HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 560/1/123    most recent jphysiol.2004.066423v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, H. Articles by Hundal, H. S Search for Related Content PubMed PubMed Citation Articles by Zhao, H. Articles by Hundal, H. S

¹ Division of Molecular Physiology, Medical Sciences Institute/Wellcome Trust Biocentre Complex, The University of Dundee, Dundee DD1 4HN, UK

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
We have investigated the expression and regulation of the Na⁺–K⁺–2Cl^– cotransporter (NKCC) by insulin and hyperosmotic stress in L6 rat skeletal muscle cells. NKCC was identified by immunoblotting as a 170 kDa protein in L6 myotubes and mediated 54% of K⁺ (⁸⁶Rb⁺) influx based on the sensitivity of ion transport to bumetanide, a NKCC inhibitor. The residual ⁸⁶Rb⁺ influx occurred via the Na⁺,K⁺-ATPase and other transporters not sensitive to bumetanide or ouabain. NKCC-mediated ⁸⁶Rb⁺ influx was enhanced significantly (1.6-fold) by acute cell exposure to insulin, but was inhibited significantly by tyrosine kinase inhibitors, wortmannin and rapamycin, consistent with a role for the insulin receptor tyrosine kinase, phosphoinositide 3 (PI3)-kinase and mTOR, respectively, in cotransporter activation. In contrast, the hormonal activation of NKCC was unaffected by inhibition of the classical Erk-signalling pathway. Subjecting L6 myotubes to an acute hyperosmotic challenge (420 mosmol l^–1) led to a 40% reduction in cell volume and was accompanied by a rapid stimulation of NKCC activity (2-fold). Intracellular volume recovered to normal levels within 60 min, but this regulatory volume increase (RVI) was prevented if bumetanide was present. Unlike insulin, activation of NKCC by hyperosmolarity did not involve PI3-kinase but was suppressed by inhibition of tyrosine kinases and the Erk pathway. While inhibition of tyrosine kinases, using genistein, led to a complete loss in NKCC activation in response to hyperosmotic stress, immunoprecipitation of NKCC revealed that the cotransporter was not regulated directly by tyrosine phosphorylation. Simultaneous exposure of L6 myotubes to insulin and hyperosmotic stress led to an additive increase in NKCC-mediated ⁸⁶Rb⁺ influx, of which, only the insulin-stimulated component was wortmannin-sensitive. Our findings indicate that L6 myotubes express a functional NKCC that is rapidly activated in response to insulin and hyperosmotic shock by distinct intracellular signalling pathways. Furthermore, activation of NKCC in response to hyperosmotic-induced cell shrinkage represents a critical component of the RVI mechanism that allows L6 muscle cells to volume regulate.

(Received 13 April 2004; accepted after revision 28 July 2004; first published online 29 July 2004)
Corresponding author H. S. Hundal: Division of Molecular Physiology, Medical Sciences Institute/Wellcome Trust Biocentre Complex, The University of Dundee, Dundee, DD1 4HN, UK. Email: h.s.hundal{at}dundee.ac.uk

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
There is increasing evidence that cellular dehydration, induced, for example, by an increase in plasma osmolarity, may have a significant impact on hormonal and nutrient-induced responses in tissues such as skeletal muscle, which represents a primary site of insulin action and a major repository for body glycogen and protein (Schliess & Haussinger, 2003). Systemic hyperosmolarity resulting from increased dietary intake or intense exercise, as well as pathological conditions such as uraemia, sepsis, burn injury and hyperglycaemia could thus be a factor in the pathogenesis of muscle insulin resistance. As with most mammalian cells, muscle cells shrink when exposed to a hyperosmotic environment and defend the loss in muscle water by stimulating the uptake of osmolytes through a process called regulatory volume increase (RVI). Of paramount importance in this RVI response is the activation of the sodium–potassium–chloride (Na⁺–K⁺–2Cl^–) cotransporter, which mediates the concerted electroneutral uptake of Na⁺, K⁺ and Cl^–. The intracellular accumulation of these ions forms an integral part of the volume regulatory mechanism as it induces the obligatory, osmotically driven, movement of water into the cell.

Two isoforms of the Na⁺–K⁺–2Cl^– (NKCC) cotransporter have been identified, NKCC1, which is expressed ubiquitously in animal cells, and NKCC2, a kidney specific isoform. Recent work has highlighted the potential role of NKCC1 (hereafter referred to as NKCC) in volume regulation of skeletal muscle (Lindinger et al. 2002; Gosmanov et al. 2003a), but precisely how changes in cell volume are sensed and how these then trigger activation of NKCC still remain poorly understood. Changes in membrane tension, cytoskeletal architecture or cytoplasmic macromolecular crowding have previously been postulated as possible mechanisms by which changes in cell volume are sensed (Hoffmann & Dunham, 1995). Alternatively, the initial sensing and transduction event may occur as a result of activation of intracellular signalling proteins, most notably members of the mitogen-activated protein kinase (MAPK) family, including p42/44 MAP (Erk) kinases (Itoh et al. 1994; Liedtke & Cole, 2002), SAPK2/p38 MAPK (Roger et al. 1999), c-Jun NH₂-terminal kinase (JNK) (Klein et al. 1999) and tyrosine kinases of the Src family (Feranchak et al. 2003). Indeed, the notion that transduction of the volume signal and activation of NKCC may involve phosphorylation is supported strongly by studies showing that inhibitors of serine/threonine protein kinases suppress the hypertonicity-induced activation of volume-regulatory transporters, and that NKCC becomes phosphorylated when activated (Flatman, 2002; Flemmer et al. 2002).

In addition to its rapid regulation by changes in osmolality there is also evidence that bumetanide-sensitive NKCC activity can be upregulated by insulin in a number of different cell types (Hallbrucker et al. 1991; Weil-Maslansky et al. 1994; Sargeant et al. 1995; Longo, 1996; Sweeney et al. 1998). In 3T3-L1 pre-adipocytes the rapid stimulation by insulin is mediated by a phosphoinositide 3-kinase (PI3K)-dependent mechanism (Sweeney et al. 1998). In contrast, it has been suggested recently that in skeletal muscle, insulin inhibits NKCC-mediated transport and that this inhibition relies upon the hormonal activation of PI3K and the p38 MAP kinase pathway (Gosmanov & Thomason, 2002). This latter finding is counter to the common view that insulin promotes potassium uptake in skeletal muscle (Clausen, 1986) and would imply that NKCC makes little, if any, contribution to insulin-stimulated potassium influx in this tissue. In an attempt to gain further insight into this issue and the role played by muscle NKCC towards volume regulation we have investigated the effects of insulin and hypertonic-induced stress on NKCC activity in cultured rat L6 skeletal muscle cells. We demonstrate that in this skeletal muscle cell line (i) NKCC makes a significant contribution to total potassium uptake (ii) the activity of the cotransporter is rapidly upregulated in response to insulin and hyperosmotic shock and (iii) the activation of NKCC by insulin and hypertonicity is additive and involves distinct intracellular signalling pathways.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials

