated with secondary horseradish peroxidase-linked goat anti-rabbit antibody (1:5,000: Santa Cruz Biotechnology), and detection was performed using enhanced chemiluminescence (Super Signal, Pierce). The specificity of the antibody was demonstrated using MDCK-SMIT2 cells, where a single band, which could be displaced by preincubation of the antibody with the immunogenic peptide, was observed (4). In addition, the band was not present when using preimmune serum (data not shown). The same characteristics were found when the antibody was used with rabbit renal tissues (31).

Immunofluorescence. Immunofluorescence detection of SMIT2 was performed on confluent MDCK-SMIT2 monolayers cultured on coverslips using a technique already described for myc-SGLT1 (5). Coverslips were rinsed with cold PBS and fixed with ice-cold formaldehyde (4%) in methanol for 4 min, a treatment that also permeabilizes the cells. After nonspecific sites were blocked with 2% BSA, coverslips were incubated for 90 min with anti-SMIT2 antibody (see Fig. 3, B and C, 1:100) or preimmune serum (see Fig. 3A, 1:100) at room temperature, rinsed, blocked again, and incubated for another 90 min with secondary antibody (Alexa fluor 488 conjugated goat anti-rabbit 1:1,000, Santa Cruz Biotechnology). Coverslips were counterstained for zonula occludens (ZO) using anti-ZO1 antibody (1:100, kind gift from Dr. J. Noël, Université de Montréal) followed by incubation with Alexa fluor 594-conjugated chicken anti-rat antibody (1:1,000, Santa Cruz Biotechnology). Coverslips were mounted using the ProLong antifade kit (Molecular Probes, Eugene, OR). Visualization was performed using an IX81 inverted microscope from Olympus using a x20 objective. SMIT2 fluorescence is displayed in green, whereas counterstain with ZO-1 is labeled in red.


Figure 3
View larger version (59K):
[in this window]
[in a new window]

  		Fig. 3. Immunofluorescence on MDCK-SMIT2 cells. Cells were treated (C) or not (A and B) for 24 h with 200 mM raffinose and probed using either preimmune serum (A) or {alpha}-SMIT2 antibody (B and C). All cell monolayers were counterstained with anti-zonula occludens-1 (red) to delineate the cell perimeters. SMIT2 fluorescence is shown in green. See MATERIALS AND METHODS for technical details.

 

Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Na+-myo-inositol (MI) cotransporter (SMIT) 1- and SMIT2-dependent uptakes of MI in Madin-Darby canine kidney (MDCK)-SMIT2 (A) and control MDCK (B) cells. Confluent cell monolayers were challenged for 24 h with hyperosmotic (hyper) shock (+200 mM raffinose) or maintained in isoosmotic (iso) condition, and specific MI uptakes were then measured. L-Fucose (100 mM) was used as a specific SMIT1 inhibitor to discriminate between SMIT1 and SMIT2 (see MATERIALS AND METHODS for details). Uptakes are means ± SD of three determinations. *Statistical significance (P < 0.05) against respective iso condition.

 
Quantitative RT-PCR. Quantification of SMIT1 and SMIT2 mRNA was performed on 0.5-µg total RNA templates purified from monolayers (TRIzol, Invitrogen) using LUX technology (Invitrogen). cRNA encoding for either SMIT1 or SMIT2 was used to generate standard curves (r ≥ 0.99) to estimate the number of copies in samples. Quality and overall efficiency of the procedure was monitored through evaluation of melting curves. Data are presented as number of molecules x 103/µg total RNA.

Data analysis. Data are presented as typical experiments and were reproduced at least three times. Values represent means ± SD of triplicates and were statistically analyzed using Student's t-test. In Figs. 1, 4, 5, and 8–11, asterisks represent statistical significance against controls (P < 0.05), whereas NS indicates absence of significance (P > 0.05). Determination of kinetic parameters was performed using Origin 6.1 software (OriginLab, Northampton, MA) using a tracer inhibition equation, including both mediated and nonspecific (Kd) fractions of transport, as described elsewhere (34).


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Susceptibility of SMIT2 induction to cycloheximide (cyclo). Confluent MDCK-SMIT2 cells were incubated for 24 h in iso or hyper conditions (200 mM raffinose), either in presence or absence of the protein synthesis inhibitor cycloheximide (2 µg/ml), and SMIT2-specific MI uptake was determined. Values are means ± SD of triplicates. *Statistical significance (P < 0.05) against iso condition.

 

Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 5. Induction profile for SMIT2. Confluent MDCK-SMIT2 cells were challenged for 24 h with various media to evaluate their capacity to induce SMIT2 activity. A: compared with control (300 mosM, iso) conditions, osmolarity of the culture media was either increased by 200 mosM (using raffinose, NaCl, or urea), decreased by one-third to 200 mosM (by diluting media with water), or incubated in the absence of MI. Values represent the SMIT2-specific proportion of MI uptake (see MATERIALS AND METHODS). *Statistical significance (P < 0.05) against iso conditions. B: time dependency of SMIT2 induction by raffinose. Confluent MDCK-SMIT2 cells were incubated in media supplemented with 200 mosM raffinose for times varying up to 48 h, and SMIT2 activity was evaluated after each interval (bullet). In addition, SMIT2 activity was evaluated in cells treated for 24 h in hyper media followed by reversal to iso conditions ({circ}) for incubation times up to 24 h. Values presented are means ± SD of triplicates.

 

Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 8. Effect of tonicity-responsive enhancer binding protein (TonEBP) inhibition on SMIT1 and SMIT2 induction. Confluent MDCK-SMIT2 cells were incubated for 24 h in iso or hyper conditions (200 mM raffinose), either in presence or absence of TonEBP inhibitors (3 µM MG-132, open bars and 20 µM lactacystin, dark gray bars), and specific SMIT1 and SMIT2 activities were measured. Values presented are means ± SD of triplicates. *Statistical significance (P < 0.05) against respective iso conditions. N.S., not significant.

 

Figure 11
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 11. Susceptibility of SMIT2 induction to MAPK inhibitors. Confluent MDCK-SMIT2 cells were maintained under iso conditions or challenged for 24 h with hyper media (+200 mM raffinose), with or without specific inhibitors of MAPK. The inhibitors used were U-0126 (against ERK, 2.5 µM), SB-203580 (against p38, 2.5 µM), and SP-600125 (against JNK, 10 µM). Values presented are means ± SD of triplicates. *Statistical significance (P < 0.05) against induction in hyper condition.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of MI uptake through hyperosmolarity. The ability of SMIT1 to respond to hyperosmotic conditions is a feature that can be easily reproduced in cell cultures, such as MDCK cells (15, 16, 40). We initially sought to examine the impact of such a procedure when applied to a MDCK cell clone that had been stably transfected with the SMIT2 transporter. The bar graph in Fig. 1A demonstrates that both SMIT1 and SMIT2 transport activities could be reliably detected in the stably transfected MDCK-SMIT2 cell line, under both iso-osmotic and hyperosmotic conditions (100 mM L-fucose was used to discriminate between the two SMIT transport activities). In isoosmotic conditions, the SMIT1-specific MI transport is only 11% the size of the SMIT2-specific transport. This is in agreement with values our laboratory has previously reported (4), showing that all MI transport can be attributed to SMIT1 or SMIT2 in this cell line. Control nontransfected MDCK cells (Fig. 1B) only express very low levels of endogenous SMIT2 (±10% of total SMIT activity). When hyperosmotic conditions were applied to SMIT2-MDCK cells for 24 h, such as by adding 200 mM raffinose, both the SMIT1 and SMIT2 transport activities increased. Throughout this series of assays, the overall stimulation of transport by hyperosmolarity varied from three- to ninefold for both transporters, depending on the initial activity level (average increase factor is 7.1 ± 2.6 for SMIT1 and 5.3 ± 1.5 for SMIT2). In Fig. 1A, SMIT1 activity increased ninefold, while SMIT2 activity increased threefold. In comparison, using control MDCK cells (Fig. 1B), stimulation of SMIT1 by hyperosmolarity is 10-fold, while SMIT2 is minimal and not significantly stimulated by hypertonicity. In accordance with other studies (26), we speculated that induction of protein targeting to the plasma membrane was responsible for the increase in transport activity in transfected cells. We further investigated this phenomenon by first measuring the kinetics of induced transport. As shown in Fig. 2A, the increased SMIT2-specific uptake essentially relies on an augmentation of the number of transporters present at the membrane, as shown by the increase in Vmax (from 11.4 ± 1.3 nmol·mg protein–1·30 min–1 in iso-osmotic conditions to 63.9 ± 4.1 nmol·mg protein–1·30 min–1 in hyperosmotic media), with no evident modification in the apparent affinity of the transporter (0.38 ± 0.07 mM in iso-osmotic against 0.47 ± 0.09 mM in hyperosmotic media). While the increase in the number of transporters could be expected to be restricted to the plasma membrane pool, we were surprised to detect by Western blot analysis (Fig. 2B) a distinct increase in the antigenic band under hyperosmotic conditions compared with the control, iso-osmotic condition in cell homogenates. The same phenomenon was also visualized through immunofluorescence staining (Fig. 3), showing an increase in specific SMIT2 staining (Fig. 3B) following 24-h treatment under hyperosmotic conditions (Fig. 3C). Only background staining for SMIT2 is found in the same cells when preimmune serum is used (Fig. 3A). We further characterized this increase in SMIT2 activity by examining its requirements for both transcriptional and translational events. As shown in Fig. 4, 24-h hyperosmotic treatment induced an eightfold increase in SMIT2 activity (from 0.24 ± 0.01 nmol·mg protein–1·30 min–1 in iso-osmotic condition to 2.00 ± 0.01 nmol·mg protein–1·30 min–1 in hyperosmotic media) but was completely abolished if performed in the presence of 10 µg/µl cycloheximide (from 0.26 ± 0.01 nmol·mg protein–1·30 min–1 in iso-osmotic condition to 0.14 ± 0.05 nmol·mg protein–1·30 min–1 in hyperosmotic media), indicating a requirement for protein synthesis. Unfortunately, addition of actinomycin D under hyperosmotic conditions induced cell death before the activation of transport could be observed, making it impossible to directly evaluate transcriptional induction in the stimulation of SMIT2 activity.