-Minimal essential medium (-MEM), fetal bovine serum, antimycotic/antibiotic solution were from Life Technologies. Sterile trypsin-EDTA, ouabain and bumetanide, wortmannin, insulin, tyrophostin AG1024 and Kodak X-OMAT film were from Sigma-Aldrich Co. Ltd. (Poole, Dorset, UK). The tyrophostin 3-amino-2,4-dicyano-5-(4-hydroxyphenyl) penta-2,4 dienonitrile (ADHD) was purchased from Tocris (Bristol, UK). Glucose and all other reagent grade chemicals for buffers were obtained from BDH (Poole, Dorset, UK). SB 203580 and PD 98059 were from Calbiochem-Novabiochem Ltd. (Nottingham, UK). ⁸⁶Rb, ³H–H₂O and ¹⁴C-inulin were obtained from New England Nuclear (Stevenage, UK). An antibody directed against NKCC (T4) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa, USA). Antibodies to PKB, p70S6K, p38 MAP kinase, Erk1/Erk2, JNK and phosphotyrosine were from New England Biolabs (Hertfordshire, UK). Antibodies to phospho-JNK (pY^183/185) were from Biosource (Nivelles Belgium). Horseradish peroxidase-conjugated to anti-rabbit IgG, anti-mouse IgG and anti-sheep/goat IgG were obtained from Cell Signalling Technology (Beverly, MA, USA). Reagents for ECL were from Pierce & Warriner (Chester, UK).

Cell culture

L6 muscle cells were cultured to myotubes as monolayers as previously described (Hajduch et al. 1998) in -minimal essential media (-MEM) containing 2% (v/v) fetal bovine serum (FBS) and 1% (v/v) antimycotic/antibiotic solution (100 units ml^–1 penicillin, 100 µg ml^–1 streptomycin, 250 ng ml^–1 amphotericin B) at 37°C with 5% CO₂. Cells were grown in 10 cm dishes for lysate preparation and in six-well plates for uptake assays. Differentiated muscle cells were serum starved for 5 h before addition of appropriate reagents for times and at concentrations indicated in the figure legends.

Preparation of cell lysates

L6 myotubes were serum starved as described above. Plates were washed three times with 0.9% (w/v) ice-cold saline and 200 µl of lysis buffer (50 mM Tris pH 7.4, 0.27 M sucrose, 1 mM Na-orthovanadate pH 10, 1 mM EDTA, 1 mM EGTA, 10 mM Na ß-glycerophosphate, 50 mM NaF, 5 mM Na pyrophosphate, 1% (w/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol, 0.1 µM microcystin-LR and protease inhibitors (Roche, Lewes, UK)) was added. Cells were scraped off the plates using a rubber policeman and homogenized by passing through a 26G hyperdermic needle prior to centrifugation (13000 g, 3°C for 5 min) and stored at –20°C. Plasma membranes were isolated from L6 cells and rat skeletal muscle as previously described (Darakhshan et al. 1998; Hajduch et al. 1998). Microsomes prepared from rat brain (Hundal et al. 1992) were used as a positive immunoreactive control for NKCC expression. Brain tissue was harvested from adult rats in accordance with current UK legislation. The protein content of membrane fractions was determined using the Bradford assay (Bradford, 1976).

SDS-PAGE and immunoblotting

Isolated membrane fractions, cell lysates and immunoprecipitates (25–50 µg protein) were subjected to SDS-PAGE and immunoblotting as previously described (Hajduch et al. 1998). Separated proteins were transferred onto nitrocellulose membranes and blocked with Tris-buffered saline (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 5% milk protein (w/v) and 0.05% (v/v) Tween-20. Membranes were probed with antibodies against NKCC (T4), PKB, p70S6K, p38 MAPK and p42/p44 MAPK (all at a dilution of 1: 1000). Following primary antibody incubation, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1: 5000) or anti-mouse IgG (1: 5000) as appropriate. Immunoreactive protein bands were visualized by enhanced chemiluminescence on Kodak X-OMAT film.

Immunoprecipitation

Following cell exposure to hyperosmotic stress L6 cells were lysed in lysis buffer (as described above) and denatured with 0.14% SDS (Lytle et al. 1995). NKCC was immunoprecipitated from 300 µg of lysate protein using 2 µg of monoclonal NKCC (T4) antibody and immune complexes captured by incubation with Protein A-Sepharose beads (Amersham Pharmacia) and extracted into Laemmli sample buffer prior to immunoblotting as described above.

⁸⁶Rb⁺ influx assay

⁸⁶Rb⁺ was used as a surrogate tracer for K⁺ (Sen et al. 1995; Sargeant et al. 1995). Cell monolayers grown on six-well multidishes were washed three times with pre-warmed (37°C) Hepes-buffered saline (HBS) 140 mM NaCl, 20 mM Hepes, 2.5 mM MgSO₄, 5 mM KCl, 1 mM CaCl₂, pH 7.4 and pre-incubated at 37°C with HBS/10 mM D-glucose containing ouabain and bumetanide in order to determine ⁸⁶Rb⁺ uptake attributable to the Na⁺,K⁺-ATPase and the Na⁺–K⁺–2Cl^– cotransporter, respectively (Sen et al. 1995; Sargeant et al. 1995). In some experiments, cells were pre-incubated in hyperosmotic medium. Buffer osmolarity (normally 300 mosmol l^–1) was manipulated by the addition of sucrose and was verified using an osmometer (Gonotec, Germany). Uptake was terminated by rapid aspiration of the radioactive incubation solution, followed by four successive washes of cells with ice-cold isotonic saline solution (0.9% NaCl, w/v). Cells were lysed with 0.05 M NaOH and 80% of the lysate was removed for quantitating cell-associated radioactivity using a Beckman LS 60001C scintillation counter. Non-specific radioactive binding was determined by assessing cell-associated radioactivity following addition and rapid removal of uptake buffer from cells maintained at 4°C. The protein content in cell lysates was determined using the method of Bradford (Bradford, 1976).

Analysis of PI3K activity in L6 cells

Following treatment with wortmannin and/or insulin, L6 cells were lysed in lysis buffer (composition as above). Cell lysates were incubated with a p85 immunoprecipitating antibody that had been bound to protein-G beads for 1 h at 4°C. Immunoprecipitates were washed and incubated with phosphatidylinositol (0.1 mg ml^–1) for 20 min in buffer containing 50 µM [³²P]ATP, 1.2 mM Na₃VO₄, 5 mM MgCl₂ and 25 mM Hepes, pH 7.4. The reaction was terminated by addition of 20 µl of 8 M HCl and 160 µl CH₃OH/CHCl₃ (1: 1, v/v). The products were separated by thin layer chromatography as previously described (Hajduch et al. 2001). Radioactivity associated with ³²P-labelled spots was quantified using a Packard InstantImager and also visualized by exposure to Kodak X-OMAT film.

Cell volume measurement

Cell volume was assayed as described by Low and colleagues (Low et al. 1996b). Monolayers of L6 myotubes were pre-incubated with HBS/10 mM D-glucose ± bumetanide (100 µM) at 37°C for 15 min. Cells were then incubated in isotonic (300 mosmol l^–1) or hypertonic (420 mosmol l^–1) media in the absence or presence of bumetanide. ³H₂O (0.1 MBq well^–1) and [¹⁴C]inulin (0.02 MBq well^–1) were added during the last 10 min of this incubation period. Intracellular volume was calculated using ³H₂O as marker for total water space and [¹⁴C]inulin as marker for extracellular water space. Data were expressed as a percentage change in intracellular volume relative to that measured in cells maintained in isotonic media.