Figure 2
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 2. Induction of SMIT2: determination of kinetic parameters. Confluent MDCK-SMIT2 cells were incubated in iso or hyper conditions (+200 mM raffinose). A: kinetic analysis, presented in an Eadie-Hofstee plot, comparing iso (bullet) against hyper media ({blacksquare}) conditions. Vmax values are 11.4 ± 1.3 nmol·mg protein–1·30 min–1 in isotonic vs. 63.9 ± 4.1 nmol·mg protein–1·30 min–1 in hypertonic, and Km values are 0.38 ± 0.07 mM in isotonic vs. 0.47 ± 0.09 mM in hyper media. B: Western blot performed on MDCK homogenates against both iso- and hyper-treated cells. The bands shown are specific SMIT2 proteins of 66-kDa molecular mass (4).

 
The increase in SMIT2 activity due to hyperosmolarity has characteristics that are similar to the increased activity reported for native SMIT1 in MDCK cells (28, 51). First of all, we incubated MDCK-SMIT2 cells with various osmolytes, looking at their ability to stimulate transport. As shown in Fig. 5A, SMIT2-specific transport is stimulated by the addition of 200 mM raffinose (mean fold of increase in 4 assays: 3.3 ± 1.4) or NaCl (4.1 ± 2.0) but not by urea (1.1 ± 0.2). Furthermore, the reduction of osmolarity by one-third (hypo-osmotic condition: 200 mosM) or incubation in the absence of MI (300 mosM total) will usually, although not systematically, induce a small reduction of MI uptake. As seen in Fig. 5B, the induction of SMIT2-specific MI transport appears quite rapidly (within 4 h) and peaks within the first 12 h of hyperosmotic treatment with raffinose. This increase in activity is sustained for at least 48 h, if raffinose remains in the culture media, and returning to iso-osmotic conditions after induction of transport (24 h) will sometimes impede (2 assays: 35% reduction) or not (2 assays) its expression within the following 24 h.

Comparing SMIT1 and SMIT2 activities. To further characterize this regulation and to better compare the two transporters, the activities of both SMIT1 and SMIT2 were monitored, while varying culture media osmolarity and induction times. Figure 6 shows the changes in activity of both transporters following increased raffinose concentrations in the culture media for 24 h. As shown in this graph, SMIT1 is induced at a low level of hyperosmolarity (+100 mosM) and reaches a plateau at 150 mosM. SMIT2 stimulation requires at least +150 mosM but continues increasing with the culture medium hyperosmolarity. Furthermore, the time dependency for induction of the two SMITs was different. As seen in Fig. 7A, SMIT2 activity is induced more rapidly (4 h) and peaks faster (12 h) than does SMIT1 activity (induction at 12 h and peaks at 24 h). As the decrease in SMIT1-mediated transport observed at 48 h (Fig. 7A) is not significant, nor is it consistently observed, both transport activities are considered to reach a plateau value that is maintained throughout the remaining time of the experiment. Parallel quantification of both SMIT mRNAs also demonstrate different profiles (Fig. 7B). SMIT2 mRNA contents increased dramatically (3.3-fold) within 4 h but then fell rapidly within 24 h to background levels. This suggests either an induction of transcription or increased stability of SMIT2 mRNA by hyperosmolarity, which could not be corroborated by use of the transcription inhibitor actinomycin B due to extensive cell death under our culture conditions (data not shown).


Figure 6
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 6. Dose dependency of SMIT1 and SMIT2 response to raffinose. Confluent MDCK-SMIT2 cells were challenged for 24 h with various concentrations of raffinose (0–200 mM), and the specific SMIT1 ({square}) and SMIT2 ({blacksquare}) activities were measured. Values presented are means ± SD of triplicates.

 

Figure 7
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 7. Time dependency of SMIT1 and SMIT2 inductions. Specific MI uptakes and mRNA contents were monitored in confluent MDCK-SMIT2 cells incubated in media supplemented with raffinose (200 mM) for times varying up to 48 h. A: SMIT1- ({square}) and SMIT2-specific ({blacksquare}) MI uptakes (30 min) were measured. B: SMIT1- and SMIT2-specific mRNA contents in same cells as in A. Determinations were performed on triplicates and presented as means ± SD.