Statistical analyses

For multiple comparisons, statistical analysis was performed using ANOVA followed by a Newman–Keuls post test. Data analysis was performed using GraphPad Prism software and considered statistically significant at P-values < 0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Expression of the Na⁺–K⁺–2Cl^– cotransporter

In order to establish the suitability of L6 myotubes as an experimental system for analysis of muscle Na⁺–K⁺–2Cl^– cotransporter function, it was first necessary to demonstrate that NKCC was expressed in this muscle cell line. Plasma membranes isolated from L6 myotubes were subjected to SDS-PAGE and Western blot analysis using T4, a monoclonal antibody against NKCC (Lytle et al. 1995). Figure 1A shows that NKCC was expressed in both L6 myoblasts and myotubes, and was detected as a single protein band of 170 kDa. The immunoreactive NKCC band observed in L6 membranes comigrated along with that from red (soleus) and white (extensor digitorum longus) rat skeletal muscle, suggesting that the cotransporter expressed in our cell line was of similar size to that present in mature adult rat skeletal muscle. A slight variation in electrophoretic mobility of the L6 immunoreactive band compared to that of rat skeletal muscle was observed. This difference may be attributable to minor variations in N-linked glycosylation of NKCC present in these membranes, as the cotransporter is known to possess two consensus glycosylation sites on the extracellular loop between transmembrane segments 7 and 8.

View larger version (22K): [in this window] [in a new window]   Figure 1.  L6 rat muscle cells express NKCC and exhibit bumetanide-sensitive 86Rb+ uptake A, plasma membranes (25 µg protein) from L6 myoblasts, L6 myotubes, red (soleus) and white (EDL) rat skeletal muscle were resolved by SDS-PAGE and subsequently immunoblotted with anti-NKCC (T4) antibody. Rat brain microsomes were run as a positive immunoreactive control. B, L6 myotubes were pre-incubated for 15 min in HBS lacking or containing bumetanide (BUM, 100 µM) or ouabain (OUA, 1 mM) prior to assay of 86Rb+ uptake for the times indicated in the absence (Total) or presence of the inhibitors. C, following pre-incubation of L6 myotubes with bumetanide at the concentrations indicated, 86Rb+ uptake was assayed in the presence of the inhibitor for 10 min. D, myotubes were pre-incubated with OUA (1 mM) and/or BUM (100 µM) as described above, followed by analysis of 86Rb+ uptake in the presence of the inhibitors for 10 min. E, the relative contribution of the Na+ pump, NKCC and ‘other’ transporters to 86Rb+ uptake was calculated from the results obtained in D. The values shown in B–D represent means + S.E.M. from between 6 and 10 experiments. *Significant change (P < 0.05) compared to the untreated control.

In order to assess the activity of NKCC in L6 myotubes, the effects of ouabain and bumetanide, which specifically inhibit potassium-mediated uptake via the Na⁺,K⁺-ATPase and NKCC, respectively, were examined. Uptake of ⁸⁶Rb⁺ (a surrogate K⁺ ion) was performed in the absence and presence of 1 mM ouabain (a concentration that maximally suppresses Na⁺,K⁺-ATPase activity in L6 cells (Sen et al. 1995)) and/or 100 µM bumetanide. Uptake was measured over 10 min for convenience, as in preliminary experiments we established that ⁸⁶Rb⁺ uptake was linear over a period of 15 min (Fig. 1B). As shown in Fig. 1C, bumetanide inhibited ⁸⁶Rb⁺ uptake in a dose-dependent manner with an IC₅₀ 1 µM. The maximum inhibition (54%) was achieved at a concentration of 100 µM bumetanide. Interestingly, however, incubation of muscle cells with ouabain alone induced a modest stimulatory effect on Rb⁺ uptake. This finding is not unprecedented and has been reported previously by others (Bakker-Grunwald et al. 1982; Aiton & Simmons, 1983; Dong et al. 1994; Coppi & Guidotti, 1997). One possible explanation for the stimulatory effect of ouabain upon Rb⁺ uptake is that Na⁺,K⁺-ATPase inhibition leads to increased K⁺ efflux via opening of K⁺ channels, and that the ensuing depletion of cellular K⁺ by this route subsequently promotes increased K⁺ uptake via NKCC. Ouabain-sensitive ⁸⁶Rb⁺ uptake was therefore assessed following inhibition of the cotransporter with bumetanide. Under these circumstances, it was found that ouabain-sensitive ⁸⁶Rb⁺ uptake accounts for 27% of total ⁸⁶Rb⁺ uptake. Figure 1E shows the relative contribution of NKCC and Na⁺-pump towards ⁸⁶Rb⁺ uptake. The bar marked as ‘other’ represents that component of ⁸⁶Rb⁺ uptake that was insensitive to both ouabain and bumetanide.

Insulin stimulates NKCC transport via a PI3 kinase-dependent pathway

To investigate the regulation of NKCC by insulin, the effect of insulin on ⁸⁶Rb⁺ uptake was examined. Muscle cells were pre-incubated in the absence and presence of bumetanide (100 µM) prior to a 15 min insulin (100 nM) incubation. Following cell exposure to the hormone, ⁸⁶Rb⁺ uptake via NKCC was increased significantly by 1.6-fold (Fig. 2A). Analysis of ouabain-sensitive ⁸⁶Rb⁺ uptake revealed that insulin caused a modest stimulation (18%) of the ion via the Na⁺,K⁺-ATPase; there was no detectable stimulation in ⁸⁶Rb⁺ uptake via routes that were insensitive to both bumetanide and ouabain (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 2.  Insulin stimulates 86Rb+ uptake in L6 myotubes and induces activation of PI3K and phosphorylation of Erk, p70 S6k and PKB L6 myotubes were pre-incubated in the absence or presence of bumetanide (100 µM) prior to incubation with insulin (100 nM, 15 min) and assay of NKCC-mediated 86Rb+ uptake. For B and C, L6 myotubes were pre-incubated with insulin (Ins, 100 nM, 15 min) in the absence or presence of wortmannin (WM, 100 nM), PD 98059 (50 µM), rapamycin (100 nM) prior to cell lysis and analyses of B, PI3K activity in p85 immunoprecipitates as described in the text (values represent means ± S.E.M. from three separate experiments) or C, SDS-PAGE and immunoblotting with antibodies against native PKB or phospho-antibodies directed against Erk1/Erk2, p70S6K or PKB. Blots are representative of three separate experiments. Bar values represent mean + S.E.M. (n = 9 for A and n = 3 for B). *Significant change from the untreated control value or between the indicated bars (P < 0.05).