 
Specificity of hyperosmolarity inductions. Although the transfected SMIT2 vector does not contain a known tonicity-responsive enhancer (TonE) sequence, we examined the possible involvement of TonEBP in the stimulation of SMIT2 by using the proteasome inhibitor MG-132, which also inhibits TonEBP activity (32, 52). As shown in Fig. 8, SMIT1-dependent MI uptake, which is increased threefold by hyperosmotic treatment (light gray bar), is abolished if 3 µM MG-132 is added to the culture media (open bar, mean inhibition 95 ± 8%, n = 11). Conversely, MG-132 has no significant inhibitory effect on the stimulation of SMIT2-specific MI uptake by hyperosmotic media, as seen by equivalent MI uptakes in either the presence or absence of inhibitor (open vs. light gray bars, mean inhibition 12 ± 10%, n = 11). It should be noted that incubation in hyperosmotic media with inhibitor MG-132 did not significantly impede cell survival within 24 h, as evaluated by protein assays. These conclusions were confirmed by using 20 µM lactacystin, another proteasome inhibitor (dark gray bar).

To evaluate the specificity of the hyperosmolarity effect, we investigated other transport activities, including both endogenous MDCK transport systems and exogenous activities, such as SGLT1, which our laboratory had previously expressed in the MDCK-mycSGLT1 cell line (5). These cells are essentially similar to those used throughout this study since the MDCK-mycSGLT1 cell line heterologously expresses myc-SGLT1 through a stably inserted vector (pJ3M-hSGLT1). As shown in Fig. 9B, SGLT1 activity, as evaluated by {alpha}-MG uptake, is also induced (3.1- to 5.1-fold in 3 assays) when submitted to the hyperosmotic treatment that had been used with MDCK-SMIT2 cells (Fig. 9A). In this assay, both transport activities are induced equally (7.2-fold for SMIT2 and 5.1-fold for SGLT1). Interestingly, the endogenous MI and {alpha}-MG transport found in nontransfected MDCK cells (MDCK), attributable to endogenous SMIT1 (since total MI uptake were measured herein) and SGLT1, respectively, are also stimulated by the hyperosmotic treatment. In Fig. 10, other endogenous transport systems of untransfected MDCK cells were tested for their response to hyperosmotic shock. The transport of both 2-DG (a specific substrate for GLUT transporters) and lactic acid was induced (2.2 ± 1.1-fold for lactic acid and 3.9 ± 1.6-fold for 2-DG). It can also be seen that inhibition of TonEBP, by the addition of MG-132 to the culture media, completely abolished osmotically induced uptakes (n = 5) for both 2-DG (100 ± 38%) and lactic acid (81 ± 28%).


Figure 9
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 9. Induction of transport in transfected MDCK. Stably transfected MDCK cells expressing SMIT2 or mycSGLT1 [Na-glucose cotransporter 1 (SGLT1)], along with nontransfected MDCK (control) cells, were cultured to confluency and kept in iso or hyper media (+200 mM raffinose) for 24 h and then tested for specific uptakes. A: specific MI uptakes in control (MDCK) and MDCK-SMIT2 cells under iso and hyper conditions. B: specific {alpha}-methyl-D-glucose ({alpha}-MG) uptakes into control (MDCK) and MDCK-SGLT1 cells under iso and hyper conditions. All values are means ± SD for triplicates. *Statistical significance (P < 0.05) against respective iso conditions.

 

Figure 10
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 10. Induction of transport for diverse substrates. Confluent, nontransfected MDCK cells were maintained under iso conditions or challenged for 24 h with hyper media (+200 mM raffinose) in the presence or absence of the TonEBP inhibitor MG-132 (3 µM), and uptakes of lactic acid and 2-deoxyglucose (2-DG) were measured. Determinations were performed on triplicates and are presented as means ± SD. *Statistical significance (P < 0.05) against respective iso condition.

 
Susceptibility to MAPK inhibitors. The TonEBP-mediated response to hyperosmotic shock has been shown to be independent of MAPK modulators such as ERK and JNK (24), with the exception of p38 (27). To evaluate any possible involvement of these pathways in the hyperosmotic upregulation of SMIT2, transport induction was performed in the presence of specific inhibitors for those three modulators. As seen in Fig. 11, the transport stimulation induced by osmotic stress (7.5-fold) is not affected by an ERK inhibitor (2.5 µM U-0126) but is reduced 40% by a p38 inhibitor (2.5 µM SB-203580) and 32% by a JNK inhibitor (10 µM SP-600125). Overall results (n = 4) confirm the absence of inhibition of ERK (4 ± 18%) along with partial inhibition of p38 (34 ± 16%), while JNK inhibition is shown to be more variable (24 ± 18%). The inhibitor concentrations used for this study are those that were shown to be effective in IMCD cell line (50).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The ability of SMIT1 to upregulate under hyperosmotic conditions has been well established (10, 22, 28, 29, 51). Its regulation occurs through the TonE/TonEBP system and essentially relies on transcriptional induction (24, 37, 40, 51). Unfortunately, little is known about the SMIT2 gene, any potential promoters for this gene, or its overall regulation. When SMIT2 is expressed by integration of an exogenous vector, as was done for the MDCK-SMIT2 cell line, the only expected regulatory features concern protein stability and recycling. This study was thus designed to evaluate the capacity of hyperosmotic conditions to modulate plasma membrane targeting of heterologously expressed SMIT2, as already reported for the BGT in a similar cell system (26). In that context, the increase in SMIT2 mRNA levels came as a considerable surprise and motivated the present study, which considers both transcriptional and translational events in the regulation of SMIT2 by hypertonicity.

As endogenous SMIT2 is insignificant in this transfected cell line compared with the heterologous form expressed in SMIT2-MDCK (one-tenth of endogenous SMIT1, which, in its turn, represents only 10–20% that of heterologously expressed SMIT2), all the activity measured herein regarding SMIT2 is attributed to the exogenous form of the transporter. In fact, the stimulation of SMIT2 in control MDCK by hypertonic treatment generates variable results (from none to 4-fold increases upon assays) that are believed to be largely contaminated with the increased activity level originating from SMIT1 (Fig. 1B), since SMIT2 activity is measured indirectly using inhibitory conditions (100 mM L-fucose). Like SMIT1, SMIT2 transport activity was induced by hyperosmolarity, as seen by increases in SMIT2-specific MI transport (Fig. 1A and the kinetics data shown in Fig. 2A). This increase was not due to recruitment of transporters to the plasma membrane from intracellular stores, contrary to what was already reported for the BGT (26), but required de novo production of the SMIT2 protein. This can be seen both by Western blot detection from whole cell homogenates (Fig. 2B) and by blockade of SMIT2 synthesis by cycloheximide (Fig. 4). Furthermore, the induction of SMIT2 through hyperosmotic treatment correlates with an increase in SMIT2 mRNA content, which was completely unexpected. This is shown in Fig. 7B, where SMIT2 mRNA is increased threefold within the first 4 h of treatment. Assays using 5 µg/µl actinomycin B to block mRNA transcription clearly showed the importance of mRNA synthesis in the adaptation to hyperosmotic treatment, as cells were unable to cope with the treatment and rapidly died within 6–8 h, while iso-osmotic cells survived in the presence of actinomycin D for >24 h (data not shown). This increase in SMIT2 mRNA content could be accounted for by either stimulation in the transcription rate or an increase in mRNA stability. Interestingly, the literature reports both conclusions when looking at TonEBP mRNA contents in relation to hyperosmotic treatment. While mRNA stability is increased in mIMCD3 cells (9), no variations are found under the same conditions in MDCK cells (51), thus leaving this question unanswered.

Surprisingly, this induction in transport function was not restricted to SMIT2, since SGLT1 activity, albeit in cells transformed with a different vector (pJ3M-hSGLT1), was also stimulated. Furthermore, the levels of transport induction were similar between SMIT2, SGLT1 (Fig. 9), and endogenous SMIT1 (Fig. 1). Still, the means by which hyperosmolarity promotes either the transcription or the mRNA stability of these three transport systems in this cell line is puzzling. It should be noted that while the induction of endogenous SMIT1 is under control of the transcriptional factor TonEBP, this transcriptional factor should have no influence on the exogenous SMIT2 transporter. Accordingly, the presence of MG-132, which inhibits TonEBP activity, did not impede the stimulation of SMIT2, as elicited by hyperosmotic treatment (Fig. 8), which was corroborated by the use of another proteasome inhibitor, lactacystin (52). In comparison, SMIT1 activity was completely abolished when hyperosmotic conditions were imposed in the presence of these inhibitors, which again demonstrates the involvement of TonEBP in SMIT1 regulation.

The induction profiles for both endogenous SMIT1 and vector-mediated SMIT2 share similarities in that both are stimulated by raffinose and NaCl while urea has no significant effect (Fig. 5A; also Refs. 28, 29, 40). Also, it is noteworthy that this exact profile is also found with other inducible proteins, such as AQP-1 (50), but more importantly with TonEBP itself (47, 51). The rationale behind this is that intracellular tonicity, as induced by the addition of raffinose, mannitol, or NaCl, but not urea, is responsible for the triggering of this regulatory system (40). Finally, it should also be noted that hyperosmotic treatment with either raffinose or NaCl, but not urea, will also modulate the activity of key MAPK proteins, such as p38, JNK, and ERK (3, 50, 54). Still, the link between activation of MAPK and induction of effectors is not clear, since, in some cases, inhibition on ERK may (50) or may not (3, 28) have any effect on activation of transport. One thus has to consider protein and cell-specific behaviors in relation to this regulatory pathway. In the present study, we find that both JNK and p38 are influential in the response of SMIT2 to hyperosmotic treatment, while ERK seems to be without effect (Fig. 11).

In this study, we have found two major differences between the induction of endogenous SMIT1 and heterologous SMIT2, which indicate dissociation between their regulation pathways. First, the sensitivities to the level of external hyperosmolarity are different, as SMIT1 was induced more effectively by low levels of external hyperosmolarity with evidence of saturation at 150 mosM, while SMIT2 induction required greater hyperosmolarity but did not seem to reach saturation with +200 mosM raffinose (Fig. 6). Second, the time courses of induction, as seen in Fig. 7, were not parallel; both SMIT2 transport and mRNA were induced more rapidly than were those of SMIT1.