Figure 3A and B show the effect of the various protein kinase inhibitors upon basal and insulin-stimulated ⁸⁶Rb⁺ transport via NKCC. With the exception of wortmanin and genistein, none of the other inhibitors tested had any significant effect on basal NKCC activity (Fig. 3A). Wortmannin and genistein reduced basal NKCC activity by 20% and 56%, respectively. The inhibitory effect of genistein is not unprecedented and has been reported previously for the ferret erythrocyte NKCC (Flatman & Creanor, 1999b). Taken together these observations imply that there may be some constitutive input from PI3K and tyrosine kinases to help maintain NKCC activity in unstimulated muscle cells. Consistent with the data shown in Fig. 2A, insulin induced a net increase in NKCC activity by 10.5 nmol min^–1 (mg protein)^–1 (representing a stimulation of 1.5-fold over control). This activation was not sensitive to PD 98059 (Erk pathway inhibitor) or SB 203580 (p38 MAP kinase inhibitor), but was reduced significantly by genistein (tyrosine kinase inhibitor) and rapamycin (mTOR inhibitor) and was effectively abolished by wortmannin (PI3K inhibitor). In separate experiments, we also assessed the effects of the tyrophostin AG1024, another tyrosine kinase inhibitor.

View larger version (18K): [in this window] [in a new window]   Figure 3.  Effects of kinase inhibitors and a constitutively active PKB on basal and insulin-stimulated NKCC-mediated 86Rb+ uptake in L6 myotubes L6 myotubes were pre-incubated for 15 min in the absence or presence of bumetanide (100 µM) prior to incubation with insulin (Ins, 100 nM, 15 min) and kinase inhibitors (wortmannin, 100 nM; PD 98059, 50 µM; rapamycin, 100 nM; genistein 50 µM). At the end of this incubation period, 86Rb+ uptake was assayed as described. The bars depict A, basal and B, insulin-stimulated NKCC-mediated 86Rb+ influx. C, insulin-stimulated 86Rb+ uptake via NKCC in L6 myotubes expressing a constitutively active membrane-targeted PKB (mPKB) or control cells transfected with the empty pCMV5 expression vector (EV). Values represent means + S.E.M. for between three and five separate experiments. *Significant change from the appropriate untreated control value in A and the insulin-stimulated value in B (P < 0.05).

PKB has been implicated in the stimulation of the slowly activating K⁺ channel KCNE1 (Embark et al. 2003), raising the possibility that it may also participate in the regulation of other K⁺ transport mechanisms. To test this possibility, we assessed NKCC activity in L6 muscle cells stably expressing a membrane-targeted form of PKB (mPKB) that is active even in the absence of insulin. We have shown previously that expressing mPKB in L6 cells mimics the stimulatory effects of insulin on GLUT4 translocation and glucose transport (Hajduch et al. 1998). However, Fig. 3C shows that expressing mPKB did not result in any such stimulatory effect on NKCC-mediated ⁸⁶Rb⁺ uptake, and, if anything, led to a marginal decrease in basal transport activity. The net increase in NKCC activity elicited by insulin was similar in muscle cells irrespective of whether they expressed mPKB or not, a finding that further excludes the likely involvement of PKB in the acute activation of NKCC by insulin.

NKCC is activated by hyperosmotic stress and plays an important role in regulatory volume increase

It has been reported that NKCC is activated by hyperosmotic-induced cell shrinkage in many cells and plays a crucial role in cell volume regulation; a process that has important implications for cell survival (reviewed in Lang et al. 2000). Shrinkage of L6 myoblasts under hyperosmotic conditions has been reported to result in a significant increase in ouabain-insensitive, bumetanide-inhibitable ⁸⁶Rb⁺ influx (Sen et al. 1995), although the underlying mechanism by which this happens in muscle cells is poorly understood. Subjecting L6 myotubes to a 10 min hyperosmotic challenge (420 mosmol l^–1) increased NKCC-mediated ⁸⁶Rb⁺ uptake by more than two-fold (Fig. 4A). Under these circumstances, hypertonicity did not have any detectable affect on ouabain-sensitive ⁸⁶Rb⁺ uptake or that component of the uptake that remains insensitive to both ouabain and bumetanide (data not shown). The increase in NKCC activity evoked by hypertonicity was just as rapid as that seen in response to insulin; NKCC was maximally stimulated by 10 min in response to both stimuli (Fig. 4B). However, peak activation was greater in response to hypertonicity than insulin, although cotransporter activation was sustained for a longer period in response to the hormone (t_1/2 for NKCC inactivation following hypertonicity 40 min; t_1/2 for NKCC inactivation following insulin >60 min) (Fig. 4B). Analysis of cell volume (Fig. 4C) revealed that muscle cells subjected to hyperosmotic stress exhibit a reduction in cell volume by 40% within 2 min, compared with cells maintained in normal iso-osmotic medium. Intracellular volume starts to recover within minutes of cells being held in hyperosmotic media and returns to near normal within 60 min, indicating that muscle cells have undergone RVI. However, in the presence of bumetanide, this cell volume recovery was not observed suggesting a crucial role for NKCC in the RVI response in L6 myotubes (Fig. 4C).

View larger version (13K): [in this window] [in a new window]   Figure 4.  Effects of hyperosmolarity on 86Rb+ uptake and intracellular volume in L6 myotubes A, L6 myotubes were pre-incubated in the absence or presence of bumetanide (100 µM) for 15 min and then either maintained in isoosmotic (300 mosmol l–1) or exposed to hyperosmotic (420 mosmol l–1) medium for 10 min ± bumetanide prior to assaying 86Rb+ uptake via NKCC. B, muscle cells were exposed to hyperosmotic media (420 mosmol l–1) or insulin (100 nM) for periods between 2 and 60 min prior to assaying bumetanide-sensitive 86Rb+ influx. The values represent the increase in NKCC-mediated uptake above that assayed at the same time points in unstimulated cells. C, changes in intracellular volume of L6 myotubes relative to that of cells maintained in normal isoosmotic media (dotted line) during a 60 min hyperosmotic challenge in the absence and presence of bumetanide (100 µM, BUM). Data points in the three panels represent means ± S.E.M. from between three and nine experiments. *Significant change (P < 0.05) compared to the isoosmotic control value.

To assess whether the increase in NKCC activity evoked by insulin and hypertonicity rely upon common signalling elements, we investigated the effects of challenging muscle cells simultaneously to both stimuli for 10 min (an incubation period that results in maximal activation of the cotransporter in response to both stimuli, Fig. 4B) in the absence and presence of wortmannin. Consistent with the data shown in Fig. 4B, hyperosmotic stress induced a modestly greater stimulation of NKCC than insulin (Fig. 5). However, when cells were incubated in hyperosmotic media in the presence of insulin, the effect on ⁸⁶Rb⁺ uptake was found to be additive. Under these conditions, wortmannin completely abolished that component of the uptake that was enhanced by insulin, but not that induced by hypertonic-induced cell shrinkage.

View larger version (26K): [in this window] [in a new window]   Figure 5.  Effects of insulin, hyperosmolarity and wortmannin on NKCC-mediated 86Rb+ uptake in L6 myotubes L6 myotubes were pre-incubated for 15 min in the absence or presence of bumetanide (100 µM) and/or wortmannin (100 nM); during the final 10 min of the pre-treatment, cells were exposed to insulin (Ins, 100 nM) and/or a hyperosmotic challenge (420 mosmol l–1) for 10 min. At the end of these pre-treatments 86Rb+ uptake via NKCC was assessed as described in the Methods. The bars depict the net stimulation of 86Rb+ uptake caused by each stimulus singularly or together over that assayed in unstimulated control cells. Values are means + S.E.M. for up to nine separate experiments. *Significant inhibition (P < 0.05) compared to the indicated value for preparations not treated with wortmannin.