We expected to find that hyperosmotic shock induced other endogenous transporters, such as the GLUT proteins (2-DG), and the transporter for lactic acid (Fig. 10). In fact, since the content of all amino acids has been reported to increase on hyperosmotic treatment (21), it is likely that a wide variety of transport systems become activated by this treatment. It remains to be seen to what extent exposure to hyperosmotic conditions globally induce transport systems as a compensatory response. Is it cell specific to MDCK, tissue specific to kidney, or a more general physiological response to hyperosmotic shock? The fact that hyperosmotic treatment can stimulate mRNA contents for a host of different transport systems, including exogenous DNA, which does not contain a TonE sequence and includes different promoters (cytomegalovirus promoter for SMIT2 and SV40 for SGLT1), suggests the possibility of different osmotic-driven promoters acting on different genes with varying time courses and sensitivities to osmolarities. The discrepancies found between endogenous SMIT1 and exogenous SMIT2 in their response to hyperosmolarity stress (Figs. 6 and 7) are indicative of this dual response, at least in MDCK cells. Our results are in accordance with others (26) showing the stimulation of an exogenous transport system by osmotic shock, early induction time of transport, and necessity for protein synthesis. Still, using Western blot and immunofluorescence data, others have concluded that the increase in betaine transport activity strictly originates from an increase in plasma membrane targeting without new BGT synthesis (26). Unfortunately, no evaluation of specific mRNA contents was performed to support this conclusion. On the other hand, in the present study, we demonstrate an increase in total SMIT2 protein that correlates with an increase in specific mRNA. The discrepancy between both studies may lie in different regulation pathways for SMIT2 and for the betaine transport systems.

Although TonE/TonEBP has been shown to be a key regulatory pathway in osmolarity adaptation, many reports have stressed the existence of a nonrelated system that precedes it. Early transcriptional induction of system A amino acid transporter, which is activated before that of betaine, a classic TonEBP-dependent transport system, has been reported (11). These results were confirmed in a study that also stressed different time frames for the hyperosmotic induction of system A (within 6 h) and BGT1 (after 24 h) (25, 26). Also, AQP-2 was shown to exhibit distinct early and late regulatory pathways in mpkCCDc14 cells (18). Lastly, a similar early induction has also been reported for the membrane proteins CD9 and β1-integrin (45). It should be noted that some of these early inductions were found to be attenuated by the presence of organic osmolytes in the culture media (11, 26, 45). Unfortunately, the nature or the mechanism responsible for the early adaptation to hyperosmotic stress is still unknown at this point.

TonEBP pathway has proven to be important in cell response to hyperosmotic stress, although it is not believed to be indispensable for survival in cell culture, since its inhibition through short interfering RNA or inhibitors such as MG-132 or cyclosporine was shown not to impede cell survival under hyperosmotic conditions (Figs. 8 and 10 and Ref. 26). If the importance of the role played by TonEBP in renal physiology is undeniable (33), the present study and others point to the fact that other tonicity-dependent stimulation mechanisms have to be active in parallel with TonEBP.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Canadian Institutes of Health Research Grant MOP-67038.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-Y. Lapointe, Groupe d'étude des protéines membranaires (GÉPROM), Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, Canada H3C 3J7 (e-mail: jean-yves.lapointe{at}umontreal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Aouameur R, Da Cal S, Bissonnette P, Coady MJ, Lapointe JY. SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine. Am J Physiol Gastrointest Liver Physiol 293: G1300–G1307, 2007.[Abstract/Free Full Text]

2. Atta MG, Dahl SC, Kwon HM, Handler JS. Tyrosine kinase inhibitors and immunosuppressants perturb the myo-inositol but not the betaine cotransporter in isotonic and hypertonic MDCK cells. Kidney Int 55: 956–962, 1999.[CrossRef][Web of Science][Medline]

3. Berl T, Siriwardana G, Ao L, Butterfield LM, Heasley LE. Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells. Am J Physiol Renal Physiol 272: F305–F311, 1997.[Abstract/Free Full Text]

4. Bissonnette P, Coady MJ, Lapointe JY. Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells. J Physiol 558: 759–768, 2004.[Abstract/Free Full Text]

5. Bissonnette P, Noel J, Coady MJ, Lapointe JY. Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes. J Physiol 520: 359–371, 1999.[Abstract/Free Full Text]

6. Bourgeois F, Coady MJ, Lapointe JY. Determination of transport stoichiometry for two cation-coupled myo-inositol cotransporters: SMIT2 and HMIT. J Physiol 563: 333–343, 2005.[Abstract/Free Full Text]

7. Burg MB. Molecular basis of osmotic regulation. Am J Physiol Renal Fluid Electrolyte Physiol 268: F983–F996, 1995.[Abstract/Free Full Text]

8. Burg MB, Kwon ED, Kultz D. Regulation of gene expression by hypertonicity. Annu Rev Physiol 59: 437–455, 1997.[CrossRef][Web of Science][Medline]

9. Cai Q, Ferraris JD, Burg MB. High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA. Am J Physiol Renal Physiol 289: F803–F807, 2005.[Abstract/Free Full Text]

10. Cha JH, Woo SK, Han KH, Kim YH, Handler JS, Kim J, Kwon HM. Hydration status affects nuclear distribution of transcription factor tonicity responsive enhancer binding protein in rat kidney. J Am Soc Nephrol 12: 2221–2230, 2001.[Abstract/Free Full Text]

11. Chen JG, Coe M, McAteer JA, Kempson SA. Hypertonic activation and recovery of system A amino acid transport in renal MDCK cells. Am J Physiol Renal Fluid Electrolyte Physiol 270: F419–F424, 1996.[Abstract/Free Full Text]

12. Coady MJ, Wallendorff B, Gagnon DG, Lapointe JY. Identification of a novel Na+/myo-inositol cotransporter. J Biol Chem 277: 35219–35224, 2002.[Abstract/Free Full Text]

13. Cohen DM, Wasserman JC, Gullans SR. Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells. Am J Physiol Cell Physiol 261: C594–C601, 1991.[Abstract/Free Full Text]

14. Handler JS, Kwon HM. Kidney cell survival in high tonicity. Comp Biochem Physiol A Physiol 117: 301–306, 1997.[Medline]

15. Handler JS, Kwon HM. Regulation of renal cell organic osmolyte transport by tonicity. Am J Physiol Cell Physiol 265: C1449–C1455, 1993.[Abstract/Free Full Text]

16. Handler JS, Kwon HM. Regulation of the myo-inositol and betaine cotransporters by tonicity. Kidney Int 49: 1682–1683, 1996.[Web of Science][Medline]

17. Handler JS, Kwon HM. Transcriptional regulation by changes in tonicity. Kidney Int 60: 408–411, 2001.[CrossRef][Web of Science][Medline]

18. Hasler U, Jeon US, Kim JA, Mordasini D, Kwon HM, Feraille E, Martin PY. Tonicity-responsive enhancer binding protein is an essential regulator of aquaporin-2 expression in renal collecting duct principal cells. J Am Soc Nephrol 17: 1521–1531, 2006.[Abstract/Free Full Text]

19. Hitomi K, Tsukagoshi N. cDNA sequence for rkST1, a novel member of the sodium ion-dependent glucose cotransporter family. Biochim Biophys Acta 1190: 469–472, 1994.[Medline]

20. Horio M, Ito A, Matsuoka Y, Moriyama T, Orita Y, Takenaka M, Imai E. Apoptosis induced by hypertonicity in Madin Darley canine kidney cells: protective effect of betaine. Nephrol Dial Transplant 16: 483–490, 2001.[Abstract/Free Full Text]

21. Horio M, Yamauchi A, Moriyama T, Imai E, Orita Y. Osmotic regulation of amino acids and system A transport in Madin-Darby canine kidney cells. Am J Physiol Cell Physiol 272: C804–C809, 1997.[Abstract/Free Full Text]

22. Ibsen L, Strange K. In situ localization and osmotic regulation of the Na(+)-myo-inositol cotransporter in rat brain. Am J Physiol Renal Fluid Electrolyte Physiol 271: F877–F885, 1996.[Abstract/Free Full Text]

23. Ito T, Fujio Y, Hirata M, Takatani T, Matsuda T, Muraoka S, Takahashi K, Azuma J. Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contributes to cytoprotection in HepG2 cells. Biochem J 382: 177–182, 2004.[CrossRef][Web of Science][Medline]

24. Jeon US, Kim JA, Sheen MR, Kwon HM. How tonicity regulates genes: story of TonEBP transcriptional activator. Acta Physiol (Oxf) 187: 241–247, 2006.[CrossRef][Medline]

25. Kempson SA. Differential activation of system A and betaine/GABA transport in MDCK cell membranes by hypertonic stress. Biochim Biophys Acta 1372: 117–123, 1998.[Medline]

26. Kempson SA, Beck JA, Lammers PE, Gens JS, Montrose MH. Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP. Biochim Biophys Acta 1712: 71–80, 2005.[Medline]

27. Ko BC, Lam AK, Kapus A, Fan L, Chung SK, Chung SS. Fyn and p38 signaling are both required for maximal hypertonic activation of the osmotic response element-binding protein/tonicity-responsive enhancer-binding protein (OREBP/TonEBP). J Biol Chem 277: 46085–46092, 2002.[Abstract/Free Full Text]

28. Kwon HM, Itoh T, Rim JS, Handler JS. The MAP kinase cascade is not essential for transcriptional stimulation of osmolyte transporter genes. Biochem Biophys Res Commun 213: 975–979, 1995.[CrossRef][Web of Science][Medline]

29. Kwon HM, Yamauchi A, Uchida S, Preston AS, Garcia-Perez A, Burg MB, Handler JS. Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J Biol Chem 267: 6297–6301, 1992.[Abstract/Free Full Text]

31. Lahjouji K, Aouameur R, Bissonnette P, Coady MJ, Bichet DG, Lapointe JY. Expression and functionality of the Na+/myo-inositol cotransporter SMIT2 in rabbit kidney. Biochim Biophys Acta 1768: 1154–1159, 2007.[Medline]