A number of intracellular signalling molecules are activated in response to hypertonic shock, including tyrosine kinases (Feranchak et al. 2003) and members of the MAPK family, such as Erk1/Erk2 (Itoh et al. 1994; Bianchini et al. 1997), p38 MAPK (Raingeaud et al. 1995) and JNK (Galcheva-Gargova et al. 1994). Using phospho-specific antibodies against Erk1/Erk2, p38 MAP kinase and JNK, we observed detectable activation of both Erk and p38 MAP kinase pathways within 5 min, a time frame within which NKCC activity is also enhanced in response to hypertonicity (Fig. 6A and B). JNK was also activated under these circumstances, however, unlike Erk1/Erk2 and p38 MAP kinase, JNK phosphorylation/activation was only noticeably enhanced between 10 and 60 min of hypertonic treatment (Fig. 6C). Since JNK activation lags the observed stimulation in NKCC activity, it is unlikely to be responsible for triggering NKCC activation, which occurs rapidly within the first 5 min of subjecting L6 cells to hypertonic media (Fig. 6C). The hypertonicity-induced activation of both Erk1/Erk2 and p38 MAP kinase was abolished by prior incubation of cells with PD 98059 and SB203580, respectively.

View larger version (31K): [in this window] [in a new window]   Figure 6.  Effects of hyperosmolarity on Erk1/Erk2, p38 MAP kinase and JNK phosphorylation and tyrosine phosphorylation of NKCC in L6 myotubes L6 myotubes were either maintained in isoosmotic or hyperosmotic (420 mosmol l–1) media for the times indicated. In some experiments cells were also pre-incubated in the absence or presence of PD 98059 (50 µM), SB 203580 (10 µM) or insulin (100 nM, 10 min). Cells were lysed at the end of these incubations and lysates (50 µg protein) resolved by SDS-PAGE prior to immunoblotting with antibodies to A, Erk1/Erk2 and phospho-Erk1/Erk2; B, p38 MAP kinase and phospho-p38 MAP kinase; and C, JNK and phospho-JNK. D, NKCC was immunoprecipitated from muscle cells following a 10 min pre-incubation in media that was either isoosmotic (300 mosmol l–1) or hyperosmotic (420 mosmol l–1) in the presence or absence of genistein (Gen, 50 µM), or which had been supplemented with insulin (100 nM). The NKCC immunoprecipitate was probed with anti-NKCC (anti-T4) (top panel) or anti-phosphotyrosine (middle panel) antibodies. The immunosupernatant was also probed for NKCC (lower panel). The blots shown are representative of three experiments.

To assess the potential involvement of Erk1/Erk2, p38 MAP kinase and tyrosine kinases in the stimulation of NKCC activity by cell shrinkage, we investigated whether inhibiting these kinases prevented NKCC activation in response to hypertonicity. Figure 7A shows that the hypertonic-induced activation of NKCC was insensitive to SB 203580 (p38 MAP kinase inhibitor) or wortmannin (PI3K inhibitor). In contrast, the enhanced transport of ⁸⁶Rb⁺ via NKCC was reduced significantly by PD 98059 (Erk pathway inhibitor), and abolished completely by the tyrosine kinase inhibitor, genistein (Fig. 7A). Since genistein can have a broad influence on cellular responses, we also investigated the effects of AG1024 and ADHD, two tyrophostins that also inhibit tyrosine kinases. Genistein and AG1024, but not ADHD, reduced significantly basal NKCC activity. However, unlike genistein, AG1024 did not suppress NKCC activation by hypertoncity, whereas this stimulation was reduced in cells treated with ADHD (a modest stimulation was still observed in ADHD-treated cells, but the spread of values rendered this increase statistically insignificant). These findings imply that NKCC is regulated by at least two different types of tyrosine kinase: one sensitive to genistein and AG1024 that regulates basal NKCC activity, and another that is responsive to changes in cell volume and which exhibits sensitivity to genistein and also, partially, to ADHD.

View larger version (22K): [in this window] [in a new window]   Figure 7.  Effect of kinase inhibitors on hyperosmotic-induced 86Rb+ uptake via NKCC in L6 myotubes L6 myotubes were pre-incubated in the absence or presence of bumetanide (100 µM). Following this initial pre-incubation period, cells were either maintained in isoosmotic (300 mosmol l–1) or incubated in hyperosmotic (420 mosmol l–1) medium for 10 min in the absence or presence of bumetanide. Cells incubated in hypertonic media were also pre-exposed to kinase inhibitors (SB 203580, 10 µM; PD 98059, 50 µM; genistein, 50 µM; wortmannin, AG1024, 100 µM; ADHD, 100 µM) for 15 min. At the end of this incubation NKCC-mediated 86Rb+ uptake was quantified. A, the increase in NKCC-mediated 86Rb+ uptake elicited by hypertonicity and the effect of different kinase inhibitors on this stimulation relative to that assayed in cells maintained in isosomotic media is shown. B, the effect of three different tyrosine kinase inhibitors on basal NKCC activity (assayed in isoosmotic media) and that induced in response to hypertonicity. Values represent means + S.E.M. from three experiments. *Significant change (P < 0.05) between the indicated bars; significant inhibition compared to the untreated isoosmotic value (P < 0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

A number of studies have shown that insulin can stimulate bumetanide-sensitive K⁺ uptake in a variety of cell types (Hallbrucker et al. 1991; Sargeant et al. 1995; Longo, 1996; Sweeney et al. 1998). This stimulation in K⁺ uptake via NKCC contributes to an increase in cell volume in response to the hormone, and has been suggested to be an important determinant of cell sensitivity to insulin; a proposition based on the observation that hypertonicity-induced cell shrinkage, or cell treatment with bumetanide impairs insulin signalling in the liver (Schliess & Haussinger, 2003). If this tenet is accepted then understanding the mechanisms by which insulin regulates K⁺ uptake via NKCC in skeletal muscle is likely to be valuable, given the substantial contribution made by this tissue to whole-body insulin action. We demonstrate here that insulin acutely stimulates Rb⁺ uptake in L6 muscle cells via NKCC, and that this is PI3K dependent. This finding is consistent with reports in the literature showing that NKCC activation is critically dependent on phosphorylation of key threonine residues within its N-terminal regulatory domain, and that this event is rate determining for NKCC activation (Darman & Forbush, 2002; Lytle, 1998). Given the crucial requirement for threonine phosphorylation, it is unlikely that PI3K (a lipid kinase that also possesses serine kinase activity) directly phosphorylates NKCC, but more probable that phosphorylation of the cotransporter is deferred to one or more downstream kinases. Indeed, the finding that NKCC activation by insulin was partially dependent on stimulation of the mTOR pathway (Fig. 3B) is consistent with such a proposition. However, a sizeable component (70%) of the insulin-stimulated NKCC activity was not sensitive to rapamycin and, moreover, could not be attributed to PKB or the classical MAP kinases (Erk1 and Erk2) implicating additional, as yet unidentified, signalling inputs to the cotransporter. Our findings are, nevertheless, broadly consistent with the work of Sweeney et al. who showed that activation of NKCC by insulin in 3T3-L1 fibroblasts was also PI3K dependent, but insensitive to inhibition by the MEK inhibitor, PD 98059 (Sweeney et al. 1998). Based on experiments using a bisindolemaleimide, these authors did, however, propose a potential role for atypical PKC in the hormonal activation of NKCC in their system. It is unlikely that this kinase contributes to cotransporter activation in L6 cells since incubation of myotubes with Gö 6850 (at concentrations that inhibit PKC) failed to prevent the hormonal activation of NKCC (data not shown).