32. Lammers PE, Beck JA, Chu S, Kempson SA. Hypertonic upregulation of betaine transport in renal cells is blocked by a proteasome inhibitor. Cell Biochem Funct 23: 315–324, 2005.[CrossRef][Web of Science][Medline]

33. Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT, Igarashi P, Rao A, Olson EN. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci USA 101: 2392–2397, 2004.[Abstract/Free Full Text]

34. Malo C, Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na(+)-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 122: 127–141, 1991.[CrossRef][Web of Science][Medline]

35. Matsuzaki T, Suzuki T, Takata K. Hypertonicity-induced expression of aquaporin 3 in MDCK cells. Am J Physiol Cell Physiol 281: C55–C63, 2001.[Abstract/Free Full Text]

36. Miyakawa H, Woo SK, Chen CP, Dahl SC, Handler JS, Kwon HM. Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity. Am J Physiol Renal Physiol 274: F753–F761, 1998.[Abstract/Free Full Text]

37. Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM. Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 96: 2538–2542, 1999.[Abstract/Free Full Text]

38. Neuhofer W, Muller E, Burger-Kentischer A, Beck FX. Hypertonicity affects heat shock protein 27 and F-actin localization in Madin-Darby canine kidney cells. Kidney Int Suppl 67: S165–S167, 1998.[Medline]

39. Neuhofer W, Vastag M, Fraek ML, Beck FX. Effect of ammonium on the expression of osmosensitive genes in Madin-Darby canine kidney cells. J Physiol 563: 497–505, 2005.[Abstract/Free Full Text]

40. Neuhofer W, Woo SK, Na KY, Grunbein R, Park WK, Nahm O, Beck FX, Kwon HM. Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity. Am J Physiol Cell Physiol 283: C1604–C1611, 2002.[Abstract/Free Full Text]

41. Padda R, Wamsley-Davis AM, Gustin MC, Ross R, Yu C, Sheikh-Hamad D. MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells. Am J Physiol Renal Physiol 291: F874–F881, 2006.[Abstract/Free Full Text]

42. Rim JS, Atta MG, Dahl SC, Berry GT, Handler JS, Kwon HM. Transcription of the sodium/myo-inositol cotransporter gene is regulated by multiple tonicity-responsive enhancers spread over 50 kilobase pairs in the 5'-flanking region. J Biol Chem 273: 20615–20621, 1998.[Abstract/Free Full Text]

43. Roll P, Massacrier A, Pereira S, Robaglia-Schlupp A, Cau P, Szepetowski P. New human sodium/glucose cotransporter gene (KST1): identification, characterization, and mutation analysis in ICCA (infantile convulsions and choreoathetosis) and BFIC (benign familial infantile convulsions) families. Gene 285: 141–148, 2002.[CrossRef][Web of Science][Medline]

44. Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts BA 3rd, Rouse D. p38 Kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J Biol Chem 273: 1832–1837, 1998.[Abstract/Free Full Text]

45. Sheikh-Hamad D, Suki WN, Zhao W. Hypertonic induction of the cell adhesion molecule beta 1-integrin in MDCK cells. Am J Physiol Cell Physiol 273: C902–C908, 1997.[Abstract/Free Full Text]

46. Takenaka M, Preston AS, Kwon HM, Handler JS. The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress. J Biol Chem 269: 29379–29381, 1994.[Abstract/Free Full Text]

47. Tian W, Cohen DM. Urea inhibits hypertonicity-inducible TonEBP expression and action. Am J Physiol Renal Physiol 280: F904–F912, 2001.[Abstract/Free Full Text]

48. Tian W, Zhang Z, Cohen DM. MAPK signaling and the kidney. Am J Physiol Renal Physiol 279: F593–F604, 2000.[Abstract/Free Full Text]

49. Uchida S, Yamauchi A, Preston AS, Kwon HM, Handler JS. Medium tonicity regulates expression of the Na(+)- and Cl(-)-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene. J Clin Invest 91: 1604–1607, 1993.[Web of Science][Medline]

50. Umenishi F, Schrier RW. Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene. J Biol Chem 278: 15765–15770, 2003.[Abstract/Free Full Text]

51. Woo SK, Dahl SC, Handler JS, Kwon HM. Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity. Am J Physiol Renal Physiol 278: F1006–F1012, 2000.[Abstract/Free Full Text]

52. Woo SK, Maouyo D, Handler JS, Kwon HM. Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity. Am J Physiol Cell Physiol 278: C323–C330, 2000.[Abstract/Free Full Text]

53. Yamauchi A, Uchida S, Preston AS, Kwon HM, Handler JS. Hypertonicity stimulates transcription of gene for Na+-myo-inositol cotransporter in MDCK cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F20–F23, 1993.[Abstract/Free Full Text]

54. Zhang Z, Cohen DM. NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1234–F1238, 1996.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/3/C791    most recent
00390.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bissonnette, P.
Right arrow Articles by Lapointe, J.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bissonnette, P.
Right arrow Articles by Lapointe, J.-Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.