The finding that the cotransporter is activated by insulin in L6 myotubes is in full accord with several reports in the literature (Hallbrucker et al. 1991; Weil-Maslansky et al. 1994; Sargeant et al. 1995; Longo, 1996; Sweeney et al. 1998), but is at odds with the work of Gosmanov and Thomason who have reported that insulin and PI3K activation actually inhibit NKCC in incubated rat muscle preparations (Gosmanov & Thomason, 2002). The reason for this apparent discrepancy is unclear. The L6 muscle cells used in this study faithfully exhibit many of the archetypal responses elicited by insulin in rat skeletal muscle such as the translocation of the GLUT4 glucose transporter (Hajduch et al. 1998), the stimulation of glucose and amino acid transport (Hajduch et al. 1998; Hyde et al. 2002) and an increase in glycogen and protein synthesis (Cross et al. 1995), so it would seem unlikely that the observed disparity can be explained solely on the basis of differences in the experimental model used, i.e. cultured rat muscle cells vis a vis an isolated rat muscle preparation. Nevertheless, since L6 muscle cells are originally derived from neonatal muscle tissue, we cannot exclude the possibility that aspects of cotransporter function/regulation in these cells may serve to fulfil specific requirements of developing muscle that may be dispensable in adult skeletal muscle.

Plasma osmolarity can increase significantly under numerous physiological and pathological circumstances, such as following a meal, in response to increased muscular activity and hyperglycaemia associated with diabetes. Loss of muscle water under these conditions would be expected to impact negatively upon the responsiveness of skeletal muscle to insulin and its ability to regulate important muscle processes such as glycogen synthesis (Low et al. 1996a; Schliess & Haussinger, 2003). To ensure that muscle sensitivity to insulin is not compromised in the face of an increase in plasma osmolarity, it is crucial that muscle dehydration is defended by activating mechanisms that restore intramuscular volume. The suggestion that skeletal muscle is capable of mounting an effective RVI response is not recent. Blinks reported nearly four decades ago that muscle fibres undergo a reduction in intramuscular volume upon exposure to hyperosmotic medium, but that fibres rapidly restore their volume (Blinks, 1965). For technical reasons, the mechanism underpinning the RVI response in skeletal muscle has not been addressed in any significant detail, but the expression of NKCC in skeletal muscle as reported here and by others (Wong et al. 1999) would imply that it represents an important component of the RVI machinery. This proposition is supported by our finding that muscle cells incubated in hypertonic media exhibit a 40% reduction in intracellular volume that can be recovered in a bumetanide-sensitive manner. Furthermore, the observation that RVI is initiated very rapidly is compatible with the acute stimulation of NKCC, which was stimulated maximally within 10 min of cells being subjected to a hypertonic challenge. The rapid onset of RVI potentially excludes the involvement or more long-term adaptive processes, such as gene transcription and protein synthesis, in the volume recovery process. Nevertheless, it is worth stressing that sustained hypertonicity has been shown to instigate an increase in the expression of membrane transporters, such as that of the system A amino acid transporter, in an attempt to accumulate organic osmolytes and to try and reverse the gradient for water flow (Dall'Asta et al. 1999). Since cotransporter activation in response to a hypertonic challenge is primarily aimed at increasing intracellular volume, it would follow that NKCC activation by insulin should also promote an increase in cell volume. Indeed, previous work has shown that exposure of rat skeletal muscle cells to insulin results in an acute increase (20%) in cell hydration (Low et al. 1996b). This observation is not restricted to muscle cells and has also been reported in rat hepatocytes (Hallbrucker et al. 1991). Whether NKCC operates in the same operational mode when activated in response to insulin and hypertonicity is presently unknown, but addressing this issue in the future may provide further insight regarding the functional importance of the cotransporter with respect to insulin sensitivity and volume control of muscle cells.

The increase in NKCC activity caused by cell shrinkage is thought to involve a volume-sensitive kinase that phosphorylates and stimulates the cotransporter (Lytle, 1998). Several kinases exhibit activation by hypertonicity, most notably members of the MAPK family, including Erk1/Erk2 (Itoh et al. 1994; Bianchini et al. 1997), p38 MAPK (Raingeaud et al. 1995) and JNK (Galcheva-Gargova et al. 1994). While there are reports demonstrating that activated JNK can phosphorylate NKCC in vitro (Klein et al. 1999), a specific volume-sensitive kinase that directly regulates NKCC in vivo has yet to be identified. Our data indicate that while both the Erk and p38 MAPK pathways are rapidly activated by cell shrinkage, only the classical Erk pathway appears to participate in the activation of NKCC in L6 cells. Previous studies in rat muscle have indicated that Erk activation is necessary for supporting NKCC activation by catecholamimes and muscle contraction (Wong et al. 2001), although, intriguingly, pharmacological inhibition of this pathway did not inhibit NKCC activation in response to mild hypertonic stress (Gosmanov et al. 2003b) or, as shown in this study, in response to insulin. The ability of the Erk pathway to regulate NKCC appears therefore to be dependent not only on the nature of the activating stimulus but perhaps also upon stimulus strength (i.e. mild versus severe hypertonic stress).

Cell shrinkage represents a potent stimulus for activating protein-tyrosine kinases, including members of the Src family of non-receptor tyrosine kinases (Lewis et al. 2002; Feranchak et al. 2003) and focal adhesion kinase (Quadri et al. 2003). The potential involvement of tyrosine kinases in the regulation of NKCC activity is highlighted by studies showing that genistein (a tyrosine kinase inhibitor) suppresses basal and arsenite-stimulated NKCC transport in ferret erythrocytes (Flatman & Creanor, 1999a; Flatman & Creanor, 1999b) and that in L6 muscle cells it not only affects basal cotransporter activity, but completely abolishes the hypertonicity-induced activation of NKCC (present study). However, our findings also suggest that NKCC is not regulated directly by tyrosine phosphorylation, an observation that is consistent with studies assessing cotransporter phosphorylation by phospho-peptide mapping and phospho-tyrosine antibodies (Lytle, 1997; Matskevich & Flatman, 2003). The lack of any direct tyrosine phosphorylation of the cotransporter would therefore imply that tyrosine kinases may instead play a critical role in the volume-sensing process and perhaps signal to other serine/threonine kinases involved in cotransporter phosphorylation/activation. Evidence in the literature demonstrates that activation of the Erk pathway by phenylephrine is dependent upon a genistein-sensitive step in rat ventricular myocytes (Thorburn & Thorburn, 1994), and it is therefore plausible that a volume-regulated tyrosine kinase may similarly act to regulate the Erk-dependent activation of NKCC during hypertonic stress in L6 muscle cells.

Previous work has shown that while NKCC can be activated by numerous stimuli or agents (cell shrinkage, cAMP, fluoride and calyculin-A), no two stimuli in combination are capable of causing greater activation and phosphorylation of NKCC than the most potent of the two stimuli acting alone (Lytle, 1997). This observation, and the finding that different stimuli generate identical patterns of NKCC phosphorylation based on phospho-peptide mapping, would support the view that cotransporter activation by diverse stimuli may be mediated by a single protein kinase (Lytle, 1997). Thus a major finding to emerge from the present study was that insulin and hypertonicity induced an additive stimulation of NKCC. Moreover, while the hormonal activation of NKCC was clearly PI3K dependent, the increase elicited by hypertonicity was not. This suggests that these two stimuli utilize distinct signalling molecules to activate NKCC and raises the possibility that the cotransporter may be activated by more than one kinase. While this proposition is supported by work from several groups (reviewed in Flatman, 2002), we cannot exclude the possibility that insulin- and volume-regulated signalling converges onto a common kinase whose activity and ability to phosphorylate NKCC may be enhanced in an additive manner. Assessing the level of NKCC phosphorylation and whether the two stimuli evoke a similar pattern of cotransporter phosphorylation may prove useful in resolving this issue, and represents an important future investigative goal.

In summary the present work has shown that L6 muscle cells express a functional NKCC transporter that is rapidly activated in response to insulin and cell shrinkage and that it plays a key role in the RVI response in these cells. Cotransporter stimulation by insulin or hyperosmolarity is additive and involves distinct postmembrane signalling cascades that are likely to modify the phosphorylation and hence activation status of the cotransporter.

	References

Top Abstract Introduction Methods Results Discussion References

 
Aiton JF & Simmons NL (1983). Effect of ouabain upon diuretic-sensitive K⁺ transport in cultured cells. Evidence for separate modes of operation of the transporter. Biochim Biophys Acta 734, 279–289.[Medline]

Bakker-Grunwald T, Ogden P & Lamb JF (1982). Effects of ouabain and osmolarity on bumetanide-sensitive potassium transport in simian virus-transformed 3T3 cells. Biochim Biophys Acta 687, 333–336.[Medline]

Bianchini L, L'Allemain G & Pouyssegur J (1997). The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na⁺/H⁺ exchanger (NHE1 isoform) in response to growth factors. J Biol Chem 272, 271–279.[Abstract/Free Full Text]

Blinks JR (1965). Influence of osmotic strength on cross section and volume of isolated single muscle fibres. J Physiol 177, 42–57.[Free Full Text]

Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Clausen T (1986). Regulation of active Na-K transport in skeletal muscle. Physiol Rev 66, 542–580.[Free Full Text]

Coppi MV & Guidotti G (1997). The alpha2L111R,N122D isoform of the Na,K-ATPase expressed in HeLa cells does not undergo an adipocyte-like increase in activity in response to insulin. Biochem Biophys Res Commun 236, 444–448.[CrossRef][Medline]

Cross DAE, Alessi DR, Cohen P, Andjelkovic M & Hemmings BA (1995). Inhibition of glycogen synthase kinase-3 by insulin-mediated protein kinase B. Nature 378, 785–789.[CrossRef][Medline]

Dall'Asta V, Bussolati O, Sala R, Parolari A, Alamanni F, Biglioli P & Gazzola GC (1999). Amino acids are compatible osmolytes for volume recovery after hypertonic shrinkage in vascular endothelial cells. Am J Physiol Cell Physiol 276, C865–C872.[Abstract/Free Full Text]

Darakhshan F, Hajduch E, Kristiansen S, Richter EA & Hundal HS (1998). Biochemical and functional characterization of the GLUT5 fructose transporter in rat skeletal muscle. Biochem J 336, 361–366.[Medline]

Darman RB & Forbush B (2002). A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1. J Biol Chem 277, 37542–37550.[Abstract/Free Full Text]

Dong J, Delamere NA & Coca-Prados M (1994). Inhibition of Na⁺-K⁺-ATPase activates Na⁺-K⁺-2Cl^– cotransporter activity in cultured ciliary epithelium. Am J Physiol Cell Physiol 266, C198–C205.[Abstract/Free Full Text]

Embark HM, Bohmer C, Vallon V, Luft F & Lang F (2003). Regulation of KCNE1-dependent K⁺ current by the serum and glucocorticoid-inducible kinase (SGK) isoforms. Pflugers Arch 445, 601–606.[Medline]

Feranchak AP, Kilic G, Wojtaszek PA, Qadri I & Fitz JG (2003). Volume-sensitive tyrosine kinases regulate liver cell volume through effects on vesicular trafficking and membrane Na⁺ permeability. J Biol Chem 278, 44632–44638.[Abstract/Free Full Text]

Flatman PW (2002). Regulation of Na-K-2Cl cotransport by phosphorylation and protein–protein interactions. Biochim Biophys Acta 1566, 140–151.[Medline]

Flatman PW & Creanor J (1999a). Regulation of Na⁺-K⁺-2Cl^– cotransport by protein phosphorylation in ferret erythrocytes. J Physiol 517, 699–708.[Abstract/Free Full Text]

Flatman PW & Creanor J (1999b). Stimulation of Na⁺-K⁺-2Cl^– cotransport by arsenite in ferret erythrocytes. J Physiol 519, 143–152.[Abstract/Free Full Text]

Flemmer AW, Gimenez I, Dowd BF, Darman RB & Forbush B (2002). Activation of the Na-K-Cl cotransporter NKCC1 detected with a phospho-specific antibody. J Biol Chem 277, 37551–37558.[Abstract/Free Full Text]

Galcheva-Gargova Z, Derijard B, Wu IH & Davis RJ (1994). An osmosensing signal transduction pathway in mammalian cells. Science 265, 806–808.[Abstract/Free Full Text]

Gosmanov AR, Lindinger MI & Thomason DB (2003a). Riding the tides: K⁺ concentration and volume regulation by muscle Na⁺-K⁺-2Cl^– cotransport activity. News Physiol Sci 18, 196–200.[Abstract/Free Full Text]

Gosmanov AR, Schneider EG & Thomason DB (2003b). NKCC activity restores muscle water during hyperosmotic challenge independent of insulin, ERK, and p38 MAPK. Am J Physiol Regul Integr Comp Physiol 284, R655–R665.[Abstract/Free Full Text]

Gosmanov AR & Thomason DB (2002). Insulin and isoproterenol differentially regulate mitogen-activated protein kinase-dependent Na⁺-K⁺-2Cl^– cotransporter activity in skeletal muscle. Diabetes 51, 615–623.[Abstract/Free Full Text]

Haas M (1994). The Na-K-Cl cotransporters. Am J Physiol Cell Physiol 267, C869–C885.[Abstract/Free Full Text]

Hajduch E, Alessi DR, Hemmings BA & Hundal HS (1998). Constitutive activation of Protein Kinase B (PKB) by membrane targeting promotes glucose and System A amino acid transport, protein synthesis and GSK3 inactivation in L6 muscle cells. Diabetes 47, 1006–1013.[Abstract]

Hajduch E, Balendran A, Batty IH, Litherland GJ, Blair AS, Downes CP & Hundal HS (2001). Ceramide impairs the insulin-dependent membrane recruitment of Protein Kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells. Diabetologia 44, 173–183.[CrossRef][Medline]

Hoffmann EK & Dunham PB (1995). Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 161, 173–262.[Medline]

Hundal HS, Marette A, Mitsumoto Y, Ramlal T, Blostein R & Klip A (1992). Insulin induces translocation of the 2 and ß1 subunits of the Na/K-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle. J Biol Chem 267, 5040–5043.[Abstract/Free Full Text]

Hyde R, Peyrollier K & Hundal HS (2002). Insulin promotes the cell surface recruitment of the SAT2/ATA2 System A amino acid transporter from an endosomal compartment in skeletal muscle cells. J Biol Chem 277, 13628–13634.[Abstract/Free Full Text]

Itoh T, Yamauchi A, Miyai A, Yokoyama K, Kamada T, Ueda N & Fujiwara Y (1994). Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest 93, 2387–2392.[Medline]

Kapus A, Di Ciano C, Sun J, Zhan X, Kim L, Wong TW & Rotstein OD (2000). Cell volume-dependent phosphorylation of proteins of the cortical cytoskeleton and cell-cell contact sites. The role of Fyn and FER kinases. J Biol Chem 275, 32289–32298.[Abstract/Free Full Text]

Klein JD, Lamitina ST & O'Neill WC (1999). JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro. Am J Physiol Cell Physiol 277, C425–C431.[Abstract/Free Full Text]

Lang F, Ritter M, Gamper N, Huber S, Fillon S, Tanneur V, Lepple-Wienhues A, Szabo I & Gulbins E (2000). Cell volume in the regulation of cell proliferation and apoptotic cell death. Cell Physiol Biochem 10, 417–428.[Medline]

Lewis A, Di Ciano C, Rotstein OD & Kapus A (2002). Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization. Am J Physiol Cell Physiol 282, C271–C279.[Abstract/Free Full Text]

Liedtke CM & Cole TS (2002). Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK. Biochim Biophys Acta 1589, 77–88.[Medline]

Lindinger MI, Hawke TJ, Lipskie SL, Schaefer HD & Vickery L (2002). K⁺ transport and volume regulatory response by NKCC in resting rat hindlimb skeletal muscle. Cell Physiol Biochem 12, 279–292.[CrossRef][Medline]

Longo N (1996). Insulin stimulates the Na⁺,K⁺-ATPase and the Na⁺/K⁺/Cl^– cotransporter of human fibroblasts. Biochim Biophys Acta 1281, 38–44.[Medline]

Low SY, Rennie MJ & Taylor PM (1996a). Modulation of glycogen synthesis in rat skeletal muscle by changes in cell volume. J Physiol 495, 299–303.[Abstract/Free Full Text]

Low SY, Taylor PM & Rennie MJ (1996b). Responses of glutamine transport in cultured rat skeletal muscle to osmotically induced changes in cell. J Physiol 492, 877–885.[Abstract/Free Full Text]

Lytle C (1997). Activation of the avian erythrocyte Na-K-Cl cotransport protein by cell shrinkage, cAMP, fluoride, and calyculin-A involves phosphorylation at common sites. J Biol Chem 272, 15069–15077.[Abstract/Free Full Text]

Lytle C (1998). A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells. Am J Physiol Cell Physiol 274, C1002–C1010.[Abstract/Free Full Text]

Lytle C, Xu JC, Biemesderfer D & Forbush B III (1995). Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269, C1496–C1505.[Abstract/Free Full Text]

Matskevich I & Flatman PW (2003). Regulation of Na⁺-K⁺-2Cl^– cotransport by threonine phosphorylation in ferret red cells. J Physiol 547P, C20.

Quadri SK, Bhattacharjee M, Parthasarathi K, Tanita T & Bhattacharya J (2003). Endothelial barrier strengthening by activation of focal adhesion kinase. J Biol Chem 278, 13342–13349.[Abstract/Free Full Text]

Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ & Davis RJ (1995). Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270, 7420–7426.[Abstract/Free Full Text]

Roger F, Martin PY, Rousselot M, Favre H & Feraille E (1999). Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory Volume increase response. J Biol Chem 274, 34103–34110.[Abstract/Free Full Text]

Sargeant RJ, Liu Z & Klip A (1995). Action of insulin on Na⁺-K⁺-ATPase and the Na⁺-K⁺-2Cl^– cotransporter in 3T3-L1 adipocytes. Am J Physiol Cell Physiol 269, C217–C225.[Abstract/Free Full Text]

Schliess F & Haussinger D (2003). Cell volume and insulin signaling. Int Rev Cytol 225, 187–228.[Medline]

Sen CK, Hanninen O & Orlov SN (1995). Unidirectional sodium and potassium flux in myogenic L6 cells: mechanisms and volume-dependent regulation. J Appl Physiol 78, 272–281.[Abstract/Free Full Text]

Standaert ML, Bandyopadhyay G, Perez L, Price D, Galloway L, Poklepovic A, Sajan MP, Cenni V, Sirri A, Moscat J, Toker A & Farese RV (1999). Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes. J Biol Chem 274, 25308–25316.[Abstract/Free Full Text]

Sweeney G, Somwar R, Ramlal T, Martin-Vasallo P & Klip A (1998). Insulin stimulation of K⁺ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta. Diabetologia 41, 1199–1204.[CrossRef][Medline]

Thorburn J & Thorburn A (1994). The tyrosine kinase inhibitor, genistein, prevents alpha-adrenergic-induced cardiac muscle cell hypertrophy by inhibiting activation of the Ras-MAP kinase signaling pathway. Biochem Biophys Res Commun 202, 1586–1591.[CrossRef][Medline]

Hallbrucker C, Lang F, Gerok W & Haussinger D (1991). Modification of liver cell Volume by insulin and glucagon. Pflugers Arch 418, 519–521.[CrossRef][Medline]

Weil-Maslansky E, Gutman Y & Sasson S (1994). Insulin activates furosemide-sensitive K⁺ and Cl^– uptake system in BC3H1 cells. Am J Physiol Cell Physiol 267, C932–C939.[Abstract/Free Full Text]

Wong JA, Fu L, Schneider EG & Thomason DB (1999). Molecular and functional evidence for Na⁺-K⁺-2Cl^– cotransporter expression in rat skeletal muscle. Am J Physiol Regul Integr Comp Physiol 277, R154–R161.[Abstract/Free Full Text]

Wong JA, Gosmanov AR, Schneider EG & Thomason DB (2001). Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle. Am J Physiol Regul Integr Comp Physiol 281, R561–R571.[Abstract/Free Full Text]

	Acknowledgements

 
We are grateful to Dr Alexander Harper for useful discussions and for providing constructive comments on the manuscript. H.Z. was supported by an ORS award from the CVCP and a University of Dundee Postgraduate Scholarship. This work was supported by Diabetes UK, Diabetes Research & Wellness Foundation and the BBSRC.

This article has been cited by other articles:

</

				Y. Jing, J. A. Dowdy, M. R. Van Scott, and J. S. Fedan Hyperosmolarity-Induced Dilation and Epithelial Bioelectric Responses of Guinea Pig Trachea in Vitro: Role of Kinase Signaling J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 186 - 195. [Abstract] [Full Text] [PDF]